Influence of indole-butyric acid and electro-pulse on in vitro rooting and development of olive (Olea europea L.) microshoots.

PubMed

Padilla, Isabel Maria Gonzalez; Vidoy, I; Encina, C L

2009-09-01

The effects of indole-butyric acid (IBA) and electro-pulses on rooting and shoot growth were studied in vitro, using olive shoot cultures. Tested shoots were obtained from seedlings belonging to three Spanish cultivars, 'Arbequina', 'Manzanilla de Sevilla' and 'Gordal Sevillana', which have easy-, medium- and difficult-to-root rooting abilities, respectively. The standard two-step rooting method (SRM), consisting of root induction in olive rooting medium supplemented with 0, 0.1 or 1 mg/l IBA followed by root elongation in the same rooting medium without IBA, was compared with a novel one-step method consisting of shoot electro-pulses of 250, 1,250 or 2,500 V in a solution of IBA (0, 0.1 or 1 mg/l) and direct transferral to root elongation medium. The rooting percentage of the seedling-derived shoots obtained with the SRM was 76% for 'Arbequina' and 'Gordal Sevillana' cultivars and 100% for 'Manzanilla de Sevilla' cultivar, whereas with the electro-pulse method, the rooting percentages were 68, 64 and 88%, respectively. IBA dipping without pulse produced 0% rooting in 'Arbequina' seedling-derived shoots. The electroporation in IBA not only had an effect on shoot rooting but also on shoot growth and development, with longer shoots and higher axillary shoot sprouting and growth after some of the treatments. These effects were cultivar-dependent. The electro-pulse per se could explain some of these effects on shoot development.

Micropropagation of Crataeva adansonii D.C. Prodr: an ornamental avenue tree.

PubMed

Tyagi, Purnima; Sharma, P K; Kothari, S L

2010-01-01

In this chapter, we describe multiplication of the superior and elite tree of Crataeva adansonii using plant tissue culture techniques. An ornamental and avenue tree, it is not available in abundance because of poor seed germination and seedling establishment. It reproduces in nature by root suckers, but that restricts its distribution to very limited areas. Efficient procedures are outlined for plant regeneration through direct shoot bud formation, indirect organogenesis, and somatic embryogenesis through callus formation. Different explants were utilized for separate pathways of regeneration. Murashige and Skoog's (MS) medium containing 3 mg/L BA and 0.05-0.1 mg/L NAA is most effective in direct induction of axillary buds from nodal explants and shoot tips. Adventitious shoots developed from leaves on MS medium containing 3 mg/L BA and 0.1 mg/L NAA. De novo shoots were obtained from the anthers on MS medium supplemented with 3 mg/L BA. Somatic embryos developed on half strength MS medium containing 0.1 mg/L 2, 4-D. Roots were induced at the cut ends of shoots on MS basal medium devoid of growth regulators. The plantlets were then transferred to pots.

Plant regeneration from hypocotyl- and anther-derived callus of berseem clover. [Trifolium alexandrium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mokhtarzedeh, A.; Constantin, M.J.

1978-01-01

Plants were regenerated from hypocotyl and anther explants of berseem clover (Trifolium alexandrinum L.) on Murashige and Skoog (MS) medium containing various combinations of plant growth regulators. The most efficient production of plants from hypocotyl explants involved: callus induction on MS medium with 1.0 mg/liter of naphthaleneacetic acid (NAA) and 1.5 mg/liter 6-furfurylaminopurine (KIN); callus increase on MS medium with 2.0 mg/liter of NAA and 0.1 mg/liter of N/sup 6/-(..delta../sup 2/-isopentenyl) adenine (2iP); induction of shoots on MS medium with 0.5 mg/liter each of NAA and KIN followed by induction of roots on MS medium with 1.0 mg/liter of indoleaceticmore » acid (IAA) and 0.1 mg/liter of 6-benzylaminopurine (BAP). Suspension cultures in liquid MS medium containing 2.0 mg/liter of NAA and 0.2 mg/liter of 2iP provided filterable cell preparations with 45% viable cells, 4% of which gave rise to colonies within 3 weeks after transfer to agar plates. Shoot development was observed when callus from the colonies was cultured on MS medium with 0.5 mg/liter of NAA and KIN. Preliminary results indicate that cells of root tips from hypocotyl- and anther-derived callus have the expected diploid and haploid number of chromosomes (2n = 16 and n = 8, respectively).« less

In vitro propagation and production of cardiotonic glycosides in shoot cultures of Digitalis purpurea L. by elicitation and precursor feeding.

PubMed

Patil, Jitendra Gopichand; Ahire, Mahendra Laxman; Nitnaware, Kirti Manik; Panda, Sayantan; Bhatt, Vijay P; Kishor, Polavarapu B Kavi; Nikam, Tukaram Dayaram

2013-03-01

Digitalis purpurea L. (Scrophulariaceae; Foxglove) is a source of cardiotonic glycosides such as digitoxin and digoxin which are commercially applied in the treatment to strengthen cardiac diffusion and to regulate heart rhythm. This investigation deals with in vitro propagation and elicited production of cardiotonic glycosides digitoxin and digoxin in shoot cultures of D. purpurea L. In vitro germinated seedlings were used as a primary source of explants. Multiple shoot formation was achieved for three explant types (nodal, internodal, and leaf) cultured on Murashige and Skoog (MS) medium with several treatments of cytokinins (6-benzyladenine-BA; kinetin-Kin; and thidiazuron-TDZ) and auxins (indole-3-acetic acid-IAA; α-naphthaleneacetic acid-NAA; and 2,4-dichlorophenoxy acetic acid-2,4-D). Maximum multiple shoots (12.7 ± 0.6) were produced from nodal explants on MS + 7.5 μM BA. Shoots were rooted in vitro on MS containing 15 μM IAA. Rooted plantlets were successfully acclimatized. To further maintain the multiple shoot induction, mother tissue was cut into four equal parts and repeatedly sub-cultured on fresh shoot induction liquid medium after each harvest. On adaptation of this strategy, an average of 18 shoots per explant could be produced. This strategy was applied for the production of biomass and glycosides digitoxin and digoxin in shoot cultures on MS medium supplemented with 7.5 μM BA and several treatments with plant growth regulators, incubation period, abiotic (salicylic acid, mannitol, sorbitol, PEG-6000, NaCl, and KCl), biotic (Aspergillus niger, Helminthosporium sp., Alternaria sp., chitin, and yeast extract) elicitors, and precursors (progesterone, cholesterol, and squalene). The treatment of KCl, mycelial mass of Helminthosporium sp., and progesterone were highly effective for the production of cardenolides. In the presence of progesterone (200 to 300 mg/l), digitoxin and digoxin accumulation was enhanced by 9.1- and 11.9-folds respectively.

Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

PubMed

Sahu, Jyoti; Khan, Shagufta; Sahu, Ram Kumar; Roy, Amit

2014-04-01

Multiple shoots of Dalbergia sissoo Roxb. (Sissoo) were incited from seeds through indirect somatic embryogenesis method. Seeds were inoculated in Murashige and Skoog's medium without any growth hormone. Than cotyledonary leaves were struck and used for callus induction on MS medium amplified with 2, 4-dichlorophenoxyacetic acid (0.5 to 4 mg mL(-1)). After 3 to 4 weeks the embryogenic callus clumps was transferred to medium supplemented with cytokinin (BAP 1 to 5 mg L(-1), kinetin 1-5.0 mg L(-1)) for embryo maturation and germination. The high-frequency shoot proliferation (82%) and maximum number of shoots per explants were recorded in MS medium containing NAA (0.5)+BAP (0.5). The findings of recent investigations have shown that, it is possible to induce indirect somatic embryogenesis in Dalbergia sissoo and plant regeneration from callus cultures derived from cotyledonary leaves as explants.

Induction of multiple shoots from leaf segments, in vitro-flowering and fruiting of a dwarf tomato (Lycopersicon esculentum).

PubMed

Rao, Kokkirala Venugopal; Kiranmayee, Kasula; Pavan, Umate; Sree, Telakalapalli Jaya; Rao, Alleni V; Sadanandam, Abbagani

2005-08-01

Multiple shoots were induced from leaf explants of Lycopersicon esculentum cultivar MicroTom, within 20-25d, on MS medium supplemented with 8.9 microM benzylaminopurine (BAP)+1.14 microM indole-3-acetic acid (IAA). For rooting, elongated microshoots were excised and transferred onto MS medium supplemented with 4.9 microM indole-3-butyric acid (IBA). Well-developed roots and flower raceme were obtained on d 7 and 13, respectively, upon transfer of the microshoots onto rooting medium. The flowers self-fertilized in vitro and produced mature fruits in additional 15-17d of culture.

A rapid method to increase the number of F₁ plants in pea (Pisum sativum) breeding programs.

PubMed

Espósito, M A; Almirón, P; Gatti, I; Cravero, V P; Anido, F S L; Cointry, E L

2012-08-16

In breeding programs, a large number of F₂ individuals are required to perform the selection process properly, but often few such plants are available. In order to obtain more F₂ seeds, it is necessary to multiply the F₁ plants. We developed a rapid, efficient and reproducible protocol for in vitro shoot regeneration and rooting of seeds using 6-benzylaminopurine. To optimize shoot regeneration, basic medium contained Murashige and Skoog (MS) salts with or without B5 Gamborg vitamins and different concentrations of 6-benzylaminopurine (25, 50 and 75 μM) using five genotypes. We found that modified MS (B5 vitamins + 25 μM 6-benzylaminopurine) is suitable for in vitro shoot regeneration of pea. Thirty-eight hybrid combinations were transferred onto selected medium to produce shoots that were used for root induction on MS medium supplemented with α-naphthalene-acetic acid. Elongated shoots were developed from all hybrid genotypes. This procedure can be used in pea breeding programs and will allow working with a large number of plants even when the F₁ plants produce few seeds.

Effect of cytokinins on in vitro multiplication of Sophora tonkinensis

PubMed Central

Jana, Sonali; Sivanesan, Iyyakkannu; Jeong, Byoung Ryong

2013-01-01

Objective To determine the effects of different cytokinins at various concentrations on in vitro shoot multiplication of an important medicinal plant. Methods Nodal explants (1.5-2.0 cm) of Sophora tonkinensis were used. Multiple shoots were induced from nodal explants cultured on the Murashige and Skoog (MS) medium supplemented with 0.0, 0.5, 1.0, 2.0, 4.0, 8.0, or 16.0 µmol 2-isopentyladenine (2iP), N6 benzyladenine, kinetin or thiadiazuron. Results Among the four investigated cytokinins, 2iP showed the best response for shoot multiplication. Maximum shoot induction (75%) was achieved on the MS medium supplemented with 2.0 µmol 2iP, with a mean number of 5.0 shoots per explant. In comparison to other cytokinins tried, 2iP showed the highest shoot elongation with a mean shoot length of 4.8 cm. Root initiation was observed within 15 d within the transfer of shoots onto the MS basal medium, and the rooting percentage was 100% with a mean number of 5.4 roots per shoot and root length of 6.2 cm over a period of 4 weeks. The healthy plants, hardened and transferred to a greenhouse for proper acclimatization, exhibited 100% survival. Conclusions It can be summarized that 2iP is the optimal plant growth regulator for Sophora multiplication. PMID:23836310

Assessment of the potentiality of TDZ on multiple shoot induction in Bauhinia tomentosa L., a woody legume.

PubMed

Naz, Ruphi; Anis, M; Aref, I M

2012-12-01

An efficient and reproducible protocol for in vitro multiplication of Bauhinia tomentosa L. was developed. Multiple shoots were regenerated from cotyledonary node and stem nodal segments excised from in vitro raised seedlings on Murashige and Skoog (MS) medium supplemented with different concentrations (0.1, 0.3, 0.5, 0.8 and 1.0 μM) of thidiazuron (TDZ). The maximum response (62.6%) was recorded on MS medium amended with 0.8 μM TDZ. A long exposure to TDZ for 8 weeks showed abnormalities such as fasciation and compact shoots formation. To avoid adverse effects of prolonged exposure to TDZ in long-term establishment, the culture were transferred to TDZ free MS medium for further multiplication and elongation. The highest number of shoots and shoot length were recorded at the end of fourth subculture passage. Ex vitro rooting was achieved when the basal cut end of regenerated shoots were dipped in 200 μM indole-3-butyric acid (IBA) for half an hour followed by their transplantation in plastic pots filled with sterile Soilrite™ where 60% plantlets grew well and all expressed normal development.

[Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis].

PubMed

Zhu, Li-Fang; Xu, Chao; Zhu, Zai-Biao; Yang, He-Tong; Guo, Qiao-Sheng; Xu, Hong-jian; Ma, Hong-Jian; Zhao, Gui-Hua

2014-08-01

To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.

The study of ascorbate peroxidase, catalase and peroxidase during in vitro regeneration of Argyrolobium roseum.

PubMed

Habib, Darima; Chaudhary, Muhammad Fayyaz; Zia, Muhammad

2014-01-01

Here, we demonstrate the micropropagation protocol of Argyrolobium roseum (Camb.), an endangered herb exhibiting anti-diabetic and immune-suppressant properties, and antioxidant enzymes pattern is evaluated. Maximum callogenic response (60 %) was observed from leaf explant at 1.0 mg L(-1) 1-nephthalene acetic acid (NAA) and 0.5 mg L(-1) 6-benzyl aminopurine (BA) in Murashige and Skoog (MS) medium using hypocotyl and root explants (48 % each). Addition of AgNO3 and PVP in the culture medium led to an increase in callogenic response up to 86 % from leaf explant and 72 % from hypocotyl and root explants. The best shooting response was observed in the presence of NAA, while maximum shoot length and number of shoots were achieved based on BA-supplemented MS medium. The regenerated shoots were rooted and successfully acclimatized under greenhouse conditions. Catalase and peroxidase enzymes showed ascending pattern during in vitro plant development from seed while ascorbate peroxidase showed descending pattern. Totally reverse response of these enzymes was observed during callus induction from three different explants. During shoot induction, catalase and peroxidase increased at high rate while there was a mild reduction in ascorbate peroxidase activity. Catalase and peroxidase continuously increased; on the other hand, ascorbate peroxidase activity decreased during root development and acclimatization states. The protocol described here can be employed for the mass propagation and genetic transformation of this rare herb. This study also highlights the importance and role of ascorbate peroxidase, catalase, and peroxidase in the establishment of A. roseum in vitro culture through callogenesis and organogenesis.

Somatic embryogenesis and plant regeneration in Carica papaya L. tissue culture derived from root explants.

PubMed

Chen, M H; Wang, P J; Maeda, E

1987-10-01

The regeneration potential of shoot tip, stem, leaf, cotyledon and root explants of two papaya cultivars (Carica papaya cv. 'Solo' and cv. 'Sunrise') were studed. Callus induction of these two cultivars of papaya showed that the shoot tips and stems are most suitable for forming callus, while leaves, cotyledons and roots are comparatively difficult to induce callus. Callus induction also varied with the varities. Somatic embryogenesis was obtained from 3-month-old root cultures. A medium containing half strength of MS inorganic salts, 160 mg/l adenine sulfate, 1.0 mg/1 NAA, 0.5 mg/1 kinetin and 1.0 mg/1 GA3 was optimal for embryogenesis. The callus maintained high regenerative capacity after two years of culture on this medium. Plants derived from somatic embryos were obtained under green-house conditions.

Combination of the Auxins NAA, IBA, and IAA with GA3 Improves the Commercial Seed-Tuber Production of Potato (Solanum tuberosum L.) under In Vitro Conditions

PubMed Central

Kumlay, Ahmet Metin

2014-01-01

The study compared the effects of 1.0 × MS medium containing various concentrations of α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), alone or in combination with gibberellic acid (GA3) in micropropagation of three potato (Solanum tuberosum L.) cultivars Pasinler, Granola, and Caspar using binodal stem cuttings. The results testified improved regeneration on 1.0 × MS medium containing variants of NAA, IAA, and IBA plus GA3 on all cultivars. The minimum days to shoot induction on three cultivars ranged 4.25–5 d on 1.0 × MS medium containing 0.25 mg L−1  GA3 + 1 mg L−1 NAA. The longest shoots (11.8 cm), maximum number of nodes (13.50), and maximum number of leaves (11.00) were recorded on cv. Caspar on 1.0 × MS medium containing 1 mg L−1  NAA + 0.25 mg L−1 GA3. The minimum time to root induction (12.25 d) was noted on cv. Pasinler on the same medium. All of the regenerated shoots could be easily rooted. The results showed that the combined effect of various concentrations of NAA, IAA, and IBA plus GA3 was more pronounced compared to the auxins used alone. The results of this research are of significant importance for potato breeders. PMID:25028654

Combination of the auxins NAA, IBA, and IAA with GA3 improves the commercial seed-tuber production of potato (Solanum tuberosum L.) under in vitro conditions.

PubMed

Kumlay, Ahmet Metin

2014-01-01

The study compared the effects of 1.0 × MS medium containing various concentrations of α-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), alone or in combination with gibberellic acid (GA3) in micropropagation of three potato (Solanum tuberosum L.) cultivars Pasinler, Granola, and Caspar using binodal stem cuttings. The results testified improved regeneration on 1.0 × MS medium containing variants of NAA, IAA, and IBA plus GA3 on all cultivars. The minimum days to shoot induction on three cultivars ranged 4.25-5 d on 1.0 × MS medium containing 0.25 mg L(-1) GA3 + 1 mg L(-1) NAA. The longest shoots (11.8 cm), maximum number of nodes (13.50), and maximum number of leaves (11.00) were recorded on cv. Caspar on 1.0 × MS medium containing 1 mg L(-1) NAA + 0.25 mg L(-1) GA3. The minimum time to root induction (12.25 d) was noted on cv. Pasinler on the same medium. All of the regenerated shoots could be easily rooted. The results showed that the combined effect of various concentrations of NAA, IAA, and IBA plus GA3 was more pronounced compared to the auxins used alone. The results of this research are of significant importance for potato breeders.

[In vitro flowering of cultures from a hybrid of Cymbidium goeringii and C. hybridium].

PubMed

Zheng, Li-Ming; Pang, Ji-Liang

2006-06-01

Wild-type female spring orchid (Cymbidium goeringii) was crossed with male Cymbidium hybridium. Over eight hundred protocorm clones were obtained from hybrid offsprings. Among them, one protocorm clone was identified to differentiate visible floral buds two months after subculture in vitro (Plate I). The protocorms and shoots derived from this clone were further used in studying the effects of abscisic acid (ABA) and paclobutrazol (PP333) pretreatment as well as different concentrations of 6-benzyladenine (6-BA) on floral bud differentiation. The optimum combination of hormones in floral bud induction was 6-BA 1.0 mg/L and NAA 0.1 mg/L, and total frequency of floral bud formation was up to 31% (Table 1). The optimum length of shoots used in floral bud induction was 1-2 cm, and the frequency of floral bud formation was 19% (Table 1). The increase in total frequency was not significant in floral bud induction from protocorms and shoots with length of 1-2 cm or 2-4 cm cultured on MS medium containing 6-BA 1.0 mg/L and NAA 0.1 mg/L after pretreatment on MS medium supplemented with ABA 0.5 mg/L and PP333 0.5 mg/L for 35 d (Table 2).

Micropropagation and Biomass Production of True-to-Type Stevia rebaudiana Bertoni.

PubMed

Modi, Arpan R; Sharma, Vikas; Patil, Ghanshyam; Singh, Amritpal S; Subhash, N; Kumar, Nitish

2016-01-01

Here we describe an efficient micropropagation protocol for Stevia rebaudiana Bertoni. We present experiments carried out to optimize the suitable media for in vitro shoot multiplication and root induction and to study the effect of culture vessel on shoot multiplication. Among all different media tested for in vitro shoot multiplication, hormone-free liquid medium is most suitable. The highest number of nodes per shoot (5.4) and length of shoot (4.76 cm) at 4 weeks after subculturing are observed when single node explants are placed on modified MS medium supplemented with 1 % sucrose and 0.7 % agar. The highest response of multiplication rate (9.56) is observed on half strength of macroelement of MS with full strength of microelement of MS and 170 mg/l KH2PO4, and 185 mg/l MgSO4 in plastic growth container. Further, RAPD marker analysis of in vitro-raised plants maintained their clonal fidelity and true-to-type without showing any somaclonal variation.

Improved tissue culture conditions for the emerging C4 model Panicum hallii.

PubMed

Grant, Joshua N; Burris, Jason N; Stewart, C Neal; Lenaghan, Scott C

2017-04-27

Panicum hallii Vasey (Hall's panicgrass) is a compact, perennial C 4 grass in the family Poaceae, which has potential to enable bioenergy research for switchgrass (Panicum virgatum L.). Unlike P. hallii, switchgrass has a large genome, allopolyploidy, self-incompatibility, a long life cycle, and large stature-all suboptimal traits for rapid genetics research. Herein we improved tissue culture methodologies for two inbred P. hallii populations: FIL2 and HAL2, to enable further development of P. hallii as a model C 4 plant. The optimal seed-derived callus induction medium was determined to be Murashige and Skoog (MS) medium supplemented with 40 mg L -1 L-cysteine, 300 mg L -1 L-proline, 3% sucrose, 1 g L -1 casein hydrolysate, 3 mg L -1 2,4-dichlorophenoxyacetic acid (2,4-D), and 45 μg L -1 6-benzylaminopurine (BAP), which resulted in callus induction of 51 ± 29% for FIL2 and 81 ± 19% for HAL2. The optimal inflorescence-derived callus induction was observed on MP medium (MS medium supplemented with 2 g L -1 L-proline, 3% maltose, 5 mg L -1 2,4-D, and 500 μg L -1 BAP), resulting in callus induction of 100 ± 0.0% for FIL2 and 84 ± 2.4% for HAL2. Shoot regeneration rates of 11.5 ± 0.8 shoots/gram for FIL2 and 11.3 ± 0.6 shoots/gram for HAL2 were achieved using seed-induced callus, whereas shoot regeneration rates of 26.2 ± 2.6 shoots/gram for FIL2 and 29.3 ± 3.6 shoots/gram for HAL2 were achieved from inflorescence-induced callus. Further, cell suspension cultures of P. hallii were established from seed-derived callus, providing faster generation of callus tissue compared with culture using solidified media (1.41-fold increase for FIL2 and 3.00-fold increase for HAL2). Aside from abbreviated tissue culture times from callus induction to plant regeneration for HAL2, we noted no apparent differences between FIL2 and HAL2 populations in tissue culture performance. For both populations, the cell suspension cultures outperformed tissue cultures on solidified media. Using the methods developed in this work, P. hallii callus was induced from seeds immediately after harvest in a shorter time and with higher frequencies than switchgrass. For clonal propagation, P. hallii callus was established from R1 inflorescences, similar to switchgrass, which further strengthens the potential of this plant as a C 4 model for genetic studies. The rapid cycling (seed-to-seed time) and ease of culture, further demonstrate the potential utility of P. hallii as a C 4 model plant.

In vitro propagation, ex vitro rooting and leaf micromorphology of Bauhinia racemosa Lam.: a leguminous tree with medicinal values.

PubMed

Sharma, Udit; Kataria, Vinod; Shekhawat, N S

2017-10-01

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l -1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l -1 ). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l -1 each of BA and Kin (Kinetin) with 0.1 mg l -1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l -1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l -1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.

Optimizing micropropagation of drought resistant Pyrus boissieriana Buhse.

PubMed

Zakavi, Maryam; Askari, Hossein; Irvani, Neda

2016-10-01

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana , which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l -1 BAP and 0.05 mg l -1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l -1 BAP and 0.07 mg l -1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50-100 mg l -1 PG with different concentrations of IBA or NAA at intervals of 30-60 days. Significant increase in shoot length was detected after 45-60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l -1 IBA and 100 mg l -1 PG. GA 3 negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l -1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25-30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.

Preculturing effect of thidiazuron on in vitro shoot multiplication and micropropagation round in Capparis decidua (Forsk.) an important multipurpose plant.

PubMed

Bukhari, Najat A W; Siddique, Iram; Perveen, Kahkashan

2016-09-01

An efficient protocol was developed for clonal multiplication of an important shrub: Capparis decidua (Forsk.) Edgew, through in vitro shoot induction and multiplication from nodal explants. Pretreatment of nodal explants in a liquid Murashige and Skoog (MS) medium augmented with various thidiazuron (TDZ) concentrations at relatively high levels (5-100 μM) for different time duration (4, 8, 12 and 16 d), proved a significant approach for in vitro shoot production. After an initial exposure time to TDZ, nodal explants were inoculated onto a MS basal medium devoid of TDZ for further induction and proliferation. The highest regeneration rate (85%), average number of shoots/explant (8.7 ± 0.22) and maximum shoot length (3.9 ± 0.33 cm) were obtained from the nodal explants exposed to 50 μM TDZ for 8 d. The nodal explants excised from the proliferated cultures of TDZ (50 μM) for 8 d were used as explants and showed an enhancement rate after next three round of in vitro propagation. Best results for rooting was obtained by ex vitro treatment of shoots with 200 μM indole-3-butyric acid (IBA) for 20 min. as it produced an average of 5.7 ± 0.41 roots per microshoot with 4.4 ± 0.39 cm root length in 84% shoots. Different planting substrates was tested for maximum survival of hardening off micropropagated plantlets and soilrite proved most effective than others as 97.1 ± 7.21 plantlets survived. All micropropagated plants grew well in natural conditions and showed similar morphology to the mother plant.

In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant

PubMed Central

Sen, Monokesh Kumer; Nasrin, Shamima; Rahman, Shahedur; Jamal, Abu Hena Mostofa

2014-01-01

Objective To study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L. Methods Sterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively. Results Sterilization treatment of 0.1% HgCl2 for 2-3 min and Bavistin 0.5% for 10-12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality. Conclusions The results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration. PMID:24144129

Somatic embryogenesis from leaf explants of Australian fan flower, Scaevola aemula R. Br.

PubMed

Wang, Y-H; Bhalla, P L

2004-01-01

Somatic embryogenesis from leaf explants of Scaevola aemula R. Br. was achieved. Somatic embryos were induced from explants cultured on MS medium supplemented with 0.2 mg/ 2,4-dichlorophenoxyacetic acid and 0.2-0.5 mg/l 6-benzylaminopurine (BAP). Various developmental stages of somatic embryos were found on this medium-from globular embryos to germinated embryos. The transfer of globular embryos to MS medium containing 0.5 mg/l BAP resulted in a high frequency of shoot regeneration. Leaf explants cultured on MS medium containing different combinations of BAP and alpha-naphthaleneacetic acid formed adventitious shoots and roots. Histological examination confirmed the process of somatic embryogenesis. Induction of somatic embryogenesis in Scaevola provides a system for studying embryogenesis in Australian native plants and will facilitate the improvement of these plants using genetic transformation techniques.

In Vitro Flower Induction from Shoots Regenerated from Cultured Axillary Buds of Endangered Medicinal Herb Swertia chirayita H. Karst.

PubMed Central

Kamal, Barkha; Srivastava, Nidhi; Dobriyal, Anoop Kumar; Jadon, Vikash Singh

2014-01-01

In vitro flowering and effective micropropagation protocol were studied in Swertia chirayita, an important medicinal plant using axillary bud explants. The Murashige and Skoog's medium (MS) supplemented with benzyl amino purine (BAP) 1.0 mg L−1 and adenine sulfate 70.0 mg L−1 was found optimum for production of multiple shoots. In the present study, incubation of flowering cultures on BAP supplemented medium (during shoot multiplication) was found necessary for flowering (6 weeks). However, concentrations of auxins-like IBA (0–2.0 mg/L) were ineffective to form reproductive buds. Subculture duration, photoperiod, and carbon source type do have influence on the in vitro flowering. The mature purple flowers were observed when the cultures were maintained in the same medium. This is the very first report that describes in vitro flowering system to overcome problems associated with flower growth and development as well as lay foundation for fruit and seed production in vitro in Swertia chirayita. PMID:24707404

Biochemical markers assisted screening of Fusarium wilt resistant Musa paradisiaca (L.) cv. puttabale micropropagated clones.

PubMed

Venkatesh; Krishna, V; Kumar, K Girish; Pradeepa, K; Kumar, S R Santosh; Kumar, R Shashi

2013-07-01

An efficient protocol was standardized for screening of panama wilt resistant Musa paradisiaca cv. Puttabale clones, an endemic cultivar of Karnataka, India. The synergistic effect of 6-benzyleaminopurine (2 to 6 mg/L) and thidiazuron (0.1 to 0.5 mg/L) on MS medium provoked multiple shoot induction from the excised meristem. An average of 30.10 +/- 5.95 shoots was produced per propagule at 4 mg/L 6-benzyleaminopurine and 0.3 mg/L thidiazuron concentrations. Elongation of shoots observed on 5 mg/L BAP augmented medium with a mean length of 8.38 +/- 0.30 shoots per propagule. For screening of disease resistant clones, multiple shoot buds were mutated with 0.4% ethyl-methane-sulfonate and cultured on MS medium supplemented with Fusarium oxysporum f. sp. cubense (FOC) culture filtrate (5-15%). Two month old co-cultivated secondary hardened plants were used for screening of disease resistance against FOC by the determination of biochemical markers such as total phenol, phenylalanine ammonia lyase, oxidative enzymes like peroxidase, polyphenol oxidase, catalase and PR-proteins like chitinase, beta-1-3 glucanase activities. The mutated clones of M. paradisiaca cv. Puttabale cultured on FOC culture filtrate showed significant increase in the levels of biochemical markers as an indicative of acquiring disease resistant characteristics to FOC wilt.

Arabidopsis alcohol dehydrogenase expression in both shoots and roots is conditioned by root growth environment

NASA Technical Reports Server (NTRS)

Chung, H. J.; Ferl, R. J.

1999-01-01

It is widely accepted that the Arabidopsis Adh (alcohol dehydrogenase) gene is constitutively expressed at low levels in the roots of young plants grown on agar media, and that the expression level is greatly induced by anoxic or hypoxic stresses. We questioned whether the agar medium itself created an anaerobic environment for the roots upon their growing into the gel. beta-Glucuronidase (GUS) expression driven by the Adh promoter was examined by growing transgenic Arabidopsis plants in different growing systems. Whereas roots grown on horizontal-positioned plates showed high Adh/GUS expression levels, roots from vertical-positioned plates had no Adh/GUS expression. Additional results indicate that growth on vertical plates closely mimics the Adh/GUS expression observed for soil-grown seedlings, and that growth on horizontal plates results in induction of high Adh/GUS expression that is consistent with hypoxic or anoxic conditions within the agar of the root zone. Adh/GUS expression in the shoot apex is also highly induced by root penetration of the agar medium. This induction of Adh/GUS in shoot apex and roots is due, at least in part, to mechanisms involving Ca2+ signal transduction.

In vitro regeneration and ploidy level analysis of Eulophia ochreata Lindl.

PubMed

Shriram, Varsha; Nanekar, Vikas; Kumar, Vinay; Kavi Kishor, P B

2014-11-01

Various parameters including explant-type, medium compositions, use of phytohormones and additives were optimized for direct and indirect regeneration of E. ochreata, a medicinal orchid under threat. Protocorm-like-bodies (PLBs) proved to be the best explants for shoot initiation, proliferation and callus induction. Murashige and Skoog's (MS) medium containing 2.5 mg L(-1) 6-benzylaminopurine (BAP), 1.0 mg L(-1) kinetin (Kin) and additives (adenine sulfate, arginine, citric acid, 30 mg L(-1) each and 50 mg L(-1) ascorbic acid) was optimal for shoot multiplication (12.1 shoots and 7.1 PLBs per explant with synchronized growth), which also produced callus. Shoot number was further increased with three successive subcultures on same media and approximately 40 shoots per explant were achieved after 3 cycles of 30 days each. Additives and casein hydrolysate (CH) showed advantageous effects on indirect shoot regeneration via protocorm-derived callus. Optimum indirect regeneration was achieved on MS containing additives, 500 mg L(-1) CH, 2.5 mg L(-1) BAP and 1.0 mg L(-1) Kin with 30 PLBs and 6 shoots per callus mass (approximately 5 mm size). The shoots were rooted (70% frequency) on one by fourth-MS medium containing 2.0 mg L(-1) indole-3-butyric acid, 200 mg L(-1) activated charcoal and additives. The rooted plantlets were hardened and transferred to greenhouse with 63% survival rate. Flow-cytometry based DNA content analysis revealed that the ploidy levels were maintained in in vitro regenerated plants. This is the first report for in vitro plant regeneration in E. ochreata.

In vitro flowering ofDendrobium candidum.

PubMed

Wang, G; Xu, Z; Chia, T F; Chua, N H

1997-02-01

Dendrobium candidum, a wild orchid species from China, normally requires three to four years of cultivation before it can produce flowers. The effects of plant hormones and polyamines on flower initiation of this species in tissue culture were investigated. The addition of spermidine, or BA, or the combination of NAA and BA to the culture medium can induce protocorms or shoots to flower within three to six months with a frequency of 31.6%-45.8%. The flowering frequency can be further increased to 82.8 % on the average by pre-treatment of protocorms in an ABA-containing medium followed by transfer onto MS medium with BA. The induction of precocious flowering depends on the developmental stage of the experimental materials (protocorms, shoots and plantlets) used, and usually occurs only when mt formation is inhibited.

Effect of adenine sulphate interaction on growth and development of shoot regeneration and inhibition of shoot tip necrosis under in vitro condition in adult Syzygium cumini L.--a multipurpose tree.

PubMed

Naaz, Afshan; Shahzad, Anwar; Anis, Mohammad

2014-05-01

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS + BA (10 μM) + GA3 (2.5 μM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 μM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.

Micropropagation of Cyclopia genistoides, an endemic South African plant of economic importance.

PubMed

Kokotkiewicz, Adam; Luczkiewicz, Maria; Hering, Anna; Ochocka, Renata; Gorynski, Krzysztof; Bucinski, Adam; Sowinski, Pawel

2012-01-01

An efficient micropropagation protocol of Cyclopia genistoides (L.) Vent., an indigenous South African shrub of economic importance, was established. In vitro shoot cultures were obtained from shoot tip fragments of sterile seedlings cultured on solid Schenk and Hildebrandt (SH) medium supplemented with 9.84 microM 6-(gamma,gamma-dimethylallylamino)purine (2iP) and 1.0 microM thidiazuron (TDZ). Maximum shoot multiplication rate [(8.2 +/- 1.3) microshoots/explant)] was observed on this medium composition. Prior to rooting, the multiplied shoots were elongated for 60 days (two 30-days passages) on SH medium with one-half sucrose concentration, supplemented with 4.92 microM indole-3-butyric acid (IBA). The rooting of explants was only possible in the case of the elongated shoots. The highest root induction rate (54.8%) was achieved on solid SH medium with one-half sucrose and one-half potassium nitrate and ammonium nitrate concentration, respectively, supplemented with 28.54 microM indole-3-acetic acid (IAA) and 260.25 microM citric acid. The plantlets were acclimatized for 30 days in the glasshouse, with the use of peat/gravel/perlite substrate (1:1:1). The highest acclimatization rate (80%) was obtained for explants rooted with the use of IAA-supplemented medium. The phytochemical profile of the regenerated plants was similar to that of the reference intact plant material. HPLC analyses showed that C. genistoides plantlets obtained by the micropropagation procedure kept the ability to produce xanthones (mangiferin and isomangiferin) and the flavanone hesperidin, characteristic of wild-growing shrubs.

Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L).

PubMed

Srinivasan, C; Liu, Zongrang; Scorza, Ralph

2011-04-01

Transgenic plants of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L) were produced by transforming with the apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KNOX1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a medium lacking cytokinin. Ectopic expression of KNOX genes retarded shoot growth by suppressing elongation of internodes in transgenic tobacco plants. Expression of each of the three KNOX1 genes induced malformation and extensive lobbing in tobacco leaves. In situ regeneration of adventitious shoots was observed from leaves and roots of transgenic tobacco plants expressing each of the three KNOX genes. In vitro culture of leaf explants and internode sections excised from in vitro grown MdKN1 expressing tobacco shoots regenerated adventitious shoots on MS (Murashige and Skoog 1962) basal medium in the absence of exogenous cytokinin. Transgenic plum plants that expressed the MdKN2 or corn KNOX1 gene grew normally but MdKN1 caused a significant reduction in plant height, leaf shape and size and produced malformed curly leaves. A high frequency of adventitious shoot regeneration (96%) was observed in cultures of leaf explants excised from corn KNOX1-expressing transgenic plum shoots. In contrast to KNOX1-expressing tobacco, leaf and internode explants of corn KNOX1-expressing plum required synthetic cytokinin (thidiazuron) in the culture medium to induce adventitious shoot regeneration. The induction of high-frequency regeneration of adventitious shoots in vitro from leaves and stem internodal sections of plum through the ectopic expression of a KNOX1 gene is the first such report for a woody perennial fruit trees.

High frequency organogenesis in hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, D K

2015-04-01

Broccoli (Brassica oleracea L. var. italica) is an important, nutritionally rich vegetable crop, but severely affected by environmental stresses, pests and diseases which cause massive yield and quality losses. Genetic manipulation is becoming an important method for broccoli improvement. In the present study, a reproducible and highly efficient protocol for obtaining organogenesis from hypocotyl, cotyledon, leaf and petiole explants of broccoli (Brassica oleracea L. var. italica cv. Solan green head) has been developed. Hypocotyl and cotyledon explants were used from 10 to 12 days old aseptically grown seedlings whereas leaf and petiole explants were excised from 18 to 20 days old green house grown seedlings and surface sterilized. These explants were cultured on shoot induction medium containing different concentration and combination of BAP and NAA. High efficiency shoot regeneration has been achieved in hypocotyl (83.33 %), cotyledon (90.11 %), leaf (62.96 %) and petiole (91.10 %) explants on MS medium supplemented with 3.5 mg/l BAP + 0.019 mg/l NAA 2.5 mg/l BAP + 0.5 mg/l NAA, 4.0 mg/l BAP + 0.5 mg/l NAA and 4.5 mg/l BAP + 0.019 mg/l NAA respectively. Petiole explants showed maximum shoot regeneration response as compared to other explants. MS medium supplemented with 0.10 mg/l NAA was found best for root regeneration (100 %) from in vitro developed shoots. The regenerated complete plantlets were transferred to the pots containing cocopeat and successfully acclimatized. This optimized regeneration protocol can be efficiently used for genetic transformation in broccoli. This is the first comparative report on multiple shoot induction using four different types of explants viz. hypocotyl, cotyledon, leaf and petiole.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinez Isaza, B.C.

Production of artemisinin was studied in both plants and tissue cultures of Artemisia annua L. Incorporation of (3{prime}-{sup 14}C) mevalonic acid sodium salt into artemisinin or arteannuin B was not found when field-grown plants were fed once with 10 or 50 {mu}Ci and harvested after 44, 144 or 288 hr. Artemisinin was not present in root organ cultures, but was present in the shoot cultures in a concentration of less than 5 mg/100 g dry weight. The content of artemisinin in a shoot culture line with elongated and indented shoots was significantly higher at p value of 0.01 from thatmore » with short and compact shoots. Induction of roots on shoot cultures was associated with increased artemisinin production. Shoot cultures that developed into plants with roots had higher artemisinin content than those shoots cultures with aerial roots, or shoots cultures with basal roots. The artemisinin content in shoot cultures apparently increased with age. Preliminary studies on the metabolism of arteannuin B demonstrated that shoot cultures absorbed the exogenous arteannuin B from the medium without an increase in artemisinin content.« less

Micropropagation of Pithecellobium dulce (Roxb.) Benth-a multipurpose leguminous tree and assessment of genetic fidelity of micropropagated plants using molecular markers.

PubMed

Goyal, Pooja; Kachhwaha, Sumita; Kothari, S L

2012-04-01

An efficient and reproducible protocol has been developed for in vitro propagation of Pithecellobium dulce (Roxb.) Benth (a multipurpose leguminous tree) from field grown nodal segments (axillary bud). Shoot bud induction occurred from nodal explants of 15-years-old tree on Murashige and Skoog (MS) basal medium supplemented with 4.4 μM 6-benzyladenine (BA) and multiplication was achieved on MS medium supplemented with 4.4 μM BA + 0.73 μM phenylacetic acid (PAA) i.e. up to 7 shoot buds in the period of 5-6 weeks. Addition of adenine sulphate (AdS) to this medium further enhanced the number of shoot buds up to 10. Proliferating shoot cultures were established by repeatedly subculturing primary culture on fresh medium (MS + 4.4 μM BA + 0.73 μM PAA) after every 25 days. In vitro rooting was achieved on MS medium supplemented with 2.46 μM Indole-3-butyric acid (IBA) + 41.63 μM activated charcoal (AC). The micropropagated shoots with well developed roots were acclimatized in green house in pots containing sand, soil and manure (1:1:1). Genetic stability of micropropagated clones was evaluated using Random amplified polymorphic DNA (RAPD) and Inter simple sequence repeat (ISSR) markers. The amplification products were monomorphic in micropropagated plants and similar to those of mother plant. No polymorphism was detected revealing the genetic uniformity of micropropagated plants. This is the first report of an efficient protocol for regeneration of P. dulce through organogenesis, which can be used for further genetic transformation and pharmaceutical purposes.

[Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration].

PubMed

Shi, Heping; Zhu, Yuanfeng; Wang, Bei; Sun, Jiangbing; Huang, Shengqin

2014-11-01

To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0-3.0 mg/L + NAA 0.1-0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L + NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30-40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.

Micropropagation of Ajuga species: a mini review.

PubMed

Park, Han Yong; Kim, Doo Hwan; Sivanesan, Iyyakkannu

2017-09-01

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82-100% survival rate.

Micropropagation of Capparis decidua (Forsk.) Edgew. - a tree of arid horticulture.

PubMed

Deora, N S; Shekhawat, N S

1995-12-01

A method for micropropagation of mature trees of Capparis decidua was developed. Multiple shoots were obtained from nodal explants on Murashige and Skoog's (1962) medium+0.1mgl(-1) NAA+5.0mgl(-1)BAP+additives (50mgl(-1) ascorbic acid and25 mgl(-1) each of adenine sulphate, L-arginine and citric acid) at 28 ± 2°C, 12 h/dphotoperiod and 35-40 μmol m(-2)s(-1) photon flux density. The shoots were multiplied by (i) subculture of nodal shoot segments onto MS +0.1 mgl(--1) IAA+1.0mgl(-1) BAPH+additives, and (ii) repeated transfer of original explant onto MS+ 0.1mgl(-1) IAA+mg l(-1) BAP+additives, at intervals of 3 weeks. Sixty to 70% of the shoots rooted when pulse treated with 100 mg l(-1) IBA in half strength MS liquid medium for 4h, and then transferred onto hormone-free half-strength agar-gelled MS basal saltmedium. Incubation in dark at 33 ± 2°C for 6d favoured root induction. In vitro hardened plants were transferred to pots.

Plant regeneration from cell suspension-derived protoplasts of Primula malacoides and Primula obconica.

PubMed

Mizuhiro, M; Kenichi, Y; Ito, K; Kadowaki, S; Ohashi, H; Mii, M

2001-05-01

Protoplasts were isolated from cell suspension cultures of Primula malacoides cv. 'Lovely Tokyo' and P. obconica cv. 'Aalsmeer Giant White'. P. obconica protoplasts were embedded in 0.1% (w/v) gellan gum-solidified discs comprising MS medium supplemented with 3 mg/l of 2,4-D or picloram, 0.1 mg/l of zeatin, 0.2 M glucose and 0.2 M mannitol, and surrounded by a liquid medium of the same composition except for the addition of 0.1% (w/v) activated charcoal. The protoplasts formed visible colonies, which were transferred to the regeneration medium containing 30 g/l of sucrose, 0.1 mg/l of picloram and 2 mg/l of zeatin for shoot induction. P. malacoides protoplasts formed visible colonies when cultured in disc culture using 0.1% (w/v) gellan gum-solidified MS medium containing 5 mg/l of 2,4-D, 1 mg/l of NAA, 0.1 mg/l of zeatin and 0.4 M glucose. Small calli were transferred to MS medium supplemented with 5 mg/l of zeatin for shoot regeneration. The shoots of both species readily rooted on plant growth regulator-free 1/2 MS medium and successfully acclimatized to greenhouse conditions. The protoplast-derived plants showed some alterations in morphological characteristics from those of the in-vitro-germinated control plants.

An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers.

PubMed

Faisal, Mohammad; Alatar, Abdulrahman A; Ahmad, Naseem; Anis, Mohammad; Hegazy, Ahmad K

2012-12-01

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

Effects of N6-benzylaminopurine and Indole Acetic Acid on In Vitro Shoot Multiplication, Nodule-like Meristem Proliferation and Plant Regeneration of Malaysian Bananas (Musa spp.)

PubMed Central

Sipen, Philip; Davey, Michael R

2012-01-01

Different concentrations of N6-benzylaminopurine (BAP) and indole acetic acid (IAA) in Murashige and Skoog based medium were assessed for their effects on shoot multiplication, nodule-like meristem proliferation and plant regeneration of the Malaysian banana cultivars Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak. BAP at 1–14 mg L−1 with or without 0.2 mg L−1 IAA, or BAP at 7–14 mg L−1 with the same concentration of IAA, was evaluated for shoot multiplication from shoot tips and the proliferation of nodule-like meristems from scalps, respectively. Plant regeneration from scalps was assessed using 1 mg L−1 BAP and 0.2 mg L−1 IAA separately, or a combination of these two growth regulators. Data on shoot multiplication, the proliferation of nodule-like meristems with associated plant regeneration were recorded after 30 days of culture. A maximum of 5 shoots per original shoot tip was achieved on medium supplemented with BAP at 5 mg L−1 (Pisang Nangka), 6 mg L−1 (Pisang Mas and Pisang Berangan), or 7 mg L−1 (Pisang Awak), with 0.2 mg L−1 IAA. BAP at 11 mg L−1 with 0.2 mg L−1 IAA induced the most highly proliferating nodule-like meristems in the four banana cultivars. Plant regeneration from scalps was optimum in all cases on medium containing 1 mg L−1 BAP and 0.2 mg L−1 IAA. This is the first report on the successful induction of highly proliferating nodule-like meristems and plant regeneration from scalps of the Malaysian banana cultivars Pisang Mas, Pisang Nangka, Pisang Berangan and Pisang Awak. PMID:24575235

Factors affecting induction and development of in vitro rooting in apple rootstocks.

PubMed

Sharma, T; Modgil, M; Thakur, M

2007-09-01

Shoots of apple rootstocks raised in vitro were transferred to various rooting media to study the effect of different factors on root initiation and development. Various concentrations of indole-3-butyric acid (IBA) initiated rooting but maximum rooting percentage was found with 2.0 and 2.5 mg l(-1) of IBA in M7 and with 1.0 mg l(-1) of IBA in MM106. The drawback was that the roots were thick, short and with profuse callus. The presence of activated charcoal (AC) in the rooting medium improved the rooting quality but reduced the rooting percentage in both the rootstocks. In high auxin dip of 70, 80 and 90 mg l(-1) IBA for 2, 2 and 1 hr showed 75-85 per cent rooting in M7, but lacked reproducibility of the results. Whereas in MM106, 66 - 70 % rooting was achieved with 70 mg l(-1) of IBA dip for 3 h. Root induction in shoots in IBA containing liquid medium (LM) in dark for few days and root elongation in IBA--free medium in light proved most effective. On the other hand, continuous light treatment showed reduced rooting. Reduction of MS salts and sucrose in root elongation medium showed decreased rooting. Plantlets from two--stage rooting procedure showed more rapid growth and satisfactory survival during hardening of plants and on transfer to field.

Micropropagation of Codiaeum variegatum (L.) Blume and regeneration induction via adventitious buds and somatic embryogenesis.

PubMed

Radice, Silvia

2010-01-01

Codiaeum variegatum (L) Blume cv. "Corazon de oro" and cv. "Norma" are successfully micropropagated when culture are initiated with explants taken from newly sprouted shoots. The establishment and multiplication steps are possible when 1 mg/L BA or 1 mg/L IAA and 3 mg/L 2iP are added to MS medium, according to the cultivar respectively selected.Adventive organogenesis and somatic embryogenesis are induced from leaf explants taken from in vitro buds of croton. On leaf-sectioned of "Corazon de oro" cultured in vitro, 1 mg/L BA stimulates continuous somatic embryos development and induces some shoots too. Replacing BA with 1 mg/L TDZ induces up to 100% bud regeneration in the same explants. On the other hand, leaf-sectioned of C. variegatum cv. Norma does not start somatic embryo differentiation if 1 mg/L TDZ is not added to the MS basal medium. Incipient callus is observed after 30 days of culture, and then, subculture to MS with 1 mg/L BA allows the same process to show on the "Corazon de oro" cultivar. Somatic embryos show growth arrest that is partially overcome by transfer to hormone-free basal medium with activated charcoal. Root induction is possible on basal medium plus 1 mg/L IBA. Plantlets in the greenhouse have variegated leaves true-to-type.

An evaluation of a new approach to the regeneration of Helichrysum italicum (Roth) G. Don, and the molecular characterization of the variation among sets of differently derived regenerants.

PubMed

Perrini, Rosaria; Alba, Vittorio; Ruta, Claudia; Morone-Fortunato, Irene; Blanco, Antonio; Montemurro, Cinzia

2009-01-01

A protocol for the induction of regeneration from leaves of Helichrysum italicum was established. Calli were found to form on the basal medium only when it was supplemented with thidiazuron (TDZ) alone or in combination with naphthalene acetic acid (NAA), with a percentage ranking of at least 80%. The hormone-free medium showed the highest percentage of shoot regeneration (62%) even though no callus formed. AFLP markers were employed to verify tissue culture-induced variation in the regenerated plantlets obtained by direct shoot regeneration or the indirect shoot regeneration process (callus formation). Seven out of the eleven AFLP primer pairs yielded polymorphic patterns. The average number of fragments per primer pair was 64.1. Singletons were represented by 12 (2.7%) fragments. Student's T-test was performed both on the average number of shared fragments and on the nucleotide diversity, and no significant statistical difference was observed between the two regeneration treatments.

Micropropagation of globe artichoke (Cynara cardunculus L. var. scolymus).

PubMed

Iapichino, Giovanni

2013-01-01

The globe artichoke (Cynara cardunculus L. var. scolymus) is a perennial plant cultivated in the Mediterranean region and the Americas for its edible young flower heads. Although vegetative propagation by offshoots or by "ovoli" (underground dormant axillary buds) has been the primary method of propagation, the potential for the diffusion of diseases and the phenotypic variability can be very high. The propagation of this species by axillary shoot proliferation from in vitro-cultured meristems produces systemic pathogen-free plants and a higher multiplication rate as compared to that obtained by conventional agamic multiplication. Axillary shoot proliferation can be induced from excised shoot apices cultured on Murashige and Skoog agar solidified medium supplemented with various concentrations of cytokinins and auxins, depending on genotype. For the production of virus-free plants, meristems, 0.3-0.8 mm long are excised from shoot apices and surface sterilized. The transfer of artichoke microshoots to a medium lacking cytokinins or with low cytokinin concentration is critical for rooting. Adventitious roots develop within 3-5 weeks after transfer to root induction MS medium containing NAA or IAA at various concentrations. However, in vitro rooting frequency rate is dependent on the genotype and the protocol used. Acclimatization of in vitro microshoots having 3-4 roots is successfully accomplished; plantlets develop new roots in ex vitro conditions and continue to grow.

Micropropagation of ornamental Prunus spp. and GF305 peach, a Prunus viral indicator.

PubMed

Kalinina, Anna; Brown, Daniel C W

2007-07-01

A micropropagation approach was developed for nine ornamental Prunus species, P. americana, P. cistena, P. glandulosa, P. serrulata 'Kwanzan', P. laurocerasus, P. sargentii, P. tomentosa, P. triloba, P. virginiana 'Schubert', commercially important in North America, and GF305 peach, commonly used for Prunus virus indexing. The micropropagation cycle based on proliferation of vegetative tissues includes establishment of tissue culture through introduction of shoot meristems in vitro, shoot proliferation, root induction and plant acclimatization steps and can be completed in 5 months. A meristem sterilization protocol minimized bacterial and fungal contamination. Multiple shoot formation in ornamental Prunus was obtained through the use of 1 mg l(-1) 6-benzyladenine. For GF305 peach, alteration in the sugar composition, fructose instead of sucrose, and addition of 1 mg l(-1 )ferulic acid had a significant impact on the shoot proliferation rate and maintenance of long-term in vitro culture. Rooting and plant acclimatization conditions were improved using a two-step protocol with a 4-day root induction in indole-3-butiric acid (IBA)-containing media with consequent 3-week root elongation in IBA-free media. One-month incubation of rooted shoots in a vermiculite-based medium resulted in additional shoot and root growth and provided better acclimatization and plant recovery. The micropropagation approach can be used for maintenance of the clonal properties for Prunus spp. as well as a protocol to support meristem therapy against viral infection.

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

PubMed Central

Aslam, Junaid; Mujib, Abdul; Sharma, Maheshwar Prasad

2012-01-01

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N6-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations. PMID:23961221

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant.

PubMed

Aslam, Junaid; Mujib, Abdul; Sharma, Maheshwar Prasad

2013-01-01

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78 μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5 μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84 μM N(6)-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38 μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.

Micropropagation of Gerbera (Gerbera jamesonii Bolus).

PubMed

Minerva, Ghani; Kumar, Surinder

2013-01-01

Gerbera (Gerbera jamesonii Bolus) is one of the most popular ornamental flowers worldwide and used both as cut flower and potted plant. Some of them show excellent agronomic characters such as color, floral diameter, stem length, and vigor, which make this plant of commercial importance. Conventionally, multiplication is done through seeds or rhizome cuttings. Rapid multiplication of elite cultivars of Gerbera, with improved agronomic traits, has been achieved by using both direct and indirect tissue culture methods. Direct shoot regeneration was accomplished from stem apices on MS medium supplemented with 1 mg/L 6-benzyladenine (BA) and 1 mg/L kinetin. Indirect shoot induction succeeded from callus differentiation has been achieved on MS medium containing 2 mg/L 2,4-dichlorophenoxyacetic acid, 0.5 mg/L indole-3-acetic acid, and 2 mg/L BA. The in vitro shoots, 4-5 cm long, were rooted by quick dipping the shoot bases for 3-5 s in 2,000 mg/L indole-3-butyric acid solution followed by transfer to the pots containing farmyard manure, soil, and sand (1:1:1 by volume). Initially, in vitro plantlets were covered with glass jars to maintain a high relative humidity (85-90%). As soon as new shoot growth begins, relative humidity is decreased by exposing them to the open environmental conditions prior transferring to the glasshouse. Indirect shoot regeneration increased the frequency of somaclonal variations. The selected somaclones were used in developing new and novel cultivars.

The Tomato (Solanum Lycopersicum cv. Micro-Tom) Natural Genetic Variation Rg1 and the DELLA Mutant Procera Control the Competence Necessary to Form Adventitious Roots and Shoots

PubMed Central

Peres, Lázaro Eustáquio Pereira

2012-01-01

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively. PMID:22915742

The tomato (Solanum lycopersicum cv. Micro-Tom) natural genetic variation Rg1 and the DELLA mutant procera control the competence necessary to form adventitious roots and shoots.

PubMed

Lombardi-Crestana, Simone; da Silva Azevedo, Mariana; e Silva, Geraldo Felipe Ferreira; Pino, Lílian Ellen; Appezzato-da-Glória, Beatriz; Figueira, Antonio; Nogueira, Fabio Tebaldi Silveira; Peres, Lázaro Eustáquio Pereira

2012-09-01

Despite the wide use of plant regeneration for biotechnological purposes, the signals that allow cells to become competent to assume different fates remain largely unknown. Here, it is demonstrated that the Regeneration1 (Rg1) allele, a natural genetic variation from the tomato wild relative Solanum peruvianum, increases the capacity to form both roots and shoots in vitro; and that the gibberellin constitutive mutant procera (pro) presented the opposite phenotype, reducing organogenesis on either root-inducing medium (RIM) or shoot-inducing medium (SIM). Mutants showing alterations in the formation of specific organs in vitro were the auxin low-sensitivity diageotropica (dgt), the lateral suppresser (ls), and the KNOX-overexpressing Mouse ears (Me). dgt failed to form roots on RIM, Me increased shoot formation on SIM, and the high capacity for in vitro shoot formation of ls contrasted with its recalcitrance to form axillary meristems. Interestingly, Rg1 rescued the in vitro organ formation capacity in proRg1 and dgtRg1 double mutants and the ex vitro low lateral shoot formation in pro and ls. Such epistatic interactions were also confirmed in gene expression and histological analyses conducted in the single and double mutants. Although Me phenocopied the high shoot formation of Rg1 on SIM, it failed to increase rooting on RIM and to rescue the non-branching phenotype of ls. Taken together, these results suggest REGENERATION1 and the DELLA mutant PROCERA as controlling a common competence to assume distinct cell fates, rather than the specific induction of adventitious roots or shoots, which is controlled by DIAGEOTROPICA and MOUSE EARS, respectively.

Establishment of an efficient plant regeneration culture protocol and achievement of successful genetic transformation in Jatropha curcas L.

PubMed

Liu, Ying; Liu, Guoxuan; Yang, Yali; Niu, Sufang; Yang, Fuguang; Yang, Shaoxia; Tang, Jianian; Chen, Jianping

2017-12-01

An efficient and reproducible protocol is described for shoot-bud regeneration and Agrobacterium tumefaciens-mediated genetic transformation of J. curcas. Treating the explants with high concentrations (5-120 mg/L) of TDZ for short durations (5-80 min) before inoculation culture increased significantly the regeneration frequency and improved the quality of the regenerated buds. The highest shoot-buds induction rate (87.35%) was achieved when petiole explants were treated with 20 mg/L TDZ solution for 20 min and inoculated on hormone-free MS medium for 30 days. Regenerated shoots of 0.5 cm or a little longer were isolated and grafted to seedling stocks of the same species, and then the grafted plantlets were planted on half-strength MS medium containing 0.1 mg/L IBA and 2 mg/L sodium nitroprusside (SNP). This grafting strategy was found to be very effective, to obtain that healthy grafted plantlets ready for acclimatization within 20 days. By the above mentioned protocol and with general Agrobacterium - mediated genetic transformation methods only 65 days were needed to obtain intact transgenic plants.

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage

PubMed Central

2010-01-01

Background Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. Results For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation. Conclusion The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin. PMID:20444255

Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage.

PubMed

Palama, Tony L; Menard, Patrice; Fock, Isabelle; Choi, Young H; Bourdon, Emmanuel; Govinden-Soulange, Joyce; Bahut, Muriel; Payet, Bertrand; Verpoorte, Robert; Kodja, Hippolyte

2010-05-05

Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus. For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l(-1)). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l(-1)) and TDZ (0.5 mg l(-1)). These calli were maintained by subculturing on BM containing IAA (0.5 mg l(-1)) and TDZ (0.3 mg l(-1)) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l(-1)). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation. The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid-protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm-like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.

In vitro plant regeneration of Aster scaber via somatic embryogenesis.

PubMed

Boo, Kyung Hwan; Cao, Dang Viet; Pamplona, Reniel S; Lee, Doseung; Riu, Key-Zung; Lee, Dong-Sun

2015-01-01

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.

Generation and multiplication of plantlets from callus derived from Haplopappus gracilus (Nutt.) Gray and their karyotype analysis

NASA Technical Reports Server (NTRS)

Kann, R. P.; O'Connor, S. A.; Levine, H. G.; Krikorian, A. D.

1991-01-01

Unopened flower heads of Haplopappus gracilis (2n = 4) provided primary explants for callus production and subsequent induction of organized growth. Callus was initiated from small (3-5 mm in length) floral buds with benzylaminopurine (BAP) (44.4 micromoles; 10 mg/l) and naphthalene acetic acid (NAA) (0.54 micromole; 0.1 mg/l). Lowering the BAP level to 4.44 micromoles (1 mg/l) but maintaining the NAA level, gave rise to organized but highly compressed shoot growing points from an otherwise undifferentiated callus mass. Shoots selected from such cultures were maintainable and could be proliferated by growing 1-1.5-cm stem tip cuttings on Murashige and Skoog basal medium (solidified with agar) containing 0.444 micromole (0.1 mg/l) BAP and 0.054 micromole (0.01 mg/l) NAA. The stem tip multiplication rates obtainable by these means permit reliable strategies for shoot multiplication or production of rooted plantlets. Prolonged subculture and maintenance of shoots on growth regulator-free medium leads to in vitro flowering and greatly reduces rooting capacity. Karyotype analysis of chromosomes from root tip cells at metaphase and chromosome measurements show that karyologically uniform plantlets (based on chromosome number and morphology) can be obtained.

In Vitro Propagation, Phytochemical Analysis, and Evaluation of Free Radical Scavenging Property of Scrophularia kakudensis Franch Tissue Extracts.

PubMed

Manivannan, Abinaya; Soundararajan, Prabhakaran; Park, Yoo Gyeong; Jeong, Byoung Ryong

2015-01-01

The current study deals with in vitro propagation, antioxidant property estimation, and assessment of acacetin content in Scrophularia kakudensis Franch. Adventitious shoot induction was achieved from the nodal explant with the highest number of adventitious shoots per explant (17.4) on Murashige and Skoog's (MS) medium fortified with 2.0 mg·L(-1) 6-benzyladenine (BA) and 0.5 mg L(-1) indole-3-acetic acid (IAA). Maximum number of roots per plant (16.5) was noted in half strength MS medium supplemented with 0.5 mg·L(-1) IAA. The regenerated plants displayed successful survival ratio (95%) in the greenhouse. The highest content of acacetin, a pharmaceutically important flavonoid, was observed in the shoot extracts (in vitro: 32.83 µg·g(-1) FW; in vivo: 30.05 µg·g(-1) FW) followed by root extracts. Total phenol and flavonoid contents along with free radical scavenging assays revealed the occurrence of larger amount of antioxidants in shoot extract in comparison with callus and root extracts of S. kakudensis. Thus, the outcome of the present study can be highly beneficial for the germplasm conservation and commercial cultivation of S. kakudensis for therapeutic purposes.

In vitro somatic embryogenesis and plant regeneration of cassava.

PubMed

Szabados, L; Hoyos, R; Roca, W

1987-06-01

An efficient and reproducible plant regeneration system, initiated in somatic tissues, has been devised for cassava (Manihot esculenta Crantz). Somatic embryogenesis has been induced from shoot tips and immature leaves of in vitro shoot cultures of 15 cassava genotypes. Somatic embryos developed directly on the explants when cultured on a medium containing 4-16 mg/l 2,4-D. Differences were observed with respect to the embryogenic capacity of the explants of different varieties. Secondary embryogenesis has been induced by subculture on solid or liquid induction medium. Long term cultures were established and maintained for up to 18 months by repeated subculture of the proliferating somatic embryos. Plantlets developed from primary and secondary embryos in the presence of 0.1 mg/l BAP, 1mg/l GA3, and 0.01 mg/l 2,4-D. Regenerated plants were transferred to the field, and were grown to maturity.

Micropropagation of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) and assessment of genetic fidelity of micropropagated plants using RAPD markers.

PubMed

Goswami, Komal; Sharma, R; Singh, P K; Singh, Govind

2013-01-01

A micropropagation protocol was developed for multiplication of seedless lemon (Citrus limon L. cv. Kaghzi Kalan) using nodal explants. The maximum shoot regeneration was observed on low level of BAP (0.1 mg l(-1)) or kinetin (0.5 mg l(-1)). BA was recorded to be better than kinetin in terms of number of days taken to bud break. The maximum number of shoots per explant was observed on 0.1 mg l(-1) BA and 0.5 mg l(-1) kinetin. Shoot proliferation decreased with increasing concentration of BA alone, but in case of a combination of BA and NAA (0.1 mg l(-1) each), it increased with increasing concentration of BA up to 10.0 mg l(-1). None of the treatments including BA or kinetin alone or BA in combination with NAA produced significantly more shoots for commercial exploitation. In the case of a combination of BA + kinetin + IBA, the maximum (5.5 shoots per explants) proliferation was observed on MS medium containing 1.0 mg l(-1) BA + 0.5 mg l(-1) kinetin + 0.5 mg l(-1) IBA or 0.25 mg l(-1) BA + 1.0 mg l(-1) kinetin + 1.0 mg l(-1) IBA. Regenerated shoots showed root induction on MS basal medium or on MS medium containing 1.0 mg l(-1) IBA. It is concluded that a five-fold increase (1.0 mg l(-1) BA + 0.5 mg l(-1) kinetin + 0.5 mg l(-1) IBA) in axillary shoot proliferation, while seven-fold increase (0.25 mg/l mg l(-1) BA + 1.0 mg l(-1) kinetin + 1.0 mg l(-1) IBA) during the second cycle of multiplication could be obtained using the two plant growth regulator combinations. PCR amplification with 14 different random primers confirmed no somaclonal variant up to two cycles of shoot multiplication.

Isatin as an auxin source favoring floral and vegetative shoot regeneration from calli produced by thin layer explants of tomato pedicel

NASA Technical Reports Server (NTRS)

Applewhite, P. B.; K-Sawhney, R.; Galston, A. W.

1994-01-01

Thin layer explants taken from the pedicels and peduncles of flowering tomato plants yielded calli with great organogenetic potential. Of the 15 cultivars tested, 7 regenerated roots, shoots and eventually entire fruit-bearing plants. Calli grown on modified Murashige-Skoog medium responded to varied auxins and cytokinins with different morphogenetic patterns. Thus, naphthaleneacetic acid yielded root-producing calli, while the auxin precursor isatin (indole 2,3-dione) caused the production of calli with vegetative and floral shoots, rarely yielding roots. This may be related to isatin's slow, steady conversion to an active auxin (Plant Physiol 41:1485-1488, 1966) in contrast with naphthaleneacetic acid's immediate presentation of a high level of active auxin. The highest incidence of vegetative shoot (100%) and flower (50%) formation was obtained with 10 micromoles isatin and 3 micromoles zeatin. A few of the flowers developed into ripe fruits. The high frequency of induction of vegetative shoots and flowers before roots with isatin suggests its utility in micropropagation from plant tissue cultures.

In vitro regeneration of Drosera burmannii Vahl.: a carnivorous plant of north-east India.

PubMed

Yanthan, J Sureni; Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

2017-06-01

An efficient in vitro regeneration protocol has been developed from shoot tips of Drosera burmannii Vahl., a carnivorous plant of north-east India. Various plant growth regulators were used to study their efficacy in the induction of multiple shoots and roots. Of the various treatments, the maximum number of shoots (28.8 ± 1.5) and roots (9.7 ± 0.6) was observed in one-fourth strength standard medium (MS with 50 mg/l citric acid and 10 mg/l ascorbic acid) supplemented with 4 mg/l 6-benzylaminopurine (BAP) and 4 mg/l α-naphthalene acetic acid (NAA) followed by 26.8 ± 1.4 shoots in one-fourth strength SM fortified with 4 mg/l kinetin (KN) and 4 mg/l NAA. The well-developed plantlets with shoots and roots were potted in small plastic glasses filled with a mixture of sand and farmyard manure (3:1); these plantlets when transferred to a glasshouse for hardening and acclimatization showed 90% survival.

In-vitro morphogenesis of corn (Zea mays L.) : I. Differentiation of multiple shoot clumps and somatic embryos from shoot tips.

PubMed

Zhong, H; Srinivasan, C; Sticklen, M B

1992-07-01

In-vitro methods have been developed to regenerate clumps of multiple shoots and somatic embryos at high frequency from shoot tips of aseptically-grown seedlings as well as from shoot apices of precociously-germinated immature zygotic embryos of corn (Zea mays L.). About 500 shoots were produced from a shoot tip after eight weeks of culture (primary culture and one subculture of four weeks) in darkness on Murashige and Skoog basal medium (MS) supplemented with 500 mg/L casein hydrolysate (CH) and 9 μM N(6)-benzyladenine (BA). In this medium, shoots formed in shoot tips as tightly packed "multiple shoot clumps" (MSC), which were composed of some axillary shoots and many adventitious shoots. When the shoot tips were cultured on MS medium containing 500 mg/L CH, 9 μM BA and 2.25 μM 2,4-dichlorophenoxyacetic acid (2,4-D), most of the shoots in the clumps were adventitious in origin. Similar shoot tips cultured on MS medium containing 500 mg/L CH, 4.5 μM BA and 2.25 μM 2,4-D regenerated many somatic embryos within eight weeks of culture. Somatic embryos were produced either directly from the shoot apical meristems or from calli derived from the shoots apices. Both the MSC and the embryos produced normal shoots on MS medium containing 2.25 μM BA and 1.8 μM indole-3-butyric acid (IBA). These shoots were rooted on MS medium containing 3.6 μM IBA, and fertile corn plants were grown in the greenhouse. The sweet-corn genotype, Honey N Pearl, was used for the experiments described above, but shoot-tip cultures from all of 19 other corn genotypes tested also formed MSC on MS medium containing 500 mg/L CH and 9 μM BA.

Unfertilized ovary: a novel explant for coconut (Cocos nucifera L.) somatic embryogenesis.

PubMed

Perera, Prasanthi I P; Hocher, Valerie; Verdeil, Jean Luc; Doulbeau, Sylvie; Yakandawala, Deepthi M D; Weerakoon, L Kaushalya

2007-01-01

Unfertilized ovaries isolated from immature female flowers of coconut (Cocos nucifera L.) were tested as a source of explants for callogenesis and somatic embryogenesis. The correct developmental stage of ovary explants and suitable in vitro culture conditions for consistent callus production were identified. The concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) and activated charcoal was found to be critical for callogenesis. When cultured in a medium containing 100 microM 2,4-D and 0.1% activated charcoal, ovary explants gave rise to 41% callusing. Embryogenic calli were sub-cultured into somatic embryogenesis induction medium containing 5 microM abscisic acid, followed by plant regeneration medium (with 5 microM 6-benzylaminopurine). Many of the somatic embryos formed were complete with shoot and root poles and upon germination they gave rise to normal shoots. However, some abnormal developments were also observed. Flow cytometric analysis revealed that all the calli tested were diploid. Through histological studies, it was possible to study the sequence of the events that take place during somatic embryogenesis including orientation, polarization and elongation of the embryos.

