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.

Cryopreservation of medicinal plants: role of melatonin

USDA-ARS?s Scientific Manuscript database

Many useful plant species found in Canada are of conservation concern. In vitro storage and cryopreservation techniques guarantees safety of these species and have potential applications which may result in sustainable agriculture. Shoot tips of in vitro-grown plantlets of American elm, St John’s Wo...

In Vitro Conservation of Date Palm Shoot-Tip Explants and Callus Cultures Under Minimal Growth Conditions.

PubMed

El-Dawayati, Maiada M

2017-01-01

Date palm fruit production has great economic significance for many countries. There is a fundamental necessity to conserve valuable date palm germplasm, but there are various problems with in vivo and ex situ conservation. In vitro storage has several advantages over conventional germplasm conservation methods. The in vitro technique offers a developed method of slow-growth storage, which is considered as an alternate solution for short- and medium-term storage of date palm germplasm under controlled conditions. Minimal growth conditions for germplasm conservation are generally achieved by reducing growth rate through modification of environmental growing conditions and culture, by using low temperatures, and the addition of growth retardants and osmotic agents. This chapter describes a protocol for short-term in vitro conservation of date palm shoot-tip and callus cultures under slow-growth storage conditions, using sucrose as an osmotic agent and abscisic acid (ABA) as a growth retardant at 15 °C for 12 months.

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.

In vitro propagation of peanut (Arachis hypogaea L.) by shoot tip culture.

PubMed

Ozudogru, Elif Aylin; Kaya, Ergun; Lambardi, Maurizio

2013-01-01

Peanut (Arachis hypogaea L.), also known as groundnut, is the most important species of Arachis genus, originating from Brazil and Peru. Peanut seeds contain high seed oil, proteins, amino acids, and vitamin E, and are consumed worldwide as edible nut, peanut butter, or candy, and peanut oil extracted from the seeds. The meal remaining after oil extraction is also used for animal feed. However, its narrow germplasm base, together with susceptibility to diseases, pathogens, and weeds, decreases yield and seed quality and causes great economic losses annually. Hence, the optimization of efficient in vitro propagation procedures would be highly effective for peanut propagation, as it would raise yield and improve seed quality and flavor. Earlier reports on traditional micropropagation methods, based on axillary bud proliferation which guarantees the multiplication of true-to-type plants, are still limited. This chapter describes a micropropagation protocol to improve multiple shoot formation from shoot-tip explants by using AgNO(3) in combination with plant growth regulators.

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 %).

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages.

PubMed

Mankessi, François; Saya, Aubin R; Favreau, Bénédicte; Doulbeau, Sylvie; Conéjéro, Geneviève; Lartaud, Marc; Verdeil, Jean-Luc; Monteuuis, Olivier

2011-10-01

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei. Copyright © Physiologia Plantarum 2011.

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.

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 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.

Production of tetraploid plants of non apomictic citrus genotypes.

PubMed

Aleza, Pablo; Juárez, José; Ollitrault, Patrick; Navarro, Luis

2009-12-01

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x-4x). These stable tetraploid plants have been used in 4x x 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.

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.

Radiation and chemical mutagen induced somaclonal variations through in vitro organogenesis of cotton (Gossypium hirsutum L.).

PubMed

Muthusamy, Annamalai; Jayabalan, Narayanasamy

2014-12-01

The purpose of the investigation was to induce somaclonal variations by gamma rays (GR), ethylmethane sulphonate (EMS) and sodium azide (SA) during in vitro organogenesis of cotton. The shoot tip explants were irradiated with 5-50 Gray (Gy) GR (Cobalt 60), 0.5-5.0 mM EMS and SA separately, and inoculated on Murashige and Skoog (MS) medium fortified with plant growth regulator (PGR) for organogenesis. The plantlets with well-developed root systems were acclimatized and transferred into the experimental field to screen the somaclonal variations during growth and development. The number of somaclonal variations was observed in growth of irradiated/treated shoot tips, multiplication, plantlet regeneration and growth in vitro and ex vitro. The lower doses/concentrations of mutagenic treatments showed significant enhancement in selected agronomical characters and they showed decreased trends with increasing doses/concentrations of mutagenic agents. The results of the present study revealed the influence of lower doses/concentrations of mutagenic treatments on in vitro and ex vitro growth of cotton plantlets and their significant improvement in agronomical characters which needs further imperative stability analysis. The present observations showed the platform to use lower doses/concentrations of mutagenic agents to induce variability for enhanced agronomical characters, resistant and tolerant cotton varieties.

Shoot regeneration and embryogenesis in lily shoot tips cryopreserved by droplet vitrification

USDA-ARS?s Scientific Manuscript database

Shoot regeneration and embryogenesis were, for the first time, achieved directly in shoot tips of Lilium Oriental hybrid ‘Siberia’ following cryopreservation by droplet-vitrification. Shoot tips (2 mm in length) including 2-3 leaf primordia were excised from 4-week-old adventitious shoots directly r...

Cultivar-Dependent Direct Organogenesis of Date Palm from Shoot Tip Explants.

PubMed

Abahmane, Larbi

2017-01-01

A number of public and private laboratories are working on date palm micropropagation to meet the increasing worldwide demand for date palm planting material. A standardized direct organogenesis protocol exists for the production of date palm plantlets to maintain the genetic fidelity of regenerated plants. Organogenesis has the advantage of using low concentrations of plant growth regulators and avoiding the callus phase. In addition, direct regeneration of vegetative buds minimizes the risk of somaclonal variation among plant regenerants. However, in vitro multiplication cycles should be limited in duration by frequent renewal of plant material. This chapter describes a simple and routine organogenesis protocol for date palm multiplication using shoot tip explants.

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 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.

Influence of cytokinins, auxins and polyamines on in vitro mass multiplication of cotton (Gossypium hirsutum L. cv. SVPR2).

PubMed

Ganesan, M; Jayabalan, N

2006-06-01

In the present investigation, the influence of different forms of cytokinins, auxins and polyamines were tested for mass multiplication and regeneration of cotton. Initially, for the identification of effective concentration for multiple shoot induction, various concentrations of BAP, Kin and 2iP along with IAA and NAA were tested. Among tested concentrations, media fortified with MS salts; B5 vitamins; 30 g/l, glucose; 2.0 mg/l, 2iP; 2.0 mg/l, IAA and 0.7 % agar showed best response for multiplication of shoot tip explants (20 shoots per shoot tip explants). In nodal explants, maximum of 18.6 shoots were obtained in the media fortified with MS salts, B5 vitamins, 30 g/l, glucose, 2.0 mg/l, 2iP, 1.0 mg/l, NAA and 0.7 % agar. Effect of different concentrations of polyamines like spermidine and putrescine were also tested along with the above said multiplication media. Among the various treatments, 20 mg/l of putrescine showed best response and the multiple of shoots were increased to 26.5 shoots per shoot tip explants and 24.5 shoots per nodal explants. Elongation of shoots was achieved on multiple shoot induction medium. Significant number of roots were initiated in the medium supplemented with MS salts, vitamin B5 and IBA (2.0 mg/l). The frequency of root induction was increased by addition of, PVP (10 mg/l) along with root induction medium and after 2 weeks, the roots reached the maximum length of 22 cm. Further, these plantlets were hardened by using sand, soil and vermiculate in 1:1:1 ratio. The hardened plants were transferred to the environmental growth chamber for proper acclimatization. The hardened plants were then transferred to field for boll yielding and they exhibited 100% survival.

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.

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.

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

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 ...

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.

Cryopreservation of in vitro-grown shoot tips of Chinese medicinal plant Atractylodes macrocephala Koidz. using a droplet-vitrification method

USDA-ARS?s Scientific Manuscript database

BACKGROUND: Atractylodes macrocephala Koidz. is an important medicinal species from China and has been used for thousands of years because of pharmacological antioxidant, hepatoprotective, anti-inflammatory, anti-allergic, antithrombotic, antiviral, and anticarcinogenic activities. OBJECTIVE: The ai...

Improvement of banana cv. Rasthali (Silk, AAB) against Fusarium oxysporum f.sp. cubense (VCG 0124/5) through induced mutagenesis: Determination of LD50 specific to mutagen, explants, toxins and in vitro and in vivo screening for Fusarium wilt resistance.

PubMed

Saraswathi, M S; Kannan, G; Uma, S; Thangavelu, R; Backiyarani, S

2016-05-01

Shoot tips and in vitro grown proliferating buds of banana cv. Rasthali (Silk, AAB) were treated with various concentrations and durations of chemical mutagens viz., EMS, NaN3 and DES. LD50 for shoot tips based on 50% reduction in fresh weight was determined as 2% for 3 h, 0.02% for 5 h and 0.15% for 5 h, while for proliferating buds, they were 0.6% for 30 min, 0.01% for 2 h and 0.06% for 2 h for the mutagens EMS, NaN3 and DES, respectively. Subsequently, the mutated explants were screened in vitro against fusarium wilt using selection agents like fusaric acid and culture filtrate. LD50 for in vitro selection agents calculated based on 50% survival of explants was 0.050 mM and 7% for fusaric acid and culture filtrate, respectively and beyond which a rapid decline in growth was observed. This was followed by pot screening which led to the identification of three putative resistant mutants with an internal disease score of 1 (corm completely clean, no vascular discolouration). The putative mutants identified in the present study have also been mass multiplied in vitro.

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.

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...

Identification of a highly successful cryopreservation method (droplet-vitrification) for petunia

USDA-ARS?s Scientific Manuscript database

Petunia (Petunia × hybrida Vilm.) is a very important crop conserved in the National Genebank of China. Petunia cultivar “Niu 2” was used to develop a droplet-vitrification protocol to cryopreserve shoot tips. Six variables (age of the in vitro plants, concentration of sucrose in the preculture solu...

Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus Americana L.)

USDA-ARS?s Scientific Manuscript database

Climate change and the global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are e...

Induction of tetraploids from petiole explants through colchicine treatments in Echinacea purpurea L.

PubMed

Nilanthi, Dahanayake; Chen, Xiao-Lu; Zhao, Fu-Cheng; Yang, Yue-Sheng; Wu, Hong

2009-01-01

Petiole explants were obtained from in vitro grown diploid (2x = 22) Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA), 0.01 mg/L naphthaleneacetic acid (NAA) and four concentrations (30, 60, 120, and 240 mg/L) of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days). The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x = 44) shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature.

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.

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet vitrification and encapsulation-dehydration procedures

USDA-ARS?s Scientific Manuscript database

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration pr...

Effect of biweekly shoot tip harvests on the growth and yield of Georgia Jet sweet potato grown hydroponically

NASA Technical Reports Server (NTRS)

Ogbuehi, Cyriacus R.; Loretan, Phil A.; Bonsi, C. K.; Hill, Walter A.; Morris, Carlton E.; Biswas, P. K.; Mortley, Desmond G.

1989-01-01

Sweet potato shoot tips have been shown to be a nutritious green vegetable. A study was conducted to determine the effect of biweekly shoot tip harvests on the growth and yield of Georgia Jet sweet potato grown in the greenhouse using the nutrient film technique (NFT). The nutrient solution consisted of a modified half Hoagland solution. Biweekly shoot tip harvests, beginning 42 days after planting, provided substantial amounts of vegetable greens and did not affect the fresh and dry foliage weights or the storage root number and fresh and dry storage root weights at final harvest. The rates of anion and cation uptake were not affected by tip harvests.

Induction of Tetraploids from Petiole Explants through Colchicine Treatments in Echinacea purpurea L.

PubMed Central

Nilanthi, Dahanayake; Chen, Xiao-Lu; Zhao, Fu-Cheng; Yang, Yue-Sheng; Wu, Hong

2009-01-01

Petiole explants were obtained from in vitro grown diploid (2x = 22) Echinacea purpurea plantlets. Shoots were regenerated by culturing the explants on MS basal medium containing 0.3 mg/L benzyladenine (BA), 0.01 mg/L naphthaleneacetic acid (NAA) and four concentrations (30, 60, 120, and 240 mg/L) of colchicine for 30 days, or 120 mg/L of colchicine for various durations (7, 14, 21, and 28 days). The regenerated shoots were induced to root on MS basal medium with 0.01 mg/L NAA, and then the root-tips of the regenerated shoots were sampled for count of chromosome number. It was found that a treatment duration of >7 days was necessary for induction of tetraploid (4x = 44) shoots, and treatment with 120 mg/L colchicine for 28 days was the most efficient for induction of tetraploids, yielding 23.5% of tetraploids among all the regenerated shoots. Chimeras were observed in almost all the treatments. However, the ratio of tetraploid to diploid cells in a chimeric plant was usually low. In comparison with diploid plants, tetraploid plants in vitro had larger stomata and thicker roots with more root branches, and had prominently shorter inflorescence stalk when mature. PMID:19696915

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...

Recovery patterns, histological observations and genetic integrity in Malus shoot tips cryopreserved using droplet-vitrification and encapsulation-dehydration procedures.

PubMed

Li, Bai-Quan; Feng, Chao-Hong; Wang, Min-Rui; Hu, Ling-Yun; Volk, Gayle; Wang, Qiao-Chun

2015-11-20

A droplet-vitrification procedure is described for cryopreservation of Malus shoot tips. Survival patterns, recovery types, histological observations, and genetic integrity were compared for Malus shoot tips cryopreserved using this droplet-vitrification procedure and an encapsulation-dehydration procedure that was previously reported by us. In both procedures, three types of shoot tip recovery were observed following cryopreservation: callus formation without shoot regrowth, leaf formation without shoot regrowth, and shoot regrowth. Three categories of histological observations were also identified in cross-sections of shoot tips recovered after cryopreservation using the two cryogenic procedures. In category 1, almost all of the cells (94-95%) in the apical dome (AD) were damaged or killed and only some cells (30-32%) in the leaf primordia (LPs) survived. In category 2, only a few cells (18-20%) in the AD and some cells (30-31%) in the LPs survived. In category 3, majority of the cells (60-62%) in the AD and some cells (30-33%) in the LPs survived. These data suggest that shoot regrowth is correlated to the presence of a majority of surviving cells in the AD after liquid nitrogen exposure. No polymorphic bands were detected by inter-simple sequence repeats or by random amplified polymorphic DNA assessments, and ploidy levels analyzed by flow cytometry were unchanged when plants recovered after cryoexposure were compared to controls. The droplet-vitrification procedure appears to be robust since seven genotypes representing four Malus species and one hybrid recovered shoots following cryopreservation. Mean shoot regrowth levels of these seven genotypes were 48% in the droplet-vitrification method, which were lower than those (61%) in the encapsulation-dehydration procedure reported in our previous study, suggesting the latter may be preferred for routine cryobanking applications for Malus shoot tips. Copyright © 2015 Elsevier B.V. All rights reserved.

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

Establishment and analysis of in vitro biomass from Salvia corrugata Vahl. and evaluation of antimicrobial activity.

PubMed

Bisio, Angela; Fraternale, Daniele; Schito, Anna Maria; Parricchi, Anita; Dal Piaz, Fabrizio; Ricci, Donata; Giacomini, Mauro; Ruffoni, Barbara; De Tommasi, Nunziatina

2016-02-01

Demethylfruticuline A and fruticuline A, the most abundant compounds from the surface extract of Salvia corrugata Vahl., have shown antibacterial, antitumor and cytotoxic activities. In order to obtain these icetexane diterpenes from in vitro cultures of S. corrugata, protocols were developed for callus production, micropropagation and shoot regeneration. Analysis of the regenerated shoots showed the presence of both icetexanes, micropropagated plants contained only fruticuline A, while the callus contained trace amounts of both diterpenes. The yield of fruticuline A was higher in the methanolic extract of regenerated shoots than in those of fresh leaves and fresh shoot tips. In addition to these diterpenes, the regenerated shoot and micropropagated plant extracts afforded seven other diterpenes, one icetexane and six abietanes, identified by UV, IR, 1D- and 2D-NMR and HR-MS analysis. Five compounds (19-acetoxy-7α-hydroxyroyleanone, 7β,20-epoxy-11,12,19-trihydroxyabieta-8,11,13-triene, 7,20-dihydrofruticuline A, 7β-acetoxy-20-hydroxy-19,20-epoxyroyleanone, 7β-ethoxy-6β,20:19,20-diepoxyroyleanone) were previously undescribed. Although the crude plant surface extract did not possess any antibacterial activity, methanolic extracts of in vitro tissues and two compounds, namely 7β-acetoxy-20-hydroxy-19,20-epoxyroyleanone and 7β-ethoxy-6β,20:19,20-diepoxyroyleanone, isolated in suitable amounts, were active in varying degrees against multidrug resistant clinical strains of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Enterococcus faecium, displaying MIC values ranging from 32, 64 to 128μg/mL. Copyright © 2015 Elsevier Ltd. All rights reserved.

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.

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.

In vitro propagation of the Garden Heliotrope, Valeriana officinalis L.: influence of pre-chilling and light on seed germination.

PubMed

Bhat, B; Sharma, V D

2015-03-01

Valeriana officinalis is an important medicinal herb commonly found in Kashmir valley. This study forms an important preliminary step for in-vitro micro propagation of V. officinalis from breaking the seed dormancy, inducing rapid seed germination and its subsequent micro propagation. We investigated the influence of pretreatment of V. officinalis seeds with reduced temperature and light on seed germination and in-vitro propagation. Culture of explants from cultivated seeds have demonstrated its potential for in vitro propagation and plantlet regeneration. Individual as well as combinations of treatments such as temperature and light availability influenced the germination of seeds variedly. Unchilled seeds of V. officinalis were given dip in GA3 (200 ppm) for 24, 48 and 120 h. Seeds treated with GA3 for 24 h and kept in darkness showed the best results, i.e. 48%. Seeds pretreated with GA3 for 120 h and incubated in dark showed 40% germination. Pre-chilling up to 72 h and kept in light showed maximum germination of 60% followed by 40% kept in darkness. Pre-chilling for 48 h resulted in 40 and 25% seed germination in light and darkness, respectively. GA3 pre-treatment for 72 h and 24 h pre chilling were most effective in inducing seed germination. Maximum shoot response was obtained on MS enriched with BAP (1 mg/L) + IAA (0.1 mg/L) combinations using shoot tips as explants. Multiple shoot regeneration from shoot apices was recorded on BAP (1 mg/L) and BAP (1 mg/L) + IAA (0.1 mg/L).

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 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.

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.

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.

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.

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.

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

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.

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).

Cultivation Versus Molecular Analysis of Banana (Musa sp.) Shoot-Tip Tissue Reveals Enormous Diversity of Normally Uncultivable Endophytic Bacteria.

PubMed

Thomas, Pious; Sekhar, Aparna Chandra

2017-05-01

The interior of plants constitutes a unique environment for microorganisms with various organisms inhabiting as endophytes. Unlike subterranean plant parts, aboveground parts are relatively less explored for endophytic microbial diversity. We employed a combination of cultivation and molecular approaches to study the endophytic bacterial diversity in banana shoot-tips. Cultivable bacteria from 20 sucker shoot-tips of cv. Grand Naine included 37 strains under 16 genera and three phyla (Proteobacteria, Actinobacteria, Firmicutes). 16S rRNA gene-ribotyping approach on 799f and 1492r PCR-amplicons to avoid plant organelle sequences was ineffective showing limited bacterial diversity. 16S rRNA metagene profiling targeting the V3-V4 hypervariable region after filtering out the chloroplast (74.2 %), mitochondrial (22.9 %), and unknown sequences (1.1 %) revealed enormous bacterial diversity. Proteobacteria formed the predominant phylum (64 %) succeeded by Firmicutes (12.1 %), Actinobacteria (9.5 %), Bacteroidetes (6.4 %), Planctomycetes, Cyanobacteria, and minor shares (<1 %) of 14 phyla including several candidate phyla besides the domain Euryarchaeota (0.2 %). Microbiome analysis of single shoot-tips through 16S rRNA V3 region profiling showed similar taxonomic richness and diversity and was less affected by plant sequence interferences. DNA extraction kit ominously influenced the phylogenetic diversity. The study has revealed vast diversity of normally uncultivable endophytic bacteria prevailing in banana shoot-tips (20 phyla, 46 classes) with about 2.6 % of the deciphered 269 genera and 1.5 % of the 656 observed species from the same source of shoot-tips attained through cultivation. The predominant genera included several agriculturally important bacteria. The study reveals an immense ecosystem of endophytic bacteria in banana shoot tissues endorsing the earlier documentation of intracellular "Cytobacts" and "Peribacts" with possible roles in plant holobiome and hologenome.

A comparative study on cryopreservation of mint (Menth asp.) shoot tips

USDA-ARS?s Scientific Manuscript database

Mint is used in natural seasoning, traditional medicine, pharmaceuticals and cosmetics. In some countries it is grown on an industrial scale due to its economic importance; hence, preserving mint genetic resources is of value. We compared three cryopreservation techniques (T) using shoot tips of thr...

CRYOTHERAPY AS A METHOD FOR REDUCING THE VIRUS INFECTION OF APPLES (Malus sp.).

PubMed

Romadanova, Natalya V; Mishustina, Svetlana A; Gritsenko, D ilyara A; Omasheva, Madina Y; Galiakparov, Nurbol N; Reed, Barbara M; Kushnarenko, Svetlana V

2016-01-01

There is an urgent need in Kazakhstan for virus-free nursery stock to reinvigorate the industry and preserve historic cultivars. An in vitro collection of apples could be used for virus testing and elimination and to provide virus-free elite stock plants to nurseries. Malus sieversii Ledeb. M. Roem. and Malus domestica Borkh. accessions were initiated in vitro for virus identification and elimination. Reverse transcription and multiplex PCR were used to test for five viruses. PVS2 vitrification was used as a tool for cryotherapy. Four viruses, Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus (ApMV) were detected in 17 accessions. Tomato ringspot virus (ToRSV) was not detected. ACLSV affected 53.8% of the accessions, ASPV 30.8%, ASGV 5.1%, and ApMV was found only in 'Aport Alexander'. Cryotherapy produced virus-free shoot tips for seven of nine cultivars tested. Six cultivars had 60-100% elimination of ACLSV. An in vitro collection of 59 accessions was established. Virus elimination using cryotherapy produced virus-free shoots for seven of nine cultivars and is a promising technique for developing a virus-free apple collection.

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.

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.

Suitability of Cryopreservation for the Long‐term Storage of Rare and Endangered Plant Species: a Case History for Cosmos atrosanguineus

PubMed Central

WILKINSON, TIM; WETTEN, ANDREW; PRYCHID, CHRISSIE; FAY, MICHAEL F.

2003-01-01

The suitability of cryopreservation for the secure, long‐term storage of the rare and endangered species Cosmos atrosanguineus was investigated. Using encapsulation/dehydration of shoot tips in alginate strips, survival rates of up to 100 % and shoot regeneration of up to 35 % were achieved. Light and electron microscopy studies indicated that cellular damage to some regions of the shoot tip during the freeze/thaw procedure was high, although cell survival in and around the meristematic region allowed shoot tip regeneration. The genetic fingerprinting technique, amplified fragment length polymorphisms (AFLPs), showed that no detectable genetic variation was present between material of C. atrosanguineus at the time of initiation into tissue culture and that which had been cryopreserved, stored in liquid nitrogen for 12 months and regenerated. Weaned plantlets that were grown under glasshouse conditions exhibited no morphological variation from non‐frozen controls. PMID:12495921

Elimination of two viruses which interact synergistically from sweetpotato by shoot tip culture and cryotherapy.

PubMed

Wang, Q C; Valkonen, J P T

2008-12-01

Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae) and Sweet potato feathery mottle virus (SPFMV; Potyviridae) interact synergistically and cause severe diseases in co-infected sweetpotato plants (Ipomoea batatas). Sweetpotato is propagated vegetatively and virus-free planting materials are pivotal for sustainable production. Using cryotherapy, SPCSV and SPCSV were eliminated from all treated single-virus-infected and co-infected shoot tips irrespective of size (0.5-1.5mm including 2-4 leaf primordia). While shoot tip culture also eliminated SPCSV, elimination of SPFMV failed in 90-93% of the largest shoot tips (1.5mm) using this technique. Virus distribution to different leaf primordia and tissues within leaf primordia in the shoot apex and petioles was not altered by co-infection of the viruses in the fully virus-susceptible sweetpotato genotype used. SPFMV was immunolocalized to all types of tissues and up to the fourth-youngest leaf primordium. In contrast, SPCSV was detected only in the phloem and up to the fifth leaf primordium. Because only cells in the apical dome of the meristem and the two first leaf primordia survived cryotherapy, all data taken together could explain the results of virus elimination. The simple and efficient cryotherapy protocol developed for virus elimination can also be used for preparation of sweetpotato materials for long-term preservation.

In vitro propagation of persimmon (Diospyros kaki Thunb.).

PubMed

Giordani, Edgardo; Naval, Mar; Benelli, Carla

2013-01-01

Persimmon (Diospyros kaki Thunb.) is a temperate fruit tree species diffused in all continents. The traditional propagation method adopted by the nursery industry is based on budding/grafting scion cultivars on seedlings from D. kaki, Diospyros lotus, and Diospyros virginiana, the most important species used as rootstock, reproduced by seeds since they are not easy to root. Furthermore, most of nonastringent cultivars of persimmon are not compatible with D. lotus, a rootstock largely utilized because of its hardiness and frost resistance. The main in vitro tissue culture techniques, developed for persimmon, deal with direct regeneration (from dormant buds and root tips), and indirect regeneration through callus from dormant buds, apexes, and leaves. The bottlenecks of micropropagation are (1) the recalcitrance of many cultivars to in vitro establishment, (2) the low multiplication ratio of D. kaki compared to other fruit tree species, (3) the very low rooting ability of ex novo microcuttings both from direct and indirect regeneration, (4) the high sensitivity to transplant from in vitro to in vivo conditions. The development of reliable in vitro regeneration procedures is likely to play a key role for production of both clonal rootstocks and self-rooted cultivars. The general protocol for micropropagation of persimmon reported here is based on the establishment of winter dormant buds in vitro, shoot development, multiplication and elongation, and shoot rooting, using cytokinins (BA or zeatin) in a MS media along with an auxinic pretreatment for rooting induction.

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.

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

Eastern Pine Shoot Borer

Treesearch

Louis F. Wilson

1978-01-01

The eastern pineshoot borer Eucosma gloriola Heinrich 2, also known as the white pine tip moth, American pine shoot moth, white pine shoot borer, and Tordeuse americaine, du pin, injures young conifers in Northeastern North America. Because it infests the new shoots of sapling conifers, this insect is particularly destructive on planted trees destined for the Christmas...

Spine micromorphology of normal and hyperhydric Mammillaria gracilis Pfeiff. (Cactaceae) shoots.

PubMed

Peharec, P; Posilović, H; Balen, B; Krsnik-Rasol, M

2010-07-01

Artificial conditions of tissue culture affect growth and physiology of crassulacean acid metabolism plants which often results in formation of hyperhydric shoots. In in vitro conditions Mammillaria gracilis Pfeiff. (Cactaceae) growth switches from organized to unorganized way, producing a habituated organogenic callus which simultaneously regenerates morphologically normal as well as altered hyperhydric shoots. In this study, influence of tissue culture conditions on morphology of cactus spines of normal and hyperhydric shoots was investigated. Spines of pot-grown Mammillaria plants and of in vitro regenerated shoots were examined with stereo microscope and scanning electron microscope. The pot-grown plants had 16-17 spines per areole. In vitro grown normal shoots, even though they kept typical shoot morphology, had lower number of spines (11-12) and altered spine morphology. This difference was even more pronounced in spine number (six to seven) and morphology of the hyperhydric shoots. Scanning electron microscopy analysis revealed remarkable differences in micromorphology of spine surface between pot-grown and in vitro grown shoots. Spines of in vitro grown normal shoots showed numerous long trichomes, which were more elongated on spines of the hyperhydric shoots; the corresponding structures on spine surface of pot-grown plants were noticed only as small protrusions. Scanning electron microscopy morphometric studies showed that the spines of pot-grown plants were significantly longer compared to the spines of shoots grown in tissue culture. Moreover, transverse section shape varies from elliptical in pot-grown plants to circular in normal and hyperhydric shoots grown in vitro. Cluster and correspondence analyses performed on the scanning electron microscope obtained results suggest great variability among spines of pot-grown plants. Spines of in vitro grown normal and hyperhydric shoots showed low level of morphological variation among themselves despite the significant difference in shoot morphology.

In vitro propagation of ginger (Zingiber officinale Rosc.) through direct organogenesis: a review.

PubMed

Seran, Thayamini H

2013-12-15

Ginger (Zingiber officinale Rosc.) is a perennial herb. It belongs to the family Zingiberaceae and commercially cultivated in most tropical regions of the world. The underground rhizomes are the planting materials in a conventional propagation of ginger however it has a low multiplication rate. It is known that there are possible methods are available for rapid vegetative propagation of ginger through direct organogenesis or somatic embryogenesis under in vitro conditions but it is necessary to find the best protocol for in vitro multiplication of ginger. Limited studies on the tissue culture technology of ginger are available in Sri Lanka. However, significant efforts have been made in the procedure for in vitro micropropagation in the other ginger growing countries. The available literature with respect to in vitro plant regeneration has been perused and this review mainly focused on the in vitro propagation via direct organogenesis from rhizome buds or shoot tips of ginger often used as explants. This review article may be an appropriate and effective guidance for establishing in vitro cultures and subsequent production of in vitro plantlets in clonal propagation of ginger.

Management of shoot boring moths from genera Rhyacionia and Eucosma with attract and kill technology

Treesearch

R. Hoffman; D. Czokajlo; G. Daterman; J. McLaughlin; J. Webster; < i> et. al.< /i>

2003-01-01

LastCall™ (LC), an attract and kill bait matrix, was deployed for the management of shoot boring moths in pine plantations and seed orchards. The targeted moths were the Western pine shoot borer, Eucosma sonomana (WPSB), European pine shoot moth, Rhyacionia buoliana (EPSM), Ponderosa pine tip moth, Rhyacionia...

Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips.

PubMed

Gross, Briana L; Henk, Adam D; Bonnart, Remi; Volk, Gayle M

2017-03-01

Transcripts related to abiotic stress, oxidation, and wounding were differentially expressed in Arabidopsis shoot tips in response to cryoprotectant and liquid nitrogen treatment. Cryopreservation methods have been implemented in genebanks as a strategy to back-up plant genetic resource collections that are vegetatively propagated. Cryopreservation is frequently performed using vitrification methods, whereby shoot tips are treated with cryoprotectant solutions, such as Plant Vitrification Solution 2 (PVS2) or Plant Vitrification Solution 3 (PVS3); these solutions remove and/or replace freezable water within the meristem cells. We used the model system Arabidopsis thaliana to identify suites of transcripts that are up- or downregulated in response to PVS2 and PVS3 treatment and liquid nitrogen (LN) exposure. Our results suggest that there are many changes in transcript expression in shoot tips as a result of cryoprotection and that these changes exceed the number detected as a result of LN exposure. In total, 180 transcripts showed significant changes in expression level unique to treatment with either the cryoprotectant or cryopreservation followed by recovery. Of these 180 transcripts, 67 were related to stress, defense, wounding, lipid, carbohydrate, abscisic acid, oxidation, temperature (cold/heat), or osmoregulation. The responses of five transcripts were confirmed using qPCR methods. The transcripts responding to PVS2 + LN suggest an oxidative response to this treatment, whereas the PVS3 + LN treatment invoked a more general metabolic response. This work shows that the choice of cryoprotectant can have a major influence on the patterns of transcript expression, presumably due to the level and extent of stress experienced by the shoot tip. As a result, there may be divergent responses of study systems to PVS2 and PVS3 treatments.

ASSESSMENT OF MOLECULAR GENETIC STABILITY BETWEEN LONG-TERM CRYOPRESERVED AND TISSUE CULTURED WASABI (Wasabia japonica) PLANTS.

PubMed

Maki, S; Hirai, Y; Niino, T; Matsumoto, T

2015-01-01

Maintaining the genetic integrity in long-term tissue cultured and cryopreserved plants is important for the conservation of plant genetic resources. In this study, the genetic stability of cryopreserved wasabi shoot tips stored for 10 years at -150 degree C was visualized using Amplified Fragment Length Polymorphism (AFLP) and Methylation Sensitive Amplified Polymorphism (MSAP). The study included plants derived from cryopreserved shoot tips after 10.5 years storage at -150 degree C (LN10yr), after 2 h storage at -196 degree C (LN2hr), cryopreservation controls (No LN cooling (TC)) and non-treated controls without LN cooling (LC). The donor plants for LN2hr, TC and LC were also maintained in vitro at 20 degree C for the same period. Neither technique detected genetic variations in either control or cryopreserved plants. Some mutations were noted in plants maintained in tissue culture for 10 years. Comparison of genome stability for TC and LN2hr plants showed only a minor change in DNA. However, when comparing the LC and Ln10yr, many differences were found. We conclude that cryopreservation is a superior conservation method compared to tissue culture in maintaining genetic stability for a long-term storage of wasabi germplasm.

Rapid in vitro propagation, conservation and analysis of genetic stability of Viola pilosa.

PubMed

Soni, Madhvi; Kaur, Rajinder

2014-01-01

A protocol for in vitro propagation was developed for Viola pilosa, a plant of immense medicinal value. To start with in vitro propagation, the sterilized explants (buds) were cultured on MS basal medium supplemented with various concentrations of growth regulators. One of the medium compositions MS basal + 0.5 mg/l BA + 0.5 mg/l TDZ + 0.5 mg/l GA3 gave best results for in vitro shoot bud establishment. Although the problem of shoot vitrification occurred on this medium but this was overcome by transferring the vitrified shoots on MS medium supplemented with 1 mg/l BA and 0.25 mg/l Kn. The same medium was found to be the best medium for further in vitro shoot multiplication. 100 % root induction from in vitro grown shoots was obtained on half strength MS medium supplemented with 1 mg/l IBA. In vitro formed plantlets were hardened and transferred to soil with 83 % survival. Additionally, conservation of in vitro multiplying shoots was also attempted using two different approaches namely slowing down the growth at low temperature and cryopreservation following vitrification. At low temperature retrieval rate was better at 10 °C than at 4 °C after conservation of in vitro multiplying shoots. In cryopreservation-vitrification studies, the vitrified shoot buds gave maximum retrieval of 41.66 % when they were precooled at 4 °C, while only 16.66 % vitrified shoots were retrieved from those precooled at 10 °C. Genetic stability of the in vitro grown plants was analysed by RAPD and ISSR markers which indicated no somaclonal variation among in vitro grown plants demonstrating the feasibility of using the protocol without any adverse genetical effects.

Sites of abscisic acid synthesis and metabolism in Ricinus communis L

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

Zeevaart, J.A.D.

1977-05-01

The sites of abscisic acid (ABA) synthesis and metabolism in Ricinus communis L. were investigated by analyzing the levels of ABA and its two metabolites phaseic acid (PA) and dihydrophaseic acid (DPA) in the shoot tips, mature leaves, and phloem sap of stressed and nonstressed plants. Water stress increased the concentration of ABA, PA, and DPA in phloem exudate and also increased the levels of all three compounds in mature leaves and in shoot tips. The latter had a very high DPA content (18.7 ..mu..g/g fresh weight) even in plants not subjected to water stress. When young and mature leavesmore » were excised and allowed to wilt, the level of ABA increased in both, demonstrating that leaves at an early stage of development have the capacity to produce ABA. These results have been interpreted to mean that in mature leaves of nonstressed Ricinus plants, ABA is synthesized and metabolized, and that ABA itself, as well as its metabolites, are translocated in the phloem to the shoot tips (sinks). Since DPA, but not ABA, accumulates in the shoot tips, it follows that ABA is metabolized rapidly in the apical region. To what extent ABA present in young leaves of nonstressed plants is the consequence of synthesis in situ and of import from older leaves remains to be determined.« less

Parasitoids of the nantucket pine tip moth (Lepidoptera: Tortricidae) in the coastal plain of Georgia

Treesearch

Kenneth W. McCravy; C. Wayne Berisford

2000-01-01

Parasitism of the Nantucket pine tip moth, Rhyacionia frustrana (Comstock), was studied for four consecutive generations in the Georgia coastal plain by collecting tip moth-infested shoots and rearing adult moths and parasitoids. Nineteen species of parasitoids were collected. Based on numbers of emerging adults, the overall tip moth parasitism rate...

Micropropagation of pear (Pyrus sp)

USDA-ARS?s Scientific Manuscript database

Establishment of shoot tip cultures, proliferation, rooting, and acclimatization of the resulting plantlets are all elements of micropropagation. The great genetic variation in Pyrus (pear)makes micropropagation challenging for many genotypes. Initiation of shoots is most successful from forced do...

Southwestern Pine Tip Moth

Treesearch

Daniel T. Jennings; Robert E. Stevens

1982-01-01

The southwestern pine tip moth, Rhyacionia neomexicana (Dyar), injures young ponderosa pines (Pinus ponderosa Dougl. ex Laws) in the Southwest, central Rockies, and midwestern plains. Larvae feed on and destroy new, expanding shoots, often seriously reducing terminal growth of both naturally regenerated and planted pines. The tip moth is especially damaging to trees on...

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.

Physiological Disorders of Pear Shoot Cultures

USDA-ARS?s Scientific Manuscript database

Physiological disorders are some of the most difficult challenges in micropropagation. Little is known of the causes of plant growth disorders which include callus formation, hyperhydricity, shoot tip necrosis, leaf lesions, epinasty, fasciation and hypertrophy. During our study of mineral nutritio...

The Sandy Hook Elementary School shooting as tipping point: "This Time Is Different".

PubMed

Shultz, James M; Muschert, Glenn W; Dingwall, Alison; Cohen, Alyssa M

2013-01-01

Among rampage shooting massacres, the Sandy Hook Elementary School shooting on December 14, 2012 galvanized public attention. In this Commentary we examine the features of this episode of gun violence that has sparked strong reactions and energized discourse that may ultimately lead toward constructive solutions to diminish high rates of firearm deaths and injuries in the United States.

The Sandy Hook Elementary School shooting as tipping point

PubMed Central

Shultz, James M; Muschert, Glenn W; Dingwall, Alison; Cohen, Alyssa M

2013-01-01

Among rampage shooting massacres, the Sandy Hook Elementary School shooting on December 14, 2012 galvanized public attention. In this Commentary we examine the features of this episode of gun violence that has sparked strong reactions and energized discourse that may ultimately lead toward constructive solutions to diminish high rates of firearm deaths and injuries in the United States. PMID:28228989

Micropropagation and in vitro flowering of Rauvolfia tetraphylla; a potent source of anti-hypertension drugs.

PubMed

Sarma, D; Sarma, S; Baruah, A

1999-04-01

A simple protocol for in vitro mass multiplication of Rauvolfia tetraphylla (Apocynaceae) has been developed. The endophytic microflora was controlled by adopting integrated measures. Multiple shoot development was achieved on MS + Kin (0.1-0.2 mg/l) + BAP (0.4-0.5 mg/l) media. Rooting from in vitro shoots occurred on NAA containing media. In vitro flowering was induced in shoot multiplication media.

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.

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.

Synchronization of Somatic Embryogenesis in Date Palm Suspension Culture Using Abscisic Acid.

PubMed

Alwael, Hussain A; Naik, Poornananda M; Al-Khayri, Jameel M

2017-01-01

Somatic embryogenesis is considered the most effective method for commercial propagation of date palm. However, the limitation of obtaining synchronized development of somatic embryos remains an impediment. The synchronization of somatic embryo development is ideal for the applications to produce artificial seeds. Abscisic acid (ABA) is associated with stress response and influences in vitro growth and development. This chapter describes an effective method to achieve synchronized development of somatic embryos in date palm cell suspension culture. Among the ABA concentrations tested (0, 1, 10, 50, 100 μM), the best synchronized growth was obtained in response to 50-100 μM. Here we provide a comprehensive protocol for in vitro plant regeneration of date palm starting with shoot-tip explant, callus initiation and growth, cell suspension establishment, embryogenesis synchronization with ABA treatment, somatic embryo germination, and rooting as well as acclimatized plantlet establishment.

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.

The Huperzia selago Shoot Tip Transcriptome Sheds New Light on the Evolution of Leaves

PubMed Central

Evkaikina, Anastasiia I.; Berke, Lidija; Romanova, Marina A.; Proux-Wéra, Estelle; Ivanova, Alexandra N.; Rydin, Catarina; Voitsekhovskaja, Olga V.

2017-01-01

Abstract Lycopodiophyta—consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)—form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls—that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap—not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY. We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants. The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509–514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined. PMID:28957460

Mechanisms of induced susceptibility to Diplodia tip blight in drought-stressed Austrian pine.

PubMed

Sherwood, Patrick; Villari, Caterina; Capretti, Paolo; Bonello, Pierluigi

2015-05-01

Plants experiencing drought stress are frequently more susceptible to pathogens, likely via alterations in physiology that create favorable conditions for pathogens. Common plant responses to drought include the production of reactive oxygen species (ROS) and the accumulation of free amino acids (AAs), particularly proline. These same phenomena also frequently occur during pathogenic attack. Therefore, drought-induced perturbations in AA and ROS metabolism could potentially contribute to the observed enhanced susceptibility. Furthermore, nitrogen (N) availability can influence AA accumulation and affect plant resistance, but its contributions to drought-induced susceptibility are largely unexplored. Here we show that drought induces accumulation of hydrogen peroxide (H2O2) in Austrian pine (Pinus nigra Arnold) shoots, but that shoot infection by the blight and canker pathogen Diplodia sapinea (Fr.) Fuckel leads to large reductions in H2O2 levels in droughted plants. In in vitro assays, H2O2 was toxic to D. sapinea, and the fungus responded to this oxidative stress by increasing catalase and peroxidase activities, resulting in substantial H2O2 degradation. Proline increased in response to drought and infection when examined independently, but unlike all other AAs, proline further increased in infected shoots of droughted trees. In the same tissues, the proline precursor, glutamate, decreased significantly. Proline was found to protect D. sapinea from H2O2 damage, while also serving as a preferred N source in vitro. Fertilization increased constitutive and drought-induced levels of some AAs, but did not affect plant resistance. A new model integrating interactions of proline and H2O2 metabolism with drought and fungal infection of plants is proposed. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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.

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.

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

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.

Studies on the tissue culture of Stevia rebaudiana and its components; (II). Induction of shoot primordia.

PubMed

Miyagawa, H; Fujioka, N; Kohda, H; Yamasaki, K; Taniguchi, K; Tanaka, R

1986-08-01

Shoot primordia, which were able to propagate vegetatively with a very high rate and to redifferentiate easily to new plants, were induced from shoot tips of Stevia rebaudiana Bertoni on Gamborg B5 medium containing 6-benzylaminopurine (BAP) and alpha-naphthaleneacetic acid (NAA) under light. The propagation of the shoot primordia of Stevia rebaudiana is rapid, and they are highly stable in chromosome number and karyotype. The shoot primordia can propagate at a high rate for a long time without differentiation. At any time, the shoot primordia readily developed into plantlets with shoots and roots within 2 or 3 weeks in static culture on B5 medium containing 0.02 mg/l BAP and 2% sucrose. The plantlets were transplanted to sterilized soil to grow to normal adult plants.

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.

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.

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 ...

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.

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.

Microclonal Multipication of Wild Cherry (Prunus Avium L.) from Shoot Tips and Root Sucker Buds

Treesearch

Branka Pevalek-Kozlina; Charles H. Michler; Sibila Jelaska

1994-01-01

The effects of different combinations and concentrations of the growth regulators: 6-benzylaminopurine (BA), 6 furfurylaminopurine (KIN), N6- (2-isopentenyl) adenine (2iP), indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and a-naphthaleneacetic acid (NAA) on axillary shoot multiplication rates for wild cherry (Prunus aviurn...

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.

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.

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.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate.

PubMed

Kou, Yaping; Yuan, Cunquan; Zhao, Qingcui; Liu, Guoqin; Nie, Jing; Ma, Zhimin; Cheng, Chenxia; Teixeira da Silva, Jaime A; Zhao, Liangjun

2016-01-01

Thidiazuron (N-phenyl-N'-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa.

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

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

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression

DOE PAGES

Xue, Liang-Jiao; Frost, Christopher J.; Tsai, Chung-Jui; ...

2016-09-19

Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism inmore » root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.« less

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.

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.

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.

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.

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.

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.

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.

Heritability of regeneration in tissue cultures of sweet potato (Ipomoea batatas L.).

PubMed

Templeton-Somers, K M; Collins, W W

1986-03-01

A population of open-pollinated progeny from 12 parents, and the 12 parents, was surveyed for in vitro growth and regeneration characteristics. Four different tissue culture procedures involving different media and the use of different explants to initiate the cultures were used. Petiole explants from young leaves were used as explants for initiation of callus cultures. These were evaluated for callus growth rate, friability, and callus color and texture, before transferring to each of three different regeneration media for evaluation of morphogenetic potential. Small shoot tips also were used to initiate callus cultures, which were evaluated for the same growth characteristics and transferred to growth-regulator free regeneration media. Regeneration occurred through root or shoot regeneration or through embryogenesis. Tissue culture treatment effects, as well as genotypic effects, were highly significant in determining: the types of callus produced, callus growth rates, color and texture on the two types of media used for the second and third subcultures. The family x treatment interaction was generally not statistically significant, affecting only callus color. Estimates of narrow sense heritability for callus growth rate in both the second and third subcultures were high enough (0.35 and 0.63, respectively) for the evaluation of parental lines for selection procedures. These characteristics were also the only early culture callus traits that were consistently correlated with later morphogenesis of the cultures. They were negatively correlated with root or shoot regeneration. The occurence of somatic embryogenesis was not correlated with early callus growth characteristics. Genetic and treatment effects were highly significant in the evaluation of morphogenetic potential, through root or shoot regeneration, or through embryogenesis. Regeneration of all types was of low frequency for all procedures, expressed in ≦ 11% of the cultures of the total population.

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.

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

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

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.

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.

A simple, cheap, one-minute method to mount insect pins for use as sharp-tipped probes in plant dissection.

PubMed

Frohlich, Michael W

2005-08-01

A new method is presented for twist mounting insect pins onto standard dissecting (teasing) needles. Insect pins, with their sharp points, are ideal for fine dissection of plants, especially of shoot tips and early developing flower buds. Twist mounting makes them convenient and effective dissecting tools to prepare specimens for SEM.

Morphogenesis in Plants: Modeling the Shoot Apical Meristem, and Possible Applications

NASA Technical Reports Server (NTRS)

Mjolsness, Eric; Gor, Victoria; Meyerowitz, Elliot; Mann, Tobias

1998-01-01

A key determinant of overall morphogenesis in flowering plants such as Arabidopsis thaliana is the shoot apical meristem (growing tip of a shoot). Gene regulation networks can be used to model this system. We exhibit a very preliminary two-dimensional model including gene regulation and intercellular signaling, but omitting cell division and dynamical geometry. The model can be trained to have three stable regions of gene expression corresponding to the central zone, peripheral zone, and rib meristem. We also discuss a space-engineering motivation for studying and controlling the morphogenesis of plants using such computational models.

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.

Picture This!

ERIC Educational Resources Information Center

McMahon, Maureen M.

2002-01-01

Outlines the development of an after school science club in which students find a science topic of interest and create a storyboard through photographs. Includes tips for shooting photographs and equipment suggestions. (DDR)

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.

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.

Micropropagation of Vaccinium sp. by in vitro axillary shoot proliferation.

PubMed

Litwińczuk, Wojciech

2013-01-01

The Vaccinium genus contains several valuable fruit and ornamental species, among others: highbush blueberry (Vaccinium × corymbosum L.), cranberry (Vaccinium macrocarpon Ait.), and lingonberry (Vaccinium vitis-idaea L.). In some most popular and valuable cultivars, the conventional propagation methods, exploiting hard or soft wood cuttings, are inefficient. The demand for nursery plants could be fulfilled only by micropropagation. In principle cultivars are propagated in vitro through similar three-stage method, based on subculture of shoot explants on different culture media supplemented with IAA (0-4 mg/L) and 2iP (5-10 mg/L), and rooting shoots in vivo. The obtained plantlets are transferred to peat substrate and grown in the glasshouse until the end of growing period. The development of adventitious shoots should be monitored and controlled during in vitro stages. Many clones have specific requirements for growing conditions and/or are recalcitrant.

Effects of NAA and BAP, double-layered media, and light distance on in vitro regeneration of Nelumbo nucifera Gaertn. (lotus), an aquatic edible plant.

PubMed

Mahmad, Noraini; Taha, Rosna Mat; Othman, Rashidi; Saleh, Azani; Hasbullah, Nor Azlina; Elias, Hashimah

2014-01-01

In vitro direct regeneration of Nelumbo nucifera Gaertn. was successfully achieved from immature explants (yellow plumule) cultured on a solid MS media supplemented with combinations of 0.5 mg/L BAP and 1.5 mg/L NAA which resulted in 16.00 ± 0.30 number of shoots per explant and exhibited a new characteristic of layered multiple shoots, while normal roots formed on the solid MS basal media. The double-layered media gave the highest number of shoots per explant with a ratio of 2 : 1 (liquid to solid) with a mean number of 16.67 ± 0.23 shoots per explant with the formation of primary and secondary roots from immature explants. In the study involving light distance, the tallest shoot (16.67 ± 0.23 mm) obtained from the immature explants was at a light distance of 200 mm from the source of inflorescent light (1000 lux). The plantlets were successfully acclimatized in clay loam soil after 8 months being maintained under in vitro conditions.

Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra

Treesearch

Jun Hyung Lee; Paula M. Pijut

2017-01-01

Black ash (Fraxinus nigra) is an endangered hardwood tree species under threat of extirpation by the emerald ash borer (EAB), an aggressive exotic phloemfeeding beetle. We have developed an efficient regeneration system through adventitious shoot organogenesis in F. nigra using in vitro-derived leaf explants. Two types of leaf...

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.

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.

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 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.

Gibberellin Biosynthesis in Developing Pumpkin Seedlings12

PubMed Central

Lange, Theo; Kappler, Jeannette; Fischer, Andreas; Frisse, Andrea; Padeffke, Tania; Schmidtke, Sabine; Lange, Maria João Pimenta

2005-01-01

A gibberellin (GA) biosynthetic pathway was discovered operating in root tips of 7-d-old pumpkin (Cucurbita maxima) seedlings. Stepwise analysis of GA metabolism in cell-free systems revealed the conversion of GA12-aldehyde to bioactive GA4 and inactive GA34. Highest levels of endogenous GA4 and GA34 were found in hypocotyls and root tips of 3-d-old seedlings. cDNA molecules encoding two GA oxidases, CmGA20ox3 and CmGA3ox3, were isolated from root tips of 7-d-old LAB150978-treated seedlings. Recombinant CmGA20ox3 fusion protein converted GA12 to GA9, GA24 to GA9, GA14 to GA4, and, less efficiently, GA53 to GA20, and recombinant CmGA3ox3 protein oxidized GA9 to GA4. Transcript profiles were determined for four GA oxidase genes from pumpkin revealing relatively high transcript levels for CmGA7ox in shoot tips and cotyledons, for CmGA20ox3 in shoot tips and hypocotyls, and for CmGA3ox3 in hypocotyls and roots of 3-d-old seedlings. Transcripts of CmGA2ox1 were mainly found in roots of 7-d-old seedlings. In roots of 7-d-old seedlings, transcripts of CmGA7ox, CmGA20ox3, and CmGA3ox3 were localized in the cap and the rhizodermis by in situ hybridization. We conclude that hypocotyls and root tips are important sites of GA biosynthesis in the developing pumpkin seedling. PMID:16126862

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.

Translocation of radiolabeled indole-3-acetic acid and indole-3-acetyl-myo-inositol from kernel to shoot of Zea mays L

NASA Technical Reports Server (NTRS)

Chisnell, J. R.; Bandurski, R. S.

1988-01-01

Either 5-[3H]indole-3-acetic acid (IAA) or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[3H]indole-3-acetic acid or 5-[3H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.

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

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.

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.

Thidiazuron Triggers Morphogenesis in Rosa canina L. Protocorm-Like Bodies by Changing Incipient Cell Fate

PubMed Central

Kou, Yaping; Yuan, Cunquan; Zhao, Qingcui; Liu, Guoqin; Nie, Jing; Ma, Zhimin; Cheng, Chenxia; Teixeira da Silva, Jaime A.; Zhao, Liangjun

2016-01-01

Thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5-ylurea; TDZ) is an artificial plant growth regulator that is widely used in plant tissue culture. Protocorm-like bodies (PLBs) induced by TDZ serve as an efficient and rapid in vitro regeneration system in Rosa species. Despite this, the mechanism of PLB induction remains relatively unclear. TDZ, which can affect the level of endogenous auxins and cytokinins, converts the cell fate of rhizoid tips and triggers PLB formation and plantlet regeneration in Rosa canina L. In callus-rhizoids, which are rhizoids that co-develop from callus, auxin and a Z-type cytokinin accumulated after applying TDZ, and transcription of the auxin transporter gene RcPIN1 was repressed. The expression of RcARF4, RcRR1, RcCKX2, RcCKX3, and RcLOG1 increased in callus-rhizoids and rhizoid tips while the transcription of an auxin response factor (RcARF1) and auxin transport proteins (RcPIN2, RcPIN3) decreased in callus-rhizoids but increased in rhizoid tips. In situ hybridization of rhizoids showed that RcWUS and RcSERK1 were highly expressed in columella cells and root stem cells resulting in the conversion of cell fate into shoot apical meristems or embryogenic callus. In addition, transgenic XVE::RcWUS lines showed repressed RcWUS overexpression while RcWUS had no effect on PLB morphogenesis. Furthermore, higher expression of the root stem cell marker RcWOX5 and root stem cell maintenance regulator genes RcPLT1 and RcPLT2 indicated the presence of a dedifferentiation developmental pathway in the stem cell niche of rhizoids. Viewed together, our results indicate that different cells in rhizoid tips acquired regeneration competence after induction by TDZ. A novel developmental pathway containing different cell types during PLB formation was identified by analyzing the endogenous auxin and cytokinin content. This study also provides a deeper understanding of the mechanisms underlying in vitro regeneration in Rosa. PMID:27200031

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.

Limited elimination of two viruses by cryotherapy of pelargonium apices related to virus distribution.

PubMed

Gallard, A; Mallet, R; Chevalier, M; Grapin, A

2011-01-01

The possibility of eradicating the pelargonium flower break virus (PFBV) and pelargonium line pattern virus (PLPV) by cryotherapy of axillary shoot apices was investigated using five Pelargonium cultivars. Viruses were detected by DAS-ELISA and their location was determined by immunolocalization. Apex culture did not permit elimination of PFBV and only 15 percent regenerated plants of 'Stellar Artic' cultivar were ELISA PLPV-negative. Plants regenerated from cryotherapy-treated apices were tested by DAS-ELISA after a 3-month in vitro culture period. Viruses were not detected in 25 percent and 50 percent of the plants tested for PFBV and PLPV, respectively. However, immunolocalization carried out on apices originating from cryopreserved shoot tips sampled from DAS-ELISA negative plants showed that they were still virus-infected. Using immunolocalization, PFBV and PLPV could be detected in Pelargonium apices, even in the meristematic dome. However, viral particles were more numerous in basal zone cells than in meristematic cells. Our results demonstrate that PFBV and PLPV are present within meristematic cells and that cryopreservation can partly reduce the quantity of these viruses in Pelargonium plants but not eliminate them totally. Additional knowledge on localization and behaviour of viruses during cryopreservation is essential to optimize cryotherapy and plant genetic resource management.

[Influence of genotype, explant type and component of culture medium on in vitro callus induction and shoot organogenesis of tomato (Solanum lycopersicum L.)].

PubMed

Khaliluev, M R; Bogoutdinova, L R; Baranova, G B; Baranova, E N; Kharchenko, P N; Dolgov, S V

2014-01-01

The influence of explant type as well as of the type of growth regulators and concentration on callus induction processes and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties possess a substantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the genotypes studied during the cultivation of cotyledons on Murashige-Skoog culture medium containing 2 mg/l of zeatin in combination with 0.1 mg/l of 3-indoleacetic acid. An effective protocol of indirect somatic organogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was developed.