Multiple shoot induction from axillary bud cultures of the medicinal orchid, Dendrobium longicornu

PubMed Central

Dohling, Stadwelson; Kumaria, Suman; Tandon, Pramod

2012-01-01

Background and aims Dendrobium longicornu, commonly known as the ‘Long-horned Dendrobium’, is an endangered and medicinally important epiphytic orchid. Over-exploitation and habitat destruction seriously threaten this orchid in Northeast India. Our objective was to develop an efficient protocol for the mass propagation of D. longicornu using axillary bud segments. Methodology and principal results Axillary buds cultured in Murashige and Skoog semi-solid medium supplemented with α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 6-benzylaminopurine (BAP) readily developed into plantlets. These formed either directly from shoot buds or from intermediary protocorm-like bodies (PLBs). The maximum explant response (86.6 %) was obtained in medium supplemented with NAA at 30 µM, while the maximum number of shoots (4.42) and maximum bud-forming capacity (3.51) were observed in medium containing 15 µM BAP and 5 µM NAA in combination. Protocorm-like bodies were obtained when the medium contained 2,4-D. The maximum number of explants forming PLBs (41.48 %) was obtained in medium containing 15 µM BAP and 15 µM 2,4-D. Well-developed plantlets obtained after 20–25 weeks of culture were acclimatized and eventually transferred to the greenhouse. Over 60 % of these survived to form plants ∼3–4 cm tall after 90 days in glasshouse conditions using a substrate of crushed brick and charcoal, shredded bark and moss. Conclusions The method described can readily be used for the rapid and large-scale regeneration of D. longicornu. Its commercial adoption would reduce the collection of this medicinally important and increasingly rare orchid from the wild. PMID:23136638

Establishment of an efficient in vitro regeneration protocol for rapid and mass propagation of Dendrobium chrysotoxum Lindl. using seed culture.

PubMed

Nongdam, Potshangbam; Tikendra, Leimapokpam

2014-01-01

An efficient in vitro regeneration protocol from seed culture has been established successfully for Dendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated more in vitro rooting, though IBA was found to be more effective in rooting induction as compared to NAA. The in vitro protocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly important Dendrobium orchid species.

Establishment of an Efficient In Vitro Regeneration Protocol for Rapid and Mass Propagation of Dendrobium chrysotoxum Lindl. Using Seed Culture

PubMed Central

2014-01-01

An efficient in vitro regeneration protocol from seed culture has been established successfully for Dendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated more in vitro rooting, though IBA was found to be more effective in rooting induction as compared to NAA. The in vitro protocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly important Dendrobium orchid species. PMID:25401154

A genetically stable rooting protocol for propagating a threatened medicinal plant—Celastrus paniculatus

PubMed Central

Phulwaria, Mahendra; Rai, Manoj K.; Patel, Ashok Kumar; Kataria, Vinod; Shekhawat, N. S.

2012-01-01

Celastrus paniculatus, belonging to the family Celastraceae, is an important medicinal plant of India. Owing to the ever-increasing demand from the pharmaceutical industry, the species is being overexploited, thereby threatening its stock in the wild. Poor seed viability coupled with low germination restricts its propagation through sexual means. Thus, alternative approaches such as in vitro techniques are highly desirable for large-scale propagation of this medicinally important plant. Nodal segments, obtained from a 12-year-old mature plant, were used as explants for multiple shoot induction. Shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoot clumps on Murashige and Skoog's (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with auxin (indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA)). The maximum number of shoots (47.75 ± 2.58) was observed on MS medium supplemented with BAP (0.5 mg L−1) and IAA (0.1 mg L−1). In vitro raised shoots were rooted under ex vitro conditions after treating them with indole-3-butyric acid (300 mg L−1) for 3 min. Over 95 % of plantlets acclimatized successfully. The genetic fidelity of the regenerated plants was assessed using random amplified polymorphic DNA. No polymorphism was detected in regenerated plants and the mother plant, revealing the genetic fidelity of the in vitro raised plantlets. The protocol discussed could be effectively employed for large-scale multiplication of C. paniculatus. Its commercial application could be realized for the large-scale multiplication and supply to the State Forest Department.

Assessment of factors affecting micropropagation and ex vitro acclimatization of Nyctanthes arbor-tristis L.

PubMed

Jahan, Anushi Arjumend; Anis, M; Aref, I M

2011-03-01

Rapid differentiation of multiple shoots was observed in 94% of nodal explants of one year old Nyctanthes arbor-tristis L. plants. Shoot bud induction and multiplication took place on Murashige and Skoog (MS) medium supplemented with two cytokinins, i.e. Benzyladenine (BA) or Kinetin (Kn) either alone or in combination with different auxins, indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) or α-naphthalene acetic acid (NAA). Between different media, pH levels and growth regulators tried, the optimum condition for maximum regenerative response was obtained on MS + Kn (2.5 μM) + N AA (0.5 μM) media at 5.8 pH, forming cultures with 23.26 ± 0.89 number of shoots and 6.36 ± 0.80 cm shoot length after 8 weeks of culture. Histological sections confirmed the formation of multiple buds from nodal explants. Rooting was achieved ex vitro by dipping the basal ends of microshoots in 200 μM IBA for 30 min followed by their transplantation in sterile soilrite. The plantlets with well-developed shoot and root system were successfully established in garden soil and grown outside in a greenhouse with a 80% survival rate.

Lippia dulcis shoot cultures as a source of the sweet sesquiterpene hernandulcin.

PubMed

Sauerwein, M; Flores, H E; Yamazaki, T; Shimomura, K

1991-04-01

The axenic shoot culture of Lippia dulcis Trev., Verbenaceae, was established on hormone-free Murashige-Skoog solid medium containing 3% sucrose. Shoots were cultured in various liquid or solid media. Woody Plant liquid medium was best for shoot multiplication, but the production of hernandulcin was relatively low. The highest hernandulcin content (2.9% dry wt) was obtained after 28 days of culture on Murashige-Skoog solid medium containing 2% sucrose. The addition of chitosan to the culture media enhanced the growth of shoots as well as the production of hernandulcin, especially with the liquid medium.

A rapid and efficient protocol for in vitro multiplication of genetically uniform Stevia rebaudiana (Bertoni).

PubMed

Khan, A; Jayanthi, M; Gantasala, Nagavara Prasad; Bhooshan, N; Rao, Uma

2016-07-01

Stevia rebaudiana (Bertoni), commonly called candy leaf or sweet leaf, endemic to South America, is an important medicinal plant. As a source of low calorie natural sweetener 'stevoside', it is used in obesity, diabetes, treatment of heartburn and tooth decay, and also serves as a food supplement. Large scale commercial propagation of S. rebaudiana demands a suitable protocol. Here, we propose an improved protocol for in vitro multiplication of S. rebaudiana from nodal explants. In this protocol, the effect of laboratory grade urea on multiple shoot induction from nodal explants was studied. The nodal explants were initially cultured on Murashige and Skoog (MS) basal media for 2 weeks which facilitated the axillary bud break. Further, culturing of these explants on MS medium fortified with 6 benzyl amninopurine (BAP) (2 mg/L) and Naphthalene acetic acid (NAA) (1 mg/L) with and .without urea (5 mg/L) for a period of 40 days revealed maximum shoot production of 44.56 from a single nodal explant in media supplemented with urea as compared to 22.44 without urea. The differences in the number of shoots produced were significant and these shoots readily rooted in MS media with NAA (4 mg/L). Primary and secondary hardening was successful in these plants. There were no visible morphological abnormalities observed in the micropropagated plantlets. Genetic analysis from random samples also revealed that these plants are genetically uniform. The advantage of the present protocol is that the complete process of multiple shoot induction, rooting and hardening could be completed within a period of 6 months as compared to the existing protocols.

1-Aminocyclopropane-1-carboxylic acid concentrations in shoot-forming and non-shoot-forming tobacco callus cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grady, K.L.; Bassham, J.A.

1982-09-01

Shoot-forming tobacco (Nicotiana tabacum var. Wisconsin 38) callus tissues contain significantly lower concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid compared to non-shoot-forming callus tissues. This difference is evident 1 day after subculture to shoot-forming or non-shoot-forming medium, and is maintained through the first week of growth. The lack of auxin in shoot-forming medium is the probable cause for this difference in ACC concentrations.

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting.

PubMed

Vibha, J B; Shekhawat, N S; Mehandru, Pooja; Dinesh, Rachana

2014-01-01

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20-25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20-21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO3)2, K2SO4, KCl, and NH4(SO4)2. The maximum shoot multiplication (29-30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2-3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.

In vitro culture and production of syringin and rutin in Saussurea involucrata (Kar. et Kir.) - an endangered medicinal plant.

PubMed

Kuo, Chao-Lin; Agrawal, Dinesh-Chandra; Chang, Hung-Chi; Chiu, Ya-Ting; Huang, Chu-Peng; Chen, Yi-Lin; Huang, Shih-Hung; Tsay, Hsin-Sheng

2015-12-01

Saussurea involucrata (Kar. et Kir.) commonly known as 'snow lotus' or 'Xue Lian' is an important plant in the traditional Chinese system of medicine. The plant contains flavonoids such as syringin and rutin. These compounds have been reported to be anti-rheumatic, anti-inflammatory and dilate blood vessels, lower blood pressure, prevent cardiovascular diseases, enhance immunity, and act as anti-aging, anti-cancer, and anti-fatigue agents. The species has become endangered due to the excessive collection of S. involucrata plants in the wild, slower plant growth and ecological destruction of natural habitats. There is a severe shortage of plant material, while the market demand is ever increasing. Hence, it is very important to apply tissue culture technique for plant propagation and production of the bioactive compounds of this species. Multiple shoot induction and proliferation in shoot base explants derived from in vitro raised seedlings of S. involucrata was achieved on 3/4 strength of Murashige and Skoog's (MS) basal medium (MSBM) supplemented with 1.0 mg/L -1 BA and 1.5 mg/L -1 NAA. Rooting was induced in 100 % shoots cultured on 1/2X MSBM supplemented with 1.0 mg/L -1 IBA for one week and then transfer to auxin free medium. The plantlets could be acclimatized successfully by sachet technique and established in the greenhouse. Maximum callus induction and proliferation in leaf segments was achieved on 1/2X MSBM supplemented with 0.5 mg/L -1 BA, 0.5 mg/L -1 NAA, 0.4 % gelrite and on incubation at 20 °C. Container closures had an influence on the quality and quantity of callus and production of the active compounds. The HPLC analysis showed much higher syringin content in in vitro shoots and callus as compared to commercially available market crude drug. The present study describes an in vitro culture protocol of Saussurea involucrata. The bioactive compounds, syringin and rutin could be produced through tissue culture technique without sacrificing the endangered Saussurea involucrata plants in the wild.

The effect of tomato juices and bean sprout extracts on vitro shoot regeneration of Physalis angulata L.

NASA Astrophysics Data System (ADS)

Mastuti, Retno; Munawarti, Aminatun; Rosyidah, Mufidatur

2017-11-01

Physalis angulata L. (Ciplukan) which belongs to Solanaceae is an important medicinal plant. In vitro culture medium contains carbon source, inorganic substance, vitamins, and plant growth regulators. However, organic growth supplements have frequently been added to improve regeneration capability of explants. This study was conducted to observe the effect of tomato juices and extract bean sprout on shoot regeneration and multiplication of in vitro nodal explants. The explants were cultured on MS basal medium + 6-benzyl amino purine (BAP) 2 mg/L + indole-3-acetic acid (IAA) 0.05 mg/L with and without organic supplements. Tomato juices (T) 5, 7.5 and 10% or bean sprout extract (B) 1.25, 2.5, and 3.75% were added as natural organic supplements. Almost all explants have produced shoots one week after culture. After six weeks of culture maximum shoot number (12.5±3.9) was produced in medium MS + T5 while maximum shoot length (10.7 ± 0.7 cm) was obtained in medium MS + T 7.5. Medium T tends to produce more shoots than the medium B and medium control. This result indicates the potential of natural organic supplements for supporting Ciplukan propagation through in vitro culture.

Micropropagation, Micromorphological Studies, and In Vitro Flowering in Rungia pectinata L.

PubMed

Shekhawat, Mahipal S; Manokari, M; Ravindran, C P

2016-01-01

A tissue culture protocol was developed for an important medicinal plant Rungia pectinata L. in the present study. Nodal shoots were used as explants and surface-sterilized with 0.1% HgCl2 solution. Murashige and Skoog (MS) medium was used to establish the cultures of R. pectinata. The bud break was reported on MS medium supplemented with 1.0 mg L(-1) 6-benzylaminopurine (BAP). About 98% response was observed with this media combination and maximum 3.2 shoots per explant with 4.3 cm length were recorded. The shoots were further multiplied using MS medium augmented with 0.5 mg L(-1) each of BAP and kinetin (Kin) + 0.1 mg L(-1) indole-3 acetic acid (IAA). Maximum 13.2 shoots per explant with 5.2 cm length were observed. All the shoots were rooted (4.9 roots per shoot with 3.5 cm length) on half strength MS medium fortified with 2.0 mg L(-1) indole-3 butyric acid (IBA). In vitro flowering was induced from the shoots on half strength MS medium supplemented with same concentrations and combinations of growth regulators used for shoot multiplication under 12/12 hr light/dark photoperiod. The plantlets were hardened in the greenhouse for two months and finally transferred to the field. The foliar micromorphological studies revealed the developmental changes in stomata, vein density, and trichomes during the culture of shoots under in vitro conditions.

Micropropagation, Micromorphological Studies, and In Vitro Flowering in Rungia pectinata L.

PubMed Central

Shekhawat, Mahipal S.; Manokari, M.; Ravindran, C. P.

2016-01-01

A tissue culture protocol was developed for an important medicinal plant Rungia pectinata L. in the present study. Nodal shoots were used as explants and surface-sterilized with 0.1% HgCl2 solution. Murashige and Skoog (MS) medium was used to establish the cultures of R. pectinata. The bud break was reported on MS medium supplemented with 1.0 mg L−1 6-benzylaminopurine (BAP). About 98% response was observed with this media combination and maximum 3.2 shoots per explant with 4.3 cm length were recorded. The shoots were further multiplied using MS medium augmented with 0.5 mg L−1 each of BAP and kinetin (Kin) + 0.1 mg L−1 indole-3 acetic acid (IAA). Maximum 13.2 shoots per explant with 5.2 cm length were observed. All the shoots were rooted (4.9 roots per shoot with 3.5 cm length) on half strength MS medium fortified with 2.0 mg L−1 indole-3 butyric acid (IBA). In vitro flowering was induced from the shoots on half strength MS medium supplemented with same concentrations and combinations of growth regulators used for shoot multiplication under 12/12 hr light/dark photoperiod. The plantlets were hardened in the greenhouse for two months and finally transferred to the field. The foliar micromorphological studies revealed the developmental changes in stomata, vein density, and trichomes during the culture of shoots under in vitro conditions. PMID:27242948

Micropropagation of pear (Pyrus sp.).

PubMed

Reed, Barbara M; Denoma, Jeanine; Wada, Sugae; Postman, Joseph

2013-01-01

Elements of micropropagation include establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets. The wide genetic variation in Pyrus makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced dormant shoots or from scions grafted onto seedling rootstocks to impose juvenility. Clean shoots are recovered after testing for contaminants at the initiation stage on ½ strength Murashige and Skoog 1962 medium (MS), at pH 6.9 for 1 week or by streaking on nutrient agar. Although pear species and cultivars are cultured on several well-known media, MS is the most commonly used. Our studies showed that multiplication and growth of shoots are best on Pear Medium with higher concentrations of calcium chloride, potassium phosphate, and magnesium sulfate than MS medium and 4.4 μM N(6) benzyladenine. Pear shoots are often recalcitrant to rooting; however, a 5 s dip in 10 mM indole-3-butyric acid or naphthalene acetic acid before planting on basal medium without plant growth regulators is effective for many genotypes. Pear shoots store well at 1-4°C, and can hold for as long as 4 years without reculture. Cryopreservation protocols are available for long-term storage of pear shoot tips. Acclimation of in vitro-rooted or micrografted shoots in a mist bed follows standard procedures.

In vitro root induction of faba bean (Vicia faba L.).

PubMed

Ismail, Roba M; Elazab, Heba E M; Hussein, Gihan M H; Metry, Emad A

2011-01-01

A major challenge for regeneration of faba bean (Vicia faba L.) plants is the difficulty of in vitro root induction. In the present study, in vitro rooting and its architecture have been studied. Adventitious root formation was successfully induced from regenerated faba bean shoots of four Egyptian cultivars, i.e., Giza 461, Giza 40, Giza 834 and Giza 716 on hormone free MS medium supplemented with 5 mg/l silver nitrate. Among the four cultivars, Giza 461 and Giza 40 were recorded as the highest root formation response (75 % and 65) followed by cultivars Giza716 and Giza843 (20%, and 10%). Anatomical study proved that the produced roots are initiated as the adventitious lateral root (LR) with tri-arch xylem strands as compared with the penta-arch of the primary roots of the intact faba bean seedling. The obtained results overcome the root induction problem in faba bean.

Protocol for efficient regulation of in vitro morphogenesis in einkorn (Triticum monococcum L.), a recalcitrant diploid wheat species

PubMed Central

Miroshnichenko, Dmitry; Chaban, Inna; Chernobrovkina, Mariya; Dolgov, Sergey

2017-01-01

Einkorn (Triticum monococcum L.) is A-genome diploid wheat that has a potential to become a useful model for understanding the biology and genomics in Triticeae. Unfortunately, the application of modern technologies such as genetic engineering, RNAi-based gene silencing and genome editing is not available for einkorn as there is no efficient in vitro tissue culture and plant regeneration system. In the present study an efficient and simple protocol for plant regeneration via direct or indirect somatic embryogenesis and organogenesis has been developed. Various auxins used as sole inductors in einkorn displayed low effect for morphogenesis (0–8%) and plant regeneration (1–2 shoots per explant). The addition of Daminozide, the inhibitor of biosynthesis of gibberellins, together with auxin significantly improved the formation of morphogenic structures, especially when Dicamba (51.4%) and Picloram (56.6%) were used for combination; furthermore, the simultaneous addition of cytokinin into induction medium significantly promoted in vitro performance. Among the tested cytokinins, the urea-type substances, such as TDZ and CPPU were more effective than the adenine type ones, BA and Zeatin, for the regulation of morphogenesis; especially, TDZ was more effective than CPPU for shoot formation (11.73 vs. 7.04 per regenerating callus). The highest morphogenic response of 90.2% with the production of more than 10 shoots per initial explant was observed when 3.0 mg/L Dicamba, 50.0 mg/L Daminozide and 0.25 mg/L TDZ were combined together. Along with the identification of appropriate induction medium, the optimal developmental stage for einkorn was found as partially transparent immature embryo in size of around 1.0 mm. Although in the present study the critical balance between plant growth regulators was established for einkorn only, we assume that further the proposed strategy could be successfully applied to other recalcitrant cereal species and genotypes. PMID:28273182

Effect of AgNO3 and BAP on Root as a Novel Explant in Date Palm (Phoenix dactylifera cv. Medjool) Somatic Embryogenesis.

PubMed

Roshanfekrrad, Marjan; Zarghami, Reza; Hassani, Hassan; Zakizadeh, Hedayat; Salari, Ali

2017-01-01

Somatic embryogenesis techniques are used for cloning a wide range of varieties of date palms around the world. The aim of the present study was to develop an efficient method with the lowest cost and the greatest potential to obtain in vitro plantlets of date palm cv. Medjool. Also, produce embryogenic callus and somatic embryos without using 2,4-dichlorophenoxyacetic acid (2,4-D). In this study, produced plantlets through somatic embryogenesis were used in vitro roots as explant cultured on Murashige and Skoog (MS) media containing three level of Silver Nitrate (AgNO3) (0, 3 and 6 mg L-1) plus two level of 6-benzylaminopurine (BAP) (0 and 2 mg L-1) plus 0.1 mg L-1 1-naphthylacetic acid (NAA) for callus induction. After 12 weeks of culture, callus induction and after 16 weeks, production of embryogenic callus and embryos were occurred from root explants. According to the results, medium containing 2 mg L-1 BAP and 3 mg L-1 silver nitrate+0.1 mg L-1 NAA showed the highest amount of embryogenic callus fresh weight (1.38 g). This treatment also cause the highest number and length of embryos by production of 90.04 embryogenic callus with length of 11.18 mm. On the other hand, shoots were appeared from germinated embryos and white roots began to appear within 8 weeks. Medium contains 3 mg L-1 BAP and 0.1 mg L-1 NAA with average of 12.27 cm shoot length and 15.48 cm root length was the best. Control treatment had the lowest average shoot (3.71 cm) and root (5.03 cm) length. This study showed that certain concentration of silver nitrate and BAP has stimulating effect on growth of produced embryonic callus from root segments of Medjool cultivar of date palm.

Metabolic changes associated with shoot formation in tobacco callus cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grady, K.L.

1982-08-01

Callus tissue derived from Nicotiana tabacum L. stem pith parenchyma cells was grown either on medium which maintains the callus in an undifferentiated state, or on medium which induces the formation of shoots. Two complementary types of studies were performed with the goal of establishing metabolic markers for the initiation of shoot formation: one designed to characterize the flow of radioactive sucrose into various metabolic pools, and one which allowed measurement of intermediary metabolite concentrations. In the former, callus tissue was incubated in (U-/sup 14/C)sucrose for periods up to one hour, and patterns of metabolite labelling in tissue grown onmore » shoot-forming and non-shoot-forming media were compared. In the latter studies, tissue was grown for an entire subculture period on non-shoot-forming medium labelled with (U-/sup 14/C)sucrose, then subcultured to labelled non-shoot-forming or shoot-forming media, and sampled at intervals during the first week of growth. 189 references.« less

Relationship between endogenous auxin and cytokinin levels and morphogenic responses inActinidia deliciosa tissue cultures.

PubMed

Centeno, M L; Rodríguez, A; Feito, I; Fernández, B

1996-11-01

Thein vitro culture ofActinidia deliciosa petioles results in a decline of cytokinin content and an increase of auxin levels. The addition of plant growth regulators (PGRs) to the medium lead to recovery of the initial auxin content, and callus induction occurs at the basal end of the explants. Endogenous auxin/cytokinin ratio was higher at this side than in the apical one, due to unequal distribution of endogenous PGRs in the cultured petioles. Some of the induced calluses showed shoot formation when they were transferred to proliferation medium. Most important differences found in hormonal content between organogenic and non-organogenic callus concerned benzyladenine levels. In this paper the relationships between explant behaviour and their hormonal content is discussed.

Improvement of Aconitum napellus micropropagation by liquid culture on floating membrane rafts.

PubMed

Watad, A A; Kochba, M; Nissim, A; Gaba, V

1995-03-01

An efficient method was developed using floating membrane rafts (Liferaft(™)) for the micropropagation of Aconitum napellus (Ranunculaceae), a cut flower crop with a low natural propagation rate. This was achieved by introducing shoot tips into culture on Murashige and Skoog's (1962) solid medium, or liquid medium-supported rafts, supplemented by different levels of benzyl adenine (BA). Optimum shoot proliferation on solid medium required 4mg/l BA, whereas for expiants supported on rafts optimal proliferation was achieved at 0.25mg/l BA. Maximum shoot proliferation was found using the floating rafts (propagation ratio of 4.2 per month), 45% higher than the maximum value on solid medium. A similar value could be obtained on solid medium after a period of 2 months. The optimal response to BA was similar for fresh weight gain and shoot length. Growth in a shallow layer of liquid in shake flasks gives a similar shoot multiplication rate to that on floating rafts; however, submerged leaves brown and die.

Micropropagation and subsequent enrichment of carotenoids, fatty acids and tocopherol contents in Sedum dasyphyllum L

NASA Astrophysics Data System (ADS)

Park, Han Yong; Saini, Ramesh Kumar; Gopal, Judy; Keum, Young-Soo; Kim, Doo Hwan; Lee, Onew; Sivanesan, Iyyakkannu

2017-10-01

A promising micropropagation protocol has been systematically established and demonstrated for the enhanced production of carotenoids, tocopherol and fatty acids in shoot tissues of Sedum dasyphyllum. Shoot tip explants were grown on Murashige and Skoog (MS) medium. Different concentrations of N6-benzyladenine (BA) or thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA) were tested in order to stimulate multiple shoot production. Ideal shoot induction (100%) and maximized shoot numbers (36.4) were obtained on explants cultured on media incorporated with 2 µM BA and 1 µM NAA combinations. The in vitro-developed shoots rooted best on half-strength MS media incorporated with 2 µM indole 3-butyric acid. Plantlets were effectively acclimatized in the greenhouse with 100% survival rate. The composition and contents of bioactive compounds such as carotenoids, tocopherol and fatty acids in shoot tissues of S. dasyphyllum were investigated using HPLC and GC-MS. The most abundant carotenoid in the shoot tissue was all-E-lutein (40.3-70.5 µg g-1 FW) followed by 9'-Z-neoxanthin (5.3-9.9 µg g-1 FW), all-E-violaxanthin (4.4-8.2 µg g-1 FW), and all-E-β-carotene (1.6-3.6 µg g-1 FW). The α-tocopherol contents of in vitro-raised shoots was 6.5-fold higher than shoots of greenhouse-grown plants. The primary fatty acids found in shoot tissues were α-linolenic acid (32.0-39.3%), linoleic acid (27.4-38.2%), palmitic acid (13.3-15.5%) and stearic acid (5.2-12.2%). In all, summarizing the findings, the micropropagated S. dasyphyllum showed significant enrichment of valuable bioactive carotenoids (92.3 µg g-1 FW), tocopherols (14.6 µg g-1 FW) and α-linolenic acid (39.3%) compared to their greenhouse counterparts. The protocol demonstrated here could be applied for the mass propagation and production of enhanced bioactive compounds from S. dasyphyllum with credibility.

Establishment of an in vitro micropropagation protocol for Boscia senegalensis (Pers.) Lam. ex Poir.

PubMed Central

Khalafalla, Mutasim M.; Daffalla, Hussien M.; Abdellatef, Eltayb; Agabna, Elsadig; El-Shemy, Hany A.

2011-01-01

This report describes in vitro micropropagation of Boscia senegalensis, so-called famine foods, that helped the people in Darfur and Kordofan, Sudan survive during the 1984–1985 famine. Four types of explants prepared from green mature zygotic embryos were cultured on Murashige and Skoog (MS) medium augmented with 1–5 mg/L 6-benzyladenine (BA). The highest number of shoots per explant (14.3±0.9) was achieved on MS medium supplemented with 3 mg/L BA, while the highest shoot length [(3.5±0.4) cm] was obtained with 1 mg/L BA. The shoot cluster, when subcultured to its same medium, significantly increased the rate of shoot multiplication by the end of the third subculture. The maximum mean number of shoots per explant (86.5±3.6) was produced after three multiplication cycles on 3 mg/L BA-supplemented medium. In vitro induced shoots were excised and rooted on half strength MS medium fortified with 0.25 mg/L indole-3-butyric acid (IBA) to obtain complete plantlets. B. senegalensis-regenerated plantlets obtained in vitro for the first time, were hardened and 95% survived under greenhouse conditions. PMID:21462387

Establishment of an in vitro micropropagation protocol for Boscia senegalensis (Pers.) Lam. ex Poir.

PubMed

Khalafalla, Mutasim M; Daffalla, Hussien M; Abdellatef, Eltayb; Agabna, Elsadig; El-Shemy, Hany A

2011-04-01

This report describes in vitro micropropagation of Boscia senegalensis, so-called famine foods, that helped the people in Darfur and Kordofan, Sudan survive during the 1984-1985 famine. Four types of explants prepared from green mature zygotic embryos were cultured on Murashige and Skoog (MS) medium augmented with 1-5 mg/L 6-benzyladenine (BA). The highest number of shoots per explant (14.3±0.9) was achieved on MS medium supplemented with 3 mg/L BA, while the highest shoot length [(3.5±0.4) cm] was obtained with 1 mg/L BA. The shoot cluster, when subcultured to its same medium, significantly increased the rate of shoot multiplication by the end of the third subculture. The maximum mean number of shoots per explant (86.5±3.6) was produced after three multiplication cycles on 3 mg/L BA-supplemented medium. In vitro induced shoots were excised and rooted on half strength MS medium fortified with 0.25 mg/L indole-3-butyric acid (IBA) to obtain complete plantlets. B. senegalensis-regenerated plantlets obtained in vitro for the first time, were hardened and 95% survived under greenhouse conditions.

Comparative effects of plant growth regulators on leaf and stem explants of Labisia pumila var. alata

PubMed Central

Ling, Anna Pick Kiong; Tan, Kinn Poay; Hussein, Sobri

2013-01-01

Objective: Labisia pumila var. alata, commonly known as ‘Kacip Fatimah’ or ‘Selusuh Fatimah’ in Southeast Asia, is traditionally used by members of the Malay community because of its post-partum medicinal properties. Its various pharmaceutical applications cause an excessive harvesting and lead to serious shortage in natural habitat. Thus, this in vitro propagation study investigated the effects of different plant growth regulators (PGRs) on in vitro leaf and stem explants of L. pumila. Methods: The capabilities of callus, shoot, and root formation were evaluated by culturing both explants on Murashige and Skoog (MS) medium supplemented with various PGRs at the concentrations of 0, 1, 3, 5, and 7 mg/L. Results: Medium supplemented with 3 mg/L indole-3-butyric acid (IBA) showed the optimal callogenesis from both leaf and stem explants with (72.34±19.55)% and (70.40±14.14)% efficacy, respectively. IBA was also found to be the most efficient PGR for root induction. A total of (50.00±7.07)% and (77.78±16.47)% of root formation were obtained from the in vitro stem and leaf explants after being cultured for (26.5±5.0) and (30.0±8.5) d in the medium supplemented with 1 and 3 mg/L of IBA, respectively. Shoot formation was only observed in stem explant, with the maximum percentage of formation ((100.00±0.00)%) that was obtained in 1 mg/L zeatin after (11.0±2.8) d of culture. Conclusions: Callus, roots, and shoots can be induced from in vitro leaf and stem explants of L. pumila through the manipulation of types and concentrations of PGRs. PMID:23825148

Efficient regeneration of sorghum, Sorghum bicolor (L.) Moench, from shoot-tip explant.

PubMed

Syamala, D; Devi, Prathibha

2003-12-01

Novel protocols for production of multiple shoot-tip clumps and somatic embryos of Sorghum bicolor (L.) Moench were developed with long-term goal of crop improvement through genetic transformation. Multiple shoot-tip clumps were developed in vitro from shoot-tip explant of one-week old seedling, cultured on MS medium containing only BA (0.5, 1 or 2 mg/l) or both BA (1 or 2 mg/l) and 2,4-D (0.5 mg/l) with bi-weekly subculture. Somatic embryos were directly produced on the enlarged dome shaped growing structures that developed from the shoot-tips of one-week old seedling explants (without any callus formation) when cultured on MS medium supplemented with both 2,4-D (0.5 mg/l) and BA (0.5 mg/l). However, the supplementation of MS medium with only 2,4-D (0.5 mg/l) induced compact callus without any plantlet regeneration. Each multiple shoot-clump was capable of regenerating more than 80 shoots via an intensive differentiation of both axillary and adventitious shoot buds, the somatic embryos were capable of 90% germination, plant conversion and regeneration. The regenerated shoots could be efficiently rooted on MS medium containing indole-3-butyric acid (IBA 1 mg/l). The plants were successfully transplanted to glasshouse and grown to maturity with a survival rate of 98%. Morphogenetic response of the explants was found to be genotypically independent.

In vitro propagation of Gentiana scabra Bunge - an important medicinal plant in the Chinese system of medicines.

PubMed

Huang, Shih-Hung; Agrawal, Dinesh Chandra; Wu, Fang-Sheng; Tsay, Hsin-Sheng

2014-12-01

Gentiana scabra Bunge commonly known as 'Long dan cao' in China has been used in traditional Chinese medicines for more than 2000 years. Dry roots and rhizome of the herb have been used for the treatment of inflammation, anorexia, indigestion and gastric infections. Iridoids and secoiridoids are the main bioactive compounds which attribute to the pharmacological properties of this plant. The species is difficult to mass propagate by seed due to the low percentage of germination and limited dormancy period. Wild populations in some locations are considered to be in the endangered category due to over exploitation. In the present study, we report an efficient micropropagation system. Shoot apices of six weeks old in vitro grown G. scabra plants were used as explants for the in vitro propagation. Induction of multiple shoots (9.1/explant) was achieved on the culture of shoot apices on half strength Murashige and Skoog's basal medium (MSBM) containing 2.0 mg/L -1 6-benzylaminopurine (BA), 3% sucrose and 0.9% Difco agar. In vitro shoots induced profuse rooting on half strength of MSBM supplemented with 0.1 mg/L -1 1-naphthaleneacetic acid (NAA), 3% sucrose and 0.3% gelrite. A two-stage ventilation closure procedure during the in vitro culture, and transparent sachet technique enhanced the survival rate of G. scabra plantlets to 96% in the greenhouse. Tissue culture plants flowered after 5 months of transfer to pots. A simple and an efficient in vitro propagation protocol of Gentiana scabra Bunge by optimizing the medium composition and ventilation closure treatments has been developed. The protocol can be very useful in germplasm conservation and commercial cultivation of G. scabra plants.

Micropropagation of Asparagus by in vitro shoot culture.

PubMed

Stajner, Nataša

2013-01-01

Asparagus officinalis is most extensively studied species within the genus Asparagus, which is well known as garden asparagus. This species is dioecious with unisexual flowers, which means that generative propagation gives roughly equal number of male and female plants. Male plants are high yielders and preferred commercially over female plants. Tissue culture techniques could efficiently promote vegetative propagation of male plants and pave the way for efficient plant breeding.This chapter describes an efficient micropropagation protocol for developing rapid growing in vitro Asparagus shoot cultures. The source of explants, inoculation, and shoot proliferation, followed by shoot propagation, rooting, and acclimatization is described. The optimal medium for Asparagus micropropagation described in this chapter is composed of MS macro- and microelements and a combination of auxins and cytokinins. Plant growth regulators NAA, kinetin, and BA were used in various concentrations. Three different media representing the whole micropropagation protocol of Asparagus are described; medium for shoot initiation, medium for shoot multiplication, and medium for root formation. By in vitro propagation of Asparagus, root initiation is difficult, but can be promoted by adding growth retardant ancymidol which also greatly promotes shoot development and suppresses callus formation.

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds.

PubMed

Kaushal, N; Modgil, M; Thakur, M; Sharma, D R

2005-06-01

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.

Apricot (Prunus armeniaca L.).

PubMed

Petri, César; Alburquerque, Nuria; Burgos, Lorenzo

2015-01-01

A protocol for Agrobacterium-mediated stable transformation of whole leaf explants of the apricot (Prunus armeniaca) cultivars 'Helena' and 'Canino' is described. Regenerated buds were selected using a two-step selection strategy with paromomycin sulfate and transferred to bud multiplication medium 1 week after they were detected for optimal survival. After buds were transferred to bud multiplication medium, antibiotic was changed to kanamycin and concentration increased gradually at each transfer to fresh medium in order to eliminate possible escapes and chimeras. Transformation efficiency, based on PCR analysis of individual putative transformed shoots from independent lines, was 5.6%. Green and healthy buds, surviving high kanamycin concentration, were transferred to shoot multiplication medium where they elongated in shoots and proliferated. Elongated transgenic shoots were rooted in a medium containing 70 μM kanamycin. Rooted plants were acclimatized following standard procedures. This constitutes the only transformation protocol described for apricot clonal tissues and one of the few of Prunus.

An efficient in vitro regeneration protocol for a natural dye yielding plant, Strobilanthes flaccidifolious Nees., from nodal explants.

PubMed

Deb, Chitta Ranjan; Arenmongla, T

2012-11-01

Adventitious shoot buds formation from axillary buds of nodal segments of S. flaccidifolious was achieved on MS medium containing sucrose (3%, w/v), and a-naphthalene acetic acid (NAA; 3 microM) and benzyl adenine (3 microM) in combination. The nodal segments were primed on 'Growtak Sieve' for 48 h on MS medium containing sucrose (2%), polyvinyl pyrollidone (200 mgL(-1)) as antioxidant. About 80% of primed nodal segments responded positively and formed approximately 12 adventitious shoot buds per explants from explants collected during October-November months of every year. The shoot buds converted into plantlets on MS medium containing sucrose (3%) and kinetin (3 microM) where approximately 7 micro shoots developed per subculture after 8 weeks of culture. The regenerated micro shoots induced average 14 roots/plant on medium containing NAA (3 microM). The regenerates were hardened for 6-7 weeks on medium with 1/2MS salt solution and sucrose (2%) under normal laboratory condition before transferring to potting mix. About 70% transplants survived after two months of transfer.

An Improved Micropropagation Protocol by Ex Vitro Rooting of Passiflora edulis Sims. f. flavicarpa Deg. through Nodal Segment Culture.

PubMed

Shekhawat, Mahipal S; Manokari, M; Ravindran, C P

2015-01-01

A procedure for rapid clonal propagation of Passiflora edulis Sims. f. flavicarpa Deg. (Passifloraceae) has been developed in this study. Nodal explants were sterilized with 0.1% HgCl2 and inoculated on Murashige and Skoog (MS) basal medium. The addition of 2.0 mgL(-1) 6-benzylaminopurine (BAP) to MS medium caused an extensive proliferation of multiple shoots (8.21 ± 1.13) primordial from the nodal meristems. Subculturing of these multiple shoots on the MS medium augmented with 1.0 mgL(-1) of each BAP and Kinetin (Kin) was successful for the multiplication of the shoots in vitro with maximum numbers of shoots (25.73 ± 0.06) within four weeks of incubation. Shoots were rooted best (7.13 ± 0.56 roots/shoots) on half strength MS medium supplemented with 2.0 mgL(-1) indole-3 butyric acid (IBA). All in vitro regenerated shoots were rooted by ex vitro method, and this has achieved 6-7 roots per shoot by pulsing of cut ends of the shoots using 200 as well as 300 mgL(-1) IBA. The plantlets were hardened in the greenhouse for 4-5 weeks. The hardened plantlets were shifted to manure containing nursery polybags after five weeks and then transferred to a sand bed for another four weeks for acclimatization before field planting with 88% survival rate.

An Improved Micropropagation Protocol by Ex Vitro Rooting of Passiflora edulis Sims. f. flavicarpa Deg. through Nodal Segment Culture

PubMed Central

Shekhawat, Mahipal S.; Manokari, M.; Ravindran, C. P.

2015-01-01

A procedure for rapid clonal propagation of Passiflora edulis Sims. f. flavicarpa Deg. (Passifloraceae) has been developed in this study. Nodal explants were sterilized with 0.1% HgCl2 and inoculated on Murashige and Skoog (MS) basal medium. The addition of 2.0 mgL−1 6-benzylaminopurine (BAP) to MS medium caused an extensive proliferation of multiple shoots (8.21 ± 1.13) primordial from the nodal meristems. Subculturing of these multiple shoots on the MS medium augmented with 1.0 mgL−1 of each BAP and Kinetin (Kin) was successful for the multiplication of the shoots in vitro with maximum numbers of shoots (25.73 ± 0.06) within four weeks of incubation. Shoots were rooted best (7.13 ± 0.56 roots/shoots) on half strength MS medium supplemented with 2.0 mgL−1 indole-3 butyric acid (IBA). All in vitro regenerated shoots were rooted by ex vitro method, and this has achieved 6-7 roots per shoot by pulsing of cut ends of the shoots using 200 as well as 300 mgL−1 IBA. The plantlets were hardened in the greenhouse for 4-5 weeks. The hardened plantlets were shifted to manure containing nursery polybags after five weeks and then transferred to a sand bed for another four weeks for acclimatization before field planting with 88% survival rate. PMID:26273489

Morphogenesis in leaf and single-cell cultures of mature Juniperus oxycedrus.

PubMed

Gomez, M P; Segura, J

1996-08-01

Single cells were mechanically isolated from leaf-derived callus of mature Juniperus oxycedrus L. These cells divided and gave rise to callus when plated on medium containing growth regulators. Best plating efficiency was obtained on a modified Schenk and Hildebrandt medium supplemented with 0.6 micro M 2,4-dichlorophenoxyacetic acid and 100 mg l(-1) casein hydrolyzate. Although single-cell-derived callus showed poor morphogenic potential, both adventitious shoots and embryogenic tissues differentiated from the callus. We also achieved induction of somatic embryogenesis in leaf explants of mature J. oxycedrus trees cultured in the presence of 6.0 or 10.0 micro M 2,4-dichlorophenoxyacetic acid or picloram. Frequency of embryogenic callus ranged from 6 to 18%; however, under the culture conditions tested, isolated embryos failed to develop into plants.

Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby.

PubMed

Bamel, Kiran; Gupta, Rajendra; Gupta, Shirish C

2016-06-02

We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10(-4) M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10(-5) M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants.

Acetylcholine suppresses shoot formation and callusing in leaf explants of in vitro raised seedlings of tomato, Lycopersicon esculentum Miller var. Pusa Ruby

PubMed Central

Bamel, Kiran; Gupta, Rajendra; Gupta, Shirish C.

2016-01-01

ABSTRACT We present experimental evidence to show that acetylcholine (ACh) causes decrease in shoot formation in leaf explants of tomato (Lycopersicon esculentum Miller var Pusa Ruby) when cultured on shoot regeneration medium. The optimum response was obtained at 10−4 M ACh-enriched medium. ACh also causes decrease in percentage of cultures forming callus and reduces the callus mass. Inhibitors of enzymatic hydrolysis of ACh, neostigmine and physostigmine, also suppresses callogenesis and caulogenesis. On the other hand, the breakdown products of Ach, choline and acetate, do not alter the morphogenic response induced on the shoot regeneration medium. Neostigmine showed optimal reduction in shoot formation at 10−5 M. The explants cultured on neostigmine augmented medium showed decline in the activity of ACh hydrolyzing enzyme acetylcholinesterase. ACh and neostigmine added together showed marked reduction in callus mass. These results strongly support the role of ACh as a natural regulator of morphogenesis in tomato plants. PMID:27348536

Micropropagation of Rubus and Ribes spp.

PubMed

Dziedzic, Ewa; Jagła, Joanna

2013-01-01

Micropropagation is the most appropriate method for large-scale production of Rubus and Ribes spp. The proliferation rate of Rubus spp. differs in shoot tips and nodal segments. The culture media used for raspberry and blackberry propagation are MS-based supplemented with different combination and ratio of plant growth regulators, depending on the stage of culture. The initiation medium containing 0.4 mg L(-1) BA and 0.1 mg L(-1) IBA is used to stabilize shoot cultures. In multiplication media, concentration of cytokinin is doubled. In vitro rooting of shoots is achieved on media supplemented with 1.0 mg L(-1) IBA. Ribes spp. cultures are initiated from shoot tips, meristem, or dormant buds on MS medium supplemented with 2.0 mg L(-1) BA, 0.5 mg L(-1) IBA, and 0.1 mg L(-1) GA(3.) After stabilization of shoot cultures in 3-4-week time, shoot multiplication is carried out on MS medium containing 1.0 mg L(-1) BA and 0.1 mg L(-1) IBA. Shoots 2 cm long are cultured to rooting on a medium amended with 2.0 mg L(-1) IBA and 5.0 mg L(-1) IAA. Rooted plantlets are transferred to universal peat substrate and acclimatized in the greenhouse.

Micropropagation and acclimatization of Stevia rebaudiana Bertoni.

PubMed

Chotikadachanarong, Kittisak; Dheeranupattana, Srisuluk

2013-09-01

Multiple shoot induction of Stevia rebaudiana Bertoni was studied by node explants that were cultured on solidified MS media and supplemented with 0, 1, 2, 3 and 4 mg L-1 kinetin for 4 weeks. The results showed the maximum amount of multiple shoot induction (9.31+/-4.17 shoots/explant) when cultured on MS media supplemented with 3 mg L-1 kinetin. In vitro shoots were rooted on solidified MS media supplemented with 0, 0.1, 0.5 and 2 mg L-1 Naphthaleneacetic Acid (NAA) for 4 weeks. The highest number of roots (11.18+/-1.34 roots/shoot) was detected on a concentration of 0.1 mg L-1 NAA while the high survival rate (80%) was obtained when the rooted plantlets were transferred to greenhouse conditions.

Shoot bending promotes flower bud formation by miRNA-mediated regulation in apple (Malus domestica Borkh.).

PubMed

Xing, Libo; Zhang, Dong; Zhao, Caiping; Li, Youmei; Ma, Juanjuan; An, Na; Han, Mingyu

2016-02-01

Flower induction in apple (Malus domestica Borkh.) trees plays an important life cycle role, but young trees produce fewer and inferior quality flower buds. Therefore, shoot bending has become an important cultural practice, significantly promoting the capacity to develop more flower buds during the growing seasons. Additionally, microRNAs (miRNAs) play essential roles in plant growth, flower induction and stress responses. In this study, we identified miRNAs potentially involved in the regulation of bud growth, and flower induction and development, as well as in the response to shoot bending. Of the 195 miRNAs identified, 137 were novel miRNAs. The miRNA expression profiles revealed that the expression levels of 68 and 27 known miRNAs were down-regulated and up-regulated, respectively, in response to shoot bending, and that the 31 differentially expressed novel miRNAs between them formed five major clusters. Additionally, a complex regulatory network associated with auxin, cytokinin, abscisic acid (ABA) and gibberellic acid (GA) plays important roles in cell division, bud growth and flower induction, in which related miRNAs and targets mediated regulation. Among them, miR396, 160, 393, and their targets associated with AUX, miR159, 319, 164, and their targets associated with ABA and GA, and flowering-related miRNAs and genes, regulate bud growth and flower bud formation in response to shoot bending. Meanwhile, the flowering genes had significantly higher expression levels during shoot bending, suggesting that they are involved in this regulatory process. This study provides a framework for the future analysis of miRNAs associated with multiple hormones and their roles in the regulation of bud growth, and flower induction and formation in response to shoot bending in apple trees. © 2015 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

In Vitro Propagation of Sambong (Blumea balsamifera Linn.).

PubMed

Soriano, Thelma L; Cangao, Evangelina C

2016-01-01

Terminal shoot tips of sambong (Blumea balsamifera Linn.) are cultured to initiate and regenerate shoots on Murashige and Skoog (MS) medium containing 1.0 mg/L benzyl adenine (BA). After 1 month, shoots, usually 4.5 cm long are separated and subcultured for multiplication. Regenerated shoots, about 6 cm long are rooted on MS medium supplemented with 1.0 mg/L naphthalene acetic acid (NAA). Exposure of shoots to high humidity for the first 2 weeks and equal proportion (1:1:1) of sterile sand, compost, and coir dust as potting mix favors the development of whole sambong plants. Young shoots from in vitro-derived sambong plants could also be used for propagation.

Light Inhibition of Shoot Regeneration Is Regulated by Endogenous Abscisic Acid Level in Calli Derived from Immature Barley Embryos

PubMed Central

Rikiishi, Kazuhide; Matsuura, Takakazu; Ikeda, Yoko; Maekawa, Masahiko

2015-01-01

Shoot regeneration in calli derived from immature barley embryos is regulated by light conditions during the callus-induction period. Barley cultivars Kanto Nijo-5 (KN5) and K-3 (K3) showed lower efficiency of shoot regeneration in a 16-h photoperiod during callus-induction than those in continuous darkness, whereas shoot regeneration was enhanced in cultures under a 16-h photoperiod in Golden Promise (GP) and Lenins (LN). These cultivars were classified as photo-inhibition type (KN5 and K3) or photo-induction type (GP and LN) according to their response to light. Contents of endogenous plant hormones were determined in calli cultured under a 16-h photoperiod and continuous darkness. In photo-inhibition type, higher accumulation of abscisic acid (ABA) was detected in calli cultured under a 16-h photoperiod, whereas calli showed lower levels of endogenous ABA in continuous darkness. However, cultivars of photo-induction type showed lower levels of ABA in calli cultured under both light conditions, similarly to photo-inhibition type in continuous darkness. Exogenous ABA inhibited the callus growth and shoot regeneration independent of light conditions in all cultivars. In photo-inhibition type, lower levels of endogenous ABA induced by ABA biosynthesis inhibitor, fluridone, reduced the photo-inhibition of shoot regeneration. Expression of ABA biosynthesis gene, HvNCED1, in calli was regulated by the light conditions. Higher expression was observed in calli cultured under a 16-h photoperiod. These results indicate that ABA biosynthesis could be activated through the higher expression of HvNCED1 in a 16-h photoperiod and that the higher accumulations of ABA inhibit shoot regeneration in the photo-inhibition type cultivars. PMID:26670930

In vitro propagation via organogenesis and synthetic seeds of Urginea altissima (L.f.) Baker: a threatened medicinal plant.

PubMed

Baskaran, Ponnusamy; Kumari, Aloka; Van Staden, Johannes

2018-01-01

Efficient in vitro propagation systems via organogenesis and synthetic seeds were developed for the first time for conservation and commercial propagation from leaf or longitudinal thin cell layer (lTCL) leaf or shoot-tip explants of Urginea altissima . Various plant growth regulators and phloroglucinol were used in semi-solid and liquid Murashige and Skoog (MS) medium to establish multiplication of shoots and roots for in vitro regeneration. Of the various treatments, the highest number of shoots (17.4 per lTCL leaf explant) was obtained on liquid MS medium supplemented with 10 µM meta -Topolin ( m T) and 2 µM benzyladenine followed by transferal to semi-solid MS media. The shoot tips were encapsulated with liquid MS medium plus 3% (w/v) sodium alginate and 100 mM calcium chloride. Adventitious shoot regeneration (91.0%; 12.6 shoots per synthetic seed) of synthetic seeds was achieved on semi-solid MS medium supplemented with 10 µM m T and 2 µM naphthaleneacetic acid (NAA) after 15 days of storage in darkness at 25 ± 2 °C. Regenerated shoots rooted (9.8 roots per shoot; 6.5 cm long) efficiently when transferred to 5 µM indole-3-butyric acid and 2.5 µM NAA. All the plantlets were successfully acclimatized (100%) in a vermiculite:soil (1:1 v/v) mixture in the greenhouse.

Micropropagation and Subsequent Enrichment of Carotenoids, Fatty Acids, and Tocopherol Contents in Sedum dasyphyllum L

PubMed Central

Park, Han Yong; Saini, Ramesh Kumar; Gopal, Judy; Keum, Young-Soo; Kim, Doo Hwan; Lee, Onew; Sivanesan, Iyyakkannu

2017-01-01

A promising micropropagation protocol has been systematically established and demonstrated for the enhanced production of carotenoids, tocopherol and fatty acids in shoot tissues of Sedum dasyphyllum. Shoot tip explants were grown on Murashige and Skoog (MS) medium. Different concentrations of N6-benzyladenine (BA) or thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA) were tested in order to stimulate multiple shoot production. Ideal shoot induction (100%) and maximized shoot numbers (36.4) were obtained on explants cultured on media incorporated with 2 μM BA and 1 μM NAA combinations. The in vitro-developed shoots rooted best on half-strength MS media incorporated with 2 μM indole 3-butyric acid. Plantlets were effectively acclimatized in the greenhouse with 100% survival rate. The composition and contents of bioactive compounds such as carotenoids, tocopherol and fatty acids in shoot tissues of S. dasyphyllum were investigated using HPLC and GC-MS. The most abundant carotenoid in the shoot tissue was all-E-lutein (40.3–70.5 μg g−1 FW) followed by 9′-Z-neoxanthin (5.3–9.9 μg g−1 FW), all-E-violaxanthin (4.4–8.2 μg g−1 FW), and all-E-β-carotene (1.6–3.6 μg g−1 FW). The α-tocopherol contents of in vitro-raised shoots was 6.5-fold higher than shoots of greenhouse-grown plants. The primary fatty acids found in shoot tissues were α-linolenic acid (32.0–39.3%), linoleic acid (27.4–38.2%), palmitic acid (13.3–15.5%), and stearic acid (5.2–12.2%). In all, summarizing the findings, the micropropagated S. dasyphyllum showed significant enrichment of valuable bioactive carotenoids (92.3 μg g−1 FW), tocopherols (14.6 μg g−1 FW), and α-linolenic acid (39.3%) compared to their greenhouse counterparts. The protocol demonstrated here could be applied for the mass propagation and production of enhanced bioactive compounds from S. dasyphyllum with credibility. PMID:29062834

Micropropagation and Subsequent Enrichment of Carotenoids, Fatty Acids, and Tocopherol Contents in Sedum dasyphyllum L.

PubMed

Park, Han Yong; Saini, Ramesh Kumar; Gopal, Judy; Keum, Young-Soo; Kim, Doo Hwan; Lee, Onew; Sivanesan, Iyyakkannu

2017-01-01

A promising micropropagation protocol has been systematically established and demonstrated for the enhanced production of carotenoids, tocopherol and fatty acids in shoot tissues of Sedum dasyphyllum . Shoot tip explants were grown on Murashige and Skoog (MS) medium. Different concentrations of N 6 -benzyladenine (BA) or thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA) were tested in order to stimulate multiple shoot production. Ideal shoot induction (100%) and maximized shoot numbers (36.4) were obtained on explants cultured on media incorporated with 2 μM BA and 1 μM NAA combinations. The in vitro -developed shoots rooted best on half-strength MS media incorporated with 2 μM indole 3-butyric acid. Plantlets were effectively acclimatized in the greenhouse with 100% survival rate. The composition and contents of bioactive compounds such as carotenoids, tocopherol and fatty acids in shoot tissues of S. dasyphyllum were investigated using HPLC and GC-MS. The most abundant carotenoid in the shoot tissue was all- E -lutein (40.3-70.5 μg g -1 FW) followed by 9'- Z -neoxanthin (5.3-9.9 μg g -1 FW), all- E -violaxanthin (4.4-8.2 μg g -1 FW), and all- E -β-carotene (1.6-3.6 μg g -1 FW). The α-tocopherol contents of in vitro -raised shoots was 6.5-fold higher than shoots of greenhouse-grown plants. The primary fatty acids found in shoot tissues were α-linolenic acid (32.0-39.3%), linoleic acid (27.4-38.2%), palmitic acid (13.3-15.5%), and stearic acid (5.2-12.2%). In all, summarizing the findings, the micropropagated S. dasyphyllum showed significant enrichment of valuable bioactive carotenoids (92.3 μg g -1 FW), tocopherols (14.6 μg g -1 FW), and α-linolenic acid (39.3%) compared to their greenhouse counterparts. The protocol demonstrated here could be applied for the mass propagation and production of enhanced bioactive compounds from S. dasyphyllum with credibility.

Alginate-encapsulation of shoot tips of jojoba [Simmondsia chinensis (Link) Schneider] for germplasm exchange and distribution.

PubMed

Kumar, Sunil; Rai, Manoj K; Singh, Narender; Mangal, Manisha

2010-12-01

Shoot tips excised from in vitro proliferated shoots derived from nodal explants of jojoba [Simmondsia chinensis (Link) Schneider] were encapsulated in calcium alginate beads for germplasm exchange and distribution. A gelling matrix of 3 % sodium alginate and 100 mM calcium chloride was found most suitable for formation of ideal calcium alginate beads. Best response for shoot sprouting from encapsulated shoot tips was recorded on 0.8 % agar-solidified full-strength MS medium. Rooting was induced upon transfer of sprouted shoots to 0.8 % agar-solidified MS medium containing 1 mg l(-1) IBA. About 70 % of encapsulated shoot tips were rooted and converted into plantlets. Plants regenerated from encapsulated shoot tips were acclimatized successfully. The present encapsulation approach could also be applied as an alternative method of propagation of desirable elite genotype of jojoba.

In vitro propagation, carotenoid, fatty acid and tocopherol content of Ajuga multiflora Bunge.

PubMed

Sivanesan, Iyyakkannu; Saini, Ramesh Kumar; Noorzai, Rafi; Zamany, Ahmad Jawid; Kim, Doo Hwan

2016-06-01

The effect of plant growth regulators on shoot proliferation from shoot tip explants of Ajuga multiflora was studied. The highest number of shoots (17.1) was observed when shoot tip explants were cultured on Murashige and Skoog (MS) medium fortified with 8.0 µM 6-Benzyladenine (BA) and 2.7 µM α-naphthaleneacetic acid (NAA). The mean number of shoots per explant was increased 1.6-fold in liquid medium as compared with semi-solid medium. Maximum rooting (100 %) with an average of 7.2 roots per shoot was obtained on MS basal medium. Rooted plantlets were successfully acclimatised in the greenhouse with 100 % survival rate. Composition of carotenoids, fatty acids and tocopherols was also studied from leaves of greenhouse-grown plants and in vitro-regenerated shoots of A. multiflora. The greatest amounts of carotenoids, fatty acids and tocopherols were obtained from leaves of in vitro-regenerated shoots cultured on MS basal medium, followed by leaves of greenhouse-grown plants and leaves of in vitro-regenerated shoots cultured on MS basal medium with 2.0 µM BA or thidiazuron. The most abundant carotenoid in A. multiflora leaves was all-E-lutein (89.4-382.6 μg g -1  FW) followed by all-E-β-carotene (32.0-156.7 μg g -1  FW), 9'-Z-neoxanthin (14.2-63.4 μg g -1  FW), all-E-violaxanthin (13.0-45.9 μg g -1  FW), all-E-zeaxanthin (1.3-2.5 μg g -1  FW) and all-E-β-cryptoxanthin (0.3-0.9 μg g -1  FW). α-Tocopherol was the predominant tocopherol in A. multiflora leaves. Linolenic acid (49.03-52.59 %) was detected in higher amounts in A. multiflora leaf samples followed by linoleic acid (18.95-21.39 %) and palmitic acid (15.79-18.66 %).

In vitro propagation of Stevia rebaudina plants using multiple shoot culture.

PubMed

Nepovím, A; Vanek, T

1998-12-01

A multiple shoot culture was induced from nodal segments on MS medium containing half concentration of macroelements, 1% sucrose, and supplemented with NAA (0.01 mg/l). A bioreactor with hormone-free MS medium (300 ml) was inoculated with 1.5 g of the multiple shoot culture and cultivated for a month. The cultivating process of the multiple shoot culture in the bioreactor and the transfer into ex vitro conditions took about 8-9 weeks and produced approx. 600 new seedlings, that could be transferred from greenhouse to field conditions.

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas).

PubMed

Mohd Din, Abd Rahman Jabir; Iliyas Ahmad, Fauziah; Wagiran, Alina; Abd Samad, Azman; Rahmat, Zaidah; Sarmidi, Mohamad Roji

2016-01-01

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L(-1)) and NAA (2 mg L(-1)) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5-1.5 mg L(-1)), BAP, NAA and 0.5 mg L(-1) of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L(-1)), Kin (1.5 mg L(-1)), NAA (0.5 mg L(-1)) and TDZ (0.5 mg L(-1)) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

PubMed Central

Mohd Din, Abd Rahman Jabir; Iliyas Ahmad, Fauziah; Wagiran, Alina; Abd Samad, Azman; Rahmat, Zaidah; Sarmidi, Mohamad Roji

2015-01-01

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L−1) and NAA (2 mg L−1) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L−1), BAP, NAA and 0.5 mg L−1 of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L−1), Kin (1.5 mg L−1), NAA (0.5 mg L−1) and TDZ (0.5 mg L−1) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots. PMID:26858569

Multiple shoot production from seedling explants of slash pine (Pinus elliottii, Engelm.).

PubMed

Burns, J A; Schwarz, O J; Schlarbaum, S E

1991-11-01

Hypocotylary explants obtained from 30- to 40-day-old slash pine (Pinus elliottii, Engelm.) seedlings treated with 6-benzylaminopurine produced multiple buds that eventually elongated into axillary shoots. The explants were pulse treated (45-s dip) with 6-benzylaminopurine (22.2, 111, 222 μM) plus a control and cultured on three different basal media containing activated charcoal (0.5% w/v). Hormonal concentration and basal medium were compared for the number and size of axillary shoots induced after 12 and 29 days. The greatest number of axillary shoots was produced by explants that were pulse treated with 111 μM 6-benzylaminopurine and cultured on Gresshoff and Doy medium. The axillary shoots were fewer in number per explant than shoots previously reported resulting from hormonally induced advantitious buds of slash pine, but the axillary shoots developed more rapidly.

A xyloglucan from seeds of the native Brazilian species Hymenaea courbaril for micropropagation of Marubakaido and Jonagored apples.

PubMed

Lima-Nishimura, N; Quoirin, M; Naddaf, Y G; Wilhelm, H M; Ribas, L L F; Sierakowski, M-R

2003-01-01

Xyloglucan was extracted from seeds of Hymenaea courbaril and mixed with agar to prepare a solid culture medium used for micropropagation of the Marubakaido apple rootstock (Malus prunifolia Borkh) and cv. Jonagored (Malus domestica). The performance on gels created from a blend of 0.4%agar and 0.2% xyloglucan (w/v) was compared with that on media gelled with a standard concentration 0.6% (w/v) of agar. The growth of shoots and the multiplication rate were higher on the modified culture medium than on the agar-gelled medium. The occurrence of hyperhydric shoots was lower on the modified medium. In the absence of auxin, shoot rooting reached 70% (Marubakaido) and 66% (Jonagored) on the agar-xyloglucan medium and 6.7% and 10.4%, respectively, on the agar medium. When 0.25 microM indole-3-butyric acid (IBA) was added to both media, the modified medium gave better results in terms of rooting percentage and quality of roots than the agar-gelled medium.

Light Requirement for Shoot Regeneration in Horseradish Hairy Roots 1

PubMed Central

Saitou, Tsutomu; Kamada, Hiroshi; Harada, Hiroshi

1992-01-01

Hairy roots of horseradish (Armoracia rusticana) were induced by inoculation with Agrobacterium rhizogenes harboring Ri plasmid and cultured on phytohormone-free Murashige and Skoog medium after eliminating the bacteria. Hairy roots grew vigorously and sometimes formed yellowish calli under dark conditions. On the other hand, growth of hairy roots stopped after several weeks of culture with light, then shoots were regenerated. Frequency of shoot formation from hairy roots increased as the culture period in light lengthened and the light intensity increased. The shoot regeneration was induced by treatment with white or red light, but not with far-red light. Shoot regeneration by red light was inhibited by following treatment with far-red light. Red and far-red light reversibly affected shoot regeneration. Excised roots of nontransformed plants grew quite slowly on phytohormone-free Murashige and Skoog medium and occasionally formed shoots under white light conditions. PMID:16669041

Light requirement for shoot regeneration in horseradish hairy roots.

PubMed

Saitou, T; Kamada, H; Harada, H

1992-08-01

Hairy roots of horseradish (Armoracia rusticana) were induced by inoculation with Agrobacterium rhizogenes harboring Ri plasmid and cultured on phytohormone-free Murashige and Skoog medium after eliminating the bacteria. Hairy roots grew vigorously and sometimes formed yellowish calli under dark conditions. On the other hand, growth of hairy roots stopped after several weeks of culture with light, then shoots were regenerated. Frequency of shoot formation from hairy roots increased as the culture period in light lengthened and the light intensity increased. The shoot regeneration was induced by treatment with white or red light, but not with far-red light. Shoot regeneration by red light was inhibited by following treatment with far-red light. Red and far-red light reversibly affected shoot regeneration. Excised roots of nontransformed plants grew quite slowly on phytohormone-free Murashige and Skoog medium and occasionally formed shoots under white light conditions.

Clonal propagation of Stevia rebaudiana Bertoni by stem-tip culture.

PubMed

Tamura, Y; Nakamura, S; Fukui, H; Tabata, M

1984-10-01

Clonal propagation of Stevia rebaudiana has been established by culturing stem-tips with a few leaf primordia on an agar medium supplemented with a high concentration (10 mg/l) of kinetin. Anatomical examination has suggested that these multiple shoots originate from a number of adventitious buds formed on the margin of the leaf. Innumerable shoots can be obtained by repeating the cycle of multiple-shoot formation from a single stem-tip of Stevia. These shoots produce roots when transferred to a medium containing NAA (0.1 mg/l) without kinetin. The regenerated plantlets can be transplanted to soil.

Optimization of Regeneration Conditions and In Vitro Propagation of Sideritis Stricta Boiss & Heldr.

PubMed

Yavuz, Dudu Özkum

2016-09-01

In this study the micropropagation of endemic species Sideritis stricta was investigated. Leaf segments and shoot explants (hypocotyl, single node and shoot tips) taken from in vitro growing plantlets and cultured on MS and B5 media containing different growth regulators combinations BAP (0.0, 1.0, 2.0 and 3.0mg/l) and NAA (0.0, 0.1 and 0.5mg/l). MS and B5 media supplemented with BAP (1.0, 2.0 and 3.0mg/l) and NAA (0.1mg/l) combinations or only BAP and kinetin (2.0 and 3.0mg/l) were used at the subculture experiments of shoots and MS and B5 media supplemented with different concentrations of IBA (0.0, 1.5, 3.0, 4.5 and 10.mg/l) were used at the rooting experiments. S. stricta seeds germinated at the rate of 100% when the seed coat was removed and endoperm with embryo part cultured on B5 medium. The single node explants taken from in vitro germinated and grown 30-40 days plantlets on B5 medium have been determined as the most successful explant at all used hormone combinations. B5 medium supplemented with 1.0mg/l BAP+0.1mg/l NAA and 2.0mg/l BAP+0.5mg/l NAA was determined as the most effective medium on shoot formation. At the first and second subculture, the highest shoot formation was maintained on medium supplemented with 1.0mg/l BAP+0.1mg/l NAA and the number of shoots per explant were 4 and 2.11, respectively. The highest multiplication rate has been determined as 33.76 at the end of second subculture. The best rooting was achieved on B5 medium supplemented with 4.5mg/l IBA. The rooted shoots were successfully acclimatized to outdoor conditions and survival rate was determined as 90%. Copyright © 2015 Elsevier B.V. All rights reserved.

Control of in vitro rooting and plant development in Corymbia maculata by silver nitrate, silver thiosulfate and thiosulfate ion.

PubMed

Steinitz, Benjamin; Barr, Nurit; Tabib, Yona; Vaknin, Yiftach; Bernstein, Nirit

2010-11-01

Plant regeneration and transformation in vitro is often improved by adding silver ion (Ag(+)) to the culture media as AgNO(3) or silver thiosulfate (STS). Ag(+) reacts with substances to form insoluble precipitates, while thiosulfate (S(2)O(3) (2-)) interferes with these reactions. We studied the implications of silver precipitation and S(2)O(3) (2-) in the medium for culture development by (1) examining formation of Ag(+) precipitates from AgNO(3) versus STS in agar gels and their possible dependence on agar type; (2) comparing Corymbia maculata culture responses to AgNO(3) and STS and determining which better suits control of culture development; (3) clarifying whether STS-dependent alterations in culture development are due to Ag(+) alone or also to a separate influence of S(2)O(3) (2-). Silver precipitates appeared in aqueous gels of four agar brands supplemented with AgNO(3), but not in Phytagel(™), which remained transparent. No precipitation was observed in gels with STS. Indole-3-butyric acid (IBA)-mediated adventitious root induction and shoot growth were higher in C. maculata shoot tips cultured on gels with STS versus AgNO(3) (6-25 μM Ag(+)). IBA-treated shoot tips exhibited enhanced adventitious root regeneration, accelerated root elongation, increased frequency of lateral root formation, and stimulated shoot growth mediated by 100-250 μM sodium thiosulfate (Na(2)S(2)O(3)) in medium without Ag(+). The potency of S(2)O(3) (2-) in facilitating culture development has never been recognized. It is inferred that superiority of STS in stimulating multiple responses of C. maculata culture results from sustained biological activity of Ag(+) through prevention of its precipitation, and from impact of S(2)O(3) (2-) on cell differentiation and growth.

Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

PubMed

Kadokura, Satoshi; Sugimoto, Kaoru; Tarr, Paul; Suzuki, Takamasa; Matsunaga, Sachihiro

2018-04-28

Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification. Copyright © 2018. Published by Elsevier Inc.

Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers.

PubMed

Sen, Ayse; Alikamanoglu, Sema

2012-01-01

Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0-75Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20Gy gamma rays. Irradiated shoot tips were sub-cultured and M(1)V(1)-M(1)V(3) generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20gl(-1) PEG6000. Of the M(1)V(3) plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20Gy gamma radiation and regenerated on selective culture media containing 10gl(-1) PEG6000 concentration, and the second cluster was further divided into five sub-clusters. Copyright © 2012 Elsevier B.V. All rights reserved.

In Vitro Development from Leaf Explants of Sugar Beet (Beta vulgaris L). Rhizogenesis and the Effect of Sequential Exposure to Auxin and Cytokinin

PubMed Central

Gürel, Ekrem; Wren, M. Jill

1995-01-01

Adventitious root development in lamina and midrib-petiole junction expiants of sugar beet cv. Primo was investigated using scanning electron microscopy and light microscopy. Primordia developed close to the vascular strands and areas of newly dividing cells (meristematic centres) were seen adjacent to the intrafascicular cambium after 2 d incubation on medium containing 30 mg 1−11-naphthalene acetic acid. Clearly defined primordia were visible at 4 d and the first roots had emerged by 6 d. A minimum of 24 h exposure to NAA was necessary for root induction. Four days on NAA caused twice as many roots to be initiated but more prolonged exposure (5 and 10 d) inhibited root development. Root initiation continued after transfer to medium containing no plant growth regulators, new primordia appearing as the older ones extended as roots. Attempts were made to modify the development of primordia by sequential culture on cytokinin after induction by auxin. Incubation on N6-benzylaminopurine within 48 h of exposure to NAA disrupted the development of primordia and roots but did not induce shoot formation. PMID:21247910

Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium.

PubMed

Liu, Yang; Zhang, Hai-Liang; Guo, He-Rong; Xie, Li; Zeng, Rui-Zhen; Zhang, Xiang-Qian; Zhang, Zhi-Sheng

2017-01-01

Cymbidium , one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium 'Xiaofeng' (CXF), which can be efficiently micropropagated, and terrestrial Cymbidium sinense 'Qijianbaimo' (CSQ), which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs) were identified between CXF and CSQ during shoot induction. Gene ontology (GO) enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid) metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium.

Influence of growth regulators and explant type on in vitro shoot propagation and rooting of red sandal wood (Pterocarpus santalinus L.).

PubMed

Arockiasamy, S; Ignacimuthu, S; Melchias, G

2000-12-01

In vitro shoot regeneration in Pterocarpus santalinus L. was achieved when detached cotyledons from in vitro germinated seedlings were cultured on MS medium containing NAA (0.1 mg/L), BA (1 mg/L) and kinetin (1 mg/L). The regenerated shoots rooted on 1/4 strength MS medium with IAA (1 mg/L) and the fully developed plantlets were successfully established in the soil.