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.

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.

In vitro shoot growth of Brugmansia x candida Pers.

USDA-ARS?s Scientific Manuscript database

The objective of this study was to improve the growth of in vitro shoot cultures of Brugmansia × candida 'Creamsickle'. Several mineral nutrient experiments were conducted to determine the effect of NH4+, NO3-, K+, FeSO4/EDTA, ZnSO4, MnSO4, and CuSO4 on quality, leaf width and length, size and weigh...

The effect of sodium hypochlorite solutions on in vitro seedling growth and shoot regeneration of flax (Linum usitatissimum)

NASA Astrophysics Data System (ADS)

Yildiz, Mustafa; Er, Celâl

2002-04-01

The aim of this study was to determine the effect of concentration (40, 60, and 80%) and temperature (0, 10, 20, and 30°C) of sodium hypochlorite (NaOCl) solutions on seed germination, in vitro viability and growth of flax seedlings and regeneration capacity of hypocotyl explants. Results showed that seed germination, seedling growth and shoot regeneration were negatively affected by increasing concentration and temperature of disinfectant. The best results in seedling growth and shoot regeneration were obtained when 40% disinfectant concentration at 10°C was used.

Plant root and shoot dynamics during subsurface obstacle interaction

NASA Astrophysics Data System (ADS)

Conn, Nathaniel; Aguilar, Jeffrey; Benfey, Philip; Goldman, Daniel

As roots grow, they must navigate complex underground environments to anchor and retrieve water and nutrients. From gravity sensing at the root tip to pressure sensing along the tip and elongation zone, the complex mechanosensory feedback system of the root allows it to bend towards greater depths and avoid obstacles of high impedance by asymmetrically suppressing cell elongation. Here we investigate the mechanical and physiological responses of roots to rigid obstacles. We grow Maize, Zea mays, plants in quasi-2D glass containers (22cm x 17cm x 1.4cm) filled with photoelastic gel and observe that, regardless of obstacle interaction, smaller roots branch off the primary root when the upward growing shoot (which contains the first leaf) reaches an average length of 40 mm, coinciding with when the first leaf emerges. However, prior to branching, contacts with obstacles result in reduced root growth rates. The growth rate of the root relative to the shoot is sensitive to the angle of the obstacle surface, whereby the relative root growth is greatest for horizontally oriented surfaces. We posit that root growth is prioritized when horizontal obstacles are encountered to ensure anchoring and access to nutrients during later stages of development. NSF Physics of Living Systems.

Micropropagation and cryopreservation of garlic (Allium sativum L.).

PubMed

Keller, E R Joachim; Senula, Angelika

2013-01-01

Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

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.

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.

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

Bryophyllum pinnatum: A Great Teaching Aid.

ERIC Educational Resources Information Center

Martin, Francis L.

1983-01-01

Suggests using Bryophyllum pinnatum to illustrate botanical principles. Includes tips for keeping and maintaining the plant in the classroom and suggests several student activities, including observing root/shoot growth, investigating apical dominance, exploring multiple leaf development, and others. (JN)

Micropropagation of Prunus species relevant to cherry fruit production.

PubMed

Druart, Philippe

2013-01-01

Cherry tree micropropagation is limited to the production of healthy cultivars of Prunus avium and Prunus cerasus, and their rootstocks; mainly the dwarfing ones. By using meristem-tip (0.1 mm long) or healthy shoot tips/nodes, four successive steps are needed to obtain whole plants capable of growing in the nursery: multiplication by axillary branching, shoot elongation, rooting, and plantlet acclimation. Along this process, several parameters have to be adjusted for each phase of the culture, including media composition, environmental culture conditions and plant handling. These parameters vary depending on genotypic response and specific vulnerability to physiological disorders such as hyperhydricity, apex necrosis, unstable propagation, and rooting rates. Based on a 40 year-long experience of study and application of culture conditions to large-scale plant production, this document summarizes the main problems (variability of the propagation rate, hyperhydricity, apex necrosis, plant re-growth) and solutions encountered to solve them, with means validated on many mericlones.

In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration.

PubMed

Park, Han Yong; Kang, Kyung Won; Kim, Doo Hwan; Sivanesan, Iyyakkannu

2018-03-01

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 μM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 μM 2,4-D and 2 μM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 μM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.

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.

Plasma membrane-targeted PIN proteins drive shoot development in a moss.

PubMed

Bennett, Tom A; Liu, Maureen M; Aoyama, Tsuyoshi; Bierfreund, Nicole M; Braun, Marion; Coudert, Yoan; Dennis, Ross J; O'Connor, Devin; Wang, Xiao Y; White, Chris D; Decker, Eva L; Reski, Ralf; Harrison, C Jill

2014-12-01

Plant body plans arise by the activity of meristematic growing tips during development and radiated independently in the gametophyte (n) and sporophyte (2n) stages of the life cycle during evolution. Although auxin and its intercellular transport by PIN family efflux carriers are primary regulators of sporophytic shoot development in flowering plants, the extent of conservation in PIN function within the land plants and the mechanisms regulating bryophyte gametophytic shoot development are largely unknown. We have found that treating gametophytic shoots of the moss Physcomitrella patens with exogenous auxins and auxin transport inhibitors disrupts apical function and leaf development. Two plasma membrane-targeted PIN proteins are expressed in leafy shoots, and pin mutants resemble plants treated with auxins or auxin transport inhibitors. PIN-mediated auxin transport regulates apical cell function, leaf initiation, leaf shape, and shoot tropisms in moss gametophytes. pin mutant sporophytes are sometimes branched, reproducing a phenotype only previously seen in the fossil record and in rare natural moss variants. Our results show that PIN-mediated auxin transport is an ancient, conserved regulator of shoot development. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Mathematical modeling and experimental validation of the spatial distribution of boron in the root of Arabidopsis thaliana identify high boron accumulation in the tip and predict a distinct root tip uptake function.

PubMed

Shimotohno, Akie; Sotta, Naoyuki; Sato, Takafumi; De Ruvo, Micol; Marée, Athanasius F M; Grieneisen, Verônica A; Fujiwara, Toru

2015-04-01

Boron, an essential micronutrient, is transported in roots of Arabidopsis thaliana mainly by two different types of transporters, BORs and NIPs (nodulin26-like intrinsic proteins). Both are plasma membrane localized, but have distinct transport properties and patterns of cell type-specific accumulation with different polar localizations, which are likely to affect boron distribution. Here, we used mathematical modeling and an experimental determination to address boron distributions in the root. A computational model of the root is created at the cellular level, describing the boron transporters as observed experimentally. Boron is allowed to diffuse into roots, in cells and cell walls, and to be transported over plasma membranes, reflecting the properties of the different transporters. The model predicts that a region around the quiescent center has a higher concentration of soluble boron than other portions. To evaluate this prediction experimentally, we determined the boron distribution in roots using laser ablation-inductivity coupled plasma-mass spectrometry. The analysis indicated that the boron concentration is highest near the tip and is lower in the more proximal region of the meristem zone, similar to the pattern of soluble boron distribution predicted by the model. Our model also predicts that upward boron flux does not continuously increase from the root tip toward the mature region, indicating that boron taken up in the root tip is not efficiently transported to shoots. This suggests that root tip-absorbed boron is probably used for local root growth, and that instead it is the more mature root regions which have a greater role in transporting boron toward the shoots. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

Mathematical Modeling and Experimental Validation of the Spatial Distribution of Boron in the Root of Arabidopsis thaliana Identify High Boron Accumulation in the Tip and Predict a Distinct Root Tip Uptake Function

PubMed Central

Shimotohno, Akie; Sotta, Naoyuki; Sato, Takafumi; De Ruvo, Micol; Marée, Athanasius F.M.; Grieneisen, Verônica A.; Fujiwara, Toru

2015-01-01

Boron, an essential micronutrient, is transported in roots of Arabidopsis thaliana mainly by two different types of transporters, BORs and NIPs (nodulin26-like intrinsic proteins). Both are plasma membrane localized, but have distinct transport properties and patterns of cell type-specific accumulation with different polar localizations, which are likely to affect boron distribution. Here, we used mathematical modeling and an experimental determination to address boron distributions in the root. A computational model of the root is created at the cellular level, describing the boron transporters as observed experimentally. Boron is allowed to diffuse into roots, in cells and cell walls, and to be transported over plasma membranes, reflecting the properties of the different transporters. The model predicts that a region around the quiescent center has a higher concentration of soluble boron than other portions. To evaluate this prediction experimentally, we determined the boron distribution in roots using laser ablation-inductivity coupled plasma-mass spectrometry. The analysis indicated that the boron concentration is highest near the tip and is lower in the more proximal region of the meristem zone, similar to the pattern of soluble boron distribution predicted by the model. Our model also predicts that upward boron flux does not continuously increase from the root tip toward the mature region, indicating that boron taken up in the root tip is not efficiently transported to shoots. This suggests that root tip-absorbed boron is probably used for local root growth, and that instead it is the more mature root regions which have a greater role in transporting boron toward the shoots. PMID:25670713

Translocation in Polytrichum commune (Bryophyta). II. Clonal integration

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

Thomas, R.J.; Schiele, E.M.; Damberg, D.T.

1990-12-01

Polytrichum commune is a perennial moss that grows by rhizomatous production of interconnected shoots. It possesses a well-developed internal system of food-conducting tissue. Liquid scintillation determinations following pulse-{sup 14}CO{sub 2} treatment of a single shoot in rhizomatously connected groups indicate that tagged photoassimilates are transported into: (1) newly developing stems during the early growing season; (2) shared rhizomes (with an increase observed in rhizome allocation toward the end of the year, as well as a year-end percentage increase in starch reserves); and (3) mature neighboring shoots. Over the course of the growing season, photoassimilate export from pulse-labeled shoots ranges betweenmore » 12.9% and 21.4% of total tagged. The amount of export is significantly influenced by defoliation, shading, stem tip removal, and 50 ppm indole-3-acetic acid treatments performed on rhizomatously connected neighbor shoots. Physiological integration within P. commune can be inferred from these results. Control is exerted by endogenous hormones or by changes in the activity of sources and sinks.« less

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.

Identification of PaCOL1 and PaCOL2, two CONSTANS-like genes showing decreased transcript levels preceding short day induced growth cessation in Norway spruce.

PubMed

Holefors, Anna; Opseth, Lars; Ree Rosnes, Anne Katrine; Ripel, Linda; Snipen, Lars; Fossdal, Carl Gunnar; Olsen, Jorunn E

2009-02-01

In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group 1a. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light.

Osmotic stress, endogenous abscisic acid and the control of leaf morphology in Hippuris vulgaris L

NASA Technical Reports Server (NTRS)

Goliber, T. E.; Feldman, L. J.

1989-01-01

Previous reports indicate that heterophyllous aquatic plants can be induced to form aerial-type leaves on submerged shoots when they are grown in exogenous abscisic acid (ABA). This study reports on the relationship between osmotic stress (e.g. the situation encountered by a shoot tip when it grows above the water surface), endogenous ABA (as measured by gas chromatography-electron capture detector) and leaf morphology in the heterophyllous aquatic plant, Hippuris vulgaris. Free ABA could not be detected in submerged shoots of H. vulgaris but in aerial shoots ABA occurred at ca. 40 ng (g fr wt)-1. When submerged shoots were osmotically stressed ABA appeared at levels of 26 to 40 ng (g fr wt)-1. These and other data support two main conclusions: (1) Osmotically stressing a submerged shoot causes the appearance of detectable levels of ABA. (2) The rise of ABA in osmotically stressed submerged shoots in turn induces a change in leaf morphology from the submerged to the aerial form. This corroborates the hypothesis that, in the natural environment, ABA levels rise in response to the osmotic stress encountered when a submerged shoot grows up through the water/air interface and that the increased ABA leads to the production of aerial-type leaves.

Quantitative determination of secoiridoid and gamma-pyrone compounds in Gentiana lutea cultured in vitro.

PubMed

Menković, N; Savikin-Fodulović, K; Momcilović, I; Grubisić, D

2000-02-01

The production of secondary metabolites was studied in shoots, roots, and hairy roots of Gentiana lutea obtained in vitro. In shoots, both secoiridoid and gamma-pyrone compounds were detected in amounts similar to those found in aerial parts of plants collected from nature. The most abundant secoiridoid was gentiopicrin while mangiferin was the main compound among the gamma-pyrones. The adventitious roots obtained in vitro showed a poor biosynthetic capacity. Upon infection with Agrobacterium rhizogenes, nine hairy root clones were established which differed in the amount of secondary metabolites.

Cryoprotectants and their components induce plasmolytic responses in sweet potato suspension cells

USDA-ARS?s Scientific Manuscript database

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove water and prevent ice crystallization, ensuring survival after liquid nitrogen expos...

In Vitro Conservation of Twenty-Three Overexploited Medicinal Plants Belonging to the Indian Sub Continent

PubMed Central

Verma, Priyanka; Mathur, Ajay Kumar; Jain, Sheetal Prasad; Mathur, Archana

2012-01-01

Twenty-three pharmaceutically important plants, namely, Elaeocarpus spharicus, Rheum emodi, Indigofera tinctoria, Picrorrhiza kurroa, Bergenia ciliata, Lavandula officinalis, Valeriana wallichii, Coleus forskohlii, Gentiana kurroo, Saussurea lappa, Stevia rebaudiana, Acorus calamus, Pyrethrum cinerariaefolium, Aloe vera, Bacopa monnieri, Salvia sclarea, Glycyrrhiza glabra, Swertia cordata, Psoralea corylifolia, Jurinea mollis, Ocimum sanctum, Paris polyphylla, and Papaver somniferum, which are at the verge of being endangered due to their overexploitation and collection from the wild, were successfully established in vitro. Collections were made from the different biodiversity zones of India including Western Himalaya, Northeast Himalaya, Gangetic plain, Western Ghats, Semiarid Zone, and Central Highlands. Aseptic cultures were raised at the morphogenic level of callus, suspension, axillary shoot, multiple shoot, and rooted plants. Synseeds were also produced from highly proliferating shoot cultures of Bacopa monnieri, Glycyrrhiza glabra, Stevia rebaudiana, Valeriana wallichii, Gentiana kurroo, Lavandula officinalis, and Papaver somniferum. In vitro flowering was observed in Papaver somniferum, Psoralea corylifolia, and Ocimum sanctum shoots cultures. Out of 23 plants, 18 plants were successfully hardened under glasshouse conditions. PMID:22593711

Three ancient hormonal cues co-ordinate shoot branching in a moss.

PubMed

Coudert, Yoan; Palubicki, Wojtek; Ljung, Karin; Novak, Ondrej; Leyser, Ottoline; Harrison, C Jill

2015-03-25

Shoot branching is a primary contributor to plant architecture, evolving independently in flowering plant sporophytes and moss gametophytes. Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis, which have a recent evolutionary origin. We show that in gametophytic shoots of Physcomitrella, lateral branches arise by re-specification of epidermal cells into branch initials. A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns, and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin, cytokinin and strigolactone. The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways. Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants, this is not so in Physcomitrella, where bi-directional transport is required to generate realistic branching patterns. Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport.

Determination of zinc oxide nanoparticles toxicity in root growth in wheat (Triticum aestivum L.) seedlings.

PubMed

Prakash, Meppaloor G; Chung, Ill Min

2016-09-01

The effect of zinc oxide nanoparticles (ZnONPs) was studied in wheat (Triticum aestivum L.) seedlings under in vitro exposure conditions. To avoid precipitation of nanoparticles, the seedlings were grown in half strength semisolid Murashige and Skoog medium containing 0, 50, 100, 200, 400 and 500 mg L(-1) of ZnONPs. Analysis of zinc (Zn) content showed significant increase in roots. In vivo detection using fluorescent probe Zynpyr-1 indicated accumulation of Zn in primary and lateral root tips. All concentrations of ZnONPs significantly reduced root growth. However, significant decrease in shoot growth was observed only after exposure to 400 and 500 mg L(-1) of ZnONPs. The reactive oxygen species and lipid peroxidation levels significantly increased in roots. Significant increase in cell-wall bound peroxidase activity was observed after exposure to 500 mg L(-1) of ZnONPs. Histochemical staining with phloroglucinol-HCl showed lignification of root cells upon exposure to 500 mg L(-1) of ZnONPs. Treatment with propidium iodide indicated loss of cell viability in root tips of wheat seedlings. These results suggest that redox imbalances, lignification and cell death has resulted in reduction of root growth in wheat seedlings exposed to ZnONPs nanoparticles.

Influence of near null magnetic field on in vitro growth of potato and wild Solanum species.

PubMed

Rakosy-Tican, Lenuta; Aurori, C M; Morariu, V V

2005-10-01

The influence of near null magnetic field on in vitro growth of different cultures of potato and related Solanum species was investigated for various exposure times and dates. Potato (Solanum tuberosum L. cv. Désirée) in vitro cultures of shoot tips or nodal segments were used. Three different exposure periods revealed either stimulation or inhibition of root, stem, or leaf in vitro growth after 14 or 28 days of exposure. In one experiment the significant stimulation of leaf growth was also demonstrated at biochemical level, the quantity of chlorophyll a and b and carotenoids increasing more than two-fold. For the wild species Solanum chacoense, S. microdontum, and S. verrucosum, standardized in vitro cultures of nodal stem segments were used. Root and stem growth was either stimulated or slightly inhibited after 9 days exposure to near null magnetic field. Callus cultures obtained from potato dihaploid line 120/19 were maintained in near null magnetic field in 2 different months. For these experiments as well as for Solanum verrucosum, callus cultures recorded either slight inhibition or no effect on fresh weight. For all experiments significant growth variation was brought about only when geomagnetic activity (AP index) showed variations at the beginning of in vitro growth and when the explant had at least one meristematic tissue. Moreover longer maintenance in near null magnetic field, 28 days as compared to 14 days or the controls, can also make a difference in plant growth in response to geomagnetic field variations when static component was reduced to zero value. These results of in vitro plant growth stimulation by variable component of geomagnetic field also sustain the so-called seasonal "window" effect. (c) 2005 Wiley-Liss, Inc.

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.

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

NASA Technical Reports Server (NTRS)

Deruere, J.; Jackson, K.; Garbers, C.; Soll, D.; Delong, A.; Evans, M. L. (Principal Investigator)

1999-01-01

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

Branch age and light conditions determine leaf-area-specific conductivity in current shoots of Scots pine.

PubMed

Grönlund, Leila; Hölttä, Teemu; Mäkelä, Annikki

2016-08-01

Shoot size and other shoot properties more or less follow the availability of light, but there is also evidence that the topological position in a tree crown has an influence on shoot development. Whether the hydraulic properties of new shoots are more regulated by the light or the position affects the shoot acclimation to changing light conditions and thereby to changing evaporative demand. We investigated the leaf-area-specific conductivity (and its components sapwood-specific conductivity and Huber value) of the current-year shoots of Scots pine (Pinus sylvestris L.) in relation to light environment and topological position in three different tree classes. The light environment was quantified in terms of simulated transpiration and the topological position was quantified by parent branch age. Sample shoot measurements included length, basal and tip diameter, hydraulic conductivity of the shoot, tracheid area and density, and specific leaf area. In our results, the leaf-area-specific conductivity of new shoots declined with parent branch age and increased with simulated transpiration rate of the shoot. The relation to transpiration demand seemed more decisive, since it gave higher R(2) values than branch age and explained the differences between the tree classes. The trend of leaf-area-specific conductivity with simulated transpiration was closely related to Huber value, whereas the trend of leaf-area-specific conductivity with parent branch age was related to a similar trend in sapwood-specific conductivity. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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 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.

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.

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.

Multitip scanning bio-Kelvin probe

NASA Astrophysics Data System (ADS)

Baikie, I. D.; Smith, P. J. S.; Porterfield, D. M.; Estrup, P. J.

1999-03-01

We have developed a novel multitip scanning Kelvin probe which can measure changes in biological surface potential ΔVs to within 2 mV and, quasisimultaneously monitor displacement to <1 μm. The control and measurement subcomponents are PC based and incorporate a flexible user interface permitting software control of each individual tip, measurement, and scan parameters. We review the mode of operation and design features of the scanning bio-Kelvin probe including tip steering, signal processing, tip calibration, and novel tip tracking/dithering routines. This system uniquely offers both tip-to-sample spacing control (which is essential to avoid spurious changes in ΔVs due to variations in mean spacing) and a dithering routine to maintain tip orientation to the biological specimen, irrespective of the latter's movement. These features permit long term (>48 h) "active" tracking of the displacement and biopotentials developed along and around a plant shoot in response to an environmental stimulus, e.g., differential illumination (phototropism) or changes in orientation (gravitropism).

Isolation and characterization of a TERMINAL FLOWER 1 homolog from Prunus serotina Ehrh.

PubMed

Wang, Ying; Pijut, Paula M

2013-08-01

Flowering control is one of the several strategies for gene containment of transgenic plants. TERMINAL FLOWER 1 (TFL1) is known to be involved in the transcriptional repression of genes for inflorescence development. Two TFL1 transcripts with different 3' UTR were cloned from black cherry (Prunus serotina Ehrh.) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Corresponding to the two TFL1 transcripts, two PsTFL1 gene sequences, 1248 bp and 1579 bp, were obtained and both contained the same 519 bp coding region which encoded a putative protein of 172 amino acid residues. The phylogenetic analysis of the amino acid sequences showed high identity of PsTFL1 to TFL1 orthologs of other Prunus species, including Yoshino cherry (Prunus × yedoensis Matsum.), peach (Prunus persica (L.) Batsch), apricot (Prunus armeniaca L.) and Japanese apricot (Prunus mume Sieb. et Zucc.). The real-time quantitative PCR detected a single copy of PsTFL1 gene sequences in the black cherry genome with two alleles. The gene expression of PsTFL1 was examined in several tissues including the stems, leaves, shoot tips, and vegetative and floral buds. The highest mRNA level was detected in shoot tips, and the lowest level in the leaves. Transgenic Arabidopsis thaliana (L.) Heynh. plants overexpressing PsTFL1 showed significantly delayed flowering. These plants also showed largely increased vegetative growth, plant height, number of nodes, trichome density, and the conversion of flower to shoot was observed at each node and shoot apex.

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.

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.

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.

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.

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

Incorporation and translocation of 2-deoxy-2-[(18)F]fluoro-D-glucose in Sorghum bicolor (L.) Moench monitored using a planar positron imaging system.

PubMed

Hattori, Etsuko; Uchida, Hiroshi; Harada, Norihiro; Ohta, Mari; Tsukada, Hideo; Hara, Yasuhiro; Suzuki, Tetsuya

2008-04-01

[(18)F]FDG (2-deoxy-2-[(18)F]fluoro-D-glucose) was fed to a sorghum plant [Sorghum bicolor (L.) Moench] from the tip of a leaf and its movement was monitored using a planar positron imaging system (PPIS). [(18)F]FDG was uptaken from the leaf tip and it was translocated to the basal part of the shoots from where it moved to the roots, the tillers and the sheaths. Autoradiographic analysis of the distribution of (18)F, [(18)F]FDG and/or its metabolites showed translocation to the roots, tillers, and to the leaves that were younger than the supplied leaf. Strong labelling was observed in the basal part of the shoots, in the sheaths, the youngest leaf and the root tips. Our results indicate that [(18)F]FDG and/or its metabolites were absorbed from the leaf and translocated to the sites where nutrients are required. This strongly suggests that [(18)F]FDG can be utilised as a tracer to study photoassimilate translocation in the living plant. This is the first report on the use of [(18)F]FDG, which is routinely used as a probe for clinical diagnosis, to study source to sink translocation of metabolites in whole plants in real time.

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.

Antioxidant and Anti-stress Compounds Improve Regrowth of Cryopreserved Rubus Shoot Tips

USDA-ARS?s Scientific Manuscript database

Regrowth of plants after cryopreservation varies and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (...

Improved droplet-vitrification and histological studies of cryopreserved shoot tips of cultivated Jerusalem artichoke genotypes

USDA-ARS?s Scientific Manuscript database

Germplasm conservation of Jerusalem artichoke (Helianthus tuberosus L.) is crucial to preserve genetic diversity and to secure materials for genetic improvement. Long-term conservation is accomplished through cryopreservation, storing cells or tissues at an ultralow temperature in liquid nitrogen (-...

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.

Three ancient hormonal cues co-ordinate shoot branching in a moss

PubMed Central

Coudert, Yoan; Palubicki, Wojtek; Ljung, Karin; Novak, Ondrej; Leyser, Ottoline; Harrison, C Jill

2015-01-01

Shoot branching is a primary contributor to plant architecture, evolving independently in flowering plant sporophytes and moss gametophytes. Mechanistic understanding of branching is largely limited to flowering plants such as Arabidopsis, which have a recent evolutionary origin. We show that in gametophytic shoots of Physcomitrella, lateral branches arise by re-specification of epidermal cells into branch initials. A simple model co-ordinating the activity of leafy shoot tips can account for branching patterns, and three known and ancient hormonal regulators of sporophytic branching interact to generate the branching pattern- auxin, cytokinin and strigolactone. The mode of auxin transport required in branch patterning is a key divergence point from known sporophytic pathways. Although PIN-mediated basipetal auxin transport regulates branching patterns in flowering plants, this is not so in Physcomitrella, where bi-directional transport is required to generate realistic branching patterns. Experiments with callose synthesis inhibitors suggest plasmodesmal connectivity as a potential mechanism for transport. DOI: http://dx.doi.org/10.7554/eLife.06808.001 PMID:25806686

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.

Development of an efficient regeneration and transformation method for the new potential oilseed crop Lepidium campestre

PubMed Central

2013-01-01

Background Lepidium campestre is an undomesticated oilseed species with a great potential to become a new crop for both food and industrial feedstocks production. Genetic modification is needed for further improving the oil quantity and quality of Lepidium. Studies on in vitro shoot regeneration of Lepidium are very limited and there is no transformation protocol available. Results We have investigated the effects of different factors, especially the type, concentration and combination of plant growth regulators (PGRs) on in vitro shoot regeneration of Lepidium. The results showed that the 2,4-D treatment was crucial to shoot regeneration from different explants. The duration of 2,4-D exposure between 2-4 days did not show significant difference in shoot regeneration, while the effect of 2,4-D concentration varied greatly depending on the type of explants and cytokinins used, for example, the low concentration of 2,4-D combined with TDZ significantly increased the regeneration frequency of hypocotyls. Cotyledon and hypocotyl explants responded differently to cytokinin, for example, TDZ was more effective than zeatin in promoting shoot regeneration from hypocotyls, but did not affect the regeneration of cotyledons which was more affected by high concentration of zeatin. The results also showed that NAA was not effective for shoot regeneration. Germination in light increased the regeneration frequency compared to that in dark. After optimization of the different conditions, an efficient regeneration protocol was developed with the regeneration efficiency of 92.7%. Using this protocol, the transformation frequency of 6% in average was achieved. The presence of transgenes in the transgenic lines was confirmed by GUS staining, PCR and Southern blot analyses. Conclusion Through systematic investigation of important factors affecting in vitro shoot regeneration, we have developed an efficient regeneration and transformation protocol for the genetic modification of Lepidium campestre. The method may also be applied to the related species. PMID:23937221

The role of silicon in plant tissue culture

PubMed Central

Sivanesan, Iyyakkannu; Park, Se Won

2014-01-01

Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production. PMID:25374578

Enhanced bacoside production in shoot cultures of Bacopa monnieri under the influence of abiotic elicitors.

PubMed

Sharma, Munish; Ahuja, Ashok; Gupta, Rajinder; Mallubhotla, Sharada

2015-01-01

The effect of different abiotic elicitors [jasmonic acid, copper sulphate (CuSO4) and salicyclic acid] at varying concentrations on the stimulation of biomass and bacoside production in in vitro Bacopa monnieri shoot culture was studied. A systematic study conducted over a period of 35 days indicated that the maximum bacoside production (6.74 mg g(-1) dry weight (DW)) was obtained after a lag of 7 days and thereafter, the content decreased gradually to again increase at 28 days (5.91 mg g(-1) DW). Therefore, elicitation experiments were carried out over a period of 3, 6 and 9 days. The shoot cultures treated with 45 mg L(-1) of CuSO4 exhibited the highest bacoside content of 8.73 mg g(-1) DW (∼1.42-fold higher) than in control cultures (6.14 mg g(-1) DW). This study indicates the effectiveness of abiotic elicitation on bacoside production in in vitro shoot cultures of this medicinally important herb known for its memory-enhancing properties.