Direct organogenesis of seaside heliotrope (Heliotropium crassavicum) using stem explants.

PubMed

Satyavani, K; Dheepak, V; Gurudeeban, S; Ramanathan, T

2013-10-15

Heliotropium crassavicum L. is a sand binder salt marsh herb with enormous traditional value and widely found in South Asia America and Europe. In the direct method of regeneration from stem explants, we observed the maximum number of shoot regeneration after four weeks culture of MS elongation medium with 2.0 mg L(-1) of 2, 4-D (17.27 +/- 0.51). It was clear that MS medium with 2.0 mg mL(-1) 2, 4-D alone suitable for shoot multiplication as well as shoot elongation then compared to other combination of auxin and cytokinin. In vitro shoots were excised from shoot clumps and transferred to rooting medium containing 2, 4-dichlorophenoxy acetic acid (0.5-3.0 mg L(-1)). The maximum number of root regeneration (6.4 +/- 0.416) and root length (6.08 +/- 0.07) were observed in MS rooting medium fortified with 2.5 mg L(-1) of 2, 4-D after 2 weeks of culture. 85% of in vitro raised plantlets with well-developed shoots and roots were transferred to ex vivo conditions into polythene bag containing sterile compost with ratio (v/v/v) of organic fertilizer: sand: peat (1:2:2; 3:1:0 or 2:2:1). Sixty five percent of acclimated plants were transferred to the pots under full sun where they grew well without any detectable phenotypic variations.

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

PubMed

Ramakrishna, Akula; Giridhar, Parvatam; Ravishankar, G A

2009-12-01

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 microM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 microM, serotonin reuptake inhibitor (Prozac) 20 microM. In another set of experiment, calcium at 5 mM, calcium ionophore (A23187) 100 microM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 microM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 microM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%-80% explants responded for organogenesis. SER or MEL along with calcium ionophore (A23187) at 100 microM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40-70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.

Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi.

PubMed

Rey, Hebe Y; Faloci, Mirta; Medina, Ricardo; Dolce, Natalia; Mroginski, Luis; Engelmann, Florent

2009-01-01

A cryopreservation protocol using the encapsulation-dehydration procedure was established for shoot tips (2-3 mm in length) and meristems (0.3-0.5 mm) sampled from in vitro plantlets of diploid and triploid cytotypes of Arachis pintoi. The optimal protocol was the following: after dissection, explants were precultured for 24 h on establishment medium (EM), encapsulated in calcium alginate beads and pretreated in liquid EM medium with daily increasing sucrose concentration (0.5, 0.75, 1.0 M) and desiccated to 22-23 percent moisture content (fresh weight basis). Explants were frozen using slow cooling (1 C per min from 25C to -30C followed by direct immersion in liquid nitrogen), thawed rapidly and post-cultured in liquid EM medium enriched with daily decreasing sucrose concentrations (0.75, 0.50, 0.1 M). Explants were then transferred to solid EM medium in order to achieve shoot regeneration, then on Murashige and Skoog medium supplemented with 0.05 microM naphthalene acetic acid to induce rooting of shoots. With this procedure, 53 percent and 56 percent of cryopreserved shoot tips of the diploid and triploid cytotypes, respectively, survived and formed plants. However, only 16 percent of cryopreserved meristems of both cytotypes regenerated plants. Using ten isozyme systems and seven RAPD profiles, no modification induced by cryopreservation could be detected in plantlets regenerated from cryopreserved material.

TDZ pulsing evaluation on the in vitro morphogenesis of peach palm.

PubMed

Graner, Erika Mendes; Oberschelp, Gustavo Pedro Javier; Brondani, Gilvano Ebling; Batagin-Piotto, Katherine Derlene; de Almeida, Cristina Vieira; de Almeida, Marcílio

2013-04-01

Peach palm (Bactris gasipaes Kunth.) cropping is an excellent alternative to native species exploitation; nevertheless, the problems with seed germination and conventional propagation justify the use of in vitro culturing. Aiming to asses TDZ pulsing effect on B. gasipaes morphogenesis, explants obtained from unarmed microplants were maintained in two treatments, half of them in MS free medium (without growth regulator) and the other half in MS with TDZ (0.36 μM). Both groups were transferred to growth regulator-free MS medium following 14 days of culture. After 84 days of culture, TDZ pulsing increased the growth and development of the shoots, restricted the growth and development of the roots, with no influence on adventitious bud induction or somatic embryogenesis. Furthermore, development of prickles, thickening of roots and chlorotic leaves were noted under TDZ pulsing. Leaf sheath histological analysis showed an epidermal origin and no vascularization of these prickles.

An efficient in vitro shoot regeneration from leaf petiolar explants and ex vitro rooting of Bixa orellana L.- A dye yielding plant.

PubMed

Mohammed, Arifullah; Chiruvella, Kishore K; Namsa, Nima D; Ghanta, Rama Gopal

2015-07-01

Bixa orellana L. (Bixaceae) is a multipurpose tree grown for the production of commercially important dyes. In the present study, an efficient, reproducible protocol was developed for direct plant regeneration from in vitro derived petiole explants of Bixa orellana L. Murashige and Skoog medium (MS) supplemented with 2-isopentenyl adenine (9.8 μM) and naphthalene acetic acid (10.7 μM) was found to be optimum for production of high frequency of shoot organogenesis. Subculturing of the shoots onto the fresh MS medium containing similar concentrations of 2-iP (9.8 μM) and NAA (10.7 μM) produced elongated shoots. Elongated shoots when placed onto MS medium supplemented with 1.7 μM indole-3-acetic acid and 14.7 μM 2-iP produced optimal rooting. Rooted plantlets were acclimatized and transplanted to the field successfully. Histological investigation revealed the origin of shoot primordia, from sub-epidermal cells of petiole explants. The regeneration protocol developed in this study can be useful for mass in vitro propagation and effective genetic transformation of commercially important edible dye yielding tree species.

Gerbera micropropagation.

PubMed

Cardoso, Jean C; Teixeira da Silva, Jaime A

2013-12-01

Gerbera jamesonii (gerbera) is an important cut-flower in the global floricultural industry. Micropropagation is the main system used to clonally propagate gerbera in vitro resulting in the production of millions of plantlets each year. Numerous types of explants and protocols for micropropagation have been established and used for gerbera. Shoot tips are the commonly used explant while adventitious shoot induction from the capitulum is also a popular method. Most papers in the literature have focused on testing the influence of different types and combinations of plant growth regulators with the aim of improving the regeneration and multiplication stage of one or few cultivars. Genotype is one of the most influential factors on the response of gerbera in vitro. Despite this, no successful universal protocol has yet been developed for multiple cultivars, limiting the usefulness of current protocols for commercial biotechnology labs. Slow-growing endogenous bacteria are one of the most important problems in gerbera micropropagation but require more studies on control and prevention. Individual shoots are normally easy to root, usually in excess of 90% of plantlets, but the acclimatization stage requires improvements and new technologies to increase the survival of plants. Epigenetic variations in micropropagated gerbera are frequently observed only with high concentrations of cytokinins in the culture medium but somaclonal variation is rare. Copyright © 2013 Elsevier Inc. All rights reserved.

Agrobacterium-mediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

PubMed Central

Nyaboga, Evans; Tripathi, Jaindra N.; Manoharan, Rajesh; Tripathi, Leena

2014-01-01

Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop. PMID:25309562

In vitro micropropagation in Polygonatum verticillatum (L.) All. an important threatened medicinal herb of Northern India.

PubMed

Bisht, Shivani; Bisht, N S; Bhandari, Snehlata

2012-01-01

An ideal micropropagation method of Polygonatum verticillatum has been developed using stem disc explants. Multiple shoots were initiated from stem disc explants on Murashige and Skoog (MS) medium fortified with different concentrations (0.25-10.0 mgl(-1)) and combinations of cytokinins (BAP, Kn and TDZ) along with (0.5-1.0 mgl(-1)) auxins (NAA/IBA/IAA). 1.0 mgl(-1) BAP with 0.5 mgl(-1) NAA was found to be the most effective in producing maximum number of shoots. Regular subculturing of these in vitro multiple shoots induced profuse growth of lateral roots in the same medium. Individual shoots were excised and rooted in vitro on half strength MS medium with 1.0 mgl(-1) NAA. Regenerants were hardened in growth chamber with high humidity and showed a high rate of survival.

An efficient, widely applicable cryopreservation of Lilium shoot tips by droplet vitrification

USDA-ARS?s Scientific Manuscript database

We report a straightforward and widely applicable cryopreservation method for Lilium shoot tips. This method uses adventitious shoots that were induced from leaf segments cultured for 4 weeks on a shoot regeneration medium containing 1 mg L-1 a-naphthaleneacetic acid (NAA) and 0.5 mg L-1 thidiazuron...

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

PubMed Central

Ramakrishna, Akula; Giridhar, Parvatam

2009-01-01

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore (A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore (A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L. PMID:20514228

High frequency early in vitro flowering of Dendrobium Madame Thong-In (Orchidaceae).

PubMed

Sim, Guek Eng; Loh, Chiang Shiong; Goh, Chong Jin

2007-04-01

We have successfully developed a method to induce early in vitro flowering of the self-pollinated seedlings of a tropical orchid hybrid, Dendrobium Madame Thong-In. Transition of vegetative shoot apical meristem to inflorescence meristem was observed when young protocorms were cultured in modified KC liquid medium. In contrast, protocorms cultured on Gelrite-solidified medium only produced axillary shoots and roots. CW was required to trigger the transitional shoot apical meristem and BA enhanced inflorescence stalk initiation and flower bud formation. However, normal flower development was deformed in liquid medium but developed fully upon transferring to two-layered (liquid over Gelrite-solidified) medium. Under optimal condition, in vitro flowering was observed about 5 months after seed sowing. Segregation of flower colours was observed in these seedlings and seedpods formed upon artificial pollination of the in vitro flowers.

In vitro propagation, micromorphological studies and ex vitro rooting of cannon ball tree (Couroupita guianensis aubl.): a multipurpose threatened species.

PubMed

Shekhawat, Mahipal S; Manokari, M

2016-01-01

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l(-1) indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l(-1) 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l(-1) each of BAP and Kinetin (Kin) + 0.5 mg l(-1) α-naphthalene acetic acid (NAA) with additives (50 mg l(-1) of ascorbic acid and 25 mg l(-1) each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l(-1) IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l(-1) of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7-8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.

Mass propagation of Rauwolfia serpentina L. Benth.

PubMed

Salma, U; Rahman, M S M; Islam, S; Haque, N; Khatun, M; Jubair, T A; Paul, B C

2008-05-01

A protocol for mass propagation through axillary bud proliferation was established for Rauwolfia serpentina L. Benth. (Apocynaceae). MS medium supplemented with 1.5 mg L(-1) BA and 0.2 mg L(-1) NAA elicited the maximum number of shoots (4 multiple shoots) from nodal explants. These adventitious shoots were best rooted on half strength MS medium supplemented with 1.0 mg L(-1) each of IBA and IAA. The in vitro raised plants were acclimatized in glass house and successfully transplanted to field condition with almost 95% survival.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of Psophocarpus tetragonolobus (L.) DC.

PubMed

Singh, Vinayak; Chauhan, Namita Singh; Singh, Mohit; Idris, Asif; Madanala, Raju; Pande, Veena; Mohanty, Chandra Sekhar

2014-01-01

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L(-1) induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L(-1)) and IAA (0.2 mg L(-1)) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations.

Establishment of an efficient and rapid method of multiple shoot regeneration and a comparative phenolics profile in in vitro and greenhouse-grown plants of psophocarpus tetragonolobus (L.) DC

PubMed Central

Singh, Vinayak; Chauhan, Namita Singh; Singh, Mohit; Idris, Asif; Madanala, Raju; Pande, Veena; Mohanty, Chandra Sekhar

2014-01-01

An in vitro method of multiple shoot induction and plant regeneration in Psophocarpus tetragonolobus (L.) DC was developed. Cotyledons, hypocotyls, epicotyls, internodal and young seedling leaves were used as explants. MS media supplemented with various concentrations of either thidiazuron (TDZ) or N6-benzylaminopurine (BAP) along with NAA or IAA combinations were used to determine their influence on multiple shoot induction. MS media supplemented with TDZ induced direct shoot regeneration when epicotyls and internodal segments were used as explants. TDZ at 3 mg L−1 induced highest rate (89.2 ± 3.28%) of regeneration with (13.4 ± 2.04) shoots per explant. MS media supplemented with BAP in combination with NAA or IAA induced callus mediated regeneration when cotyledons and hypocotyls were used as explants. BAP (2.5 mg L−1) and IAA (0.2 mg L−1) induced highest rate (100 ± 2.66%) of regeneration with (23.2 ± 2.66) shoots per explant. Mature plants produced from regenerated shoots were transferred successfully to the greenhouse. In a comparative study, the phenolics contents of various parts of greenhouse-grown plants with that of in vitro-raised plants showed significant variations. PMID:25482808

In vitro propagation of Homalomena aromatica Schott., an endangered aromatic medicinal herb of Northeast India.

PubMed

Raomai, Shiveirou; Kumaria, Suman; Tandon, Pramod

2013-04-01

A successful report on the in vitro propagation of Homalomena aromatica via rhizome axillary bud multiplication is presented. Rhizome bud explants were cultured on Murashige and Skoog medium supplemented with various concentrations of cytokinins to induce multiple shoot formation for micropropagation. The highest number of shoots was achieved in MS medium supplemented with 2.0 mg l(-1) 6-benzylaminopurine. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 0.5 mg l(-1) α-naphthalene acetic acid. The regenerated plantlets showed no morphological differences from the parent plant. This protocol takes approximately 6 months to reach the acclimatization stage from the initiation stage and facilitates commercial and rapid propagation of H. aromatica.

Biochemical characterization of embryogenic calli of Vanilla planifolia in response to two years of thidiazuron treatment.

PubMed

Kodja, Hippolyte; Noirot, Michel; Khoyratty, Shahnoo S; Limbada, Hafsah; Verpoorte, Robert; Palama, Tony Lionel

2015-11-01

Vanilla planifolia embryogenic calli were cultured for two years on a medium containing thidiazuron (TDZ). Due to the presence of TDZ, these calli were under permanent chemical treatment and the differentiation of adventitious shoots from protocorm-like-bodies (PLBs) was blocked. When embryogenic calli were transferred onto a medium without TDZ, shoot organogenesis and plantlet regeneration occurred. To gain better knowledge about the biochemical and molecular processes involved in the morphoregulatory role of TDZ, hormonal and metabolomic analyses were performed. Our results indicate that in the presence of TDZ, embryogenic calli contained a high amount of abscisic acid (ABA) essentially metabolized into abscisic acid glucosyl ester (ABAGE) and phaseic acid (PA), which was the most abundant. When transferred onto a medium without TDZ, shoot regeneration and development take place in four stages that include: embryogenic calli growth, differentiation of PLBs from meristmatic cells zones (MCZ), shoot organogenesis from PLBs and the elongation of well-formed shoots. From a hormonal perspective, the significant reduction in ABA metabolism and its readjustment in the ABAGE pathway triggered PLBs formation. However, this first morphogenesis was stimulated by a strong reduction in IAA metabolism. The organogenesis of PLBs into shoots is associated with an increase in ABA catabolism and a gradual shift in cellular metabolism towards shoot differentiation. Thus, the initiation of the elongation process in shoots is correlated with an alteration in metabolite composition, including an increase in energy reserves (sucrose/starch) and a rapid decrease in alanine content. Our data highlighted the relationship between endogenous hormone signalling, carbohydrate metabolism and shoot organogenesis in Orchid plants. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Transcriptomic and Hormonal Analyses Reveal that YUC-Mediated Auxin Biogenesis Is Involved in Shoot Regeneration from Rhizome in Cymbidium

PubMed Central

Liu, Yang; Zhang, Hai-Liang; Guo, He-Rong; Xie, Li; Zeng, Rui-Zhen; Zhang, Xiang-Qian; Zhang, Zhi-Sheng

2017-01-01

Cymbidium, one of the most important orchid genera in horticulture, can be classified into epiphytic and terrestrial species. Generally, epiphytic Cymbidium seedlings can be easily propagated by tissue culture, but terrestrial seedlings are difficult to propagate. To date, the molecular mechanisms underlying the differences in the ease with which terrestrial and epiphytic cymbidiums can be propagated are largely unknown. Using RNA-sequencing, quantitative reverse transcription PCR and enzyme-linked immunosorbent assay, Cymbidium ‘Xiaofeng’ (CXF), which can be efficiently micropropagated, and terrestrial Cymbidium sinense ‘Qijianbaimo’ (CSQ), which has a low regeneration ability, were used to explore the molecular mechanisms underlying the micropropagation ability of Cymbidium species. To this end, 447 million clean short reads were generated, and 31,264 annotated unigenes were obtained from 10 cDNA libraries. A total of 1,290 differentially expressed genes (DEGs) were identified between CXF and CSQ during shoot induction. Gene ontology (GO) enrichment analysis indicated that the DEGs were significantly enriched in auxin pathway-related GO terms. Further analysis demonstrated that YUC and GH3 family genes, which play crucial roles in the regulation of auxin/IAA (indole-3-acetic acid) metabolism, acted quickly in response to shoot induction culture in vitro and were closely correlated with variation in shoot regeneration between CXF and CSQ. In addition, the study showed that IAA accumulated rapidly and significantly during shoot induction in CXF compared to that in CSQ; in contrast, no significant changes in other hormones were observed between CXF and CSQ. Furthermore, shoot regeneration in CXF was inhibited by a yucasin-auxin biosynthesis inhibitor, indicating that increased IAA level is required for high-frequency shoot regeneration in CXF. In conclusion, our study revealed that YUC-mediated auxin biogenesis is involved in shoot regeneration from rhizome in Cymbidium. PMID:29163591

Microspore culture in Corchorus olitorius: effect of growth regulators, temperature and sucrose on callus formation.

PubMed

Ali, M A; Jones, J K

2000-06-01

Culture of isolated microspores and of anthers on media containing IAA directed free microspore development to an embryogenic pathway in C. olitorius. The first division of microspores on transfer to culture media was symmetrical in contrast to the asymmetrical division seen in normal development in vivo. Initially, 10-30% microspores divided symmetrically, but only 0.2-1% of the dividing microspores continued dividing and produced multicellular microcalli. About 30% of these microcalli produced callus but only on medium with 2.0 mg/L zeatin and 0.1 mg/L IAA. Incubation in the dark at temperatures of 35 degrees C for 1 day and then 25 degrees C was found effective for induction of first embryonic division in Corchorus. The frequency of microspore callus formation was higher on medium containing either 3% or 5% sucrose. Addition of colchicine and addition of activated charcoal to the above medium did not enhance microspore division in Corchorus olitorius. On transfer to different media most calli produced roots but regeneration of shoots and embryos was not induced.

Influence of organic supplements on production of shoot and callus biomass and accumulation of bacoside in Bacopa monniera (L.) Pennell.

PubMed

Parale, Anuradha; Barmukh, Rajkumar; Nikam, Tukaram

2010-04-01

Production of valuable secondary metabolites through plant cell or organ culture is the best suited alternative to extraction of whole plant material and to increase production of secondary metabolites in in-vitro systems, feeding precursor or intermediate metabolites is an obvious and popular approach. The present investigation was aimed to study the influence of feeding of organic supplements, glycine (0-125 μM), ferulic acid (0-200 μM), phenylalanine (0-200 μM), α-ketoglutaric acid (0-200 μM) and pyruvic acid (0-200 μM) on production of bacoside-A (a triterpenoid type secondary metabolite responsible for cognition effects) in shoot and callus biomass of Bacopa monniera (L.) Pennell. The shoots were raised in liquid Murashige and Skoog's (MS) medium fortified with 5 μM 6-benzyladenine (BA) and callus biomass on agar solidified MS medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4 -D) in conjunction with 5 μM 1-napthaleneacetic acid (NAA). Among the organic supplements used, 100 μM pyruvic acid effectively enhanced the production of bacoside-A in shoot as well as callus biomass. The bacoside-A content in in-vitro raised shoot biomass was 4.0 and 1.2 times higher as compared to control and shoot biomass of naturally grown plants respectively. Inclusion of pyruvic acid in MS medium for in-vitro shoot cultures of B. monniera, can be adapted for enhanced production of bacoside-A.

Plant regeneration from protoplasts ofVicia narbonensis via somatic embryogenesis and shoot organogenesis.

PubMed

Tegeder, M; Kohn, H; Nibbe, M; Schieder, O; Pickardt, T

1996-11-01

Protoplasts ofVicia narbonensis isolated from epicotyls and shoot tips of etiolated seedlings were embedded in 1.4% sodium-alginate at a final density of 2.5×10(5) protoplasts/ml and cultivated in Kao and Michayluk-medium containing 0.5 mg/I of each of 2,4- dichlorophenoxyacetic acid, naphthylacetic acid and 6 -benzylaminopurine. A division frequency of 36% and a plating efficiency of 0.40-0.5% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred onto gelrite-solidified Murashige and Skoog-media containing various growth regulators. Regeneration of plants was achieved via two morphologically distinguishable pathways. A two step protocol (initially on medium with a high auxin concentration followed by a culture phase with lowered auxin amount) was used to regenerate somatic embryos, whereas cultivation on medium containing thidiazuron and naphthylacetic acid resulted in shoot morphogenesis. Mature plants were recovered from both somatic embryos as well as from thidiazuron-induced shoots.

An in vitro method for rapid regeneration of a monopodial orchid hybrid Aranda Deborah using thin section culture.

PubMed

Lakshmanan, P; Loh, C S; Goh, C J

1995-05-01

A thin section culture system for rapid regeneration of the monopodial orchid hybrid Aranda Deborah has been developed. Thin sections (0.6-0.7mm thick) obtained by transverse sectioning of a single shoot tip (6-7mm), when cultured in Vacin and Went medium enriched with coconut water (20% v/v), produced an average 13.6 protocorm-like bodies (PLB) after 45 days, compared to 2.7 PLB formed by a single 6-7 mm long shoot tip under same culture condition. Addition of α-naphthaleneacetic acid to Vacin and Went medium enriched with coconut water further increased PLB production by thin sections. PLB developed into plantlets on solid Vacin and Went medium containing 10% (v/v) coconut water and 0.5 g l(-1) activated charcoal. With this procedure, more than 80,000 plantlets could be produced from thin sections obtained from a single shoot tip in a year as compared to nearly 11,000 plantlets produced by the conventional shoot tip method.

Developmental anatomy of blueberry (Vaccinium corymbosum L. ‘Aurora’) shoot regeneration

USDA-ARS?s Scientific Manuscript database

The culture of Vaccinium corymbosum L. ’Aurora’ leaves on regeneration medium results in the regeneration of adventitious shoots. We present anatomical evidence that these new shoot apices are directly regenerated from the cultured blades. Mounds of densely staining cells, which formed from epidermi...

Improved regeneration and transformation protocols for three strawberry cultivars

PubMed Central

Zakaria, Hossam; Hussein, Gihan M; Abdel-Hadi, Abdel-Hadi A; Abdallah, Naglaa A

2014-01-01

Strawberry (Fragaria × ananassa) is an economically important soft fruit crop with polyploid genome which makes the breeding of new cultivars difficult. Simple and efficient method for transformation and regeneration is required for cultivars improvement in strawberry. In the present study, adventitious shoot regeneration has been investigated in three cultivated strawberry plants, i.e., Festival, Sweet Charly and Florida via direct organogenesis using the in vitro juvenile leaves as explants. Explants were collected after sub-culturing on a propagation medium composed of MS supplemented with 0.5 mg/l BA; 0.1 mg/l GA3 and 0.1 mg/l IBA. To select the suitable organogenesis, the explants of the three cultivars were cultured on MS medium supplemented with different concentrations of TDZ (1, 2, 3, and 4 mg/l), then incubated at a temperature of 22 °C ± 2. Medium containing 2 mg/l TDZ revealed the best regeneration efficiency with the three cultivars (72% for Festival, and 73% for Sweet Charly and Florida). After 4 weeks, the produced shoots were cultured on MS medium with different concentrations of BA and Kin to enhance shoot elongation. Results showed that the medium containing 1.5 mg/l BA and 0.5 mg/l Kin revealed highest elongation efficiency (88% and 94%) for Festival and Sweet Charly, respectively. On the other hand, medium containing 1.5 mg/l BA and 0.1 mg/l Kin showed highest elongation efficiency (90%) in Florida. Elongated shoots were successfully rooted on MS medium containing 1.5 mg/l NAA. Furthermore, transformation of the two cultivars, Festival and Sweet Charly, has been established via Agrobacterium strain LBA44404 containing the plasmid pISV2678 with gus-intron and bar genes. Three days post co-cultivation, GUS activity was screening using the histochemical assay. The results showed 16% and 18% of the tested plant materials has changed into blue color for Festival and Sweet Charly, respectively. Out of 120 explants only 13 shoots were developed on bialaphos medium for each cultivar, representing 10.8% bialaphos resistant strawberry shoot. The presence of the both genes bar and uid A was detected by PCR and Northern giving a transformation efficiency of 5%. PMID:24322545

In vitro plantlet regeneration from nodal segments and shoot tips of Capsicum chinense Jacq. cv. Naga King Chili.

PubMed

Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

2012-03-01

An in vitro regeneration protocol was developed for Capsicum chinense Jacq. cv. Naga King Chili, a very pungent chili cultivar and an important horticultural crop of Nagaland (Northeast India). Maximum number of shoot (13 ± 0.70) was induced with bud-forming capacity (BFC) index of 10.8, by culturing nodal segments in Murashige and Skoog (MS) medium supplemented with 18.16 μM Thidiazuron (TDZ) followed by 35.52 μM 6-benzylaminopurine (BAP). Using shoot tips as explants, multiple shoot (10 ± 0.37) (BFC 8.3) was also induced in MS medium fortified with either 18.16 μM TDZ or 35.52 μM BAP. Elongated shoots were best rooted in MS medium containing 5.70 μM indole-3-acetic acid (IAA). Rooted plantlets thus developed were hardened in 2-3 weeks time in plastic cups containing potting mixture of a 1:1 mix of soil and cow dung manure and then subsequently transferred to earthen pots. The regenerated plants did not show any variation in the morphology and growth as compared to the parent plant.

Effect of reverse photoperiod on in vitro regeneration and piperine production in Piper nigrum L.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Fazal, Hina; Khan, Mubarak Ali; Afridi, Muhammad Siddique

2014-01-01

In this study, a novel approach for in vitro regeneration of Piper nigrum L. has been applied in order to increase healthy biomass, phytochemicals and piperine production via reverse photoperiod (16hD/8hL). Leaf portions of the seed-derived plants were placed on an MS-medium fortified with different PGRs. Under 16hD/8hL, thidiazuron (TDZ; 4.0 mg L⁻¹) and BA (1.5 mg L⁻¹) was found to be the most effective (<90%) in callus induction. Two concentrations (1.5, 2.0 mg L⁻¹) of the IBA produced>80% shoots from callus cultures. Healthy shoots were transferred to rooting medium and higher percentage of rooting (<90%) was observed on IBA (1.5 mg L⁻¹). These in vitro tissues were subjected to amino acid analysis, spectrophotometry, and HPLC. ARG, SER, THR, and TYR were the most abundant components out of 17 amino acids. Higher amino acid production was observed under normal photoperiod (16hL/8hD) than under reverse photoperiod (16hD/8hL). The highest total phenolic content (TPC; 9.91 mg/g-DW) and flavonoid content (7.38 mg/g-DW) were observed in callus cultures incubated under 16hL/8hD than other tissues incubated under 16hD/8hL photoperiod. Higher DPPH and PoMo activities were observed in tissues incubated under 16hL/8hD photoperiod, while ABTS and Fe²⁺ chelating activities were found higher in tissues incubated under reverse photoperiod. Significant quantities of piperine content were observed in all tissues except callus cultures. These results suggest that reverse photoperiod is a promising approach for callus induction, phytochemicals and piperine production for commercial applications. Copyright © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.

Biophysicochemical evaluation of wild hilly biotypes of Jatropha curcas for biodiesel production and micropropagation study of elite plant parts.

PubMed

Verma, K C; Verma, S K

2015-01-01

Depleting reserves of fossil fuel and increasing effects of environmental pollution from petrochemicals demands eco-friendly alternative fuel sources. Jatropha curcas oil, an inedible vegetable oil, can be a substitute feedstock for traditional food crops in the production of environment-friendly and renewable fuel. Jatropha oil is looked up in terms of availability and cost and also has several applications and enormous economic benefits. The seed oils of various jatropha biotypes from hilly regions were screened out and evaluated for their physiochemical parameters, viz, seed index(520-600 g), oil content (15-42 %), biodiesel yield (71-98 %), moisture content (2.3-6.5 %), ash content (3.2-5.6 %), acid value (4.2-26), density (0.9172-0.9317 g/cm(3)), viscosity (5-37 mm(2)/s), saponification value (195.8-204.2 mg/g), iodine value (106.6-113.6 mg/g), flash point (162-235 °C), cetane value (46.70-50.06 °C), free fatty acid value (2.5-10.2 %), and refractive index (1.4600-1.4710). Fatty acid profiling of jatropha resembles as edible oilseeds. NAA with BAP was found to be superior for callus induction (up to 87 %), as well as for shoot regeneration (up to12 shoots). Root induction (90-100 %) was successfully obtained in MS medium with or without phytoregulators. Grown plantlets were successfully transferred from lab to field with a survival rate of 80 %.

Surface disinfection procedure and in vitro regeneration of grapevine (Vitis vinifera L.) axillary buds.

PubMed

Lazo-Javalera, M F; Troncoso-Rojas, R; Tiznado-Hernández, M E; Martínez-Tellez, M A; Vargas-Arispuro, I; Islas-Osuna, M A; Rivera-Domínguez, M

2016-01-01

Establishment of an efficient explants surface disinfection protocol is essential for in vitro cell and tissue culture as well as germplasm conservation, such as the case of Grapevine (Vitis spp.) culture. In this research, different procedures for disinfection and regeneration of field-grown grapevine cv. 'Flame seedless' axillary buds were evaluated. The buds were disinfected using either NaOCl or allyl, benzyl, phenyl and 2-phenylethyl isothiocyanates. Two different media for shooting and four media for rooting were tested. Shoot and root development per buds were registered. The best disinfection procedure with 90 % of tissue survival involved shaking for 60 min in a solution containing 20 % Clorox with 50 drops/L Triton(®) X-100. These tissues showed the potential to regenerate a complete plant. Plant regeneration was conducted using full strength Murashigue and Skoog (MS) medium supplemented with 8 µM benzyl aminopurine for shoot induction and multiplication, whereas rooting was obtained on half strength MS supplemented with 2 mg L(-1) of indole-3-butyric acid and 200 mg L(-1) of activated charcoal. In this work, it was designed the protocols for obtaining sterile field-grown grapevine buds and in vitro plant development. This methodology showed potential to produce vigorous and healthy plants in 5 weeks for clonal grapevine propagation. Regenerated plants were successfully established in soil.

Growth and carbon balance are differently regulated by tree and shoot fruiting contexts: an integrative study on apple genotypes with contrasted bearing patterns.

PubMed

Pallas, Benoît; Bluy, Sylvie; Ngao, Jérôme; Martinez, Sébastien; Clément-Vidal, Anne; Kelner, Jean-Jacques; Costes, Evelyne

2018-01-09

In plants, carbon source-sink relationships are assumed to affect their reproductive effort. In fruit trees, carbon source-sink relationships are likely to be involved in their fruiting behavior. In apple, a large variability in fruiting behaviors exists, from regular to biennial, which has been related to the within-tree synchronization vs desynchronization of floral induction in buds. In this study, we analyzed if carbon assimilation, availability and fluxes as well as shoot growth differ in apple genotypes with contrasted behaviors. Another aim was to determine the scale of plant organization at which growth and carbon balance are regulated. The study was carried out on 16 genotypes belonging to three classes: (i) biennial, (ii) regular with a high production of floral buds every year and (iii) regular, displaying desynchronized bud fates in each year. Three shoot categories, vegetative and reproductive shoots with or without fruits, were included. This study shows that shoot growth and carbon balance are differentially regulated by tree and shoot fruiting contexts. Shoot growth was determined by the shoot fruiting context, or by the type of shoot itself, since vegetative shoots were always longer than reproductive shoots whatever the tree crop load. Leaf photosynthesis depended on the tree crop load only, irrespective of the shoot category or the genotypic class. Starch content was also strongly affected by the tree crop load with some adjustments of the carbon balance among shoots since starch content was lower, at least at some dates, in shoots with fruits compared with the shoots without fruits within the same trees. Finally, the genotypic differences in terms of shoot carbon balance partly matched with genotypic bearing patterns. Nevertheless, carbon content in buds and the role of gibberellins produced by seeds as well as the distances at which they could affect floral induction should be further analyzed. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of cytokinins on in vitro multiplication, volatiles composition and rosmarinic acid content of Thymus leucotrichus Hal. shoots.

PubMed

Bekircan, Tuba; Yaşar, Ahmet; Yıldırım, Sercan; Sökmen, Münevver; Sökmen, Atalay

2018-03-01

An efficient in vitro multiplication protocol was designed to Thymus leucotrichus , a subshrub and perennial herb growing naturally in the Northwest of Turkey. Of all basal media studied, Murashige and Skoog medium was found to be superior to the others, providing higher shoot formation and the maximum shoot length. Varying concentrations of cytokinins, i.e., 6-benzyladenine, thidiazuron, 2-isopentenyladenine and kinetin were supplemented in the nutrient media to observe their effects on shoot development and biomass. Rosmarinic acid content and volatile compositions of both naturally growing plants and in vitro multiplied plantlets were also evaluated. 6-benzyladenine (1.0 mg/L) and kinetin (0.5 mg/L) were found to be optimum for shoot number and shoot elongation, respectively. Thidiazuron (1.0 mg/L) was superior for biomass production. Rosmarinic acid content of in vitro multiplied plants was found to be higher than that of wild plants, reaching a maximum with 0.5 mg/L 2-isopentenyladenine, which yielded 10.15 mg/g dry weight. The highest thymol content was obtained with 1.0 mg/L kinetin (55.82%), while thidiazuron (0.1 mg/L) increased carvacrol production (12.53%). Overall, Murashige and Skoog medium supplemented with 1.0 mg/L kinetin was determined to be the most favorable medium studied.

Somatic embryogenesis, pigment accumulation, and synthetic seed production in Digitalis davisiana Heywood.

PubMed

Verma, Sandeep Kumar; Sahin, Gunce; Gurel, Ekrem

2016-04-01

Digitalis davisiana, commonly called Alanya foxglove, from Turkey, is an important medicinal herb as the main source of cardiac glycosides, cardenolides, anthraquinones, etc. It is also known in the Indian Medicine for treatment of wounds and burns. It has ornamental value as well. Overexploitation of D. davisiana has led this species to be declared protected, and thereby encouraged various methods for its propagation. In this study, an optimized and efficient plant tissue culture protocol was established using cotyledonary leaf, hypocotyl and root explants of D. davisiana. Callus tissues were obtained from the cotyledonary leaf, hypocotyl and root segments cultured on Murashige and Skoog's (MS) medium containing different plant growth regulators. The maximum number of somatic embryos were achieved by the MS medium containing 6-benzyladenine (1.0 mg/L BAP) or 2,4-dichlorophenoxy acetic acids (0.1 mg/L 2,4-D), which produced an average of 8.3 ± 1.5 or 5.3 ± 1.5 embryos per cotyledonary leaf, respectively. After 3 wk of culture in MS medium supplemented with 1.0 mg/L 2,4-D, callus showed a clear accumulation of orange pigmentation. Shoot regeneration was remarkably higher (14.3 indirect shoots) in a combination of α-naphthalene acetic acid (0.25 mg/L NAA) plus 3.0 mg/L BAP than 2.0 mg/L zeatin (10.3 ± 0.5 direct shoots) alone. The shoots were successfully rooted on MS medium supplemented with NAA (0.1-1.0 mg/L). In addition, synthetic seeds were produced by encapsulating shoot tips in 4% sodium alginate solution. Maximum conversion frequency of 76.6% was noted from encapsulated shoot tips cultured on 0.25 mg/L NAA with 1.0 mg/L BAP. The encapsulated shoot tips could be stored up to 60 days at 4 °C. Regenerated plantlets of D. davisiana were successfully acclimatized and transferred to soil. This study has demonstrated successful preservation of elite genotypes of D. davisiana.

Efficient in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood) and assessment of genetic fidelity of micropropagated plants.

PubMed

Shinde, Smita; Sebastian, Joseph Kadanthottu; Jain, Jyothi Ramesh; Hanamanthagouda, Manohar Shirugumbi; Murthy, Hosakatte Niranjana

2016-10-01

A reliable protocol has been established for in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood), a valuable medicinal plant from India. A highly proliferating organogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 2.5 µM IAA when nodal explants were cultured on MS medium supplemented with various growth regulators. Further, highest regeneration frequency (83.3 %) of adventitious shoots was observed, when the callus was sub-cultured on MS medium supplemented with 6-benzylaminopurine (BAP; 2.5 µM) along with 7.5 µM 2-isopentenyl adenine (2-iP). An optimal of 10.16 ± 2.24 shoots were regenerated on medium supplemented with 2.5 µM BAP + 7.5 µM 2-iP. Quarter strength MS medium supplemented with 10 µM IBA was effective for rooting of the shoots. Ex-vitro plants were normal and were established successfully. Cytological and molecular marker studies showed that regenerated plants showed genetic stability in micro-propagated plants.

High frequency plant regeneration from leaf derived callus of high Δ9-tetrahydrocannabinol yielding Cannabis sativa L.

PubMed

Lata, Hemant; Chandra, Suman; Khan, Ikhlas A; Elsohly, Mahmoud A

2010-10-01

An efficient in vitro propagation protocol for rapidly producing Cannabis sativa plantlets from young leaf tissue was developed. Using gas chromatography-flame ionization detection (GC-FID), high THC yielding elite female clone of a drug-type CANNABIS variety (MX) was screened and its vegetatively propagated clones were used for micropropagation. Calli were induced from leaf explant on Murashige and Skoog medium supplemented with different concentrations (0.5, 1.0, 1.5, and 2.0 µM) of indole- 3-acetic acid (IAA), indole- 3- butyric acid (IBA), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxy-acetic acid (2,4-D) in combination with 1.0 µM of thidiazuron (TDZ) for the production of callus. The optimum callus growth and maintenance was in 0.5 µM NAA plus 1.0 µM TDZ. The two-month-old calli were subcultured to MS media containing different concentrations of cytokinins (BAP, KN, TDZ). The rate of shoot induction and proliferation was highest in 0.5 µM TDZ. Of the various auxins (IAA, IBA, and NAA) tested, regenerated shoots rooted best on half strength MS medium (1/2 - MS) supplemented with 2.5 µM IBA. The rooted plantlets were successfully established in soil and grown to maturity with no gross variations in morphology and cannabinoids content at a survival rate of 95 % in the indoor growroom. © Georg Thieme Verlag KG Stuttgart · New York.

In vitro propagation of two Iranian commercial pomegranates (Punica granatum L.) cvs. 'Malas Saveh' and 'Yusef Khani'.

PubMed

Valizadehkaji, Babak; Ershadi, Ahmad; Tohidfar, Masoud

2013-10-01

An efficient in vitro propagation is described for Punica granatum L. using shoot tip and nodal explants. The influence of two basal medium, WPM and MS, and different plant growth regulators was investigated on micropropagation of the Iranian pomegranate cultivars, 'Malas Saveh' and 'Yousef Khani'. For proliferation stage, media supplemented with different concentrations (2.3, 4.7, 9.2 and 18.4 μM) of kinetin along with 0.54 μM NAA was used. WPM proved to be more efficient medium compared to MS. The best concentrations of kinetin were 4.7 μM for 'Malas Saveh' and 9.2 μM for 'Yousef Khani', resulting in the highest number of shoots per explants, shoot length and leaf number. For both cultivars, half-strength WPM medium supplemented with 5.4 μM NAA was most effective for rooting of shoots. Rooted plantlets were successfully acclimatized and transferred into soil. The micropropagated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants.

Optimization of an efficient semi-solid culture protocol for sterilization and plant regeneration of Centella asiatica (L.) as a medicinal herb.

PubMed

Moghaddam, Sina Siavash; Jaafar, Hawa Binti; Aziz, Maheran Abdul; Ibrahim, Rusli; Rahmat, Asmah Bt; Philip, Elizabeth

2011-01-01

The present study investigates the effects of different concentrations, as well as type of plant growth regulators (PGRs) and medium (MS, Duchefa) on the growth and development of Centella asiatica in semi-solid culture. In addition, a protocol for successful sterilization of C.asiatica explants prepared from field-grown plants highly exposed to fungal and bacterial contamination was determined. Results for sterilization treatments revealed that applying HgCl₂ and Plant Preservative Mixture (PPM) with cetrimide, bavistin and trimethoprim which were included after washing with tap water, followed by the addition of PPM in the medium, produced a very satisfactory result (clean culture 90 ± 1.33%) and TS5 (decon + cetrimide 1% + bavistin 150 mg/L + trimethoprim 50 mg/L + HgCl₂0.1% + PPM 2% soak and 2 mL/L in medium) was hence chosen as the best method of sterilization for C.asiatica. The synergistic combination of 6 benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in concentrations of 2 mg/L and 0.1 mg/L, respectively, in Duchefa medium compared with MS induced the most optimal percentage of sprouted shoots (93 ± 0.667), number of shoots (5.2 ± 0.079) and nodes (4 ± 0.067) per explant, leaf per explant (14 ± 0.107) and shoot length (4.1 ± 0.67 cm). Furthermore, optimum rooting frequency (95.2 ± 0.81%), the number of roots/shoot (7.5 ± 0.107) and the mean root length (4.5 ± 0.133 cm) occurred for shoots that were cultured on full-strength MS medium containing 0.5 mg/L indole-3-butyric acid (IBA). In this study, the acclimatized plantlets were successfully established with almost 85% survival. The findings of this study have proven an efficient medium and PGR concentration for the mass propagation of C.asiatica. These findings would be useful in micropropagation and ex situ conservation of this plant.

The promoter of the cereal VERNALIZATION1 gene is sufficient for transcriptional induction by prolonged cold.

PubMed

Alonso-Peral, Maria M; Oliver, Sandra N; Casao, M Cristina; Greenup, Aaron A; Trevaskis, Ben

2011-01-01

The VERNALIZATION1 (VRN1) gene of temperate cereals is transcriptionally activated by prolonged cold during winter (vernalization) to promote flowering. To investigate the mechanisms controlling induction of VRN1 by prolonged cold, different regions of the VRN1 gene were fused to the GREEN FLUORESCENT PROTEIN (GFP) reporter and expression of the resulting gene constructs was assayed in transgenic barley (Hordeum vulgare). A 2 kb segment of the promoter of VRN1 was sufficient for GFP expression in the leaves and shoot apex of transgenic barley plants. Fluorescence increased at the shoot apex prior to inflorescence initiation and was subsequently maintained in the developing inflorescence. The promoter was also sufficient for low-temperature induction of GFP expression. A naturally occurring insertion in the proximal promoter, which is associated with elevated VRN1 expression and early flowering in some spring wheats, did not abolish induction of VRN1 transcription by prolonged cold, however. A translational fusion of the promoter and transcribed regions of VRN1 to GFP, VRN1::GFP, was localised to nuclei of cells at the shoot apex of transgenic barley plants. The distribution of VRN1::GFP at the shoot apex was similar to the expression pattern of the VRN1 promoter-GFP reporter gene. Fluorescence from the VRN1::GFP fusion protein increased in the developing leaves after prolonged cold treatment. These observations suggest that the promoter of VRN1 is targeted by mechanisms that trigger vernalization-induced flowering in economically important temperate cereal crops.

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.-an important multipurpose plant of the Pacific traditional Medicine.

PubMed

Ahmed, Rafique; Anis, Mohammad

2014-07-01

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 μM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 μM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.

Ethylene induced shikonin biosynthesis in shoot culture of Lithospermum erythrorhizon.

PubMed

Touno, Kaori; Tamaoka, Jin; Ohashi, Yuko; Shimomura, Koichiro

2005-02-01

Lithospermum erythrorhizon shoots, cultured on phytohormone-free Murashige and Skoog solid medium, produced shikonin derivatives, whereas shoots cultured in well-ventilated petri dishes, produced small amount. Analysis by gas chromatography revealed the presence of ethylene in non-ventilated petri dishes where the shoots, producing shikonin derivatives, were cultured. Therefore, the possible involvement of ethylene in shikonin biosynthesis of shoot cultures was investigated. Treatment of ethylene or the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, resulted in increasing shikonin derivatives contents in cultured shoots. Silver ion, an ethylene-response inhibitor, or aminoethoxyvinylglycine, an ethylene biosynthesis inhibitor, decreased production of shikonin derivatives in cultured shoots. Our results indicate that ethylene is one of the regulatory elements of shikonin biosynthesis in L. erythrorhizon shoot culture.

Development of In Vitro Systems for Switchgrass (Panicum virgatum) - Final Report for 1992 to 2002

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conger, B.V.

2003-01-16

Our project began on July 1, 1992, with the objective of developing systems that could be used in biotechnological approaches to switchgrass improvement. Within six months after initiation of the project, we had worked out protocols in which plants could be regenerated from callus cultures through both organogenesis and somatic embryogenesis. Documentation for both modes of regeneration was provided in our progress reports and in publications. One thousand regenerated plants were established in the field during the first year. We found that Alamo (lowland type) was much more amenable to in vitro culture, and plants could be regenerated much moremore » easily than from Cave-in-Rock (upland type). During the first three years of the project, we studied the influence of genotype, culture medium components, explant type, etc., on regeneration. As mentioned, we found that the lowland cultivars Alamo and Kanlow were much easier to regenerate than upland cultivars, such as Trailblazer, Blackwell, and Cave-in-Rock. For callus induction, we initially used mature caryopses, young leaf tissue, and portions of seedlings. We were successful in inducing callus and regenerating plants from all explants. Two other systems developed during the 4th to 6th year period of the project included multiple shoot formation initiated from germinated seedlings and regenerable suspension cultures. The latter were initiated from embryogenic calluses produced from in vitro developed inflorescences. An important factor for producing multiple shoots was the presence of thidiazuron in the medium. The shoots could be easily rooted and numerous plantlets produced. The last 3 to 4 years of the project focused on anther and microspore culture experiments to produce haploid plants and on genetic transformation. Although thousands of putative haploid plants were produced from a few anthers, they were very weak and difficult to keep alive. Chromosome counts revealed the gametic number in cells where it was possible to count chromosomes. The isolated microspore culture experiments were not successful.« less

Transformation of miniature potted rose (Rosa hybrida cv. Linda) with P( SAG12 )-ipt gene delays leaf senescence and enhances resistance to exogenous ethylene.

PubMed

Zakizadeh, Hedayat; Lütken, Henrik; Sriskandarajah, Sridevy; Serek, Margrethe; Müller, Renate

2013-02-01

KEY MESSAGE : The P ( SAG12 ) -ipt gene was transferred to miniature rose, as the first woody species, resulting in increased ethylene resistance due to specific up-regulation of the ipt gene under senescence promoting conditions. Transgenic plants of Rosa hybrida 'Linda' were obtained via transformation with Agrobacterium tumefaciens strain harboring the binary vector pSG529(+) containing the P( SAG12 )-ipt construct. A. tumefaciens strains AGL1, GV3850 and LBA4404 (containing P(35S)-INTGUS gene) were used for transformation of embryogenic callus, but transgenic shoots were obtained only when AGL1 was applied. The highest transformation frequency was 10 % and it was achieved when half MS medium was used for the dilution of overnight culture of Agrobacterium. Southern blot confirmed integration of 1-6 copies of the nptII gene into the rose genome in the tested lines. Four transgenic lines were obtained which were morphologically true-to-type and indistinguishable from Wt shoots while they were in in vitro cultures. Adventitious root induction was more difficult in transgenic shoots compared to the Wt shoots, however, one of the transgenic lines (line 6) was rooted and subsequently analyzed phenotypically. The ipt expression levels were determined in this line after exposure to exogenous ethylene (3.5 μl l(-1)) and/or darkness. Darkness resulted in twofold up-regulation of ipt expression, whereas darkness combined with ethylene caused eightfold up-regulation in line 6 compared to Wt plants. The transgenic line had significantly higher content of chlorophyll at the end of the treatment period compared to Wt plants.

Micropropagation of Origanum acutidens (HAND.-MAZZ.) IETSWAART using stem node explants.

PubMed

Yildirim, Mehmet Ugur

2013-01-01

Origanum acutidens (HAND.-MAZZ.) IETSWAART is a promising ornamental plant that can be widely used in landscape management. It is endemic to Eastern Anatolian region of Turkey. Tissue culture has not been used to micropropagate it. The study reports stem node explants from one-week-old seedlings of the plant for successful micropropagation. The stem nodes were cultured on MS medium containing 0.6, 1.2, 1.8, and 2.4 mg/L BAP with 0.2 mg/L NAA. Visible effects of culture media on shoot proliferation were recorded. Shoot regeneration rate was maximum on MS medium containing 1.80 mg/L BAP-0.2 mg/L NAA. The micropropagated shoots were rooted on MS medium containing 0.2 mg/L NAA. All microrooted plantlets survived during acclimatisation on peat moss. It was concluded that O. acutidens can be successfully micropropagated under in vitro conditions.

Micropropagation of Origanum acutidens (HAND.-MAZZ.) IETSWAART Using Stem Node Explants

PubMed Central

Yildirim, Mehmet Ugur

2013-01-01

Origanum acutidens (HAND.-MAZZ.) IETSWAART is a promising ornamental plant that can be widely used in landscape management. It is endemic to Eastern Anatolian region of Turkey. Tissue culture has not been used to micropropagate it. The study reports stem node explants from one-week-old seedlings of the plant for successful micropropagation. The stem nodes were cultured on MS medium containing 0.6, 1.2, 1.8, and 2.4 mg/L BAP with 0.2 mg/L NAA. Visible effects of culture media on shoot proliferation were recorded. Shoot regeneration rate was maximum on MS medium containing 1.80 mg/L BAP-0.2 mg/L NAA. The micropropagated shoots were rooted on MS medium containing 0.2 mg/L NAA. All microrooted plantlets survived during acclimatisation on peat moss. It was concluded that O. acutidens can be successfully micropropagated under in vitro conditions. PMID:23983625

Two-stage culture procedure using thidiazuron for efficient micropropagation of Stevia rebaudiana, an anti-diabetic medicinal herb.

PubMed

Singh, Pallavi; Dwivedi, Padmanabh

2014-08-01

Stevia rebaudiana Bertoni, member of Asteraceae family, has bio-active compounds stevioside and rebaudioside which taste about 300 times sweeter than sucrose. It regulates blood sugar, prevents hypertension and tooth decay as well as used in treatment of skin disorders having high medicinal values, and hence there is a need for generating the plant on large scale. We have developed an efficient micropropagation protocol on half strength Murashige and Skoog (MS) media, using two-stage culture procedures. Varying concentrations of cytokinins, i.e., benzylaminopurine, kinetin and thidiazuron (TDZ) were supplemented in the nutrient media to observe their effects on shoot development. All the cytokinins promoted shoot formation, however, best response was observed in the TDZ (0.5 mg/l). The shoots from selected induction medium were sub-cultured on the multiplication media. The media containing 0.01 mg/l TDZ produced maximum number of shoot (11.00 ± 0.40) with longer shoots (7.17 ± 0.16) and highest number of leaves (61.00 ± 1.29). Rooting response was best observed in one-fourth strength on MS media supplemented with indole-3-butyric acid (1.0 mg/l) and activated charcoal (50 mg/l) with (11.00 ± 0.40) number of roots. The plantlets thus obtained were hardened and transferred to the pots with soil and sand mixture, where the survival rate was 80 % after 2 months. Quantitative analysis of stevioside content in leaves of in vivo mother plant and in vitro plantlets was carried out by high performance liquid chromatography. A remarkable increase in stevioside content was noticed in the in vitro-raised plants as compared to in vivo grown plants. The protocol reported here might be useful in genetic improvement and high stevioside production.

Early events in geotropism of seedling shoots

NASA Technical Reports Server (NTRS)

Pickard, B. G.

1985-01-01

Developments during the first ten minutes of geotropic stimulation in plant seedling shoots are reviewed. Topics include induction and curvature; early processes; the relationship between auxin, electric field, calcium, and differential growth; gravity reception leading to Went-Cholodny transport; and comparison of root and shoot. Early processes reviewed are sedimentation of amyloplasts, release of ethylene, rise of electrical and auxin asymmetry, redistribution of calcium, asymmetric vascular transport, increase in tendency to deposit callose, and simulation of putative exocytotic voltage transients.

Honokiol and magnolol production by in vitro micropropagated plants of Magnolia dealbata, an endangered endemic Mexican species.

PubMed

Domínguez, Fabiola; Chávez, Marco; Garduño-Ramírez, María Luisa; Chávez-Avila, Víctor M; Mata, Martín; Cruz-Sosa, Francisco

2010-02-01

An efficient protocol for the in vitro propagation of Magnolia dealbata Zucc., an important medicinal plant that is the source of the anxiolytic and anticancer compounds honokiol and magnolol, was established. This plant is wild-crafted, and conservationists have expressed concerns with regard to the sustainability of production. In the present work, two factors were found to be of importance for the regeneration of M. dealbata and the production of honokiol and magnolol. These factors were the type of explants and the combination and concentration of plant-growth regulators. Green, compact, nodular organogenic callus was obtained from leaf explants in a medium fortified with Murashige and Skoog salts and supplemented with 1.5 mg/L 2,4-dicholorophenoxyacetic acid and 1.5 mg/L kinetin. Shoots multiplication from callus cultures was achieved in the Murashige and Skoog (MS) medium with 1.5 mg/L thidiazuron (TDZ). Phenol secretion was controlled by the addition of 250 mg/L of activated charcoal. For rooting, shoots were transferred to MS medium supplemented with several auxins. After root induction, the plants were hardened in earthen pots containing sand, soil, and vermiculite. The contents of honokiol (HK) and magnolol (MG) were determined in different plant materials by high-performance liquid chromatography-diode-array detection techniques. This analysis revealed that the honokiol and magnolol content in aerial and underground parts of micropropagated M. dealbata were higher than that observed in wild plants (both 6 months old). Our results suggest that conservation of M. dealbata is possible by means of in vitro multiplication of leaf-derived callus. The usefulness of M. dealbata regeneration and production of HK and MG may be attributed to the proper selection of explant sourcing and identification of the correct growth medium to support adequate growth. This careful selection of explants and growth medium leads to a very useful source of plant material for pharmacological and phytomedicinal screening applications and, above all, would safeguard this plant species from the threat of extinction.

In Vitro Propagation and Conservation of Bacopa monnieri L.

PubMed

Sharma, Neelam; Singh, Rakesh; Pandey, Ruchira

2016-01-01

Bacopa monnieri L. (common name brahmi) is a traditional and renowned Indian medicinal plant with high commercial value for its memory revitalizer potential. Demand for this herb has further escalated due to popularization of various brahmi-based drugs coupled with reported anticancer property. Insufficient seed availability and problems associated with seed propagation including short seed viability are the major constraints of seed conservation in the gene banks. In vitro clonal propagation, a prerequisite for in vitro conservation by enhanced axillary branching was standardized. We have developed a simple, single step protocol for in vitro establishment, propagation and medium-term conservation of B. monnieri. Single node explants, cultured on Murashige and Skoog's medium supplemented with BA (0.2 mg/L), exhibited shoot proliferation without callus formation. Rooting was achieved on the same medium. The in vitro raised plants were successfully transferred to soil with ~80 % survival. On the same medium, shoots could also be conserved for 12 months with high survival and genetic stability was maintained as revealed by molecular markers. The protocol optimized in the present study has been applied for culture establishment, shoot multiplication and medium-term conservation of several Bacopa germplasm, procured from different agro-ecological regions of India.

Potential link between biotic defense activation and recalcitrance to induction of somatic embryogenesis in shoot primordia from adult trees of white spruce (Picea glauca)

PubMed Central

2013-01-01

Background Among the many commercial opportunities afforded by somatic embryogenesis (SE), it is the ability to clonally propagate individual plants with rare or elite traits that has some of the most significant implications. This is particularly true for many long-lived species, such as conifers, but whose long generation times pose substantive challenges, including increased recalcitrance for SE as plants age. Identification of a clonal line of somatic embryo-derived trees whose shoot primordia have remained responsive to SE induction for over a decade, provided a unique opportunity to examine the molecular aspects underpinning SE within shoot tissues of adult white spruce trees. Results Microarray analysis was used to conduct transcriptome-wide expression profiling of shoot explants taken from this responsive genotype following one week of SE induction, which when compared with that of a nonresponsive genotype, led to the identification of four of the most differentially expressed genes within each genotype. Using absolute qPCR to expand the analysis to three weeks of induction revealed that differential expression of all eight candidate genes was maintained to the end of the induction treatment, albeit to differing degrees. Most striking was that both the magnitude and duration of candidate gene expression within the nonresponsive genotype was indicative of an intense physiological response. Examining their putative identities further revealed that all four encoded for proteins with similarity to angiosperm proteins known to play prominent roles in biotic defense, and that their high-level induction over an extended period is consistent with activation of a biotic defense response. In contrast, the more temperate response within the responsive genotype, including induction of a conifer-specific dehydrin, is more consistent with elicitation of an adaptive stress response. Conclusions While additional evidence is required to definitively establish an association between SE responsiveness and a specific physiological response, these results suggest that biotic defense activation may be antagonistic, likely related to the massive transcriptional and metabolic reprogramming that it elicits. A major issue for future work will be to determine how and if suppressing biotic defense activation could be used to promote a physiological state more conducive to SE induction. PMID:23937238

Successful micropropagation of the cadmium hyperaccumulator Viola baoshanensis (Violaceae).

PubMed

Li, Jin-Tian; Deng, Dong-Mei; Peng, Guang-Tian; Deng, Jin-Chuan; Zhang, Jun; Liao, Bin

2010-01-01

Viola baoshanensis is one of the most rare cadmium (Cd) hyperaccumulators, however, it is hard to propagate. Micropropagation has been applied to solve the problems with propagation of a few heavy metal hyperaccumulators. Therefore there is a high likelihood that micropropagation may offer a suitable method for large-scale propagation of V. baoshanensis To test this hypothesis, three types of explants were used for shoot regeneration and various combinations of four plant growth regulators were used to improve shoot regeneration efficiency from leaflet of V. baoshanensis. Best shoot regeneration efficiency was obtained by incubating leaflet in a 1/2 MS medium supplemented with 2.5 oM BA + 2.5 microM IBA, therein shoot regeneration rate was 70.9% and the number of shoots formation per explant was 22.4. Rooting was achieved from almost all regenerated shoot growing on 1/2 MS medium without plant growth regulator. Micropropagated seedlings were acclimatized under greenhouse conditions and 95% of them survived and showed no visible morphological variation compared to their donor plant. Furthermore, there were no significant differences between regenerated and seed-germinated V. baoshanensis in Cd tolerance and accumulation. These results suggested that an efficient and rapid micropropogation system was successfully developed for V. baoshanensis.

Agrobacterium tumefaciens-mediated transformation of Campanula carpatica: factors affecting transformation and regeneration of transgenic shoots.

PubMed

Sriskandarajah, Sridevy; Frello, Stefan; Jørgensen, Kirsten; Serek, Margrethe

2004-08-01

An efficient transformation system for Campanula carpatica was developed using Agrobacterium tumefaciens strains LBA4404 (harbouring the plasmid pBI121), and AGL0 (harbouring the plasmid pBEO210). This is the first report on the transformation of C. carpatica. Various factors affecting the transformation efficiency and subsequent regeneration were identified. The age of seedlings from which the explants for transformation studies were taken, and the growth conditions under which the seedlings were grown had a significant influence on the production of transformed shoots. Hypocotyls taken from 12-day-old seedlings grown in the dark were the most productive, with up to 25% of hypocotyls producing transformed shoots. Explants taken from 5-week-old seedlings produced only transformed callus. The medium used for co-cultivation and incubation also had a significant influence on transformation frequency and shoot regeneration. The cultivar "Blue Uniform" was more responsive than "White Uniform". Both bacterial strains and plasmids were equally effective in producing transformed tissue. Transformed shoots were selected on kanamycin medium, and the presence of the uidA and nptII genes in those selected shoots was confirmed by beta-glucuronidase and ELISA analyses, respectively.

In vitro propagation and analysis of secondary metabolites in Glossogyne tenuifolia (Hsiang-Ju) - a medicinal plant native to Taiwan.

PubMed

Chen, Chia-Chen; Chang, Hung-Chi; Kuo, Chao-Lin; Agrawal, Dinesh Chandra; Wu, Chi-Rei; Tsay, Hsin-Sheng

2014-12-01

Glossogyne tenuifolia Cassini (Hsiang-Ju in Chinese) is a perennial herb native to Penghu Islands, Taiwan. The herb is a traditional anti-pyretic and hepatoprotective used in Chinese medicine. Several studies on G. tenuifolia have demonstrated its pharmacological values of antioxidation, anti-inflammation, immunomodulation, and cytotoxicity on several human cancer cell lines. Active compounds, oleanolic acid and luteolin in G. tenuifolia are affected by several factors, including climatic change, pathogens and agricultural practices. Plant population of G. tenuifolia has been severely affected and reduced considerably in natural habitat due to the use of herbicides by farmers. Also, collection of plant material from the natural habitat is restricted to a few months in a year. Therefore, the objective of the present study was to develop an efficient micropropagation protocol for G. tenuifolia. The study also aimed to investigate the influence of in vitro growth environment on the active compounds in in vitro shoots, tissue culture raised greenhouse plants; compare the values with wild plants and commercially available crude drug. Half-strength MS (Murashige and Skoog) basal medium supplemented with 0.1 mg/L 6-benzyladenine (BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA) induced the maximum average number of shoots (7.3) per shoot tip explant excised from in vitro grown seedlings. Induction of rooting in cent percent in vitro shoots with an average number of 6.6 roots/shoot was achieved on ½ strength MS medium supplemented with 3.0 mg/L indole-3-acetic acid (IAA). The rooted plantlets acclimatized successfully in the greenhouse with a 100% survival rate. HPLC analysis revealed that the quantity of oleanolic acid and luteolin in in vitro shoots, tissue culture plants in the greenhouse, wild type plants and commercial crude drug varied depending upon the source. The oleanolic acid and luteolin contents were found to be significantly higher (16.89 mg/g and 0.84 mg/g, respectively) in 3-month old tissue culture raised plants in greenhouse compared to commercially available crude drug (6.51 mg/g, 0.13 mg/g, respectively). We have successfully developed an in vitro propagation protocol for G. tenuifolia which can expedite its plant production throughout the year. The contents of oleanolic acid and luteolin in the tissue culture raised plants in the greenhouse were significantly higher than the marketed crude drug demonstrating the practical application of the tissue culture technology. These findings may be very useful in micropropagation, germplasm conservation and commercial cultivation of G. tenuifolia. So far, there is no published report on tissue culture propagation of this important medicinal plant species.

Dress-Related Responses to the Columbine Shootings: Other-Imposed and Self-Designed.

ERIC Educational Resources Information Center

Ogle, Jennifer Paff; Eckman, Molly

2002-01-01

An inductive content analysis approach was used to examine 155 dress-related newspaper articles following the Columbine High School shootings in 1999. Analysis revealed two dress-related responses: (1) other-imposed regulation to protect students and deter them from expressing hatred and (2) self-designed acts of resistance for grieving. (Contains…

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [ Cajanus cajan (L.) Millsp.] cv. JKR105.

PubMed

Krishna, Gaurav; Reddy, P Sairam; Ramteke, Pramod W; Rambabu, Pogiri; Sohrab, Sayed S; Rana, Debashis; Bhattacharya, Parthasarathi

2011-10-01

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5 mg L(-1) 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2 % sucrose and 0.5 mg L(-1) 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.

Augmenting in vitro shoot multiplication by vipul (triacontanol) and adventitious rhizogenesis by rice bran extract in Dendrocalamus strictus.

PubMed

Mishra, Y; Rana, P K; Shirin, F; Ansari, S A

2001-02-01

Like other bamboo species, Dendrocalamus strictus flowers gregariously after a prolonged intermast period of 48 years and constitutes an ideal material for in vitro clonal propagation. In this study, MS liquid medium containing 0.5, 1.0 and 2.0 mL/L vipul (Godrej Agrovet, Ltd., Sachin, India), a commercial formulation of triacontanol, with or without BA (3.0 mg/L) was tested for in vitro shoot multiplication and 1.0, 2.5 and 5.0 mL/L of 20% (w/v) alcoholic/aqueous rice bran extract (alone or in combination) with NAA (3 mg/L) used for in vitro adventitious rhizogenesis in single node culture derived shoots of Dendrocalamus strictus.. After a multiplication cycle for 4-5 week, vipul (0.5 mL/L) with BA (3.0 mg/L) in the culture medium induced 4.59 fold shoot multiplication rate whereas application of BA and vipul alone had corresponding values of 3.29 and 0.53 fold respectively. Maximum vipul concentration (2 mL/L) with BA (3 mg/L) exhibited shoot multiplication higher than (or equal to) that of BA alone. Maximum in vitro rooting percentage (55.66%) was obtained on half MS medium enriched with alcoholic rice bran extract (2.5 mL/L) and NAA (3 mg/L). This is the first investigation reporting amelioration of in vitro shoot multiplication rate by triacontanol and rooting percentage by rice bran extract in explants from mature bamboo culms. The protocol is economical and rapid for in vitro clonal propagation of Dendrocalamus strictus.

Carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2006-01-01

Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/Lthidiazuron (TDZ), 0.1 mg/L alpha-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 microM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants.

Gelling agents and culture vessels affect in vitro multiplication of banana plantlets.

PubMed

Kaçar, Y A; Biçen, B; Varol, I; Mendi, Y Y; Serçe, S; Cetiner, S

2010-03-09

Agar is the most commonly used gelling agent in media for plant tissue culture. Because of the high price of tissue-culture-grade agar, attempts have been made to identify suitable alternatives. The type of culture vessel and lid also affects the gaseous composition inside the vessel as well as light penetration. In turn, the vessel affects growth parameters, such as shoot elongation, proliferation and fresh weight, as well as hyperhydric degradation processes. We examined the effects of different culture vessels, including commercial glass jars, magenta boxes, and disposable containers, as well as different gelling agents (agar-agar, Agargel, Phytagel, and plant agar) on the micropropagation of Dwarf Cavendish bananas in an effort to find a combination that yields large numbers of high-quality seedlings. The different culture vessels did not significantly affect seedling culture success. The medium significantly affected shoot weight. Phytagel resulted in the highest shoot weight (overall mean = 2.4 g), while agar, Agargel and plant agar resulted in 1.7, 2.2 and 2.2 g, respectively. Disposable container/Phytagel and Magenta/Agargel combinations yielded the highest shoot weights (2.9 and 3.0 g, respectively). Mean shoot length increased progressively with subculture (four subcultures were made). The highest mean shoot length was obtained with Phytagel and Agargel media (6.4 and 6.3 cm, respectively). Shoot number was significantly affected by medium only at subculture 4. Overall, the highest mean shoot length was obtained with the Magenta/Agargel combination (8.5 cm). Phytagel and plant agar gave higher mean shoot number than agar and Agargel (2.1, 2.1 and 1.7 and 1.9, respectively). The costs of the media and of the culture vessels need to be taken into account for final choice of the banana shoot culture system.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

PubMed

Omar, Ahmad A; Murata, Mayara M; El-Shamy, Hesham A; Graham, James H; Grosser, Jude W

2018-04-01

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants.

PubMed

Ceasar, S Antony; Ignacimuthu, S

2011-09-01

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM L: -cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R(1) progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R(1) progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.

Clonal propagation of Phyllanthus amarus: A hepatoprotector

PubMed Central

Xavier, Janifer R.; Gnanam, Ramaswamy; Murugan, Muthiah P.; Pappachan, Anju

2012-01-01

Background: The micropropagation protocol for Phyllanthus amarus, an important medicinal herb used widely for the treatment of hepatitis in ethnomedicinal systems, was standardized with shoot tip and single node explants. Materials and Methods: The micropropagation was carried out for the hyperproducing ecotype (phyllanthin content 463.828 ppm; hypophyllanthin content: 75.469 ppm) collected from Aanaikatti, Coimbatore, and grown in mist chamber, CPMB, TNAU. For micropropagation studies, the leaves were trimmed off and the shoot tips (6 mm long) and nodal segments (single node) were used for initiation. Results: Shoot tips and single node explants gave a maximum of 6.00 and 7.00 multiple shoots per explant with Benzyl Amino Purine (BAP) (1.0mg/L mg/L). Upon subculturing, a shoot length of around 7 cm with an average of eight internodes per shoot was observed after 20 days in the elongation medium supplemented with BAP (0.2 mg/Lmg/L) and Indole Acetic Acid (IAA) (2.0 mg/L). Seven to ten adventitious roots developed when the elongated microshoots were cultured in half strength MS medium with Indole Butyric Acid (IBA) (2.0 mg/Lmg/L) and NAA (1.0 mg/L mg/L) in 15-20 days after transfer. The rooted shoots acclimatized successfully to field conditions. Conclusion: A method for successful micropropagation of the valuable medicinal plant was established which will provide a better source for continuous supply of plants for manufacturing drugs. PMID:22438668

Cloning: plants – micropropagation/tissue culture

USDA-ARS?s Scientific Manuscript database

Clonal micropropagation is the multiplication of the buds and shoots that occur in leaf axils on a defined nutrient medium in an aseptic in vitro environment. The resulting shoots are either subdivided for continued multiplication or rooted and acclimatized to the greenhouse or field. Micropropagati...

An improved micropropagation of Arnebia hispidissima (Lehm.) DC. and assessment of genetic fidelity of micropropagated plants using DNA-based molecular markers.

PubMed

Phulwaria, Mahendra; Rai, Manoj K; Shekhawat, N S

2013-07-01

An efficient and improved in vitro propagation method has been developed for Arnebia hispidissima, a medicinally and pharmaceutically important plant species of arid and semiarid regions. Nodal segments (3-4 cm) with two to three nodes obtained from field grown plants were used as explants for shoot proliferation. Murashige and Skoog's (MS) medium supplemented with cytokinins with or without indole-3-acetic acid (IAA) or naphthalene acetic acid was used for shoot multiplication. Out of different PGRs combinations, MS medium containing 0.5 mg l(-1) 6-benzylaminopurine and 0.1 mg l(-1) IAA was optimal for shoot multiplication. On this medium, explants produced the highest number of shoots (47.50 ± 0.38). About 90 % of shoots rooted ex vitro on sterile soilrite under the greenhouse condition when the base (2-4 mm) of shoots was treated with 300 mg l(-1) of indole-3-butyric acid for 5 min. The plantlets were hardened successfully in the greenhouse with 85-90 % survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro-regenerated plants of A. hispidissima. Out of 40 (25 RAPD and 15 ISSR) primers screened, 15 RAPD and 7 ISSR primers produced a total number of 111 (77 RAPD and 34 ISSR) reproducible amplicons. The amplified products were monomorphic across all the micropropagated plants and were similar to the mother plant. To the best of our knowledge, it is the first report on the assessment of the genetic fidelity in micropropagated plants of A. hispidissima.

Micropropagation of Hedychium coronarium J. Koenig through rhizome bud.

PubMed

Mohanty, Pritam; Behera, Shashikanta; Swain, Swasti S; Barik, Durga P; Naik, Soumendra K

2013-10-01

An optimized protocol was developed for in vitro plant regeneration of a medicinally important herb Hedychium coronarium J. Koenig using sprouted buds of rhizomes. The rhizomes with sprouted bud were inoculated on Murashige and Skoog (Physiol Plant 15:473-497, 1962) medium (MS) supplemented with either N(6)-benzyladenine (BA) alone (1.0-4.0 mg L(-1)) or in combination with 0.5 mg L(-1) naphthalene acetic acid (NAA). Of these combinations, MS supplemented with a combination of 2.0 mg L(-1) BA and 0.5 mg L(-1) NAA was most effective. In this medium, best shoots (3.6) and roots (4.0) regeneration was observed simultaneously with an average shoot and root length of 4.7 cm and 4.2 cm respectively. Regeneration of shoots and roots in the same medium at the same time (One step shoot and root regeneration) reduced the time for production of in vitro plantlets and eliminates the media cost of rooting. Cent-percent (100 %) success in plant establishment was observed in both gradual acclimatization process as well as when plants were directly transferred to outdoor in clay pots containing a mixture of garden soil and sand (2:1) without any sequential acclimatization stage.

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1

PubMed Central

Saha, Soumen; Adhikari, Sinchan; Dey, Tulsi; Ghosh, Parthadeb

2015-01-01

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62 ± 0.01), with average length 4.19 ± 0.01 cm, was initially achieved with medium containing 0.5 mg/l N6-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0 mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies. PMID:26693403

Systemic Induction of Photosynthesis via Illumination of the Shoot Apex Is Mediated Sequentially by Phytochrome B, Auxin and Hydrogen Peroxide in Tomato1[OPEN

PubMed Central

Guo, Zhixin; Ahammed, Golam Jalal; Wang, Mengmeng; Zhou, Jie; Xia, Xiaojian; Shi, Kai; Yin, Xueren; Chen, Kunsong; Yu, Jingquan; Zhou, Yanhong

2016-01-01

Systemic signaling of upper leaves promotes the induction of photosynthesis in lower leaves, allowing more efficient use of light flecks. However, the nature of the systemic signals has remained elusive. Here, we show that preillumination of the tomato (Solanum lycopersicum) shoot apex alone can accelerate photosynthetic induction in distal leaves and that this process is light quality dependent, where red light promotes and far-red light delays photosynthetic induction. Grafting the wild-type rootstock with a phytochome B (phyB) mutant scion compromised light-induced photosynthetic induction as well as auxin biosynthesis in the shoot apex, auxin signaling, and RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1)-dependent hydrogen peroxide (H2O2) production in the systemic leaves. Light-induced systemic H2O2 production in the leaves of the rootstock also was absent in plants grafted with an auxin-resistant diageotropica (dgt) mutant scion. Cyclic electron flow around photosystem I and associated ATP production were increased in the systemic leaves by exposure of the apex to red light. This enhancement was compromised in the systemic leaves of the wild-type rootstock with phyB and dgt mutant scions and also in RBOH1-RNA interference leaves with the wild type as scion. Silencing of ORANGE RIPENING, which encodes NAD(P)H dehydrogenase, compromised the systemic induction of photosynthesis. Taken together, these results demonstrate that exposure to red light triggers phyB-mediated auxin synthesis in the apex, leading to H2O2 generation in systemic leaves. Enhanced H2O2 levels in turn activate cyclic electron flow and ATP production, leading to a faster induction of photosynthetic CO2 assimilation in the systemic leaves, allowing plants better adaptation to the changing light environment. PMID:27550998

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

PubMed

Fatima, Nigar; Ahmad, Naseem; Ahmad, Iqbal; Anis, Mohammad

2015-09-01

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 μM 6-benzyladenine (BA) and 0.5 μM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 μM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 μM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

Micropropagation of annatto (Bixa orellana L.) from mature tree and assessment of genetic fidelity of micropropagated plants with RAPD markers.

PubMed

Siril, E A; Joseph, Nisha

2013-01-01

An in vitro propagation technique based on axillary bud proliferation was developed for the first time to mature annatto (Bixa orellana L.) tree. Nodal segments cultured on Murashige and Skoog (MS) medium supplemented with 1.0 μM benzyl adenine (BA) and tender coconut water (10 %) showed significantly high (P < 0.05) explant response (67.0 %), development of elongated shoots (3.36), shoot buds (8.9) and shoot elongation (3.53 cm). Cytokinins like zeatin, isopentenyl adenine (2-iP), kinetin, or thidiazuron (TDZ) were inferior to BA to induce multiple shoots. Seasonal variations significantly affected the in vitro response of nodal explants. In vitro rooting experiments have showed 55.6 % rooting on MS medium containing 15 μM indole-3-butyric acid (IBA). Alternatively, in vitro raised shoots were rooted (61.1 %) ex vitro, by 10 mM indole-3-butyric acid (IBA) for 30 s. The results of the RAPD marker system revealed the genetic stability among the micropropagated plants. The present protocol in brief, can be used for the clonal propagation of the superior genotype and preservation of germplasm.

Conservation and multiplication of encapsulated micro shoots of Rauvolfia vomitoria--an endangered medicinal tree: ISSR and RAPD based evaluation of genetic fidelity of converted plantlets.

PubMed

Mehrotra, Shakti; Rahman, Liaq Ur; Mishra, Jahnvi; Kukreja, Arun K

2012-12-01

The in vitro grown axillary micro shoots of Rauvolfia vomitoria were encapsulated in alginate beads. Following 6 months of normal storage at 25 +/- 2 degrees C the regrowth of encapsulated micro shoots, reached 95.2% within 40 days of incubation on MS medium containing 1.0 mg/L BAP and 0.1 mg/L NAA. Among the responding encapsulated explants 69.6% showed emergence of multiple shoots. The developing shoots showed rhizogenesis in two weeks following their transfer to rooting medium. Healthy plants were established in a glass house with 95% survival. Of the 50 RAPD primers tested, 10 produced 23 clear and reproducible amplicons, with an average of 2.3 bands per primer. Eleven ISSR primers produced a total of 42 bands, with a size range of 0.1-1.9 kb. The number of scorable bands for each primer varied from 2 to 6, with an average of 3.81. The similarity matrix, calculated individually from the results obtained from ISSR and RAPD analysis, showed similarity coefficients ranging from 1.0 for RAPD and 0.85 to 1.0 for ISSR.

Establishment of the regeneration system for Vicia faba L.

PubMed

Bahgat, Shimaa; Shabban, Omer A; El-Shihy, Osama; Lightfoot, David A; El-Shemy, Hany A

2009-01-01

A reliable regeneration system for faba bean has been difficult to establish and therefore, the genetic improvement of Vicia faba L. was delayed. The paper describes a method of somatic embryo induction in callus of V. faba. Two Egyptian faba bean cultivars 'Giza 2' and '24 Hyto' were used. Callus was induced from epicotyls and shoot tips cultured on MS or Gamborg medium supplemented with 3% sucrose and 0.025% (w/v) for each of ascorbic and citric acid, 0.8% agar and different concentrations of 10 mg/l BAP, 0.5 mg/l of each NAA and 2,4-dichlorophenoxyacetic acid (M1) and 1 mg/l BAP and 0.5 mg/l NAA (M2) . The media with BAP, NAA and 2,4-D were optimal for embryogenic callus induction. Somatic embryos developed after transfer of the callus to 1/2 B5 medium with no plant growth regulators. There were various stages of somatic embryo development present including globular, heart-shaped, torpedo, and cotyledonary stages. Embryos developed into plantlets and plants were regenerated. RAPD analyses were performed to investigate the genetic stability of the regenerated plants obtained from different treatments and different explants. The cultivar Giza 2 exhibited more genetic stability than cultivar 24 Hyto. In conclusion, a regeneration system was established suitable for both gene transformation and the isolation of somaclonal mutants. The regeneration system will be used in order to improve the nutritional value of faba bean.

Induction of mRNA for Phosphoenolpyruvate Carboxylase Is Correlated with a Decrease in Shoot Water Content in Well-Irrigated Mesembryanthemum crystallinum 1

PubMed Central

Schmitt, Jürgen M.; Piepenbrock, Mechtild

1992-01-01

The abundance of mRNA specific for phosphoenolpyruvate carboxylase (PEPCase) was measured in leaves from well-watered plants of Mesembryanthemum crystallinum. Plants grown side by side in pots of four different volumes (0.16, 0.74, 2.6, 6.5 liters) were compared. The time of increase in the steady-state level of PEPCase mRNA in well-watered plants was dependent on soil volume. The larger the pot, the later PEPCase transcripts were increased. PEPCase mRNA induction started when shoot water content decreased to well below 4000% of dry weight. No positive correlation with the developmental status of the plants could be found. The data indicate that PEPCase mRNA induction in well-watered plants up to 10 weeks of age is controlled environmentally rather than developmentally. ImagesFigure 2 PMID:16668951

In vitro propagation of northern red oak (Quercus rubra L.)

Treesearch

G. Vengadesan; Paula M. Pijut

2009-01-01

In vitro propagation of northern red oak (Quercus rubra) shoots was successful from cotyledonary node explants excised from 8-wk-old in vitro grown seedlings. Initially, four shoots per explant were obtained on Murashige and Skoog (MS) medium supplemented with 4.4 µM 6-benzylaminopurine (BA), 0.45 ...

[The possibilities for determining the shooting distance by means of inductively coupled plasma optical emission spectrometry].

PubMed

Svetlolobov, D Yu; Luzanova, I S; Zorin, Yu V; Makarov, I Yu; Lorents, A S

The objective of the present study was to evaluate the possibilities for determining the shooting distance for the MR-79-9 Makarych non-lethal pistol (diameter 9 mm, rubber bullet, shot energy 50 J) by means of inductively coupled plasma optical emission spectrometry. The experiments were carried under the conditions of a ballistic shooting range making the shots from a distance of 0 to 120 cm. The 15×15 cm pieces of muslin fabric and biomaterials (leather) were used as the targets. The morphological signs of the damages inflicted to the targets were evaluated either with the unassisted eye, a criminalistical magnifying glass or the SMT-4 binocular stereoscopic microscope (Germany). The shot products, the area and boundaries of their dispersion were determined in reflected IR and filtered UV rays. Inductively coupled plasma optical emission spectrometry was applied for the qualitative and quantitative analysis of various shot products from the entry hole zone with the contamination (wipedown) bands and contusion collars being 0.2-0.5 cm (group 1) and 2-3 cm (group 2) in width, with special reference to the identification of Ba, Cu, Cr, Fe, K, Ni, Pb, Sb, Sn and Zn. The results of the study give evidence that the detection of Ba, Pb, and Sb among the products of a shot fired from the MR-79-9 Makarych non-lethal pistol is of especially high informative value for determining the shooting distance whereas the detection of Cr, K, Sn and Ni is of a minimum value for this purpose.

Assessing the response of indigenous loquat cultivar Mardan to phytohormones for in vitro shoot proliferation and rooting*

PubMed Central

Abbasi, Nadeem Akhtar; Pervaiz, Tariq; Hafiz, Ishfaq Ahmed; Yaseen, Mehwish; Hussain, Azhar

2013-01-01

In vitro cultures of loquat cultivar Mardan were established using shoot apices after treating with NaOCl (5%, 7%, 10%, 12%, 14% (v/v)) for 12 min and HgCl2 (0.01%, 0.05%, 0.10%, 0.20%, 0.25% (w/v)) for 2 min. A maximum survival rate of 70% was recorded after surface sterilization with 10% NaOCl. Caulogenic response was assessed on Murashige and Skoog (MS) medium fortified with assorted combinations of the cytokinins, benzylaminopurine (BAP), kinetin, and N6-(2-isopentyl)adenine (2iP). Treatment of BAP 1.5 mg/L combined with 2iP 9.0 mg/L and kinetin 1.5 mg/L was found to be optimum for shoot morphogenesis in terms of the number and subsequent growth of shoots, while the highest shoot length was yielded by the combination of BAP 0.5 mg/L, kinetin 0.5 mg/L, and 2iP 3 mg/L. Higher levels of cytokinins induced callogenesis, vitrification and stunted growth to some extent. For rhizogenesis, uniform sized micro-shoots were excised and transferred to half-strength MS medium containing auxins. The best rooting expression was observed with naphthaleneacetic acid (NAA) 1 mg/L combined with indole-3-butyric acid (IBA) 2 mg/L and paclobutrazol (PBZ) 1 mg/L. PMID:24009197

Micropropagation of chokeberry by in vitro axillary shoot proliferation.

PubMed

Litwińczuk, Wojciech

2013-01-01

The black chokeberry-aronia (Aronia melanocarpa Elliot) is a shrub native to North America although nowadays well known in Eastern Europe. The fruits are regarded as the richest source of antioxidant phytonutrients among fruit crops and vegetables. Chokeberries can be easily propagated by seeds but this method is not recommended. Micropropagation is far more efficient than other conventional cloning methods like layering or softwood cuttings. Aronia clones are propagated in vitro through four- or three-stage method based on subculturing of shoot explants. The double diluted MS or full strength MS medium with elevated 50% Ca(2+) and Mg(2+) content are used in the initiation and proliferation chokeberry in vitro cultures, respectively. They are supplemented with 0.5-1.0 mg LBA, and 0.05 mg LIBA. The double-phase medium is recommended in the last passage before shoot rooting. The regenerated shoots could be rooted both in vitro on double diluted MS with 0.05 mg L(-1) IBA or in vivo in peat and perlite substrate and subsequently grown in the greenhouse.

Effect of activated charcoal on callus growth and shoot organogenesis in tobacco. [Nicotiana tabacum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Constantin, M.J.; Henke, R.R.; Mansur, M.A.

1977-01-01

Incorporating activated charcoal (AC) in culture media has been shown to affect growth and development of various organisms. Since AC stimulates the development of tobacco haploid plantlets from cultured anthers, research was conducted to determine the effect of activated charcoal on pith-derived callus growth and shoot development in Nicotiana tabacum cv. Wisconsin 38. Our results indicate that the hormones required for callus growth and shoot development in Wisconsin-38 tobacco are adsorbed by AC, thereby inhibiting callus growth and prohibiting shoot development. This effect was observed even when AC was removed from the medium by filtration prior to culturing the callus.

LONG-TERM PRECONDITIONING OF PLANTLETS: A PRACTICAL METHOD FOR ENHANCING SURVIVAL OF PINEAPPLE (Ananas comosus Merr.) SHOOT TIPS CRYOPRESERVED USING VITRIFICATION.

PubMed

Hu, W H; Liu, S F; Liaw, S I

2015-01-01

The purpose of this study was to develop an efficient cryopreservation protocol for pineapple (Ananas comosus Merr.) shoot tips. The optimal state of pineapple plantlets was investigated by using sucrose preconditioning to enhance survival after cryostorage. To achieve a suitable state of plantlets before cryopreservation, 0.2 M to 0.4 M sucrose concentrations combined with short- (0-7 days), medium- (15-30 days), and long-term (75-150 days) preconditioning periods were compared. The highest survival (100 %) was achieved using the following procedure: intact plantlets underwent long-term preconditioning with 0.2 M sucrose for 135 days, dissected shoot tips were treated with a loading solution containing 2.0 M glycerol + 0.4 M sucrose for 60 min at 25 degree and the shoot tips were dehydrated in PVS2 for 2h at 0 degree C before being plunged in liquid nitrogen. Rewarming was conducted in a water-bath for 30 s at 40 degree C and PVS2 was replaced with a 1.2 M sucrose solution for 30 min at 25 degree C. The shoot tips were transferred on semisolid medium and left in the dark for 1 week, then in dim light for 3 weeks.

A high-frequency in vitro multiplication, micromorphological studies and ex vitro rooting of Cadaba fruticosa (L.) Druce (Bahuguni): a multipurpose endangered medicinal shrub.

PubMed

Lodha, Deepika; Patel, Ashok Kumar; Shekhawat, N S

2015-07-01

An efficient and reproducible in vitro propagation protocol has been established for Cadaba fruticosa (L.) Druce. Surface-sterilized nodal stem segments of mature plant were used as explants for culture establishment. Multiple shoots were optimally differentiated from the nodal stem explants through bud breaking on Murashige and Skoog (1962) medium containing 3.0 mg l(-1) benzyladenine (BA). The effect of different plant growth regulators and minerals were studied on different stages of micropropagation procedure (i.e., explant establishment, shoot multiplication/growth and ex vitro rooting). Additionally, for enhancing shoot multiplication during subculture, MS medium was modified (MMS) with higher levels of magnesium, potassium and sulphate ions. Out of these, MMS3 medium containing 0.25 mg l(-1) each of BA and Kin (N6-furfuryladenine), with 0.1 mg l(-1) NAA (α-naphthalene acetic acid) was found the best for shoot multiplication (42.45 ± 3.82 per culture vessel). The in vitro regenerated shoots were rooted under ex vitro conditions on treating the shoot base with 500 mg l(-1) of IBA (indole-3 butyric acid) for 3 min on sterile Soilrite®. The ex vitro rooted plants were hardened in the greenhouse and transferred to the field with ≈85 % survival rate. There were not any visual differences between wild and micropropagated plants in the field, although the later underwent significant changes during acclimatization. Micromorphological changes on leaf surface characters from in vitro to acclimatized plantlets were studied in terms of development of glandular trichomes, changes in vein spacing and vein structure in order to understand the nature of plant responses towards environmental conditions. The method developed and defined can be applied for commercial cultivation, which may be important for extraction of bioactive compounds and may facilitate conservation of this multipurpose endangered medicinal shrub.

Clarifying the effects of dwarfing rootstock on vegetative and reproductive growth during tree development: a study on apple trees.

PubMed

Costes, E; García-Villanueva, E

2007-08-01

Despite the widespread use of dwarfing rootstocks in the fruit-tree industry, their impact on tree architectural development and possible role in the within-tree balance between growth and flowering are still poorly understood, in particular during the early years of growth. The present study addressed this question in apple trees, through a detailed analysis of shoot populations, i.e. both vegetative and flowering shoots, during tree development. Architectural databases were constructed for trees of two cultivars that were either own-rooted or grafted on dwarfing rootstock. Within-tree shoot demographics and annual shoot characteristics, i.e. their dimensions, number of laterals and flowering, were observed from the first to the fifth year of growth and compared among scion/root system combinations. Differences in axis demographics appeared among scion/root system combinations after the second year of growth. Differences were found (a) in the number of long axes and (b) the number of medium axes. Dwarfing rootstock reduced the total number of axes developed in a tree, and this reduction resulted from proportionally more medium axes and spurs than long axes. The life span of spurs was also shortened. These phenomena appeared after an increase in flowering that started in the second year of growth and involved both axillary and terminal positions. Flowering regularity was also increased in grafted trees. These results confirm that the number of long shoots and flowering potential depend on the cultivar. They indicate that tree architectural plasticity in response to its root system mainly derives from the number of medium shoots developed and follows priorities within the whole tree axis population. There was also evidence for dwarfing rootstock involvement in adjusting the flowering abundance and that differences in flowering occurrence take precedence over those regarding vegetative growth during tree development.

In vitro propagation, encapsulation, and genetic fidelity analysis of Terminalia arjuna: a cardioprotective medicinal tree.

PubMed

Gupta, Amit K; Harish; Rai, Manoj K; Phulwaria, Mahendra; Agarwal, Tanvi; Shekhawat, N S

2014-07-01

The present study described an improved and reproducible in vitro regeneration system for Terminalia arjuna using nodal segment explants obtained from a mature plant. Shoot tips excised from in vitro proliferated shoots were encapsulated in 3 % sodium alginate and 100 mM CaCl2[Symbol: see text]2H2O for the development of synthetic seeds which may be applicable in short-term storage and germplasm exchange of elite genotype. Shoot multiplication was significantly influenced by a number of factors, namely types and concentrations of plant growth regulators, medium composition, repeated transfer of mother explants, subculturing of in vitro regenerated shoot clumps, agar concentrations, and temperature. Maximum numbers of shoots (16.50 ± 3.67) were observed on modified Murashige and Skoog (MMS) medium containing 0.5 mg l(-1) of benzylaminopurine (BAP) and 0.1 mg l(-1) of naphthalene acetic acid (NAA). To shortening the regeneration pathway, rooting of micropropagated shoots under in vitro condition was excluded and an experiment on ex vitro rooting was conducted and it was observed that the highest percentage of shoots rooted ex vitro when treated with indole-3-butyric acid (IBA, 250 mg l(-1)) + 2-naphthoxy acetic acid (NOA, 250 mg l(-1)) for 5 min. The well-developed ex vitro rooted shoots were acclimatized successfully in soilrite under greenhouse conditions with 80 % survival of plants. Randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants were genetically identical to the mother plant, suggesting the absence of detectable genetic variation in the regenerated plantlets. To the best of our knowledge, this is the first report on synthetic seed production as well as ex vitro rooting and genetic fidelity assessment of micropropagated shoots of T. arjuna.

Soil type affects Pinus ponderosa var. scopulorum (Pinaceae) seedling growth in simulated drought experiments.

PubMed

Lindsey, Alexander J; Kilgore, Jason S

2013-08-01

Effects of drought stress and media type interactions on growth of Pinus ponderosa var. scopulorum germinants were investigated. • Soil properties and growth responses under drought were compared across four growth media types: two native soils (dolomitic limestone and granite), a soil-less industry standard conifer medium, and a custom-mixed conifer medium. After 35 d of growth, the seedlings under drought stress (reduced watering) produced less shoot and root biomass than watered control seedlings. Organic media led to decreased root biomass, but increased root length and shoot biomass relative to the mineral soils. • Media type affected root-to-shoot biomass partitioning of P. ponderosa var. scopulorum, which may influence net photosynthetic rates, growth, and long-term seedling survival. Further work should examine how specific soil properties like bulk density and organic matter influence biomass allocation in greenhouse studies.

Soil type affects Pinus ponderosa var. scopulorum (Pinaceae) seedling growth in simulated drought experiments1

PubMed Central

Lindsey, Alexander J.; Kilgore, Jason S.

2013-01-01

• Premise of the study: Effects of drought stress and media type interactions on growth of Pinus ponderosa var. scopulorum germinants were investigated. • Methods and Results: Soil properties and growth responses under drought were compared across four growth media types: two native soils (dolomitic limestone and granite), a soil-less industry standard conifer medium, and a custom-mixed conifer medium. After 35 d of growth, the seedlings under drought stress (reduced watering) produced less shoot and root biomass than watered control seedlings. Organic media led to decreased root biomass, but increased root length and shoot biomass relative to the mineral soils. • Conclusions: Media type affected root-to-shoot biomass partitioning of P. ponderosa var. scopulorum, which may influence net photosynthetic rates, growth, and long-term seedling survival. Further work should examine how specific soil properties like bulk density and organic matter influence biomass allocation in greenhouse studies. PMID:25202578

In Vitro and Cryopreservation Techniques for Conservation of Snow Mountain Garlic.

PubMed

Mahajan, Ritu

2016-01-01

Garlic is an important medicinal herb of culinary value by imparting its flavors and odors to the food. Allicin, a notable flavonoid in garlic, is a powerful antibiotic and antifungal compound. Due to poor bioavailability, garlic is of limited use for oral human consumption. Being sexually sterile, propagation of garlic is done by individual cloves from a bulb which increases the chances of transfer of viral diseases. In this chapter, an efficient and improved regeneration protocol for explant establishment and shoot multiplication under in vitro conditions is described. A high rate of shoot multiplication is obtained on MS medium supplemented with 0.5 mg/l BAP, 1.0 mg/l KN, and 2.0 mg/l GA3. Addition of 1.0 mg/l NAA to MS medium resulted in rooting at the shoot bases. A detailed method for encapsulation of explant in sodium alginate beads and their cryopreservation using encapsulation-dehydration is also described.

Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation.

PubMed

Uchendu, Esther E; Leonard, Scott W; Traber, Maret G; Reed, Barbara M

2010-01-01

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more alpha- and gamma-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.

In vitro propagation of fraser photinia using Azospirillum-mediated root development.

PubMed

Llorente, Berta E; Larraburu, Ezequiel E

2013-01-01

Fraser photinia (Photinia × fraseri Dress.) is a woody plant of high ornamental value. The traditional propagation system for photinia is by rooting apical cuttings using highly concentrated auxin treatments. However, photinia micropropagation is an effective alternative to traditional in vivo propagation which is affected by the seasonal supply of cuttings, the long time required to obtain new plants, and the difficulties in rooting some clones.A protocol for in vitro propagation of fraser photinia using the plant growth-promoting ability of some rhizobacteria is described here. Bacterial inoculation is a new tool in micropropagation protocols that improves plant development in in vitro culture. Shoots culture on a medium containing MS macro- and microelements, Gamborg's vitamins (BM), N (6)-benzyladenine (BA, 11.1 μM), and gibberellic acid (1.3 μM) produce well-established explants. Proliferation on BM medium supplemented with 4.4 μM BA results in four times the number of shoots per initial shoot that develops monthly. Consequently, there is a continuous supply of plant material since shoot production is independent of season. Azospirillum brasilense inoculation, after 49.2 μM indole-3-butyric acid pulse treatment, stimulates early rooting of photinia shoots and produces significant increase in root fresh and dry weights, root surface area, and shoot fresh and dry weights in comparison with controls. Furthermore, inoculated in vitro photinia plants show anatomical and morphological changes that might lead to better adaptation in ex vitro conditions after transplanting, compared with the control plants.

Environmental responses of the FT/TFL1 gene family and their involvement in flower induction in Fragaria × ananassa.

PubMed

Nakano, Yoshihiro; Higuchi, Yohei; Yoshida, Yuichi; Hisamatsu, Tamotsu

2015-04-01

Flowering time control is important for fruit production in Fragaria × ananassa. The flowering inhibition pathway has been extensively elucidated in the woodland strawberry, Fragaria vesca, whereas the factors involved in its promotion remain unclear. In this study, we investigated the environmental responses of F. × ananassa FT and TFL1-like genes, which are considered key floral promoters and repressors in many plants, respectively. A putative floral promoter, FaFT3, was up-regulated in the shoot tip under short-day and/or low growth temperature, in accordance with the result that these treatments promoted flowering. FaFT3 mRNA accumulated before induction of a floral meristem identity gene, FaAP1. FaFT2, a counterpart of FvFT2, expressed in the flower bud of F. vesca, was not induced in the shoot tip differentiating sepal or stamen, suggesting that this gene works at a later stage than stamen formation. In F. vesca, FvFT1 transmits the long-day signal perceived in the leaves to the shoot tip, and induces the potent floral inhibitor FvTFL1. FaFT1 was expressed in the leaves under long-day conditions in F. × ananassa. Expression of FaTFL1 was higher in the shoot tip under long-day than short-day conditions. Independent of day-length, FaTFL1 expression was higher under high temperature than low temperature conditions. These results suggest that FaFT3 induction by short-day or low temperature stimuli is a key step for flowering initiation. As in F. vesca, F. × ananassa floral inhibition pathways depend on FaTFL1 regulation by day-length via FaFT1, and by temperature. Copyright © 2015 Elsevier GmbH. All rights reserved.

Gentiana dinarica Beck hairy root cultures and evaluation of factors affecting growth and xanthone production

USDA-ARS?s Scientific Manuscript database

The induction and establishment of hairy root cultures of Gentiana dinarica using two strains of Agrobacterium rhizogenes (A4M70GUS and 15834/PI) is reported for the first time. Hairy roots were formed from the shoots 25 days after inoculation, and strain 15834/PI had higher induction rate of hairy ...

In vitro propagation and withaferin A production in Withania ashwagandha, a rare medicinal plant of India.

PubMed

Mir, Bilal Ahmad; Mir, Shabir Ahmad; Koul, Sushma

2014-07-01

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.

In vitro propagation and conservation of Satureja avromanica Maroofi-an indigenous threatened medicinal plant of Iran.

PubMed

Mozafari, Ali Akbar; Vafaee, Yavar; Karami, Edris

2015-07-01

An efficient and rapid in vitro propagation system for Satureja avromanica, a rare and endangered folk medicinal plant of Iran was developed through the shoot tip and leaf disc explants. Nodal and leaf explants from wild plants were established on MS and WPM media supplemented with BA, BAP and TDZ (0, 0.1, 0.5, 1, 1.5, 2, 5 and 10 mgl(-1)) alone or by application of BA and TDZ (0, 2, 5 and 10 mgl(-1)) in combination with IBA and 2,4-D (0, 0.1, 0.5 and 1 mgl(-1)), respectively. Based on results, the highest mean shoot number (6.21) was obtained on MS medium supplemented with 2 mgl(-1) BA. Regarding the shoot elongation, MS supplemented with 2 mgl(-1) TDZ and MS containing 5 mgl(-1) BA showed the longest shoots (4.82 and 4.39 cm, respectively) after 6 weeks of culture. As a matter of fact, increasing all three tested cytokinins levels led to enhancement of explant response frequency and regenerated shoot number. On the other side, WPM medium supplemented with 0.1 mgl(-1) IBA was found suitable for rooting of regenerated shoots. RAPD molecular analysis revealed genetic stability of in vitro raised plants. In conclusion, individual application of BA, BAP and TDZ were in favor of S. avromanica direct shoot regeneration while treatment media with a combination of IBA and BA as well as 2,4-D and TDZ resulted in callogenesis in most explants. Finally, the in vitro raised plantlets were acclimatized and successfully established in the greenhouse conditions. Our developed protocol can be employed for the large-scale micropropagation and conservation of S. avromanica as a threatened medicinal plant.

Rapid in vitro multiplication and restoration of Celastrus paniculatus Willd. sub sp. paniculatus (Celastraceae), a medicinal woody climber.

PubMed

Nair, L G; Seeni, S

2001-07-01

Nodes, shoot tips, internodes and leaf bases (approximately 1.0 cm) excised from young vines of the flowering woody climber, Celastrus paniculatus WilId. sub. sp. paniculatus (Celastraceae) were cultured in Murashige and Skoog (MS) medium containing agar (0.6%), sucrose (3%) and varied concentrations of 6-benzyl aminopurine (BAP) and kinetin. All the explant types were regenerative and maximum number (3.6) and frequency (94%) of axillary shoot formation of (5.08 cm long) was recorded in the nodes cultured in BAP (1 mg L(-1)) after 6 weeks. Combinations of BAP (1 mg L(-1)) and indole-3-acetic acid/l-naphthalene acetic acid (0.01-1 mg L(-1); IAA/NAA) tested with nodes induced formation of less number (3 and 2.2) of shoots at same frequency (94%). All the explant types viz. node, shoot tip, internode and leaf base of in vitro derived shoots responded earlier and better in lower concentrations of BAP (0.5-2 mg L(-1)) with formation of 8, 3.1, 6.4 and 1.8 shoots respectively during the same period. In spite of the advanced and increased caulogenic responses, differences in cytokinin requirements between different explants observed during culture initiation still persisted with the nodes, shoot tips, internodes and petiole segments responding best at 0.5, 1 and 2 mg L(-1) BAP, respectively. The repeated reculture up to 10 cycles of the nodes from the shoot cultures each at 6-week intervals enabled multiplication and stocking of shoots without decline. Rooting of 3-7 cm shoot cuttings was induced in half-strength MS liquid medium containing IAA (1 mg L(-1)) with formation of 7.25 roots of 2.41 cm length within 6 weeks. Rooted plants were established at 84-96% rate in community pots without hardening, the least value (84%) being obtained with NAA- induced thick and calloid rooted plants. Four month old community potted plants were reintroduced into native forest habitats at 95% efficiency and 8 months after restoration, the plants were uniform in morphological, growth, cytological and peroxidase and esterase isozyme characteristics.

Improved efficiency of plant regeneration from protoplasts of eggplant Solanum melongena L.

PubMed

Guri, A; Izhar, S

1984-12-01

Eggplant (Solanum melongena L.) mesophyll protoplasts were obtained from in vitro growing plants of line 410 and cv. 'Classic'. Relatively high (15%) plating efficiency was achieved using petri dishes with alternate quadrants containing reservoir medium (R medium + 1% activated charcoal) and culture medium. Shoot regeneration occurred within 6 weeks following initiation of protoplast culture.

Development of disease-resistant marker-free tomato by R/RS site-specific recombination.

PubMed

Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2011-06-01

The selection marker genes, imparting antibiotic or herbicide resistance, in the final transgenics have been criticized by the public and considered a hindrance in their commercialization. Multi-auto-transformation (MAT) vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators (PGRs). In the study reported here, isopentenyltransferase (ipt) gene was used as a selection marker and wasabi defensin (WD) gene, isolated from Wasabia japonica as a target gene. WD was cloned from the binary vector, pEKH-WD to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledons of tomato cv. Reiyo were cultured on PGR- and antibiotic-free MS medium. Adventitious shoots were developed by the explants infected with the pMAT21/wasabi defensin. The same PGR- and antibiotic-free MS medium was used in subcultures of the adventitious shoot lines (ASLs) to produce ipt and normal shoots. Approximately, 6 months after infection morphologically normal shoots were produced. Molecular analyses of the developed shoots confirmed the integration of gene of interest (WD) and excision of the selection marker (ipt). Expression of WD was confirmed by Northern blot and Western blot analyses. The marker-free transgenic plants exhibited enhanced resistance against Botrytis cinerea (gray mold), Alternaria solani (early blight), Fusarium oxysporum (Fusarium wilt) and Erysiphe lycopersici (powdery mildew).

Development and plasticity of endangered shrub Lindera melissifolia (Lauraceae) seedlings under contrasting light regimes

Treesearch

Brian R Lockhart; Emile S Gardiner; Theran Stautz; Theodor D. Leininger

2012-01-01

Lindera melissifolia (Walt.) Blume seedlings were raised in a growth chamber to determine the effects of light availability on shoot growth pattern, and basic leaf and stem growth. Lindera melissifolia seedlings exhibited a sympodial shoot growth pattern for 3 months following emergence from the soil medium, but this pattern was characterized by a reduction in leaf...

Transformation of PRT6 RNAi construct into tomato (Solanum lycopersicum) cv. Micro-Tom

NASA Astrophysics Data System (ADS)

Suka, Intan Elya; Chew, Bee Lynn; Goh, Hoe-Han; Isa, Nurulhikma Md

2018-04-01

PROTEOLYSIS 6 plays major role in the N-end rule pathway as N-recognin which functions as E3 ligase enzyme. It mediates ubiquitin processes that lead to degradation of unstable substrate protein. The aim of the current study is to transform the PRT6 gene into tomato (Solanum lycopersicum) from the cultivar Micro-Tom and to investigate its function in regulating ripening in tomato fruits. The PRT6_RNAi construct was successfully transformed into Agrobacterium C58 via heat shock method and transformed into seven days old cotyledon explants. Factors affecting transformation efficiency such as co-cultivation time and type of plant growth regulator combination were evaluated. Results from this study found that pre-cultured cotyledons from seven days old seedlings incubated for 2 days in co-cultivation medium increased shoot regeneration. Plant growth hormones zeatin combine with auxin produced a higher number of callus formation but lower shoot proliferation and transformation frequency compared to treatments of single plant hormone in the selection medium. Polymerase chain reaction (PCR) was performed on the regenerated shoots to confirm the integration of PRT6 fragment into the genome of transgenic plants. Based on PCR analysis, all putative shoots were positive transformants.

The biology and in vitro propagation of the ornamental aquatic plant, Aponogeton ulvaceus.

PubMed

Kam, Melissa Yit Yee; Chai, Li Chin; Chin, Chiew Foan

2016-01-01

Aponogeton ulvaceus Baker (Aponogetonaceae) is a commercially important ornamental aquatic plant species with traditional medicinal uses. Due to the low survival rate of seedlings, propagation by conventional means has been met with many difficulties. In this study, botanical aspects of A. ulvaceus were examined with regards to the morphology, anatomy and physiology of the plant and an efficient protocol for its in vitro propagation using immature tuber explants has been established. The existence of glandular trichomes on the leaves was discovered and the occurrence of circumnutation in A. ulvaceus has been demonstrated. Immature tuber segments with meristems were cultured on MS medium supplemented with various combinations (0, 1, 2, and 3 mg/L) of BAP and NAA for callus induction. The highest percentage of callus production (100 %) was obtained in two different treatments: 1 mg/L BAP and 3 mg/L NAA, and 2 mg/L BAP and 3 mg/L NAA. For shoot and root organogenesis, the combination of 1 mg/L BAP and 1 mg/L NAA was shown to be significant for A. ulvaceus regeneration when compared to control, which yields a mean shoot and root number of 22.50 and 29.50 respectively. The current protocol is the first reported successful establishment of in vitro clonal propagation of A. ulvaceus .

In vitro propagation and assessment of the genetic fidelity of Musa acuminata (AAA) cv. Vaibalhla derived from immature male flowers.

PubMed

Hrahsel, Lalremsiami; Basu, Adreeja; Sahoo, Lingaraj; Thangjam, Robert

2014-02-01

An efficient in vitro propagation method has been developed for the first time for Musa acuminata (AAA) cv. Vaibalhla, an economically important banana cultivar of Mizoram, India. Immature male flowers were used as explants. Murashige and Skoog's (MS) medium supplemented with plant growth regulators (PGRs) were used for the regeneration process. Out of different PGR combinations, MS medium supplemented with 2 mg L(-1) 6-benzylaminopurine (BAP) + 0.5 mg L(-1) α-naphthalene acetic acid (NAA) was optimal for production of white bud-like structures (WBLS). On this medium, explants produced the highest number of buds per explant (4.30). The highest percentage (77.77) and number (3.51) of shoot formation from each explants was observed in MS medium supplemented with 2 mg L(-1) kinetin + 0.5 mg L(-1) NAA. While MS medium supplemented with a combination of 2 mg L(-1) BAP + 0.5 mg L(-1) NAA showed the maximum shoot length (14.44 cm). Rooting efficiency of the shoots was highest in the MS basal medium without any PGRs. The plantlets were hardened successfully in the greenhouse with 96% survival rate. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were employed to assess the genetic stability of in vitro regenerated plantlets of M. acuminata (AAA) cv. Vaibalhla. Eight RAPD and 8 ISSR primers were successfully used for the analysis from the 40 RAPD and 30 ISSR primers screened initially. The amplified products were monomorphic across all the regenerated plants and were similar to the mother plant. The present standardised protocol will find application in mass production, conservation and genetic transformation studies of this commercially important banana.

Carbon: Nitrogen Interaction Regulates Expression of Genes Involved in N-Uptake and Assimilation in Brassica juncea L.

PubMed Central

Goel, Parul; Bhuria, Monika; Kaushal, Mamta

2016-01-01

In plants, several cellular and metabolic pathways interact with each other to regulate processes that are vital for their growth and development. Carbon (C) and Nitrogen (N) are two main nutrients for plants and coordination of C and N pathways is an important factor for maintaining plant growth and development. In the present work, influence of nitrogen and sucrose (C source) on growth parameters and expression of genes involved in nitrogen transport and assimilatory pathways was studied in B. juncea seedlings. For this, B. juncea seedlings were treated with four combinations of C and N source viz., N source alone (-Suc+N), C source alone (+Suc-N), with N and C source (+Suc+N) or without N and C source (-Suc-N). Cotyledon size and shoot length were found to be increased in seedlings, when nitrogen alone was present in the medium. Distinct expression pattern of genes in both, root and shoot tissues was observed in response to exogenously supplied N and C. The presence or depletion of nitrogen alone in the medium leads to severe up- or down-regulation of key genes involved in N-uptake and transport (BjNRT1.1, BjNRT1.8) in root tissue and genes involved in nitrate reduction (BjNR1 and BjNR2) in shoot tissue. Moreover, expression of several genes, like BjAMT1.2, BjAMT2 and BjPK in root and two genes BjAMT2 and BjGS1.1 in shoot were found to be regulated only when C source was present in the medium. Majority of genes were found to respond in root and shoot tissues, when both C and N source were present in the medium, thus reflecting their importance as a signal in regulating expression of genes involved in N-uptake and assimilation. The present work provides insight into the regulation of genes of N-uptake and assimilatory pathway in B. juncea by interaction of both carbon and nitrogen. PMID:27637072

Rapid in vitro production of cloned plants of Uraria picta (Jacq.) DC-A rare medicinal herb in long-term culture.

PubMed

Rai, Santosh Kumar; Sharma, Meena; Jain, Madhu; Awasthi, Abhishek; Purshottam, Dharmendra Kumar; Nair, Narayanan Kuttanpillai; Sharma, Ashok Kumar

2010-11-01

An efficient in vitro process for rapid production of cloned plants of Uraria picta has been developed employing nodal stem segments taken from field-grown plants. Explants showed bud-break followed by regeneration of shoots with restricted growth within 12 days on modified Murashige and Skoog's medium supplemented with 0.25 mg l(-1) each of 6-benzylaminopurine and indole-3-acetic acid and 25 mg l(-1) adenine sulfate. Normal growth of shoots with good proliferation rate was achieved by reducing the concentrations of 6-benzylaminopurine and indole-3-acetic acid to 0.1 mg l(-1) each and incorporating 0.5 mg l(-1) gibberellic acid in the medium in which, on an average, 19.6 shoots per explant were produced. Further, during successive subcultures, increased concentrations of adenine sulfate (50 mg l(-l)) and gibberellic acid (2 mg l(-l)) along with the addition of 20 mg l(-l)  DL: -tryptophan were found conducive to control the problem of necrosis of shoots. In this treatment, several "crops" of shoots were obtained from single culture by repeated subculturing of basal portion of stalk in long-term. Isolated shoots rooted 100% in 0.25 mg l(-1) indole-3-butyric acid. In vitro-raised plants after hardening in inorganic salt solution grew normally in soil and came to flowering. Genetic fidelity of in vitro-raised plants was ascertained by rapid amplified polymorphic DNA (RAPD) markers. Also, quantitative estimation of two isoflavonones in their root extracts further confirmed true-to-type nature of plantlets.

Improved recovery of cryotherapy-treated shoot tips following thermotherapy of in vitro-grown stock shoots of raspberry (Rubus idaeus L.).

PubMed

Wang, Qiaochun; Valkonen, Jari P T

2009-01-01

Raspberry bushy dwarf virus (RBDV) can be efficiently eradicated from raspberry plants (Rubus idaeus) by a procedure combining thermotherapy and cryotherapy. However, the bottleneck of this procedure is that, following thermotherapy, cryopreserved shoot tips become chlorotic during regrowth and eventually die after several subcultures. In addition, survival of heat-treated stock shoots and recovery of cryopreserved shoot tips following thermotherapy are low. The present study focused towards improving regrowth of cryopreserved raspberry shoot tips following thermotherapy. Results showed that preconditioning stock shoots with salicylic acid (SA; 0.01-0.1 mM) markedly increased survival of stock shoots after 4 weeks of thermotherapy. Regrowth of cryopreserved shoot tips following thermotherapy was also significantly enhanced when SA (0.05-0.1 mM) was used for preconditioning stock shoots. Addition of either Fe-ethylenediaminetetracetic acid (Fe-EDTA, 50 mg per L) or Fe-ethylenediaminedi(o)hydroxyphenylacetic acid (Fe-EDDHA, 50 mg per L) to post-culture medium strongly promoted regrowth and totally prevented chlorosis of shoots regenerated from cryopreserved shoot tips following thermotherapy. Using the parameters optimized in the present study, about 80 percent survival of heat-treated stock shoots and about 33 percent regrowth of cryopreserved shoot tips following thermotherapy were obtained. Morphology of plants regenerated from cryopreserved shoot tips following thermotherapy was identical to that of control plants, based on observations of leaf shape and size, internode length and plant height. Optimization of the thermotherapy procedure followed by cryotherapy will facilitate the wider application of this technique to eliminate viruses which can invade meristems.

Somatic Embryogenesis in Peach Palm Using the Thin Cell Layer Technique: Induction, Morpho-histological Aspects and AFLP Analysis of Somaclonal Variation

PubMed Central

Steinmacher, D. A.; Krohn, N. G.; Dantas, A. C. M.; Stefenon, V. M.; Clement, C. R.; Guerra, M. P.

2007-01-01

Background and Aims The thin cell layer (TCL) technique is based on the use of very small explants and has allowed enhanced in vitro morphogenesis in several plant species. The present study evaluated the TCL technique as a procedure for somatic embryo production and plantlet regeneration of peach palm. Methods TCL explants from different positions in the shoot apex and leaf sheath of peach palm were cultivated in MS culture medium supplemented with 0–600 µm Picloram in the presence of activated charcoal. The production of primary calli and embryogenic calli was evaluated in these different conditions. Histological and amplified fragment length polymorphism (AFLP) analyses were conducted to study in vitro morphogenetic responses and genetic stability, respectively, of the regenerated plantlets. Key Results Abundant primary callus induction was observed from TCLs of the shoot meristem in culture media supplemented with 150–600 µm Picloram (83–97 %, respectively). The production of embryogenic calli depends on Picloram concentration and explant position. The best response observed was 43 % embryogenic callus production from shoot meristem TCL on 300 µm Picloram. In maturation conditions, 34 ± 4 somatic embryos per embryogenic callus were obtained, and 45·0 ± 3·4 % of these fully developed somatic embryos were converted, resulting in plantlets ready for acclimatization, of which 80 % survived. Histological studies revealed that the first cellular division events occurred in cells adjacent to vascular tissue, resulting in primary calli, whose growth was ensured by a meristematic zone. A multicellular origin of the resulting somatic embryos arising from the meristematic zone is suggested. During maturation, histological analyses revealed bipolarization of the somatic embryos, as well as the development of new somatic embryos. AFLP analyses revealed that 92 % of the regenerated plantlets were true to type. The use of TCL explants considerably improves the number of calli and somatic embryos produced in comparison with previously described protocols for in vitro regeneration of peach palm. Conclusions The present study suggests that the TCL somatic embryogenesis protocol developed is feasible, although it still requires further optimization for in vitro multiplication of peach palm, especially the use of similar explants obtained from adult palm trees. PMID:17670751

Regeneration of Acer caudatifolium Hayata plantlets from juvenile explants.

PubMed

Durkovic, J

2003-07-01

Juvenile and fully mature Acer caudatifolium Hayata explants were assayed for their organogenic capacity. A protocol for multiple shoot culture formation and in vitro plant regeneration was developed for juvenile axillary bud cultures. Mature explants failed in shoot regeneration. Shoot multiplication was achieved by releasing apical dominance of the single elongated shoot on woody plant medium (WPM) supplemented with 0.7 mg l(-1) 6-benzylaminopurine and 0.05 mg l(-1) alpha-naphthaleneacetic acid. The highest rooting percentage was recorded on half-strength WPM containing 1.0 mg l(-1) indole-3-butyric acid. Regenerated plantlets were successfully hardened to ex vitro conditions and continued to grow after transfer to soil. No morphological aberrations were observed in the regenerates.

In Vitro Regeneration of Endangered Medicinal Plant Heliotropium kotschyi (Ramram).

PubMed

Sadeq, Manal Ahmed; Pathak, Malabika Roy; Salih, Ahmed Ali; Abido, Mohammed; Abahussain, Asma

2016-01-01

Heliotropium kotschyi (Ramram) is an important endangered medicinal plant distributed in the Kingdom of Bahrain. Plant tissue culture technique is applied for ex situ conservation study. Nodal stem segments are cultured in modified MS media supplemented with various combination and concentration of plant growth regulators (PGRs). Plants are regenerated via shoot organogenesis from the nodal meristems. Plants are regenerated in three different steps: initial shoot development, shoot multiplication, and rooting. After 4 weeks of culture, 100 % explants respond to shoot initiation on the medium containing 8.88 μM BAP and 5.71 μM IAA. The highest frequency of shoot regeneration is observed in the same media after second subculture of shoots. The highest rooting frequency is observed in the presence of 2.85 μM IAA. After root development, the plantlets are transferred to pots filled with soil and 60 % of plants survived after 45 days. This plant regeneration protocol is of great value for rapid desert plant propagation program.

Auxin Synthesis-Encoding Transgene Enhances Grape Fecundity1[OA

PubMed Central

Costantini, Elisa; Landi, Lucia; Silvestroni, Oriana; Pandolfini, Tiziana; Spena, Angelo; Mezzetti, Bruno

2007-01-01

Grape (Vitis vinifera) yield is largely dependent on the fecundity of the cultivar. The average number of inflorescences per shoot (i.e. shoot fruitfulness) is a trait related to fecundity of each grapevine. Berry number and weight per bunch are other features affecting grape yield. An ovule-specific auxin-synthesizing (DefH9-iaaM) transgene that increases the indole-3-acetic acid content of grape transgenic berries was transformed into cultivars Silcora and Thompson Seedless, which differ in the average number of inflorescences per shoots. Thompson Seedless naturally has very low shoot fruitfulness, whereas Silcora has medium shoot fruitfulness. The average number of inflorescences per shoot in DefH9-iaaM Thompson Seedless was doubled compared to its wild-type control. Berry number per bunch was increased in both transgenic cultivars. The quality and nutritional value of transgenic berries were substantially equivalent to their control fruits. The data presented indicate that auxin enhances fecundity in grapes, thus enabling to increase yield with lower production costs. PMID:17337528

Development of guayule (Parthenium argentatum) research in cell culture

NASA Technical Reports Server (NTRS)

Ball, E. A.

1981-01-01

Utilizing the lateral buds of known high rubber producing plants as explants in culture medium specifically designed to engender shoot development and to prevent callus formation, unlimited numbers of replicate plants can be produced. Each has the same genotype as the parent. This procedure has long been used to rid plants of virus, the latter generally does not occur in the embryonic tissues of the bud; it also, by virtue of its axenic nature, eliminates all microorganisms characteristic of the parent plant. Auxins were found essential to callus formation, but since the latter is known to bring about chromosomal aberrations, it was avoided. The cytokinin benzylaminopurine strongly stimulated shoot growth, and the number of regenerated buds on the inoculum was proportional to its concentration. These buds produced shoots several centimeters in length which were caused to root on medium containing indolebutyric acid. Transferred to the septic condition of soil, the plantlets were gradually brought into full sunlight where they showed a brief vegetative growth with production of mature leaves, and flowered.

Do Si/As ratios in growth medium affect arsenic uptake, arsenite efflux and translocation of arsenite in rice (Oryza sativa)?

PubMed

Zhang, Min; Zhao, Quanli; Xue, Peiying; Zhang, Shijie; Li, Bowen; Liu, Wenju

2017-10-01

Silicon (Si) may decrease the uptake and accumulation of arsenic (As) in rice. However, the effects of Si/As ratios in growth medium on arsenic uptake, arsenite efflux to the external medium and translocation of arsenite in rice are currently unclear. Rice seedlings (Oryza sativa L.) were exposed to nutrient solutions with 10 μM arsenite [As(III)] or 10 μM arsenate [As(V)] to explore the influence of different silicic acid concentrations (0, 10, 100, 1000 μM) on arsenic uptake and translocation of arsenite with or without 91 μM phosphate for 24 h. Arsenic speciation was determined in nutrient solutions, roots, and shoots. In the arsenite treatments, different Si/As ratios (1:1, 10:1, 100:1) did not affect As(III) uptake by rice roots, however they did inhibit translocation of As(III) from roots to shoots significantly (P < 0.001) in the absence of P. In the arsenate treatments, a Si/As ratio of 100:1 significantly decreased As(V) uptake and As(III) efflux compared with the control (Si/As at 0:1), accounting for decreases of 27.4% and 15.1% for -P treatment and 47.8% and 61.1% for + P treatment, respectively. As(III) is the predominant species of arsenic in rice roots and shoots. A Si/As ratio of 100:1 reduced As(III) translocation from roots to shoots markedly without phosphate. When phosphate was supplied, As(III) translocation from roots to shoots was significantly inhibited by Si/As ratios of 10:1 and 100:1. The results indicated that in the presence of P, different silicic acid concentrations did not impact arsenite uptake and transport in rice when arsenite was supplied. However, a Si/As ratio of 100:1 inhibited As(V) uptake, as well as As(III) efflux and translocation from roots to shoots when arsenate was supplied. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effects of ABA application on cessation of shoot elongation in long-day grown Norway spruce seedlings.

PubMed

Heide, O M

1986-06-01

Abscisic acid (ABA) was applied in lanolin to apical buds of Norway spruce (Picea abies (L.) Karst.) seedlings actively growing in a 24 h photoperiod. At a rate of 100 microg per plant, ABA suspended shoot elongation for about three weeks in the majority of plants but failed to induce normal winter buds. The role of ABA in the induction of dormancy is thus uncertain in conifers as well as in deciduous woody plants.

Effects of PEG-induced osmotic stress on growth and dhurrin levels of forage sorghum.

PubMed

O'Donnell, Natalie H; Møller, Birger Lindberg; Neale, Alan D; Hamill, John D; Blomstedt, Cecilia K; Gleadow, Roslyn M

2013-12-01

Sorghum (Sorghum bicolor L. Moench) is a valuable forage crop in regions with low soil moisture. Sorghum may accumulate high concentrations of the cyanogenic glucoside dhurrin when drought stressed resulting in possible cyanide (HCN) intoxication of grazing animals. In addition, high concentrations of nitrate, also potentially toxic to ruminants, may accumulate during or shortly after periods of drought. Little is known about the degree and duration of drought-stress required to induce dhurrin accumulation, or how changes in dhurrin concentration are influenced by plant size or nitrate metabolism. Given that finely regulating soil moisture under controlled conditions is notoriously difficult, we exposed sorghum plants to varying degrees of osmotic stress by growing them for different lengths of time in hydroponic solutions containing polyethylene glycol (PEG). Plants grown in medium containing 20% PEG (-0.5 MPa) for an extended period had significantly higher concentrations of dhurrin in their shoots but lower dhurrin concentrations in their roots. The total amount of dhurrin in the shoots of plants from the various treatments was not significantly different on a per mass basis, although a greater proportion of shoot N was allocated to dhurrin. Following transfer from medium containing 20% PEG to medium lacking PEG, shoot dhurrin concentrations decreased but nitrate concentrations increased to levels potentially toxic to grazing ruminants. This response is likely due to the resumption of plant growth and root activity, increasing the rate of nitrate uptake. Data presented in this article support a role for cyanogenic glucosides in mitigating oxidative stress. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Efficient Micropropagation of Highly Economic, Medicinal and Ornamental Plant Lallemantia iberica (Bieb.) Fisch. and C. A. Mey

PubMed Central

Ozdemir, Fethi Ahmet; Yildirim, Mehmet Ugur; Pourali Kahriz, Mahsa

2014-01-01

Lallemantia iberica (Bieb.) Fisch. and C. A. Mey is high valued annual ornamental and medicinal plant from Lamiaceae family that prefers dry sunny hillsides, roadsides, slopes, and fallow fields over an altitude of 500–2150 m. It bears beautiful white flowers and bloom from April to June each year. This study reports L. iberica micropropagation using cotyledon node explants isolated from 15-day-old in vitro regenerated plantlets. The cotyledon node explants were cultured on MS medium containing 0.50, 1.00 plus 2.00 mg/L BAP, 0.00, 0.01, and 0.02 mg/L NAA. Maximum shoot regeneration was noted on MS medium containing 0.50 mg/L BAP. Well-developed micropropagated shoots were rooted on MS medium containing 1.00 mg/L IBA. The rooted plants were easily hardened in the growth chamber and acclimatised in greenhouse. PMID:25247175

Efficient micropropagation of highly economic, medicinal and ornamental plant Lallemantia iberica (Bieb.) Fisch. and C. A. Mey.

PubMed

Ozdemir, Fethi Ahmet; Yildirim, Mehmet Ugur; Pourali Kahriz, Mahsa

2014-01-01

Lallemantia iberica (Bieb.) Fisch. and C. A. Mey is high valued annual ornamental and medicinal plant from Lamiaceae family that prefers dry sunny hillsides, roadsides, slopes, and fallow fields over an altitude of 500-2150 m. It bears beautiful white flowers and bloom from April to June each year. This study reports L. iberica micropropagation using cotyledon node explants isolated from 15-day-old in vitro regenerated plantlets. The cotyledon node explants were cultured on MS medium containing 0.50, 1.00 plus 2.00 mg/L BAP, 0.00, 0.01, and 0.02 mg/L NAA. Maximum shoot regeneration was noted on MS medium containing 0.50 mg/L BAP. Well-developed micropropagated shoots were rooted on MS medium containing 1.00 mg/L IBA. The rooted plants were easily hardened in the growth chamber and acclimatised in greenhouse.

High efficiency induction of callus and regeneration of sporophytes of Laminaria japonica (Phaeophyta)

NASA Astrophysics Data System (ADS)

Wang, Xi-Hua; Qin, Song; Li, Xin-Ping; Jiang, Peng; Zeng, Cheng-Kui; Qin, Mei

1998-03-01

Four media (PESI solid, MS liquid, MS solid and ASP-C-I solid medium) were used to induce callus from excised tissues of the kelp Laminaria japonica. Only PESI solid medium and MS solid medium produced calli. Modified MS solid medium supplemented with mannitol (3%,W/V), yeast extract (0.1%, W/V), VB2 (0.5 mg/ml), VB12 (0.5 mg/ml), kinetin (0.108 μg/ml) and NAA (1.860μg/ml) showed much better effect on callus induction than non-modified MS solid medium. After 24 days of induction 75.5% of tissues in PESI solid medium showed callus formation. For modified MS solid medium, after three months of induction 67.3% of tissues dedifferentiated into calli. No callus could be found after five months of induction in either MS liquid or ASP-C-I solid medium. When calli were squashed and cultured in N-P enriched autoclaved seawater, MS liquid medium and ASP12-NTA liquid medium (both modified with kelp extract), differentiation of cells and regeneration of sporophytes were only observed in ASP12-NTA medium supplemented with kelp extract. Gametophyte-like filaments formed first, then eggs were released. It was suggested that sporophyte formation could be a process of parthenogenesis. Sterilization techniques in tissue culture of L. japonica were also tested in this study.

Arsenate tolerance mechanism of Oenothera odorata from a mine population involves the induction of phytochelatins in roots.

PubMed

Kim, Dae-Yeon; Park, Hyun; Lee, Sang-Hwan; Koo, Namin; Kim, Jeong-Gyu

2009-04-01

We investigated the arsenate tolerance mechanisms of Oenothera odorata by comparing two populations [i.e., one population from the mine site (MP) and the other population from an uncontaminated site (UP)] via the exposure of hydroponic solution containing arsenate (i.e., 0-50 microM). The MP plants were significantly more tolerant to arsenate than UP plants. The UP plants accumulated more As in their shoots and roots than did the MP plants. The UP plants translocated up to 21 microg g(-1) of As into shoots, whereas MP plants translocated less As (up to 4.5 microg g(-1)) to shoots over all treatments. The results of lipid peroxidation indicated that MP plants were less damaged by oxidative stress than were UP plants. Phytochelatin (PC) content correlated linearly with root As concentration in the MP (i.e., [PCs](root)=1.69x[As](root), r(2)=0.945) and UP (i.e., [PCs](root)=0.89x[As](root), r(2)=0.979) plants. This relationship means that increased PC to As ratio may be associated with increased tolerance. Our results suggest that PC induction in roots plays a critical role in As tolerance of O. odorata.

NAA-Induced Direct Organogenesis from Female Immature Inflorescence Explants of Date Palm.

PubMed

Khierallah, Hussam S M; Bader, Saleh M; Al-Khafaji, Makki A

2017-01-01

Micropropagation has great potential for the multiplication of female and male date palms of commercially grown cultivars by using inflorescences. This approach is simple, convenient, and much faster than the conventional method of using shoot-tip explants. We describe here a stepwise micropropagation procedure using inflorescence explants of Iraqi date palm cultivar Maktoom. Cultured explants were derived from 0.5-cm-long spike segments excised from 8 to 10-cm-long spathes. About 70% formed adventitious buds on Murashige and Skoog (MS) medium supplemented with 2 mg/L naphthalene acetic acid (NAA), 4 mg/L benzylaminopurine (BAP), and 40 g/L sucrose and maintained in the dark for 16 weeks before transferring to normal light conditions. The best multiplication rate was achieved with 3 mg/L 2ip and 2 mg/L; for shoot elongation, the best medium is MS containing 0.5 mg/L BAP, 0.5 mg/L 2ip, and 1 mg/L GA 3 . Well-developed shoots were cultured for rooting in half MS medium amended with 1 mg/L NAA and 45 g/L sucrose. Plantlets with well-developed roots were successfully hardened in the greenhouse. Inflorescence explants proved to be a promising alternative explant source for micropropagation of date palm cultivars.

Plant regeneration of Erigeron breviscapus (vant.) Hand. Mazz. and its chromatographic fingerprint analysis for quality control.

PubMed

Liu, Chun-Zhao; Gao, Min; Guo, Bin

2008-01-01

An efficient micropropagation system for Erigeron breviscapus (vant.) Hand. Mazz., an important medicinal plant for heart disease, has been developed. Shoot organogenesis occurred from E. breviscapus leaf explants inoculated on a medium supplemented with a combination of plant growth regulators. On average, 17 shoots per leaf explant were produced after 30 days when they were cultured on MS basal salts and vitamin medium containing 5 microM 6-benzylaminopurine (BAP) and 5 microM 1-naphthaleneacetic acid (NAA). All the regenerated shoots formed complete plantlets on a medium containing 2.5-10 microM indole-3-butyric acid (IBA) within 30 days, and 80.2% of the regenerated plantlets survived and grew vigorously in field conditions. Based on the variation in common peaks and the produced amount of the most important bioactive component, scutellarin, a high performance liquid chromatography (HPLC) fingerprinting system was developed for quality control of these micropropagated plants. Chemical constituents in E. breviscapus micropropagated plants varied during plant development from regeneration to maturation, the latter of which showed the most similar phytochemical profile in comparison with mother plants. The regeneration protocol and HPLC fingerprint analysis developed here provided a new approach to quality control of micropropagated plants producing secondary metabolites with significant implications for germplasm conservation.

In Vitro propagation of Jasminum officinale L.: a woody ornamental vine yielding aromatic oil from flowers.

PubMed

Bhattacharya, Sabita; Bhattacharyya, Sanghamitra

2010-01-01

The growing demand for flower extracts in perfume trade can primarily be met by increasing flower production and multiplying planting material. The major commercial aromatic flower yielding plants including Jasminum officinale L., a member of the Family Oleaceae have drawn the attention of a large section of the concerned sectors leading to a thrust upon developing advanced propagation technologies for these floral crops, in addition to conventional nature-dependent agro-techniques. This chapter describes concisely and critically, a protocol developed for in vitro propagation of Jasminum officinale by shoot regeneration from existing as well as newly developed adventitious axillary buds via proper phytohormonal stimulation. To start with nodal segments as explants, March-April is the most ideal time of the year when planting material suitable for in vitro multiplication is abundantly available. Prior to inoculation of explants in the culture medium, special care is needed to reduce microbial contamination by spraying on selected spots of the donor plant with anti-microbial agents 24 h prior to collection; treatment with antiseptic solution after final cleaning and surface sterilization by treating explants with mercuric chloride. Inoculated explants are free from brown leaching from cut ends by two consecutive subcultures within 48 h in MS basal medium. Multiplication of shoots, average 4-5 at each node, takes place in MS medium containing 4.0 mg/L BAP, 0.1 mg/L NAA, and 40 g/L sucrose over a period of 8 weeks. For elongation of regenerated shoots, cultures are transferred to MS medium, supplemented with a single growth hormone, kinetin at 2.0 mg/L. Emergence and elongation of roots from shoot base is facilitated by placing on the notch of a filter paper bridge. The hardened in vitro propagated plants are able to grow normally in soil like other conventionally propagated Jasminum officinale.

Callus induction and flavonoid production on the immature seed of Stelechocarpus burahol

NASA Astrophysics Data System (ADS)

Habibah, N. A.; Moeljopawiro, S.; Dewi, K.; Indrianto, A.

2018-03-01

Stelechocarpus burahol [(Bl.) Hook. f. & Th.] is one of the medicinal plants. In vitro callus induction studies on S. burahol were carried out to determine phytohormone requirement for optimum callus induction. Immature seed explants were cultured on MS medium by adding different kinds and different concentrations of plant growth regulators (picloram and 2,4-D) under light and dark conditions. The results showed that callus formation was initiated on the 18,50th to the 55th days. The best condition for optimum callus induction was found on MS medium, which was supplemented with 7.5 mg/L picloram and was maintained in the dark condition. The callus induction varied from 60% to 100%. The callus that produced the highest flavonoid was grown on the medium with the addition of 10 mg/L of 2,4-D. In conclusion, the results represented a suitable medium for S.burahol callus induction.

Vitrification-based cryopreservation of shoot-tips of Pinus kesiya Royle ex. Gord.

PubMed

Kalita, V; Choudhury, H; Kumaria, S; Tandon, P

2012-01-01

The present investigation was aimed at developing a protocol for long-term preservation of germplasm of Pinus kesiya Royle ex. Gord. through vitrification. Some of the critical components affecting explant tolerance to cryopreservation, such as effects of preculture, vitrification solutions, exposure time to vitrification solutions, volume of vitrification solution and its toxicity, washing of vitrified tissues after thawing, were analysed. The results showed that shoot regrowth of P. kesiya shoot-tips was considerably affected when exposed to cryoprotectants for longer periods of time (longer than 10 min). Among different vitrification solutions studied, maximum survival (76 percent) of shoot-tips was achieved with mVSL (using 0.6 ml of the solution) in MS basal medium containing 4.0 mg l-1 N6-benzyladenine (BA).

Protocols for In Vitro Mass Multiplication and Analysis of Medicinally Important Phenolics of a Salep Orchid, Satyrium nepalense D.Don ("Salam Mishri").

PubMed

Babbar, Shashi B; Singh, Deepak K

2016-01-01

Satyrium nepalense is a rare and threatened medicinal orchid, populations of which in its native habitats are dwindling because of indiscriminate collections and habitat destruction, thus necessitating the development of methods for its in situ and ex situ conservation. Because of non-endospermous nature of the seeds and the immature embryos at seed dispersal stage, orchids cannot be seed-propagated as other plants. Micropropagation, using plant tissue culture techniques, offers an effective method for the multiplication of orchids. In this chapter, a five-step efficient reproducible protocol for large-scale in vitro multiplication of Satyrium nepalense is described. The first step involves asymbiotic germination of seeds isolated from immature green pods and cultured on Mitra's medium (M) gelled with 0.8 % agar and supplemented with 2 % sucrose and 1 % peptone (hereafter referred to as basal medium, BM). On this medium, seeds start germinating after a week of culture. Protocorms developed from the seeds are sub-cultured on BM fortified with 4 μM kinetin (Kn) after 8 weeks, for shoot differentiation and multiplication. The shoots developed on Kn-supplemented medium are transferred to BM alone for their elongation for the same period. The elongated shoots are transferred to the rooting medium, comprising BM supplemented with 0.5 or 1.0 μM indole-3-butyric acid, for further 8 weeks. The regenerated plantlets are transferred to a potting mix of sand and vermiculite (1:1) for acclimatization. The tubers and leaves excised from both in vitro-developed plants and those from their native habitats are analyzed and compared for the contents and concentration of medicinally important phenolics using high-performance liquid chromatography (HPLC), details of which are provided in this chapter.

Growth medium alterations improve in vitro cold storage of pear germplasm.

PubMed

Kovalchuk, I; Zhumagulova, Z; Turdiev, T; Reed, B M

2014-01-01

Development of new fruit cultivars is dependent on genetic resource collections such as those at the Pomological Garden of the Institute of Horticulture and Viticulture near Almaty, Kazakhstan. The pear germplasm collection of the Pomological Garden contains 615 cultivars and three species. In vitro cold storage of the collection would provide additional security to the field collection. This study was designed to improve medium-term in vitro storage of pear germplasm. Shoots of seven pear cultivars (Pyrus communis L.) were stored in plastic five-section bags at 4 degree C and a 10-h photoperiod (7 μmol/m2/s). Treatments included medium with four carbohydrate sources (3% sucrose, 2% or 3% mannitol, or 2% sucrose + 2% mannitol) with 0.5 mg/l BAP and 0.1 mg/l IBA or without plant growth regulators (PGRs) and at three Murashige and Skoog (MS) nitrogen concentrations (100%, 50% or 25%). Pear shoots remained viable for 9 to 15 months without repropagation on the control MS medium with 3% sucrose without PGRs. There were significant impacts of cultivar and treatment on the duration of cold storage. Shoots of 'Mramornaya' remained viable (rating of ≥ 2) for 27 months with PGRs and 2% sucrose + 2% mannitol compared to 12 months for the PGR + 3% sucrose treatment. Talgarskaya Krasaviza stored for 18 months on 2% sucrose + 2% mannitol while all other treatments lasted only 6 to 9 months. Treatments with 0.5 or 1 mg/l abscisic acid (ABA) with 3% sucrose increased storage duration as did reducing the concentration of nitrogen in the medium to 25% without PGRs and with 3% sucrose.