The Complete Step-by-Step Guide to Designing and Teaching Online Courses

ERIC Educational Resources Information Center

Thormann, Joan; Zimmerman, Isa Kaftal

2012-01-01

In this valuable resource, experts share deep knowledge including practical "how-to" and preventive trouble-shooting tips. Instructors will learn about course design and development, instructional methods for online teaching, and student engagement and community building techniques. The book contains successful teaching strategies, guidance for…

Challenges in the development of a widely applicable method for sugarcane (Saccharum spp.) shoot tip cryopreservation

USDA-ARS?s Scientific Manuscript database

The USDA-ARS National Plant Germplasm System (NPGS) maintains 946 accessions of sugarcane (Saccharum spp.) in the field at the Subtropical Horticulture Research Station in Miami, Florida. These accessions are particularly vulnerable to hurricanes, diseases, and other threats. We sought to a identify...

Implementation of Citrus Shoot Tip Cryopreservation in the USDA-ARS National Plant Germplasm System

USDA-ARS?s Scientific Manuscript database

The USDA-ARS National Plant Germplasm System (NPGS) maintains 540 Citrus cultivars and crop wild relatives as duplicate clones in a screenhouse at the National Clonal Germplasm Repository for Citrus and Dates (NCGRCD) in Riverside, California. These 540 accessions are pathogen-tested and apparently ...

Carbohydrate metabolism of vegetative and reproductive sinks in the late-maturing peach cultivar 'Encore'

Treesearch

Riccardo Lo Bianco; Mark Rieger; Shi-Jean S. Sung

1999-01-01

Activities of NAD+-dependent sorbitol dehydrogenase (SDH), sorbitol oxidase (SOX), sucrose synthase (SS), acid invertase (AI), and neutral invertase (NI) in ?Encore? peach (Prunus persica L.) fruits and developing shoot tips were assayed during the growing season to determine whether carbohydrate metabolizing enzymes could...

Evaluation of twig pre-harvest temperature for effective cryopreservation of Vaccinium dormant buds

USDA-ARS?s Scientific Manuscript database

Cryopreservation of plant material by dormant buds is less expensive than using shoot tips; however currently, dormant buds are used only for preservation of selected temperate tree and shrub species. Using dormant buds could be an efficient strategy for long-term preservation of blueberry (Vacciniu...

Sporulation of Phytophthora ramorum and P. kernoviae on asymptomatic foliage and fruit

Treesearch

S. Denman; E. Moralejo; S.A. Kirk; E. Orton; A. Whybrow

2008-01-01

Phytophthora ramorum and P. kernoviae are newly discovered invasive Phytophthoras causing leaf necrosis, shoot tip dieback (mostly on ornamental and forest understorey host species) and bleeding cankers on tree trunks of a wide range of plant species. Both pathogens are now present in south-west England....

Droplet-vitrification and morphohistological studies of cryopreserved shoot tips of cultivated and wild pineapple genotypes

USDA-ARS?s Scientific Manuscript database

Germplasm conservation of pineapple [Ananas comosus (L.) Mer.] is crucial to preserve the genus’ genetic diversity to secure material for genetic improvement and to support innovative and new research. Long-term conservation is accomplished through cryopreservation that is done by storing cells or t...

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.

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.

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.

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.

Effects of GhWUS from upland cotton (Gossypium hirsutum L.) on somatic embryogenesis and shoot regeneration.

PubMed

Xiao, Yanqing; Chen, Yanli; Ding, Yanpeng; Wu, Jie; Wang, Peng; Yu, Ya; Wei, Xi; Wang, Ye; Zhang, Chaojun; Li, Fuguang; Ge, Xiaoyang

2018-05-01

The WUSCHEL (WUS) gene encodes a plant-specific homeodomain-containing transcriptional regulator, which plays important roles during embryogenesis, as well as in the formation of shoot and flower meristems. Here, we isolated two homologues of Arabidopsis thaliana WUS (AtWUS), GhWUS1a_At and GhWUS1b_At, from upland cotton (Gossypium hirsutum). Domain analysis suggested that the two putative GhWUS proteins contained a highly conserved DNA-binding HOX domain and a WUS-box. Expression profile analysis showed that GhWUSs were predominantly expressed during the embryoid stage. Ectopic expression of GhWUSs in Arabidopsis could induce somatic embryo and shoot formation from seedling root tips. Furthermore, in the absence of exogenous hormone, overexpression of GhWUSs in Arabidopsis could promote shoot regeneration from excised roots, and in the presence of exogenous auxin, excised roots expressing GhWUS could be induced to produce somatic embryo. In addition, expression of the chimeric GhWUS repressor in cotton callus inhibited embryogenic callus formation. Our results show that GhWUS is an important regulator of somatic embryogenesis and shoot regeneration. Copyright © 2018 Elsevier B.V. All rights reserved.

Mathematical Modeling and Optimizing of in Vitro Hormonal Combination for G × N15 Vegetative Rootstock Proliferation Using Artificial Neural Network-Genetic Algorithm (ANN-GA)

PubMed Central

Arab, Mohammad M.; Yadollahi, Abbas; Ahmadi, Hamed; Eftekhari, Maliheh; Maleki, Masoud

2017-01-01

The efficiency of a hybrid systems method which combined artificial neural networks (ANNs) as a modeling tool and genetic algorithms (GAs) as an optimizing method for input variables used in ANN modeling was assessed. Hence, as a new technique, it was applied for the prediction and optimization of the plant hormones concentrations and combinations for in vitro proliferation of Garnem (G × N15) rootstock as a case study. Optimizing hormones combination was surveyed by modeling the effects of various concentrations of cytokinin–auxin, i.e., BAP, KIN, TDZ, IBA, and NAA combinations (inputs) on four growth parameters (outputs), i.e., micro-shoots number per explant, length of micro-shoots, developed callus weight (CW) and the quality index (QI) of plantlets. Calculation of statistical values such as R2 (coefficient of determination) related to the accuracy of ANN-GA models showed a considerably higher prediction accuracy for ANN models, i.e., micro-shoots number: R2 = 0.81, length of micro-shoots: R2 = 0.87, CW: R2 = 0.88, QI: R2 = 0.87. According to the results, among the input variables, BAP (19.3), KIN (9.64), and IBA (2.63) showed the highest values of variable sensitivity ratio for proliferation rate. The GA showed that media containing 1.02 mg/l BAP in combination with 0.098 mg/l IBA could lead to the optimal proliferation rate (10.53) for G × N15 rootstock. Another objective of the present study was to compare the performance of predicted and optimized cytokinin–auxin combination with the best optimized obtained concentrations of our other experiments. Considering three growth parameters (length of micro-shoots, micro-shoots number, and proliferation rate), the last treatment was found to be superior to the rest of treatments for G × N15 rootstock in vitro multiplication. Very little difference between the ANN predicted and experimental data confirmed high capability of ANN-GA method in predicting new optimized protocols for plant in vitro propagation. PMID:29163583

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.

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

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.

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.

Developmental decline in height growth in Douglas-fir.

Treesearch

Barbara J. Bond; Nicole M. Czarnomski; Clifton Cooper; Michael E. Day; Michael S. Greenwood

2007-01-01

The characteristic decline in height growth that occurs over a tree's lifespan is often called "age-related decline." But is the reduction in height growth in aging trees a function of age or of size? We grafted shoot tips across different ages and sizes of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) trees to determine whether...

Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips

USDA-ARS?s Scientific Manuscript database

Cryopreservation methods have been implemented in genebanks worldwide as a strategy to ensure long-term, secure back-ups of critical collections of plant genetic resources. Effective and efficient cryopreservation methods are particularly necessary for collections of cultivars that are propagated ve...

Use of protoplast, cell, and shoot tip culture in an elm germ plasm improvement program

Treesearch

R. Daniel Lineberger; M.B. Sticklen; P.M. Pijut; Mark A. Kroggel; C.V.M. Fink; S.C. Domir

1990-01-01

An elm germplasm improvement program was established using three distinct approaches: (1) development of protoplast regeneration protocols with the goal of attempting somatic hybridization between Ulmus americana and disease resistant hybrids; (2) evaluation of the extent of somaclonal variation in plants regenerated from protoplasts; and (3)...

75 FR 34322 - Citrus Greening and Asian Citrus Psyllid; Quarantine and Interstate Movement Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-17

... leaves. High populations feeding on a citrus shoot can kill the growing tip. ACP is currently present in... the public the availability of an environmental assessment, titled ``Movement of Regulated Articles... greening is a high-risk pathway for the spread of the disease. For example, a tourist visiting a...

Pheromone-based disruption of Eucosma sonomana and Rhyacionia zozana (Lepidoptera: Tortricidae) using aerially applied microencapsulated pheromone

Treesearch

Nancy E. Gillette; John D. Stein; Donald R. Owen; Jeffrey N. Webster; Sylvia R. Mori

2006-01-01

Two aerial applications of microencapsulated pheromone were conducted on five 20.2 ha plots to disrupt western pine shoot borer (Eucosma sonomana Kearfott) and ponderosa pine tip moth (Rhyacionia zowna (Kearfott): Lepidoptera: Tortricidae) orientation to pheromones and oviposition in ponderosa pine plantations in 2002 and 2004...

Altered invertase activities of symptomatic tissues on Beet severe curly top virus (BSCTV) infected Arabidopsis thaliana.

PubMed

Park, Jungan; Kim, Soyeon; Choi, Eunseok; Auh, Chung-Kyun; Park, Jong-Bum; Kim, Dong-Giun; Chung, Young-Jae; Lee, Taek-Kyun; Lee, Sukchan

2013-09-01

Arabidopsis thaliana infected with Beet severe curly top virus (BSCTV) exhibits systemic symptoms such as stunting of plant growth, callus induction on shoot tips, and curling of leaves and shoot tips. The regulation of sucrose metabolism is essential for obtaining the energy required for viral replication and the development of symptoms in BSCTV-infected A. thaliana. We evaluated the changed transcript level and enzyme activity of invertases in the inflorescence stems of BSCTV-infected A. thaliana. These results were consistent with the increased pattern of ribulose-1,5-bisphosphate carboxylase/oxygenase activity and photosynthetic pigment concentration in virus-infected plants to supply more energy for BSCTV multiplication. The altered gene expression of invertases during symptom development was functionally correlated with the differential expression patterns of D-type cyclins, E2F isoforms, and invertase-related genes. Taken together, our results indicate that sucrose sensing by BSCTV infection may regulate the expression of sucrose metabolism and result in the subsequent development of viral symptoms in relation with activation of cell cycle regulation.

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.

In vitro co-cultures of Pinus pinaster with Bursaphelenchus xylophilus: a biotechnological approach to study pine wilt disease.

PubMed

Faria, Jorge M S; Sena, Inês; Vieira da Silva, Inês; Ribeiro, Bruno; Barbosa, Pedro; Ascensão, Lia; Bennett, Richard N; Mota, Manuel; Figueiredo, A Cristina

2015-06-01

Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all β- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

PubMed

Duarte-Aké, Fátima; Castillo-Castro, Eduardo; Pool, Felipe Barredo; Espadas, Francisco; Santamaría, Jorge M; Robert, Manuel L; De-la-Peña, Clelia

2016-12-01

Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

New potential markers of in vitro tomato morphogenesis identified by mRNA differential display.

PubMed

Torelli, A; Soragni, E; Bolchi, A; Petrucco, S; Ottonello, S; Branca, C

1996-12-01

The identification of plant genes involved in early phases of in vitro morphogenesis can not only contribute to our understanding of the processes underlying growth regulator-controlled determination, but also provide novel markers for evaluating the outcome of in vitro regeneration experiments. To search for such genes and to monitor changes in gene expression accompanying in vitro regeneration, we have adapted the mRNA differential display technique to the comparative analysis of a model system of tomato cotyledons that can be driven selectively toward either shoot or callus formation by means of previously determined growth regulator supplementations. Hormone-independent transcriptional modulation (mainly down-regulation) has been found to be the most common event, indicating that a non-specific reprogramming of gene expression quantitatively predominates during the early phases of in vitro culture. However, cDNA fragments representative of genes that are either down-regulated or induced in a programme-specific manner could also be identified, and two of them (G35, G36) were further characterized. One of these cDNA fragments, G35, corresponds to an mRNA that is down-regulated much earlier in callus- (day 2) than in shoot-determined explants (day 6). The other, G36, identifies an mRNA that is transiently expressed in shoot-determined explants only, well before any macroscopic signs of differentiation become apparent, and thus exhibits typical features of a morphogenetic marker.

Final Scientific/Technical Report for DOE Award No. DE-FG02-03ER15426: Role of Arabidopsis PINHEAD gene in meristem function

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

Dr. M. Kathryn Barton

2011-11-29

The shoot apical meristems of land plants are small mounds of hundreds of cells located at the tips of branches. It is from these small clusters of cells that essentially all above ground plant biomass and therefore much of our energy supply originates. Several key genes have been discovered that are necessary for cells in the shoot apical meristem to take on stem cell properties. The goal of this project is to understand how the synthesis and accumulation of the mRNAs and proteins encoded by these genes is controlled. A thorough understanding of the molecules that control the growth ofmore » shoot apical meristems in plants will help us to manipulate food, fiber and biofuel crops to better feed, clothe and provide energy for humans.« less

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.

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.

Silver Uptake, Distribution, and Effect on Calcium, Phosphorus, and Sulfur Uptake 1

PubMed Central

Koontz, Harold V.; Berle, Karen L.

1980-01-01

Bean, corn, and tomato plants were grown in a nutrient solution labeled with 32P, 45Ca, or 35S and varying concentrations of AgNO3. Following a 6-hour treatment period, plants were harvested and analyzed. A low Ag+ concentration (50 nanomolar) inhibited the shoot uptake of the ions investigated. In the roots, Ca uptake increased whereas P and S uptake decreased. Autoradiograms of bean and corn plants, using 110mAg, showed that Ag+ was uniformly deposited in the bean shoot, but corn shoots had regions of high activity along the leaf margins and at the tips where guttation had occurred. Roots were heavily labeled and shoots (especially the new growth) continued to accumulate Ag+ even after the intact plant was returned to Ag-free solution. Silver was believed to be phloem-mobile since it was exported from a treated leaf. Bean plants removed one-half the Ag+ from 4 liters of nutrient solution containing 50 nanomolar AgNO3 within 1.5 hours, but took 16 hours for 20 liters of solution. Images PMID:16661185

The FANTASTIC FOUR proteins influence shoot meristem size in Arabidopsis thaliana.

PubMed

Wahl, Vanessa; Brand, Luise H; Guo, Ya-Long; Schmid, Markus

2010-12-22

Throughout their lives plants produce new organs from groups of pluripotent cells called meristems, located at the tips of the shoot and the root. The size of the shoot meristem is tightly controlled by a feedback loop, which involves the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA (CLV) proteins. This regulatory circuit is further fine-tuned by morphogenic signals such as hormones and sugars. Here we show that a family of four plant-specific proteins, encoded by the FANTASTIC FOUR (FAF) genes, has the potential to regulate shoot meristem size in Arabidopsis thaliana. FAF2 and FAF4 are expressed in the centre of the shoot meristem, overlapping with the site of WUS expression. Consistent with a regulatory interaction between the FAF gene family and WUS, our experiments indicate that the FAFs can repress WUS, which ultimately leads to an arrest of meristem activity in FAF overexpressing lines. The finding that meristematic expression of FAF2 and FAF4 is under negative control by CLV3 further supports the hypothesis that the FAFs are modulators of the genetic circuit that regulates the meristem. This study reports the initial characterization of the Arabidopsis thaliana FAF gene family. Our data indicate that the FAF genes form a plant specific gene family, the members of which have the potential to regulate the size of the shoot meristem by modulating the CLV3-WUS feedback loop.

A rapid and efficient in vitro regeneration system for lettuce (Lactuca sativa L.).

PubMed

Armas, Isabel; Pogrebnyak, Natalia; Raskin, Ilya

2017-01-01

Successful biotechnological improvement of crop plants requires a reliable and efficient in vitro regeneration system. Lettuce ( Lactuca sativa L.), one the most important vegetable crops worldwide, is strongly genotype-dependent in terms of regeneration capacity, limiting the potential for biotechnological improvement of cultivars which show recalcitrance under currently available protocols. The effect of different nutrient sources, plant hormone combinations and activated charcoal supplementation on shoot induction efficiency was evaluated on the cultivar 'RSL NFR', which had previously shown poor regeneration efficiency. Multiple shoot organogenesis from cotyledon explants was recorded at the highest frequency and speed on Murashige and Skoog regeneration medium supplemented with 200 mg/l of activated charcoal, 3% sucrose, 10 mg/l benzylaminopurine and 0.5 mg/l naphthaleneacetic acid, which induced shoots through direct regeneration in 90.8 ± 7.9% of explants. High shoot induction efficiency was also observed, albeit not quantified, when using this medium on some other cultivars. This activated charcoal-containing regeneration medium might offer a rapid and efficient option for direct shoot induction in some lettuce genotypes that do not respond well to common lettuce regeneration protocols. This is also the first report of the effect of activated charcoal in lettuce tissue culture.

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.

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.

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.

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.

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.

Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

PubMed

Whitehill, Justin G A; Henderson, Hannah; Schuetz, Mathias; Skyba, Oleksandr; Yuen, Macaire Man Saint; King, John; Samuels, A Lacey; Mansfield, Shawn D; Bohlmann, Jörg

2016-08-01

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. © 2015 John Wiley & Sons Ltd.

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-...

Control of Arabidopsis leaf morphogenesis through regulation of the YABBY and KNOX families of transcription factors

USDA-ARS?s Scientific Manuscript database

The patterning of initiating organs along specific axes of polarity is critical for the proper development of all higher organisms. Plant lateral organs, such as leaves, are derived from the shoot apical meristems located at the growing tips. After initiation, the leaf primordia of species such as A...

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.

Elimination of five sugarcane viruses from sugarcane using in vitro culture of axillary bud and apical meristem

USDA-ARS?s Scientific Manuscript database

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus el...

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.

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.

Brassinolide Increases Potato Root Growth In Vitro in a Dose-Dependent Way and Alleviates Salinity Stress

PubMed Central

Xia, Shitou; Su, Yi; Wang, Huiqun; Luo, Weigui; Su, Shengying

2016-01-01

Brassinosteroids (BRs) are steroidal phytohormones that regulate various physiological processes, such as root development and stress tolerance. In the present study, we showed that brassinolide (BL) affects potato root in vitro growth in a dose-dependent manner. Low BL concentrations (0.1 and 0.01 μg/L) promoted root elongation and lateral root development, whereas high BL concentrations (1–100 μg/L) inhibited root elongation. There was a significant (P < 0.05) positive correlation between root activity and BL concentrations within a range from 0.01 to 100 μg/L, with the peak activity of 8.238 mg TTC·g−1 FW·h−1 at a BL concentration of 100 μg/L. Furthermore, plants treated with 50 μg/L BL showed enhanced salt stress tolerance through in vitro growth. Under this scenario, BL treatment enhanced the proline content and antioxidant enzymes' (superoxide dismutase, peroxidase, and catalase) activity and reduced malondialdehyde content in potato shoots. Application of BL maintain K+ and Na+ homeostasis by improving tissue K+/Na+ ratio. Therefore, we suggested that the effects of BL on root development from stem fragments explants as well as on primary root development are dose-dependent and that BL application alleviates salt stress on potato by improving root activity, root/shoot ratio, and antioxidative capacity in shoots and maintaining K+/Na+ homeostasis in potato shoots and roots. PMID:27803931

The FANTASTIC FOUR proteins influence shoot meristem size in Arabidopsis thaliana

PubMed Central

2010-01-01

Background Throughout their lives plants produce new organs from groups of pluripotent cells called meristems, located at the tips of the shoot and the root. The size of the shoot meristem is tightly controlled by a feedback loop, which involves the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA (CLV) proteins. This regulatory circuit is further fine-tuned by morphogenic signals such as hormones and sugars. Results Here we show that a family of four plant-specific proteins, encoded by the FANTASTIC FOUR (FAF) genes, has the potential to regulate shoot meristem size in Arabidopsis thaliana. FAF2 and FAF4 are expressed in the centre of the shoot meristem, overlapping with the site of WUS expression. Consistent with a regulatory interaction between the FAF gene family and WUS, our experiments indicate that the FAFs can repress WUS, which ultimately leads to an arrest of meristem activity in FAF overexpressing lines. The finding that meristematic expression of FAF2 and FAF4 is under negative control by CLV3 further supports the hypothesis that the FAFs are modulators of the genetic circuit that regulates the meristem. Conclusion This study reports the initial characterization of the Arabidopsis thaliana FAF gene family. Our data indicate that the FAF genes form a plant specific gene family, the members of which have the potential to regulate the size of the shoot meristem by modulating the CLV3-WUS feedback loop. PMID:21176196

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.

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.

Pigments for natural dye-sensitized solar cells from in vitro grown shoot cultures

NASA Astrophysics Data System (ADS)

Di Bari, Chiara; Forni, Cinzia; Di Carlo, Aldo; Barrajón-Catalán, Enrique; Micol, Vicente; Teoli, Federico; Nota, Paolo; Matteocci, Fabio; Frattarelli, Andrea; Caboni, Emilia; Lucioli, Simona

2017-04-01

In vitro grown shoots cultures (Prunus salicina × Prunus persica), elicited by methyl jasmonate (MJ), are reported here for the first time to prepare a natural dye for dye-sensitized solar cells (DSSC). Redox properties of the dye, its photostability, and light absorption properties suggested it as a candidate as natural photosensitizers for TiO2 photoelectrodes. Redox properties of the dye influence the DSSC production of photocurrent, thus three antioxidant assays were performed in order to characterize the antioxidant potential of this dye. The dye exhibited a high antioxidant activity in all the assays performed. Photostability assay revealed that the dye was quite stable to light. The power conversion efficiency that we obtained (0.53%) was comparable to the data by other authors with anthocyanins-based dyes from in vivo grown plants. Finally, we compared the dye with the partially purified one as photosensitizer in DSSC. The results indicated that the raw pigment from in vitro shoot cultures of P. salicina × P. persica elicited with MJ can be proposed without the needing of any other chemicals, thermal or purification process, or pH adjustments, as a dye for natural sensitized solar cells.

Ethylene-associated phase change from juvenile to mature phenotype of daylily (Hemerocallis) in vitro

NASA Technical Reports Server (NTRS)

Smith, D. L.; Kelly, K.; Krikorian, A. D.

1989-01-01

Hemerocallis plantlets maintained in vitro for extended periods of time in tightly closed culture vessels frequently show a phenotype, albeit on a miniaturized scale, typical of more mature, field-grown plants. The positive relationship of elevated ethylene in the headspace of such vessels to the phase shift from juvenile to mature form is established. Rigorous restriction in air exchange with the external environment by means of silicone grease seals hastens the phase change and improves uniformity of response. Although some plantlets may take longer to accumulate enough ethylene in sealed jars to undergo change, added ethylene and ethylene-releasing agents promote it. Ethylene adsorbants (e.g. mercuric perchlorate) block the shift of juvenile to mature form. Critical ambient ethylene level for the shift is ca 1 microliter l-1. Levels up to 1000 microliters l-1 do not hasten the response but are not toxic. The phase change is fully reversible when air exchange permits ethylene to drop below 1 microliter l-1. At least 1 microliter l-1 ethylene is required to sustain the mature phenotype. The ethylene synthesis inhibitor aminoethoxyvinylglycine (AVG) prevents the phase change, while the ethylene biosynthesis intermediate 1-aminocyclopropanecarboxylic acid (ACC) improves it. KOH, as a CO2 absorbent, does not prevent the phase change. Histology sections demonstrate subtle changes in the form of shoot tips of plantlets undergoing phase change.

Comparative Assessment of Response to Cadmium in Heavy Metal-Tolerant Shrubs Cultured In Vitro.

PubMed

Wiszniewska, A; Hanus-Fajerska, E; Muszyńska, E; Smoleń, S

2017-01-01

Two species of Pb-adapted shrubs, Alyssum montanum and Daphne jasminea , were evaluated in vitro for their tolerance to elevated concentrations of cadmium. Shoot cultures were treated with 0.5, 2.5, and 5.0 μM CdCl 2 for 16 weeks and analyzed for their organogenic response, biomass accretion, pigment content, and macronutrient status. Cadmium accumulation and its root-to-shoot translocation were also determined. In both species, rooted microplantlets, suitable for acclimatization, were obtained in the presence of Cd applied as selection agent. In A. montanum , low and moderate dose of Cd stimulated multiplication, rooting, and biomass production. Growth tolerance index (GTI) in Cd-treated shoots ranged from 120 to 215%, while in the roots 51-202%. In turn, in Cd-treated D. jasminea proliferation and rooting were inhibited, and GTI for shoots decreased with increasing doses of Cd. However, roots exposed to Cd had higher biomass accretion. Both species accumulated Cd in developed organs, and its content increased with increasing CdCl 2 dose. Interestingly, D. jasminea accumulated higher amounts of Cd in the roots than A. montanum and immobilized this metal in the root system. On the contrary, A. montanum translocated some part of accumulated Cd to the shoots, but with low efficiency. In the presence of Cd, A. montanum maintained macronutrient homeostasis and synthesized higher amounts of phytosynthetic pigments in the shoots. D. jasminea accumulated root biomass, immobilized Cd, and restricted its translocation at the expense of nutrient balance. Considering remediation potential, A. montanum could be exploited in phytoextraction, while D. jasminea in phytostabilization of polluted substrate.

Turning Your Life Around: Tips from an Ex-Juvenile Delinquent

ERIC Educational Resources Information Center

Brown, Waln K.

2005-01-01

In this article, a former troubled youth who is now an expert on resilience shares his experience and provides youth a guided exercise in turning their own lives around. The ex-juvenile delinquent stresses the importance of setting goals claiming that goals are things to shoot for, like getting your chores done or saving your money to buy a car.…

Effect of drought on sorbitol and sucrose metabolism in sinks and sources of peach

Treesearch

Riccardo Lo Bianco; Mark Rieger; Shi-Jean S. Sung

2000-01-01

In peach (Prunus persica [L.] Batsch.), sorbitol and sucrose are the two main forms of photosynthetic and translocated carbon and may have different functions depending on the organ of utilization and its developmental stage. The role and interaction of sorbitol and sucrose metabolism was studied in mature leaves (source) and shoot tips (sinks) of...

Fluoride toxicity effects in onion (Allium cepa L.) grown in contaminated soils.

PubMed

Jha, S K; Nayak, A K; Sharma, Y K

2009-07-01

A pot experiment was carried out under controlled condition to investigate the accumulation, uptake and toxicity effects of fluoride (F) in onion (Allium cepa L.) grown on the soil contaminated by inorganic fluoride (NaF). Six different levels of soil contamination were used by adding 0, 100, 200, 400, 600 and 800 mg NaFkg(-1) to the soil. The F concentration in shoot, bulb and root varied between 16.3 and 109.1mg Fkg(-1), 15.8 and 54.3mg Fkg(-1) and 18.6 and 151.6 mg Fkg(-1), respectively. The visible symptoms of F toxicity in terms of tip burning and death of the plant was noticed in highly contaminated soils (>400mg NaFkg(-1) soil). The phyto-toxic threshold limit (LC(50)) in onion shoot was found to be 55 mg Fkg(-1), beyond which the biomass yield decreased by 50%. It was also inferred from the study that there is a partitioning of F in onion, with more accumulation in roots and shoots than in bulbs. The order of retention of fluoride in onion found to be roots>shoot>bulb.

Effect of simulated climate warming on the morphological and physiological traits of Elsholtzia haichowensis in copper contaminated soil.

PubMed

Guan, Ming; Jin, Zexin; Li, Junmin; Pan, Xiaocui; Wang, Suizi; Li, Yuelin

2016-01-01

The aim of this study was to investigate the effects of temperature and Cu on the morphological and physiological traits of Elsholtzia haichowensis grown in soils amended with four Cu concentrations (0, 50, 500, and 1000 mg kg(-1)) under ambient temperature and slight warming. At the same Cu concentration, the height, shoot dry weight, total plant dry weight, and root morphological parameters such as length, surface area and tip number of E. haichowensis increased due to the slight warming. The net photosynthetic rate, stomatal conductance, transpiration, light use efficiency were also higher under the slight warming than under ambient temperature. The increased Cu concentrations, total Cu uptake, bioaccumulation factors and tolerance indexes of shoots and roots were also observed at the slight warming. The shoot dry weight, root dry weight, total plant dry weight and the bioaccumulation factors of shoots and roots at 50 mg Cu kg(-1) were significantly higher than those at 500 and 1000 mg Cu kg(-1) under the slight warming. Therefore, the climate warming may improve the ability of E. haichowensis to phytoremediate Cu-contaminated soil, and the ability improvement greatly depended on the Cu concentrations in soils.

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.

Production of Pharmaceuticals from Papaver Cultivars In Vitro

USDA-ARS?s Scientific Manuscript database

A methodology to clonally proliferate Iranian poppy (Papaver bracteatum Lindl.) and opium poppy (P. somniferum L.) shoots is presented employing an in vitro hydroponics system (i.e., automated plant culture system (APCS)). Temperature had a profound effect on growth and alkaloid production after 8-...

Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting.

PubMed

Jakobsson, Lars; Franco, Claudio A; Bentley, Katie; Collins, Russell T; Ponsioen, Bas; Aspalter, Irene M; Rosewell, Ian; Busse, Marta; Thurston, Gavin; Medvinsky, Alexander; Schulte-Merker, Stefan; Gerhardt, Holger

2010-10-01

Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection. We find that endothelial cells compete for the tip cell position through relative levels of Vegfr1 and Vegfr2, demonstrating a biological role for differential Vegfr regulation in individual endothelial cells. Differential Vegfr levels affect tip selection only in the presence of a functional Notch system by modulating the expression of the ligand Dll4. Time-lapse microscopy imaging of mosaic sprouts identifies dynamic position shuffling of tip and stalk cells in vitro and in vivo, indicating that the VEGFR-Dll4-Notch signalling circuit is constantly re-evaluated as cells meet new neighbours. The regular exchange of the leading tip cell raises novel implications for the concept of guided angiogenic sprouting.

AUTOMOTIVE DIESEL MAINTENANCE 1. UNIT II, MAINTAINING THE AIR SYSTEM--DETROIT DIESEL ENGINES.

ERIC Educational Resources Information Center

Human Engineering Inst., Cleveland, OH.

THIS MODULE OF A 30-MODULE COURSE IS DESIGNED TO DEVELOP AN UNDERSTANDING OF THE OPERATION AND MAINTENANCE OF THE DIESEL ENGINE AIR SYSTEM. TOPICS ARE (1) OPERATION AND FUNCTION, (2) AIR CLEANER, (3) AIR SHUT-DOWN HOUSING, (4) EXHAUST SYSTEM, (5) BLOWER, (6) TURBOCHARGER, AND (7) TROUBLE-SHOOTING TIPS ON THE AIR SYSTEM. THE MODULE CONSISTS OF A…

Micropropagation of apple--a review.

PubMed

Dobránszki, Judit; da Silva, Jaime A Teixeira

2010-01-01

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.

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

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.

Effect of different in vitro culture extracts of black pepper (Piper nigrum L.) on toxic metabolites-producing strains.

PubMed

Ahmad, Nisar; Abbasi, Bilal Haider; Fazal, Hina

2016-03-01

In the present study, the effect of different in vitro cultures (callus, in vitro shoots) and commercially available peppercorn extract was investigated for its activity against toxic metabolite-producing strains (Escherichia coli, Pseudomonas aeroginosa, Salmonella typhi, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Candida albicans). These in vitro cultures were extracted with ethanol, hexane, and chloroform, and the antipathogenic activity was determined by well-diffusion method. Hexane extract of callus showed 22 mm zone of inhibition against B. cereus, 23 mm against S. aureus, while regenerated shoots and seeds have shown 24.3 and 26 mm zones of inhibition. The ethanolic extracts of regenerated Piper shoots have shown 25 mm activity against S. aureus, 21 mm against B. cereus, and 16 mm in the case of C. albicans in comparison with standard antibiotics. Peppercorn extracts in chloroform and ethanol had shown activities against B. cereus (23.6 mm) and B. subtilis (23.5 mm). During in vitro organogenesis and morphogenesis, cells and tissues produced a comparable phytochemicals profile like mother plant. Morphogenesis is critically controlled by the application of exogenous plant-growth regulators. Such addition alters the hormonal transduction pathways, and cells under in vitro conditions regenerate tissues, which are dependant on the physiological state of cells, and finally enhance the production of secondary metabolites. To the best of our knowledge, this is the first report to compare the antimicrobial potential of in vitro regenerated tissues and peppercorn with standard antibiotics. In conclusion, most of the extracts showed pronounced activities against all the pathogenic microbes. This is a preliminary work, and the minimum inhibitory concentration values needs to be further explored. Regenerated tissues of P. nigrum are a good source of biologically active metabolites for antimicrobial activities, and callus culture presented itself as a good candidate for such activities. © The Author(s) 2013.

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.

Rapid in vitro shoot multiplication of the recalcitrant species Juglans nigra L.

Treesearch

Micah E. Stevens; Paula M. Pijut

2018-01-01

Black walnut (Juglans nigra L.) has long been prized for its timber, leading to commercial cultivation and significant breeding efforts for improving marketable traits. Vegetative and in vitro black walnut propagation techniques, however, are variable and highly genotype dependent. Optimizing plant growth regulator type and...

Production of Brugmansia plants free of Colombian datura virus by in vitro ribavirin chemotherapy

USDA-ARS?s Scientific Manuscript database

Brugmansia x candida Pers ‘Creamsickle’ plants produced by in vitro treatment with ribavirin, and no thermal therapy, remained polymerase chain reaction (PCR-) negative for Columbian datura virus (CDV) after one year. The plants were produced by establishing B. x candida ‘Creamsickle’ shoot cultures...

Screening larch in vitro for resistance to Mycosphaerella laricina

Treesearch

M.E. Ostry; Paula M. Pijut; D.D. Skilling

1991-01-01

Needle blight of larch caused by Mycosphaerella laricina seriously limits the productivity of susceptible trees in the north central and northeastern United States. Adventitious shoots, derived from cotyledon tissue culture, of selected European larch (Larix decidua) and a hybrid larch were inoculated in vitro with three isolates...

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.

Inactivation of Plasma Membrane–Localized CDPK-RELATED KINASE5 Decelerates PIN2 Exocytosis and Root Gravitropic Response in Arabidopsis[C][W

PubMed Central

Rigó, Gábor; Ayaydin, Ferhan; Tietz, Olaf; Zsigmond, Laura; Kovács, Hajnalka; Páy, Anikó; Salchert, Klaus; Darula, Zsuzsanna; Medzihradszky, Katalin F.; Szabados, László; Palme, Klaus; Koncz, Csaba; Cséplő, Ágnes

2013-01-01

CRK5 is a member of the Arabidopsis thaliana Ca2+/calmodulin-dependent kinase-related kinase family. Here, we show that inactivation of CRK5 inhibits primary root elongation and delays gravitropic bending of shoots and roots. Reduced activity of the auxin-induced DR5–green fluorescent protein reporter suggests that auxin is depleted from crk5 root tips. However, no tip collapse is observed and the transcription of genes for auxin biosynthesis, AUXIN TRANSPORTER/AUXIN TRANSPORTER-LIKE PROTEIN (AUX/LAX) auxin influx, and PIN-FORMED (PIN) efflux carriers is unaffected by the crk5 mutation. Whereas AUX1, PIN1, PIN3, PIN4, and PIN7 display normal localization, PIN2 is depleted from apical membranes of epidermal cells and shows basal to apical relocalization in the cortex of the crk5 root transition zone. This, together with an increase in the number of crk5 lateral root primordia, suggests facilitated auxin efflux through the cortex toward the elongation zone. CRK5 is a plasma membrane–associated kinase that forms U-shaped patterns facing outer lateral walls of epidermis and cortex cells. Brefeldin inhibition of exocytosis stimulates CRK5 internalization into brefeldin bodies. CRK5 phosphorylates the hydrophilic loop of PIN2 in vitro, and PIN2 shows accelerated accumulation in brefeldin bodies in the crk5 mutant. Delayed gravitropic response of the crk5 mutant thus likely reflects defective phosphorylation of PIN2 and deceleration of its brefeldin-sensitive membrane recycling. PMID:23673979

NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis

PubMed Central

Wang, Yuqi; Li, Ruihong; Li, Demou; Jia, Xiaomin; Zhou, Dangwei; Li, Jianyong; Lyi, Sangbom M.; Hou, Siyu; Huang, Yulan

2017-01-01

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. PMID:28439024

From laboratory to field studies - The assessment of Biscutella laevigata suitability to biological reclamation of areas contaminated with lead and cadmium.

PubMed

Muszyńska, Ewa; Hanus-Fajerska, Ewa; Piwowarczyk, Barbara; Augustynowicz, Joanna; Ciarkowska, Krystyna; Czech, Tomasz

2017-08-01

The aim of the work was to evaluate the usefulness of the in vitro multiplication of Biscutella laevigata calamine ecotype for in situ reclamation of post-flotation wastes polluted with Pb and Cd. The experiment was conducted on three steps: (i) plant shoots' production under in vitro condition, (ii) establishment of the material in greenhouse experiment, and finally (iii) field cultivation directly on the mining-waste heap of Olkusz Ore-Bearing Region, Poland. This region is known to be one of the most chemically-degraded area in central Europe. The laboratory-set in vitro analysis enabled to obtain the high-quality plant shoots, which multiply the most effectively (with growth tolerance index 130-150%) on medium containing 5.0μM CdCl 2 and 0.5mM Pb(NO 3 ) 2 . These plants were used for the next two ex vitro experiments. Several biometric and physiological analysis (i.e. of photosystem II activity F v /F m and PI, photosynthetic pigment contents) were done to indicate plant physiological status during these experiments. The main novelty of the work was to prove that in vitro-multiplied shoots of B. laevigata - the representative of native flora from Olkusz Ore-Bearing Region - can be successfully implemented in situ for the restoration of these degraded area. Moreover, the addition of sewage sludge as a source of organic compounds significantly improved plants' growth and development what is especially important due to the lack of other legal solutions for the management of the sewage sludge in some countries. Copyright © 2017 Elsevier Inc. All rights reserved.

Effect of a longitudinally applied voltage upon the growth of Zea mays seedlings

NASA Technical Reports Server (NTRS)

Desrosiers, M. F.; Bandurski, R. S.

1988-01-01

The electrical parameters that affect young seedling growth were investigated. Voltages ranging from 5 to 40 volts were applied longitudinally along the mesocotyl region of 4-day old Zea mays L. (cv Silver Queen) seedlings for periods of 3 or 4 hours. It was determined that: (a) making the tips of the seedlings electrically positive relative to the base strongly inhibited shoot growth at 5 volts, whereas the reverse polarity had no effect; (b) at higher voltages, making the tip of the seedlings negative caused less growth inhibition than the reverse polarity at each voltage level; (c) the higher the applied voltage the greater the degree of inhibition; and, (d) the more growth inhibition experienced by the plants the poorer, and slower, their recovery. Previous observations of a relationship between the amount of free indole-3-acetic acid in the mesocotyl cortex and the growth rate of the mesocotyl and of gravitropism-induced movement of labeled indole-3-acetic acid from the seed to the shoot lead to the prediction of a voltage-dependent gating of the movement of indole-3-acetic acid from the stele to the cortex. This provided the basis for attempting to alter the growth rate of seedlings by means of an applied voltage.

Effect of a longitudinally applied voltage upon the growth of Zea mays seedlings.

PubMed

Desrosiers, M F; Bandurski, R S

1988-01-01

The electrical parameters that affect young seedling growth were investigated. Voltages ranging from 5 to 40 volts were applied longitudinally along the mesocotyl region of 4-day old Zea mays L. (cv Silver Queen) seedlings for periods of 3 or 4 hours. It was determined that: (a) making the tips of the seedlings electrically positive relative to the base strongly inhibited shoot growth at 5 volts, whereas the reverse polarity had no effect; (b) at higher voltages, making the tip of the seedlings negative caused less growth inhibition than the reverse polarity at each voltage level; (c) the higher the applied voltage the greater the degree of inhibition; and, (d) the more growth inhibition experienced by the plants the poorer, and slower, their recovery. Previous observations of a relationship between the amount of free indole-3-acetic acid in the mesocotyl cortex and the growth rate of the mesocotyl and of gravitropism-induced movement of labeled indole-3-acetic acid from the seed to the shoot lead to the prediction of a voltage-dependent gating of the movement of indole-3-acetic acid from the stele to the cortex. This provided the basis for attempting to alter the growth rate of seedlings by means of an applied voltage.

Effect of a Longitudinally Applied Voltage Upon the Growth of Zea mays Seedlings 1

PubMed Central

Desrosiers, Mark F.; Bandurski, Robert S.

1988-01-01

The electrical parameters that affect young seedling growth were investigated. Voltages ranging from 5 to 40 volts were applied longitudinally along the mesocotyl region of 4-day old Zea mays L. (cv Silver Queen) seedlings for periods of 3 or 4 hours. It was determined that: (a) making the tips of the seedlings electrically positive relative to the base strongly inhibited shoot growth at 5 volts, whereas the reverse polarity had no effect; (b) at higher voltages, making the tip of the seedlings negative caused less growth inhibition than the reverse polarity at each voltage level; (c) the higher the applied voltage the greater the degree of inhibition; and, (d) the more growth inhibition experienced by the plants the poorer, and slower, their recovery. Previous observations of a relationship between the amount of free indole-3-acetic acid in the mesocotyl cortex and the growth rate of the mesocotyl and of gravitropism-induced movement of labeled indole-3-acetic acid from the seed to the shoot lead to the prediction of a voltage-dependent gating of the movement of indole-3-acetic acid from the stele to the cortex. This provided the basis for attempting to alter the growth rate of seedlings by means of an applied voltage. Images Fig. 1 PMID:11537877

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures

PubMed Central

Wesley-Smith, James; Berjak, Patricia; Pammenter, N. W.; Walters, Christina

2014-01-01

Background and Aims Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Methods Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Key Results Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm2 in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. Conclusions The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches. PMID:24368198

Intracellular ice and cell survival in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum: an ultrastructural study of factors affecting cell and ice structures.

PubMed

Wesley-Smith, James; Berjak, Patricia; Pammenter, N W; Walters, Christina

2014-03-01

Cryopreservation is the only long-term conservation strategy available for germplasm of recalcitrant-seeded species. Efforts to cryopreserve this form of germplasm are hampered by potentially lethal intracellular freezing events; thus, it is important to understand the relationships among cryo-exposure techniques, water content, structure and survival. Undried embryonic axes of Acer saccharinum and those rapidly dried to two different water contents were cooled at three rates and re-warmed at two rates. Ultrastructural observations were carried out on radicle and shoot tips prepared by freeze-fracture and freeze-substitution to assess immediate (i.e. pre-thaw) responses to cooling treatments. Survival of axes was assessed in vitro. Intracellular ice formation was not necessarily lethal. Embryo cells survived when crystal diameter was between 0·2 and 0·4 µm and fewer than 20 crystals were distributed per μm(2) in the cytoplasm. Ice was not uniformly distributed within the cells. In fully hydrated axes cooled at an intermediate rate, the interiors of many organelles were apparently ice-free; this may have prevented the disruption of vital intracellular machinery. Intracytoplasmic ice formation did not apparently impact the integrity of the plasmalemma. The maximum number of ice crystals was far greater in shoot apices, which were more sensitive than radicles to cryo-exposure. The findings challenge the accepted paradigm that intracellular ice formation is always lethal, as the results show that cells can survive intracellular ice if crystals are small and localized in the cytoplasm. Further understanding of the interactions among water content, cooling rate, cell structure and ice structure is required to optimize cryopreservation treatments without undue reliance on empirical approaches.

Human embryonic lung epithelial tips are multipotent progenitors that can be expanded in vitro as long-term self-renewing organoids

PubMed Central

Nikolić, Marko Z; Caritg, Oriol; Jeng, Quitz; Johnson, Jo-Anne; Sun, Dawei; Howell, Kate J; Brady, Jane L; Laresgoiti, Usua; Allen, George; Butler, Richard; Zilbauer, Matthias; Giangreco, Adam; Rawlins, Emma L

2017-01-01

The embryonic mouse lung is a widely used substitute for human lung development. For example, attempts to differentiate human pluripotent stem cells to lung epithelium rely on passing through progenitor states that have only been described in mouse. The tip epithelium of the branching mouse lung is a multipotent progenitor pool that self-renews and produces differentiating descendants. We hypothesized that the human distal tip epithelium is an analogous progenitor population and tested this by examining morphology, gene expression and in vitro self-renewal and differentiation capacity of human tips. These experiments confirm that human and mouse tips are analogous and identify signalling pathways that are sufficient for long-term self-renewal of human tips as differentiation-competent organoids. Moreover, we identify mouse-human differences, including markers that define progenitor states and signalling requirements for long-term self-renewal. Our organoid system provides a genetically-tractable tool that will allow these human-specific features of lung development to be investigated. DOI: http://dx.doi.org/10.7554/eLife.26575.001 PMID:28665271

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

Tonoplast-Bound Protein Kinase Phosphorylates Tonoplast Intrinsic Protein 1

PubMed Central

Johnson, Kenneth D.; Chrispeels, Maarten J.

1992-01-01

Tonoplast intrinsic protein (TIP) is a member of a family of putative membrane channels found in bacteria, animals, and plants. Plants have seed-specific, vegetative/reproductive organ-specific, and water-stress-induced forms of TIP. Here, we report that the seed-specific TIP is a phosphoprotein whose phosphorylation can be monitored in vivo by allowing bean cotyledons to take up [32P]orthophosphate and in vitro by incubating purified tonoplasts with γ-labeled [32P]ATP. Characterization of the in vitro phosphorylation of TIP indicates that a membrane-bound protein kinase phosphorylates TIP in a Ca2+-dependent manner. The capacity of the isolated tonoplast membranes to phosphorylate TIP declined markedly during seed germination, and this decline occurred well before the development-mediated decrease in TIP occurs. Phosphoamino acid analysis of purified, radiolabeled TIP showed that serine is the major, if not only, phosphorylated residue, and cyanogen bromide cleavage yielded a single radioactive peptide peak on a reverse-phase high-performance liquid chromatogram. Estimation of the molecular mass of the cyanogen bromide phosphopeptide by laser desorption mass spectroscopy led to its identification as the hydrophilic N-terminal domain of TIP. The putative phosphate-accepting serine residue occurs in a consensus phosphorylation site for serine/threonine protein kinases. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:16653198

Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots.

PubMed

Wang, Shengyin; Ren, Xiaoyan; Huang, Bingru; Wang, Ge; Zhou, Peng; An, Yuan

2016-07-20

The objective of this study was to investigate Al(3+)-induced IAA transport, distribution, and the relation of these two processes to Al(3+)-inhibition of root growth in alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or 100 μM AlCl3 and were foliar sprayed with water or 6 mg L(-1) IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell shapes, altered cell structure, and a shorter length of the meristematic zone in root tips. Aluminium stress significantly decreased the IAA concentration in apical buds and root tips. The distribution of IAA fluorescence signals in root tips was disturbed, and the IAA transportation from shoot base to root tip was inhibited. The highest intensity of fluorescence signals was detected in the apical meristematic zone. Exogenous application of IAA markedly alleviated the Al(3+)-induced inhibition of root growth by increasing IAA accumulation and recovering the damaged cell structure in root tips. In addition, Al(3+) stress up-regulated expression of AUX1 and PIN2 genes. These results indicate that Al(3+)-induced reduction of root growth could be associated with the inhibitions of IAA synthesis in apical buds and IAA transportation in roots, as well as the imbalance of IAA distribution in root tips.

Aluminium-induced reduction of plant growth in alfalfa (Medicago sativa) is mediated by interrupting auxin transport and accumulation in roots

PubMed Central

Wang, Shengyin; Ren, Xiaoyan; Huang, Bingru; Wang, Ge; Zhou, Peng; An, Yuan

2016-01-01

The objective of this study was to investigate Al3+-induced IAA transport, distribution, and the relation of these two processes to Al3+-inhibition of root growth in alfalfa. Alfalfa seedlings with or without apical buds were exposed to 0 or 100 μM AlCl3 and were foliar sprayed with water or 6 mg L−1 IAA. Aluminium stress resulted in disordered arrangement of cells, deformed cell shapes, altered cell structure, and a shorter length of the meristematic zone in root tips. Aluminium stress significantly decreased the IAA concentration in apical buds and root tips. The distribution of IAA fluorescence signals in root tips was disturbed, and the IAA transportation from shoot base to root tip was inhibited. The highest intensity of fluorescence signals was detected in the apical meristematic zone. Exogenous application of IAA markedly alleviated the Al3+-induced inhibition of root growth by increasing IAA accumulation and recovering the damaged cell structure in root tips. In addition, Al3+ stress up-regulated expression of AUX1 and PIN2 genes. These results indicate that Al3+-induced reduction of root growth could be associated with the inhibitions of IAA synthesis in apical buds and IAA transportation in roots, as well as the imbalance of IAA distribution in root tips. PMID:27435109

Effect of polyvinyl alcohol on in vitro rooting capacity of shoots in pear clones (Pyrus communis L.) of different ploidy

USDA-ARS?s Scientific Manuscript database

Poor adventitious root formation is a major obstacle in micropropagation. In this study, intense efforts have been made for improvement of rooting procedures for triploid, tetraploid, and mixploid clones of the pear cultivar, 'Fertility', obtained by in vitro colchicine treatment. An efficient roo...

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.

Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens.

PubMed

Cheel, José; Schmeda-Hirschmann, Guillermo; Jordan, Miguel; Theoduloz, Cristina; Rodríguez, Jaime A; Gerth, André; Wilken, Dirk

2007-01-01

An in vitro propagation system was developed to obtain shoot and root cultures from the Andean spice Sanicula graveolens (Apiaceae). Propagation of shoots, roots and plantlets was achieved by the temporary immersion system. The free radical scavenging effect of the methanol/water (7:3 v/v) extracts was determined by the discoloration of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH). Total phenolic, flavonoid, chlorogenic acid (CA) and quercetin 3-O-glucoside content in the samples was assessed by spectrophotometry and DAD-HPLC analysis, respectively. On a dry weight basis, the crude extracts showed total phenolic values ranging from 3.57 to 6.93%, with highest content for the root culture sample. Total flavonoid content ranged from 1.23 to 2.23% and was lower for the root culture. Chlorogenic acid and neochlorogenic acid were identified by TLC in all samples. Highest free radical scavenging effect was observed for the root culture which also presented the highest CA content. Two of the shoot culture samples, with similar IC50 values in the DPPH discoloration assay, also presented close quercetin-3-O-glucoside content.

Involvements of PCD and changes in gene expression profile during self-pruning of spring shoots in sweet orange (Citrus sinensis).

PubMed

Zhang, Jin-Zhi; Zhao, Kun; Ai, Xiao-Yan; Hu, Chun-Gen

2014-10-13

Citrus shoot tips abscise at an anatomically distinct abscission zone (AZ) that separates the top part of the shoots into basal and apical portions (citrus self-pruning). Cell separation occurs only at the AZ, which suggests its cells have distinctive molecular regulation. Although several studies have looked into the morphological aspects of self-pruning process, the underlying molecular mechanisms remain unknown. In this study, the hallmarks of programmed cell death (PCD) were identified by TUNEL experiments, transmission electron microscopy (TEM) and histochemical staining for reactive oxygen species (ROS) during self-pruning of the spring shoots in sweet orange. Our results indicated that PCD occurred systematically and progressively and may play an important role in the control of self-pruning of citrus. Microarray analysis was used to examine transcriptome changes at three stages of self-pruning, and 1,378 differentially expressed genes were identified. Some genes were related to PCD, while others were associated with cell wall biosynthesis or metabolism. These results strongly suggest that abscission layers activate both catabolic and anabolic wall modification pathways during the self-pruning process. In addition, a strong correlation was observed between self-pruning and the expression of hormone-related genes. Self-pruning plays an important role in citrus floral bud initiation. Therefore, several key flowering homologs of Arabidopsis and tomato shoot apical meristem (SAM) activity genes were investigated in sweet orange by real-time PCR and in situ hybridization, and the results indicated that these genes were preferentially expressed in SAM as well as axillary meristem. Based on these findings, a model for sweet orange spring shoot self-pruning is proposed, which will enable us to better understand the mechanism of self-pruning and abscission.

Effect of red and blue light emitting diodes "CRB-LED" on in vitro organogenesis of date palm (Phoenix dactylifera L.) cv. Alshakr.

PubMed

Al-Mayahi, Ahmed Madi Waheed

2016-10-01

The objective of the present study is to determine the effect of light source on enhancement of shoot multiplication, phytochemicals, as well as, antioxidant enzyme activities of in vitro cultures of date palm cv. Alshakr. In vitro-grown buds were cultured on Murashige and Skoog (MS) medium and incubated under a conventional white fluorescent light (control), and combinations of red + blue light emitting diode (18:2) (CRB-LED). Results revealed that the treatment of CRB-LED showed a significant increase in the number of shoots compared with the white florescent light. Total soluble carbohydrate "TSCH" (7.10 mg g(-1) DW.), starch (1.63 mg g(-1) DW.) and free amino acids (2.90 mg g(-1) DW.) were significantly higher in CRB-LED (p < 0.05). Additionally, CRB-LED induced a higher peroxidase activity (25.50 U ml(-1)) compared with the white fluorescent light treatment (19.74 U ml(-1)) as control treatment. Potassium, magnesium and sodium contents in (3.62, 13.99 and 2.76 mg g(-1) DW.) were increased in in vitro shoots under CRB-LED treatment in comparison with fluorescent light (p < 0.05). Protein profile showed the appearance of newly bands with the molecular weight of 38 and 60 kDa at the treatment CRB-LED compared with control treatment. Our results demonstrate the positive effects of CRB-LED light during the course of date palm tissue cultures.

The influence of different hormone concentration and combination on callus induction and regeneration of Rauwolfia serpentina L. Benth.

PubMed

Salma, U; Rahman, M S M; Islam, S; Haque, N; Jubair, T A; Haque, A K M F; Mukti, I J

2008-06-15

The influence of media composition on callus induction and subsequent regeneration of Rauwolfia serpentina L. Benth has been studied. High frequency (96.43%) callus induction was obtained when nodal segments from in vitro raised shoots were cultured on MS medium supplemented with 0.5 mg L(-1) BA and 2.0 mg L(-1) NAA. The callus differentiated into adventitious shoots when it was subcultured on MS medium supplemented with 2.0 mg L(-1) BA with 0.2 mg L(-1) NAA. Regenerated shoots were best rooted on half-strength MS medium with 1.0 mg L(-1) each of IBA and IAA.

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.

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.

In vitro propagation of Cichorium intybus L. and quantification of enhanced secondary metabolite (esculin).

PubMed

Rafsanjani, Mehrnaz S Ohadi; Alvari, Amene; Mohammad, Anis; Abdin, M Z; Hejazi, M A

2011-12-01

In this report, rapid and effective shoot as well as root regeneration system through direct multiplication was successfully developed for Cichorium intybus L. Furthermore, the effect of exogenous growth regulators (TDZ and IAA) at different concentrations on the regulation process of the plant was also studied. Enhanced production of esculin in developed C. intybus L. was evaluated using leaf extract. Only on the expense of 20 days, regeneration was seen and very low dose of TDZ was seen to be more effective. When 0.02 mg/L of TDZ was combined with 1.5mg/L of IAA, nearly 100% of explants produced shoots with the highest number of regenerated shoots (85.37). With further increase in concentration (≥ 0.05 mg/L), the number of shoots per explants get decreased. A lower NAA to IBA ratio (1.0mg/L of IBA and 0.5mg/L of NAA) seemed to be more effective for root generation and considered to be the most effective combination among the tried groups. IBA was more effective in root development than NAA, but both were comparatively effective. On quantitative analysis by RP-HPLC, the 76.23% of Esculin were found in leaf extract of the in vitro developed C. intybus L. This amount was 26.77% higher than normal grown plants.