Bud structure, position and fate generate various branching patterns along shoots of closely related Rosaceae species: a review

PubMed Central

Costes, Evelyne; Crespel, Laurent; Denoyes, Béatrice; Morel, Philippe; Demene, Marie-Noëlle; Lauri, Pierre-Eric; Wenden, Bénédicte

2014-01-01

Branching in temperate plants is closely linked to bud fates, either floral or vegetative. Here, we review how the fate of meristematic tissues contained in buds and their position along a shoot imprint specific branching patterns which differ among species. Through examples chosen in closely related species in different genera of the Rosaceae family, a panorama of patterns is apparent. Patterns depend on whether vegetative and floral buds are borne individually or together in mixed buds, develop as the shoot grows or after a rest period, and are located in axillary or terminal positions along the parent shoot. The resulting branching patterns are conserved among varieties in a given species but progressively change with the parent shoot length during plant ontogeny. They can also be modulated by agronomic and environmental conditions. The existence of various organizations in the topology and fate of meristematic tissues and their appendages in closely related species questions the between-species conservation of physiological and molecular mechanisms leading to bud outgrowth vs. quiescence and to floral induction vs. vegetative development. PMID:25520729

Micropropagation of Phalaenopsis and Doritaenopsis by culturing shoot tips of flower stalk buds.

PubMed

Tokuhara, K; Mii, M

1993-11-01

Green Protocorm-like Bodies (PLB) with high multiplication capacity were induced from shoot tips of flower stalk buds having 1 or 2 leaf primordia using New Dogashima Medium (NDM) containing 0.1 mg l(-1) α-naphthaleneacetic acid (NAA) and 1 mg 1(-1) 6-benzylaminopurine (BAP). These PLB were subcultured on the same medium. More than 10,000 PLBs were obtained from a few buds on a single flower stalk within one year. After transfer onto NDM containing no plant growth regulator (PGR), the PLB developed into plantlets. The micropropagation method formulated in this study was applicable to 12 different genotypes. These results suggest that the methodology could be used on a commercial scale for vegetative propagation of Phalaenopsis and Doritaenopsis.

Micropropagation of African violet (Saintpaulia ionantha Wendl.).

PubMed

Shukla, Mukund; Sullivan, J Alan; Jain, Shri Mohan; Murch, Susan J; Saxena, Praveen K

2013-01-01

Micropropagation is an important tool for rapid multiplication and the creation of genetic variability in African violets (Saintpaulia ionantha Wendl.). Successful in vitro propagation depends on the specific requirements and precise manipulation of various factors such as the type of explants used, physiological state of the mother plant, plant growth regulators in the culture medium, and growth conditions. Development of cost-effective protocols with a high rate of multiplication is a crucial requirement for commercial application of micropropagation. The current chapter describes an optimized protocol for micropropagation of African violets using leaf explants obtained from in vitro grown plants. In this process, plant regeneration occurs via both somatic embryogenesis and shoot organogenesis simultaneously in the explants induced with the growth regulator thidiazuron (TDZ; N-phenyl-N'-1,2,3-thidiazol-5-ylurea). The protocol is simple, rapid, and efficient for large-scale propagation of African violet and the dual routes of regeneration allow for multiple applications of the technology from simple clonal propagation to induction or selection of variants to the production of synthetic seeds.

Transformation of pecan and regeneration of transgenic plants.

PubMed

McGranahan, G H; Leslie, C A; Dandekar, A M; Uratsu, S L; Yates, I E

1993-09-01

A gene transfer system developed for walnut (Juglans regia L.) was successfully applied to pecan (Carya illinoensis [Wang] K. Koch). Repetitively embryogenic somatic embryos derived from open-pollinated seed of 'Elliott', 'Wichita', and 'Schley' were co-cultivated with Agrobacterium strain EHA 101/pCGN 7001, which contains marker genes for beta-glucuronidase activity and resistance to kanamycin. Several modifications of the standard walnut transformation techniques were tested, including a lower concentration of kanamycin and a modified induction medium, but these treatments had no measurable effect on efficiency of transformation. Nineteen of the 764 viable inoculated embryos produced transgenic subclones; 13 of these were from the line 'Elliott'6, 3 from 'Schley'5/3, and 3 from 'Wichita'9. Transgenic embryos of 'Wichita'9 germinated most readily and three subclones were successfully micropropagated. Three transgenic plants of one of these subclones were obtained by grafting the tissue cultured shoots to seedling pecan rootstock in the greenhouse. Gene insertion, initially detected by GUS activity, was confirmed by detection of integrated T-DNA sequences using Southern analysis.

Adaptation to rhizosphere acidification is a necessary prerequisite for wheat (Triticum aestivum L.) seedling resistance to ammonium stress.

PubMed

Wang, Feng; Gao, Jingwen; Tian, Zhongwei; Liu, Yang; Abid, Muhammad; Jiang, Dong; Cao, Weixing; Dai, Tingbo

2016-11-01

Because soil acidification accompanies ammonium (NH 4 + ) stress, the tolerance of higher plants to ammonium is associated with their adaptation to root medium acidification. However, the underlying mechanisms of this adaptation have not been fully elucidated. The objective of this study was thus to elucidate the effect of rhizosphere pH on NH 4 + tolerance in different winter wheat cultivars (Triticum aestivum L.). Hydroponic experiments were carried out on two wheat cultivars: AK58 (an NH 4 + -sensitive cultivar) and XM25 (an NH 4 + -tolerant cultivar). Four pH levels resembling acidified (4.0, 5.0, 6.0 and 7.0) were tested and 5 mM NH 4 + nitrogen (AN) was used as a stress treatment, with 5 mM nitrate nitrogen used as a control. The addition of AN led to a severe reduction in biomass and an increase in free NH 4 + , amino acids, and the activities of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in the shoots and roots of the two wheat cultivars. Further decreases in growth medium pH led to further increases in free NH 4 + , but decreases in total amino acids and the activities of GS and NADH-dependent glutamate synthase (NADH-GDH). However, there was less of an increase in free NH 4 + and less of a reduction in the activities of GS and NADH-GDH in the cultivar XM25 compared with AK58. In addition, total soluble sugar content and the root-to-shoot soluble sugar ratio were also decreased by AN treatment, except in the shoots of XM25. Decreasing pH resulted in lower root-to-shoot soluble sugar ratios with greater reductions in the AK58 cultivar. These results indicate that wheat growth was inhibited significantly by the addition of NH 4 + combined with low pH. Low medium pH reduced the capacity for nitrogen assimilation and interrupted carbohydrate transport between the shoot and root. The NH 4 + -tolerant cultivar XM25 was better adapted to low rhizosphere pH due to its increased capacity for assimilating NH 4 + efficiently and thereby avoiding toxic levels of intracellular NH 4 + at low medium pH. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm.

PubMed

Danso, K E; Ford-Lloyd, B V

2003-04-01

We report the encapsulation of in vitro-derived nodal cuttings or shoot tips of cassava in 3% calcium alginate for storage and germplasm exchange purposes. Shoot regrowth was not significantly affected by the concentration of sucrose in the alginate matrix while root formation was. In contrast, increasing the sucrose concentration in the calcium chloride polymerisation medium significantly reduced regrowth from encapsulated nodal cuttings of accession TME 60444. Supplementing the alginate matrix with increased concentrations of 6-benzylaminopurine and alpha-naphthaleneacetic acid enhanced complete plant regrowth within 2 weeks. Furthermore, plant regrowth by encapsulated nodal cuttings and shoot tips was significantly affected by the duration of the storage period as shoot recovery decreased from almost 100% to 73.3% for encapsulated nodal cuttings and 94.4% to 60% for shoot tips after 28 days of storage. The high frequency of plant regrowth from alginate-coated micropropagules coupled with high viability percentage after 28 days of storage is highly encouraging for the exchange of cassava genetic resources. Such encapsulated micropropagules could be used as an alternative to synthetic seeds derived from somatic embryos.

Generation of selectable marker-free transgenic eggplant resistant to Alternaria solani using the R/RS site-specific recombination system.

PubMed

Darwish, Nader Ahmed; Khan, Raham Sher; Ntui, Valentine Otang; Nakamura, Ikuo; Mii, Masahiro

2014-03-01

Marker-free transgenic eggplants, exhibiting enhanced resistance to Alternaria solani , can be generated on plant growth regulators (PGRs)- and antibiotic-free MS medium employing the multi-auto-transformation (MAT) vector, pMAT21 - wasabi defensin , wherein isopentenyl transferase ( ipt ) gene is used as a positive selection marker. Use of the selection marker genes conferring antibiotic or herbicide resistance in transgenic plants has been considered a serious problem for environment and the public. Multi-auto-transformation (MAT) vector system has been one of the tools to excise the selection marker gene and produce marker-free transgenic plants. Ipt gene was used as a selection marker gene. Wasabi defensin gene, isolated from Wasabia japonica (a Japanese horseradish which has been a potential source of antimicrobial proteins), was used as a gene of interest. Wasabi defensin gene was cloned from the binary vector, pEKH-WD, to an ipt-type MAT vector, pMAT21, by gateway cloning technology and transferred to Agrobacterium tumefaciens strain EHA105. Infected cotyledon explants of eggplant were cultured on PGRs- and antibiotic-free MS medium. Extreme shooty phenotype/ipt shoots were produced by the explants infected with the pMAT21-wasabi defensin (WD). The same PGRs- and antibiotic-free MS medium was used in subcultures of the ipt shoots. Subsequently, morphologically normal shoots emerged from the Ipt shoots. Molecular analyses of genomic DNA from transgenic plants confirmed the integration of the WD gene and excision of the selection marker (ipt gene). Expression of the WD gene was confirmed by RT-PCR and Northern blot analyses. In vitro whole plant and detached leaf assay of the marker-free transgenic plants exhibited enhanced resistance against Alternaria solani.

[Effects of combined pollution of lead and benzo[a] pyrene on seed growth of wheat in soils].

PubMed

Wang, Hong-Qi; Wang, Shuai; Ning, Shao-Wei; Sun, Yan-Ling; Hou, Ze-Qing

2011-03-01

Seed germination, root elongation, shoot elongation and ratio of shoot to root of wheat in soils polluted by lead (Pb) and benzo (a)pyrene (B[a] P) with medium-low concentrations were studied to reveal the ecological effects of combined pollution and screen the indicative markers. Results indicated that seed germination was not sensitive to single or combined pollution of Pb or B[a] P. Root elongation was inhibited by single pollution of Pb or B[a]P to different extents. Extensive interactions between Pb and B[a]P occurred to root elongation of wheat, including synergistic-stimulatory effect and antagonistic-inhibitory effect. The joint action was mainly antagonistic. Single pollution of B [a] P had an inhibitory effect on shoot elongation. Under combined pollution conditions, the shoot elongation of wheat correlated well with Pb contents (p < 0.01). B[a] P or the interactions between pollutants had little effect on shoot elongation of wheat. The joint action on shoot elongation was consistently antagonistic. The response pattern of the ratio of shoot to root was similar to the response pattern of shoot elongation. However, the former had better correlation than the latter, indicating it as a more suitable indicative marker for Pb pollution. If lead acetate was employed instead of lead nitrate, longer root elongation, shorter shoot elongation and no effect on ratio of shoot to root were found. Therefore, the forms of Pb salt had significant influence on seed growth of wheat in soils.

Efficient protocol for rapid Aloe vera micropropagation.

PubMed

Molsaghi, Mozhgan; Moieni, Ahmad; Kahrizi, Danial

2014-06-01

Aloe vera Linn. (Liliaceae) is a medicinal plant and has a number of curative properties. Vegetative propagation has not enough potential for supplying market demand. However, via in vitro propagation makes possible the mass production of Aloe plants. The current study was conducted to investigate growth regulators' effects on proliferation of A. vera. In this study, for comparison of plant growth regulators' effects on proliferation, the shoot tips and auxiliary buds of A. vera were cultured in the Murashige and Skoog (MS) medium. Rooted plantlets were transferred to garden soil, compost, and sand in the proportion of 1:1:1, respectively, after hardening. The maximum number of shoots was obtained on the medium supplemented with 1 mg/L IAA+4 mg/L BAP and 0.2 mg/L IAA+0.8 BAP mg/L. Rooting was also achieved in the same media composition proliferation of shoot. The acclimatized plants showed 100% of survival. The regenerated plants looked healthy, and they were morphologically similar to that of stock plants. These results suggest that in vitro culture may be used as a technique for rapid propagation of A. vera.

A Genetic Transformation Method for Cadmium Hyperaccumulator Sedum plumbizincicola and Non-hyperaccumulating Ecotype of Sedum alfredii

PubMed Central

Liu, Huan; Zhao, Haixia; Wu, Longhua; Xu, Wenzhong

2017-01-01

The present study demonstrates the development of an Agrobacterium-mediated genetic transformation method for species of the Sedum genus, which includes the Cd/Zn hyperaccumulator Sedum plumbizincicola and the non-hyperaccumulating ecotype of S. alfredii. Multiple shoots were induced from stem nodes of two Sedum plants using Murashige and Skoog (MS) medium containing 0.1 mg/L cytokinin 6-benzyladenine (6-BA) and 1.0 mg/L auxin 1-naphthaleneacetic acid (NAA). The shoot primordia were used as direct targets for Agrobacterium infection. Selection on hygromycin was highly effective in generating Agrobacterium-transformed explants. This callus-free procedure allowed us to obtain transgenic plantlets after rooting hygromycin-resistant shoots on phytohormone-free MS medium containing the antibiotic. The presence and expression of the reporter genes gusA and GFP in transgenic plants were confirmed by a real-time polymerase chain reaction, histochemical GUS assays, and confocal microscopy. This reliable method for genetic transformation of Sedum plants will help us to understand gene functions and the molecular mechanisms underlying Cd hypertolerance and hyperaccumulation in these species. PMID:28670322

Periodic acid‑Schiff staining method for function detection of liver cells is affected by 2% horse serum in induction medium.

PubMed

Hui, Hui; Ma, Wenjun; Cui, Jiejie; Gong, Mengjia; Wang, Yi; Zhang, Yuanyuan; He, Tongchuan; Bi, Yang; He, Yun

2017-12-01

Developing a thorough understanding of experimental methods of hepatic differentiation in hepatic progenitor cells (HPCs) should expand the knowledge of hepatocyte induction in vitro and may help to develop cell transplantation therapies for the clinical usage of HPCs in liver diseases. A previous induction method effectively induced differentiation and metabolic abilities in HPCs. Periodic acid‑Schiff (PAS) staining is used to identify glycogen synthesis and hepatocyte function; however, this method failed to detect induced hepatocytes. The present study aimed to investigate the possible factors affecting the previous confusing results of PAS staining. Removal of single induction factors, including dexamethasone, hepatic growth factor and fibroblast growth factor 4 from the induction media did not restore PAS staining, whereas replacement of 2% horse serum (HS) with 10% fetal bovine serum (FBS) significantly increased the number of PAS positive cells. Following 12 days of basal induction, replacing the induction medium with media containing 10% FBS for 12‑72 h significantly improved PAS staining, but did not influence indocyanine green uptake. Furthermore, incubation in induction medium with 10% FBS following 12 days of normal induction did not affect the expression of hepatic markers and mature function of HPCs. Therefore, the present study suggested that 2% HS in the induction medium did not affect the hepatic function of induced cells, but did affect glycogen storage, whereas replacement of medium with 10% FBS in advance of PAS staining may restore the failure of PAS staining in low serum concentrations of induced hepatocytes.

MicroRNAs, polyamines, and the activities antioxidant enzymes are associated with in vitro rooting in white pine (Pinus strobus L.).

PubMed

Fei, Yunjun; Xiao, Bo; Yang, Man; Ding, Qiong; Tang, Wei

2016-01-01

Molecular mechanism of in vitro rooting in conifer is not fully understood. After establishment of a regeneration procedure in eastern white pine (Pinus strobus L.) using mature embryos as explants to induce shoot formation on medium containing 3 μM IAA, 6 μM BA and 6 μM TDZ and induce root formation on medium containing 0.001-0.05 μM IAA, 0.001-0.05 μM IBA, 0.001-0.05 μM TDZ, we have investigated the changes of polyamine content and the activities of antioxidant enzymes during in vitro rooting in P. strobus. Our results demonstrated that putrescine (Put), spermidine (Spd), and spermine (Spm) did not increase in P. strobus during the first week of rooting on medium supplemented with 0.01 μM indole-3-acetic acid (IAA), whereas the levels of Put, Spd, and Spm increased during the 1st-3rd week of culture on medium with IAA, and then decreased on medium with IAA. No such a change in Put, Spd, and Spm was observed on medium without IAA. Measurement of antioxidant enzyme activity demonstrated that the activities of polyphenol oxidase, catalase, and peroxidase slightly increased in the first week of culture and reached to the highest peak in the 3rd-5th week of culture. Quantitative RT-PCR results indicated that miR160 was increased on the 7th day, miR162, miR397, and miR408 was increased from the 21th to 35th day, miR857 was increased on the 35th day, and miR827 was increased on the 49th day. These results demonstrated that enhanced polyamine biosynthesis, antioxidant enzyme activity, and microRNAs are correlated with the root induction and formation in P. strobus.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss.

PubMed

Srivastava, Priyanka; Chaturvedi, Rakhi

2011-07-01

Present report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 μM), 2,4-D (1 μM) and NAA (1 μM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 μM) + NAA (0.05 μM) medium, were elongated on MS + BAP (0.5 μM) and multiplied on MS + BAP (1 μM) + CH (250 mg/l). Thereafter, the shoots were rooted on ¼ MS + IBA (0.5 μM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n = x = 12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 μg/g DW) was detected in calli supporting best shoot proliferation while least (49 μg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss

PubMed Central

Srivastava, Priyanka

2011-01-01

Present report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on ¼ MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n = x = 12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier. PMID:21701249

In Vitro Plant Regeneration from Commercial Cultivars of Soybean

PubMed Central

Raza, Ghulam; Singh, Mohan B.

2017-01-01

Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60–87%, 50–100%, and 75–100%, and regeneration rate ranged within 4.2–10, 2.7–4.2, and 2.6–10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested. PMID:28691031

In Vitro Plant Regeneration from Commercial Cultivars of Soybean.

PubMed

Raza, Ghulam; Singh, Mohan B; Bhalla, Prem L

2017-01-01

Soybean, a major legume crop, is the source of vegetable oil and protein. There is a need for transgenic approaches to breeding superior soybean varieties to meet future climate challenges. Efficient plant regeneration is a prerequisite for successful application of genetic transformation technology. Soybean cultivars are classified into different maturity groups based on photoperiod requirements. In this study, nine soybean varieties belonging to different maturity group were regenerated successfully from three different explants: half split hypocotyl, complete hypocotyl, and cotyledonary node. All the genotypes and explant types responded by producing adventitious shoots. Shoot induction potential ranged within 60-87%, 50-100%, and 75-100%, and regeneration rate ranged within 4.2-10, 2.7-4.2, and 2.6-10.5 shoots per explant using half split hypocotyl, complete hypocotyl, and cotyledonary explants, respectively, among all the tested genotypes. Bunya variety showed the best regeneration response using half split and complete hypocotyl explants and the PNR791 with cotyledonary node. The regenerated shoots were successfully rooted and acclimatized to glasshouse conditions. This study shows that commercial varieties of soybean are amenable to shoot regeneration with high regeneration frequencies and could be exploited for genetic transformation. Further, our results show no correlation between shoots regeneration capacity with the maturity grouping of the soybean cultivars tested.

Micropropagation of Salvia wagneriana Polak and hairy root cultures with rosmarinic acid production.

PubMed

Ruffoni, Barbara; Bertoli, Alessandra; Pistelli, Laura; Pistelli, Luisa

2016-01-04

Salvia wagneriana Polak is a tropical species native to Central America, well adapted to grow in the Mediterranean basin for garden decoration. Micropropagation has been assessed from axillary shoots of adult plants using a Murashige and Skoog basal medium, with the addition of 1.33-μM 6-benzylaminopurine for shoot proliferation; the subsequent rooting phase occurred in plant growth regulator-free medium. The plants were successfully acclimatised with high survival frequency. Hairy roots were induced after co-cultivation of leaf lamina and petiole fragments with Agrobacterium rhizogenes and confirmed by PCR. The establishment and proliferation of the selected HRD3 line were obtained in hormone-free liquid medium and the production of rosmarinic acid (RA) was evaluated after elicitation. The analysis of RA was performed by LC-ESI-DAD-MS in the hydroalcoholic extracts. The addition of casein hydrolysate increased the RA production, whereas no enrichment was observed after the elicitation with jasmonic acid.

Kinetic Induction of Oat Shoot Pulvinus Invertase mRNA by Gravistimulation and Partial cDNA Cloning by the Polymerase Chain Reaction

NASA Technical Reports Server (NTRS)

Wu, Liu-Lai; Song, Il; Karuppiah, Nadarajah; Kaufman, Peter B.

1993-01-01

An asymmetric (top vs. bottom halves of pulvini) induction of invertase mRNA by gravistimulation was analyzed in oat shoot pulvini. Total RNA and poly(A)(+) RNA, isolated from oat pulvini, and two oli-gonucleotide primers, corresponding to two conserved amino acid sequences (NDPNG and WECPD) found in invertase from other species, were used for the polymerase chain reaction (PCR). A partial length cDNA (550 bp) was obtained and characterized. A 62% nucleotide sequence homology and 58% deduced amino acid sequence homology, as compared to beta-fructosidase of carrot cell wall, was found. Northern blot analysis showed that there was an obviously transient induction of invertase mRNA by gravistimulation in the oat pulvinus system. The mRNA was rapidly induced to a maximum level at 1 hour after gravistimulation treatment and gradually decreased afterwards. The mRNA level in the bottom half of the oat pulvinus was significantly higher than that in the top half of the pulvinus tissue. The kinetic induction of invertase mRNA was consistent with the transient accumulation of invertase activity during the graviresponse of the pulvinus. This indicates that the expression of the invertase gene(s) could be regulated by gravistimulation at the transcriptional level. Southern blot analysis showed that there were two to three genomic DNA fragments which hybridized with the partial-length invertase cDNA.

Micropropagation of Ajuga bracteosa, a medicinal herb.

PubMed

Kaul, Shivanee; Das, Sandip; Srivastava, P S

2013-04-01

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25 ± 2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.

Effect of Antioxidants, Amino Acids and Plant Growth Regulators on in vitro Propagation of Rosa centifolia.

PubMed

Akhtar, Gulzar; Jaskani, Muhammad Jafar; Sajjad, Yasar; Akram, Ahsan

2016-03-01

Rosa centifoliais commercially propagated by asexual means but in vitro propagation ensure the production of disease free and healthy plants and browning of explants creates hurdle in their multiplication. The aim was to reduce oxidative browning of shoots of R. centifolia in MS medium during in vitro propagation. Axillary buds of R. centifolia were sterilized with 70% ethyl alcohol for 4 min and 5% sodium hypochlorite for 2 min followed by three washing with sterilized double distilled water. In order to control oxidative browning, Ascorbic acid (100 mg.L -1 ), citric acid (100 mg.L -1 ) and activated charcoal (3 g.L -1 ) were used while to control withering of shoots, different concentrations (3.0 mg.L -1 , 6.0 mg.L -1 , 9.0 mg.L -1 ) of either glutamine, asparagine and proline were put into trial. Different concentrations of Benzyl aminopurine (BAP) and naphthalene acetic acid (NAA) were used for in vitro shoot and root formation. Minimum browning percentage (20%) was achieved in the presence of activated charcoal (3.0 g.L -1 ) and pretreatment of explants with running tap water. Asparagin (9.0 mg.L -1 ) produced maximum shooting (93%), minimum withering (6.67%), and it took longer period (27 days) for shoots to wither. BAP (3.0 mg.L -1 ) + NAA (0.5 mg.L -1 ) was produced the highest number of shoots (1.63), in a shortest periods (9 days). For root production, NAA (1.5 mg.L -1 ) + BAP (0.5 mg.L -1 ) reduced the time to 11 days with maximum number of roots (4.33) and root length (4.20 cm). The supplement of activated charcoal (3.0 g.L -1 ), a sparagin (9.0 mg.L -1 ) and combination of BAP and NAA in the MS medium is effective for in vitro propagation of R. centifolia.

Cryopreservation of in vitro grown shoot tips of Diospyros kaki thunb. using different methods.

PubMed

Niu, Y L; Luo, Z R; Zhang, Y F; Zhang, Q L

2012-01-01

The objective of this study was to compare the potential of different cryopreservation strategies for in vitro shoot tips of Diospyros kaki Thunb. The treatments consisted of three different cryopreservation methods: vitrification, droplet-vitrification and modified droplet-vitrification. The following variables were assessed: cold acclimation, sucrose concentration in the preculture medium and PVS2 treatment time. A higher average survival level was obtained using the modified droplet-vitrification method compared to the other two methods.

Non-Darcy Forchheimer flow of ferromagnetic second grade fluid

NASA Astrophysics Data System (ADS)

Hayat, T.; Ahmad, Salman; Khan, M. Ijaz; Alsaedi, A.

This article discusses impacts of thermal radiation, viscous dissipation and magnetic dipole in flow of second grade fluid saturating porous medium. Porous medium is characterized by nonlinear Darcy-Forchheimer relation. Relevant nonlinear ordinary differential systems after using appropriate transformations are solved numerically. Shooting technique is implemented for the numerical treatment. Temperature, velocity, skin fraction and Nusselt number are analyzed.

Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina)

Treesearch

Xiaomei Liu; Paula M. Pijut

2008-01-01

A regeneration system was developed for Prunus serotina from a juvenile (F) and two mature genotypes (#3 and #4). Adventitious shoots regenerated from leaves of in vitro cultures on woody plant medium with thidiazuron (TDZ) and naphthaleneacetic acid (NAA). The best regeneration for genotype F (91.4%) was observed on medium with 9.08 µM TDZ...

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert.

PubMed

Lodha, Deepika; Rathore, Nisha; Kataria, Vinod; Shekhawat, N S

2014-07-01

Ephedra foliata Boiss. & Kotschy ex Boiss., (family - Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l(-1) of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR's) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l(-1) ammonium sulphate {(NH4) 2SO4} (MMS3) + BA (0.25 mg l(-1)), Kinetin (Kin; 0.25 mg l(-1)), Indole-3-acetic acid (IAA; 0.1 mg l(-1)) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l(-1) of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.

In vitro propagation and cell cultures of memory tonic herb Evolvulus alsinoides: a best source for elicited production of scopoletin.

PubMed

Naikawadi, Vikas Bandu; Ahire, Mahendra Laxman; Lahiri, Anindita; Nikam, Tukaram Dayaram

2016-04-01

Evolvulus alsinoides L. is used for preparation of 'Shankhapushpi', an important popular ayurvedic drug that contributes considerably to the improvement of memory power. The improvement is attributed to the presence of furanocoumarin scopoletin, a metabolite with a wide range of biological activities. This report describes, for the first time, an in vitro culture system for propagation and enhanced production of scopoletin. Different concentrations of auxins and cytokinins individually and in combination were used in Murashige and Skoog (MS) medium to induce shoot regeneration in cotyledonary nodal explants and callus formation in leaf explants. The best response was achieved in MS medium fortified with 5.0 μM 6-benzyladenine (BA) in which 96 % of cultures produced 7.6 ± 0.6 shoots per explant. Regenerated shoots were rooted on MS medium with 5.0 μM indole-3-acetic acid (IAA). Plantlets were successfully acclimatized and established in soil. MS medium fortified with 10 μM BA + 5.0 μM IAA showed maximum growth and accumulation of scopoletin in cell cultures. Cell cultures could be maintained over 24 months. The influences of auxins, cytokinins, organic acids, amino acids, and fungal-derived elicitors on production of scopoletin were studied. Presence of either L-arginine, sodium pyruvate, or yeast extract highly promoted scopoletin production as compared with control and achieved 75.02-, 72.13-, and 57.98-fold higher accumulation, respectively. The results presented herein have laid solid foundation for large-scale production of scopoletin and further investigation of its purification and utilization as a novel pharmaceutical drug.

Organogenesis from internode-derived nodules of Humulus lupulus var. Nugget (Cannabinaceae): histological studies and changes in the starch content.

PubMed

Fortes, A M; Pais, M S

2000-07-01

The sequence of histological and histochemical events occurring during organogenesis from Humulus lupulus var. Nugget internode-derived nodules was studied. Sections were made and studies were carried out from the start of culture treatment until the development of shoot buds. Cell division was observed in both cambial and cortical regions during the first week of culture establishment. Cell division in cortical cells led to the formation of an incipient callus tissue. From the calluses prenodular structures of cambial origin appeared and gave rise to nodules from which shoot buds formed. Nodules kept separating into "daughter nodules" from which arose an increasing number of shoot buds. Iodide staining showed a strong starch accumulation in callus tissue and in prenodular structures. During shoot-bud primordia formation starch content decreased in nodules. Some starch was also noted in control explants (cultured on basal medium), however at a lower level than that observed in explants cultured on media with growth regulators. Shoot-bud regeneration was not observed in control explants.

A new approach for in vitro regeneration of tomato plants devoid of exogenous plant growth hormones.

PubMed

Plana, Dagmara; Fuentes, Alejandro; Alvarez, Marta; Lara, Regla M; Alvarez, Félix; Pujol, Merardo

2006-10-01

Many available methodologies for in vitro regeneration of commercial tomato varieties promote not only the production of normal shoots but also individual leaves, shoots without apical meristems and vitrified structures. All these abnormal formations influence and diminish the regeneration efficiency. At the basis of this phenomenon lies callus development. We optimized an alternative procedure by which the regeneration occurs without abnormal shoot formation. The portion including the proximal part of hypocotyls and the radicle was cultured on medium consisting of Murashige and Skoog salts, 4 mg/L thiamine, 100 mg/L mio-inositol and 3% sucrose. After two-three weeks, 60% explants showed adventitious shoot formation. No changes in the morphological characteristics of regenerated plants and fruits were observed as compared with parents. Karyotypic analysis of regenerated plants showed no variations in chromosome number. The optimized procedure offers the advantage of tomato plant regeneration avoiding callus formation, which enables normal plant recovery with an efficiency ranging from 1.45 +/- 0.05 to 2.57 +/- 0.06 shoots per explant in Campbell-28, Amalia, Lignon, and Floradel cultivars.

Factors influencing axillary shoot proliferation and adventitious budding in cedar.

PubMed

Renau-Morata, Begoña; Ollero, Javier; Arrillaga, Isabel; Segura, Juan

2005-04-01

We developed procedures for in vitro cloning of Cedrus atlantica Manetti and C. libani A. Rich explants from juvenile and mature plants. Explant size was one determinant of the frequency of axillary bud break in both species. Shoot tips and nodal explants mainly developed calli, whereas bud sprouting occurred in defoliated microcuttings cultured on a modified Murashige and Skoog medium without growth regulators. Isolation and continuous subculture of sprouted buds on the same medium allowed cloning of microcuttings from C. atlantica and C. libani seedlings and bicentennial C. libani trees, thus providing a desirable alternative for multiplying mature trees that have demonstrated superior characteristics. We also report adventitious bud differentiation from isolated embryos of C. atlantica. Neither auxin treatments nor other methods tested, including infection with Agrobacterium rhizogenes, were effective in inducing root initiation.

Effect of nitrogen sources on some morphological characteristics of in vitro stevia rebaudiana Bertoni.

PubMed

Akbari, F; Arminian, A; Kahrizi, D; Fazeli, A

2017-02-28

Stevia rebaudiana Bertoni belongs to Asteraceae family that leaves 200-300 times sweeter than sugar. Low seed fertility is one of the most important problems in Stevia production. So, Plant tissue culture is an efficient method for mass propagation of Stevia. In this research, we studied the effect of various concentrations of nitrogen on some morphological traits of stevia under in vitro conditions. We used axillary nodes as explants and they were cultured on Murashige and Skoog (MS) medium containing inorganic nitrogen sources i.e. NH4NO3(0, 825 and 1650 mg/l), KNO3(0, 950 and 1900 mg/l) were observed. The cultures were kept for 4 weeks at a temperature of 25±2°C with a photoperiod of 16/8 hour low light/dark each day. Maximum shoot length (89.33 mm), dry weight of plants (0.10 mg) and leaf fresh weight (0.42 mg) was observed on MS medium with 1650 mg/l NH4NO3 and 950 mg/l KNO3. Minimum shoot length (6.13 mm), root length (6.60 mm), leaf number (4.26), leaf dry weight (0.01 mg), leaf fresh weight (0.05 mg), total dry and fresh weight (0.02 and 0.15 mg) and growth rate was observed on a MS medium without nitrogen sources. Moreover, presence of nitrogen sources increases both shooting and rooting in Stevia rebaudiana Bertoni.

Hydrolyzable tannins of tamaricaceous plants. IV: Micropropagation and ellagitannin production in shoot cultures of Tamarix tetrandra.

PubMed

Orabi, Mohamed A A; Taniguchi, Shoko; Terabayashi, Susumu; Hatano, Tsutomu

2011-11-01

Shoot cultures of Tamarix tetrandra on Linsmaier-Skoog (LS) agar medium with 30 g l(-1) sucrose, 2.13 mg l(-1) indoleacetic acid and 2.25 mg l(-1) benzyl adenine produced ellagitannins found in intact plants of the Tamaricaceae. This was demonstrated by the isolation of 14 monomeric-tetrameric ellagitannins from the aq. Me2CO extract of the cultured tissues. This is the first report on the production of ellagitannin tetramers by plant tissue culture. The effects of light and certain medium constituents on tissue growth and ellagitannin production were examined. The contents of representative tannins of different types [i.e., tellimagrandin II (monomer), hirtellin A (linear GOG-type dimer), hirtellin B (hellinoyl-type dimer), hirtellin C (macrocyclic-type dimer), and hirtellin T1 (linear GOG-type trimer)] in the resultant tissues in response to these factors were estimated by HPLC, and the optimal condition for production of these tannins were established. Shoots cultured on LS hormone-free medium promoted root development, and regenerated plants could adapt to ordinary soil and climate. Acclimatized and intact T. tetrandra plants that were collected in November and May, respectively, demonstrated seasonal differences in individual ellagitannin contents. HPLC comparison of individual ellagitannin contents in different plant materials (i.e., leaves, stems, and roots) of intact T. tetrandra plants is also reported. The results are discussed with respect to cellular deposition and biosynthetic relationship of tannins. Copyright © 2011 Elsevier Ltd. All rights reserved.

Factors affecting in vitro plant regeneration of the critically endangered Mediterranean knapweed ( Centaurea tchihatcheffii Fisch et. Mey)

NASA Astrophysics Data System (ADS)

Ozel, Cigdem Alev; Khawar, Khalid Mahmood; Mirici, Semra; Ozcan, Sebahattin; Arslan, Orhan

2006-10-01

Habitat destruction has resulted in the extinction of many plant species from the earth, and many more face extinction. Likely, the annual endemic Mediterranean knapweed ( Centaurea tchihatcheffii) growing in the Golbasi district of Ankara, Turkey is facing extinction and needs urgent conservation. Plant tissue culture, a potentially useful technique for ex situ multiplication, was used for the restoration of this ill-fated plant through seed germination, micropropagation from stem nodes, and adventitious shoot regeneration from immature zygotic embryos. The seeds were highly dormant and very difficult to germinate. No results were obtained from the micropropagation of stem nodes. However, immature zygotic embryos showed the highest adventitious shoot regeneration on Murashige and Skoog (MS) medium, containing 1 mg l-1 kinetin and 0.25 mg l-1 NAA. Regenerated shoots were best rooted on MS medium containing 1 mg l-1 IBA and transferred to the greenhouse for flowering and seed set. As such, the present work is the first record of in vitro propagation of critically endangered C. tchihatcheffii, using immature zygotic embryos, and is a step forward towards conservation of this indigenous species.

Somatic embryogenesis and massive shoot regeneration from immature embryo explants of tef.

PubMed

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2-0.35 mm embryo explants on a medium containing KBP minerals, 9.2-13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar "DZ-01-196" and the landrace "Fesho", the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each "DZ-01-196" explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef.

Chemical Growth Regulators for Guayule Plants

NASA Technical Reports Server (NTRS)

Dastoor, M. N.; Schubert, W. W.; Petersen, G. R.

1982-01-01

Test Tubes containing Guayule - tissue cultures were used in experiments to test effects of chemical-growth regulators. The shoots grew in response to addition of 2-(3,4-dichlorophenoxy)-triethylamine (triethylamine (TEA) derivative) to agar medium. Preliminary results indicate that a class of compounds that promotes growth in soil may also promote growth in a culture medium. Further experiments are needed to define the effect of the TEA derivative.

Toxicity of arsenic (III) and (V) on plant growth, element uptake, and total amylolytic activity of mesquite (Prosopis juliflora x P. velutina).

PubMed

Mokgalaka-Matlala, Ntebogeng S; Flores-Tavizón, Edith; Castillo-Michel, Hiram; Peralta-Videa, Jose R; Gardea-Torresdey, Jorge L

2008-01-01

The effects of arsenite [As(III)] and arsenate [As(V)] on the growth of roots, stems, and leaves and the uptake of arsenic (As), micro- and macronutrients, and total amylolytic activity were investigated to elucidate the phytotoxicity of As to the mesquite plant (Prosopis juliflora x P. velutina). The plant growth was evaluated by measuring the root and shoot length, and the element uptake was determined using inductively coupled plasma optical emission spectroscopy. The root and leaf elongation decreased significantly with increasing As(III) and As(V) concentrations; whereas, stem elongation remained unchanged. The As uptake increased with increasing As(III) or As(V) concentrations in the medium. Plants treated with 50 mg/L As(III) accumulated up to 920 mg/kg dry weight (d wt) in roots and 522 mg/kg d wt in leaves, while plants exposed to 50 mg/L As(V) accumulated 1980 and 210 mg/kg d wt in roots and leaves, respectively. Increasing the As(V) concentration up to 20 mg/L resulted in a decrease in the total amylolytic activity. On the contrary, total amylolytic activity in As(III)-treated plants increased with increasing As concentration up to 20 mg/L. The macro- and micronutrient concentrations changed in As-treated plants. In shoots, Mo and K were reduced but Ca was increased, while in roots Fe and Ca were increased but K was reduced. These changes reduced the size of the plants, mainly in the As(III)-treated plants; however, there were no visible sign of As toxicity.

Micropropagation of tulip: production of virus-free stock plants.

PubMed

Podwyszyńska, Małgorzata; Sochacki, Dariusz

2010-01-01

We describe here a new tulip micropropagation method based on the cyclic shoot multiplication in presence of the thidiazuron (TDZ), which enables the production of virus-free stock plants, speeds up breeding, and provides new genotypes for the market. In our novel protocol, cyclic shoot multiplication can be performed for 2-3 years by using TDZ instead of other cytokinins, as 6-benzylaminopurine (BAP) and N(6)-(-isopentyl)adenine (2iP). It makes possible to produce 500-2,000 microbulbs from one healthy plant. There are six main stages of tulip micropropagation. Stage 0 is the selection of true-to-type and virus-free plants, confirmed by ELISA. Fragments of flower stems isolated from bulbs are used as initial explants. Shoot multiplication is based on the regeneration of adventitious shoots, which are sub-cultured every 8 weeks. In the Stage 3, the specially prepared shoots are induced by low temperature treatment to form bulbs which finally develop on a sucrose-rich medium at 20 degrees C. Bulbs are then dried for 6 weeks and rooted in vivo. The number of multiplication subcultures should be limited to 5-10 cycles in order to lower the risk of mutation. Virus indexing should be repeated 3-4 times, at the initial stage and then during shoot multiplication. Genetic stability of micropropagated shoots can be confirmed using molecular markers.

[Induction on callus culture and regeneration of Orostachyis fimbriatae].

PubMed

Su, Rui-jun; Zou, Li-juan; Wu, Qing-gui; Luo, Ming-hua

2014-01-01

To explore the effects of different hormonal combinations on induction, proliferation and differentiation of Orostachyis fimbriatae callus culture. Aseptic seedling leaves were used as explants,the different concentrations of IAA,NAA, 6-BA and KT on induction proliferation of callus were optimized by orthogonal test to explore the optimum medium for differentiation of callus by tissue culture techniques. The best medium for induction was MS + IAA 1.0 mg/L + NAA 0.5 mg/L + KT 1.0 mg/L, and the best hormonal combination for proliferation was MS + IAA 0.5 mg/L + 6-BA 0.5 mg/I. + KT 1.0 mg/L. The best medium for differentiation was MS + IAA 0.1 mg/L + KT 2.0 mg/L, and 1/2MS + IAA 0.2 mg/L was the optimum medium for rooting culture. The system of regeneration of Orostachyis fimbriatae is establishd by tissue culture techniques in this study.

Agrobacterium-mediated genetic transformation of pineapple (Ananas comosus L., Merr.).

PubMed

Mhatre, Minal

2013-01-01

Pineapple (Ananas comosus L., Merr.) is a commercially important crop, grown in the tropical and subtropical regions. However, the crop is faced with postharvest damage and poor varietal and nutritional improvement. Being a vegetatively propagated crop, conventional breeding programs take longer time for genetic improvement, which may not necessarily successfully develop an improved cultivar. Hence, the genetic modification of pineapple is an alternative handy approach to improve pineapple. We have established an Agrobacterium-mediated transformation system using leaf bases from in vitro-grown pineapple plants. Being a monocot, acetosyringone is added to the culture medium for overnight growth of Agrobacterium and transformation to transfer a gene of interest MSI99 soybean ferritin. Leaf bases isolated from in vitro shoot cultures are treated with Agrobacterium suspension at two dilutions, 10× and 20×, for 30 min. Explants are subsequently blot dried and cultured on gelrite solidified hormone-free Pin1 medium for 2 days (cocultivation). Periodic transfer is first done to the regeneration medium (Pin1) containing cefotaxime for the suppression of Agrobacterium growth. The transformants are selected by culturing on Pin1 medium containing cefotaxime and kanamycin. Multiple shoots, regenerated in leaf bases, are further multiplied and individually rooted in the liquid RM medium amended with antibiotics to recover plants. Putative transformants are analyzed for transgene integration and expression using standard molecular biological methods of PCR, RT-PCR, and genomic Southern.

Assessment of phytoremediation ability of Coriander sativum for soil and water co-contaminated with lead and arsenic: a small-scale study.

PubMed

Gaur, Nisha; Kukreja, Aayush; Yadav, Mahavir; Tiwari, Archana

2017-07-01

A study was conducted to access the phytoremediation potential of Coriandrum sativum for lead (Pb) and Arsenic (As). Metal tolerance index and pot experiment were conducted. Viable seeds were spread on filter paper and planted in soil placed in pots. The amount of Pb and As in control and in tailing soil was 0.27, 0.141, 1.77, and 0.35 ppm. The study was carried out in triplicates for a period of 4 weeks under natural conditions. The physico-chemical properties of soil were determined using the standard methods. Germination of seeds of Coriander sativum was inhibited more rigorously in filter paper as compared to soil medium. Shoot height and root length were significantly reduced in filter paper medium under Pb and As stress. These were inhibited by 33 and 40%, respectively, from the first to fourth weeks. Seedling growth was less affected in soil medium while greatly reduced in filter paper medium. Soil sustained almost equal stress in the fourth week as compared to the third week in filter paper medium. Shoot height was enormously affected by Pb and As compared to root length in filter paper medium, whereas slight inhibition of growth was observed in soil medium. Coriander sativum grown in pots was effective in removing Pb and As from control and tailing soils in comparison with seeds grown on filter paper. On this basis, it could be used in restoring soil polluted with Pb and As.

Heavy metal accumulation and growth of seedlings of five forest species as influenced by soil cadmium level

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kelly, J.M.; Parker, G.R.; McFee, W.W.

1979-07-01

White pine (Pinus strobus L.), loblolly pine (P. taeda L.), yellow poplar (Liriodendron tulipifera L.), yellow birch (Betula alleghaniensis Britt.), and choke cherry (Prunus virginiana L.) were grown from seeds in the greenhouse for 17 weeks. Plainfield fine sand, with pretreatment cadmium, lead, copper, and zinc concentrations of 0.6, 11.4, 2.0, and 20.6 ppM, respectively, was used as the growth medium. This soil was amended with CdCl/sub 2/ to produce cadmium addition levels of 0, 15, and 100 ppM. Shoot elongation and root and shoot dry weights were reduced with increasing levels of soil cadmium. All species exhibited increased cadmiummore » content in roots and shoots in response to increased soil cadmium levels; however, content varied according to species. Root and shoot accumulations of lead, copper, and zinc also varied with species as well as cadmium concentration in the soil.« less

Uptake of CeO2 nanoparticles and its effect on growth of Medicago arborea In vitro plantlets.

PubMed

Gomez-Garay, Aranzazu; Pintos, Beatriz; Manzanera, Jose Antonio; Lobo, Carmen; Villalobos, Nieves; Martín, Luisa

2014-10-01

The present study analyzes some effects of nano-CeO2 particles on the growth of in vitro plantlets of Medicago arborea when the nanoceria was added to the culture medium. Various concentrations of nano-CeO2 and bulk ceric oxide particles in suspension form were introduced to the agar culture medium to compare the effects of nanoceria versus ceric oxide bulk material. Germination rate and shoot dry weight were not affected by the addition of ceric oxide to the culture media. Furthermore, no effects were observed on chlorophyll content (single-photon avalanche diode (SPAD) measurements) due to the presence of either nano- or micro-CeO2 in the culture medium. When low concentrations of nanoceria were added to the medium, the number of trifoliate leaves and the root length increased but the root dry weight decreased. Also the values of maximum photochemical efficiency of PSII (F(v)/F m) showed a significant decrease. Dark-adapted minimum fluorescence (F 0) significantly increased in the presence of 200 mg L(-1) nanoceria and 400 mg L(-1) bulk material. Root tissues were more sensitive to nanoceria than were the shoots at lower concentrations of nanoceria. A stress effect was observed on M. arborea plantlets due to cerium uptake.

Biolistic transformation of Carrizo citrange (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.).

PubMed

Wu, Hao; Acanda, Yosvanis; Jia, Hongge; Wang, Nian; Zale, Janice

2016-09-01

The development of transgenic citrus plants by the biolistic method. A protocol for the biolistic transformation of epicotyl explants and transgenic shoot regeneration of immature citrange rootstock, cv. Carrizo (Citrus sinensis Osb. × Poncirus trifoliata L. Raf.) and plant regeneration is described. Immature epicotyl explants were bombarded with a vector containing the nptII selectable marker and the gfp reporter. The number of independent, stably transformed tissues/total number of explants, recorded by monitoring GFP fluorescence 4 weeks after bombardment was substantial at 18.4 %, and some fluorescing tissues regenerated into shoots. Fluorescing GFP, putative transgenic shoots were micro-grafted onto immature Carrizo rootstocks in vitro, confirmed by PCR amplification of nptII and gfp coding regions, followed by secondary grafting onto older rootstocks grown in soil. Southern blot analysis indicated that all the fluorescing shoots were transgenic. Multiple and single copies of nptII integrations were confirmed in five regenerated transgenic lines. There is potential to develop a higher throughput biolistics transformation system by optimizing the tissue culture medium to improve shoot regeneration and narrowing the window for plant sampling. This system will be appropriate for transformation with minimal cassettes.

Enhancement of in vitro Guayule propagation

NASA Technical Reports Server (NTRS)

Dastoor, M. N.; Schubert, W. W.; Petersen, G. R. (Inventor)

1982-01-01

A method for stimulating in vitro propagation of Guayule from a nutrient medium containing Guayule tissue by adding a substituted trialkyl amine bioinducing agent to the nutrient medium is described. Selective or differentiated propagation of shoots or callus is obtained by varying the amounts of substituted trialky amine present in the nutrient medium. The luxuriant growth provided may be processed for its poly isoprene content or may be transferred to a rooting medium for production of whole plants as identical clones of the original tissue. The method also provides for the production of large numbers of Guayule plants having identical desirable properties such as high polyisoprene levels.

Micropropagation and validation of genetic and biochemical fidelity amongst regenerants of Cassia angustifolia Vahl employing RAPD marker and HPLC.

PubMed

Chetri, Siva K; Sardar, Pratima Rani; Agrawal, Veena

2014-10-01

In vitro protocol has been established for clonal propagation of Cassia angustifolia Vahl which is an important source of anticancerous bioactive compounds, sennoside A and B. Nodal explants excised from field raised elite plant (showing optimum level of sennoside A and B) of C. angustifolia when reared on Murashige and Skoog's medium augmented with different cytokinins, viz. N(6)-benzyladenine (BA), N(6)-(2-isopentenyl) adenine (2iP) and 6-furfuryl aminopurine (Kn) differentiated multiple shoots in their axils. Of the three cytokinins, BA at 5 μM proved optimum for differentiating multiple shoots in 95 % cultures with an average of 9.14 shoots per explant within 8 weeks of culture. Nearly, 95 % of the excised in vitro shoots rooted on half strength MS medium supplemented with 10 μM indole-3-butyric acid (IBA). The phenotypically similar micropropagated plants were evaluated for their genetic fidelity employing random amplified polymorphic DNA (RAPD) markers. Eleven individuals, randomly chosen amongst a population of 120 regenerants were compared with the donor plant. A total of 36 scorable bands, ranging in size from 100 to 1,000 bp were generated amongst them by the RAPD primers. All banding profiles from micropropagated plants were monomorphic and similar to those of mother plant proving their true to the type nature. Besides, high performance liquid chromatography evaluation of the sennoside A and B content amongst leaves of the mature regenerants and the elite mother plant too revealed consistency in their content.

Enhanced micropropagation and tiller formation in sugarcane through pretreatment of explants with thidiazuron (TDZ).

PubMed

Kumari, Kavita; Lal, Madan; Saxena, Sangeeta

2017-10-01

An efficient, simple and commercially applicable protocol for rapid micropropagation of sugarcane has been designed using variety Co 05011. Pretreatment of shoot tip explants with thidiazuron (TDZ) induced high frequency regeneration of shoot cultures with improved multiplication ratio. The highest frequency (80%) of shoot initiation in explants pretreated with 10 mg/l of TDZ was obtained during the study. Maximum 65% shoot cultures could be established from the explants pretreated with TDZ as compared to minimum 40% establishment in explants without pretreatment. The explants pretreated with 10 mg/l of TDZ required minimum 40 days for the establishment of shoot cultures as compared to untreated explants which required 60 days. The highest average number of shoots per culture (19.1) could be obtained from the explants pretreated with 10 mg/l of TDZ, indicating the highest multiplication ratio (1:6). Highest rooting (over 94%) was obtained in shoots regenerated from pretreated explants on ½ strength MS medium containing 5.0 mg/l of NAA and 50 g/l of sucrose within 15 days. Higher number of tillers/clump (15.3) could be counted in plants regenerated from pretreated explants than untreated ones (10.9 tillers/clump) in field condition, three months after transplantation. Molecular analysis using RAPD and DAMD markers suggested that the pretreatment of explants with TDZ did not adversely affect the genetic stability of regenerated plants and maintained high clonal purity.

Better Rooting Procedure to Enhance Survival Rate of Field Grown Malaysian Eksotika Papaya Transformed with 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Gene

PubMed Central

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

2013-01-01

A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets. PMID:25969786

Better rooting procedure to enhance survival rate of field grown malaysian eksotika papaya transformed with 1-aminocyclopropane-1-carboxylic Acid oxidase gene.

PubMed

Sekeli, Rogayah; Abdullah, Janna Ong; Namasivayam, Parameswari; Muda, Pauziah; Abu Bakar, Umi Kalsom

2013-01-01

A high survival rate for transformed papaya plants when transferred to the field is useful in the quest for improving the commercial quality traits. We report in this paper an improved rooting method for the production of transformed Malaysian Eksotika papaya with high survival rate when transferred to the field. Shoots were regenerated from embryogenic calli transformed with antisense and RNAi constructs of 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) genes using the Agrobacterium tumefaciens-mediated transformation method. Regenerated transformed shoots, each measuring approximately 3-4 cm in height, were cultured in liquid half-strength Murashige and Skoog (MS) medium or sterile distilled water, and with either perlite or vermiculite supplementation. All the culturing processes were conducted either under sterile or nonsterile condition. The results showed that rooting under sterile condition was better. Shoots cultured in half-strength MS medium supplemented with vermiculite exhibited a 92.5% rooting efficiency while perlite showed 77.5%. The survival rate of the vermiculite-grown transformed papaya plantlets after transfer into soil, contained in polybags, was 94%, and the rate after transfer into the ground was 92%. Morpho-histological analyses revealed that the tap roots were more compact, which might have contributed to the high survival rates of the plantlets.

Somatic Embryogenesis and Massive Shoot Regeneration from Immature Embryo Explants of Tef

PubMed Central

Gugsa, Likyelesh; Kumlehn, Jochen

2011-01-01

Tef (Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2–0.35 mm embryo explants on a medium containing KBP minerals, 9.2–13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar “DZ-01-196” and the landrace “Fesho”, the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each “DZ-01-196” explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef. PMID:22028975

Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.

PubMed

Bakshi, Souvika; Saha, Bedabrata; Roy, Nand Kishor; Mishra, Sagarika; Panda, Sanjib Kumar; Sahoo, Lingaraj

2012-06-01

A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T₀ transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

Investigation of plant hormone level changes in shoot tips of longan (Dimocarpus longan Lour.) treated with potassium chlorate by liquid chromatography-electrospray ionization mass spectrometry.

PubMed

Susawaengsup, Chanthana; Rayanakorn, Mongkon; Wongpornchai, Sugunya; Wangkarn, Sunanta

2011-08-15

The endogenous levels of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA) and cytokinins (CKs) and their changes were investigated in shoot tips of ten longan (Dimocarpus longan Lour.) trees for off-season flowering until 60 days after potassium chlorate treatment in comparison with those of ten control (untreated) longan trees. These analytes were extracted and interfering matrices removed with a single mixed-mode solid phase extraction under optimum conditions. The recoveries at three levels of concentration were in the range of 72-112%. The endogenous plant hormones were separated and quantified by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). Detection limits based on the signal-to-noise ratio ranged from 10 ng mL(-1) for gibberellin A4 (GA4) to 200 ng mL(-1) for IAA. Within the first week after potassium chlorate treatment, dry weight (DW) amounts in the treated longan shoot tips of four gibberellins, namely: gibberellin A1(GA1), gibberellic acid (GA3), gibberellin A19 (GA19) and gibberellin A20 (GA20), were found to increase to approximately 25, 50, 20 and 60 ng g(-1) respectively, all of which were significantly higher than those of the controls. In contrast, gibberellin A8 (GA8) obtained from the treated longan was found to decrease to approximately 20 ng g(-1)DW while that of the control increased to around 80 ng g(-1)DW. Certain CKs which play a role in leaf bud induction, particularly isopentenyl adenine (iP), isopentenyl adenosine (iPR) and dihydrozeatin riboside (DHZR), were found to be present in amounts of approximately 20, 50 and 60 ng g(-1)DW in the shoot tips of the control longan. The analytical results obtained from the two-month off-season longan flowering period indicate that high GA1, GA3, GA19 and GA20 levels in the longan shoot tips contribute to flower bud induction while high levels of CKs, IAA and ABA in the control longan contribute more to the vegetative development. Copyright © 2011 Elsevier B.V. All rights reserved.

Determining nitrate and ammonium requirements for optimal in vitro response of diverse pear species

USDA-ARS?s Scientific Manuscript database

Inorganic nitrate (NO3-) and ammonium (NH4+) are the two major components in nitrogen (N) nutrition of typical tissue culture growth media, and the total amounts and ratios influence both shoot induction and differentiation. This study was designed to determine the optimal N requirements and NH4+ to...

Micropropagation and assessment of genetic fidelity of Dendrocalamus strictus (Roxb.) nees using RAPD and ISSR markers.

PubMed

Goyal, Arvind Kumar; Pradhan, Sushen; Basistha, Bharat Chandra; Sen, Arnab

2015-08-01

Dendrocalamus strictus popularly known as 'Male bamboo' is a multipurpose bamboo which is extensively utilized in pharmaceutical, paper, agricultural and other industrial implements. In this study, in vitro regeneration of D. strictus through nodal culture has been attempted. Murashige and Skoog's medium supplemented with 4 mg/l BAP was found to be most effective in shoot regeneration with 3.68 ± 0.37 shoots per explant. The effect of Kn was found to be moderate. These hormones also had considerable effect on the shoot length. The highest shoot length after 6 weeks (3.11 ± 0.41 cm) was noted with 5 mg/l BAP followed by 3.07 ± 0.28 cm with 5 mg/l Kn, while decrease in the shoot length was noted with other treatments. The effect of IBA and NAA individually or in combination at different concentrations on rooting was evaluated. The highest number of root (1.36 ± 0.04) was regenerated on full-strength MS medium supplemented with 3 mg/l NAA, while maximum length of 1.64 ± 0.03 cm of roots was recorded with combination of 1 mg/l IBA and 3 mg/l NAA. Tissue-cultured plants thus obtained were successfully transferred to the soil. The clonal fidelity among the in vitro-regenerated plantlets was assessed by RAPD and ISSR markers. The ten RAPD decamers produced 58 amplicons, while nine ISSR primers generated a total of 66 bands. All the bands generated were monomorphic. These results confirmed the clonal fidelity of the tissue culture-raised D. strictus plantlets and corroborated the fact that nodal culture is perhaps the safest mode for multiplication of true to type plants.

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop.

PubMed

Choudhary, Sumitra Kumari; Patel, Ashok Kumar; Harish; Shekhawat, Smita; Shekhawat, Narpat S

2017-07-01

Momordica dioica Roxb. ex Willd., is a perennial and dioecious (2n = 28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30 ± 3.83) with an average length 6.52 ± 0.89 cm were differentiated on MS medium containing 0.5 mg L -1 BAP, 0.1 mg L -1 IAA and additives (50 mg L -1 ascorbic acid, 25 mg L -1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250 mg L -1 IBA for 5 min produced 12.3 ± 1.33 with a mean length 5.4 ± 0.73 cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica , a popular summer vegetable crop.

Effect of lanthanum on rooting of in vitro regenerated shoots of Saussurea involucrata Kar. et Kir.

PubMed

Guo, Bin; Xu, Ling-Ling; Guan, Zhen-Jun; Wei, Ya-Hui

2012-06-01

In present study, the effect of lanthanum (La) on the rooting of regenerated shoots of Saussurea involucrata Kar. et Kir was analyzed. Rooting occurred from regenerated shoots inoculated on a medium supplemented with La, the plant rooting hormone indole-3-acetic acid (IAA), or both La and IAA together. The highest rooting efficiency (96%), root number/shoot (8.5), and root length (63 mm) were recorded in shoots cultured on medium containing 2.5 μM IAA combined with 100 μM La(3+). In order to elucidate the mechanism of rooting enhancement by La, we examined dynamic changes in antioxidant enzyme activities in plant tissue over time in culture. We found that the activities of peroxidase (POX) and superoxide dismutase (SOD) were significantly higher in plant tissue cultured in IAA plus La than in La or IAA alone. At the same time, the highest H(2)O(2) content was detected in plant tissue in the presence of 2.5 μM IAA plus 100 μM La(3+). In light of these data and previous results, we speculate that La enhanced IAA-induced rooting by acting as a mild abiotic stress to stimulate POX and SOD activities in plant cells. Then, IAA reacted with oxygen and POX to form the ternary complex enzyme-IAA-O(2) that dissociated into IAA radicals and O(2)(-). Subsequently, IAA-induced O(2)(-) readily converted to hydroxyl radical (HO·) via SOD-catalyzed dismutation. Finally, cell wall loosening and cell elongation occurred as a consequence of HO-dependent scission of wall components, leading to root growth. The treatment of IAA combined with La resulted in the highest plantlet survival (80%) compared to single treatments with IAA or La alone. These data suggest that rare earth elements enhance root morphogenesis and the growth of S. involucrata.

Simulation of carbon allocation and organ growth variability in apple tree by connecting architectural and source–sink models

PubMed Central

Pallas, Benoît; Da Silva, David; Valsesia, Pierre; Yang, Weiwei; Guillaume, Olivier; Lauri, Pierre-Eric; Vercambre, Gilles; Génard, Michel; Costes, Evelyne

2016-01-01

Background and aims Plant growth depends on carbon availability and allocation among organs. QualiTree has been designed to simulate carbon allocation and partitioning in the peach tree (Prunus persica), whereas MappleT is dedicated to the simulation of apple tree (Malus × domestica) architecture. The objective of this study was to couple both models and adapt QualiTree to apple trees to simulate organ growth traits and their within-tree variability. Methods MappleT was used to generate architectures corresponding to the ‘Fuji’ cultivar, accounting for the variability within and among individuals. These architectures were input into QualiTree to simulate shoot and fruit growth during a growth cycle. We modified QualiTree to account for the observed shoot polymorphism in apple trees, i.e. different classes (long, medium and short) that were characterized by different growth function parameters. Model outputs were compared with observed 3D tree geometries, considering shoot and final fruit size and growth dynamics. Key Results The modelling approach connecting MappleT and QualiTree was appropriate to the simulation of growth and architectural characteristics at the tree scale (plant leaf area, shoot number and types, fruit weight at harvest). At the shoot scale, mean fruit weight and its variability within trees was accurately simulated, whereas the model tended to overestimate individual shoot leaf area and underestimate its variability for each shoot type. Varying the parameter related to the intensity of carbon exchange between shoots revealed that behaviour intermediate between shoot autonomy and a common assimilate pool was required to properly simulate within-tree fruit growth variability. Moreover, the model correctly dealt with the crop load effect on organ growth. Conclusions This study provides understanding of the integration of shoot ontogenetic properties, carbon supply and transport between entities for simulating organ growth in trees. Further improvements regarding the integration of retroaction loops between carbon allocation and the resulting plant architecture are expected to allow multi-year simulations. PMID:27279576

Efficient and reproducible in vitro regeneration of Solanum lycopersicum and assessment genetic uniformity using flow cytometry and SPAR methods.

PubMed

Alatar, Abdulrahman A; Faisal, Mohammad; Abdel-Salam, Eslam M; Canto, Tomas; Saquib, Quaiser; Javed, Saad B; El-Sheikh, Mohamed A; Al-Khedhairy, Abdulaziz A

2017-09-01

In the present study, we develop an efficient and reproducible in vitro regeneration system for two cultivars viz. , Jamila and Tomaland of Solanum lycopersicum L., an economically important vegetable crop throughout the world. Sterilization of seeds with 2.5% (v/v) NaOCl was found to be most effective, about 97% of seeds germinated on cotton in magenta box moistened with sterile half strength (½)Murashige and Skoog (MS) medium. Regeneration efficiency of cotyledonary leaf (CL) and cotyledonary node (CN) explants derived from 08 days old aseptic seedling were assessed on MS medium supplemented with different concentrations of auxins and cytokinin. CL explants were found more responsive in comparison to CN in both the cultivars. Types of basal media were also assessed and found to have a significant effect on shoot regeneration. Highest regeneration frequency and maximum number of shoots were standardized from CL explants on MS medium supplied with 6-benzyl adenine (BA; 5.0 µM), indole-3-butyric acid (IBA; 2.5 µM) and Kinetin (Kin; 10.0 µM). In vitro regenerated microshoots were rooted on ½MS medium containing 0.5 µM indole-3-butyric acid (IBA). Regenerated plantlets with well-developed roots and shoot system were successfully acclimated to ex vitro condition. Genetic uniformity of tissue culture raised plantlets was first time evaluated using flow cytometry and single primer amplification reaction (SPAR) methods viz ., DAMD and ISSR. No significant changes in ploidy level and nuclear DNA content profile were observed between in vitro propagated plants and normal plants of both the cultivars. Similarly, the SPAR analysis also revealed monomorphic banding patterns in regenerated plantlets of S. lycopersicum verifying their genetic uniformity and clonal fidelity. This efficient regeneration system can be used as a fast and reproducible method for genetic transformation of this important vegetable crop.

Hyperhydricity in in vitro eggplant regenerated plants: structural characteristics and involvement of BiP (Binding Protein).

PubMed

Picoli, E A.T.; Otoni, W C.; Figueira, M L.; Carolino, S M.B.; Almeida, R S.; Silva, E A.M.; Carvalho, C R.; Fontes, E P.B.

2001-04-01

The hyperhydricity in eggplant (Solanum melongena L.) plants was monitored by the induction of the ER-luminal resident protein BiP. Although tissue culture conditions may induce BiP synthesis, the accumulation of BiP in hyperhydric shoots was consistently higher than in non-hyperhydric shoots. The leaf and stem anatomy in non-hyperhydric and hyperhydric eggplant was investigated aiming to identify structural changes associated with this phenomenon. In non-hyperhydric organs there were smaller and more organized cells, besides a more differentiated vascular system when compared with its hyperhydric counterpart. Scanning electron microscopy of leaves showed that leaf surface and stomata differentiation were also affected in hyperhydric plants.

Identification of genes differentially expressed during adventitious shoot induction in Pinus pinea cotyledons by subtractive hybridization and quantitative PCR.

PubMed

Alonso, Pablo; Cortizo, Millán; Cantón, Francisco R; Fernández, Belén; Rodríguez, Ana; Centeno, Maria L; Cánovas, Francisco M; Ordás, Ricardo J

2007-12-01

As part of a study aimed at understanding the physiological and molecular mechanisms involved in adventitious shoot bud formation in pine cotyledons, we conducted a transcriptome analysis to identify early-induced genes during the first phases of adventitious caulogenesis in Pinus pinea L. cotyledons cultured in the presence of benzyladenine. A subtractive cDNA library with more than 700 clones was constructed. Of these clones, 393 were sequenced, analyzed and grouped according to their putative function. Quantitative real-time PCR analysis was performed to confirm the differential expression of 30 candidate genes. Results are contrasted with available data for other species.

[Hydroxyproline: Rich glycoproteins of the plant and cell wall]. Annual technical progress report, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varner, J.E.

1993-06-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H{sub 2}O{sub 2} production reinforce the earlier ideas of others that H{sub 2}O{sub 2} is involved in normal lignification.« less

[Hydroxyproline: Rich glycoproteins of the plant and cell wall

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varner, J.E.

1993-01-01

Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a numbermore » of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.« less

Influence of Cytokinins in Combination with GA3 on Shoot Multiplication and Elongation of Tea Clone Iran 100 (Camellia sinensis (L.) O. Kuntze)

PubMed Central

Gonbad, Reza Azadi; Mohamad, Rosfarizan

2014-01-01

The use of in vitro culture has been accepted as an efficient technique for clonal propagation of many woody plants. In the present research, we report the results of a number of experiments aimed at optimizing micropropagation protocol for tea (Camellia sinensis (L.) O. Kuntze) (clone Iran 100) using nodal segments as the explant. The effect of different combinations and concentrations of plant growth regulators (PGR) (BAP, TDZ, GA3) on shoot multiplication and elongation was assessed. The influence of exposure to IBA in liquid form prior to transfer to solid media on rooting of tea microshoots was investigated. The results of this study showed that the best treatment for nodal segment multiplication in terms of the number of shoot per explant and shoot elongation was obtained using 3 mg/L BAP in combination with 0.5 mg/L GA3. TDZ was found to be inappropriate for multiplication of tea clone Iran 100 as it resulted in hyperhydricity especially at concentrations higher than 0.05 mg/L. Healthy shoots treated with 300 mg/L IBA for 30 min followed by transfer to 1/2 strength MS medium devoid of PGR resulted in 72.3% of shoots producing roots and upon transferring them to acclimatization chamber 65% survival was obtained prior to field transfer. PMID:24605069

Uptake, localization, and speciation of cobalt in Triticum aestivum L. (wheat) and Lycopersicon esculentum M. (tomato).

PubMed

Collins, Richard N; Bakkaus, Estelle; Carrière, Marie; Khodja, Hicham; Proux, Olivier; Morel, Jean-Louis; Gouget, Barbara

2010-04-15

The root-to-shoot transfer, localization, and chemical speciation of Co were investigated in a monocotyledon (Triticum aestivum L., wheat) and a dicotyledon (Lycopersicon esculentum M., tomato) plant species grown in nutrient solution at low (5 muM) and high (20 muM) Co(II) concentrations. Cobalt was measured in the roots and shoots by inductively coupled plasma-mass spectrometry. X-ray absorption spectroscopy measurements were used to identify the chemical structure of Co within the plants and Co distribution in the leaves was determined by micro-PIXE (particle induced X-ray emission). Although the root-to-shoot transport was higher for tomato plants exposed to excess Co, both plants appeared as excluders. The oxidation state of Co(II) was not transformed by either plant in the roots or shoots and Co appeared to be present as Co(II) in a complex with carboxylate containing organic acids. Cobalt was also essentially located in the vascular system of both plant species indicating that neither responded to Co toxicity via sequestration in epidermal or trichome tissues as has been observed for other metals in metal hyperaccumulating plants.

Development of guayule (parthenium argentatum)

NASA Technical Reports Server (NTRS)

Ball, E. A.

1981-01-01

The cytokinin benzylaminopurine strongly stimulated shoot growth, and the number of regenerated buds on the inoculum was proportional to its concentration. These buds produced shoots several centimeters in length which were caused to root on medium containing indolebutyric acid. Transferred to the septic condition of soil, the plantlets were gradually brought into full sunlight where they showed a brief vegetative growth with production of mature type leaves, and flowered. In contrast, seedlings of the same age remained vegetative. Chromosome studies of root smears from the tissue cultured plantlets showed that 2n = 36, the normal number for sexually reproducing guayules.

The arbuscular mycorrhizal fungus Rhizophagus irregularis differentially regulates the copper response of two maize cultivars differing in copper tolerance.

PubMed

Merlos, Miguel A; Zitka, Ondrej; Vojtech, Adam; Azcón-Aguilar, Concepción; Ferrol, Nuria

2016-12-01

Arbuscular mycorrhiza can increase plant tolerance to heavy metals. The effects of arbuscular mycorrhiza on plant metal tolerance vary depending on the fungal and plant species involved. Here, we report the effect of the arbuscular mycorrhizal fungus Rhizophagus irregularis on the physiological and biochemical responses to Cu of two maize genotypes differing in Cu tolerance, the Cu-sensitive cv. Orense and the Cu-tolerant cv. Oropesa. Development of the symbiosis confers an increased Cu tolerance to cv. Orense. Root and shoot Cu concentrations were lower in mycorrhizal than in non-mycorrhizal plants of both cultivars. Shoot lipid peroxidation increased with soil Cu content only in non-mycorrhizal plants of the Cu-sensitive cultivar. Root lipid peroxidation increased with soil Cu content, except in mycorrhizal plants grown at 250mg Cu kg -1 soil. In shoots of mycorrhizal plants of both cultivars, superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase activities were not affected by soil Cu content. In Cu-supplemented soils, total phytochelatin content increased in shoots of mycorrhizal cv. Orense but decreased in cv. Oropesa. Overall, these data suggest that the increased Cu tolerance of mycorrhizal plants of cv. Orense could be due to an increased induction of shoot phytochelatin biosynthesis by the symbiosis in this cultivar. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Accumulation and distribution of Zn in the shoots and reproductive structures of the halophyte plant species Kosteletzkya virginica as a function of salinity.

PubMed

Han, Ruiming; Quinet, Muriel; André, Emilie; van Elteren, Johannes Teun; Destrebecq, Florence; Vogel-Mikuš, Katarina; Cui, Guangling; Debeljak, Marta; Lefèvre, Isabelle; Lutts, Stanley

2013-09-01

Kosteletzkya virginica is a wetland halophyte that is a good candidate for rehabilitation of degraded salt marshes and production of oil as biodiesel. Salt marshes are frequently contaminated by heavy metals. The distribution of Zn in vegetative and reproductive organs of adult plants, and the NaCl influence on this distribution remain unknown and were thus explored in the present study. Plants were cultivated in a nutrient film technique system, from seedling stage until seed maturation in a control, Zn (100 μM), NaCl (50 mM) or Zn + NaCl medium. Photosynthesis, ion nutrition, malondialdehyde and non-protein thiol concentrations were quantified. Zinc distribution in reproductive organs was estimated by a laser ablation-inductively coupled plasma-mass spectrometry procedure (LA-ICP-MS). Adult plants accumulated up to 2 mg g(-1) DW Zn in the shoots. Zinc reduced plant growth, inhibited photosynthesis and reduced seed yield. Zinc accumulation in the seeds was only two times higher in Zn-treated plants than in controls. Exogenous NaCl neutralized the damaging action of Zn and modified the Zn distribution through a preferential accumulation of toxic ions in older leaves. Zinc was present in seed testa, endosperm and, to a lower extent, in embryo. Additional NaCl induced a chalazal retention of Zn during seed maturation and reduced final Zn seed content. It is concluded that NaCl 50 mM had a positive impact on the response of K. virginica to Zn toxicity and acts through a modification in Zn distribution rather than a decrease in Zn absorption.

Alginate Encapsulation of Begonia Microshoots for Short-Term Storage and Distribution

PubMed Central

Sakhanokho, Hamidou F.; Pounders, Cecil T.; Blythe, Eugene K.

2013-01-01

Synthetic seeds were formed from shoot tips of two in vitro grown Begonia cultivars using 3% sodium alginate in Murashige and Skoog medium (MS) salt solution as the gel matrix and 100 mM calcium chloride for complexation. Synthetic seed formation was achieved by releasing the sodium alginate/explant combination into 100 mM calcium chloride (CaCl2 ·H2O) solution for 30 or 45 min. Both control and encapsulated shoots were transferred into sterile Petri dishes and stored at 4°C or 22°C for 0, 2, 4, 6, or 8 weeks. Conversion of synthetic seeds into plantlets for both storage environments was assessed in MS medium or peat-based substrate. No significant difference was found between the 30 and 45 min CaCl2 ·H2O treatments or the two cultivars. Encapsulation of explants improved survival rate over time irrespective of the medium type or storage environment. Survival rates of 88, 53, 28, and 11% for encapsulated microshoots versus 73, 13, 0, and 0% for control explants were achieved in microshoots stored for 2, 4, 6, and 8 weeks, respectively. The best results were obtained when synthetic seeds were stored at 4°C and germinated on MS medium. Regenerated plantlets were successfully established in potting soil. PMID:24396296

Micropropagation of Araucaria excelsa R. Br. var. glauca Carrière from orthotropic stem explants.

PubMed

Sarmast, Mostafa Khoshhal; Salehi, Hassan; Khosh-Khui, Morteza

2012-07-01

The objectives of the present work were in vitro propagation of Araucaria excelsa R. Br. var. glauca Carrière (Norfolk Island pine) with focus on the evaluation of the mean number of shoots per explant (MNS/E) and mean length of shoots per explants (MLS/E) produced by different parts of the orthotropic stem of A. excelsa R. Br. var. glauca in response to plant growth regulators. Norfolk Island pine axillary meristems responded very well to the 2-iso-pentenyl adenine (2iP) and thidiazuron (TDZ) levels. Explants taken from stem upper segments in the media containing 2iP had a higher MNS/E (3.47) and MLS/E (6.27 mm) in comparison to those taken from stem lower segments, which were 0.71 and 0.51 mm, respectively. Using 0.045 μM TDZ in the MS medium not only resulted in 4.60 MNS/E with 7.08 mm MLS/E but proliferated shoots showed a good performance as well. Investigating the best position of stem explant on mother plant as well as the best concentrations of growth regulators were performed which were useful for efficient micropropagation of this plant. Thirty three percent of explants were rooted in the MS medium containing 3 % sucrose, supplemented with 7.5 μM of both NAA and IBA for 2 weeks before transferring to a half strength MS medium without any growth regulator. Plantlets obtained were acclimatized and transferred to the greenhouse with less than 20 % mortality. This procedure considered the first successful report for regeneration and acclimatization of A. excelsa R. Br. var. glauca plantlet through main stem explants.

Transformation of apple (Malus × domestica) using mutants of apple acetolactate synthase as a selectable marker and analysis of the T-DNA integration sites.

PubMed

Yao, Jia-Long; Tomes, Sumathi; Gleave, Andrew P

2013-05-01

Apple acetolactate synthase mutants were generated by site-specific mutagenesis and successfully used as selection marker in tobacco and apple transformation. T-DNA/Apple genome junctions were analysed using genome-walking PCR and sequencing. An Agrobacterium-mediated genetic transformation system was developed for apple (Malus × domestica), using mutants of apple acetolactate synthase (ALS) as a selectable marker. Four apple ALS mutants were generated by site-specific mutagenesis and subsequently cloned under the transcriptional control of the CaMV 35S promoter and ocs 3' terminator, in a pART27-derived plant transformation vector. Three of the four mutations were found to confer resistance to the herbicide Glean(®), containing the active agent chlorsulfuron, in tobacco (Nicotiana tabacum) transformation. In apple transformation, leaf explants infected with Agrobacterium tumefaciens EHA105 containing one of the three ALS mutants resulted in the production of shoots on medium containing 2-8 μg L(-1) Glean(®), whilst uninfected wild-type explants failed to regenerate shoots or survive on medium containing 1 and 3 μg L(-1) Glean(®), respectively. Glean(®)-resistant, regenerated shoots were further multiplied and rooted on medium containing 10 μg L(-1) Glean(®). The T-DNA and apple genome-DNA junctions from eight rooted transgenic apple plants were analysed using genome-walking PCR amplification and sequencing. This analysis confirmed T-DNA integration into the apple genome, identified the genome integration sites and revealed the extent of any vector backbone integration, T-DNA rearrangements and deletions of apple genome DNA at the sites of integration.

The influence of L-phenylalanine, methyl jasmonate and sucrose concentration on the accumulation of phenolic acids in Exacum affine Balf. f. ex Regel shoot culture.

PubMed

Skrzypczak-Pietraszek, Ewa; Słota, Joanna; Pietraszek, Jacek

2014-01-01

Phenolic acids are an important group of plant secondary metabolites with various, valuable therapeutic properties. Apart from plants growing in the open air, tissue cultures can be an alternative source of the secondary metabolites. The yield of their accumulation in in vitro cultures can be increased by different methods, including culture medium supplementation with precursors, elicitors and changing the standard amounts of the medium components. The purpose of this study was to investigate the influence of the precursor (L-phenylalanine), the elicitor (methyl jasmonate) and a higher sucrose concentration on the phenolic acids accumulation in the agitated shoot cultures of Exacum affine Balf. f. ex Regel (Gentianaceae). Qualitative and quantitative analyses of the phenolic acids in methanolic extracts from the biomass were conducted by applying the HPLC method. Fourteen phenolic acids and cinnamic acid were found in all samples. The total content of free phenolic acids increased from approximately 0.242% to 0.635% (2.6-fold) and the total content of the whole phenolic acids (free and bound) - from 0.712% to 1.160% (1.6-fold). The studies show that the best variant for the accumulation of most of the identified phenolic acids contained 6% of sucrose (double the standard amount), L-phenylalanine 1.6 gL(-1) of medium and methyl jasmonate 100 μM. The analysis of the results in the experiment presented here showed that it is possible to increase the accumulation of the phenolic acids in Exacum affine shoot cultures - by adding the precursor (L-phenylalanine), the elicitor (methyl jasmonate) and by increasing the sucrose concentration.

Enhanced iron and zinc accumulation in genetically engineered pineapple plants using soybean ferritin gene.

PubMed

Mhatre, Minal; Srinivas, Lingam; Ganapathi, Thumballi R

2011-12-01

Pineapple (Ananas comosus L. Merr., cv. "Queen") leaf bases were transformed with Agrobacterium tumefaciens strain EHA 105 harboring the pSF and pEFESF plasmids with soybean ferritin cDNA. Four to eight percent of the co-cultivated leaf bases produced multiple shoots 6 weeks after transfer to Murashige and Skoog's medium supplemented with α-naphthalene acetic acid 1.8 mg/l, indole-3-butyric acid 2.0 mg/l, kinetin 2.0 mg/l, cefotaxime 400 mg/l, and kanamycin 50 mg/l. Putatively transformed shoots (1-2 cm) were selected and multiplied on medium of the same composition and elongated shoots (5 cm) were rooted on liquid rooting medium supplemented with cefotaxime 400 mg/l and kanamycin 100 mg/l. The rooted plants were analyzed through PCR, genomic Southern analysis, and reverse transcription PCR. The results clearly confirmed the integration and expression of soybean ferritin gene in the transformed plants. Atomic absorption spectroscopic analysis carried out with six independently transformed lines of pSF and pEFE-SF revealed a maximum of 5.03-fold increase in iron and 2.44-fold increase in zinc accumulation in the leaves of pSF-transformed plants. In pEFE-SF-transformed plants, a 3.65-fold increase in iron and 2.05-fold increase in zinc levels was observed. Few of the transgenic plants were hardened in the greenhouse and are being grown to maturity to determine the enhanced iron and zinc accumulation in the fruits. To the best of our knowledge this is the first report on the transformation of pineapple with soybean ferritin for enhanced accumulation of iron and zinc content in the transgenic plants.

Gibberellin mediates daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria × ananassa Duch)

PubMed Central

Hytönen, Timo; Elomaa, Paula; Moritz, Thomas; Junttila, Olavi

2009-01-01

Background Differentiation of long and short shoots is an important developmental trait in several species of the Rosaceae family. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of gibberellin (GA) in regulation of shoot differentiation in strawberry (Fragaria × ananassa Duch.) cv. Korona. In strawberry, differentiation of axillary buds to runners (long shoot) or to crown branches (short shoot) is promoted by long-day and short-day conditions, respectively. Formation of crown branches is a prerequisite for satisfactory flowering because inflorescences are formed from the apical meristems of the crown. Results We found that both prohexadione-calcium and short photoperiod inhibited runner initiation and consequently led to induction of crown branching. In both cases, this correlated with a similar decline in GA1 level. Exogenous GA3 completely reversed the effect of prohexadione-calcium in a long photoperiod, but was only marginally effective in short-day grown plants. However, transfer of GA3-treated plants from short days to long days restored the normal runner formation. This did not occur in plants that were not treated with GA3. We also studied GA signalling homeostasis and found that the expression levels of several GA biosynthetic, signalling and target genes were similarly affected by prohexadione-calcium and short photoperiod in runner tips and axillary buds, respectively. Conclusion GA is needed for runner initiation in strawberry, and the inhibition of GA biosynthesis leads to the formation of crown branches. Our findings of similar changes in GA levels and in GA signalling homeostasis after prohexadione-calcium and short-day treatments, and photoperiod-dependent responsiveness of the axillary buds to GA indicate that GA plays a role also in the photoperiod-regulated differentiation of axillary buds. We propose that tightly regulated GA activity may control induction of cell division in subapical tissues of axillary buds, being one of the signals determining bud fate. PMID:19210764

Herbivore-induced volatiles in the perennial shrub, Vaccinium corymbosum, and their role in inter-branch signaling.

PubMed

Rodriguez-Saona, Cesar R; Rodriguez-Saona, Luis E; Frost, Christopher J

2009-02-01

Herbivore feeding activates plant defenses at the site of damage as well as systemically. Systemic defenses can be induced internally by signals transported via phloem or xylem, or externally transmitted by volatiles emitted from the damaged tissues. We investigated the role of herbivore-induced plant volatiles (HIPVs) in activating a defense response between branches in blueberry plants. Blueberries are perennial shrubs that grow by initiating adventitious shoots from a basal crown, which produce new lateral branches. This type of growth constrains vascular connections between shoots and branches within plants. While we found that leaves within a branch were highly connected, vascular connectivity was limited between branches within shoots and absent between branches from different shoots. Larval feeding by gypsy moth, exogenous methyl jasmonate, and mechanical damage differentially induced volatile emissions in blueberry plants, and there was a positive correlation between amount of insect damage and volatile emission rates. Herbivore damage did not affect systemic defense induction when we isolated systemic branches from external exposure to HIPVs. Thus, internal signals were not capable of triggering systemic defenses among branches. However, exposure of branches to HIPVs from an adjacent branch decreased larval consumption by 70% compared to those exposed to volatiles from undamaged branches. This reduction in leaf consumption did not result in decreased volatile emissions, indicating that leaves became more responsive to herbivory (or "primed") after being exposed to HIPVs. Chemical profiles of leaves damaged by gypsy moth caterpillars, exposed to HIPVs, or non-damaged controls revealed that HIPV-exposed leaves had greater chemical similarities to damaged leaves than to control leaves. Insect-damaged leaves and young HIPV-exposed leaves had higher amounts of endogenous cis-jasmonic acid compared to undamaged and non-exposed leaves, respectively. Our results show that exposure to HIPVs triggered systemic induction of direct defenses against gypsy moth and primed volatile emissions, which can be an indirect defense. Blueberry plants appear to rely on HIPVs as external signals for inter-branch communication.

High density growth of T7 expression strains with auto-induction option

DOEpatents

Studier, F. William

2010-07-20

A bacterial growth medium for promoting auto-induction of transcription of cloned DNA in cultures of bacterial cells grown batchwise is disclosed. The transcription is under the control of a lac repressor. Also disclosed is a bacterial growth medium for improving the production of a selenomethionine-containing protein or polypeptide in a bacterial cell, the protein or polypeptide being produced by recombinant DNA techniques from a lac or T7lac promoter, the bacterial cell encoding a vitamin B12-dependent homocysteine methylase. Finally, disclosed is a bacterial growth medium for suppressing auto-induction of expression in cultures of bacterial cells grown batchwise, said transcription being under the control of lac repressor.

Shooting for Stars: Cross-Sector Lessons for Retaining High-Performing Educators. Building an Opportunity Culture for America's Teachers

ERIC Educational Resources Information Center

Ableidinger, Joe; Kowal, Julie

2010-01-01

As the United States continues to grapple with unacceptable education results, "a great teacher for every student" has risen to a national imperative. In response, many districts have increased retention efforts through teacher induction programs, professional development, mentoring, and other strategies. But education actually has lower…

Micropropagation and non-steroidal anti-inflammatory and anti-arthritic agent boswellic acid production in callus cultures of Boswellia serrata Roxb.

PubMed

Nikam, Tukaram D; Ghorpade, Ravi P; Nitnaware, Kirti M; Ahire, Mahendra L; Lokhande, Vinayak H; Chopra, Arvind

2013-01-01

Micropropagation through cotyledonary and leaf node and boswellic acid production in stem callus of a woody medicinal endangered tree species Boswellia serrata Roxb. is reported. The response for shoots, roots and callus formation were varied in cotyledonary and leafy nodal explants from in vitro germinated seeds, if inoculated on Murshige and Skoog's (MS) medium fortified with cytokinins and auxins alone or together. A maximum of 8.0 ± 0.1 shoots/cotyledonary node explant and 6.9 ± 0.1 shoots/leafy node explants were produced in 91 and 88 % cultures respectively on medium with 2.5 μM 6-benzyladenine (BA) and 200 mg l(-1) polyvinylpyrrolidone (PVP). Shoots treated with 2.5 μM IBA showed the highest average root number (4.5) and the highest percentage of rooting (89 %). Well rooted plantlets were acclimatized and 76.5 % of the plantlets showed survival upon transfer to field conditions. Randomly amplified polymorphic DNA (RAPD) analysis of the micropropagated plants compared with mother plant revealed true-to-type nature. The four major boswellic acid components in calluses raised from root, stem, cotyledon and leaf explants were analyzed using HPLC. The total content of four boswellic acid components was higher in stem callus obtained on MS with 15.0 μM IAA, 5.0 μM BA and 200 mg l(-1) PVP. The protocol reported can be used for conservation and exploitation of in vitro production of medicinally important non-steroidal anti-inflammatory metabolites of B. serrata.

Hormonal induction and antihormonal inhibition of tracheary element differentiation in Zinnia cell cultures

NASA Technical Reports Server (NTRS)

Church, D. L.; Galston, A. W.

1988-01-01

Mechanically isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate to tracheary elements when cultured in inductive medium containing sufficient auxin and cytokinin. Tracheary element differentiation was induced by the three auxins (alpha-naphthaleneacetic acid, indole-3-acetic acid, and 2,4-dichlorophenoxyacetic acid) and four cytokinins (6-benzyladenine, kinetin, 2-isopentenyladenine and zeatin) tested. Tracheary element formation is inhibited or delayed if the inductive medium is supplemented with an anticytokinin, antiauxin, or inhibitor of auxin transport.

Improved Mineral Nutrition for Pear Shoot Cultures

USDA-ARS?s Scientific Manuscript database

The USDA-ARS National Clonal Germplasm Repository has over 200 pear accessions in culture, encompassing 18 species. Plant growth response on standard tissue culture medium varies widely due to the wide horticultural diversity of this collection. We used a systematic response-surface approach to eva...

SYSTEMATIC SCANNING ELECTRON MICROSCOPY TECHNIQUE FOR EVALUATING COMBINED BIOLOIGCAL/GRANULAR ACTIVATED CARBON TREATMENT PROCESSES

EPA Science Inventory

A systematic scanning election microscope analytical technique has been developed to examine granular activated carbon used a a medium for biomass attachment in liquid waste treatment. The procedure allows for the objective monitoring, comparing, and trouble shooting of combined ...

Chemical inducible promoter used to obtain transgenic plants with a silent marker and organisms and cells and methods of using same for screening for mutations

DOEpatents

Zuo, Jianru [New York, NY; Chua, Nam-Hai [Scarsdale, NY

2007-06-12

Disclosed is a chemically inducible promoter for transforming plants or plant cells with genes which are regulatable by adding the plants or cells to a medium containing an inducer or by removing them from such medium. The promoter is inducible by a glucocorticoid, estrogen or inducer not endogenous to plants. Such promoters may be used with any plant genes that can promote shoot regeneration and development to induce shoot formation in the presence of a glucocorticoid, estrogen or inducer. The promoter may be used with antibiotic or herbicide resistance genes or other genes which are regulatable by the presence or absence of a given inducer. Also presented are organisms or cells comprising a gene wherein the natural promoter of the gene is disrupted and the gene is placed under the control of a transgenic inducible promoter. These organisms and cells and their progeny are useful for screening for conditional gain of function and loss of function mutations.

Effects of long-term hypergravity on growth of Arabidopsis seedlings

NASA Astrophysics Data System (ADS)

Karahara, Ichirou; Ando, Naoko; Tamaoki, Daisuke; Kamisaka, Seiichiro

Effects of altered gravity on growth of plant root are not yet well understood compared to that of shoot organ such as stem, epicotyl or hypocotyl. And besides, its effect on growth is not yet examined at cellular level either in the root or the shoot. In the present study, we examined effects of long-term hypergravity on growth not only of the root but also the shoot at cellular level. Seeds of Arabidopsis were sown on gelrite containing Murashige-Skoog medium and were started to be exposed to hypergravity before germination. Growth of the hypocotyl had been inhibited since 3 d after the onset of hypergravity treatment at both 100 and 300 G while that of the root was not at either gravity. Longitudinal length of epidermal cells in one cell file decreased in response to hypergravity at 300 G in 3 d old hypocotyls while the number of the epidermal cells did not.

Antimony (SbIII) reduces growth, declines photosynthesis, and modifies leaf tissue anatomy in sunflower (Helianthus annuus L.).

PubMed

Vaculík, Marek; Mrázová, Anna; Lux, Alexander

2015-12-01

The role of antimony (Sb)--a non-essential trace metalloid--in physiological processes running in crops is still poorly understood. Present paper describes the effect of Sb tartrate (SbIII) on growth, Sb uptake, photosynthesis, photosynthetic pigments, and leaf tissue organization in young sunflower plants grown in hydroponics. We found that growth of below- and aboveground part was reduced with increasing concentration of Sb in the medium. Although Sb was mostly taken up by sunflower roots and only small part (1-2%) was translocated to the shoots, decline in photosynthesis, transpiration, and decreased content of photosynthetic pigments were observed. This indicates that despite relatively low mobility of Sb in root-shoot system, Sb in shoot noticeably modifies physiological status and reduced plant growth. Additionally, leaf anatomical changes indicated that Sb reduced the size of intercellular spaces and made leaf tissue more compact.

In vitro conservation and sustained production of breadfruit ( Artocarpus altilis, Moraceae): modern technologies for a traditional tropical crop

NASA Astrophysics Data System (ADS)

Murch, Susan J.; Ragone, Diane; Shi, Wendy Lei; Alan, Ali R.; Saxena, Praveen K.

2008-02-01

Breadfruit ( Artocarpus altilis, Moraceae) is a traditionally cultivated, high-energy, high-yield crop, but widespread use of the plant for food is limited by poor quality and poor storage properties of the fruit. A unique field genebank of breadfruit species and cultivars exists at the National Tropical Botanical Garden in the Hawaiian Islands and is an important global resource for conservation and sustainable use of breadfruit. However, this plant collection could be damaged by a random natural disaster such as a hurricane. We have developed a highly efficient in vitro plant propagation system to maintain, conserve, mass propagate, and distribute elite varieties of this important tree species. Mature axillary shoot buds were collected from three different cultivars of breadfruit and proliferated using a cytokinin-supplemented medium. The multiple shoots were maintained as stock cultures and repeatedly used to develop whole plants after root differentiation on a basal or an auxin-containing medium. The plantlets were successfully grown under greenhouse conditions and were reused to initiate additional shoot cultures for sustained production of plants. Flow cytometry was used to determine the nuclear deoxyribonucleic acid content and the ploidy status of the in vitro grown population. The efficacy of the micropropagation protocols developed in this study represents a significant advancement in the conservation and sustained mass propagation of breadfruit germplasm in a controlled environment free from contamination.

Statistical optimization of medium composition and culture condition for the production of recombinant anti-lipopolysaccharide factor of Eriocheir sinensis in Escherichia coli

NASA Astrophysics Data System (ADS)

Jiang, Shan; Liu, Mei; Wang, Baojie; Jiang, Keyong; Wang, Lei

2011-11-01

Anti-lipopolysaccharide factors (ALFs) are important antimicrobial peptides that are isolated from some aquatic species. In a previous study, we isolated ALF genes from Chinese mitten crab, Eriocheir sinensis. In this study, we optimized the production of a recombinant ALF by expressing E. sinensis ALF genes in Escherichia coli maintained in shake-flasks. In particular, we focused on optimization of both the medium composition and the culture condition. Various medium components were analyzed by the Plackett-Burman design, and two significant screened factors, (NH4)2SO4 and KH2PO4, were further optimized via the central composite design (CCD). Based on the CCD analysis, we investigated the induction start-up time, the isopropylthio-D-galactoside (IPTG) concentration, the post-induction time, and the temperature by response surface methodology. We found that the highest level of ALF fusion protein was achieved in the medium containing 1.89 g/L (NH4)2SO4 and 3.18 g/L KH2PO4, with a cell optical density of 0.8 at 600 nm before induction, an IPTG concentration of 0.5 mmol/L, a post-induction temperature of 32.7°C, and a post-induction time of 4 h. Applying the whole optimization strategy using all optimal factors improved the target protein content from 6.1% (without optimization) to 13.2%. We further applied the optimized medium and conditions in high cell density cultivation, and determined that the soluble target protein constituted 10.5% of the total protein. Our identification of the economic medium composition, optimal culture conditions, and details of the fermentation process should facilitate the potential application of ALF for further research.

High Concentration of Benzyladenine Solution Stimulates Anthers for Inducing Callus in Ricinus Communis L.

NASA Astrophysics Data System (ADS)

Liu, Ying; Yan, Shuying; Yang, Fuguang; Li, Dongliang; Tang, Jianian; Liu, Guoxuan; Lin, Shiwan; Niu, Sufang; Yang, Yali

2017-12-01

An high-frequency protocol for induction of callus from anther explants of Ricinus communis was described. When anther explants of R. communis was cultured directly onto medium containing 6-benzylaminopurine (BA) induced formation of only poor quality callus that had a low induction frequency of anther callus (10.67%). However, treating the anther explants with high concentrations (7.5-120 mg/L) of BA solution for short time periods (5-80 min) helped to improve the induction frequency and enhance the quality of the callus formation significantly. The best callus induction (41.25%) was observed when anther explants were treated with 15 mg/L BA solution for 10 min before being inoculated onto hormone-free Murashige and Skoog (MS) medium for 30 days. In order to further optimize the culture system, after treated with 15 mg/L BA for 10 min, anther explants were inoculated on the hormone-free MS medium contained concentrations of sodium nitroprusside (SNP). The results showed that SNP significantly promoted the response of callus induction, especially when 8 mg/L SNP was applied, the the highest percentage of callus induction (60.37%) were gained.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells.

PubMed

Vielba, Jesús M; Díaz-Sala, Carmen; Ferro, Enrique; Rico, Saleta; Lamprecht, María; Abarca, Dolores; Ballester, Antonio; Sánchez, Conchi

2011-10-01

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

Molecular Regulation of Temperature-Dependent Floral Induction in Tulipa gesneriana.

PubMed

Leeggangers, Hendrika A C F; Nijveen, Harm; Bigas, Judit Nadal; Hilhorst, Henk W M; Immink, Richard G H

2017-03-01

The vegetative-to-reproductive phase change in tulip ( Tulipa gesneriana ) is promoted by increasing temperatures during spring. The warm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis ( Arabidopsis thaliana ), very little is known about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencing was performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start of reproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip gene with a high similarity to Arabidopsis APETALA1 Gene Ontology enrichment analysis of differentially expressed genes showed the overrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expression analysis revealed that TERMINAL FLOWER1 ( TgTFL1 ) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1 ( TgSOC1-like1 ) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the flowering time-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field. Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1 being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long before morphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increased ambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before the commencement of the phase change. © 2017 American Society of Plant Biologists. All Rights Reserved.

Control of fire blight (Erwinia amylovora) on apple trees with trunk-injected plant resistance inducers and antibiotics and assessment of induction of pathogenesis-related protein genes

PubMed Central

Aćimović, Srđan G.; Zeng, Quan; McGhee, Gayle C.; Sundin, George W.; Wise, John C.

2015-01-01

Management of fire blight is complicated by limitations on use of antibiotics in agriculture, antibiotic resistance development, and limited efficacy of alternative control agents. Even though successful in control, preventive antibiotic sprays also affect non-target bacteria, aiding the selection for resistance which could ultimately be transferred to the pathogen Erwinia amylovora. Trunk injection is a target-precise pesticide delivery method that utilizes tree xylem to distribute injected compounds. Trunk injection could decrease antibiotic usage in the open environment and increase the effectiveness of compounds in fire blight control. In field experiments, after 1–2 apple tree injections of either streptomycin, potassium phosphites (PH), or acibenzolar-S-methyl (ASM), significant reduction of blossom and shoot blight symptoms was observed compared to water injected control trees. Overall disease suppression with streptomycin was lower than typically observed following spray applications to flowers. Trunk injection of oxytetracycline resulted in excellent control of shoot blight severity, suggesting that injection is a superior delivery method for this antibiotic. Injection of both ASM and PH resulted in the significant induction of PR-1, PR-2, and PR-8 protein genes in apple leaves indicating induction of systemic acquired resistance (SAR) under field conditions. The time separating SAR induction and fire blight symptom suppression indicated that various defensive compounds within the SAR response were synthesized and accumulated in the canopy. ASM and PH suppressed fire blight even after cessation of induced gene expression. With the development of injectable formulations and optimization of doses and injection schedules, the injection of protective compounds could serve as an effective option for fire blight control. PMID:25717330

Molecular Regulation of Temperature-Dependent Floral Induction in Tulipa gesneriana1

PubMed Central

Leeggangers, Hendrika A.C.F.; Bigas, Judit Nadal

2017-01-01

The vegetative-to-reproductive phase change in tulip (Tulipa gesneriana) is promoted by increasing temperatures during spring. The warm winters of recent years interfere with this process and are calling for new adapted cultivars. A better understanding of the underlying molecular mechanisms would be of help, but unlike the model plant Arabidopsis (Arabidopsis thaliana), very little is known about floral induction in tulip. To shed light on the gene regulatory network controlling flowering in tulip, RNA sequencing was performed on meristem-enriched tissue collected under two contrasting temperature conditions, low and high. The start of reproductive development correlated with rounding of the shoot apical meristem and induction of TGSQA expression, a tulip gene with a high similarity to Arabidopsis APETALA1. Gene Ontology enrichment analysis of differentially expressed genes showed the overrepresentation of genes potentially involved in floral induction, bulb maturation, and dormancy establishment. Expression analysis revealed that TERMINAL FLOWER1 (TgTFL1) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1-like1 (TgSOC1-like1) might be repressors, whereas TgSOC1-like2 likely is an activator, of flowering. Subsequently, the flowering time-associated expression of eight potential flowering time genes was confirmed in three tulip cultivars grown in the field. Additionally, heterologous functional analyses in Arabidopsis resulted in flowering time phenotypes in line with TgTFL1 being a floral repressor and TgSOC1-like2 being a floral activator in tulip. Taken together, we have shown that long before morphological changes occur in the shoot apical meristem, the expression of floral repressors in tulip is suppressed by increased ambient temperatures, leading either directly or indirectly to the activation of potential flowering activators shortly before the commencement of the phase change. PMID:28104719

The α-Amylase Induction in Endosperm during Rice Seed Germination Is Caused by Gibberellin Synthesized in Epithelium1

PubMed Central

Kaneko, Miyuki; Itoh, Hironori; Ueguchi-Tanaka, Miyako; Ashikari, Motoyuki; Matsuoka, Makoto

2002-01-01

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3β-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909–8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3β-hydroxylase genes and also an α-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and α-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3β-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3β-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for α-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for α-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced α-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not. PMID:11950975

Micropropagation and maintenance of phytoplasmas in tissue culture.

PubMed

Bertaccini, Assunta; Paltrinieri, Samanta; Martini, Marta; Tedeschi, Mara; Contaldo, Nicoletta

2013-01-01

Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1-3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and macroelements and 0.12 mg/L benzylaminopurine. The continued presence of phytoplasmas in infected shoots of periwinkle that have been maintained in micropropagation for up to 20 years can be shown by diagnostic methods such as nested PCR tests using the 16S rDNA gene (see Chapters 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,and 26 for phytoplasma diagnostic methods).

In vitro induction of tetraploid plants from diploid Zizyphus jujuba Mill. cv. Zhanhua.

PubMed

Gu, X F; Yang, A F; Meng, H; Zhang, J R

2005-12-01

Tetraploid plants of Zizyphus jujuba Mill. cv. Zhanhua were obtained with in vitro colchicine treatment. Shoot tips from in vitro-grown plants were treated with five different concentrations of colchicine (0.01, 0.03, 0.05, 0.1, 0.3%) in liquid MS medium (Murashige and Skoog 1962), and shaken (100 rpm) at 25 degrees C in darkness for 24, 48, 72 or 96 h, respectively. Tetraploids were obtained at a frequency of over 3% by using 0.05% colchicine (48 h, 72 h) and 0.1% colchicine (24 h, 48 h) treatment as determined by flow cytometry. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The chromosome number of diploid plants was 24 and that of tetraploid plants was 48. The stomata sizes of tetraploid plants were significantly larger than those of diploid plants, while the frequency of stomata were reduced significantly. Similarly, the chloroplast number of guard cells of tetraploid plants increased significantly. The selected tetraploid plants were grafted onto mature trees of Z. jujuba Mill. cv. Zhanhua in the field, resulted in thicker stems, rounder and succulent leaves, larger flowers and a delay in florescence time (3-4 days later) than diploid plants.

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget.

PubMed

Fortes, Ana M; Testillano, Pilar S; Del Carmen Risueño, Maria; Pais, Maria S

2002-09-01

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.

Microarray Analyses and Comparisons of Upper or Lower Flanks of Rice Shoot Base Preceding Gravitropic Bending

PubMed Central

Zang, Aiping; Chen, Haiying; Dou, Xianying; Jin, Jing; Cai, Weiming

2013-01-01

Gravitropism is a complex process involving a series of physiological pathways. Despite ongoing research, gravitropism sensing and response mechanisms are not well understood. To identify the key transcripts and corresponding pathways in gravitropism, a whole-genome microarray approach was used to analyze transcript abundance in the shoot base of rice (Oryza sativa sp. japonica) at 0.5 h and 6 h after gravistimulation by horizontal reorientation. Between upper and lower flanks of the shoot base, 167 transcripts at 0.5 h and 1202 transcripts at 6 h were discovered to be significantly different in abundance by 2-fold. Among these transcripts, 48 were found to be changed both at 0.5 h and 6 h, while 119 transcripts were only changed at 0.5 h and 1154 transcripts were changed at 6 h in association with gravitropism. MapMan and PageMan analyses were used to identify transcripts significantly changed in abundance. The asymmetric regulation of transcripts related to phytohormones, signaling, RNA transcription, metabolism and cell wall-related categories between upper and lower flanks were demonstrated. Potential roles of the identified transcripts in gravitropism are discussed. Our results suggest that the induction of asymmetrical transcription, likely as a consequence of gravitropic reorientation, precedes gravitropic bending in the rice shoot base. PMID:24040303

Analysis of two potential long-distance signaling molecules, LjCLE-RS1/2 and jasmonic acid, in a hypernodulating mutant too much love.

PubMed

Magori, Shimpei; Kawaguchi, Masayoshi

2010-04-01

Legume plants tightly control the number and development of root nodules. This is partly regulated by a long-distance signaling known as auto-regulation of nodulation (AON). AON signaling involves at least two potential long-distance signals: root-derived signal and shoot-derived signal. However, their molecular characteristics and the mode of action remain unclear. In our recent study, we isolated a novel Lotus japonicus hypernodulating mutant too much love (tml). Based on several grafting experiments, we concluded that its causative gene TML functions as a receptor of the shoot-derived signal. This finding prompted us to ask how the candidates of the long-distance signal molecules, LjCLE-RS1/2 and jasmonic acid (JA), are affected in tml mutants. Expression analysis revealed that rapid induction of LjCLE-RS1/2 upon rhizobial inoculation is still intact in tml, supporting that TML plays a role in reception of the shoot-derived signal but not in generation of the root-derived signal. Furthermore, physiological analysis showed that JA, a candidate of the shoot-derived signal, can suppress tml hypernodulation. Therefore, contrary to the previous report, JA might not be a component of AON signaling.

Heavy metals accumulation by Athyrium yokoscence in a mine area, Southwestern Japan

NASA Astrophysics Data System (ADS)

Prasetia, Hendra; Sakakibara, Masayuki; Takehara, Akinari; Sueoka, Yuri

2017-06-01

Mine dumps pose environmental problems worldwide. Phytoremediation is a remediation technique that uses plants to clean polluted environments. Athyrium yokoscence, a fern of the family Aspidiaceae, is a well-known Cd hyperaccumulator used in phytoremediation. The aim of the current study is to determine the extent of heavy metal absorption by A. yokoscence and evaluate its potential use in phytoremediation. The shoots, stems, and roots of A. yokoscence were analysed by inductively coupled plasma-optical emission spectrometry (ICP-OES) to determine the concentrations of Cd, As, Pb, Cu, and Zn, yielding maximum concentrations in the shoots of 851, 215, 192, 60.9, and 769 mg/kg-DW, in the stems of 481, 140, 881, 28.9, and 495 mg/kg-DW, and in the roots of 1210, 1868, 6473, 2484, and 5446 mg/kg-DW, respectively. The results indicate that A. yokoscence is a hyperaccumulator of Cd and can be used for the translocation of As, Pb, Cu, and Zn within the shoot tissue. The roots translocate heavy metals through the stems and they finally reside within the shoot tissue. The results suggest that A. yokoscence has considerable potential use as an environmental assessment tool for Cd, As, Pb, Cu, and Zn, and in helping to remediate mine dumps.

High-resolution transcript profiling reveals shoot abscission process of spruce dwarf mistletoe Arceuthobium sichuanense in response to ethephon

PubMed Central

Wang, Yonglin; Xiong, Dianguang; Jiang, Ning; Li, Xuewu; Yang, Qiqing; Tian, Chengming

2016-01-01

Arceuthobium (dwarf mistletoes) are hemiparasites that may cause great damage to infected trees belonging to Pinaceae and Cupressaceae. Currently, dwarf mistletoe control involves the use of the ethylene-producing product ethephon (ETH), which acts by inducing dwarf mistletoe shoot abscission. However, the process by which ETH functions is mostly unknown. Therefore, the transcriptome of the ETH-exposed plants was compared to non-exposed controls to identify genes associated with the response to ethephon. In this study, the reference transcriptome was contained 120,316 annotated unigenes, with a total of 21,764 ETH-responsive differentially expressed unigenes were identified. These ETH-associated genes clustered into 20 distinctly expressed pattern groups, providing a view of molecular events with good spatial and temporal resolution. As expected, the greatest number of unigenes with changed expression were observed at the onset of abscission, suggesting induction by ethylene. ETH also affected genes associated with shoot abscission processes including hormone biosynthesis and signaling, cell wall hydrolysis and modification, lipid transference, and more. The comprehensive transcriptome data set provides a wealth of genomic resources for dwarf mistletoe communities and contributes to a better understanding of the molecular regulatory mechanism of ethylene-caused shoots abscission. PMID:27941945

Direct and indirect organogenesis of Alpinia galanga and the phytochemical analysis.

PubMed

Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Gandi, Suryakala; Mangamoori, Lakshmi Narasu; Giri, Archana

2011-11-01

Alpinia galanga is a rhizomatous herb rich in essential oils and various other significant phytoconstituents. Rapid direct regeneration was obtained from the rhizome explants (15.66 ± 0.57 shoots) on MS media supplemented with zeatin at a concentration of 2 mg/l. The callus cultures of A. galanga were initiated from the rhizome explants on MS media supplemented with 2 mg/l each of BAP, 2,4-D, and NAA. The callus was analyzed for the presence of a vital phytoconstituent--acetoxychavicol acetate (ACA) associated with various biological properties. ACA was detected in the young friable callus as well as the stationary phase callus. Moreover, the induction of morphogenetic response in callus resulted in higher accumulation of ACA. The phytohormone withdrawal from the propagation media and the subsequent transfer of callus to BAP (2 mg/l) containing MS media has resulted in multiple shoot induction. The regenerated (indirect) plants have shown 1.6-fold higher ACA content (1.253%) when compared to the control plant (0.783%). Micropropagation of such conventionally propagated plants is very essential to meet the commercial demand as well as to ensure easy storage and transportation of disease free stocks.

Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-Glucanase (CenC) from Ruminiclostridium thermocellum in a Mesophilic Host Through Various Cultivation and Induction Strategies.

PubMed

Haq, Ikram Ul; Akram, Fatima

2017-09-01

Commonly, unintentional induction and inadvertently preparing medium for engineered Escherichia coli BL21 CodonPlus (DE3)-RIPL, give poor or variable yields of heterologous proteins. Therefore, to enhance the activity and production of an industrially relevant recombinant processive endo-1,4-β-glucanase (CenC) propagated in Escherichia coli BL21 CodonPlus(DE3)-RIPL through various cultivation and induction strategies. Investigation of various growth media and induction parameters revealed that high-cell-density and optimal CenC expression were obtained in ZYBM9 medium induced either with 0.5 mM IPTG/150 mM lactose, after 6 h induction at 37 °C; and before induction, bacterial cells were given heat shock (42 °C) for 1 h when culture density (OD 600nm ) reached at 0.6. Intracellular enzyme activity was enhanced by 6.67 and 3.20-fold in ZYBM9 and 3×ZYBM9 medium, respectively, under optimal conditions. Using YNG auto-induction medium, activity was 2.5-fold increased after 10 h incubation at 37 °C. Approximately similar results were obtained by transferring the optimized process at the bioreactor level. Results showed that the effective process strategy is essential to enhance recombinant bacterial cell mass and enzyme production from small to large-scale. To the best of our knowledge, this is the first ever report on enhanced production of thermostable processive endo-1,4-β-glucanase cloned from Ruminiclostridium thermocellum, which is a suitable candidate for industrial applications. Graphical Abstract Flow Chart Summary of Enhanced Production of a Recombinant Multidomain Thermostable GH9 Processive Endo-1,4-β-glucanase from Ruminiclostridium thermocellum.

Micropropagation and assessment of genetic fidelity of Henckelia incana: an endemic and medicinal Gesneriad of South India.

PubMed

Prameela, J; Ramakrishnaiah, H; Krishna, V; Deepalakshmi, A P; Naveen Kumar, N; Radhika, R N

2015-07-01

Henckelia incana is an endemic medicinal plant used for the treatment of fever and skin allergy. In the present study shoot regeneration was evaluated on Murashige and Skoog's (MS) medium supplemented with auxins, Indole-3-acetic acid (IAA), Indole-3- butyric acid (IBA), 1-Naphthaleneacetic acid (NAA), 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and cytokinins, 6-Benzylaminopurine (BAP) and Kinetin (Kn) at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mgl(-1). MS medium with IBA (18.08), NAA (17.83) and IAA (17.58) at 0.5 mgl(-1) concentrations showed efficient regeneration. Regenerated shoots were rooted on half-strength MS medium with and without 0.5 mgl(-1) IBA or NAA. The plantlets were successfully hardened in rooting trays (peat, vermiculite and sand) and transferred to field mileu. The genetic fidelity of in vitro raised plants was assessed by using three different single primer amplification reaction (SPAR) markers namely random amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR) and direct amplification of mini-satellite DNA region (DAMD). The results consistently demonstrated true-to-true type propagation. This is the first report of in vitro propagation and establishment of true-to-true type genetic fidelity in H. incana.

Micropropagation of Calophyllum brasiliense (Cambess.) from nodal segments.

PubMed

Silveira, S S; Cordeiro-Silva, R; Degenhardt-Goldbach, J; Quoirin, M

2016-05-03

Micropropagation of Calophyllum brasiliense Cambess. (Clusiaceae) is a way to overcome difficulties in achieving large-scale plant production, given the recalcitrant nature of the seeds, irregular fructification and absence of natural vegetative propagation of the species. Cultures were established using nodal segments 2 cm in length, obtained from 1-2 year old seedlings, maintained in a greenhouse. Mercury chloride and Plant Preservative Mixture™ were used in the surface sterilizing stage, better results being achieved with Plant Preservative Mixture™ incorporation in culture medium, at any concentration. Polyvinylpyrrolidone, activated charcoal, cysteine, ascorbic acid or citric acid were added to the culture medium to avoid oxidation. After 30 days of culture, polyvinylpirrolidone and ascorbic acid gave better results, eliminating oxidation in most explants. For shoot multiplication, benzylaminopurine was used in concentrations of 4.4 and 8.8 µM in Woody Plant Medium, resulting in an average of 4.43 and 4.68 shoots per explant, respectively, after 90 days. Indole-3-butyric acid and α-naphthalene acetic acid were used to induce root formation, reaching a maximum rooting rate of 24% with 20µM α-naphthalene acetic acid. For acclimatization. the rooted plants were transferred to Plantmax® substrate and cultured in a greenhouse, reaching 79% of survival after 30 days and 60% after one year.

Tissue-Culture Method of Cloning Rubber Plants

NASA Technical Reports Server (NTRS)

Ball, E. A.

1983-01-01

Guayule plant, a high-yield rubber plant cloned by tissue-culture method to produce multiple new plants that mature quickly. By adjusting culture medium, excised shoot tip produces up to 50 identical guayule plants. Varying concentration of cytokinin, single excised tip produces either 1 or several (up to 50) new plants.

Micropropagation of the endangered shrub pondberry (Lindera melissifolia [Walt.] Blume)

Treesearch

Tracy S. Hawkins; Nathan M. Schiff; Emile s. Gardiner; Theodore Leininger; Margaret S. Devall; A. Dan Wilson; Paul Hamel; Deborah D. McCown; Kristina Connor

2007-01-01

A micropropagation protocol using shoot cultures is described for Lindera melissifolia, a federally listed endangered shrub endemic to the southeastern United States. Stock plants were harvested from native L. melissifolia populations growing in the lower Mississippi Alluvial Valley. In vitro proliferation was on woody plant medium...

Data Collection from Videotaped Experiments in a Biology Laboratory

ERIC Educational Resources Information Center

Russock, Howard I.

1977-01-01

Describes individualized television instruction involving active student participation, where experiments on air breathing in fish and shoot growth were videotaped. Students collected data directly from the television screen. Student accuracy was found to be excellent; reactions to the medium were found to be apathetic. Reasons for apathy are…

Response simulation and theoretical calibration of a dual-induction resistivity LWD tool

NASA Astrophysics Data System (ADS)

Xu, Wei; Ke, Shi-Zhen; Li, An-Zong; Chen, Peng; Zhu, Jun; Zhang, Wei

2014-03-01

In this paper, responses of a new dual-induction resistivity logging-while-drilling (LWD) tool in 3D inhomogeneous formation models are simulated by the vector finite element method (VFEM), the influences of the borehole, invaded zone, surrounding strata, and tool eccentricity are analyzed, and calibration loop parameters and calibration coefficients of the LWD tool are discussed. The results show that the tool has a greater depth of investigation than that of the existing electromagnetic propagation LWD tools and is more sensitive to azimuthal conductivity. Both deep and medium induction responses have linear relationships with the formation conductivity, considering optimal calibration loop parameters and calibration coefficients. Due to the different depths of investigation and resolution, deep induction and medium induction are affected differently by the formation model parameters, thereby having different correction factors. The simulation results can provide theoretical references for the research and interpretation of the dual-induction resistivity LWD tools.

INDUCTION OF 6-THIOGUANINE RESISTANCE IN SYNTHRONIZED HUMAN FIBROBLAST CELLS TREATED WITH METHYL METHANESULFONATE, N-ACETOXY-2-ACETHYLAMINOFLUORENE AND N-METHYL-N'-NITRO-N-NITROSOGUANIDINE

EPA Science Inventory

Chemical induction of 6-thioguanine resistance was studied in synchronized human fibroblast cells. Cells initially grown in a medium lacking arginine and glutamine for 24 h ceased DNA synthesis and failed to enter the S phase. After introduction of complete medium, the cells prog...

Light quanta modulated characteristics of Ni uptake by Brassica juncea seedlings: the interdependence of plant metal concentration and biomass.

PubMed

Dasgupta-Schubert, N; Whelan, T; Reyes, M A; Lloren, C; Brandt, T T; Persans, M W

2007-01-01

The relationships between the concentration of metal in the growth medium, Cs, the concentration of metal absorbed by the plant, Cp, and the total biomass achieved, M, all of which are factors relevant to the efficiency of metal uptake and tolerance by the plant, have been investigated via the physiological response of Brassica juncea seedlings to Ni stress. The factorial growth experiments treated the Ni concentration in agar medium and the diurnal light quanta as independently variable parameters. Observations included the evidence of light enhancement of Ni toxicity in the root, as well as at the whole-plant level. The shoot mass index possibly is an indicator of the amount of shoot metal sequestration in B. juncea, as are the logarithmic variation of Cp with Cs and the power-law dependence of M on Cp. The sum total of these observations indicates that, for the Ni accumulating plant B. juncea, the overall metabolic allocation to either growth or metal tolerance of the plant is important. Neither a rapid biomass increase nor a high metal absorbed concentration favored the removal of high metal mass from the medium. Rather, the plants with a moderate rate of biomass growth and a moderate absorbed metal concentration demonstrated the ability to remove the maximum mass of metal from the medium. The implication of these results as related to the extant model of phyoextraction efficiency is discussed.

Cardenolides, induced responses, and interactions between above- and belowground herbivores of milkweed (Asclepias spp.).

PubMed

Rasmann, Sergio; Agrawal, Anurag A; Cook, Susan C; Erwin, Alexis C

2009-09-01

Theory has long predicted allocation patterns for plant defense against herbivory, but only recently have both above- and belowground plant defenses been considered simultaneously. Milkweeds in the genus Asclepias are a classic chemically defended clade of plants with toxic cardenolides (cardiac glycosides) and pressurized latex employed as anti-herbivore weapons. Here we combine a comparative approach to investigate broadscale patterns in allocation to root vs. shoot defenses across species with a species-specific experimental approach to identify the consequences of defense allocational shifts on a specialist herbivore. Our results show phylogenetic conservatism for inducibility of shoot cardenolides by an aboveground herbivore, with only four closely related tropical species showing significant induction; the eight temperate species examined were not inducible. Allocation to root and shoot cardenolides was positively correlated across species, and this relationship was maintained after accounting for phylogenetic nonindependence. In contrast to long-standing theoretical predictions, we found no evidence for a trade-off between constitutive and induced cardenolides; indeed the two were positively correlated across species in both roots and shoots. Finally, specialist root and shoot herbivores of common milkweed (A. syriaca) had opposing effects on latex production, and these effects had consequences for caterpillar growth consistent with latex providing resistance. Although cardenolides were not affected by our treatments, A. syriaca allocated 40% more cardenolides to shoots over roots. We conclude that constitutive and inducible defenses are not trading off across plant species, and shoots of Asclepias are more inducible than roots. Phylogenetic conservatism cannot explain the observed patterns of cardenolide levels across species, but inducibility per se was conserved in a tropical clade. Finally, given that above- and belowground herbivores can systemically alter the defensive phenotype of plants, we concur with recent calls for a whole-plant perspective in testing models of plant defense allocation.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants.

PubMed

Gnanaraj, Wesely Edward; Antonisamy, Johnson Marimuthu; R B, Mohanamathi; Subramanian, Kavitha Marappampalyam

2012-01-01

To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants. Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3-4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration. Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field. The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.

Artificial Neural Network Genetic Algorithm As Powerful Tool to Predict and Optimize In vitro Proliferation Mineral Medium for G × N15 Rootstock

PubMed Central

Arab, Mohammad M.; Yadollahi, Abbas; Shojaeiyan, Abdolali; Ahmadi, Hamed

2016-01-01

One of the major obstacles to the micropropagation of Prunus rootstocks has, up until now, been the lack of a suitable tissue culture medium. Therefore, reformulation of culture media or modification of the mineral content might be a breakthrough to improve in vitro multiplication of G × N15 (garnem). We found artificial neural network in combination of genetic algorithm (ANN-GA) as a very precise and powerful modeling system for optimizing the culture medium, So that modeling the effects of MS mineral salts (NH4+, NO3-, PO42-, Ca2+, K+, SO42-, Mg2+, and Cl−) on in vitro multiplication parameters (the number of microshoots per explant, average length of microshoots, weight of calluses derived from the base of stem explants, and quality index of plantlets) of G × N15. Showed high R2 correlation values of 87, 91, 87, and 74 between observed and predicted values were found for these four growth parameters, respectively. According to the ANN-GA results, among the input variables, NH4+ and NO3- had the highest values of VSR in data set for the parameters studied. The ANN-GA showed that the best proliferation rate was obtained from medium containing (mM) 27.5 NO3-, 14 NH4+, 5 Ca2+, 25.9 K+, 0.7 Mg2+, 1.1 PO42-, 4.7 SO42-, and 0.96 Cl−. The performance of the medium optimized by ANN-GA, denoted as YAS (Yadollahi, Arab and Shojaeiyan), was compared to that of standard growth media for all Prunus rootstock, including the Murashige and Skoog (MS) medium, (specific media) EM, Quoirin and Lepoivre (QL) medium, and woody plant medium (WPM) Prunus. With respect to shoot length, shoot number per cultured explant and productivity (number of microshoots × length of microshoots), YAS was found to be superior to other media for in vitro multiplication of G × N15 rootstocks. In addition, our results indicated that by using ANN-GA, we were able to determine a suitable culture medium formulation to achieve the best in vitro productivity. PMID:27807436

Changes in Canada thistle (Cirsium arvense) shoot density following inoculation with Puccinia punctiformis and induction of symptomatic and asymptomatic systemic disease

USDA-ARS?s Scientific Manuscript database

Canada thistle (Cirsium arvense, CT) is one of the worst weeds in temperate areas of the world. The rust fungus Puccinia punctiformis was first proposed as a biological control agent for CT in 1893. The rust causes systemic disease which ultimately kills CT plants. In 2013, it was demonstrated in fo...

Identification and elimination of bacterial contamination during in vitro propagation of Guadua angustifolia Kunth.

PubMed

Nadha, Harleen Kaur; Salwan, Richa; Kasana, Ramesh Chand; Anand, Manju; Sood, Anil

2012-04-01

Guadua angustifolia Kunth is a very important bamboo species with significant utility in pharmaceutical, paper, charcoal, and construction industries. Microbial contamination is a major problem encountered during establishment of in vitro cultures of Guadua. This study has been designed to analyze the identity of contaminating bacteria and to develop the strategy to eliminate them during micropropagation of Guadua. We isolated and consequently analyzed partial sequence analysis of the 16S rRNA gene to identify two contaminating bacteria as (1) Pantoea agglomerans and (2) Pantoea ananatis. In addition, we also- performed antibiotic sensitivity testing on these bacterial isolates. We identified kanamycin and streptomycin sulfate as potentially useful antibiotics in eliminating the contaminating bacteria. We grew shoots on multiplication medium containing BAP (2 mg/l) and adenine sulfate (10 mg/l) supplemented with kanamycin (10 μg/ml) for 10 days and transferred them to fresh medium without antibiotics and found that bacterial growth was inhibited. Moreover, we observed intensive formation of high-quality shoots. Streptomycin sulfate also inhibited bacterial growth but at higher concentration. We also demonstrated that shoots grown in streptomycin sulfate tended to be shorter and had yellow leaves. Thus, we have developed a novel strategy to identify and inhibit intriguing microbial contaminations of (1) Pantoea agglomerans and (2) Pantoea ananatis during establishment of in vitro cultures of Guadua. This would improve in vitro establishment of an important bamboo, Guadua angustifolia Kunth for large scale propagation.

Search for a plant for phytoremediation--what can we learn from field and hydroponic studies?

PubMed

Zabłudowska, E; Kowalska, J; Jedynak, L; Wojas, S; Skłodowska, A; Antosiewicz, D M

2009-10-01

The main aim of the study was to evaluate the strategies for coping with arsenic toxicity developed by the mine species (Calamagrostis arundinacea, Fragaria vesca, Stachys sylvatica, and Epilobium parviflorum), and to compare results obtained from plants exposed to arsenic present in contaminated soil (2000-3500 mg/kg dw) and in hydroponic solution (2 microM and 12 microM arsenate). Here we report basic differences in plant responses to arsenic depending on growth conditions (hydroponic/soil) with respect to uptake, root-to-shoot translocation, distribution, and detoxification/speciation. Calamagrostis has the highest level of As-tolerance among the tested species. When grown in soil, it accumulated the highest amount of As in roots and shoots relative to other species, however, when exposed to arsenic in hydroponics, it had lower As concentrations. The efficiency of arsenic root-to-shoot translocation was also different, being less effective in soil-grown Calamagrostis compared with hydroponics. Furthermore, in Calamagrostis exposed to arsenate in liquid medium, As(III) was the predominant arsenic form, in contrast to plants grown in As-contaminated soil, in which As(V) predominated. In addition, comparison of the level of phytochelatins showed that only PC2 was detected in plants from hydroponics, whereas in those from soil, additionally PC3 and PC4 were found. The results show that the basic components of a plant's response to arsenic, including uptake, accumulation as well as detoxification, change depending on the experimental conditions (arsenic in liquid medium or contaminated soil).

Efficiency of plant growth-promoting P-solubilizing Bacillus circulans CB7 for enhancement of tomato growth under net house conditions.

PubMed

Mehta, Preeti; Walia, Abhishek; Kulshrestha, Saurabh; Chauhan, Anjali; Shirkot, Chand Karan

2015-01-01

P-solubilizing bacterial isolate CB7 isolated from apple rhizosphere soil of Himachal Pradesh, India was identified as Bacillus circulans on the basis of phenotypic characteristics, biochemical tests, fatty acid methyl esters analysis, and 16S rRNA gene sequence. The isolate exhibited plant growth-promoting traits of P-solubilization, auxin, 1-aminocyclopropane-1-carboxylate deaminase activity, siderophore, nitrogenase activity, and antagonistic activity against Dematophora necatrix. In vitro studies revealed that P-solubilization and other plant growth-promoting traits were dependent on the presence of glucose in PVK medium and removal of yeast extract had no significant effect on plant growth-promoting traits. Plant growth-promoting traits of isolate CB7 were repressed in the presence of KH2 PO4 . P-solubilization activity was associated with the release of organic acids and a drop in the pH of the Pikovskaya's medium. HPLC analysis detected gluconic and citric acid as major organic acids in the course of P-solubilization. Remarkable increase was observed in seed germination (22.32%), shoot length (15.91%), root length (25.10%), shoot dry weight (52.92%) and root dry weight (31.4%), nitrogen (18.75%), potassium (57.69%), and phosphorus (22.22%) content of shoot biomass over control. These results demonstrate that isolate CB7 has the promising PGPR attributes to be developed as a biofertilizer to enhance soil fertility and promote plant growth. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Nitrate uptake and utilization is modulated by exogenous gamma-aminobutyric acid in Arabidopsis thaliana seedlings.

PubMed

Barbosa, Jose M; Singh, Narendra K; Cherry, Joe H; Locy, Robert D

2010-06-01

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO(3)(-)) in the growth medium. At NO(3)(-) concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO(3)(-), the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO(3)(-) uptake at low NO(3)(-), while GABA inhibited NO(3)(-) uptake at high NO(3)(-). Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.

Micropropagation and cytogenetic assessment of Zingiber species of Northeast India.

PubMed

Das, Archana; Kesari, Vigya; Rangan, Latha

2013-12-01

An improved micropropagation protocol was developed for Zingiber moran and Z. zerumbet, two wild species of the genus Zingiber, found in Northeast India. The effects of growth regulators, sugar concentrations, and nutrients were tested on the rate of shoot initiation and multiplication. An increase in proliferation and multiplication occurred in modified Murashige and Skoog (MS) medium supplemented with benzyladenine and kinetin. About 2 % sucrose and 0.7 % agar were found to be the optimum for shoot multiplication and regeneration. Naphthalene acetic acid at 0.5 mg/L produced the best rooting response for both the species. Regenerated plantlets were acclimatized successfully and cytogenetic stability was confirmed by RAPD profiling and ploidy checks.

Cell Type-Specific Gene Expression Analyses by RNA Sequencing Reveal Local High Nitrate-Triggered Lateral Root Initiation in Shoot-Borne Roots of Maize by Modulating Auxin-Related Cell Cycle Regulation1[OPEN

PubMed Central

Yu, Peng; Eggert, Kai; von Wirén, Nicolaus; Li, Chunjian; Hochholdinger, Frank

2015-01-01

Plants have evolved a unique plasticity of their root system architecture to flexibly exploit heterogeneously distributed mineral elements from soil. Local high concentrations of nitrate trigger lateral root initiation in adult shoot-borne roots of maize (Zea mays) by increasing the frequency of early divisions of phloem pole pericycle cells. Gene expression profiling revealed that, within 12 h of local high nitrate induction, cell cycle activators (cyclin-dependent kinases and cyclin B) were up-regulated, whereas repressors (Kip-related proteins) were down-regulated in the pericycle of shoot-borne roots. In parallel, a ubiquitin protein ligase S-Phase Kinase-Associated Protein1-cullin-F-box proteinS-Phase Kinase-Associated Protein 2B-related proteasome pathway participated in cell cycle control. The division of pericycle cells was preceded by increased levels of free indole-3-acetic acid in the stele, resulting in DR5-red fluorescent protein-marked auxin response maxima at the phloem poles. Moreover, laser-capture microdissection-based gene expression analyses indicated that, at the same time, a significant local high nitrate induction of the monocot-specific PIN-FORMED9 gene in phloem pole cells modulated auxin efflux to pericycle cells. Time-dependent gene expression analysis further indicated that local high nitrate availability resulted in PIN-FORMED9-mediated auxin efflux and subsequent cell cycle activation, which culminated in the initiation of lateral root primordia. This study provides unique insights into how adult maize roots translate information on heterogeneous nutrient availability into targeted root developmental responses. PMID:26198256

Callus Induction from Various Organs of Dragon Fruit, Apple and Tomato on some Mediums.

PubMed

Rumiyati; Sismindari; Semiarti, Endang; Milasari, Asri Fajar; Sari, Dheatika Karina; Fitriana, Nia; Galuh, Sekar

2017-01-01

Dragon fruit (Hylocereus spp.), apple (Malus sylvestris Mill.) and tomato (Solanum lycopersicum L.) are high potential sources of antioxidant compounds such as phenolics. The compounds have the capability of protecting cells and tissues against free radicals. Secondary metabolite produced by callus cell culture from plant organs also acts as a source of antioxidants. This study aimed to determine the optimal ratio of sucrose and 2,4-D in Murashige and Skoog (MS) medium for callus induction from different plant organ explants. With all of characteristic, callus can be used further for the development of natural cell regeneration agent. This study was conducted using analytical technique. Suitable explants were obtained. They were developed in various concentrations of combination between MS medium and 2,4-D. Callus growth, including their weight and surface was then measured and analyzed by using one-way analysis of variance (ANOVA). Callus was able to grow from its explants in 5-7 days after induction process. They were clear in color and had friable texture. The highest value of fresh weight of dragon fruit callus was obtained through MS supplemented with 1 μL L-1 2,4-D and 30 g sucrose. However, apple and tomato callus induction and growth maintenance reached optimal medium on MS supplemented with 30 g sucrose and 2 μL L-1 2,4-D. Callus of apple, dragon fruit and tomato was maintained upon MS supplemented with 30-40 g sucrose and 1-2 μL L-1 2,4-D for optimum induction and growth. The optimization of growth medium will give advantages for further development of natural cell regeneration agent.

Temporary Immersion System for Date Palm Micropropagation.

PubMed

Othmani, Ahmed; Bayoudh, Chokri; Sellemi, Amel; Drira, Noureddine

2017-01-01

The temporary immersion system (TIS) is being used with tremendous success for automation of micropropagation of many plant species. TIS usually consists of a culture vessel comprising two compartments, an upper one with the plant material and a lower one with the liquid culture medium and an automated air pump. The latter enables contact between all parts of the explants and the liquid medium by setting overpressure to the lower part of the container. These systems are providing the most satisfactory conditions for date palm regeneration via shoot organogenesis and allow a significant increase of multiplication rate (5.5-fold in comparison with that regenerated on agar-solidified medium) and plant material quality, thereby reducing production cost.

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

Treesearch

Ningxia Du; Paula M. Pijut

2008-01-01

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 µM6-benzylaminopurine (BA) plus 4.5 µM thidiazuron (TDZ), and 22.2 µM...

Production and short-term of synthetic seeds from encapsulated begonia

USDA-ARS?s Scientific Manuscript database

Synthetic seeds were formed from in vitro grown Begonia (cvs ‘Sweetheart Mix’ and ‘Baby Wing White’) shoot tips using 3% sodium alginate in Murashige and Skoog (1962) medium (MS) salt solution as the gel matrix and 100 mM calcium chloride for complexation. Synthetic seed formation was achieved by re...

Alginate encapsulation of Begonia microshoots for short-term storage and distribution

USDA-ARS?s Scientific Manuscript database

Synthetic seeds were formed from in vitro grown Begonia cultivars (Sweetheart Mix and BabyWing White) shoot tips using 3% sodium alginate in Murashige and Skoog medium (MS) salt solution as the gel matrix and 100 mM calcium chloride for complexation. Synthetic seed formation was achieved by releasin...

Optimizing shoot culture media for Rubus germplasm: the effects of NH4+, NO3-, and total nitrogen

USDA-ARS?s Scientific Manuscript database

The nitrogen components of Murashige and Skoog (MS) medium were significant factors for improved growth in our earlier study that modeled the effects of mineral nutrition on growth and development of micropropagated red raspberry(Rubus idaeus L.). In this study, a mixture component design was applie...

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration.

PubMed

Matsumoto, T; Sakai, A; Yamada, K

1994-05-01

In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.

GLABROUS INFLORESCENCE STEMS modulates the regulation by gibberellins of epidermal differentiation and shoot maturation in Arabidopsis.

PubMed

Gan, Yinbo; Kumimoto, Rod; Liu, Chang; Ratcliffe, Oliver; Yu, Hao; Broun, Pierre

2006-06-01

As a plant shoot matures, it transitions through a series of growth phases in which successive aerial organs undergo distinct developmental changes. This process of phase change is known to be influenced by gibberellins (GAs). We report the identification of a putative transcription factor, GLABROUS INFLORESCENCE STEMS (GIS), which regulates aspects of shoot maturation in Arabidopsis thaliana. GIS loss-of-function mutations affect the epidermal differentiation of inflorescence organs, causing a premature decrease in trichome production on successive leaves, stem internodes, and branches. Overexpression has the opposite effect on trichome initiation and causes other heterochronic phenotypes, affecting flowering and juvenile-adult leaf transition and inducing the formation of rosette leaves on inflorescence stems. Genetic and gene expression analyses suggest that GIS acts in a GA-responsive pathway upstream of the trichome initiation regulator GLABROUS1 (GL1) and downstream of the GA signaling repressor SPINDLY (SPY). GIS mediates the induction of GL1 expression by GA in inflorescence organs and is antagonized in its action by the DELLA repressor GAI. The implication of GIS in the broader regulation of phase change is further suggested by the delay in flowering caused by GIS loss of function in the spy background. The discovery of GIS reveals a novel mechanism in the control of shoot maturation, through which GAs regulate cellular differentiation in plants.

Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number.

PubMed

Khuong, Thi Thu Huong; Crété, Patrice; Robaglia, Christophe; Caffarri, Stefano

2013-09-01

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions. Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.

Cold-Specific Induction of a Dehydrin Gene Family Member in Barley.

PubMed Central

Van Zee, K.; Chen, F. Q.; Hayes, P. M.; Close, T. J.; Chen, THH.

1995-01-01

An interval on barley (Hordeum vulgare L.) chromosome 7 accounting for significant quantitative trait locus effects for winter hardiness were detected in a winter (Dicktoo) x spring (Morex) barley population (P.M. Hayes, T. Blake, T.H.H. Chen, S. Tragoonrung, F. Chen, A. Pan, and B. Liu [1993] Genome 36: 66-71). Two members of the barley dehydrin gene family, Dhn1 and Dhn2, were located within the region defining the winter hardiness quantitative trait locus effect (A. Pan, P.M. Hayes, F. Chen, T. Blake, T.H.H. Chen, T.T.S. Wright, I. Karsai, Z. Bedo [1994] Theor Appl Genet 89: 900-910). To investigate the possible role of Dhn1 and Dhn2 in winter hardiness, we examined the expression pattern of six barley dehydrin gene family members in shoot tissue in response to cold temperature. Incubation of 3-week-old barley plants at 2[deg]C resulted in a rapid induction of a single 86-kD polypeptide that was recognized by an antiserum against a peptide conserved in the dehydrin gene family. Northern blot analysis confirmed the induction of an mRNA corresponding to Dhn5. The expression patterns of cold-induced dehydrins in shoot tissue for Dicktoo and Morex were identical under the conditions studied, in spite of the known phenotypic differences in their winter hardiness. These results, together with the allelic structure of selected high- and low-survival lines, suggest that the Dicktoo alleles at the Dhn1 and Dhn2 may not be the primary determinants of winter hardiness in barley. PMID:12228540

Engineering high Zn in tomato shoots through expression of AtHMA4 involves tissue-specific modification of endogenous genes.

PubMed

Kendziorek, Maria; Klimecka, Maria; Barabasz, Anna; Borg, Sören; Rudzka, Justyna; Szczęsny, Paweł; Antosiewicz, Danuta Maria

2016-08-12

To increase the Zn level in shoots, AtHMA4 was ectopically expressed in tomato under the constitutive CaMV 35S promoter. However, the Zn concentration in the shoots of transgenic plants failed to increase at all tested Zn levels in the medium. Modification of Zn root/shoot distribution in tomato expressing 35S::AtHMA4 depended on the concentration of Zn in the medium, thus indicating involvement of unknown endogenous metal-homeostasis mechanisms. To determine these mechanisms, those metal-homeostasis genes that were expressed differently in transgenic and wild-type plants were identified by microarray and RT-qPCR analysis using laser-assisted microdissected RNA isolated from two root sectors: (epidermis + cortex and stele), and leaf sectors (upper epidermis + palisade parenchyma and lower epidermis + spongy parenchyma). Zn-supply-dependent modification of Zn root/shoot distribution in AtHMA4-tomato (increase at 5 μM Zn, no change at 0.5 μM Zn) involved tissue-specific, distinct from that in the wild type, expression of tomato endogenous genes. First, it is suggested that an ethylene-dependent pathway underlies the detected changes in Zn root/shoot partitioning, as it was induced in transgenic plants in a distinct way depending on Zn exposure. Upon exposure to 5 or 0.5 μM Zn, in the epidermis + cortex of the transgenics' roots the expression of the Strategy I Fe-uptake system (ethylene-dependent LeIRT1 and LeFER) was respectively lower or higher than in the wild type and was accompanied by respectively lower or higher expression of the identified ethylene genes (LeNR, LeACO4, LeACO5) and of LeChln. Second, the contribution of LeNRAMP2 expression in the stele is shown to be distinct for wild-type and transgenic plants at both Zn exposures. Ethylene was also suggested as an important factor in a pathway induced in the leaves of transgenic plants by high Zn in the apoplast, which results in the initiation of loading of the excess Zn into the mesophyll of "Zn accumulating cells". In transgenic tomato plants, the export activity of ectopically expressed AtHMA4 changes the cellular Zn status, which induces coordinated tissue-specific responses of endogenous ethylene-related genes and metal transporters. These changes constitute an important mechanism involved in the generation of the metal-related phenotype of transgenic tomato expressing AtHMA4.

Micropropagation and in vitro conservation of the rare and threatened plants Ramonda serbica and Ramonda nathaliae.