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.

National Academy of Sciences: Helping Scientists Navigate & Troubleshoot Visa Issues

NASA Astrophysics Data System (ADS)

Bailey, Kathie

2014-03-01

The International Visitors Office (IVO) is a program operated by the Board on International Scientific Organizations of the National Academy of Sciences. The IVO serves as a resource on visa-related issues for scientists and students traveling to the United States for professional activities. The speaker will address visa issues for international scientists wishing to visit the United States, tips for trouble-shooting visa issues, and statistics on the current visa system.

Impact of the arundo wasp, Tetramesa romana (Hymenoptera: Eurytomidae) on biomass of the invasive weed, Arundo donax (Poaceae: Arundinoideae) and on revegetation of riparian habitat along the Rio Grande in Texas

USDA-ARS?s Scientific Manuscript database

An invasive grass, Arundo donax, occupies thousands of hectares of arid riparian habitat along the Rio Grande and was the first perennial grass to be targeted with biological control, due to the great negative impacts of this weed on water resources and riparian ecosystems. The shoot-tip galling was...

Comparative in vitro culture of white and green ash from seed to plantlet production

Treesearch

J. W. Van Sambeek; John E. Preece; Nadia E. Navarrete-Tindall

2002-01-01

In vitro procedures have already been reported for white ash (Fraxinus americana L.) to establish cut dormant seeds, force axillary shoot proliferation, and induce rapid rooting to produce clonal plantlets (Preece et al., 1987, Navarrete et al., 1989, Preece et al., 1989, Preece et al., 1995). Hypothetically, a production cycle from seed to...

Zinc and copper tolerance of Agrostis stolonifera L. in tissue culture

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

Wu, L.; Antonovics, J.

1978-03-01

Callus tissue was induced from shoot meristematic tissue and root tips of a clone of the grass Agrostis stolonifera tolerant to both zinc and copper, and from a control clone tolerant to neither metal. Growth of the callus tissue on media containing zinc and copper showed that tolerance to both metals was maintained in tissue culture. The pattern of metal uptake in tissue culture resembled uptake by whole plants in that tolerant tissue took up more metal than nontolerant tissue. Plants regenerated from callus had the same copper and zinc tolerance as the original parental clones regardless of time ofmore » growth in tissue culture and shoot or root origin of the tissue. The results support previous evidence that metal tolerance is genetically determined and acts at the cellular level.« less

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.

Defining toxicological tipping points in neuronal network development.

PubMed

Frank, Christopher L; Brown, Jasmine P; Wallace, Kathleen; Wambaugh, John F; Shah, Imran; Shafer, Timothy J

2018-02-02

Measuring electrical activity of neural networks by microelectrode array (MEA) has recently shown promise for screening level assessments of chemical toxicity on network development and function. Important aspects of interneuronal communication can be quantified from a single MEA recording, including individual firing rates, coordinated bursting, and measures of network synchrony, providing rich datasets to evaluate chemical effects. Further, multiple recordings can be made from the same network, including during the formation of these networks in vitro. The ability to perform multiple recording sessions over the in vitro development of network activity may provide further insight into developmental effects of neurotoxicants. In the current study, a recently described MEA-based screen of 86 compounds in primary rat cortical cultures over 12 days in vitro was revisited to establish a framework that integrates all available primary measures of electrical activity from MEA recordings into a composite metric for deviation from normal activity (total scalar perturbation). Examining scalar perturbations over time and increasing concentration of compound allowed for definition of critical concentrations or "tipping points" at which the neural networks switched from recovery to non-recovery trajectories for 42 compounds. These tipping point concentrations occurred at predominantly lower concentrations than those causing overt cell viability loss or disrupting individual network parameters, suggesting tipping points may be a more sensitive measure of network functional loss. Comparing tipping points for six compounds with plasma concentrations known to cause developmental neurotoxicity in vivo demonstrated strong concordance and suggests there is potential for using tipping points for chemical prioritization. Published by Elsevier Inc.

Methyl jasmonate mediates upregulation of bacoside A production in shoot cultures of Bacopa monnieri.

PubMed

Sharma, Poojadevi; Yadav, Sheetal; Srivastava, Anshu; Shrivastava, Neeta

2013-07-01

Methyl jasmonate (MJ) enhances the production of a range of secondary metabolites including triterpenoid saponins in a variety of plant species. Here, it enhanced production of bacoside A, a valuable triterpenoid saponin having nootropic therapeutic activity in in vitro shoot cultures of Bacopa monnieri, the only known source of bacoside A. The highest yield was with 50 μM MJ giving 4.4 mg bacoside A/g dry wt; an 1.8-fold increase (compared to control) after 1 week.

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.

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.

Distribution and phenology of Dasineura oxycoccana (Diptera: Cecidomyiidae) in Michigan blueberries.

PubMed

Hahn, Noel G; Isaacs, Rufus

2012-06-01

The blueberry gall midge, Dasineura oxycoccana Johnson, is a serious pest of rabbiteye blueberries in Florida, Georgia, and Mississippi, and a potential pest of southern and northern highbush blueberries. Its damage has been observed with increasing frequency in highbush blueberry plantings in the Great Lakes region, including in Wisconsin and in Michigan. Unlike in rabbiteye blueberry plantings, where blueberry gall midge primarily damages flowering buds, it is found to damage only the vegetative shoots of northern highbush blueberry. In this study, farms throughout Michigan were surveyed for the presence of blueberry gall midge and it was found in 43 of 46 sampled farms in 11 counties. From 2009-2011, several monitoring techniques, including yellow sticky traps, emergence traps, observational sampling, and vegetative shoot dissections were used to determine the ecology of this species in blueberry fields in southwest Michigan. Emergence traps were most useful in early detection of blueberry gall midge in April, and observational sampling for damage symptoms and vegetative shoot dissections revealed multiple population peaks throughout July and August. Infestation was detected in vegetative shoot tips in all parts of the bushes, with initial infestation greatest at the base of bushes. Degree day accumulations until first midge detection and peak infestation suggest some potential for predicting key events in the pest's phenology. This information about the distribution and timing of infestation will be useful in developing management strategies for blueberry gall midge infestation.

Optimization of culture conditions (sucrose, pH, and photoperiod) for in vitro regeneration and early detection of somaclonal variation in ginger lime (Citrus assamensis).

PubMed

Yaacob, Jamilah Syafawati; Mahmad, Noraini; Mat Taha, Rosna; Mohamed, Normadiha; Mad Yussof, Anis Idayu; Saleh, Azani

2014-01-01

Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5-2.0 mg L(-1)) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mg L(-1) NAA and 2.0 mg L(-1) BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL(-1) sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.

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.

Variable Levels of Glutathione S-Transferases Are Responsible for the Differential Tolerance to Metolachlor between Maize (Zea mays) Shoots and Roots.

PubMed

Li, Dongzhi; Xu, Li; Pang, Sen; Liu, Zhiqian; Wang, Kai; Wang, Chengju

2017-01-11

Glutathione S-transferases (GSTs) play important roles in herbicide tolerance. However, studies on GST function in herbicide tolerance among plant tissues are still lacking. To explore the mechanism of metolachlor tolerance difference between maize shoots and roots, the effects of metolachlor on growth, GST activity, and the expression of the entire GST gene family were investigated. It was found that this differential tolerance to metolachlor was correlated with contrasting GST activity between the two tissues and can be eliminated by a GST inhibitor. An in vitro metolachlor-glutathione conjugation assay confirmed that the transformation of metolachlor is 2-fold faster in roots than in shoots. The expression analysis of the GST gene family revealed that most GST genes are expressed much higher in roots than shoots, both in control and in metolachlor-treated plants. Taken together, higher level expression of most GST genes, leading to higher GST activity and faster herbicide transformation, appears to be responsible for the higher tolerance to metolachlor of maize roots than shoots.

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.

The Nutritional Facts of Bamboo Shoots and Their Usage as Important Traditional Foods of Northeast India

PubMed Central

Nongdam, P.; Tikendra, Leimapokpam

2014-01-01

Bamboo shoots are considered as one of the useful health foods because of their rich contents of proteins, carbohydrates, vitamins, fibres, and minerals and very low fat. Though bamboo shoots provide lots of health benefits, their consumption is confined mostly to Southeast Asian and East Asian countries. The acceptability of bamboo shoots as popular vegetable crop is very less due to their high pungent smell and bitter acidic taste. The use of bamboo as food in India is mainly restricted to Northeastern part of the country where they form an indispensable part of several traditional speciality dishes. The different ethnic communities take fresh or fermented bamboo shoot as one of most preferred traditional food items. Some of the important bamboo based traditional foods are ushoi, soibum, rep, mesu, eup, ekhung, hirring, and so forth. Bamboo shoots should be properly processed before they are consumed as freshly harvested shoots have high content of toxic cyanogenic glycosides which may pose serious health problems. The prospect of bamboo shoot industry in Northeast India is bright due to its rich genetic resources of bamboos. However, habitat destruction and extensive use of bamboos for food, handicraft, and construction purposes have resulted in severe depletion of natural bamboo resources. This review stresses upon the high nutritive values and health benefits of bamboo shoots and their usage as important traditional foods in Northeast India. The bamboo market potential of the region and use of in vitro plant micropropagation methods as effective means of bamboo conservation are also emphasized in this paper. PMID:27433496

Mevalonate kinase activity during different stages of plant regeneration from nodular callus cultures in white pine (Pinus strobus).

PubMed

Tang, Wei; Newton, Ronald J

2006-02-01

Mevalonate kinase (MK) catalyzes a step in the isoprenoid biosynthetic pathway, which leads to a huge number of compounds that play important roles in plant growth and development. Here, we report on changes in MK activity in white pine (Pinus strobus L.) during plant regeneration by adventitious shoot organogenesis from cotyledons of mature embryos, including nodular callus induction, shoot formation and rooting. Nodular calli were induced from Pinus strobus (PS) embryos by culture in nodular callus induction medium in a 0-, 8- or 16-h photoperiod. Mevalonate kinase activity peaked in nodular calli after three weeks of culture on nodular callus induction medium in a 16-h photoperiod, whereas frequency of nodular callus formation peaked after 4 weeks of culture on nodular callus induction medium in darkness. During adventitious shoot formation, MK activity peaked in shoots derived from dark-grown nodular calli after 3 weeks on bud formation medium, and frequency of shoot formation was highest in dark-grown nodular calli cultured on bud formation medium for 4 weeks. During rooting, MK activity peaked 2 weeks after transfer of adventitious shoots to rooting medium and rooting frequency was highest in adventitious shoots after 3 weeks on rooting medium. Although during nodular callus induction in darkness MK activity was inversely related to frequency of nodular callus formation, MK activity was highly correlated with frequency of shoot formation and with rooting frequency. The observed increase in MK activity preceding rooting suggests that MK could serve as a marker for rooting of white pine shoots in vitro.

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 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.

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

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...

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.

CASIROZ: Root parameters and types of ectomycorrhiza of young beech plants exposed to different ozone and light regimes.

PubMed

Zeleznik, P; Hrenko, M; Then, C; Koch, N; Grebenc, T; Levanic, T; Kraigher, H

2007-03-01

Tropospheric ozone (O(3)) triggers physiological changes in leaves that affect carbon source strength leading to decreased carbon allocation below-ground, thus affecting roots and root symbionts. The effects of O(3) depend on the maturity-related physiological state of the plant, therefore adult and young forest trees might react differently. To test the applicability of young beech plants for studying the effects of O(3) on forest trees and forest stands, beech seedlings were planted in containers and exposed for two years in the Kranzberg forest FACOS experiment (Free-Air Canopy O(3) Exposure System, http://www.casiroz.de ) to enhanced ozone concentration regime (ambient [control] and double ambient concentration, not exceeding 150 ppb) under different light conditions (sun and shade). After two growing seasons the biomass of the above- and below-ground parts, beech roots (using WinRhizo programme), anatomical and molecular (ITS-RFLP and sequencing) identification of ectomycorrhizal types and nutrient concentrations were assessed. The mycorrhization of beech seedlings was very low ( CA. 5 % in shade, 10 % in sun-grown plants), no trends were observed in mycorrhization (%) due to ozone treatment. The number of Cenococcum geophilum type of ectomycorrhiza, as an indicator of stress in the forest stands, was not significantly different under different ozone treatments. It was predominantly occurring in sun-exposed plants, while its majority share was replaced by Genea hispidula in shade-grown plants. Different light regimes significantly influenced all parameters except shoot/root ratio and number of ectomycorrhizal types. In the ozone fumigated plants the number of types, number of root tips per length of 1 to 2 mm root diameter, root length density per volume of soil and concentration of Mg were significantly lower than in control plants. Trends to a decrease were found in root, shoot, leaf, and total dry weights, total number of root tips, number of vital mycorrhizal root tips, fine root (mass) density, root tip density per surface, root area index, concentration of Zn, and Ca/Al ratio. Due to the general reduction in root growth indices and nutrient cycling in ozone-fumigated plants, alterations in soil carbon pools could be predicted.

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.

In vitro grown thickened taproots, a new type of soil transplanting source in Panax ginseng.

PubMed

Kim, Jong Youn; Kim, Dong Hwi; Kim, Young Chang; Kim, Kee Hong; Han, Jung Yeon; Choi, Yong Eui

2016-10-01

The low survival rate of in vitro regenerated Panax ginseng plantlets after transfer to soil is the main obstacle for their successful micropropagation and molecular breeding. In most cases, young plantlets converted from somatic embryos are transferred to soil. In vitro thickened taproots, which were produced after prolonged culture of ginseng plantlets, were transferred to soil. Taproot thickening of plantlets occurred near hypocotyl and primary roots. Elevated concentration of sucrose in the medium stimulated the root thickening of plantlets. Senescence of shoots occurred following the prolonged culture of plantlets. Once the leaves of plantlets senesced, the buds on taproots developed a dormant tendency. Gibberellic acid treatment was required for dormancy breaking of the buds. Analysis of endogenous abscisic acid revealed that the content of abscisic acid in taproots with senescent shoots was comparatively higher than that of taproots with green shoots. Thickened taproots were transferred to soil, followed by exposure to gibberellic acid or a cold temperature of 2°C for 4 mo. Cold treatment of roots at 2°C for 4 mo resulted in bud sprouting in 84% of roots. Spraying of 100 mg/L gibberellic acid also induced the bud sprouting in 81% roots. Soil transfer of dormant taproots of P. ginseng has advantages since they do not require an acclimatization procedure, humidity control of plants, and photoautotrophic growth, and a high soil survival rate was attained.

Dealing with school shootings, violence: how Jonesboro and Denver hospitals met this new challenge to emergency preparedness.

PubMed

1999-08-01

Acts of violence at schools across the country committed by gun-wielding students in recent years have all too frequently, as we know by now, resulted in multiple casualties and widespread community grieving. Two of the shooting rampages noted in this report that attracted national and international media attention--one at West-side Middle School, Jonesboro, AR, on March 24, 1998, and the other at Columbine High School, Littleton, CO, on April 20, 1999--illustrate the importance of hospital preparedness and quick implementation of emergency disaster plans. In both instances, officials say their administrative, clinical, and security personnel were well prepared to handle the physical and emotional trauma caused by the tragedies. Meanwhile, a leading criminologist warns that the trend toward school violence likely will continue and provides tips for hospitals and their security directors.

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.

Localization of needle tip with color doppler during pericardiocentesis: In vitro validation and initial clinical application

NASA Technical Reports Server (NTRS)

Armstrong, G.; Cardon, L.; Vilkomerson, D.; Lipson, D.; Wong, J.; Rodriguez, L. L.; Thomas, J. D.; Griffin, B. P.

2001-01-01

This study evaluates a new device that uses color Doppler ultrasonography to enable real-time image guidance of the aspirating needle, which has not been possible until now. The ColorMark device (EchoCath Inc, Princeton, NJ) induces high-frequency, low-amplitude vibrations in the needle to enable localization with color Doppler. We studied this technique in 25 consecutive patients undergoing pericardiocentesis, and in vitro, in a urethane phantom with which the accuracy of color Doppler localization of the needle tip was compared with that obtained by direct measurement. Tip localization was excellent in vitro; errors axial to the ultrasound beam (velocity Doppler -0.13 +/- 0.90 mm, power Doppler -0.05 +/- 1.7 mm) were less than lateral errors (velocity -0.36 +/- 1.8 mm, power -0.02 +/- 2.8 mm). In 18 of 25 patients, the needle was identified and guided into the pericardial space with the ColorMark technique, and it allowed successful, uncomplicated drainage of fluid. Initial failures were the result of incorrect settings on the echocardiographic machine and inappropriate combinations of the needle puncture site and imaging window. This study demonstrates a novel color Doppler technique that is highly accurate at localizing a needle tip. The technique is feasible for guiding pericardiocentesis. Further clinical validation of this technique is required.

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.

Evaluating chemical safety: ToxCast, Tipping Points and Virtual Tissues (Tamburro Symposium)

EPA Science Inventory

This presentation provides an overview of high-throughput toxicology at the NCCT using high-content imaging and computational models for analyzing chemical safety. In In particular, this work outlines the derivation of toxicological "tipping points" from in vitro concentration- a...

Survival and genetic stability of Dendranthema grandiflora Tzvelev shoot apices after cryopreservation by vitrification and encapsulation-dehydration.

PubMed

Martín, Carmen; González-Benito, M Elena

2005-12-01

The aim of this study was to compare the genetic stability of chrysanthemum (cv. Pasodoble) apices cryopreserved using two different methods: encapsulation-dehydration and vitrification. The assessment of the genetic stability was developed using RAPDs markers. Assessment of stability was evaluated in pot-cultivated mother plants (from which buds were excised for micropropagation), in shoots (leave tissue) from which apices were extracted for cryopreservation, and in shoots regenerated from cryopreserved apices 30 days after recovery and after further 3 months in culture. Throughout the process the origin of the apices (in vitro-shoot from which they were excised) was recorded. Twenty one regenerants cryopreserved by vitrification and 25 by encapsulation-dehydration were assessed. Only one cryopreserved regenerant from the encapsulation-dehydration method showed a different band pattern. These results support the necessity of monitoring the genetic stability of the regenerants obtained after cryopreservation, as this is a very useful technique for the conservation of plant genetic resources.

Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L. (Scrophulariaceae)-an ethnomedicinal herb

PubMed Central

Premkumar, G; Sankaranarayanan, R; Jeeva, S; Rajarathinam, K

2011-01-01

Objective To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis (S. dulcis) L. Methods Explants were inoculated on MS basal medium supplemented with kinetin and 6-benzylaminopurine for shoot bud induction. To enhance the shoot induction, various auxins like 3-indoleacetic acid or 3-indolebutyric acid or α-naphthylacetic acid were tested along with 2.32 M KI and 4.44 µM BAP. The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA, IBA or NAA. After roots were developed, the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%–80% humidity under 16 h photoperiod. After acclimatization, the plantlets were transferred to the garden and survival percentage was calculated. Data were statistically analyzed and means were compared using Duncan's multiple range test (P<0.05). Results An in vitro method was developed to induce high frequency shoots regeneration from stem, mature leaf and young leaf explants of S. dulcis. Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins (2.32 µM KI and 4.44 µM BAP) 2.85 µM IAA, 10% CM and 1 483.79 µM adenine sulfate. A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP. Conclusions Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S. dulcis. PMID:23569752

Cytokinin induced shoot regeneration and flowering of Scoparia dulcis L. (Scrophulariaceae)-an ethnomedicinal herb.

PubMed

Premkumar, G; Sankaranarayanan, R; Jeeva, S; Rajarathinam, K

2011-06-01

To develop an improved protocol for micropropagation of ethnomedicinally important Scoparia dulcis (S. dulcis) L. Explants were inoculated on MS basal medium supplemented with kinetin and 6-benzylaminopurine for shoot bud induction. To enhance the shoot induction, various auxins like 3-indoleacetic acid or 3-indolebutyric acid or α-naphthylacetic acid were tested along with 2.32 M KI and 4.44 µM BAP. The regenerated shoots were rooted in half strength MS medium supplemented with various concentrations of IAA, IBA or NAA. After roots were developed, the plantlets were transplanted to pots filled with vermiculate and sand and kept in growth chamber with 70%-80% humidity under 16 h photoperiod. After acclimatization, the plantlets were transferred to the garden and survival percentage was calculated. Data were statistically analyzed and means were compared using Duncan's multiple range test (P<0.05). An in vitro method was developed to induce high frequency shoots regeneration from stem, mature leaf and young leaf explants of S. dulcis. Shoot induction on young leaf explants was most successful in MS medium supplemented with combination of two cytokinins (2.32 µM KI and 4.44 µM BAP) 2.85 µM IAA, 10% CM and 1 483.79 µM adenine sulfate. A single young leaf explant was capable of producing 59 shoots after 13 days of culture. Flower was induced in medium supplemented with combination of KI and BAP. Cytokinins are the key factor to induce the direct shoot regeneration and flowering of S. dulcis.

Abiotic stresses modulate expression of major intrinsic proteins in barley (Hordeum vulgare).

PubMed

Ligaba, Ayalew; Katsuhara, Maki; Shibasaka, Mineo; Djira, Gemechis

2011-02-01

In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed. Copyright © 2010 Académie des sciences. Published by Elsevier SAS. All rights reserved.

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.

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.

Micropropagation of Iris sp.

PubMed

Jevremović, Slađana; Jeknić, Zoran; Subotić, Angelina

2013-01-01

Irises are perennial plants widely used as ornamental garden plants or cut flowers. Some species accumulate secondary metabolites, making them highly valuable to the pharmaceutical and perfume industries. Micropropagation of irises has successfully been accomplished by culturing zygotic embryos, different flower parts, and leaf base tissues as starting explants. Plantlets are regenerated via somatic embryogenesis, organogenesis, or both processes at the same time depending on media composition and plant species. A large number of uniform plants are produced by somatic embryogenesis, however, some species have decreased morphogenetic potential overtime. Shoot cultures obtained by organogenesis can be multiplied for many years. Somatic embryogenic tissue can be reestablished from leaf bases of in vitro-grown shoots. The highest number of plants can be obtained by cell suspension cultures. This chapter describes effective in vitro plant regeneration protocols for Iris species from different types of explants by somatic embryogenesis and/or organogenesis suitable for the mass propagation of ornamental and pharmaceutical irises.

Static biofilm removal around ultrasonic tips in vitro.

PubMed

Thurnheer, Thomas; Rohrer, Elodie; Belibasakis, Georgios N; Attin, Thomas; Schmidlin, Patrick R

2014-09-01

This study aims to investigate the biofilm removal capacity of two ultrasonic tips under standardized conditions using a multi-species biofilm model. Six-species biofilms were grown on hydroxyapatite discs for 64.5 h and were treated for 15 s with a standardized load of 40 g with a piezoelectric or magnetostrictive device. Tips were applied either with the tip end or with the side facing downwards. Detached bacteria were determined in the supernatant and colony-forming units (CFUs) counted after 72 h of incubation. Untreated specimens served as controls. Moreover, the biofilms remaining on the hydroxyapatite surface after treatment were stained using the Live/Dead stain, and the pattern of their detachment was assessed by confocal laser scanning microscopy (CLSM). As compared to the untreated control, it was found that only a side application of the magnetostrictive device was able to remove efficiently the biofilm. In contrast, its tip application as well as both applications of the piezoelectric device removed significantly less bacteria from the biofilm structure. These findings were corroborated by CLSM observation. Both ultrasonic tips under investigations led to bacterial detachment, but the action mode as well as the tip configuration and adaptation appeared to be influenced by the biofilm removal effectiveness. Biofilm removal remains a main goal of ultrasonic debridement. This should be reflected in respective laboratory investigations. The presented combination of methods applied on a multi-species biofilm model in vitro allows the evaluation of the effectiveness of different ultrasonic scaler applications.

Cryobiotechnology of apple (Malus spp.): development, progress and future prospects.

PubMed

Wang, Min-Rui; Chen, Long; Teixeira da Silva, Jaime A; Volk, Gayle M; Wang, Qiao-Chun

2018-05-01

Cryopreservation provides valuable genes for further breeding of elite cultivars, and cryotherapy improves the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. Apple (Malus spp.) is one of the most economically important temperate fruit crops. Wild Malus genetic resources and existing cultivars provide valuable genes for breeding new elite cultivars and rootstocks through traditional and biotechnological breeding programs. These valuable genes include those resistant to abiotic factors such as drought and salinity, and to biotic factors such as fungi, bacteria and aphids. Over the last three decades, great progress has been made in apple cryobiology, making Malus one of the most extensively studied plant genera with respect to cryopreservation. Explants such as pollen, seeds, in vivo dormant buds, and in vitro shoot tips have all been successfully cryopreserved, and large Malus cryobanks have been established. Cryotherapy has been used for virus eradication, to obtain virus-free apple plants. Cryopreservation provided valuable genes for further breeding of elite cultivars, and cryotherapy improved the production of virus-free plants in Malus spp., thus assisting the sustainable development of the apple industry. This review provides updated and comprehensive information on the development and progress of apple cryopreservation and cryotherapy. Future research will reveal new applications and uses for apple cryopreservation and cryotherapy.

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.

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.

An efficient regeneration and rapid micropropagation protocol for Almond using dormant axillary buds as explants.

PubMed

Choudhary, Ravish; Chaudhury, Rekha; Malik, Surendra Kumar; Sharma, Kailash Chandra

2015-07-01

An efficient in vitro protocol was standardized for Almond (Prunus dulcis) propagation using dormant axillary buds as explants. Explants were cultured on Murashige and Skoog (MS) and woody plant medium (WPM) supplemented with different concentration/combination(s) of phytohormones. MS basal medium showed lowest shoot induction and took longest duration for shoot initiation. Multiple shoots were induced in MS medium supplemented with the combination of BAP (0.5 mgL(-1)). Cultures showed poor response for rooting in all combinations of plant growth regulators (PGRs) and took 90 days for initiation. Rooting was higher in half strength of MS than in full-strength. The highest root induction (33.33%) was recorded in half MS medium supplemented with 0.1 mgL(-1) IBA (indole-3-butyric acid) followed by full strength of MS medium (20%) supplemented with IBA (0.1 mgL(-1)). α-Naphthalene acetic acid (NAA) was less effective for rooting than IBA. The highest root induction (25%) was found in half strength of MS medium supplemented with 0.1 mgL(-1) NAA followed by full strength of MS medium (20%). The protocol developed would be of use in mass propagation of almond and also support in vitro conservation.

[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).

Light Spectral Quality Effects on the Growth of Potato (Solanum Tuberosum L.) Nodal Cutttings in Vitro

NASA Technical Reports Server (NTRS)

Wilson, Deborah A.; Weigel, Russell, C.; Wheeler, Raymond M.; Sager, John C.

1993-01-01

The effects of light spectral quality on the growth of in vitro nodal cutting of potato (Solanum tuberosum) cultivars Norland, Superior, Kennebec, and Denali were examined. The different light spectra were provided by Vita-Lite fluorescent (VF) (a white light control), blue fluorescent (BF), red fluorescent (RF), low-pressure sodium (LPS), and a combination of low-pressure sodium plus cool-white fluorescent lamp (LPS/CWF). Results suggested that shoot morphologic development of in vitro grown potato plants can be controlled by controlling irradiant spectral quality.

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

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.

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.

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.

The effect of piezoelectric ultrasonic instrumentation on titanium discs: a microscopy and trace elemental analysis in vitro study.