PubMed

Gashi, Bekim; Abdullai, Kasamedin; Sota, Valbona; Kongjika, Efigjeni

2015-01-01

Ramonda serbica and Ramonda nathaliae are rare and endemo relict plant species from Balkan Peninsula. An efficient micro propagation and in vitro conservation method via direct and indirect organogenesis from seed and leaf explants, respectively, was established in this study. The seed of both Ramonda species were collected from different populations in Kosovo, and were germinated in nutrient media JG-B without any phytohormone. The highest number of shoots and multiplication rate was observed on JG-B medium supplemented with BAP and IAA (0.5 mg l(-1) each), whereas the highest number of leaves per plantlets was found on WPM and RA medium supplemented with BAP and IAA (0.1 mg l(-1) each). During this stage of micro propagation some significant differences were observed in plantlets from different populations. The indirect organogenesis from parts of leaves of natural plants was not successful due to unavailability of established protocol for disinfections of the plant material. On other hand, parts of leaves from micro propagated plantlets, cultured on MS medium supplemented with different ratio of BAP and NAA, resulted in the highest efficiency for shoot regeneration. In vitro conservation of micro propagated plants at the lower temperature (4 °C) had a significantly positive effect for storage of more than 12 months.

Cryopreservation of in vitro shoot apices of Oxalis tuberosa Mol.

PubMed

Gonzalez-Benito, M E; Mendoza-Condori, V H; Molina-Garcia, A D

2007-01-01

Oca (Oxalis tuberosa Mol.) is an under-utilized tuber crop from the Andean region. Cryopreservation would allow the safe and long-term preservation of the genetic resources of this crop. A protocol for the cryopreservation of in vitro grown shoots has been developed using the vitrification solution PVS2. Two genotypes were studied (G1 and G27). Nodal segments were cultured on MS medium and incubated at 10 degree C with 16 h photoperiod and 10 mol per square meter per second irradiance, for two weeks. Apices were then excised and cultured on MS+0.15 M sucrose for 3 days at 5 degree C in darkness. Subsequently, apices were immersed in a loading solution (liquid MS medium+2 M glycerol+0.4 M sucrose), and then treated with the vitrification solution PVS2 for 0 to 40 minutes. Cryovials were then immersed in liquid nitrogen. Four weeks after rewarming and culture on recovery medium, genotype G1 showed approximately 60 percent recovery (normal growth) with 20 min PVS2 treatment. Genotype G27 showed lower recovery (30 percent). Differential scanning calorimetry yielded a Tg midpoint for PSV2 solution of ca. -120 degree C. Calorimetric studies on apices at different stages of the cryopreservation protocol showed a change in calorimetric parameters consistent with a decrease in the amount of frozen water as the protocol advanced.

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

PubMed

Babur, Betul Kul; Futrega, Kathryn; Lott, William B; Klein, Travis Jacob; Cooper-White, Justin; Doran, Michael Robert

2015-09-01

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

Joint enhancement of lead accumulation in Brassica plants by EDTA and ammonium sulfate in sand culture.

PubMed

Xiong, Zhi-ting; Lu, Ping

2002-04-01

When EDTA was added alone in the Pb-contaminated sand, the plant biomass and the total Pb amount in Plant decreased in both species, Brassica pekinensis and B. juncea var. multiceps, though the shoot Pb amount increased. In contrast, when (NH4)2SO4 was added alone in the Pb-contaminated sand, little effect was observed on the shoot Pb amount, though the root Pb amount was significantly increased in B. juncea var. multiceps. When amending EDTA and (NH4)2SO4 in combination, however, the shoot Pb amount in both species substantially increased, being, on an average, 2 times and 9 times higher than that in EDTA alone or (NH4)2SO4 alone amended treatment, respectively. The two amendments showed antagonism for plant growth, but synergism for Pb bioaccumulation. B. pekinensis showed its highest level of shoot and total Pb amount in the treatment amended with EDTA and (NH4)2SO4 only a half as much as in the other treatments. It is inferred that the mechanisms responsible for the joint-enhanced Pb accumulation might be concerned with the acidification of the growth medium, cation exchange reaction and relieving EDTA induced toxicity as results by amending ammonium sulfate.

Reduction and Coordination of Arsenic in Indian Mustard1

PubMed Central

Pickering, Ingrid J.; Prince, Roger C.; George, Martin J.; Smith, Robert D.; George, Graham N.; Salt, David E.

2000-01-01

The bioaccumulation of arsenic by plants may provide a means of removing this element from contaminated soils and waters. However, to optimize this process it is important to understand the biological mechanisms involved. Using a combination of techniques, including x-ray absorption spectroscopy, we have established the biochemical fate of arsenic taken up by Indian mustard (Brassica juncea). After arsenate uptake by the roots, possibly via the phosphate transport mechanism, a small fraction is exported to the shoot via the xylem as the oxyanions arsenate and arsenite. Once in the shoot, the arsenic is stored as an AsIII-tris-thiolate complex. The majority of the arsenic remains in the roots as an AsIII-tris-thiolate complex, which is indistinguishable from that found in the shoots and from AsIII-tris-glutathione. The thiolate donors are thus probably either glutathione or phytochelatins. The addition of the dithiol arsenic chelator dimercaptosuccinate to the hydroponic culture medium caused a 5-fold-increased arsenic level in the leaves, although the total arsenic accumulation was only marginally increased. This suggests that the addition of dimercaptosuccinate to arsenic-contaminated soils may provide a way to promote arsenic bioaccumulation in plant shoots, a process that will be essential for the development of an efficient phytoremediation strategy for this element. PMID:10759512

High accumulation of anthocyanins via the ectopic expression of AtDFR confers significant salt stress tolerance in Brassica napus L.

PubMed

Kim, Jihye; Lee, Won Je; Vu, Tien Thanh; Jeong, Chan Young; Hong, Suk-Whan; Lee, Hojoung

2017-08-01

The ectopic expression of AtDFR results in increased accumulation of anthocyanins leading to enhanced salinity and drought stress tolerance in B. napus plants. Flavonoids with antioxidant effects confer many additional benefits to plants. Evidence indicates that flavonoids, including anthocyanins, protect tissues against oxidative stress from various abiotic stressors. We determined whether increases in anthocyanins increased abiotic stress tolerance in Brassica napus, because the values of B. napus L. and its cultivation area are increasing worldwide. We overexpressed Arabidopsis dihydroflavonol-4-reductase (DFR) in B. napus. Increased DFR transcript levels for AtDFR-OX B. shoots correlated with higher anthocyanin accumulation. AtDFR-OX Brassica shoots exhibited lower reactive oxygen species (ROS) accumulation than wild-type (WT) shoots under high NaCl and mannitol concentrations. This was corroborated by 3,3-diaminobenzidine staining for ROS scavenging activity in 1,1-diphenyl-2-picryl-hydrazyl assays. Shoots of the AtDFR-OX B. napus lines grown in a high salt medium exhibited enhanced salt tolerance and higher chlorophyll content than similarly grown WT plants. Our observations suggested that the AtDFR gene can be effectively manipulated to modulate salinity and drought stress tolerance by directing to high accumulation of anthocyanins in oilseed plants.

Micropropagation of juvenile and mature american beech

Treesearch

Melanie J. Barker; Paula M. Pijut; Michael E. Ostry; David R. Houston

1997-01-01

The purpose of this study was to micropropagate juvenile and mature American beech (Fagus grandifolia Ehrh.) resistant to beech bark disease. Shoot tips (from juvenile seedlings and root sprouts of mature trees) and buds from branches of mature trees, were cultured and multiplied on aspen culture medium supplemented with 0.89 ?M 6-benzyladenine, 0.27 ?M a-...

Adventitious shoot regeneration and rooting of Prunus serotina in vitro cultures

Treesearch

Ana Carolina Espinosa; Paula M. Pijut; Charles H. Michler

2006-01-01

A complete regeneration protocol was developed for Prunus serotina Ehrh., an important hardwood species for timber and sawlog production in the central and eastern United States. Nodal sections were cultures on Murashige and Skoog (MS) medium supplemented with 4.44 µM 6-benzylaminopurine (BA), 0.49 µM indole-3-butyric acid (IBA),...

Girdling and Applying Chemicals Promote Rapid Rooting of Sycamore Cuttings

Treesearch

Robert C. Hare

1975-01-01

Shoots of 6- and 13-year-old sycamore (Platanus occidentalis L.) were girdled and treated with rooting powder 4 weeks before cuttings were taken. The powder, which contained auxins, sucrose, and cap tan, was also applied basally to nongirdled cuttings immediately before iwertion in a rooting medium. Thirteen days later, 100 percent of the...

Antioxidative efficiency of Triticum aestivum L. exposed to chromium stress.

PubMed

Dey, Surjendu Kumar; Jena, Priyanka Priyadarshani; Kundu, Satyajit

2009-07-01

Wheat (Triticum aestivum L. cv Sonalika) seedlings were grown in presence of K2Cr2O7 (10, 50 and 100 ppm) for 7 days and growth, total chlorophyll, activities of antioxidative enzymes like superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6) and guaiacol peroxidase (POX; EC 1.11.1.7) and lipid peroxidation were determined in root and shoot tissues. Growth of the seedlings was significantly (p < or = 0.05) depressed and at 100 ppm, root length was reduced by 63% and shoot length by 44% in comparison to the respective controls. Total chlorophyll loss in shoots was about 46% at 10 ppm of K2Cr2O7 which further increased to 80% at 100 ppm. Both in root and shoot tissues, activities of SOD and CAT declined with increase of metal in growth medium and it was significant (p < or = 0.05) even at lowest concentration of the metal tested. But POX activity showed a different trend. In root tissues it was decreased whereas in shoots, there was many fold increase in the activity (about 370% over control at 100 ppm). Malondialdehyde (MDA) content increased both in root and shoot tissues, but it reached significant (p < 0.05) level at 50 ppm in roots and at 100 ppm in shoot tissues. Even though antioxidative enzyme activities were not assayed in germinating embryos, inhibition in germination percentage (by 40% at 100 ppm) and increase in lipid peroxidation level (by 71% over control at 100 ppm) were observed in 2-day-old embryos, germinated in presence of K2Cr2O7 (10, 50 and 100 ppm). The results indicated the imposition of oxidative stress situations both during germination and early stages of seedling growth by Cr6 stress, which might be one of the probable reasons behind Cr toxicity in plants.

Auto-induction for high level production of biologically active reteplase in Escherichia coli.

PubMed

Fathi-Roudsari, Mehrnoosh; Maghsoudi, Nader; Maghsoudi, Amirhossein; Niazi, Sepideh; Soleiman, Morvarid

2018-06-07

Reteplase is a third generation tissue plasminogen activator (tPA) with a modified structure and prolonged half-life in comparison to native tPA. As a non-glycosylated protein, reteplase is expressed in Escherichia coli. Due to presence of several disulfide bonds, high level production of reteplase is complicated and needs extra steps for conversion to biologically active form. Auto-induction represents a method for high-yield growth of bacterial cells and higher expression of recombinant proteins. Here we have tried to optimize the auto-induction procedure for soluble and active expression of reteplase in E. coli. Results showed that using auto-induction strategy at 37 °C, Rosetta-gami (DE3) had the highest level of active and soluble reteplase production in comparison to E. coli strains BL21 (DE3), and Shuffel T7. Temperature dominantly affected the level of active reteplase production. Decreasing the temperature to 25 and 18 °C increased the level of active reteplase by 20 and 60%, respectively. The composition of auto-induction medium also dramatically changed the active production of reteplase in cytoplasm. Using higher enriched auto-induction medium, super broth base including trace elements, significantly increased biologically active reteplase by 30%. It is demonstrated here that auto-induction is a powerful method for expression of biologically active reteplase in oxidative cytoplasm of Rosetta-gami. Optimizing expression condition by decreasing temperature and using an enriched auto-induction medium resulted in at least three times higher level of active reteplase production. Production of correctly folded and active reteplase in spite of its complex structure helps for removal of inefficient and cumbersome step of refolding. Copyright © 2018. Published by Elsevier Inc.

Early histological, hormonal, and molecular changes during pineapple (Ananas comosus (L.) Merrill) artificial flowering induction.

PubMed

Espinosa, Maita Eulalia Ávila; Moreira, Rafael Oliveira; Lima, André Almeida; Ságio, Solange Aparecida; Barreto, Horllys Gomes; Luiz, Sara Lazara Pérez; Abreu, Carlos Eduardo Aragón; Yanes-Paz, Ermis; Ruíz, Yanelis Capdesuñer; González-Olmedo, Justo Lorenzo; Chalfun-Júnior, Antonio

2017-02-01

Natural flowering can cause serious scheduling problems in the pineapple (Ananas comosus) industry and increase harvest costs. Pineapple flowering is thought to be triggered by increased ethylene levels and artificial forcing of pineapple flowering is a common practice to promote flowering synchronisation. However, little is known about the early hormonal and molecular changes of pineapple flowering induction and development. Here, we aimed to analyse the molecular, hormonal, and histological changes during artificial pineapple flowering by Ethrel ® 48 treatment. Histological analyses of the shoot apical meristem, leaf gibberellic acid (GA 3 ), and ethylene quantification were carried out during the first 72h after Ethrel ® 48 treatment. Expression profiles from ethylene biosynthesis (AcACS2 and AcACO1), gibberellin metabolism (AcGA2-ox1 and AcDELLA1), and flower development (FT-like gene (AcFT), LFY-like gene (AcLFY), and a PISTILLATA-like gene (AcPI)) genes were analysed during the first 24h after Ethrel ® 48 treatment. Differentiation processes of the shoot apical meristem into flower buds were already present in the first 72h after Ethrel ® 48 treatment. Ethrel ® 48 lead to a reduction in GA 3 levels, probably triggered by elevated ethylene levels and the positive regulation AcGA2-ox1. AcLFY activation upon Ethrel ® 48 may also have contributed to the reduction of GA 3 levels and, along with the up-regulation of AcPI, are probably associated with the flower induction activation. AcFT and AcDELLA1 do not seem to be regulated by GA 3 and ethylene. Decreased GA 3 and increased ethylene levels suggest an accumulation of AcDELLA1, which may display an important role in pineapple flowering induction. Thus, this study shows that molecular, hormonal, and histological changes are present right after Ethrel ® 48 treatment, providing new insights into how pineapple flowering occurs under natural conditions. Copyright © 2016 Elsevier GmbH. All rights reserved.

Regeneration of Stevia Plant Through Callus Culture

PubMed Central

Patel, R. M.; Shah, R. R.

2009-01-01

Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions. PMID:20177455

Effects of carbon ion beam irradiation on the shoot regeneration from in vitro axillary bud explants of the Impatiens hawkeri

NASA Astrophysics Data System (ADS)

Zhou, Libin; Zhou, Libin; Li, Wenjian; Li, Ping; Dong, Xicun; Qu, Ying; Ma, Shuang; Li, Qiang

Accelerated ion beams is an excellent mutagen in plant breeding which can induce higher mutation frequencies and wider mutation spectrum than those of low linear energy transfer (LET) irradiations, such as X-rays (Okamura et al. 2003, Yamaguchi et al. 2003). Mutation breeding operation of two Saintpaulia ionahta cultivars using the method combining plant tissue culture technique and carbon ion beam irradiations were set out at Institute of Modern Physics from 2005 (Zhou et al. 2006). The effects of 960 MeV carbon ion beam and 8 MeV X-ray irradiations on regenerated shoots of Impatiens hawkeri from another kind of explants named in vitro axillary buds explants were studied recently. The biology endpoints in this study included relative number of roots (RNR), relative length of roots (RLR), relative height of shoots (RHS), relative number of nodes (RNN), survival fraction (SF) and morphology changes in the regenerated shoots. The experimental results showed that carbon ion beams inhibited the root and stem developments of axillary bud explants more severely than X-rays did. And the 50% lethal dose (LD50 ) is about 23.3 Gy for the carbon ion beam and 49.1 Gy for the X-rays, respectively. Relative biological effectiveness (RBE) of Impatiens hawkeri with respect to X-rays according to 50% SF was about two. Secondly, the percentage of shoots regenerated with malformed shoots including curliness, carnification, nicks in all Impatiens hawkeri axillary bud explants irradiated with carbon ion beam at 20 Gy accounted for 55.6%, while the highest number for the 40 Gy X-ray irradiation was 40%. Last, many regenerated shoots whose vascular bundle fused together were obtained only from explants irradiated with carbon ion beams. Based on the results above, it can be concluded that the effect of mutation induction by carbon ion beam irradiation on the axillary explants of Impatiens hawkeri is better than that by X-ray irradiation; and the optimal mutagenic dose varies from 20 Gy for carbon ion beam irradiation.

Somatic embryogenesis and plant regeneration in tissue cultures of sweet potato (Ipomea batatas Poir.).

PubMed

Liu, J R; Cantliffe, D J

1984-06-01

Leaf, shoot-tip, stem, and root explants of sweet potato (Ipomea batatas Poir.) gave rise to two kinds of callus on nutrient agar medium containing 0.5 to 2.0 mg/l 2,4-D. One callus, bright- to pale-yellow, was compact and organized, while the other was dull-yellow and friable. The former callus gave rise to numerous globular and heart-shaped embryoids. When transferred onto hormone-free medium, the embryoids readily developed into a torpedo-shape before germination. The plantlets were transplanted to soil where they flowered and formed storage roots at maturity.

Optimising Sterilisation Techniques and Callus Induction of Nodes Durio Zibethinus Murr in Vitro Method with Various Media

NASA Astrophysics Data System (ADS)

Hermayani, N.; Retnoningsih, A.; Rahayu, E. S.

2017-04-01

The application of in vitro propagation method needs an aseptic or sterile condition. The objective of the study was to get an optimal sterilisation techniques and medium of callus induction of Durio zibethinus. Sterilisation treatments studied were NaClO and Ca(ClO)2. The three kind of callus induction medium studied were Gamborg (B5), Woody Plant Medium (WPM), and Murashige and Skoog (MS) with the addition of auxin and cytokinin. The experiment unit was three bottles with nodes as explant. Cultures were kept in the culture room for 16 hours daily by LED light intensity of 1000 lux. Parameters investigated of sterilisation technique development was the percentage of contamination, the percentage of explant browning, and the percentage of life explants; whereas the callus induction measured were the rate of callus formation, percentage of callus covered, callus texture, colour and diameter of callus. The results showed that the code of C3 is soaking in calcium hypochlorite (Ca(ClO)2) 50% for 3 minutes, calcium hypochlorite 40% for 2 minutes, and 70% alcohol for 30 seconds separately were suitable for sterilisation of nodes explant. The development of callus on B5 medium with the addition of auxin (2,4-D) 2 ppm and cytokinin (Thidiazuron) 1 ppm was best compared to the other.

Artificial Neural Network Genetic Algorithm As Powerful Tool to Predict and Optimize In vitro Proliferation Mineral Medium for G × N15 Rootstock.

PubMed

Arab, Mohammad M; Yadollahi, Abbas; Shojaeiyan, Abdolali; Ahmadi, Hamed

2016-01-01

One of the major obstacles to the micropropagation of Prunus rootstocks has, up until now, been the lack of a suitable tissue culture medium. Therefore, reformulation of culture media or modification of the mineral content might be a breakthrough to improve in vitro multiplication of G × N15 (garnem). We found artificial neural network in combination of genetic algorithm (ANN-GA) as a very precise and powerful modeling system for optimizing the culture medium, So that modeling the effects of MS mineral salts ([Formula: see text], [Formula: see text], [Formula: see text], Ca 2+ , K + , [Formula: see text], Mg 2+ , and Cl - ) on in vitro multiplication parameters (the number of microshoots per explant, average length of microshoots, weight of calluses derived from the base of stem explants, and quality index of plantlets) of G × N15. Showed high R 2 correlation values of 87, 91, 87, and 74 between observed and predicted values were found for these four growth parameters, respectively. According to the ANN-GA results, among the input variables, [Formula: see text] and [Formula: see text] had the highest values of VSR in data set for the parameters studied. The ANN-GA showed that the best proliferation rate was obtained from medium containing (mM) 27.5 [Formula: see text], 14 [Formula: see text], 5 Ca 2+ , 25.9 K + , 0.7 Mg 2+ , 1.1 [Formula: see text], 4.7 [Formula: see text], and 0.96 Cl - . The performance of the medium optimized by ANN-GA, denoted as YAS (Yadollahi, Arab and Shojaeiyan), was compared to that of standard growth media for all Prunus rootstock, including the Murashige and Skoog (MS) medium, (specific media) EM, Quoirin and Lepoivre (QL) medium, and woody plant medium (WPM) Prunus . With respect to shoot length, shoot number per cultured explant and productivity (number of microshoots × length of microshoots), YAS was found to be superior to other media for in vitro multiplication of G × N15 rootstocks. In addition, our results indicated that by using ANN-GA, we were able to determine a suitable culture medium formulation to achieve the best in vitro productivity.

Diazotrophic bacteria isolated from wild rice Oryza glumaepatula (Poaceae) in the Brazilian Amazon.

PubMed

Júnior, Paulo Ivan Fernandes; Pereira, Gilmara Maria Duarte; Perin, Liamara; da Silva, Luana Mesquita; Baraúna, Alexandre Cardoso; Alvess, Francilene Muniz; Passos, Samuel Ribeiro; Zilli, Jerri Edson

2013-06-01

The association of wild grasses with diazotrophic bacteria in Brazilian biomes is poorly understood. The isolation and characterization of bacteria associated with wild grasses can contribute to understand the diazotrophic ecology as well as to identify bacteria with biotechnological applications. In this study, we isolated and characterized diazotrophic bacterial isolates from Oryza glumaepatula collected in Cerrado and Forest areas of the Amazon in Roraima State, Brazil. Healthy O. glumepatula plants were collected at five sampling sites at Forest and seven at Cerrado, respectively. The plants were collected at the Cerrado areas in September 2008 while the Forest plants were collected in June/2008 and April/2009. The plants and the soil adhering to the roots were transferred to pots and grown for 35 days in greenhouse conditions. During the harvest, the shoots and the roots were crushed separately in a saline solution; the suspension was diluted serially and inoculated in Petri dishes containing Dyg's medium. All distinct bacterial colonies were purified in the same medium. The diazotrophic capacity of each bacterium in microaerophilic conditions was assessed in semisolid BMGM medium. In addition, the pellicles forming bacterial isolates were also evaluated by PCR amplification for nifH gene. The diversity of nifH bacteria was analyzed by Box-PCR fingerprinting. For selected strains, the growth promoting capacity of O. sativa as a model plant was also evaluated. A total of 992 bacterial isolates were obtained. Fifty-one bacteria were able to form pellicles in the semisolid medium and 38 also positively amplified the 360 bp nifH gene fragment. Among the 38 nifH+ isolates, 24 were obtained from the shoots, while 14 originated from the roots. The Box-PCR profiles showed that the bacterial isolates obtained in this study presented a low similarity with the reference strains belonging to the Herbaspirillum, Azospirillum and Burkholderia genus. The growth-promoting ability was confirmed for at least five isolates. For these bacteria, the root and shoot growing results showed higher increases when compared to those observed in plants inoculated with the evaluated reference strains. These results indicate that O. glumaepatula is colonized by a high diverse diazotrophic community in the Brazilian Amazon. Further investigations are now being carried out to determine the taxonomic positions of these isolates and their growth promoting mechanisms.

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni)

PubMed Central

Khalil, Shahid Akbar; Zamir, Roshan; Ahmad, Nisar

2014-01-01

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l−1). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l−1) and gibberellic acid (GA3; 0.5 mg l−1). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%). PMID:25473365

Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni).

PubMed

Khalil, Shahid Akbar; Zamir, Roshan; Ahmad, Nisar

2014-12-01

Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Pakistan. That is why people did not know the exact time of propagation. The main objective of the present study was to establish feasible propagation methods for healthy biomass production. In the present study, seed germination, stem cuttings and micropropagation were investigated for higher productivity. Fresh seeds showed better germination (25.51-40%) but lost viability after a few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth regulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium augmented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg l(-1)). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg l(-1)) and gibberellic acid (GA3; 0.5 mg l(-1)). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51-40%) and stem cutting (60%).

Histological and transcript analyses of intact somatic embryos in an elite maize (Zea mays L.) inbred line Y423.

PubMed

Liu, Beibei; Su, Shengzhong; Wu, Ying; Li, Ying; Shan, Xiaohui; Li, Shipeng; Liu, Hongkui; Dong, Haixiao; Ding, Meiqi; Han, Junyou; Yuan, Yaping

2015-07-01

Intact somatic embryos were obtained from an elite maize inbred line Y423, bred in our laboratory. Using 13-day immature embryos after self-pollination as explants, and after 4-5 times subculture, a large number of somatic embryos were detected on the surface of the embryonic calli on the medium. The intact somatic embryos were transferred into the differential medium, where the plantlets regenerated with shoots and roots forming simultaneously. Histological analysis and scanning electron micrographs confirmed the different developmental stages of somatic embryogenesis, including globular-shaped embryo, pear-shaped embryo, scutiform embryo, and mature embryo. cDNA-amplified fragment length polymorphism (cDNA-AFLP) was used for comparative transcript profiling between embryogenic and non-embryogenic calli of a new elite maize inbred line Y423 during somatic embryogenesis. Differentially expressed genes were cloned and sequenced. Gene Ontology analysis of 117 candidate genes indicated their involvement in cellular component, biological process and molecular function. Nine of the candidate genes were selected. The changes in their expression levels during embryo induction and regeneration were analyzed in detail using quantitative real-time PCR. Two full-length cDNA sequences, encoding ZmSUF4 (suppressor of fir 4-like protein) and ZmDRP3A (dynamin-related protein), were cloned successfully from intact somatic embryos of the elite inbred maize line Y423. Here, a procedure for maize plant regeneration from somatic embryos is described. Additionally, the possible roles of some of these genes during the somatic embryogenesis has been discussed. This study is a systematic analysis of the cellular and molecular mechanism during the formation of intact somatic embryos in maize. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Occupational exposure to airborne lead in Brazilian police officers.

PubMed

Rocha, Ernesto Díaz; Sarkis, Jorge E Souza; Carvalho, Maria de Fátima H; Santos, Gerson Vechio Dos; Canesso, Claudemir

2014-07-01

Shooting with lead-containing ammunition in indoor firing ranges is a known source of lead exposure in adults. Police officers may be at risk of lead intoxication when regular training shooting exercises are yearly mandatory to law enforcement officers. Effects on health must be documented, even when low-level elemental (inorganic) lead exposure is detected. Forty police officers (nineteen cadets and twenty-one instructors) responded to a questionnaire about health, shooting habits, and potential lead exposure before a training curse. Blood samples were collected and analyzed for blood lead level (BLL) before and after a three days training curse. The mean BLL for the instructors' group was 5.5 μg/dL ± 0.6. The mean BLL for the cadets' group before the training was 3.3 μg/dL ± 0.15 and after the training the main BLL was 18.2 μg/d L± 1.5. Samples were analyzed by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). All the participants in the training curse had significantly increased BLL (mean increment about 15 μg/dL) after the three days indoor shooting season. In conclusion, occupational lead exposure in indoor firing ranges is a source of lead exposure in Brazilian police officers, and appears to be a health risk, especially when heavy weapons with lead-containing ammunition are used in indoor environments during the firing training seasons. Copyright © 2013 Elsevier GmbH. All rights reserved.

Induction of root Fe(lll) reductase activity and proton extrusion by iron deficiency is mediated by auxin-based systemic signalling in Malus xiaojinensis.

PubMed

Wu, Ting; Zhang, Heng-Tao; Wang, Yi; Jia, Wen-Suo; Xu, Xue-Feng; Zhang, Xin-Zhong; Han, Zhen Hai

2012-01-01

Iron is a critical cofactor for a number of metalloenzymes involved in respiration and photosynthesis, but plants often suffer from iron deficiency due to limited supplies of soluble iron in the soil. Iron deficiency induces a series of adaptive responses in various plant species, but the mechanisms by which they are triggered remain largely unknown. Using pH imaging and hormone localization techniques, it has been demonstrated here that root Fe(III) reductase activity and proton extrusion upon iron deficiency are up-regulated by systemic auxin signalling in a Fe-efficient woody plant, Malus xiaojinensis. Split-root experiments demonstrated that Fe-deprivation in a portion of the root system induced a dramatic increase in Fe(III) reductase activity and proton extrusion in the Fe-supplied portion, suggesting that the iron deficiency responses were mediated by a systemic signalling. Reciprocal grafting experiments of M. xiaojinensis with Malus baccata, a plant with no capability to produce the corresponding responses, indicate that the initiation of the systemic signalling is likely to be determined by roots rather than shoots. Iron deficiency induced a substantial increase in the IAA content in the shoot apex and supplying exogenous IAA analogues (NAA) to the shoot apex could mimic the iron deficiency to trigger the corresponding responses. Conversely, preventing IAA transport from shoot to roots blocked the iron deficiency responses. These results strongly indicate that the iron deficiency-induced physiological responses are mediated by systemic auxin signalling.

Expression Profiling of FLOWERING LOCUS T-Like Gene in Alternate Bearing ‘Hass' Avocado Trees Suggests a Role for PaFT in Avocado Flower Induction

PubMed Central

Ziv, Dafna; Zviran, Tali; Zezak, Oshrat; Samach, Alon; Irihimovitch, Vered

2014-01-01

In many perennials, heavy fruit load on a shoot decreases the ability of the plant to undergo floral induction in the following spring, resulting in a pattern of crop production known as alternate bearing. Here, we studied the effects of fruit load on floral determination in ‘Hass' avocado (Persea americana). De-fruiting experiments initially confirmed the negative effects of fruit load on return to flowering. Next, we isolated a FLOWERING LOCUS T-like gene, PaFT, hypothesized to act as a phloem-mobile florigen signal and examined its expression profile in shoot tissues of on (fully loaded) and off (fruit-lacking) trees. Expression analyses revealed a strong peak in PaFT transcript levels in leaves of off trees from the end of October through November, followed by a return to starting levels. Moreover and concomitant with inflorescence development, only off buds displayed up-regulation of the floral identity transcripts PaAP1 and PaLFY, with significant variation being detected from October and November, respectively. Furthermore, a parallel microscopic study of off apical buds revealed the presence of secondary inflorescence axis structures that only appeared towards the end of November. Finally, ectopic expression of PaFT in Arabidopsis resulted in early flowering transition. Together, our data suggests a link between increased PaFT expression observed during late autumn and avocado flower induction. Furthermore, our results also imply that, as in the case of other crop trees, fruit-load might affect flowering by repressing the expression of PaFT in the leaves. Possible mechanism(s) by which fruit crop might repress PaFT expression, are discussed. PMID:25330324

Expression profiling of FLOWERING LOCUS T-like gene in alternate bearing 'Hass' avocado trees suggests a role for PaFT in avocado flower induction.

PubMed

Ziv, Dafna; Zviran, Tali; Zezak, Oshrat; Samach, Alon; Irihimovitch, Vered

2014-01-01

In many perennials, heavy fruit load on a shoot decreases the ability of the plant to undergo floral induction in the following spring, resulting in a pattern of crop production known as alternate bearing. Here, we studied the effects of fruit load on floral determination in 'Hass' avocado (Persea americana). De-fruiting experiments initially confirmed the negative effects of fruit load on return to flowering. Next, we isolated a FLOWERING LOCUS T-like gene, PaFT, hypothesized to act as a phloem-mobile florigen signal and examined its expression profile in shoot tissues of on (fully loaded) and off (fruit-lacking) trees. Expression analyses revealed a strong peak in PaFT transcript levels in leaves of off trees from the end of October through November, followed by a return to starting levels. Moreover and concomitant with inflorescence development, only off buds displayed up-regulation of the floral identity transcripts PaAP1 and PaLFY, with significant variation being detected from October and November, respectively. Furthermore, a parallel microscopic study of off apical buds revealed the presence of secondary inflorescence axis structures that only appeared towards the end of November. Finally, ectopic expression of PaFT in Arabidopsis resulted in early flowering transition. Together, our data suggests a link between increased PaFT expression observed during late autumn and avocado flower induction. Furthermore, our results also imply that, as in the case of other crop trees, fruit-load might affect flowering by repressing the expression of PaFT in the leaves. Possible mechanism(s) by which fruit crop might repress PaFT expression, are discussed.

Involvement of alternative oxidase (AOX) in adventitious rooting of Olea europaea L. microshoots is linked to adaptive phenylpropanoid and lignin metabolism.

PubMed

Santos Macedo, E; Sircar, D; Cardoso, H G; Peixe, A; Arnholdt-Schmitt, B

2012-09-01

Alternative oxidase (AOX) has been proposed as a functional marker candidate in a number of events involving cell differentiation, including rooting efficiency in semi-hardwood shoot cuttings of olive (Olea europaea L.). To ascertain the general importance of AOX in olive rooting, the auxin-induced rooting process was studied in an in vitro system for microshoot propagation. Inhibition of AOX by salicylhydroxamic acid (SHAM) significantly reduced rooting efficiency. However, the inhibitor failed to exhibit any effect on the preceding calli stage. This makes the system appropriate for distinguishing dedifferentiation and de novo differentiation during root induction. Metabolite analyses of microshoots showed that total phenolics, total flavonoids and lignin contents were significantly reduced upon SHAM treatment. It was concluded that the influence of alternative respiration on root formation was associated to adaptive phenylpropanoid and lignin metabolism. Transcript profiles of two olive AOX genes (OeAOX1a and OeAOX2) were examined during the process of auxin-induced root induction. Both genes displayed stable transcript accumulation in semi-quantitative RT-PCR analysis during all experimental stages. In contrary, when the reverse primer for OeAOX2 was designed from the 3'-UTR instead of the ORF, differential transcript accumulation was observed suggesting posttranscriptional regulation of OeAOX2 during metabolic acclimation. This result confirms former observations in olive semi-hardwood shoot cuttings on differential OeAOX2 expression during root induction. It further points to the importance of future studies on the functional role of sequence and length polymorphisms in the 3'-UTR of this gene. The manuscript reports the general importance of AOX in olive adventitious rooting and the association of alternative respiration to adaptive phenylpropanoid and lignin metabolism.

Epigenomics and bolting tolerance in sugar beet genotypes.

PubMed

Hébrard, Claire; Peterson, Daniel G; Willems, Glenda; Delaunay, Alain; Jesson, Béline; Lefèbvre, Marc; Barnes, Steve; Maury, Stéphane

2016-01-01

In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Endophytic Penicillium citrinum Thom. from Scoparia dulcis Linn.

PubMed

Mathew, Annie J; Jayachandran, K; Mathew, Jyothis

2010-10-01

Scoparia dulcis of Scrophulariaceae is an annual herb distributed through out the tropics. Penicillium citrinum was obtained from apparently healthy roots, stem, leaves and fruits of this plant. Callus and multiple shoots produced during micropropagation from various explants were also symptomless but showed occurrence of Penicillium citrinum when cultured in Murashige & Skoog liquid medium for the production of secondary metabolites.

Shoot regeneration and plantlet formation by cascade huckleberry, mountain huckleberry, and in oval-leaf bilberry on a zeatin-containing nutrient medium

USDA-ARS?s Scientific Manuscript database

A plant regeneration protocol was developed for Cascade huckleberry (Vaccinium deliciosum Piper), mountain huckleberry (V. membranaceum Douglas ex Hooker) and for oval-leaf bilberry (V. ovalifolium Smith) clones. The effects of zeatin concentrations (0, 4.6, 9.1 and 13.7 µM) and explant type (leaf a...

Reduction in pathogen populations at grapevine wound sites is associated with the mechanism underlying the biological control of crown gall by rhizobium vitis strain ARK-1.

PubMed

Kawaguchi, Akira

2014-09-17

A nonpathogenic strain of Rhizobium (=Agrobacterium) vitis, ARK-1, limited the development of grapevine crown gall. A co-inoculation with ARK-1 and the tumorigenic strain VAT07-1 at a 1:1 cell ratio resulted in a higher population of ARK-1 than VAT07-1 in shoots without tumors, but a significantly lower population of ARK-1 than VAT07-1 in grapevine shoots with tumors. ARK-1 began to significantly suppress the VAT07-1 population 2 d after the inoculation. This result indicated that ARK-1 reduced the pathogen population at the wound site through biological control. Although ARK-1 produced a zone of inhibition against other tumorigenic Rhizobium spp. in in vitro assays, antibiosis depended on the culture medium. ARK-1 did not inhibit the growth of tumorigenic R. radiobacter strain AtC1 in the antibiosis assay, but suppressed the AtC1-induced formation of tumors on grapevine shoots, suggesting that antibiosis by ARK-1 may not be the main mechanism responsible for biological control.

The endophytic bacterium Serratia sp. PW7 degrades pyrene in wheat.

PubMed

Zhu, Xuezhu; Wang, Wanqing; Crowley, David E; Sun, Kai; Hao, Shupeng; Waigi, Michael Gatheru; Gao, Yanzheng

2017-03-01

This research was conducted to isolate polycyclic aromatic hydrocarbon-degrading (PAH-degrading) endophytic bacteria and investigate their potential in protecting plants against PAH contamination. Pyrene-degrading endophytic bacteria were isolated from plants grown in PAH-contaminated soil. Among these endophytic bacteria, strain PW7 (Serratia sp.) isolated from Plantago asiatica was selected to investigate the suppression of pyrene accumulation in Triticum aestivum L. In the in vitro tests, strain PW7 degraded 51.2% of the pyrene in the media within 14 days. The optimal biodegradation conditions were pH 7.0, 30 °C, and MS medium supplemented with additional glucose, maltose, sucrose, and peptones. In the in vivo tests, strain PW7 successfully colonized the roots and shoots of inoculated (E + ) wheat plants, and its colonization decreased pyrene accumulation and pyrene transportation from roots to shoots. Remarkably, the concentration of pyrene in shoots decreased much more than that in roots, suggesting that strain PW7 has the potential for protecting wheat against pyrene contamination and mitigating the threat of pyrene to human health via food consumption.

Micropropagation of Helleborus through axillary budding.

PubMed

Beruto, Margherita; Viglione, Serena; Bisignano, Alessandro

2013-01-01

Helleborus genus, belonging to the Ranunculaceae family, has 20 species of herbaceous perennial flowering plants. The commercial exploitation of this plant is dependent on the selection and propagation of appropriate lines. High propagation rate could be accomplished by using a suitable tissue culture method enabling the rapid introduction of valuable selections in the market. However, in vitro cultivation of Helleborus is still very difficult. Thereby the development of reliable in vitro propagation procedures is crucial for future production systems. Axillary buds cultured on agar-solidified Murashige and Skoog medium supplemented with 1 mg/L benzyladenine, 0.1 mg/L β-naphthoxyacetic acid, and 2 mg/L isopentenyl adenine develop shoots after 16 weeks of culture under 16 h light regime, 50-60 μmol/s/m(2), and 19 ± 1°C. The multiplication rate ranges from 1.4 to 2.1. However, the genotype and the number of subcultures affect the efficiency of the micropropagation process. The rooting of shoots is about 80% in solidified MS medium containing 1 mg/L 1-naphthaleneacetic acid and 3 mg/L indole-3-butyric acid. The described protocol provides information which can contribute to the commercial production of Helleborus plants.

Organic amendments enhance Pb tolerance and accumulation during micropropagation of Daphne jasminea.

PubMed

Wiszniewska, Alina; Muszyńska, Ewa; Hanus-Fajerska, Ewa; Smoleń, Sylwester; Dziurka, Michał; Dziurka, Kinga

2017-01-01

The study investigated the effects of organic amendments: pineapple pulp (PP) and agar hydrolyzate (AH), on micropropagation and Pb bioaccumulation and tolerance in a woody shrub Daphne jasminea cultured in vitro. The amendments were analyzed for their content of carbohydrates, phenolic acids, and phytohormones and added at a dose of 10 mL L -1 to the medium containing 1.0 mM lead nitrate. Micropropagation coefficient increased by 10.2-16.6 % in PP and AH variants, respectively. Growth tolerance index increased by 22.9-31.8 % for the shoots and by 60.1-82.4 % for the roots. In the absence of Pb, the additives inhibited multiplication and growth of microplantlets. PP and AH facilitated Pb accumulation in plant organs, especially in the roots. PP enhanced bioconcentration factor and AH improved Pb translocation to the shoots. Adaptation to Pb was associated with increased accumulation of phenolics and higher radical scavenging activity. Medium supplementation, particularly with AH, enhanced antiradical activity of Pb-adapted lines but reduced the content of phenolic compounds. The study results indicated that supplementation with organic amendments may be beneficial in in vitro selection against lead toxicity.

Photography in Dermatologic Surgery: Selection of an Appropriate Camera Type for a Particular Clinical Application.

PubMed

Chen, Brian R; Poon, Emily; Alam, Murad

2017-08-01

Photographs are an essential tool for the documentation and sharing of findings in dermatologic surgery, and various camera types are available. To evaluate the currently available camera types in view of the special functional needs of procedural dermatologists. Mobile phone, point and shoot, digital single-lens reflex (DSLR), digital medium format, and 3-dimensional cameras were compared in terms of their usefulness for dermatologic surgeons. For each camera type, the image quality, as well as the other practical benefits and limitations, were evaluated with reference to a set of ideal camera characteristics. Based on these assessments, recommendations were made regarding the specific clinical circumstances in which each camera type would likely be most useful. Mobile photography may be adequate when ease of use, availability, and accessibility are prioritized. Point and shoot cameras and DSLR cameras provide sufficient resolution for a range of clinical circumstances, while providing the added benefit of portability. Digital medium format cameras offer the highest image quality, with accurate color rendition and greater color depth. Three-dimensional imaging may be optimal for the definition of skin contour. The selection of an optimal camera depends on the context in which it will be used.

High-frequency transformation of Lobelia erinus L. by Agrobacterium-mediated gene transfer.

PubMed

Tsugawa, H; Kagami, T; Suzuki, M

2004-05-01

A highly efficient transformation procedure was developed for Lobelia erinus. Leaf or cotyledon discs were inoculated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector plasmid pIG121Hm, which contains a beta-glucuronidase gene with an intron as a reporter gene and both the neomycin phosphotransferase II and hygromycin phosphotransferase genes as selectable markers. The hygromycin-resistant calli produced on the selection medium were transferred to MS medium supplemented with 0.5 mg/l benzyladenine and 0.2 mg/l indole-3-acetic acid for regeneration of adventitious shoots. Transgenic plants were obtained as a result of the high regeneration rate of the transformed calli, which was as high as 83%. In contrast, no transgenic plant was obtained by the procedure of direct shoot formation following inoculation with A. tumefaciens. Transgenic plants flowered 3-4 months after transformation. Integration of the transgenes was detected using PCR and Southern blot analysis, which revealed that one to several copies were integrated into the genomes of the host plants. The transformation frequency at the stage of whole plants was very high--45% per inoculated disc. Copyright 2004 Springer-Verlag

Induction and identification of rabbit peripheral blood derived dendritic cells

NASA Astrophysics Data System (ADS)

Zhou, Jing; Yang, FuYuan; Chen, WenLi

2012-03-01

Purpose: To study a method of the induction of dendritic cells (DCs) from rabbit peripheral blood. Methods: Peripheral blood cells were removed from rabbit, filtered through nylon mesh. Peripheral blood mononuclear cells (PBMC) were isolated from the blood cells by Ficoll-Hypaque centrifugation (density of 1.077g/cm3).To obtain DCs, PBMC were cultured in RPMI1640 medium containing 10% fetal calf serum, 50U/mL penicillin and streptomycin, referred to subsequently as complete medium, at 37°C in 5% CO2 atmosphere for 4 hours. Nonadherent cells were aspirated, adherent cells were continued incubated in complete medium, supplemented with granulocyte/macrophage colony-stimulating factor (GM-CSF, 50ng/ml),and interleukin 4 (IL-4, 50ng/ml) for 9 days. Fluorescein labeled antibodies(anti-CD14, anti-HLA-DR, anti-CD86) were used to sign cells cultured for 3,6,9 days respectively, Then flow cytometry was performed. Results: Ratio of anti-HLA-DR and anti-CD86 labeled cells increased with induction time extension, in contrast with anti-CD14. Conclusion: Dendritic cells can be effectively induced by the method of this experiment, cell maturation status increased with induction time extension.

In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity

PubMed Central

Dakah, Abdulkarim; Zaid, Salim; Suleiman, Mohamad; Abbas, Sami; Wink, Michael

2014-01-01

Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants. The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA. DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC50 was 0.307 and 0.369 mg/ml, respectively, while the IC50 of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity. PMID:25183942

In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity.

PubMed

Dakah, Abdulkarim; Zaid, Salim; Suleiman, Mohamad; Abbas, Sami; Wink, Michael

2014-09-01

Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants. The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA. DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC50 was 0.307 and 0.369 mg/ml, respectively, while the IC50 of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.

Evaluation of hyperaccumulator plant species grown in metalliferous sites in Albania

NASA Astrophysics Data System (ADS)

Babani, F.; Civici, N.; Mullaj, A.; Kongjika, E.; Ylli, A.

2007-04-01

Heavy metal contamination of soils causes serious problems to our society. A small number of interesting plant species have been identified that can grow in soils containing high levels of heavy metals, and can also accumulate these metals to high concentrations in the shoot. The heavy metal contents in root, shoot, leaves and flowers of spontaneous plants grown in metalliferous sites in Albania together with the elemental composition of the native soils were determined by X-ray fluorescence spectrometry. Efficiency of photosynthetic apparatus of analyzed ecotypes was evaluated via chlorophyll fluorescence imaging during induction kinetics. Response of plant root system to the presence of metals, the available pools of metals to plants, effect of plant biomass to phytoextraction, photosynthetic pigment metabolism and chlorophyll fluorescence signature of leaves allowed to characterize hyperaccumulator properties and to detect the variation between selected ecotypes to heavy metal accumulation.

Flowering in Xanthium strumarium: INITIATION AND DEVELOPMENT OF FEMALE INFLORESCENCE AND SEX EXPRESSION.

PubMed

Leonard, M; Kinet, J M; Bodson, M; Havelange, A; Jacqmard, A; Bernier, G

1981-06-01

Vegetative plants of Xanthium strumarium L. grown in long days were induced to flower by exposure to one or several 16-hour dark periods. The distribution of male and female inflorescences on the flowering shoot was described, and a scoring system was designed to assess the development of the female inflorescences. The time of movement of the floral stimulus out of the induced leaf and the timing of action of high temperature were shown to be similar for both the apical male and lateral female inflorescences.Strong photoperiodic induction of the plants favored female sex expression, while maleness was enhanced by exogenous gibberellic acid. The problem of the control of sex expression in Xanthium is discussed in relation to the distribution pattern of male and female inflorescences on the flowering shoot and to the state of the meristem at the time of the arrival of the floral stimulus.

Flowering in Xanthium strumarium

PubMed Central

Leonard, Maggy; Kinet, Jean-Marie; Bodson, Monique; Havelange, Andrée; Jacqmard, Annie; Bernier, Georges

1981-01-01

Vegetative plants of Xanthium strumarium L. grown in long days were induced to flower by exposure to one or several 16-hour dark periods. The distribution of male and female inflorescences on the flowering shoot was described, and a scoring system was designed to assess the development of the female inflorescences. The time of movement of the floral stimulus out of the induced leaf and the timing of action of high temperature were shown to be similar for both the apical male and lateral female inflorescences. Strong photoperiodic induction of the plants favored female sex expression, while maleness was enhanced by exogenous gibberellic acid. The problem of the control of sex expression in Xanthium is discussed in relation to the distribution pattern of male and female inflorescences on the flowering shoot and to the state of the meristem at the time of the arrival of the floral stimulus. Images PMID:16661844

Phytoremediation of Cadmium by Native Plants Grown on Mining Soil.

PubMed

Palutoglu, Mahmut; Akgul, Bunyamin; Suyarko, Vasily; Yakovenko, Myroslava; Kryuchenko, Nataliya; Sasmaz, Ahmet

2018-02-01

The Gümüsköy mining area is located about 25 km west of Kutahya and is the largest silver deposit in Turkey. The present study investigated translocation and accumulation of cadmium (Cd) from the soil into 11 native plants. Plant and soil samples were collected from the field, and Cd concentrations were analyzed by inductively coupled plasma mass spectroscopy. Mean Cd values in the soil, root, and shoot of native plants in the study area were 82.8 ± 5, 55.4 ± 6, and 43.5 ± 4 mg kg - 1 , respectively. Plants were separated into several groups according to the enrichment coefficients for shoot and root values of plants. These groups showed Carduus nutans and Phlomis could be potentially bioaccumulator plants useful for phytoremediation of mining soils contaminated by Cd.

Somatic embryogenesis and plant regeneration from cell suspension cultures of Cucumis sativus L.

PubMed

Chee, P P; Tricoli, D M

1988-06-01

A procedure for the regeneration of whole cucumber plants (Cucumis sativus L. cv. Poinsett 76) by embryogenesis from cell suspension cultures is described. Embryogenic callus was initiated from the primary leaves of 14-17 day old plants. Suspension cultures of embryogenic cells were grown in liquid Murashige and Skoog basal medium containing 5 uM 2,4,5-trichlorophenoxyacetic acid and 4 uM 6-benzylaminopurine. Suspension cultures were composed of a population of cells that were densely cytoplasmic and potentially embryogenic. Differentiation of embryos was enhanced by washing the suspension culture cells with MS basal medium containing 0.5% activated charcoal and twice with MS basal medium followed by liquid shake cultures in MS basal medium. Sixty to 70 percent of the embryos prewashed with activated charcoal germinated into plantlets with normal morphology. Embryos obtained from suspension cultured cells without prewashing with activated charcoal organized into plantlets with abnormal primary leaves. Morphologically normal plantlets were obtained by excising the shoot tips and transferring them to fresh medium.

Liquid in vitro culture for the propagation of Arundo donax.

PubMed

Herrera-Alamillo, Miguel Angel; Robert, Manuel L

2012-01-01

We describe a simple and inexpensive plant micropropagation system for giant reed (Arundo donax L.) that uses axillary buds from the lateral stems of elite plants selected from field- or nursery-grown plants. The buds, attached to the stems are cultured in stationary liquid MS culture medium, supplemented with indole 3-acetic acid and kinetin. This formulation is the only one required for all the stages. Contrary to what happens in semisolid medium where roots are not formed, the plants cultured in liquid medium are whole plants with shoots and roots that develop at the same time. The survival rate of these plants when transferred to soil is close to 100% during acclimatization. A clonal line of 900 plants from a single mother plant can be produced in 4 months.

Enhanced formation of aerenchyma and induction of a barrier to radial oxygen loss in adventitious roots of Zea nicaraguensis contribute to its waterlogging tolerance as compared with maize (Zea mays ssp. mays).

PubMed

Abiko, Tomomi; Kotula, Lukasz; Shiono, Katsuhiro; Malik, Al Imran; Colmer, Timothy David; Nakazono, Mikio

2012-09-01

Enhancement of oxygen transport from shoot to root tip by the formation of aerenchyma and also a barrier to radial oxygen loss (ROL) in roots is common in waterlogging-tolerant plants. Zea nicaraguensis (teosinte), a wild relative of maize (Zea mays ssp. mays), grows in waterlogged soils. We investigated the formation of aerenchyma and ROL barrier induction in roots of Z. nicaraguensis, in comparison with roots of maize (inbred line Mi29), in a pot soil system and in hydroponics. Furthermore, depositions of suberin in the exodermis/hypodermis and lignin in the epidermis of adventitious roots of Z. nicaraguensis and maize grown in aerated or stagnant deoxygenated nutrient solution were studied. Growth of maize was more adversely affected by low oxygen in the root zone (waterlogged soil or stagnant deoxygenated nutrient solution) compared with Z. nicaraguensis. In stagnant deoxygenated solution, Z. nicaraguensis was superior to maize in transporting oxygen from shoot base to root tip due to formation of larger aerenchyma and a stronger barrier to ROL in adventitious roots. The relationships between the ROL barrier formation and suberin and lignin depositions in roots are discussed. The ROL barrier, in addition to aerenchyma, would contribute to the waterlogging tolerance of Z. nicaraguensis. © 2012 Blackwell Publishing Ltd.

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

Hormonal interactions during cluster-root development in phosphate-deficient white lupin (Lupinus albus L.).

PubMed

Wang, Zhengrui; Rahman, A B M Moshiur; Wang, Guoying; Ludewig, Uwe; Shen, Jianbo; Neumann, Günter

2015-04-01

This study addresses hormonal interactions involved in cluster-root (CR) development of phosphate (Pi)-deficient white lupin (Lupinus albus), which represents the most efficient plant strategy for root-induced mobilisation of sparingly soluble soil phosphorus (P) sources. Shoot-to-root translocation of auxin was unaffected by P-limitation, while strong stimulatory effects of external sucrose on CR formation, even in P-sufficient plants, suggest sucrose, rather than auxins, acts as a shoot-borne signal, triggering the induction of CR primordia. Ethylene may act as mediator of the sucrose signal, as indicated by moderately increased expression of genes involved in ethylene biosynthesis in pre-emergent clusters and by strong inhibitory effects of the ethylene antagonist CoCl2 on CR formation induced by sucrose amendments or P-limitation. As reported in other plants, moderately increased production of brassinosteroids (BRs) and cytokinin, in pre-emergent clusters, may be required for the formation of auxin gradients necessary for induction of CR primordia via interference with auxin biosynthesis and transport. The well-documented inhibition of root elongation by high doses of ethylene may be involved in the growth inhibition of lateral rootlets during CR maturation, indicated by a massive increased expression of gene involved in ethylene production, associated with a declined expression of transcripts with stimulatory effects (BR and auxin-related genes). Copyright © 2014 Elsevier GmbH. All rights reserved.

An Efficient In Vitro Propagation Protocol of Cocoyam [Xanthosoma sagittifolium (L) Schott

PubMed Central

Sama, Anne E.; Hughes, Harrison G.; Abbas, Mohamed S.; Shahba, Mohamed A.

2012-01-01

Sprouted corm sections of “South Dade” white cocoyam were potted and maintained in a greenhouse for 8 weeks. Shoot tips of 3–5 mm comprising the apical meristem with 4–6 leaf primordial, and approximately 0.5 mm of corm tissue at the base. These explants were treated to be used into the culture medium. A modified Gamborg's B5 mineral salts supplemented with 0.05 μM 1-naphthaleneacetic acid (NAA) were used throughout the study. Thidiazuron (TDZ) solution containing 0.01% dimethyl sulfoxide (DMSO) was used. Erlenmeyer flasks and test tubes were used for growing cultures. The effect of different media substrate, thidiazuron, and the interaction between TDZ and Benzylaminopurine (BAP) on cocoyam culture were tested. Results indicated that cocoyam can be successfully micropropagated in vitro through various procedures. All concentrations tested (5–20 μM BAP and 1–4 μM TDZ) produced more axillary shoots per shoot tip than the control without cytokinins. Greater proliferation rates were obtained through the use of 20 μM BAP and 2 μM TDZ, respectively, 12 weeks from initiation. Shoots produced with BAP were larger and more normal in appearance than those produced with TDZ, which were small, compressed, and stunted. The use of stationary liquid media is recommended for economic reasons. PMID:22666109

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

PubMed Central

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

PubMed

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

Artificial neural network-based model for the prediction of optimal growth and culture conditions for maximum biomass accumulation in multiple shoot cultures of Centella asiatica.

PubMed

Prasad, Archana; Prakash, Om; Mehrotra, Shakti; Khan, Feroz; Mathur, Ajay Kumar; Mathur, Archana

2017-01-01

An artificial neural network (ANN)-based modelling approach is used to determine the synergistic effect of five major components of growth medium (Mg, Cu, Zn, nitrate and sucrose) on improved in vitro biomass yield in multiple shoot cultures of Centella asiatica. The back propagation neural network (BPNN) was employed to predict optimal biomass accumulation in terms of growth index over a defined culture duration of 35 days. The four variable concentrations of five media components, i.e. MgSO 4 (0, 0.75, 1.5, 3.0 mM), ZnSO 4 (0, 15, 30, 60 μM), CuSO 4 (0, 0.05, 0.1, 0.2 μM), NO 3 (20, 30, 40, 60 mM) and sucrose (1, 3, 5, 7 %, w/v) were taken as inputs for the ANN model. The designed model was evaluated by performing three different sets of validation experiments that indicated a greater similarity between the target and predicted dataset. The results of the modelling experiment suggested that 1.5 mM Mg, 30 μM Zn, 0.1 μM Cu, 40 mM NO 3 and 6 % (w/v) sucrose were the respective optimal concentrations of the tested medium components for achieving maximum growth index of 1654.46 with high centelloside yield (62.37 mg DW/culture) in the cultured multiple shoots. This study can facilitate the generation of higher biomass of uniform, clean, good quality C. asiatica herb that can efficiently be utilized by pharmaceutical industries.

Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.

PubMed

Ntui, Valentine Otang; Thirukkumaran, Gunaratnam; Azadi, Pejman; Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2010-09-01

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.

Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marusich, W.C.; Jensen, R.A.; Zamir, L.O.

Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than in complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-phenylalanine will also induce lyase synthesis during exponential growth in minimal medium inmore » which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared fom cultures induced with doubly labeled (U-/sup 14/C; ring-4-/sup 3/H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate.« less

Shikonin Production by Callus Culture of Onosma bulbotrichom as Active Pharmaceutical Ingredient

PubMed Central

Bagheri, Fereshteh; Tahvilian, Reza; Karimi, Naser; Chalabi, Maryam; Azami, Mahsa

2018-01-01

The objective of this research was in-vitro germination and callus induction of Onosma bulbotrichum (O. bulbotrichum) as a medicinal herb which belongs to Boraginaceae family. For germination, the seeds were cultured on growth regulator-free MS medium and for callus induction, seeds were sown on modified MS medium containing different concentrations of kinetin (kn)- Indole-3-acetic acid (IAA) and kn- 2,4-D (2,4-dichlorophenoxyacetic acid), respectively. The plates were maintained in the dark at growth chamber. After 7 days seed germination on hormone-free medium and after 10 days callus initiation on modified medium in the presence of hormones was occurred. The maximum pigmented callus (100%) was observed on modified MS medium with a combination of 0.2 mg.L-1 IAA + 2.10 mg.L-1 kn. Shikonin determination was performed by HPLC method. In addition, total hydroxynaphtoquinons as polyphenols in sum of callus and culture medium were measured by spectrophotometric method and revealed that total naphtoquinones content at IAA was more than 2, 4-D. PMID:29881407

In vitro regeneration of solanum aethiopicum L. (scarlet eggplant), an african vegetable crop with potential ornamental value

USDA-ARS?s Scientific Manuscript database

Successful in vitro regeneration of plantlets was obtained from shoot tips of five Solanum aethiopicum (African eggplants) accessions evaluated in two media, M1 and M2. The M1 medium consisted of Murashige and Skoog (MS) basal salt mixture supplemented with 20 g/L sucrose, 0.75 g/L MgCl2, and 2 g/L ...

Exogenous Supplementation of Silicon Improved the Recovery of Hyperhydric Shoots in Dianthus caryophyllus L. by Stabilizing the Physiology and Protein Expression

PubMed Central

Soundararajan, Prabhakaran; Manivannan, Abinaya; Cho, Yoon S.; Jeong, Byoung R.

2017-01-01

Hyperhydricity is one of the major problems hindering in vitro propagation of Dianthus caryophyllus L. Silicon (Si) is a well-known beneficial element renowned for its stress amelioration properties in plants. This study has demonstrated the physiological and molecular mechanism behind the Si-mediated recovery from hyperhydricity in D. caryophyllus L. ‘Green Beauty’. Four weeks old hyperhydric shoots obtained from temporary immersion system were cultured on the Murashige and Skoog medium supplemented with 0 (control), 1.8 mM, or 3.6 mM of potassium silicate (K2SiO3). After 2 weeks of culture, we observed only 20% of hyperhydric shoots were recovered in control. On the other hand hyperhydricity, shoot recovery percentage in 1.8 mM and 3.6 mM of Si were 44% and 36%, respectively. Shoots in control possessed higher lipid peroxidation rate compared to the Si treatments. Similarly, damaged stomata were detected in the control, while Si treatments restored the normal stomatal development. Expressions of superoxide dismutase, guaiacol peroxidase, and catalase varied between the control and Si treatments. Furthermore, a proteomic analysis showed that as compared with the control Si up-regulated 17 and 10 protein spots in abundance at 1.8 and 3.6 mM of Si, respectively. In comparison to the 3.6 mM, 1.8 mM of Si treatment up-regulated 19 proteins and down-regulated 7 proteins. Identified proteins were categorized into six groups according to their biological roles such as ribosomal binding, oxido-reduction, hormone/cell signaling, metal/ion binding, defense, and photosynthesis. The proteomic results revealed that Si actively involved in the various metabolisms to accelerate the recovery of the shoots from hyperhydricity. Thus, the outcomes of this study can be utilized for addressing the molecular insight of hyperhydricity and its recovery mechanism by the supplementation of Si. Therefore, we conclude that active involvement of Si in the regulation and signaling process of proteins at 1.8 mM concentration could be efficient to trigger the reclamation process of hyperhydric carnation shoots. PMID:28533793

Optimization of in vitro regeneration and Agrobacterium tumefaciens-mediated transformation with heat-resistant cDNA in Brassica oleracea subsp. italica cv. Green Marvel.

PubMed

Ravanfar, Seyed Ali; Aziz, Maheran Abdul; Saud, Halimi Mohd; Abdullah, Janna Ong

2015-11-01

An efficient system for shoot regeneration and Agrobacterium tumefaciens-mediated transformation of Brassica oleracea cv. Green Marvel cultivar is described. This study focuses on developing shoot regeneration from hypocotyl explants of broccoli cv. Green Marvel using thidiazuron (TDZ), zeatin, and kinetin, the optimization of factors affecting Agrobacterium-mediated transformation of the hypocotyl explants with heat-resistant cDNA, followed by the confirmation of transgenicity of the regenerants. High shoot regeneration was observed in 0.05-0.1 mg dm(-3) TDZ. TDZ at 0.1 mg dm(-3) produced among the highest percentage of shoot regeneration (96.67 %) and mean number of shoot formation (6.17). The highest percentage (13.33 %) and mean number (0.17) of putative transformant production were on hypocotyl explants subjected to preculture on shoot regeneration medium (SRM) with 200 µM acetosyringone. On optimization of bacterial density and inoculation time, the highest percentage and mean number of putative transformant production were on hypocotyl explants inoculated with a bacterial dilution of 1:5 for 30 min. Polymerase chain reaction (PCR) assay indicated a transformation efficiency of 8.33 %. The luciferase assay showed stable integration of the Arabidopsis thaliana HSP101 (AtHSP101) cDNA in the transgenic broccoli regenerants. Three out of five transgenic lines confirmed through PCR showed positive hybridization bands of the AtHSP101 cDNA through Southern blot analysis. The presence of AtHSP101 transcripts in the three transgenic broccoli lines indicated by reverse transcription-PCR (RT-PCR) confirmed the expression of the gene. In conclusion, an improved regeneration system has been established from hypocotyl explants of broccoli followed by successful transformation with AtHSP101 for resistance to high temperature.

Interactions of phytochromes A, B1 and B2 in light-induced competence for adventitious shoot formation in hypocotyl of tomato (Solanum lycopersicum L.).

PubMed

Lercari, B; Bertram, L

2004-02-01

The interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phytochrome B2 (phyB2) in light-dependent shoot regeneration from the hypocotyl of tomato was analysed using all eight possible homozygous allelic combinations of the null mutants. The donor plants were pre-grown either in the dark or under red or far-red light for 8 days after sowing; thereafter hypocotyl segments (apical, middle and basal portions) were transferred onto hormone-free medium for culture under different light qualities. Etiolated apical segments cultured in vitro under white light showed a very high frequency of regeneration for all of the genotypes tested besides phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants. Evidence is provided of a specific interference of phyB2 with phyA-mediated HIR to far-red and blue light in etiolated explants. Pre-treatment of donor plants by growth under red light enhanced the competence of phyB1phyB2, phyAphyB1 and phyAphyB1phyB2 mutants for shoot regeneration, whereas pre-irradiation with far-red light enhanced the frequency of regeneration only in the phyAphyB1 mutant. Multiple phytochromes are involved in red light- and far-red light-dependent acquisition of competence for shoot regeneration. The position of the segments along the hypocotyl influenced the role of the various phytochromes and the interactions between them. The culture of competent hypocotyl segments under red, far-red or blue light reduced the frequency of explants forming shoots compared to those cultured under white light, with different genotypes having different response patterns.

The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage.

PubMed

Halmagyi, A; Surducan, E; Surducan, V

2017-09-01

Two distinct microwave power levels and techniques have been studied in two cases: low-power microwave (LPM) irradiation on in vitro Sequoia plants and high-power microwave (HPM) exposure on recovery rates of cryostored (-196°C) Sequoia shoot apices. Experimental variants for LPM exposure included: (a) in vitro plants grown in regular conditions (at 24 ± 1°C during a 16-h light photoperiod with a light intensity of 39.06 μEm -2 s -1 photosynthetically active radiation), (b) in vitro plants grown in the anechoic chamber with controlled environment without microwave irradiation, and (c) in vitro plants grown in the anechoic chamber with LPM irradiation for various times (5, 15, 30, 40 days). In comparison to control plants, significant differences in shoot multiplication and growth parameters (length of shoots and roots) were observed after 40 days of LPM exposure. An opposite effect was achieved regarding the content of total soluble proteins, which decreased with increasing exposure time to LPM. HPM irradiation was tested as a novel rewarming method following storage in liquid nitrogen. To our knowledge, this is the first report using this type of rewarming method. Although, shoot tips subjected to HPM exposure showed 28% recovery following cryostorage compared to 44% for shoot tips rewarmed in liquid medium at 22 ± 1 °C, we consider that the method represent a basis and can be further improved. The results lead to the overall conclusion that LPM had a stimulating effect on growth and multiplication of in vitro Sequoia plants, while the HPM used for rewarming of cryopreserved apices was not effective to achieve high rates of regrowth after liquid nitrogen exposure.

In vitro propagation of carnation (Dianthus caryophyllus L.).

PubMed

Casas, Jose L; Olmos, Enrique; Piqueras, Abel

2010-01-01

Carnation (Dianthus caryophyllus L.) is one of the most popular ornamental plants worldwide and also among the most studied ones, mainly in cut flower postharvest physiology. Several protocols for the in vitro propagation of this species including nodal segment culture, somatic embryogenesis, and adventitious shoot induction are described in this chapter. The presence of hyperhydricity as an abnormality during micropropagation of carnation plants has also been the object of research for many years and different strategies to overcome this problem are also included in this study.

Induction of lead-binding phytochelatins in vetiver grass [Vetiveria zizanioides (L.)].

PubMed

Andra, Syam S; Datta, Rupali; Sarkar, Dibyendu; Makris, Konstantinos C; Mullens, Conor P; Sahi, Shivendra V; Bach, Stephan B H

2009-01-01

Elevated lead (Pb) concentrations in residential houseyards around house walls painted with Pb-based pigments pose serious human health risks, especially to children. Vetiver grass (Vetiveria zizanioides L.) has shown promise for use in in situ Pb phytoremediation efforts. However, little is known about the biochemical mechanisms responsible for the observed high Pb tolerance by vetiver. We hypothesized that vetiver exposure to Pb induced the synthesis of phytochelatins (PC(n)) and the formation of Pb-PC(n) complexes, alleviating the phytotoxic effects of free Pb ions. Our main objective was to identify PC(n) and Pb-PC(n) complexes in root and shoot compartments of vetiver grass using high-performance liquid chromatography coupled to electrospray mass spectrometry (HPLC-ES-MS). After 7 d of exposure to Pb, vetiver accumulated up to 3000 mg Pb kg(-1) in shoot tissues, but much higher Pb concentrations were measured in root ( approximately 20,000 mg kg(-1)), without phytotoxic symptoms. Scanning electron micrographs showed Pb deposition in the vascular tissues of root and shoot, suggesting Pb translocation to shoot. Collision-induced dissociation analyses in MS/ MS mode during HPLC-ES-MS analysis allowed for the confirmation of four unique PC(n) (n = 1-4) based on their respective amino acid sequence. The high tolerance of vetiver grass to Pb was attributed to the formation of PC(n) and Pb-PC(n) complexes within the plant tissues, using ES-MS and Pb mass isotopic patterns. These data illustrate the mechanism of high Pb tolerance by vetiver grass, suggesting its potential usefulness for the remediation of Pb-contaminated residential sites.

Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants

PubMed Central

Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K.

2016-01-01

Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. PMID:26685186

Genetic transformation of Begonia tuberhybrida by Ri rol genes.

PubMed

Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H

1996-04-01

We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

Agrobacterium rhizogenes-mediated DNA transfer to Aesculus hippocastanum L. and the regeneration of transformed plants.

PubMed

Zdravković-Korać, S; Muhovski, Y; Druart, P; Calić, D; Radojević, L

2004-04-01

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 microM 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 microM BA. Following elongation on MS medium supplemented with 1 microM BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.

Performance of fenugreek bioinoculated with Rhizobium meliloti strains under semi-arid condition.

PubMed

Singh, N K; Patel, D B

2016-01-01

Rhizobium meliloti strains were isolated from the fields of S.D. Agricultural University (Gujarat, India) and were maintained in the Congo Red Yeast Extract Mannitol Agar medium. These strains were tested for their effectiveness for fenugreek crop grown under semi-arid condition. Among the six Rhizobium strains, FRS-7 strain showed best plant growth parameters like shoot length, shoot dry weight, shoot total nitrogen, root length, root dry weight, root total nitrogen, seed yield, 1000 grain weight, number of root nodules, and nodules fresh and dry weight. The performance of this strain was better as compared to 20 kgN ha(-1) treatment through urea and was even far better over control plot. Seed yields obtained with FRS-7 during two years were 10.14 and 9.66 q ha(-1); which was about 36.8% and 45.9% high over control. This strain resulted in saving of about 20 kgN ha(-1) accompanied with better crop yield and soil health. Results of the present experiments can be utilized in integrated nutrient management for cultivation of fenugreek in semi-arid areas to provide sustainability to agricultural productivity in such regions.

A new species of Brevianthus (Brevianthaceae, Marchantiophyta) from New Caledonia with unusual underleaf production.