PubMed

Tawse-Smith, A; Atieh, M A; Tompkins, G; Duncan, W J; Reid, M R; Stirling, C H

2016-08-01

To evaluate in vitro topographical and composition changes by piezoelectric ultrasonic instrumentation with metallic and plastic tips on machined and moderately roughened titanium surfaces. Twenty machined and moderately roughened laser-marked titanium discs were ultrasonically instrumented with metallic and plastic tips. Surface instrumentation was carried out with controlled pressure for 20 and 30 seconds at two power settings. For each time and power setting, instrumentation was repeated four times with one instrumentation per disc quadrant. Surface topography analysis was performed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Surface roughness measurements were compared between instrumented and non-instrumented surfaces. Surface element composition and rinsing solutions were evaluated using energy-dispersive spectroscopy (EDS) and trace elemental analysis using inductively coupled plasma mass spectrometry (ICPMS), respectively. SEM photomicrographs and CLSM 3D surface plot images of instrumented machined and moderately roughened surfaces demonstrated severe surface topographical alterations with metallic tips and mild to moderate changes for plastic tip instrumented sites. ICPMS analysis of the rinsing solutions identified titanium and other metal traces with the use of metallic tips, and mainly titanium and carbon when plastic tips were used. Surface EDS analysis showed elemental traces of the ultrasonic tips. Ultrasonic instrumentation with metallic or plastic tips created surface topographical and compositional changes. Different changes in surface topography were noted between the surfaces, as the roughness of the machined surfaces increased while the extent of roughness of the moderately roughened surfaces decreased. The clinical relevance of these changes is yet to be determined. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

Cytokinin-induced promotion of root meristem size in the fern Azolla supports a shoot-like origin of euphyllophyte roots.

PubMed

de Vries, Jan; Fischer, Angela Melanie; Roettger, Mayo; Rommel, Sophie; Schluepmann, Henriette; Bräutigam, Andrea; Carlsbecker, Annelie; Gould, Sven Bernhard

2016-01-01

The phytohormones cytokinin and auxin orchestrate the root meristem development in angiosperms by determining embryonic bipolarity. Ferns, having the most basal euphyllophyte root, form neither bipolar embryos nor permanent embryonic primary roots but rather an adventitious root system. This raises the questions of how auxin and cytokinin govern fern root system architecture and whether this can tell us something about the origin of that root. Using Azolla filiculoides, we characterized the influence of IAA and zeatin on adventitious fern root meristems and vasculature by Nomarski microscopy. Simultaneously, RNAseq analyses, yielding 36,091 contigs, were used to uncover how the phytohormones affect root tip gene expression. We show that auxin restricts Azolla root meristem development, while cytokinin promotes it; it is the opposite effect of what is observed in Arabidopsis. Global gene expression profiling uncovered 145 genes significantly regulated by cytokinin or auxin, including cell wall modulators, cell division regulators and lateral root formation coordinators. Our data illuminate both evolution and development of fern roots. Promotion of meristem size through cytokinin supports the idea that root meristems of euphyllophytes evolved from shoot meristems. The foundation of these roots was laid in a postembryonically branching shoot system. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Automorphosis of higher plants on a 3-D clinostat

NASA Astrophysics Data System (ADS)

Hoson, T.; Kamisaka, S.; Yamashita, M.; Masuda, Y.

On a three-dimensional (3-D) clinostat, various plant organs developed statocytes capable of responding to the gravity vector. The graviresponse of primary roots of garden cress and maize grown on the clinostat was the same as the control roots, whereas that of maize coleoptiles was reduced. When maize seedlings were grown in the presence of 10^-4 M gibberellic acid and kinetin, the graviresponse of both roots and shoots was suppressed. The corresponding suppression of amyloplast development was observed in the clinostatted and the hormone-treated seedlings. Maize roots and shoots showed spontaneous curvatures in different portions on the 3-D clinostat. The hormone treatment did not significantly influence such an automorphic curvature. When the root cap was removed, maize roots did not curve gravitropically. However, the removal suppressed the automorphic curvatures only slightly. On the other hand, the removal of coleoptile tip did not influence its graviresponse, whereas the spontaneous curvature of decapitated coleoptiles on the clinostat was strongly suppressed. Also, cytochalasin B differently affected the gravitropic and the automorphic curvatures of maize roots and shoots. From these results it is concluded that the graviperception and the early processes of signal transmission are unnecessary for automorphoses under simulated microgravity conditions. Moreover, the results support the view that the amyloplasts act as statoliths probably via an interaction with microfilaments.

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.

Soil-plant-atmosphere ammonia exchange associated with calluna vulgaris and deschampsia flexuosa

NASA Astrophysics Data System (ADS)

Schjoerring, Jan K.; Husted, Søren; Poulsen, Mette M.

Ammonia fluxes and compensation points at atmospheric NH 3 concentrations corresponding to those occurring under natural growth conditions (0-26 nmol NH 3 mol air -1) were measured for canopies of two species native to heathland in N.W. Europe, viz. Calluna vulgaris (L.) Hull and Deschampsia flexuosa (L.) Trin. The NH 3 compensation point in 2 yr-old C. vulgaris plants, in which current year's shoots had just started growing, was below the detection limit (0.1 nmol mol -1 at 8°C). Fifty days later, when current year's shoots were elongating and flowers developed, the NH 3 compensation point was approximately 6±2.0 nmol mol -1 at 22°C (0.8±0.3 nmol mol -1 at 8°C). The plants in which the shoot tips had just started growing were characterized by a low N concentration in the shoot dry matter (5.8 mg N g -1 shoot dry weight) and a low photosynthetic CO 2 assimilation compared to the flowering plants in which the average dry matter N concentration in old shoots and woody stems was 7.4 and in new shoots 9.5 mg N g -1 shoot dry weight. Plant-atmosphere NH 3 fluxes in C. vulgaris responded approximately linearly to changes in the atmospheric NH 3 concentration. The maximum net absorption rate at 26 nmol NH 3 mol -1 air was 12 nmol NH 3 m -2 ground surface s -1 (equivalent to 13.3 pmol NH 3 g -1 shoot dry matter s -1). Ammonia absorption in Deschampsia flexuosa plants increased approximately linearly with increasing NH 3 concentrations up to 20 nmol mol -1. The maximum NH 3 absorption was 8.5 nmol m -2 ground surface s -1 (30.4 pmol g -1 shoot dry weight s -1). The NH 3 compensation point at 24°C was 3.0±1.1, and at 31°C 7.5±0.6 nmol mol air -1. These values correspond to a NH 3 compensation point of 0.45±0.15 at 8°C. The soil used for cultivation of C. vulgaris (peat soil with pH 6.9) initially adsorbed NH 3 at a rate which exceeded the absorption by the plant canopy. During a 24 d period following the harvest of the plants soil NH 3 adsorption declined and the soil NH 3 compensation point increased from below the detection limit to 8.0±1.8 nmol NH 3 mol air -1 (22°C). No detectable NH 3 exchange took place between the D. flexuosa soil (sandy soil with pH 6.8) and the atmosphere.

In vitro performance of DIAGNOdent laser fluorescence device for dental calculus detection on human tooth root surfaces.

PubMed

Rams, Thomas E; Alwaqyan, Abdulaziz Y

2017-10-01

This study assessed the reproducibility of a red diode laser device, and its capability to detect dental calculus in vitro on human tooth root surfaces. On each of 50 extracted teeth, a calculus-positive and calculus-free root surface was evaluated by two independent examiners with a low-power indium gallium arsenide phosphide diode laser (DIAGNOdent) fitted with a periodontal probe-like sapphire tip and emitting visible red light at 655 nm wavelength. Laser autofluorescence intensity readings of examined root surfaces were scored on a 0-99 scale, with duplicate assessments performed using the laser probe tip directed both perpendicular and parallel to evaluated tooth root surfaces. Pearson correlation coefficients of untransformed measurements, and kappa analysis of data dichotomized with a >40 autofluorescence intensity threshold, were calculated to assess intra- and inter-examiner reproducibility of the laser device. Mean autofluorescence intensity scores of calculus-positive and calculus-free root surfaces were evaluated with the Student's t -test. Excellent intra- and inter-examiner reproducibility was found for DIAGNOdent laser autofluorescence intensity measurements, with Pearson correlation coefficients above 94%, and kappa values ranging between 0.96 and 1.0, for duplicate readings taken with both laser probe tip orientations. Significantly higher autofluorescence intensity values were measured when the laser probe tip was directed perpendicular, rather than parallel, to tooth root surfaces. However, calculus-positive roots, particularly with calculus in markedly-raised ledges, yielded significantly greater mean DIAGNOdent laser autofluorescence intensity scores than calculus-free surfaces, regardless of probe tip orientation. DIAGNOdent autofluorescence intensity values >40 exhibited a stronger association with calculus (36.6 odds ratio) then measurements of ≥5 (20.1 odds ratio) when the laser probe tip was advanced parallel to root surfaces. Excellent intra- and inter-examiner reproducibility of autofluorescence intensity measurements was obtained with the DIAGNOdent laser fluorescence device on human tooth roots. Calculus-positive root surfaces exhibited significantly greater DIAGNOdent laser autofluorescence than calculus-free tooth roots, even with the laser probe tip directed parallel to root surfaces. These findings provide further in vitro validation of the potential utility of a DIAGNOdent laser fluorescence device for identifying dental calculus on human tooth root surfaces.

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.

Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.

PubMed

Sun, Yingli; Du, Fengxia

2017-01-01

The activation of ATM is critical in the DNA double strand breaks repair pathway. Acetylation of ATM by Tip60 histone acetyltransferase (HAT) plays a key role in the activation of ATM kinase activity in response to DNA damage. ATM forms a stable complex with Tip60 through the FATC domain of ATM. Tip60 acetylates lysine3016 of ATM, and this acetylation induces the activation of ATM. Several techniques are included in the study of ATM acetylation by Tip60, such as in vitro kinase assay, systematic mutagenesis, western blots. Here, we describe how to study the acetylation of ATM using acetylation-specific antibodies.

miRNA and Degradome Sequencing Reveal miRNA and Their Target Genes That May Mediate Shoot Growth in Spur Type Mutant “Yanfu 6”

PubMed Central

Song, Chunhui; Zhang, Dong; Zheng, Liwei; Zhang, Jie; Zhang, Baojuan; Luo, Wenwen; Li, Youmei; Li, Guangfang; Ma, Juanjuan; Han, Mingyu

2017-01-01

The spur-type growth habit in apple trees is characterized by short internodes, increased number of fruiting spurs, and compact growth that promotes flowering and facilitates management practices, such as pruning. The molecular mechanisms responsible for regulating spur-type growth have not been elucidated. In the present study, miRNAs and the expression of their potential target genes were evaluated in shoot tips of “Nagafu 2” (CF) and spur-type bud mutation “Yanfu 6” (YF). A total of 700 mature miRNAs were identified, including 202 known apple miRNAs and 498 potential novel miRNA candidates. A comparison of miRNA expression in CF and YF revealed 135 differentially expressed genes, most of which were downregulated in YF. YF also had lower levels of GA, ZR, IAA, and ABA hormones, relative to CF. Exogenous applications of GA promoted YF shoot growth. Based on the obtained results, a regulatory network involving plant hormones, miRNA, and their potential target genes is proposed for the molecular mechanism regulating the growth of YF. miRNA164, miRNA166, miRNA171, and their potential targets, and associated plant hormones, appear to regulate shoot apical meristem (SAM) growth. miRNA159, miRNA167, miRNA396, and their potential targets, and associated plant hormones appear to regulate cell division and internode length. This study provides a foundation for further studies designed to elucidate the mechanism underlying spur-type apple architecture. PMID:28424721

Proteomic analysis of two divergently responding potato genotypes (Solanum tuberosum L.) following osmotic stress treatment in vitro.

PubMed

Bündig, Christin; Jozefowicz, Anna Maria; Mock, Hans-Peter; Winkelmann, Traud

2016-06-30

Starch potatoes (Solanum tuberosum L.) are of interest for production of starch, ethanol, and biopolymers. Due to the predicted increase in drought periods, the breeding of starch potatoes for drought tolerance is essential. This study aims to elucidate the physiological mechanisms that give rise to drought tolerance. Two genotypes contrasting in drought tolerance were compared. We applied osmotic stress which is a known component of drought stress under in vitro conditions. Shoot tips were harvested after 11days of culture on control medium and medium supplied with 0.2M sorbitol. Their proteomes were analyzed using two-dimensional isoelectric focussing sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D-IEF/SDS-PAGE). Of a total of 679 distinct protein spots, 118 and 20 spots with differential abundance were found in the sensitive and the tolerant genotype, respectively, after the application of stress. Using mass spectrometry, the proteins in 100 differentially abundant spots were identified; a majority of these proteins were from the chloroplast. For the sensitive genotype, an increase in the abundance of proteinase inhibitors and their precursors, changes in stress responsive proteins and an altered RNA/DNA-binding response were observed. The differentially abundant spots of the tolerant genotype comprised one chaperone and one hydrogen peroxide detoxifying protein. Our findings reveal that the two genotypes have different responses to osmotic stress in terms of protein degradation and reactive oxygen species (ROS) scavenging and production. Our data suggest that the tolerant genotype might adjust to the applied stress more quickly. A comparative temporal analysis might provide further insights into these rapid changes and assist in the development of biomarkers. Copyright © 2016 Elsevier B.V. All rights reserved.

Adaptation to the Local Environment by Modifications of the Photoperiod Response in Crops

PubMed Central

Nakamichi, Norihito

2015-01-01

Flowering plants produce a meristem at the shoot tip where specialized tissue generates shoot apical meristems at the appropriate time to differentiate into reproductive structures, pollinate and efficiently generate seeds. The complex set of molecular and phenological events culminating in development of a flowering meristem is referred to as ‘flowering time’. Flowering time affects plant productivity because plants dedicate energy to produce flowers and seeds rather than vegetative tissue once the molecular decision to initiate flowering has been taken. Thus, initiation of flowering time is an important decision in plants, especially in annual plants including crops. Humans have introduced crops into latitudes and climate areas far from their origin or natural ecosystem, requiring in many cases modification of native flowering times. Recent molecular–genetic studies shed light on the genetic basis related to such introductions. In this review, recent progress regarding crop introductions and their genetic bases are summarized, as well as the potential of other agricultural plants to be introduced into different climatic zones. PMID:25432974

Micropropagation and validation of genetic and biochemical fidelity among regenerants of Nothapodytes nimmoniana (Graham) Mabb. employing ISSR markers and HPLC.

PubMed

Prakash, Lokesh; Middha, Sushil Kumar; Mohanty, Sudipta Kumar; Swamy, Mallappa Kumara

2016-12-01

An in vitro protocol has been established for clonal propagation of Nothapodytes nimmoniana which is an important source of Camptothecin (CPT). Elite source was identified based on the chemical potency to accumulate the optimum level of CPT. Different types and concentrations of plant growth regulators were used to study their effect on inducing multiple shoots from the explants regenerated from embryos of N. nimmoniana. Of these, a combination of N6-benzyladenine (0.2 mg L -1 ) and Indole-3-butyric acid (IBA) (0.1 mg L -1 ) proved optimum for differentiating multiple shoots in 90.6 % of the cultures with an average of 10.24 shoots per explant obtained within 8 weeks of inoculation. Nearly, 92 % of the excised in vitro shoots rooted on half strength Murashige and Skoog (MS) medium containing 0.05 % activated charcoal, supplemented with 1-naphthaleneacetic acid and IBA at 0.1 mg L -1 each. The micropropagated plants were evaluated for their genetic fidelity by employing inter simple sequence repeats (ISSR) markers. Ten individuals, randomly chosen from a population of 145 regenerants, were compared with the donor plant. The regenerated plants were also evaluated for their chemical potency using high-performance liquid chromatography (HPLC) analysis of CPT content. The true-to-type nature of the micropropagated plants was confirmed based on their monomorphic banding profiles with that of the mother plants using ISSR markers. Besides, HPLC evaluation of the CPT content confirmed the existence of chemical uniformity among the regenerated plants and the elite mother plant.

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.

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

Arabidopsis florigen FT binds to diurnally oscillating phospholipids that accelerate flowering.

PubMed

Nakamura, Yuki; Andrés, Fernando; Kanehara, Kazue; Liu, Yu-chi; Dörmann, Peter; Coupland, George

2014-04-04

Arabidopsis FT protein is a component of florigen, which transmits photoperiodic flowering signals from leaf companion cells to the shoot apex. Here, we show that FT specifically binds phosphatidylcholine (PC) in vitro. A transgenic approach to increase PC levels in vivo in the shoot meristem accelerates flowering whereas reduced PC levels delay flowering, demonstrating that PC levels are correlated with flowering time. The early flowering is related to FT activity, because expression of FT-effector genes is increased in these plants. Simultaneous increase of FT and PC in the shoot apical meristem further stimulates flowering, whereas a loss of FT function leads to an attenuation of the effect of increased PC. Specific molecular species of PC oscillate diurnally, and night-dominant species are not the preferred ligands of FT. Elevating night-dominant species during the day delays flowering. We suggest that FT binds to diurnally changing molecular species of PC to promote flowering.

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.

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.

Antioxidants and phenolic secretion in sugarcane genotypes shoot culture

USDA-ARS?s Scientific Manuscript database

The secretion of phenolic compounds is a major limitation for sugarcane in vitro culture, causing a loss of regenerative capacity and subsequent cell death. In this study, micropropagation and phenolic secretion of four Saccharum genotypes in the presence of different antioxidants were evaluated. As...

In vitro propagation of olive (Olea europaea L.) by nodal segmentation of elongated shoots.

PubMed

Lambardi, Maurizio; Ozudogru, Elif Aylin; Roncasaglia, Romano

2013-01-01

Olive (Olea europaea L.), long-living, ever-green fruit tree of the Old World, has been part of a traditional landscape in the Mediterranean area for centuries. Both the fruits consumed after processing and the oil extracted from the fruits are among the main components of the Mediterranean diet, widely used for salads and cooking, as well as for preserving other food. Documentations show that the ancient use of this beautiful tree also includes lamp fuel production, wool treatment, soap production, medicine, and cosmetics. However, unlike the majority of the fruit species, olive propagation is still a laborious practice. As regards traditional propagation, rooting of cuttings and grafting stem segments onto rootstocks are possible, former being achieved only when the cuttings are collected in specific periods (spring or beginning of autumn), and latter only when skilled grafters are available. In both the cases, performance of the cultivars varies considerably. The regeneration of whole plants from ovules, on the other hand, is used only occasionally. Micropropagation of olive is not easy mainly due to explant oxidation, difficulties in explant disinfection, and labor-oriented establishment of in vitro shoot cultures. However today, the progress in micropropagation technology has made available the complete protocols for several Mediterranean cultivars. This chapter describes a micropropagation protocol based on the segmentation of nodal segments obtained from elongated shoots.

Bioavailability of coated and uncoated ZnO nanoparticles to cucumber in soil with or without organic matter.

PubMed

Moghaddasi, Sahar; Fotovat, Amir; Khoshgoftarmanesh, Amir Hossein; Karimzadeh, F; Khazaei, Hamid Reza; Khorassani, Reza

2017-10-01

There is a gap of knowledge for the fate, effects and bioavailability of coated and uncoated ZnO nanoparticles (NPs) in soil. Moreover, little is known about the effects of soil properties on effects of NPs on plants. In this study, the availability ZnO NPs in two soils with different organic matter content (one treated with cow manure (CM) and the other as untreated) was compared with their bulk particles. Results showed that coated and uncoated ZnO NPs can be more bioaccessible than their bulk counterpart and despite their more positive effects at low concentration (< 100mgkg -1 ), they were more phytotoxic for plants compared to the bulk ZnO particles at high concentration (1000mgkg -1 ) in the soil untreated with CM. The concentration of 1000mgkg -1 of ZnO NPs, decreased shoot dry biomass (52%) in the soil untreated with CM but increased shoot dry biomass (35%) in CM-treated soil compared to their bulk counterpart. In general, plants in the CM-treated soil showed higher Zn concentration in their tissues compared with those in untreated soil. The difference in shoot Zn concentration between CM-treated and untreated soil for NPs treatments was more than bulk particles treatment. This different percentage at 100mgkg -1 of bulk particles was 20.6% and for coated and uncoated NPs were 37% and 32%, respectively. Generally, the distribution of ZnO among Zn fractions in soil (exchangeable, the metal bound to carbonates, Fe-Mn oxides, organic matter and silicate minerals and the residual fraction) changed based on applied Zn concentration, Zn source and soil organic matter content. The root tip deformation under high concentration of NPs (1000mgkg -1 treatment ) was observed by light microscopy in plants at the soil untreated with CM. It seems that root tip deformation is one of the specific effects of NPs which in turn inhibits plant growth and nutrients uptake by root. The transmission electron microcopy image showed the aggregation of NPs inside the plant cytoplasm and their accumulation adjacent to the cell membrane. Copyright © 2017. Published by Elsevier Inc.

Proteomic analysis indicates massive changes in metabolism prior to the inhibition of growth and photosynthesis of grapevine (Vitis vinifera L.) in response to water deficit

PubMed Central

2013-01-01

Background Cabernet Sauvignon grapevines were exposed to a progressive, increasing water defict over 16 days. Shoot elongation and photosynthesis were measured for physiological responses to water deficit. The effect of water deficit over time on the abundance of individual proteins in growing shoot tips (including four immature leaves) was analyzed using nanoflow liquid chromatography - tandem mass spectrometry (nanoLC-MS/MS). Results Water deficit progressively decreased shoot elongation, stomatal conductance and photosynthesis after Day 4; 2277 proteins were identified by shotgun proteomics with an average CV of 9% for the protein abundance of all proteins. There were 472 out of 942 (50%) proteins found in all samples that were significantly affected by water deficit. The 472 proteins were clustered into four groups: increased and decreased abundance of early- and late-responding protein profiles. Vines sensed the water deficit early, appearing to acclimate to stress, because the abundance of many proteins changed before decreases in shoot elongation, stomatal conductance and photosynthesis. Predominant functional categories of the early-responding proteins included photosynthesis, glycolysis, translation, antioxidant defense and growth-related categories (steroid metabolism and water transport), whereas additional proteins for late-responding proteins were largely involved with transport, photorespiration, antioxidants, amino acid and carbohydrate metabolism. Conclusions Proteomic responses to water deficit were dynamic with early, significant changes in abundance of proteins involved in translation, energy, antioxidant defense and steroid metabolism. The abundance of these proteins changed prior to any detectable decreases in shoot elongation, stomatal conductance or photosynthesis. Many of these early-responding proteins are known to be regulated by post-transcriptional modifications such as phosphorylation. The proteomics analysis indicates massive and substantial changes in plant metabolism that appear to funnel carbon and energy into antioxidant defenses in the very early stages of plant response to water deficit before any significant injury. PMID:23514573

Molecular and biochemical characterization in Rauvolfia tetraphylla plantlets grown from synthetic seeds following in vitro cold storage.

PubMed

Faisal, Mohammad; Alatar, Abdularhaman A; Hegazy, Ahmad K

2013-01-01

Synseed technology is one of the most important applications of plant biotechnology for in vitro conservation and regeneration of medicinal and aromatic plants. In the present investigation, synseeds of Rauvolfia tetraphylla were produced using in vitro-proliferated shoots upon complexation of 3 % sodium alginate and 100 mM CaCl(2). The encapsulated buds were stored at 4, 8, 12, and 16 °C and high conversion was observed in synseeds stored at 4 °C for 4 weeks. The effect of different medium strength on in vitro conversion response of synseed was evaluated and the maximum conversion (80.6 %) into plantlets was recorded on half-strength woody plant medium supplemented with 7.5 μM 6-benzyladenine and 2.5 μM α-naphthalene acetic acid after 8 weeks of culture. Plantlets with well-developed shoot and roots were hardened and successfully transplanted in field condition. After 4 weeks of transfer to ex vitro conditions, the performance of synseed-derived plantlets was evaluated on the basis of some physiological and biochemical parameters and compared with the in vivo-grown plants. Short-term storage of synthetic seeds at low temperature had no negative impact on physiological and biochemical profile of the plants that survived the storage process. Furthermore, clonal fidelity of synseed-derived plantlets was also assessed and compared with mother plant using rapid amplified polymorphic DNA and inter-simple sequence repeats analysis. No changes in molecular profiles were found among the regenerated plantlets and comparable to mother plant, which confirm the genetic stability among clones. This synseed protocol could be useful for in vitro clonal multiplication, conservation, and short-term storage and exchange of germplasm of this antihypertensive drug-producing plant.

Agrobacterium-medicated transformation of mature Prunus serotina (black cherry) and regeneration of trangenic shoots

Treesearch

Xiaomei Liu; Paula Pijut

2010-01-01

A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced...

An Efficient In Vitro Regeneration System for Ornamental Ginger (Hedychium spp.)

USDA-ARS?s Scientific Manuscript database

An improved and efficient regeneration protocol was established for Hedychium via somatic embryogenesis. The plant material used consisted of 11 species and 9 cultivars of Hedychium. The explants consisted of young leaves taken from lateral or terminal shoots of mature greenhouse grown plants. These...

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.

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.

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

Allele compensation in tip60+/- mice rescues white adipose tissue function in vivo.

PubMed

Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

2014-01-01

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/- mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/- mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/- displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/- mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice.

Functional analysis of the GmESR1 gene associated with soybean regeneration

PubMed Central

Chen, Qingshan; Liu, Ming; Xin, Dawei; Qi, Zhaoming; Li, Sinan; Ma, Yanlong; Wang, Lingshuang; Jin, Yangmei; Li, Wenbin; Wu, Xiaoxia; Su, An-yu

2017-01-01

Plant regeneration can occur via in vitro tissue culture through somatic embryogenesis or de novo shoot organogenesis. Transformation of soybean (Glycine max) is difficult, hence optimization of the transformation system for soybean regeneration is required. This study investigated ENHANCER OF SHOOT REGENERATION 1 (GmESR1), a soybean transcription factor that targets regeneration-associated genes. Sequence analysis showed that GmESR1 contained a conserved 57 amino acid APETALA 2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) DNA-binding domain. The relative expression level of GmESR1 was highest in young embryos, flowers and stems in the soybean cultivar ‘Dongnong 50’. To examine the function of GmESR1, transgenic Arabidopsis (Arabidopsis thaliana) and soybean plants overexpressing GmESR1 were generated. In Arabidopsis, overexpression of GmESR1 resulted in accelerated seed germination, and seedling shoot and root elongation. In soybean overexpression of GmESR1 also led to faster seed germination, and shoot and root elongation. GmESR1 specifically bound to the GCC-box. The results provide a foundation for the establishment of an efficient and stable transformation system for soybean. PMID:28403182

Characterizing the walnut genome through analyses of BAC end sequences

USDA-ARS?s Scientific Manuscript database

Persian walnut (Juglans regia L.) is an economically important tree for its nut crop and timber. To gain insight into the structure and evolution of the walnut genome, we constructed two bacterial artificial chromosome (BAC) libraries, containing a total of 129,024 clones, from in vitro-grown shoots...

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...

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.

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.

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.

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.

Oxidation of Proline by Mitochondria Isolated from Water-Stressed Maize Shoots 1

PubMed Central

Sells, Gary D.; Koeppe, David E.

1981-01-01

Proline oxidation and coupled phosphorylation were measured in mitochondria after isolation from shoots of water-stressed, etiolated maize (Zea mays L.) seedlings. Both state III and state IV rates of proline oxidation decreased as a logarithmic function of increased seedling water stress between −5 and −10 bars. Proline oxidation rates decreased 62% (state III) and 58% (state IV) as seedling water potentials were decreased from −5 to −10 bars. By comparison, oxidation of succinate, exogenous NADH, or malate + pyruvate decreased only 10 to 15% in this stress range. These decreases were a linear function of increased stress and were comparable to oxidation rates of mitochondria subjected to varying in vitro osmotic potentials. Osmotically induced in vitro stress reduced proline oxidation rates linearly with more negative osmotic potentials, a decrease that was similar to the responses of the other substrates to more negative osmotic potentials. Some decrease in coupling, with all substrates as determined by ADP/O ratios, was observed under osmotic stress. Mitochondria were also isolated from shoot tissue that had been stressed and then rewatered. On a percentage basis, the recovery of proline oxidation was greater than that of the other substrates. The decreases in the proline oxidase activity of mitochondria after only slight stress indicate a mitochondrial sensitivity to water stress at significantly less negative water potentials than previously reported for measurements of maize membrane permeability and respiratory activity. PMID:16662051

Inducible growth mode switches influence Valonia rhizoid differentiation.