PubMed

Renner, Matt A M; Engel, John J; Patzak, Simon D F; Heinrichs, Jochen

2015-01-01

Brevianthus is a distinctive genus of leafy liverwort in its succubously inserted, entire leaves, lack of underleaves, restriction of sexual organs to lateral-intercalary branches, scattered rhizoids and dense leaf-surface ornamentation. The sole species, Brevianthusflavus, is divided into two subspecies, one in Tasmania the other in New Zealand. A second species, Brevianthushypocanthidium, is described as new and is the first record of the genus for New Caledonia. Among its distinguishing characters are its shallowly bilobed leaves, and triangular underleaves present on small to medium-sized shoot sectors, the lack of a hyaline leaf margin, and the crenulate leaf margin formed by heavily thickened external cell walls. The most unusual features of the new species are the presence of underleaves between lateral leaf insertion lines that reach the ventral stem mid-line, and the absence of underleaves from larger shoots. To explain these features we propose a competitive model of shoot formation wherein the ventral merophyte progressively loses vigor as its relative stature decreases, and its derivative cells become discontinuous and isolated along the ventral stem surface, with intervening areas occupied by derivatives of the more vigorous lateral merophytes.

An effective protocol for micropropagation of edible bamboo species (Bambusa tulda and Melocanna baccifera) through nodal culture.

PubMed

Waikhom, Sayanika Devi; Louis, Bengyella

2014-01-01

High demand for edible bamboo shoots of Bambusa tulda and Melocanna baccifera in many Asian ethnic groups has led to the need for developing intensive bamboo farming. To achieve this, in vitro regeneration of bamboo plantlets is needed due to the long and irregular bamboo flowering cycle and scarcity of bamboo seeds. An effective protocol for plantlets regeneration in B. tulda and M. baccifera from nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is described. Effective axillary bud breaking was achieved at 3 mg/L of 6-benzylaminopurine (BAP) in MS medium. Importantly, combining 2 mg/L of kinetin (Kn) with 3 mg/L of BAP produced a synergistic effect for shoot multiplication in B. tulda and M. baccifera. Under optimized conditions in half-strength MS medium supplemented with 3 mg/L of indole-3-butyric acid (IBA), 10 mg/L of coumarin, and 3% sucrose, profuse production of dark-brown rhizome in B. tulda and abundant rooting (81.67%, P < 0.05, F = 15.46) for M. baccifera within 30 days were achieved. The established protocol and the validation of the reported species at the molecular level will be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity.

Micropropagation of onion (Allium cepa L.) from immature inflorescences.

PubMed

Marinangeli, Pablo

2013-01-01

In vitro plant production by direct organogenesis from immature flower heads is an ideal approach for clonal propagation of onions (Allium cepa L.). This technique ensures genetic stability, high propagation rate, and maintains donor plant of explants with an advantage over other means of in vitro regeneration. Onion micropropagation is usually applied in breeding programs, maintenance, and multiplication of cytoplasmic-male sterile lines for hybrid production, germplasm conservation, and as a tool for the application of other biotechnologies. For in vitro culture, mature onion bulbs are induced to reproductive phase by vernalization and forced to inflorescence initiation. Immature umbels are dissected from bulbs or cut directly when they appear from the pseudostem among the leaves. Disinfected inflorescences are cultivated in BDS basal medium supplemented with 30 g/L sucrose, 0.1 mg/L naphthalene acetic acid, 1 mg/L N (6)-benzyladenine, and 8 g/L agar, pH 5.5, under 16 h photoperiod white fluorescent light (PPD: 50-70 μmol/m(2)s) for 35 days. The regenerated shoot clumps are divided and subculture under the same conditions. For bulbification phase, the individual shoots are cultured in BDS basal medium containing 90 g/L sucrose, without plant growth regulators, pH 5.5, under 16 h photoperiod. Microbulbs can be directly cultivated ex vitro without acclimation.

In vitro propagation and cryopreservation of Aerides odorata Lour. (Orchidaceae).

PubMed

Hongthongkham, J; Bunnag, S

2014-05-01

An efficient method for in vitro propagation and cryopreservation of Aerides odorata was established. Leaf segments were cultured on New Dogashima (ND) mediums supplemented with various concentrations of Benzyladenine (BA) (0-5 mg L(-1)) combined with Naphthaleneacetic Acid (NAA) (0-2 mg L(-1)). The optimal treatment for inducing Protocorm-like Bodies (PLBs) from leaf segments was obtained from the combination of 1 or 3 mg L(-1) BA and 0.5 or 1 mg L(-1) NAA; whereas, the addition of BA or NAA alone induced shoot and/or root initiation rather than PLB or callus formation. Shoots rapidly developed on ND mediums containing 5 mg L(-1) BA. Cryopreservation of leaf segment-derived PLBs was successful using the encapsulation-dehydration method. The maximum survival percentage of Cryopreserved (Cryp) PLBs was achieved by encapsulating PLBs with 2% Na-alginate combined with 2 M glycerol and 0.4 M sucrose. The encapsulated PLBs were then precultured in 0.75 M sucrose for 24 h and dehydrated for 6 h before plunging into liquid nitrogen. Genetic stability of Cryp PLBs after regrowth was assessed by flow cytometry. The findings showed no different patterns of ploidy levels and morphology between Cryp and non-cryopreserved (Ncryp) control plantlets.

Ex situ conservation of Phyllanthus fraternus Webster and evaluation of genetic fidelity in regenerates using DNA-based molecular marker.

PubMed

Upadhyay, Richa; Kashyap, Sarvesh Pratap; Singh, Chandra Shekhar; Tiwari, Kavindra Nath; Singh, Karuna; Singh, Major

2014-11-01

Germplasm storage of Phyllanthus fraternus by using synseed technology has been optimized. Synseeds were prepared from nodal segments taken from in vitro-grown plantlets. An encapsulation matrix of 3 % sodium alginate and 100 mM calcium chloride with polymerization duration up to 15 min was found most suitable for synseed formation. Maximum plantlet conversion (92.5 ± 2.5 %) was obtained on a growth regulator-free ½-strength solid Murashige and Skoog (MS) medium. Multiple shoot proliferation was optimum on a ½ MS medium containing 0.5 mg/l 6-benzylaminopurine (BAP). Shoots were subjected to rooting on MS media containing 1 mg/l α-naphthaleneacetic acid (NAA) and acclimatized successfully. Encapsulated nodal segments can be stored for up to 90 days with a survival frequency of 47.33 %. The clonal fidelity of synseed-derived plantlets was also assessed and compared with that of the mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeat analysis. No changes in molecular profiles were observed among the synseed-derived plantlets and mother plant, which confirms the genetic stability of regenerates. This synseed production protocol could be useful for in vitro multiplication, short-term storage, and exchange of germplasm of this important antiviral and hepatoprotective plant.

Cryopreservation of chayote (Sechium edule JACQ. SW.) zygotic embryos and shoot-tips from in vitro plantlets.

PubMed

Abdelnour-Esquivel, Ana; Engelmann, Florent

2002-01-01

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.

Biofertilization with Azospirillum brasilense improves in vitro culture of Handroanthus ochraceus, a forestry, ornamental and medicinal plant.

PubMed

Llorente, Berta E; Alasia, María A; Larraburu, Ezequiel E

2016-01-25

Biofertilization with plant growth-promoting rhizobacteria is a potential alternative to plant productivity. Here, in vitro propagation of Handroanthus ochraceus (yellow lapacho), a forest crop with high economic and environmental value, was developed using the Azospirillum brasilense strains Cd and Az39 during rhizogenesis. Epicotiles of in vitro plantlets were multiplied in Woody Plant Medium (WPM). For rooting, elongated shoots were transferred to auxin-free Murashige-Skoog medium with Gamborg's vitamins and WPM, both at half salt concentration (½MSG and ½WPM), and inoculated with Cd or Az39 at the base of each shoot. Anatomical studies were performed using leaves cleared and stained with safranin for optical microscopy and leaves and roots metalized with gold-palladium for scanning electron microscopy (SEM). In ½WPM auxin-free medium, A. brasilense Cd inoculation produced 55% of rooting, increased root fresh and dry weight (45% and 77%, respectively), and led to lower stomata size and density with similar proportion of open and closed stomata. Both strains selectively increased the size or density of glandular trichomes in ½MSG. Moreover, bacteria were detected on the root surface by SEM. In conclusion, the difference in H. ochraceus response to A. brasilense inoculation depends on the strain and the plant culture media. Cd strain enhanced rooting in auxin-free ½WPM and produced plantlets with features similar to those expected in ex vitro plants. This work presents an innovative in vitro approach using beneficial plant-microorganism interaction as an ecologically compatible strategy in plant biotechnology. Copyright © 2015 Elsevier B.V. All rights reserved.

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1.

PubMed Central

Soler, C; Felipe, A; Casado, F J; McGivan, J D; Pastor-Anglada, M

1993-01-01

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms. PMID:8435065

Analysis of physiological and miRNA responses to Pi deficiency in alfalfa (Medicago sativa L.).

PubMed

Li, Zhenyi; Xu, Hongyu; Li, Yue; Wan, Xiufu; Ma, Zhao; Cao, Jing; Li, Zhensong; He, Feng; Wang, Yufei; Wan, Liqiang; Tong, Zongyong; Li, Xianglin

2018-03-01

The induction of miR399 and miR398 and the inhibition of miR156, miR159, miR160, miR171, miR2111, and miR2643 were observed under Pi deficiency in alfalfa. The miRNA-mediated genes involved in basic metabolic process, root and shoot development, stress response and Pi uptake. Inorganic phosphate (Pi) deficiency is known to be a limiting factor in plant development and growth. However, the underlying miRNAs associated with the Pi deficiency-responsive mechanism in alfalfa are unclear. To elucidate the molecular mechanism at the miRNA level, we constructed four small RNA (sRNA) libraries from the roots and shoots of alfalfa grown under normal or Pi-deficient conditions. In the present study, alfalfa plants showed reductions in biomass, photosynthesis, and Pi content and increases in their root-to-shoot ratio and citric, malic, and succinic acid contents under Pi limitation. Sequencing results identified 47 and 44 differentially expressed miRNAs in the roots and shoots, respectively. Furthermore, 909 potential target genes were predicted, and some targets were validated by RLM-RACE assays. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed prominent enrichment in signal transducer activity, binding and basic metabolic pathways for carbohydrates, fatty acids and amino acids; cellular response to hormone stimulus and response to auxin pathways were also enriched. qPCR results verified that the differentially expressed miRNA profile was consistent with sequencing results, and putative target genes exhibited opposite expression patterns. In this study, the miRNAs associated with the response to Pi limitation in alfalfa were identified. In addition, there was an enrichment of miRNA-targeted genes involved in biological regulatory processes such as basic metabolic pathways, root and shoot development, stress response, Pi transportation and citric acid secretion.

Uptake and Transformation of Methylated and Inorganic Antimony in Plants.

PubMed

Ji, Ying; Mestrot, Adrien; Schulin, Rainer; Tandy, Susan

2018-01-01

Used as a hardening agent in lead bullets, antimony (Sb) has become a major contaminant in shooting range soils of some countries including Switzerland. Soil contamination by Sb is also an environmental problem in countries with Sb-mining activities such as China and Bolivia. Because of its toxicity and relatively high mobility, there is concern over the risk of Sb transfer from contaminated soils into plants, and thus into the food chain. In particular there is very little information on the environmental behavior of methylated antimony, which can be produced by microbial biomethylation of inorganic Sb in contaminated soils. Using a new extraction and high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method, we investigated antimony speciation in roots and shoots of wheat, fescue, rye, and ryegrass plants exposed to trimethyl antimony(V) (TMSb), antimonite (Sb(III)), and antimonate (Sb(V)) in hydroponics. The total root Sb concentrations followed the order Sb(III) treatment > Sb(V) treatment > TMSb treatment, except for fescue. Shoot Sb concentrations, however, did not differ among the three treatments. In the Sb(V) treatment small quantities of TMSb were found in the roots, whereas no TMSb was detected in the roots of Sb(III)-treated plants. In contrast, similar concentrations of TMSb were found in the shoots in both inorganic Sb treatments. The results indicate that biomethylation of Sb may occur in plants. In the TMSb treatment TMSb was the major Sb species, but the two inorganic Sb species were also found both in shoots and roots along with some unknown Sb species, suggesting that also TMSb demethylation may occur within plant tissues. The results furthermore indicate that methylated Sb is more mobile in plants than inorganic Sb species. Knowledge about this is important in risk assessments of Sb-contaminated sites, as methylation may render Sb more toxic than inorganic Sb, as it is known for arsenic (As).

Uptake and Transformation of Methylated and Inorganic Antimony in Plants

PubMed Central

Ji, Ying; Mestrot, Adrien; Schulin, Rainer; Tandy, Susan

2018-01-01

Used as a hardening agent in lead bullets, antimony (Sb) has become a major contaminant in shooting range soils of some countries including Switzerland. Soil contamination by Sb is also an environmental problem in countries with Sb-mining activities such as China and Bolivia. Because of its toxicity and relatively high mobility, there is concern over the risk of Sb transfer from contaminated soils into plants, and thus into the food chain. In particular there is very little information on the environmental behavior of methylated antimony, which can be produced by microbial biomethylation of inorganic Sb in contaminated soils. Using a new extraction and high-performance liquid chromatography inductively coupled plasma mass spectrometry (HPLC-ICP-MS) method, we investigated antimony speciation in roots and shoots of wheat, fescue, rye, and ryegrass plants exposed to trimethyl antimony(V) (TMSb), antimonite (Sb(III)), and antimonate (Sb(V)) in hydroponics. The total root Sb concentrations followed the order Sb(III) treatment > Sb(V) treatment > TMSb treatment, except for fescue. Shoot Sb concentrations, however, did not differ among the three treatments. In the Sb(V) treatment small quantities of TMSb were found in the roots, whereas no TMSb was detected in the roots of Sb(III)-treated plants. In contrast, similar concentrations of TMSb were found in the shoots in both inorganic Sb treatments. The results indicate that biomethylation of Sb may occur in plants. In the TMSb treatment TMSb was the major Sb species, but the two inorganic Sb species were also found both in shoots and roots along with some unknown Sb species, suggesting that also TMSb demethylation may occur within plant tissues. The results furthermore indicate that methylated Sb is more mobile in plants than inorganic Sb species. Knowledge about this is important in risk assessments of Sb-contaminated sites, as methylation may render Sb more toxic than inorganic Sb, as it is known for arsenic (As). PMID:29487607

Calcium and calmodulin are involved in blue light induction of the gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas.

PubMed Central

Im, C S; Matters, G L; Beale, S I

1996-01-01

The Chlamydomonas reinhardtii nuclear gene gsa, which encodes the early chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase (GSAT), is specifically induced by blue light in cells synchronized in a 12-hr-light and 12-hr-dark regime. Light induction required the presence of a nitrogen source in the incubation medium. Maximal induction also required acetate. However, in the absence of acetate, partial induction occurred when Ca2+ was present in the medium at concentrations of > or = 1 microM. The Ca2+ channel-blocking agents Nd3+ and nifedipine partially inhibited the external Ca(2+)-supported induction of GSAT mRNA but did not inhibit acetate-supported induction. The calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide inhibited both external Ca(2+)-supported and acetate-supported induction. The Ca2+ ionophore A23187 caused a transient induction in the dark. These results suggest that Ca2+ and calmodulin are involved in the signal transduction pathway linking blue light perception to the induction of GSAT mRNA. The electron transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited acetate-supported induction of GSAT mRNA but did not inhibit external Ca(2+)-supported induction. It is proposed that in the presence of acetate, an internal pool of Ca2+ can be mobilized as a second message, whereas in the absence of acetate, internal Ca2+ is not available but the requirement for Ca2+ can be partially met by an external Ca2+ source. The mobilization of internal Ca2+ may require energy derived from metabolism of acetate. PMID:8989881

Use of the cryptogein gene to stimulate the accumulation of Bacopa saponins in transgenic Bacopa monnieri plants.

PubMed

Majumdar, Sukanya; Garai, Saraswati; Jha, Sumita

2012-10-01

Genetic transformation of the Indian medicinal plant, Bacopa monnieri, using a gene encoding cryptogein, a proteinaceous elicitor, via Ri and Ti plasmids, were established and induced bioproduction of bacopa saponins in crypt-transgenic plants were obtained. Transformed roots obtained with A. rhizogenes strain LBA 9402 crypt on selection medium containing kanamycin (100 mg l(-1)) dedifferentiated forming callus and redifferentiated to roots which, spontaneously showed shoot bud induction. Ri crypt-transformed plants thus obtained showed integration and expression of rol genes as well as crypt gene. Ti crypt-transformed B. monnieri plants were established following transformation with disarmed A. tumefaciens strain harboring crypt. Transgenic plants showed significant enhancement in growth and bacopa saponin content. Bacopasaponin D (1.4-1.69 %) was maximally enhanced in transgenic plants containing crypt. In comparison to Ri-transformed plants, Ri crypt-transformed plants showed significantly (p ≤ 0.05) enhanced accumulation of bacoside A(3), bacopasaponin D, bacopaside II, bacopaside III and bacopaside V. Produced transgenic lines can be used for further research on elicitation in crypt-transgenic plants as well as for large scale production of saponins. Key message The cryptogein gene, which encodes a proteinaceous elicitor is associated with increase in secondary metabolite accumulation-either alone or in addition to the increases associated with transformation by A. rhizogenes.

Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

PubMed Central

Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping

2011-01-01

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121

A stability analysis on forced convection boundary layer stagnation-point slip flow in Darcy-Forchheimer porous medium towards a shrinking sheet

NASA Astrophysics Data System (ADS)

Bakar, Shahirah Abu; Arifin, Norihan Md; Ali, Fadzilah Md; Bachok, Norfifah; Nazar, Roslinda

2017-08-01

The stagnation-point flow over a shrinking sheet in Darcy-Forchheimer porous medium is numerically studied. The governing partial differential equations are transformed into ordinary differential equations using a similarity transformation, and then solved numerically by using shooting technique method with Maple implementation. Dual solutions are observed in a certain range of the shrinking parameter. Regarding on numerical solutions, we prepared stability analysis to identify which solution is stable between non-unique solutions by bvp4c solver in Matlab. Further we obtain numerical results or each solution, which enable us to discuss the features of the respective solutions.

Chemometric classification of gunshot residues based on energy dispersive X-ray microanalysis and inductively coupled plasma analysis with mass-spectrometric detection

NASA Astrophysics Data System (ADS)

Steffen, S.; Otto, M.; Niewoehner, L.; Barth, M.; Bro¿żek-Mucha, Z.; Biegstraaten, J.; Horváth, R.

2007-09-01

A gunshot residue sample that was collected from an object or a suspected person is automatically searched for gunshot residue relevant particles. Particle data (such as size, morphology, position on the sample for manual relocation, etc.) as well as the corresponding X-ray spectra and images are stored. According to these data, particles are classified by the analysis-software into different groups: 'gunshot residue characteristic', 'consistent with gunshot residue' and environmental particles, respectively. Potential gunshot residue particles are manually checked and - if necessary - confirmed by the operating forensic scientist. As there are continuing developments on the ammunition market worldwide, it becomes more and more difficult to assign a detected particle to a particular ammunition brand. As well, the differentiation towards environmental particles similar to gunshot residue is getting more complex. To keep external conditions unchanged, gunshot residue particles were collected using a specially designed shooting device for the test shots revealing defined shooting distances between the weapon's muzzle and the target. The data obtained as X-ray spectra of a number of particles (3000 per ammunition brand) were reduced by Fast Fourier Transformation and subjected to a chemometric evaluation by means of regularized discriminant analysis. In addition to the scanning electron microscopy in combination with energy dispersive X-ray microanalysis results, isotope ratio measurements based on inductively coupled plasma analysis with mass-spectrometric detection were carried out to provide a supplementary feature for an even lower risk of misclassification.

Reducing properties, energy efficiency and carbohydrate metabolism in hyperhydric and normal carnation shoots cultured in vitro: a hypoxia stress?

PubMed

Saher, Shady; Fernández-García, Nieves; Piqueras, Abel; Hellín, Eladio; Olmos, Enrique

2005-06-01

Hyperhydricity is considered as a physiological disorder that can be induced by different stressing conditions. In the present work we have studied the metabolic and energetic states of hyperhydric carnation shoots. We have evaluated the hypothesis that hypoxia stress is the main factor affecting the metabolism of hyperhydric leaves. Our results indicate a low level of ATP in hyperhydric tissues, but only slight modifications in pyridine nucleotide contents. Concurrently, the glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) activity in hyperhydric leaves was increased but glucokinase (GK; EC 2.7.1.2) activity was unchanged. We have observed that the metabolism of pyruvate was altered in hyperhydric tissues by the induction of pyruvate synthesis via NADP-dependent malic enzyme (EC 1.1.1.40). The enzymes of the fermentative metabolism pyruvate decarboxylase (PDC; EC 4.1.1.1) and alcohol dehydrogenase (ADH; EC 1.1.1.1) were highly increased in hyperhydric leaves. Sucrose metabolism was modified in hyperhydric leaves with a high increase in the activity of both synthesis and catabolic enzymes. The analysis of the sucrose, glucose and fructose contents indicated that all of these sugars were accumulated in hyperhydric leaves. However, the pinitol content was drastically decreased in hyperhydric leaves. We consider that these results suggest that hyperhydric leaves of carnation have adapted to hypoxia stress conditions by the induction of the oxidative pentose phosphate and fermentative pathways.

Natural variation reveals relationships between pre-stress carbohydrate nutritional status and subsequent responses to xenobiotic and oxidative stress in Arabidopsis thaliana.

PubMed

Ramel, Fanny; Sulmon, Cécile; Gouesbet, Gwenola; Couée, Ivan

2009-12-01

Soluble sugars are involved in responses to stress, and act as signalling molecules that activate specific or hormone cross-talk transduction pathways. Thus, exogenous sucrose treatment efficiently induces tolerance to the herbicide atrazine in Arabidopsis thaliana plantlets, at least partially through large-scale modifications of expression of stress-related genes. Availability of sugars in planta for stress responses is likely to depend on complex dynamics of soluble sugar accumulation, sucrose-starch partition and organ allocation. The question of potential relationships between endogenous sugar levels and stress responses to atrazine treatment was investigated through analysis of natural genetic accessions of A. thaliana. Parallel quantitative and statistical analysis of biochemical parameters and of stress-sensitive physiological traits was carried out on a set of 11 accessions. Important natural variation was found between accessions of A. thaliana in pre-stress shoot endogenous sugar levels and responses of plantlets to subsequent atrazine stress. Moreover, consistent trends and statistically significant correlations were detected between specific endogenous sugar parameters, such as the pre-stress end of day sucrose level in shoots, and physiological markers of atrazine tolerance. These significant relationships between endogenous carbohydrate metabolism and stress response therefore point to an important integration of carbon nutritional status and induction of stress tolerance in plants. The specific correlation between pre-stress sucrose level and greater atrazine tolerance may reflect adaptive mechanisms that link sucrose accumulation, photosynthesis-related stress and sucrose induction of stress defences.

Enhanced astaxanthin accumulation in Haematococcus pluvialis using high carbon dioxide concentration and light illumination.

PubMed

Christian, David; Zhang, Jun; Sawdon, Alicia J; Peng, Ching-An

2018-05-01

In this study, an economical two-stage method was proposed for the production of natural astaxanthin from Haematococcus pluvialis without a medium replacement step. In stage 1, H. pluvialis were grown under low light illumination until they reached optimal biomass. In stage 2, cells were switched to astaxanthin induction conditions utilizing the combination of high light illumination and elevated carbon dioxide levels (5 or 15%). The introduction of CO 2 altered the C/N balance creating a nutrient deficiency without a change of media. The resulting astaxanthin yield was 2-3 times that of using either stressor alone. This astaxanthin induction method has many advantages over current methods including no medium replacement and a short induction time of less than four days. Copyright © 2018 Elsevier Ltd. All rights reserved.

Ectopic expression of class 1 KNOX genes induce and adventitious shoot regeneration and alter growth and development of tobacco (Nicotiana tabacum L) and European plum (Prunus domestica L)

USDA-ARS?s Scientific Manuscript database

Transgenic plants of tobacco (Nicotiana tabacum L) and plum (Prunus domestica L) were produced by transforming with apple class 1 KNOX genes (MdKN1 and MdKN2) or corn KN1 gene. Transgenic tobacco plants were regenerated in vitro from transformed leaf discs cultured in a tissue medium lacking cytoki...

Production of haploid plantlets in anther cultures of Albizzia lebbeck L.

PubMed

Gharyal, P K; Rashid, A; Maheshwari, S C

1983-12-01

Anthers of Albizzia lebbeck on B5 medium (BM) supplemented with kinetin (2 mg/l) and 2, 4-D (0.5 mg/l) showed callus initiation from microspores. Differentiation of embryoids and shoots was obtained on BM + BAP (1 mg/l) + IAA (0.5 mg/l) and of roots on BM. Root tip squashes of the regenerated plantlets showed the haploid chromosome number (n=13), confirming the microspore origin of the regenerants.

Influence of Salt Stress on Growth and Frost Resistance of Three Winter Cereals

NASA Astrophysics Data System (ADS)

Matuszak-Slamani, Renata; Brzóstowicz, Aleksander

2015-04-01

This paper presents results of a study on the influence of 0-150 mmol NaCl dm-3 Hoagland solution on growth, chlorophyll content, photosynthesis and frost resistance of seedlings of three winter cereals: wheat - cv. Almari, rye - cv. Amilo, and triticale - cv. Tornado. Sodium chloride at 25 mmol dm-3 caused better growth of wheat shoots and roots, both of fresh and dry matter. Higher concentrations of NaCl in the medium decreased the biomass of the tested seedlings. The influence of NaCl on the chlorophyll content in the seedlings varied. The conductometry method showed that the resistance of the cell walls of wheat and rye to low temperature decreased in the presence of NaCl in the growth medium. Luminescence has shown that seedlings that grew in NaCl-containing medium indicated an impediment of electron flow at a lower temperature than the control plants.

Optimization of multi-epitopic HIV-1 recombinant protein expression in prokaryote system and conjugation to mouse DEC-205 monoclonal antibody: implication for in-vivo targeted delivery of dendritic cells

PubMed Central

Rahimi, Roghayeh; Ebtekar, Massoumeh; Moazzeni, Seyed Mohammad; Mostafaie, Ali; Mahdavi, Mehdi

2015-01-01

Objective(s): Multi-epitopic protein vaccines and direction of vaccine delivery to dendritic cells (DCs) are promising approaches for enhancing immune responses against mutable pathogens. Escherichia coli is current host for expression of recombinant proteins, and it is important to optimize expression condition. The aim of this study was the optimization of multi-epitopic HIV-1 tat/pol/gag/env recombinant protein (HIVtop4) expression by E. coli and conjugation of purified protein to anti DEC-205 monoclonal antibody as candidate vaccine. Materials and Methods: In this study, expression was induced in BL21 (DE3) E. coli cells by optimization of induction condition, post induction incubation time, temperature and culture medium formula. Some culture mediums were used for cell culture, and isopropyl-beta-D-thiogalactopyranoside was used for induction of expression. Protein was purified by Ni-NTA column chromatography and confirmed against anti-His antibody in western-blotting. To exploit DCs properties for immunization purposes, recombinant protein chemically coupled to αDEC-205 monoclonal antibody and confirmed against anti-His antibody in western-blotting. Results: The optimum condition for expression was 1 mM IPTG during 4 hr cultures in 2XYT medium, and final protein produced in soluble form. Conjugation of purified protein to αDEC-205 antibody resulted in smears of protein: antibodies conjugate in different molecular weights. Conclusion: The best cultivation condition for production of HIVtop4 protein is induction by 1 mM IPTG during 4 hr in 2XYT medium. The final concentration of purified protein was 500 µg/ml. PMID:25810888

Piper nigrum: micropropagation, antioxidative enzyme activities, and chromatographic fingerprint analysis for quality control.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Rahman, Inayat ur; Fazal, Hina

2013-04-01

A reliable in vitro regeneration system for the economical and medicinally important Piper nigrum L. has been established. Callus and shoot regeneration was encouraged from leaf portions on Murashige and Skoog (MS) medium augmented with varied concentrations of plant growth regulators. A higher callus production (90 %) was observed in explants incubated on MS medium incorporated with 1.0 mg L(-1) 6-benzyladenine (BA) along with 0.5 mg L(-1) gibberellic acid after 4 weeks of culture. Moreover, a callogenic response of 85 % was also recorded for 1.0 mg L(-1) BA in combination with 0.25 mg L(-1) α-naphthalene acetic acid (NAA) and 0.25 mg L(-1) 2,4-dichlorophenoxyacetic acid or 0.5 mg L(-1) indole butyric acid (IBA) along with 0.25 mg L(-1) NAA and indole acetic acid. Subsequent sub-culturing of callus after 4 weeks of culture onto MS medium supplemented with 1.5 mg L(-1) thiodiazoran or 1.5 mg L(-1) IBA induced 100 % shoot response. Rooted plantlets were achieved on medium containing varied concentrations of auxins. The antioxidative enzyme activities [superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX)] revealed that significantly higher SOD was observed in regenerated plantlets than in other tissues. However, POD, CAT, and APX were higher in callus than in other tissues. A high-performance liquid chromatography (HPLC) fingerprint analysis protocol was established for quality control in different in vitro-regenerated tissues of P. nigrum L. During analysis, most of the common peaks represent the active principle "piperine." The chemical contents, especially piperine, showed variation from callus culture to whole plantlet regeneration. Based on the deviation in chromatographic peaks, the in vitro-regenerated plantlets exhibit a nearly similar piperine profile to acclimated plantlets. The in vitro regeneration system and HPLC fingerprint analysis established here brought a novel approach to the quality control of in vitro plantlets, producing metabolites of interest with substantial applications for the conservation of germplasm.

Influence of Culture Medium Composition and Light Conditions on the Accumulation of Bioactive Compounds in Shoot Cultures of Scutellaria lateriflora L. (American Skullcap) Grown In Vitro.

PubMed

Kawka, Beata; Kwiecień, Inga; Ekiert, Halina

2017-12-01

Methanolic extracts from in vitro grown Scutellaria lateriflora shoots cultured on five Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) under different light conditions (monochromatic light, white light and no light) were analysed by HPLC for three groups of metabolites: flavonoids (26 compounds), phenolic acids and their precursors (19+2) and phenylethanoid glycosides (2). The analyses revealed the presence of baicalein, baicalin, wogonin, wogonoside, 3,4-dihydroxyphenylacetic acid and verbascoside. There was clear evidence of the influence of plant growth regulators and light conditions on the accumulation of the analysed groups of secondary metabolites. The amounts of the compounds changed within a wide range-for the total flavonoid content, 30.2-fold (max. 1204.3 mg·100 g -1 dry weight (DW)); for 3,4-dihydroxyphenylacetic acid, 5.5-fold (max. 33.56 mg·100 g -1 DW); and for verbascoside, 1.5-fold (169.15 max. mg·100 g -1 DW). The best medium for the production of most of the compounds was the Murashige and Skoog variant with 1 mg l -1 BAP and 1 mg l -1 NAA. For verbascoside, the best 'productive' medium was the MS variant supplemented with 0.5 mg l -1 BAP and 2 mg l -1 NAA. The accumulation of the metabolites was stimulated to the greatest extent by blue light, under which the extracts were found to contain the highest total amount of flavonoids and the highest amounts of flavonoid glucuronides, baicalin and wogonoside, as well as of verbascoside. Their amounts were, respectively, 1.54-, 1.49-, 2.05- and 1.86-fold higher than under the control white light.

Knocking down mitochondrial iron transporter (MIT) reprograms primary and secondary metabolism in rice plants.

PubMed

Vigani, Gianpiero; Bashir, Khurram; Ishimaru, Yasuhiro; Lehmann, Martin; Casiraghi, Fabio Marco; Nakanishi, Hiromi; Seki, Motoaki; Geigenberger, Peter; Zocchi, Graziano; Nishizawa, Naoko K

2016-03-01

Iron (Fe) is an essential micronutrient for plant growth and development, and its reduced bioavailability strongly impairs mitochondrial functionality. In this work, the metabolic adjustment in the rice (Oryza sativa) mitochondrial Fe transporter knockdown mutant (mit-2) was analysed. Biochemical characterization of purified mitochondria from rice roots showed alteration in the respiratory chain of mit-2 compared with wild-type (WT) plants. In particular, proteins belonging to the type II alternative NAD(P)H dehydrogenases accumulated strongly in mit-2 plants, indicating that alternative pathways were activated to keep the respiratory chain working. Additionally, large-scale changes in the transcriptome and metabolome were observed in mit-2 rice plants. In particular, a strong alteration (up-/down-regulation) in the expression of genes encoding enzymes of both primary and secondary metabolism was found in mutant plants. This was reflected by changes in the metabolic profiles in both roots and shoots of mit-2 plants. Significant alterations in the levels of amino acids belonging to the aspartic acid-related pathways (aspartic acid, lysine, and threonine in roots, and aspartic acid and ornithine in shoots) were found that are strictly connected to the Krebs cycle. Furthermore, some metabolites (e.g. pyruvic acid, fumaric acid, ornithine, and oligosaccharides of the raffinose family) accumulated only in the shoot of mit-2 plants, indicating possible hypoxic responses. These findings suggest that the induction of local Fe deficiency in the mitochondrial compartment of mit-2 plants differentially affects the transcript as well as the metabolic profiles in root and shoot tissues. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

CuO and ZnO nanoparticles: phytotoxicity, metal speciation, and induction of oxidative stress in sand-grown wheat

NASA Astrophysics Data System (ADS)

Dimkpa, Christian O.; McLean, Joan E.; Latta, Drew E.; Manangón, Eliana; Britt, David W.; Johnson, William P.; Boyanov, Maxim I.; Anderson, Anne J.

2012-09-01

Metal oxide nanoparticles (NPs) are reported to impact plant growth in hydroponic systems. This study describes the impact of commercial CuO (<50 nm) and ZnO (<100 nm) NPs on wheat ( Triticum aestivum) grown in a solid matrix, sand. The NPs contained both metallic and non-metallic impurities to different extents. Dynamic light scattering and atomic force microscopy (AFM) assessments confirmed aggregation of the NPs to submicron sizes. AFM showed transformation of ZnO NPs from initial rhomboid shapes in water to elongated rods in the aqueous phase of the sand matrix. Solubilization of metals occurred in the sand at similar rates from CuO or ZnO NPs as their bulk equivalents. Amendment of the sand with 500 mg Cu and Zn/kg sand from the NPs significantly ( p = 0.05) reduced root growth, but only CuO NPs impaired shoot growth; growth reductions were less with the bulk amendments. Dissolved Cu from CuO NPs contributed to their phytotoxicity but Zn release did not account for the changes in plant growth. Bioaccumulation of Cu, mainly as CuO and Cu(I)-sulfur complexes, and Zn as Zn-phosphate was detected in the shoots of NP-challenged plants. Total Cu and Zn levels in shoot were similar whether NP or bulk materials were used. Oxidative stress in the NP-treated plants was evidenced by increased lipid peroxidation and oxidized glutathione in roots and decreased chlorophyll content in shoots; higher peroxidase and catalase activities were present in roots. These findings correlate with the NPs causing increased production of reactive oxygen species. The accumulation of Cu and Zn from NPs into edible plants has relevance to the food chain.

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis.

PubMed

Benelli, Carla; De Carlo, Anna; Engelmann, Florent

2013-01-01

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at -5 °C, and then cooled slowly to -30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation-dehydration, vitrification, encapsulation-vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants. Copyright © 2012 Elsevier Inc. All rights reserved.

Induction of L-phenylalanine ammonia-lyase during utilization of phenylalanine as a carbon or nitrogen source in Rhodotorula glutinis.

PubMed Central

Marusich, W C; Jensen, R A; Zamir, L O

1981-01-01

Rhodotorula glutinis is a convenient source of L-phenylalanine ammonia-lyase, an enzyme that is useful as a biochemical reagent in the assay of L-phenylalanine. There have been previous descriptions of induced lyase production in complex medium where induction occurs late in exponential growth, suggesting a role in secondary metabolism such as is the case in higher plants. A higher specific activity of L-phenylalanine ammonia-lyase (sixfold higher than a complex medium) can be obtained during midexponential growth in a defined medium containing L-phenylalanine as the sole source of carbon. L-Phenylalanine will also induce lyase synthesis during exponential growth in minimal in which L-phenylalanine is the sole source of nitrogen. The appearance of lyase in complex medium supplemented with L-phenylalanine is probably triggered fortuitously by exhaustion late in growth of a prime source of nitrogen. In this study, R. glutinis appeared to express a single lyase enzyme, regardless of whether induction was nitrogen signaled or carbon signaled. Thin-layer chromatographic analysis of ether extracts prepared from cultures induced with doubly labeled (U-14C; ring-4-3H) L-phenylalanine provided evidence of a catabolic sequence containing cinnamic acid, benzoic acid, and 4-hydroxybenzoic acid as degradative intermediates. 3,4-Dihydroxybenzoic acid was not identified as a catabolic intermediate. PMID:7195398

[Enhanced phytoextraction of heavy metal contaminated soil by chelating agents and auxin indole-3-acetic acid].

PubMed

Zhou, Jian-min; Dang, Zhi; Chen, Neng-chang; Xu, Sheng-guang; Xie, Zhi-yi

2007-09-01

The environmental risk of chelating agents such as EDTA application to the heavy metals polluted soils and the stress on plant roots due to the abrupt increase metals concentration limit the wide commercial use of chelate-induced phytoextraction. Chelating agent ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) and auxin indole-3-acetic acid (IAA) were used for enhancing heavy metals uptake from soils by Zea mays L. (corn) in pot experiments. The metals content in plant tissues was quantified using an inductively coupled plasma mass spectrometer (ICP-MS). The results showed that the combination of IAA and EDTA increased the biomass by about 40.0% and the contents of Cu, Zn, Cd and Pb in corn shoots by 27.0%, 26.8%, 27.5% and 32.8% respectively, as compared to those in EDTA treatment. While NTA&IAA treatment increased the biomass by about 29.9% and the contents of Cu, Zn, Cd and Pb in corn shoots by 31.8%, 27.6%, 17.0% and 26.9% respectively, as compared to those in NTA treatment. These results indicated that corn growth was promoted, and the biomass and the accumulation of heavy metals in plant shoots were increased significantly with the addition of IAA, which probably helps to change the cell membrane properties and the biomass distribution, resulting in the alleviation of the phytotoxicity of metals and the chelating agents.

Growth and recombinant protein expression with Escherichia coli in different batch cultivation media.

PubMed

Hortsch, Ralf; Weuster-Botz, Dirk

2011-04-01

Parallel operated milliliter-scale stirred tank bioreactors were applied for recombinant protein expression studies in simple batch experiments without pH titration. An enzymatic glucose release system (EnBase), a complex medium, and the frequently used LB and TB media were compared with regard to growth of Escherichia coli and recombinant protein expression (alcohol dehydrogenase (ADH) from Lactobacillus brevis and formate dehydrogenase (FDH) from Candida boidinii). Dissolved oxygen and pH were recorded online, optical densities were measured at-line, and the activities of ADH and FDH were analyzed offline. Best growth was observed in a complex medium with maximum dry cell weight concentrations of 14 g L(-1). EnBase cultivations enabled final dry cell weight concentrations between 6 and 8 g L(-1). The pH remained nearly constant in EnBase cultivations due to the continuous glucose release, showing the usefulness of this glucose release system especially for pH-sensitive bioprocesses. Cell-specific enzyme activities varied considerably depending on the different media used. Maximum specific ADH activities were measured with the complex medium, 6 h after induction with IPTG, whereas the highest specific FDH activities were achieved with the EnBase medium at low glucose release profiles 24 h after induction. Hence, depending on the recombinant protein, different medium compositions, times for induction, and times for cell harvest have to be evaluated to achieve efficient expression of recombinant proteins in E. coli. A rapid experimental evaluation can easily be performed with parallel batch operated small-scale stirred tank bioreactors.

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway

PubMed Central

Lin, Fei-xiang; Du, Shi-xin; Liu, De-zhong; Hu, Qin-xiao; Yu, Guo-yong; Wu, Chu-cheng; Zheng, Gui-zhou; Xie, Da; Li, Xue-dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway. PMID:27904711

Naringin promotes osteogenic differentiation of bone marrow stromal cells by up-regulating Foxc2 expression via the IHH signaling pathway.

PubMed

Lin, Fei-Xiang; Du, Shi-Xin; Liu, De-Zhong; Hu, Qin-Xiao; Yu, Guo-Yong; Wu, Chu-Cheng; Zheng, Gui-Zhou; Xie, Da; Li, Xue-Dong; Chang, Bo

2016-01-01

Naringin is an active compound extracted from Rhizoma Drynariae, and studies have revealed that naringin can promote proliferation and osteogenic differentiation of bone marrow stromal cells (BMSCs). In this study, we explored whether naringin could promote osteogenic differentiation of BMSCs by upregulating Foxc2 expression via the Indian hedgehog (IHH) signaling pathway. BMSCs were cultured in basal medium, basal medium with naringin, osteogenic induction medium, osteogenic induction medium with naringin and osteogenic induction medium with naringin in the presence of the IHH inhibitor cyclopamine (CPE). We examined cell proliferation by using a WST-8 assay, and differentiation by Alizarin Red S staining (for mineralization) and alkaline phosphatase (ALP) activity. In addition, we detected core-binding factor α1 (Cbfα1), osteocalcin (OCN), bone sialoprotein (BSP), peroxisome proliferation-activated receptor gamma 2 (PPARγ2) and Foxc2 expression by using RT-PCR. We also determined Foxc2 and IHH protein levels by western blotting. Naringin increased the mineralization of BMSCs, as shown by Alizarin red S assays, and induced ALP activity. In addition, naringin significantly increased the mRNA levels of Foxc2, Cbfα1, OCN, and BSP, while decreasing PPARγ2 mRNA levels. Furthermore, the IHH inhibitor CPE inhibited the osteogenesis-potentiating effects of naringin. Naringin increased Foxc2 and stimulated the activation of IHH, as evidenced by increased expression of proteins that were inhibited by CPE. Our findings indicate that naringin promotes osteogenic differentiation of BMSCs by up-regulating Foxc2 expression via the IHH signaling pathway.

Evidence of root zone hypoxia in Brassica rapa L. grown in microgravity.

PubMed

Stout, S C; Porterfield, D M; Briarty, L G; Kuang, A; Musgrave, M E

2001-03-01

A series of experiments was conducted aboard the U.S. space shuttle and the Mir space station to evaluate microgravity-induced root zone hypoxia in rapid-cycling Brassica (Brassica rapa L.), using both root and foliar indicators of low-oxygen stress to the root zone. Root systems from two groups of plants 15 and 30 d after planting, grown in a phenolic foam nutrient delivery system on the shuttle (STS-87), were harvested and fixed for microscopy or frozen for enzyme assays immediately postflight or following a ground-based control. Activities of fermentative enzymes were measured as indicators of root zone hypoxia and metabolism. Following 16 d of microgravity, ADH (alcohol dehydrogenase) activity was increased in the spaceflight roots 47% and 475% in the 15-d-old and 30-d-old plants, respectively, relative to the ground control. Cytochemical localization showed ADH activity in only the root tips of the space-grown plants. Shoots from plants that were grown from seed in flight in a particulate medium on the Mir station were harvested at 13 d after planting and quick-frozen and stored in flight in a gaseous nitrogen freezer or chemically fixed in flight for subsequent microscopy. When compared to material from a high-fidelity ground control, concentrations of shoot sucrose and total soluble carbohydrate were significantly greater in the spaceflight treatment according to enzymatic carbohydrate analysis. Stereological analysis of micrographs of sections from leaf and cotyledon tissue fixed in flight and compared with ground controls indicated no changes in the volume of protoplast, cell wall, and intercellular space in parenchyma cells. Within the protoplasm, the volume occupied by starch was threefold higher in the spaceflight than in the ground control, with a concomitant decrease in vacuolar volume in the spaceflight treatment. Both induction of fermentative enzyme activity in roots and accumulation of carbohydrates in foliage have been repeatedly shown to occur in response to root zone oxygen deprivation. These results indicate that root zone hypoxia is a persistent challenge in spaceflight plant growth experiments and may be caused by microgravity-induced changes in fluid and gas distribution.

Evidence of root zone hypoxia in Brassica rapa L. grown in microgravity

NASA Technical Reports Server (NTRS)

Stout, S. C.; Porterfield, D. M.; Briarty, L. G.; Kuang, A.; Musgrave, M. E.

2001-01-01

A series of experiments was conducted aboard the U.S. space shuttle and the Mir space station to evaluate microgravity-induced root zone hypoxia in rapid-cycling Brassica (Brassica rapa L.), using both root and foliar indicators of low-oxygen stress to the root zone. Root systems from two groups of plants 15 and 30 d after planting, grown in a phenolic foam nutrient delivery system on the shuttle (STS-87), were harvested and fixed for microscopy or frozen for enzyme assays immediately postflight or following a ground-based control. Activities of fermentative enzymes were measured as indicators of root zone hypoxia and metabolism. Following 16 d of microgravity, ADH (alcohol dehydrogenase) activity was increased in the spaceflight roots 47% and 475% in the 15-d-old and 30-d-old plants, respectively, relative to the ground control. Cytochemical localization showed ADH activity in only the root tips of the space-grown plants. Shoots from plants that were grown from seed in flight in a particulate medium on the Mir station were harvested at 13 d after planting and quick-frozen and stored in flight in a gaseous nitrogen freezer or chemically fixed in flight for subsequent microscopy. When compared to material from a high-fidelity ground control, concentrations of shoot sucrose and total soluble carbohydrate were significantly greater in the spaceflight treatment according to enzymatic carbohydrate analysis. Stereological analysis of micrographs of sections from leaf and cotyledon tissue fixed in flight and compared with ground controls indicated no changes in the volume of protoplast, cell wall, and intercellular space in parenchyma cells. Within the protoplasm, the volume occupied by starch was threefold higher in the spaceflight than in the ground control, with a concomitant decrease in vacuolar volume in the spaceflight treatment. Both induction of fermentative enzyme activity in roots and accumulation of carbohydrates in foliage have been repeatedly shown to occur in response to root zone oxygen deprivation. These results indicate that root zone hypoxia is a persistent challenge in spaceflight plant growth experiments and may be caused by microgravity-induced changes in fluid and gas distribution.

Orbital experiment ``Gravisensor'': phototropic reactions of the moss Physcomitrella patens to different types of LED lighting.

NASA Astrophysics Data System (ADS)

Nikitin, Vladimir; Berkovich, Yuliy A.; Skripnikov, Alexander; Zyablova, Natalya; Mukhoyan, Makar; Emelianov, Grigory

The experiment was conducted on Russian Biological Satelite Bion-M #1 19.04-19.05 2013. Five transparent plastic cultural flasks were placed in five light isolated sections of Biocont-B2 cylindrical container with inner diameter of 120 mm and height of 230 mm. In four sections the flasks could be illuminated by top or side LED with wavelength of 458 nm, 630 nm, 730 nm, and white (color temperature 5000° K, peaks 453, 559 nm). Photon flux in each variant was 15 umol/(m2c). In the fifth section the flask with the shoots was in conditions of constant dark. Each section was equipped with its own video camera module. Cameras, video recorder and lighting were managed by micro controller. 12 days before launch, 5 tips of the moss shoots were explanted at each of the five flasks on the agar medium with nutrient components and were cultivated under white fluorescent lamps at 12 hour photo period till the launch. After entering the orbit and during next 14 days of flight top LEDs were turned on above the flasks. Then for the following 14 days of flight the side LEDs of similar wavelength were turned on. The moss gametophores were cultivated at 12-h photoperiod. During the experiment on an hourly basis a video recording of the moss was performed. Similar equipment was used for ground control. After the experiment video files were used to produce separate time-lapse films for each flask using AviSynth program. In flight the shoots demonstrated the maximum growth speed with far red lighting and slower speed with white lighting. With blue and red lighting after switching to side light stimuli the growth of shoots almost stopped. In the dark the shoots continued to grow until the 13 day after launch of the satellite, then their growth stopped. In ground control the relation of growth rate with various LEDs remained basically the same, with the exception of side blue lighting, where the shoots demonstrated considerable vertical growth. In flight the angle of inclination towards the light source was maximal (about 90º) with white lighting, and somewhat smaller with 730 nm. Under red and blue light the angle of phototropic inclination was difficult to measure due to poor growth of the shoots.In ground control the growth rate under blue light was several times higher, than in flight and final degree of inclination of the shoot tip came to about 10º. In ground control under side red lighting the growth was weak, while demonstrating a pronounced phototropic bend of 90º. In ground control in the dark a vertical growth of one shoot was observed with the rate somewhat larger, than in flight variant. Data on the dynamics of inclination of experimental and control plants are presented. The acquired data will be used to analyse the mechanisms of phototropic growth changes of moss shoots.

Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply.

PubMed

Nie, Zhaojun; Zhao, Peng; Wang, Jia; Li, Jinfeng; Liu, Hongen

2017-01-01

Nitrogen (N) is critical for zinc (Zn) absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat ( Triticum aestivum L.), an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn 10 ) treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate ( V max ), root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N 7.5 ) treatment were higher, but the Michaelis constant ( K m ) and minimum equilibrium concentrations ( C min ) in this treatment were lower than those in the low (N 0.05 ) and high (N 15 ) N treatments, when Zn was supplied at a high level (Zn 10 ). Meanwhile, there were no pronounced differences in the above root traits between the N 0.05 Zn 0 and N 7.5 Zn 10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root development in winter wheat. An increase in N supply was beneficial in terms of achieving a balanced distribution of Zn subcellular fractions, thus enhancing Zn translocation to shoots, while maintaining normal metabolism.

Absorption Kinetics and Subcellular Fractionation of Zinc in Winter Wheat in Response to Nitrogen Supply

PubMed Central

Nie, Zhaojun; Zhao, Peng; Wang, Jia; Li, Jinfeng; Liu, Hongen

2017-01-01

Nitrogen (N) is critical for zinc (Zn) absorption into plant roots; this in turn allows for Zn accumulation and biofortification of grain in winter wheat (Triticum aestivum L.), an important food crop. However, little is known about root morphology and subcellular Zn distribution in response to N treatment at different levels of Zn supply. In this study, two nutrient solution culture experiments were conducted to examine Zn accumulation, Zn absorption kinetics, root morphology, and Zn subcellular distribution in wheat seedlings pre-cultured with different N concentrations. The results showed positive correlations between N and Zn concentrations, and N and Zn accumulation, respectively. The findings suggested that an increase in N supply enhanced root absorption and the root-to-shoot transport of Zn. Nitrogen combined with the high Zn (Zn10) treatment increased the Zn concentration and consequently its accumulation in both shoots and roots. The maximum influx rate (Vmax), root length, surface area, and volume of 14-d-old seedlings, and root growth from 7 to 14 d in the medium N (N7.5) treatment were higher, but the Michaelis constant (Km) and minimum equilibrium concentrations (Cmin) in this treatment were lower than those in the low (N0.05) and high (N15) N treatments, when Zn was supplied at a high level (Zn10). Meanwhile, there were no pronounced differences in the above root traits between the N0.05Zn0 and N7.5Zn10 treatments. An increase in N supply decreased Zn in cell walls and cell organelles, while it increased Zn in the root soluble fraction. In leaves, an increase in N supply significantly decreased Zn in cell walls and the soluble fraction, while it increased Zn in cell organelles under Zn deficiency, but increased Zn distribution in the soluble fraction under medium and high Zn treatments. Therefore, a combination of medium N and high Zn treatments enhanced Zn absorption, apparently by enhancing Zn membrane transport and stimulating root development in winter wheat. An increase in N supply was beneficial in terms of achieving a balanced distribution of Zn subcellular fractions, thus enhancing Zn translocation to shoots, while maintaining normal metabolism. PMID:28868060

In vitro propagation and assessment of genetic stability of acclimated plantlets of Cornus alba L. using RAPD and ISSR markers.

PubMed

Ilczuk, Agnieszka; Jacygrad, Ewelina

2016-01-01

Cornus alba L. (white dogwood) is an important ornamental shrub having a wide range of applications such as reforestation programs and soil retention systems. The vegetative propagation of dogwood by cuttings may be slow, difficult, and cultivar dependent; therefore, an improved micropropagation method was developed. Nodal stem segments of C. alba cultivars 'Aurea' and 'Elegantissima' were cultured on media enriched with six different sources of macronutrients. Media were supplemented with either N 6 -benzyladenine (BA) or thidiazuron (TDZ) in combination with 1-naphthaleneacetic acid (NAA). Regardless of the cultivar, the best shoot proliferation was observed on Lloyd and McCown medium (woody plant medium (WPM)) at pH 6.2, containing 1.0 mg L -1 BA, 0.1 mg L -1 NAA, and 20-30 g L -1 sucrose. Rooting of regenerated shoots was achieved by an in vitro method when different concentrations of NAA or indole-3-butyric acid (IBA) were tested. Microcuttings were rooted for 8 wk on medium enriched with 0.25 mg L -1 NAA and potted into P9 containers in the greenhouse. The final survival rate of the plants after 20 wk was 80% for 'Aurea' and 90% for 'Elegantissima'. Genetic stability of the micropropagated plants was confirmed by using two DNA-based molecular marker techniques. A total of 30 random amplified polymorphic DNA (RAPD) and 20 inter-simple sequence repeat (ISSR) primers resulted in 197-199 and 184-187 distinct and reproducible band classes, respectively, in 'Aurea' and 'Elegantissima' plantlets. All of the RAPD and ISSR profiles were monomorphic and comparable with the mother plant.

Growth and antioxidant defense responses of wheat seedlings to di-n-butyl phthalate and di (2-ethylhexyl) phthalate stress.

PubMed

Gao, Minling; Dong, Youming; Zhang, Ze; Song, Wenhua; Qi, Yun

2017-04-01

Phthalate acid esters (PAEs) are vital environmental hormone-like chemicals that are noxious to plants, animals, and human beings. In this study, the influences of di-n-butyl phthalate (DBP) and di (2-ethylhexyl) phthalate (DEHP) on the seed germination, root morphology, and various physiological changes of wheat seedlings were investigated by analyzing superoxide anion (O 2 - ) accumulation, antioxidant enzyme activity, and lipid peroxidation. DBP and DEHP were found to obviously inhibit germination only at high concentrations, but significantly affected root morphology even at lower concentrations. Their toxic effects were the most severe on root elongation, followed by shoot elongation, and were the least severe on germination rate, indicating that root elongation was the best index for evaluating DBP and DEHP eco-toxicity. DBP and DEHP also enhanced O 2 - and malondialdehyde levels and membrane permeability, as well as produced changes in the antioxidant status and PAE content in the stem and leaf (combined tissues, hereafter shoot) and root tissues. The activities of superoxide dismutase, catalase, and peroxidase increased at low and medium DBP and DEHP concentrations, but declined at high PAE concentrations. These results indicated that PAEs could exert oxidative damage in the early development stage of wheat, particularly at higher concentrations. DBP and DEHP accumulation was higher in the roots than in the shoot tissues, and their levels in these tissues increased with increasing PAE concentrations, supporting their more-serious toxic effects on roots than those on shoots. Further, the physicochemical properties of DBP rendered it more harmful than DEHP. Copyright © 2017 Elsevier Ltd. All rights reserved.

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine.

PubMed

Manganaris, G A; Economou, A S; Boubourakas, I N; Katis, N I

2003-10-01

The plum pox virus (PPV) and prunus necrotic ringspot virus (PNRSV) cause serious disease problems in stone-fruit trees. In this work, the possibility of obtaining plant material free from these viruses through thermotherapy and meristem-tip culture from infected nectarine shoots (Prunus persica var. nectarina Max, cv. 'Arm King') was studied. In addition, the detection of these viruses in in vitro cultures and young acclimatized plantlets with double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was studied. Meristem-tip explants (0.8-1.3 mm) derived from sprouted buds of winter wood and spring shoots from field grown plants had a 2-5% regeneration response. However, application of thermotherapy to potted nectarine trees (3 weeks at a maximum temperature of 35 degrees C) facilitated excision of longer meristem tips (1.3-2.0 mm) that resulted in a significantly higher regeneration response (38%) in woody plant medium (WPM) without plant growth regulators. Such explants formed multiple shoots with the addition of 8 microM benzylaminopurine and 0.8 microM indoleacetic acid. When they were tested for the presence of PPV and PNRSV, 86% and 81% were found to be virus-free as detected by DAS-ELISA and multiplex RT-PCR, respectively. Individual shoots excised from virus-free cultures readily rooted in vitro (half-strength WPM plus 2 microM indolebutyric acid) and grew to plantlets. The combination of an efficient protocol for virus elimination and the establishment of highly sensitive diagnostics resulted in the production of nectarine plants free from PPV and PNRSV.

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification.

PubMed

Sajini, K K; Karun, A; Amamath, C H; Engelmann, F

2011-01-01

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.

Polysaccharides obtained from bamboo shoots (Chimonobambusa quadrangularis) processing by-products: New insight into ethanol precipitation and characterization.

PubMed

Zhang, Fusheng; Ran, ChunXia; Zheng, Jiong; Ding, Yongbo; Chen, Guangjing

2018-06-01

Chimonobambusa quadrangularis polysaccharides (CPS) were extracted by ultrasonic-assisted extraction from bamboo shoots (C. quadrangularis) processing by-products. Three polysaccharide fractions, CPS70, CPS75 and CPS80, were obtained by precipitation at final ethanol concentrations of 70%, 75% and 80%, respectively. The physicochemical characterization and chemical antioxidant activities of the three polysaccharide fractions were compared on the basis of HPLC, FT-IR, XRD, TGA, and antioxidant measurements in vitro. The results suggested that ethanol concentrations used for precipitation of CPS can affect its physicochemical and associated functional properties, and antioxidant activities. Compared with CPS70 and CPS80, CPS75 had lower glucose content, higher total sugar content, and higher protein and uronic acid contents. The CPS70 and CPS80 were composed of Man, Rha, GlcA, Glc, Gal, Xyl and Ara, but none of them were found to contain GalA. In contrast, CPS75 consisted of Man, Rha, GlcA, GalA, Glc, Gal, Xyl and Ara. CPS75 had the lowest medium-high-molecular-weight value (116.53-118.18kDa) and the highest medium-low-molecular-weight value (21.30-22.68kDa). Meanwhile, CPS75 exhibited better functional properties including the repose angle, swelling capacity (SC), water retention capacity (WRC), and oil retention capacity (ORC). Moreover, CPS75 possessed higher scavenging capacities on DPPH, hydroxyl and ABTS radicals, higher oxygen radical absorbance capacity (OARC), higher metal chelating activity, and more significant reducing power. According to the results above, a final ethanol concentration of 75% could be chose to precipitate polysaccharides from bamboo shoots (C. quadrangularis) processing by-products. In summary, it is strongly recommended that the ethanol concentration employed in precipitation of natural polysaccharides could be optimized in advance. Copyright © 2018 Elsevier B.V. All rights reserved.

Salt-dependent regulation of a CNG channel subfamily in Arabidopsis.

PubMed

Kugler, Annette; Köhler, Barbara; Palme, Klaus; Wolff, Patricia; Dietrich, Petra

2009-11-27

In Arabidopsis thaliana, the family of cyclic nucleotide-gated channels (CNGCs) is composed of 20 members. Previous studies indicate that plant CNGCs are involved in the control of growth processes and responses to abiotic and biotic stresses. According to their proposed function as cation entry pathways these channels contribute to cellular cation homeostasis, including calcium and sodium, as well as to stress-related signal transduction. Here, we studied the expression patterns and regulation of CNGC19 and CNGC20, which constitute one of the five CNGC subfamilies. GUS, GFP and luciferase reporter assays were used to study the expression of CNGC19 and CNGC20 genes from Arabidopsis thaliana in response to developmental cues and salt stress. CNGC19 and CNGC20 were differentially expressed in roots and shoots. The CNGC19 gene was predominantly active in roots already at early growth stages. Major expression was observed in the phloem. CNGC20 showed highest promoter activity in mesophyll cells surrounding the veins. Its expression increased during development and was maximal in mature and senescent leaves. Both genes were upregulated in the shoot in response to elevated NaCl but not mannitol concentrations. While in the root, CNGC19 did not respond to changes in the salt concentration, in the shoot it was strongly upregulated in the observed time frame (6-72 hours). Salt-induction of CNGC20 was also observed in the shoot, starting already one hour after stress treatment. It occurred with similar kinetics, irrespective of whether NaCl was applied to roots of intact plants or to the petiole of detached leaves. No differences in K and Na contents of the shoots were measured in homozygous T-DNA insertion lines for CNGC19 and CNGC20, respectively, which developed a growth phenotype in the presence of up to 75 mM NaCl similar to that of the wild type. Together, the results strongly suggest that both channels are involved in the salinity response of different cell types in the shoot. Upon salinity both genes are upregulated within hours. CNGC19 and CNGC20 could assist the plant to cope with toxic effects caused by salt stress, probably by contributing to a re-allocation of sodium within the plant.

Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir ( Pseudotsuga menziesii) shoot cultures

PubMed Central

Chen, Chien-Chih; Bates, Rick; Carlson, John

2015-01-01

The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change on explant growth performance and 3) assess the effects of adding a pH stabilizer, 2-(N-morpholino)ethanesulfonic acid (MES) that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth. PMID:26535110

An Effective Protocol for Micropropagation of Edible Bamboo Species (Bambusa tulda and Melocanna baccifera) through Nodal Culture

PubMed Central

Waikhom, Sayanika Devi; Louis, Bengyella

2014-01-01

High demand for edible bamboo shoots of Bambusa tulda and Melocanna baccifera in many Asian ethnic groups has led to the need for developing intensive bamboo farming. To achieve this, in vitro regeneration of bamboo plantlets is needed due to the long and irregular bamboo flowering cycle and scarcity of bamboo seeds. An effective protocol for plantlets regeneration in B. tulda and M. baccifera from nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is described. Effective axillary bud breaking was achieved at 3 mg/L of 6-benzylaminopurine (BAP) in MS medium. Importantly, combining 2 mg/L of kinetin (Kn) with 3 mg/L of BAP produced a synergistic effect for shoot multiplication in B. tulda and M. baccifera. Under optimized conditions in half-strength MS medium supplemented with 3 mg/L of indole-3-butyric acid (IBA), 10 mg/L of coumarin, and 3% sucrose, profuse production of dark-brown rhizome in B. tulda and abundant rooting (81.67%, P < 0.05, F = 15.46) for M. baccifera within 30 days were achieved. The established protocol and the validation of the reported species at the molecular level will be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity. PMID:24967429

Plant regeneration from leaf protoplasts of Solanum torvum.

PubMed

Guri, A; Volokita, M; Sink, K C

1987-07-01

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10(4) per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1-2 weeks and 4 weeks later p-calli were 1-3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.

Hormetic Response by Silver Nanoparticles on In Vitro Multiplication of Sugarcane (Saccharum spp. Cv. Mex 69-290) Using a Temporary Immersion System.

PubMed

Bello-Bello, Jericó J; Chavez-Santoscoy, Rocío A; Lecona-Guzmán, Carlos A; Bogdanchikova, Nina; Salinas-Ruíz, Josafhat; Gómez-Merino, Fernando Carlos; Pestryakov, Alexey

2017-01-01

Hormesis is considered a dose-response phenomenon characterized by growth stimulation at low doses and inhibition at high doses. The hormetic response by silver nanoparticles (AgNPs) on in vitro multiplication of sugarcane was evaluated using a temporary immersion system. Sugarcane shoots were used as explants cultured in Murashige and Skoog medium with AgNPs at concentrations of 0, 25, 50, 100, and 200 mg/L. Shoot multiplication rate and length were used to determine hormetic response. Total content of phenolic compounds of sugarcane, mineral nutrition, and reactive oxygen species (ROS) was determined. Results were presented as a dose-response curve. Stimulation phase growth was observed at 50 mg/L AgNPs, whereas inhibition phase was detected at 200 mg/L AgNPs. Mineral nutrient analysis showed changes in macronutrient and micronutrient contents due to the effect of AgNPs. Moreover, AgNPs induced ROS production and increased total phenolic content, with a dose-dependent effect. Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNP-induced hormesis and that phenolic accumulation was obtained as a response of the plant to stress produced by high doses of AgNPs. Therefore, small doses of AgNPs in the culture medium could be an efficient strategy for commercial micropropagation.

Hormetic Response by Silver Nanoparticles on In Vitro Multiplication of Sugarcane (Saccharum spp. Cv. Mex 69-290) Using a Temporary Immersion System

PubMed Central

Chavez-Santoscoy, Rocío A.; Lecona-Guzmán, Carlos A.; Bogdanchikova, Nina; Salinas-Ruíz, Josafhat; Gómez-Merino, Fernando Carlos; Pestryakov, Alexey

2017-01-01

Background: Hormesis is considered a dose–response phenomenon characterized by growth stimulation at low doses and inhibition at high doses. The hormetic response by silver nanoparticles (AgNPs) on in vitro multiplication of sugarcane was evaluated using a temporary immersion system. Methods: Sugarcane shoots were used as explants cultured in Murashige and Skoog medium with AgNPs at concentrations of 0, 25, 50, 100, and 200 mg/L. Shoot multiplication rate and length were used to determine hormetic response. Total content of phenolic compounds of sugarcane, mineral nutrition, and reactive oxygen species (ROS) was determined. Results: Results were presented as a dose–response curve. Stimulation phase growth was observed at 50 mg/L AgNPs, whereas inhibition phase was detected at 200 mg/L AgNPs. Mineral nutrient analysis showed changes in macronutrient and micronutrient contents due to the effect of AgNPs. Moreover, AgNPs induced ROS production and increased total phenolic content, with a dose-dependent effect. Conclusion: Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNP-induced hormesis and that phenolic accumulation was obtained as a response of the plant to stress produced by high doses of AgNPs. Therefore, small doses of AgNPs in the culture medium could be an efficient strategy for commercial micropropagation. PMID:29238274

Thiol peptides induction in the seagrass Thalassia testudinum (Banks ex König) in response to cadmium exposure.

PubMed

Alvarez-Legorreta, Teresa; Mendoza-Cozatl, David; Moreno-Sanchez, Rafael; Gold-Bouchot, Gerardo

2008-01-20

Trace metal accumulation and thiol compounds synthesis as induced by cadmium exposure was studied in the seagrass Thalassia testudinum. Shoots were exposed for 24, 48, 96 and 144 h to several CdCl(2) concentrations (0, 30, 50 and 70 microM). Levels of cadmium, cysteine, glutathione (GSH), gamma-glutamylcysteine (gamma-EC), and phytochelatin-like peptides were determined in green blades, live sheaths and root/rhizomes tissues. Metal accumulation was dependent on Cd concentration and type of tissue, with green blades showing the highest content followed by live sheaths and root/rhizomes. All tissues experienced an increase in thiol-containing compounds as a response to cadmium exposure. Live sheaths showed the highest levels of cysteine, GSH and gamma-EC. This is the first report of induction of thiol peptides, presumably phytochelatins, by a trace metal in a sea grass species.

Induction of differentiation of human embryonic stem cells into functional hair-cell-like cells in the absence of stromal cells.

PubMed

Ding, Jie; Tang, Zihua; Chen, Jiarong; Shi, Haosong; Chen, Jianling; Wang, Cuicui; Zhang, Cui; Li, Liang; Chen, Ping; Wang, Jinfu

2016-12-01

Sensorineural hearing loss and vestibular dysfunction have become the most common forms of sensory defects. Stem cell-based therapeutic strategies for curing hearing loss are being developed. Several attempts to develop hair cells by using chicken utricle stromal cells as feeder cells have resulted in phenotypic conversion of stem cells into inner ear hair-cell-like cells. Here, we induced the differentiation of human embryonic stem cells (hESCs) into otic epithelial progenitors (OEPs), and further induced the differentiation of OEPs into hair-cell-like cells using different substrates. Our results showed that OEPs cultured on the chicken utricle stromal cells with the induction medium could differentiate into hair-cell-like cells with stereociliary bundles. Co-culture with stromal cells, however, may be problematic for subsequent examination of the induced hair-cell-like cells. In order to avoid the interference from stromal cells, we cultured OEPs on laminin with different induction media and examined the effects of the induction medium on the differentiation potentials of OEPs into hair-cell-like cells. The results revealed that the culture of OEPs on laminin with the conditioned medium from chicken utricle stromal cells supplemented with EGF and all-trans retinoic acid (RA) could promote the organization of cells into epithelial clusters displaying hair-cell-like cells with stereociliary bundles. These cells also displayed the expected electrophysiological properties. Copyright © 2015 Elsevier Ltd. All rights reserved.

Induction of insulin-producing cells from human pancreatic progenitor cells.

PubMed

Noguchi, H; Naziruddin, B; Shimoda, M; Fujita, Y; Chujo, D; Takita, M; Peng, H; Sugimoto, K; Itoh, T; Tamura, Y; Olsen, G S; Kobayashi, N; Onaca, N; Hayashi, S; Levy, M F; Matsumoto, S

2010-01-01

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells. Copyright 2010 Elsevier Inc. All rights reserved.

Carbon source dependent somatic embryogenesis and plant regeneration in cotton, Gossypium hirsutum L. cv. SVPR2 through suspension cultures.

PubMed

Ganesan, M; Jayabalan, N

2005-10-01

Highly reproducible and simple protocol for cotton somatic embryogenesis is described here by using different concentrations of maltose, glucose, sucrose and fructose. Maltose (30 g/l) is the best carbon source for embryogenic callus induction and glucose (30 g/l) was suitable for induction, maturation of embryoids and plant regeneration. Creamy white embryogenic calli of hypocotyl explants were formed on medium containing MS basal salts, myo-inositol (100 mg/l), thiamine HCI (0.3 mg/l), picloram (0.3 mg/l), Kin (0.1 mg/l) and maltose (30 g/l). During embryo induction and maturation, accelerated growth was observed in liquid medium containing NH3NO4 (1 g/l), picloram (2.0 mg/l), 2 ip (0.2 mg/l), Kin (0.1 mg/l) and glucose (30 g/l). Before embryoid induction, large clumps of embryogenic tissue were formed. These tissues only produced viable embryoids. Completely matured somatic embryos were germinated successfully on the medium fortified with MS salts, myo-inositol (50 mg/l), thiamine HCl (0.2 mg/l), GA3 (0.2 mg/l), BA (1.0 mg/l) and glucose (30 g/l). Compared with earlier reports, 65% of somatic embryo germination was observed. The abnormal embryo formation was highly reduced by using glucose (30 g/l) compared to other carbon sources. The regenerated plantlets were fertile but smaller in height than the seed derived control plants.