PubMed

Elvira, Paul Rommel; Sekida, Satoko; Okuda, Kazuo

2013-02-01

Cell differentiation and cell type commitment are an integral part of plant growth and development. Investigations on how environmental conditions affect the formation of shoots, roots, and rhizoids can help illustrate how plants determine cell fate and overall morphology. In this study, we evaluated the role of substratum and light on rhizoid differentiation in the coenocytic green alga, Valonia aegagropila. Elongating rhizoids displayed varying growth modes and cell shape upon exposure to different substrata and light conditions. It was found that soft substrata and dark incubation promoted rhizoid elongation via tip growth while subsequent exposure to light prevented tip growth and instead induced swelling in the apical region of rhizoids. Swelling was accompanied by the accumulation of protoplasm in the rhizoid tip through expansion of the cell wall and uninhibited cytoplasmic streaming. Subsequent diffuse growth led to the transformation from slender, rod-shaped rhizoids into spherical thallus-like structures that required photosynthesis. Further manipulation of light regimes caused vacillating cell growth redirections. An elongating V. aegagropila rhizoid cell thus appears capable of growth mode switching that is regulated by immediate environmental conditions thereby influencing ultimate cell shape and function. This is the first description of inducible, multiple growth mode shifts in a single intact plant cell that directly impact its differentiation.

Allele Compensation in Tip60+/− Mice Rescues White Adipose Tissue Function In Vivo

PubMed Central

Gao, Yuan; Hamers, Nicole; Rakhshandehroo, Maryam; Berger, Ruud; Lough, John; Kalkhoven, Eric

2014-01-01

Adipose tissue is a key regulator of energy homestasis. The amount of adipose tissue is largely determined by adipocyte differentiation (adipogenesis), a process that is regulated by the concerted actions of multiple transcription factors and cofactors. Based on in vitro studies in murine 3T3-L1 preadipocytes and human primary preadipocytes, the transcriptional cofactor and acetyltransferase Tip60 was recently identified as an essential adipogenic factor. We therefore investigated the role of Tip60 on adipocyte differentiation and function, and possible consequences on energy homeostasis, in vivo. Because homozygous inactivation results in early embryonic lethality, Tip60+/− mice were used. Heterozygous inactivation of Tip60 had no effect on body weight, despite slightly higher food intake by Tip60+/− mice. No major effects of heterozygous inactivation of Tip60 were observed on adipose tissue and liver, and Tip60+/− displayed normal glucose tolerance, both on a low fat and a high fat diet. While Tip60 mRNA was reduced to 50% in adipose tissue, the protein levels were unaltered, suggesting compensation by the intact allele. These findings indicate that the in vivo role of Tip60 in adipocyte differentiation and function cannot be properly addressed in Tip60+/− mice, but requires the generation of adipose tissue-specific knock out animals or specific knock-in mice. PMID:24870614

Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri

PubMed Central

Vernot, Jean-Paul; Perdomo-Arciniegas, Ana María; Pérez-Quintero, Luis Alberto; Martínez, Diego Fernando

2015-01-01

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts. PMID:26539553

Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip from Herpesvirus saimiri.

PubMed

Vernot, Jean-Paul; Perdomo-Arciniegas, Ana María; Pérez-Quintero, Luis Alberto; Martínez, Diego Fernando

2015-01-01

The Lck interacting protein Tip of Herpesvirus saimiri is responsible for T-cell transformation both in vitro and in vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human and Aotus sp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

Tetraploidy Enhances Boron-Excess Tolerance in Carrizo Citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.).

PubMed

Ruiz, Marta; Quiñones, Ana; Martínez-Alcántara, Belén; Aleza, Pablo; Morillon, Raphaël; Navarro, Luis; Primo-Millo, Eduardo; Martínez-Cuenca, Mary-Rus

2016-01-01

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport. Tetraploidy enhances B excess tolerance in citrange CarrizoExpression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidiesB tolerance is attributed to root anatomical modifications induced by genome duplicationThe rootstock 4x citrange carrizo may prevent citrus trees from B excess.

Tetraploidy Enhances Boron-Excess Tolerance in Carrizo Citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.)

PubMed Central

Ruiz, Marta; Quiñones, Ana; Martínez-Alcántara, Belén; Aleza, Pablo; Morillon, Raphaël; Navarro, Luis; Primo-Millo, Eduardo; Martínez-Cuenca, Mary-Rus

2016-01-01

Tetraploidy modifies root anatomy which may lead to differentiated capacity to uptake and transport mineral elements. This work provides insights into physiological and molecular characters involved in boron (B) toxicity responses in diploid (2x) and tetraploid (4x) plants of Carrizo citrange (Citrus sinensis L. Osb. × Poncirus trifoliata L. Raf.), a widely used citrus rootstock. With B excess, 2x plants accumulated more B in leaves than 4x plants, which accounted for their higher B uptake and root-to-shoot transport rates. Ploidy did not modify the expression of membrane transporters NIP5 and BOR1 in roots. The cellular allocation of B excess differed between ploidy levels in the soluble fraction, which was lower in 4x leaves, while cell wall-linked B was similar in 2x and 4x genotypes. This correlates with the increased damage and stunted growth recorded in the 2x plants. The 4x roots were found to have fewer root tips, shorter specific root length, longer diameter, thicker exodermis and earlier tissue maturation in root tips, where the Casparian strip was detected at a shorter distance from the root apex than in the 2x roots. The results presented herein suggest that the root anatomical characters of the 4x plants play a key role in their lower B uptake capacity and root-to-shoot transport. Highlights Tetraploidy enhances B excess tolerance in citrange Carrizo Expression of NIP5 and BOR1 transporters and cell wall-bounded B are similar between ploidies B tolerance is attributed to root anatomical modifications induced by genome duplication The rootstock 4x citrange carrizo may prevent citrus trees from B excess. PMID:27252717

Regulation, overexpression, and target gene identification of Potato Homeobox 15 (POTH15) – a class-I KNOX gene in potato

PubMed Central

Mahajan, Ameya S.; Kondhare, Kirtikumar R.; Rajabhoj, Mohit P.; Kumar, Amit; Ghate, Tejashree; Ravindran, Nevedha; Habib, Farhat; Siddappa, Sundaresha; Banerjee, Anjan K.

2016-01-01

Potato Homeobox 15 (POTH15) is a KNOX-I (Knotted1-like homeobox) family gene in potato that is orthologous to Shoot Meristemless (STM) in Arabidopsis. Despite numerous reports on KNOX genes from different species, studies in potato are limited. Here, we describe photoperiodic regulation of POTH15, its overexpression phenotype, and identification of its potential targets in potato (Solanum tuberosum ssp. andigena). qRT-PCR analysis showed a higher abundance of POTH15 mRNA in shoot tips and stolons under tuber-inducing short-day conditions. POTH15 promoter activity was detected in apical and axillary meristems, stolon tips, tuber eyes, and meristems of tuber sprouts, indicating its role in meristem maintenance and leaf development. POTH15 overexpression altered multiple morphological traits including leaf and stem development, leaflet number, and number of nodes and branches. In particular, the rachis of the leaf was completely reduced and leaves appeared as a bouquet of leaflets. Comparative transcriptomic analysis of 35S::GUS and two POTH15 overexpression lines identified more than 6000 differentially expressed genes, including 2014 common genes between the two overexpression lines. Functional analysis of these genes revealed their involvement in responses to hormones, biotic/abiotic stresses, transcription regulation, and signal transduction. qRT-PCR of selected candidate target genes validated their differential expression in both overexpression lines. Out of 200 randomly chosen POTH15 targets, 173 were found to have at least one tandem TGAC core motif, characteristic of KNOX interaction, within 3.0kb in the upstream sequence of the transcription start site. Overall, this study provides insights to the role of POTH15 in controlling diverse developmental processes in potato. PMID:27217546

Transcriptomic Analysis Implies That GA Regulates Sex Expression via Ethylene-Dependent and Ethylene-Independent Pathways in Cucumber (Cucumis sativus L.).

PubMed

Zhang, Yan; Zhao, Guiye; Li, Yushun; Mo, Ning; Zhang, Jie; Liang, Yan

2017-01-01

Sex differentiation of flower buds is an important developmental process that directly affects fruit yield of cucumber ( Cucumis sativus L.). Plant hormones, such as gibberellins (GAs) and ethylene can promote development of male and female flowers, respectively, however, the regulatory mechanisms of GA-induced male flower formation and potential involvement of ethylene in this process still remain unknown. In this study, to unravel the genes and gene networks involved in GA-regulated cucumber sexual development, we performed high throughout RNA-Seq analyses that compared the transcriptomes of shoot tips between GA 3 treated and untreated gynoecious cucumber plants. Results showed that GA 3 application markedly induced male flowers but decreased ethylene production in shoot tips. Furthermore, the transcript levels of M ( CsACS2 ) gene, ethylene receptor CsETR1 and some ethylene-responsive transcription factors were dramatically changed after GA 3 treatment, suggesting a potential involvement of ethylene in GA-regulated sex expression of cucumber. Interestingly, GA 3 down-regulated transcript of a C-class floral homeotic gene, CAG2 , indicating that GA may also influence cucumber sex determination through an ethylene-independent process. These results suggest a novel model for hormone-mediated sex differentiation and provide a theoretical basis for further dissection of the regulatory mechanism of male flower formation in cucumber. Statement: We reveal that GA can regulate sex expression of cucumber via an ethylene-dependent manner, and the M ( CsACS2 ), CsETR1 , and ERFs are probably involved in this process. Moreover, CAG2 , a C-class floral homeotic gene, may also participate in GA-modulated cucumber sex determination, but this pathway is ethylene-independent.

In vitro experiments of cerebral blood flow during aspiration thrombectomy: potential effects on cerebral perfusion pressure and collateral flow.

PubMed

Lally, Frank; Soorani, Mitra; Woo, Timothy; Nayak, Sanjeev; Jadun, Changez; Yang, Ying; McCrudden, John; Naire, Shailesh; Grunwald, Iris; Roffe, Christine

2016-09-01

Mechanical thrombectomy with stent retriever devices is associated with significantly better outcomes than thrombolysis alone in the treatment of acute ischemic stroke. Thrombus aspiration achieves high patency rates, but clinical outcomes are variable. The aim of this study was to examine the effect of different suction conditions on perfusate flow during aspiration thrombectomy. A computational fluid dynamics model of an aspiration device within a patent and occluded blood vessel was used to simulate flow characteristics using fluid flow solver software. A physical particulate flow model of a patent vessel and a vessel occluded by thrombus was then used to visualize flow direction and measure flow rates with the aspiration catheter placed 1-10 mm proximal of the thrombus, and recorded on video. The mathematical model predicted that, in a patent vessel, perfusate is drawn from upstream of the catheter tip while, in an occluded system, perfusate is drawn from the vessel proximal to the device tip with no traction on the occlusion distal of the tip. The in vitro experiments confirmed the predictions of this model. In the occluded vessel aspiration had no effect on the thrombus unless the tip of the catheter was in direct contact with the thrombus. These experiments suggest that aspiration is only effective if the catheter tip is in direct contact with the thrombus. If the catheter tip is not in contact with the thrombus, aspirate is drawn from the vessels proximal of the occlusion. This could affect collateral flow in vivo. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

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...

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.

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.

Micropropagation of Lavandula spp.

PubMed

Gonçalves, Sandra; Romano, Anabela

2013-01-01

Lavandula species are some of the most popular ornamental and medicinal plants with great economic values. These species are vegetative propagated by stem cuttings. However, the poor rooting ability and vulnerability of plantlets to contamination are major limiting factors for propagation. In vitro culture methods are suitable to overcome these limitations. This chapter describes protocols for in vitro propagation of Lavandula viridis L'Hér and Lavandula vera DC. Nodal shoot proliferation of L. viridis and plant regeneration from leaf-derived callus of L. vera by an "open culture system" are highlighted.

Micropropagation of Ficus religiosa L. via leaf explants and comparative evaluation of acetylcholinesterase inhibitory activity in the micropropagated and conventionally grown plants.

PubMed

Siwach, Priyanka; Gill, Anita Rani

2014-10-01

A high-frequency, season-independent, in vitro regeneration of Ficusreligiosa was developed, followed by comparative acetylcholinesterase inhibitory (AChEI) activity assay of the in vitro raised and conventionally grown plants. The use of AChEI activity is the most accepted strategy for the treatment of Alzheimer disease. Fully expanded, mature leaves were cut into different segments to initiate the cultures. The middle section of the leaf in vertical orientation with cut portion inserted inside the medium was found most suitable for direct shoot regeneration. Leaf explants responded with nearly consistent frequency (60-66.67 %) throughout the year. To obtain high frequency response with enhanced shoot multiplication rate, 32 plant growth regulator regimes were screened amongst which benzylaminopurine at 5.0 mg/l was found most suitable, yielding 100 % response and maximum number of shoots per explant (7.93); same concentration was also most supportive for repeated multiplication (6.53 shoots). The quality of the shoots and multiplication rate could be significantly enhanced (24.35 shoots) when adenine sulphate, glutamine and phloroglucinol, in an optimised concentration, were additionally supplemented. The clonal nature of the micropropagated plants was confirmed by random amplified polymorphic DNA analysis. A comparative analysis of AChEI activity was carried out amongst the methanolic extracts of stem segments of the mother plant, randomly selected seedlings of different age (4 and 6 months old) of the same mother plant and randomly selected micropropagated plants of different age (3 and 6 months age). The mother plant sample showed effective AChEI activity, with IC 50 of 66.46 μg/ml while seedlings, of different age groups, performed poorly (6-month-old seedlings, Se-1 6M , yielded IC 50 of 20,538.46 μg/ml, while two randomly selected 4 months' aged seedlings, Se-2 4M and Se-3 4M exhibited IC 50 of 19,341.03 and 24,281.70 μg/ml). On the other hand, various micropropagated plants, 2 of 3 months (MiP-1 3M , MiP-2 3M ) and 2 of 6 months (MiP-3 6M and MiP-4 6M ) age behaved like the mother plant, exhibiting IC 50 values of 71.87, 72.91, 67.65 and 69.65 μg/ml, respectively.

Metabolite profiling of flavonols and in vitro antioxidant activity of young shoots of wild Humulus lupulus L. (hop).

PubMed

Maietti, Annalisa; Brighenti, Virginia; Bonetti, Gianpiero; Tedeschi, Paola; Prencipe, Francesco Pio; Benvenuti, Stefania; Brandolini, Vincenzo; Pellati, Federica

2017-08-05

Humulus lupulus L., commonly named hop, is well-known for its sedative and estrogenic activity. While hop cones are widely characterized, only few works have been carried out on the young shoots of this plant. In the light of this, the aim of this study was to identify for the first time the flavonoids present in young hop shoots and to compare the composition of samples harvested from different locations in Northern Italy with their antioxidant activity. The samples were extracted by means of dynamic maceration with methanol. The HPLC-UV/DAD, HPLC-ESI-MS and MS 2 analysis were carried out by using an Ascentis C 18 column (250×4.6mm I.D., 5μm), with a mobile phase composed of 0.1M formic acid in both water and acetonitrile, under gradient elution. Quercetin and kaempferol glycosides were the main compounds identified and quantified in hop shoot extracts. Total flavonols ranged from 2698±185 to 517±48μg/g (fresh weight). The antioxidant activity was determined by means of the radical scavenging activity assay against diphenylpicrylhydrazyl (DPPH) and by using a photochemiluscence assay with a Photochem ® apparatus. The results showed that hop shoots represent a new source of flavonols; therefore, they can be useful for a possible incorporation in the diet as a functional food or applied in the nutraceutical ambit. Copyright © 2017 Elsevier B.V. All rights reserved.

A comparative study of baby immature and adult shoots of Aloe vera on UVB-induced skin photoaging in vitro.

PubMed

Hwang, Eunson; Kim, Su Hyeon; Lee, Sarah; Lee, Choong Hwan; Do, Seon-Gil; Kim, Jinwan; Kim, Sun Yeou

2013-12-01

Ultraviolet (UV) irradiation induces photo-damage of the skin, which in turn causes depletion of the dermal extracellular matrix and chronic alterations in skin structure. Skin wrinkle formations are associated with collagen synthesis and matrix metalloproteinase (MMP) expression. The production of type I procollagen is regulated by transforming growth factor-β1 (TGF-β1) expression; the activation of MMP is also correlated with an increase of interleukin-6 (IL-6). Aloe barbadensis M. (Aloe vera) is widely used in cosmetic and pharmaceutical products. In this study, we examined whether baby aloe shoot extract (BAE, immature aloe extract), which is from the one-month-old shoots of Aloe vera, and adult aloe shoot extract (AE), which is from the four-month-old shoots of Aloe vera, have a protective effect on UVB-induced skin photoaging in normal human dermal fibroblasts (NHDFs). The effects of BAE and AE on UVB-induced photoaging were tested by measuring the levels of reactive oxygen species, MMP-1, MMP-3, IL-6, type I procollagen, and TGF-β1 after UVB irradiation. We found that NHDF cells treated with BAE after UVB-irradiation suppressed MMP-1, MMP-3, and IL-6 levels compared to the AE-treated cells. Furthermore, BAE treatment elevated type I procollagen and TGF-β1 levels. Our results suggest that BAE may potentially protect the skin from UVB-induced damage more than AE. Copyright © 2013 John Wiley & Sons, Ltd.

Evaluating the bioreducing potential of the leaves, knobs and roots of Zanthoxylum capense (small knobwood) for the synthesis of silver nanoparticles, applicable to in vitro fungal contamination control

NASA Astrophysics Data System (ADS)

Bodede, Olusola; Shaik, Shakira; Govinden, Roshini; Moodley, Roshila

2017-12-01

In this study we report on the green synthesis of silver nanoparticles using extracts from selected morphological parts of Zanthoxylum capense. UV-vis spectra of the biosynthesised silver nanoparticles (AgNPs) revealed absorption peaks at around 450 nm, indicative of the nanoparticles’ surface plasmon resonance, whilst infrared vibrational frequencies indicated the presence of flavonoids, alkaloids, and free and bonded sugars which could be responsible for the reduction and stabilisation of the AgNPs. 1H-NMR fingerprinting of the aqueous knob extract confirmed the active bio-reducing phytochemical of the knobs to be 6-O-p-coumaroyl-β-D-glucopyranoside. The nature, shape and morphology of the biosynthesised AgNPs were examined using transmission electron microscopy (TEM), selected area electron diffraction (SAED), scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis. Z. capense AgNPs were mostly spherical in shape with particle sizes in the range of 4-28 nm, 7-20 nm and 4-32 nm for leaves, knobs and roots, respectively. Leaf extracts were the most efficient in the synthesis of AgNPs with an average yield of 0.027 g AgNPs per g of plant (dry mass). The AgNPs were more effective than sodium hypochlorite (NaOCl) and sodium dichloroisocyanurate (NaDCC) in the control of in vitro fungal contamination in nodal explants of Z. capense up to two weeks. Shoots induced from the surface sterilised explants were further used for shoot multiplication on benzyl aminopurine (BAP) and kinetin (KIN). BAP at 0.5 mg l-1 gave the highest percentage (88.6%) of explants bearing shoots with an average of 4.78 shoots per explant. A total of 15 fungal endophyte strains associated with Z. capense were identified using molecular methods.

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

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

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

In Vitro Susceptibility Testing Methods for Caspofungin against Aspergillus and Fusarium Isolates

PubMed Central

Arikan, Sevtap; Lozano-Chiu, Mario; Paetznick, Victor; Rex, John H.

2001-01-01

We investigated the relevance of prominent reduction in turbidity macroscopically (MIC) and formation of aberrant hyphal tips microscopically (minimum effective concentration; MEC) in measuring the in vitro activity of caspofungin against Aspergillus and Fusarium. Caspofungin generated low MICs and MECs against Aspergillus, but not for Fusarium. While MICs increased inconsistently when the incubation time was prolonged, MEC appeared as a stable and potentially relevant endpoint in testing in vitro caspofungin activity. PMID:11120990

In Vitro Propagation and Branching Morphogenesis from Single Ureteric Bud Cells.

PubMed

Yuri, Shunsuke; Nishikawa, Masaki; Yanagawa, Naomi; Jo, Oak D; Yanagawa, Norimoto

2017-02-14

A method to maintain and rebuild ureteric bud (UB)-like structures from UB cells in vitro could provide a useful tool for kidney regeneration. We aimed in our present study to establish a serum-free culture system that enables the expansion of UB progenitor cells, i.e., UB tip cells, and reconstruction of UB-like structures. We found that fibroblast growth factors or retinoic acid (RA) was sufficient for the survival of UB cells in serum-free condition, while the proliferation and maintenance of UB tip cells required glial cell-derived neurotrophic factor together with signaling from either WNT-β-catenin pathway or RA. The activation of WNT-β-catenin signaling in UB cells by endogenous WNT proteins required R-spondins. Together with Rho kinase inhibitor, our culture system facilitated the expansion of UB tip cells to form UB-like structures from dispersed single cells. The UB-like structures thus formed retained the original UB characteristics and integrated into the native embryonic kidneys. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Micro-PIXE studies of elemental distribution in Cd-accumulating Brassica juncea L.

NASA Astrophysics Data System (ADS)

Schneider, Thorsten; Haag-Kerwer, Angela; Maetz, Mischa; Niecke, Manfred; Povh, Bogdan; Rausch, Thomas; Schüßler, Arthur

1999-10-01

Brassica juncea L. is a high biomass producing crop plant, being able to accumulate Cd and other heavy metals in their roots and shoots. It is a good candidate for efficient phytoextraction of heavy metals - such as Cd - from polluted soils. PIXE and STIM analyses were applied to investigate Cd-uptake in roots and the resulting effects on the elemental distribution of Cd stressed plants. The axial distribution of trace elements as a function of distance from the root tip as well as the radial distribution within cross-sections were analysed. The results are compared with the elemental distribution in control plants.

Photoshop tips and tricks every facial plastic surgeon should know.

PubMed

Hamilton, Grant S

2010-05-01

Postprocessing of patient photographs is an important skill for the facial plastic surgeon. Postprocessing is intended to optimize the image, not change the surgical result. This article refers to use of Photoshop CS3 (Adobe Systems Incorporated, San Jose, CA, USA) for descriptions, but any recent version of Photoshop is sufficiently similar. Topics covered are types of camera, shooting formats, color balance, alignment of preoperative and postoperative photographs, and preparing figures for publication. Each section presents step-by-step guidance and instructions along with a graphic depiction of the computer screen and Photoshop tools under discussion. Copyright 2010 Elsevier Inc. All rights reserved.

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.

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 .

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),...

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.

Three-dimensional analyses of ultrasonic scaler oscillations.

PubMed

Lea, Simon C; Felver, Bernhard; Landini, Gabriel; Walmsley, A Damien

2009-01-01

It is stated that the oscillation patterns of dental ultrasonic scalers are dependent upon whether the instrument is of a magnetostrictive or piezoelectric design. These patterns are then linked to differences in root surface debridement in vitro. Piezoelectric (A, P) and magnetostrictive (Slimline, TFI-3) ultrasonic scalers (three of each) were evaluated, loaded (100 g/200 g) and unloaded with a 3D laser vibrometer. Loads were applied to the probe tips via teeth mounted in a load-measuring device. Elliptical motion was demonstrated for all probes under loaded and unloaded conditions. Loading flattened the elliptical motion along the length of the probe. Unloaded, Slimline tip 1 was significantly different to tips 2 and 3 (p<0.0001). There were no differences between the A-tips (p>0.207). All TFI-3 tips were different to each other (p<0.0001). P-tips 1 and 2 were different to each other (p=0.046). Loaded, Slimline tips were different to each other (p<0.001). There were no differences between the P probes (p>0.867). Generator power increased all Slimline and P tip vibrations (p<0.0001). Probe oscillation patterns are independent of ultrasound production mechanism and are dependent upon probe shape and generator power. Loaded probes oscillated with an elliptical pattern.

Automated On-tip Affinity Capture Coupled with Mass Spectrometry to Characterize Intact Antibody-Drug Conjugates from Blood

NASA Astrophysics Data System (ADS)

Li, Ke Sherry; Chu, Phillip Y.; Fourie-O'Donohue, Aimee; Srikumar, Neha; Kozak, Katherine R.; Liu, Yichin; Tran, John C.

2018-05-01

Antibody-drug conjugates (ADCs) present unique challenges for ligand-binding assays primarily due to the dynamic changes of the drug-to-antibody ratio (DAR) distribution in vivo and in vitro. Here, an automated on-tip affinity capture platform with subsequent mass spectrometry analysis was developed to accurately characterize the DAR distribution of ADCs from biological matrices. A variety of elution buffers were tested to offer optimal recovery, with trastuzumab serving as a surrogate to the ADCs. High assay repeatability (CV 3%) was achieved for trastuzumab antibody when captured below the maximal binding capacity of 7.5 μg. Efficient on-tip deglycosylation was also demonstrated in 1 h followed by affinity capture. Moreover, this tip-based platform affords higher throughput for DAR characterization when compared with a well-characterized bead-based method.

Micropropagation and organogenesis of Anthurium andreanum Lind cv Rubrun.

PubMed

Maira, Oropeza; Alexander, Mejías; Vargas, Teresa Edith

2010-01-01

Tissue culture techniques are routinely used for mass propagation and the establishment of disease free stock material. Virtually all pot type Anthuriums available in the market today are produced by tissue culture. In this chapter, we describe an efficient protocol to obtain Anthurium andreanum cv Rubrun vitro plants through micropropagation and organogenesis. Seeds from plant spadixes were germinated on MS medium supplemented with 0.5 mg/L BA. Micro-cuttings from in vitro germinated seedlings were subcultured on MS medium containing 2 mg/L BA and 0.5 mg/L NAA. Four-week-old in vitro plants obtained from microcuttings, showed callus proliferation at the stem base. The development of shoots and plantlets was observed from callus tissue. We also describe a detailed method for the histological analysis of callus tissue and a vitro plants acclimatization protocol.

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.

Inhibition of low-density lipoprotein oxidation and up-regulation of low-density lipoprotein receptor in HepG2 cells by tropical plant extracts.

PubMed

Salleh, Mohd Nizar; Runnie, Irine; Roach, Paul D; Mohamed, Suhaila; Abeywardena, Mahinda Y

2002-06-19

Twelve edible plant extracts rich in polyphenols were screened for their potential to inhibit oxidation of low-density lipoprotein (LDL) in vitro and to modulate LDL receptor (LDLr) activity in cultured HepG2 cells. The antioxidant activity (inhibition of LDL oxidation) was determined by measuring the formation of conjugated dienes (lag time) and thiobarbituric acid reagent substances (TBARS). Betel leaf (94%), cashew shoot (63%), Japanese mint (52%), semambu leaf (50%), palm frond (41%), sweet potato shoot, chilli fruit, papaya shoot, roselle calyx, and maman showed significantly increased lag time (>55 min, P < 0.05) and inhibition of TBARS formation (P < 0.05) compared to control. LDLr was significantly up-regulated (P < 0.05) by Japanese mint (67%), semambu (51%), cashew (50%), and noni (49%). Except for noni and betel leaf, most plant extracts studied demonstrated a positive association between antioxidant activity and the ability to up-regulate LDL receptor. Findings suggest that reported protective actions of plant polyphenols on lipoprotein metabolism might be exerted at different biochemical mechanisms.

Phosphorylation regulates the water channel activity of the seed-specific aquaporin alpha-TIP.

PubMed

Maurel, C; Kado, R T; Guern, J; Chrispeels, M J

1995-07-03

The vacuolar membrane protein alpha-TIP is a seed-specific protein of the Major Intrinsic Protein family. Expression of alpha-TIP in Xenopus oocytes conferred a 4- to 8-fold increase in the osmotic water permeability (Pf) of the oocyte plasma membrane, showing that alpha-TIP forms water channels and is thus a new aquaporin. alpha-TIP has three putative phosphorylation sites on the cytoplasmic side of the membrane (Ser7, Ser23 and Ser99), one of which (Ser7) has been shown to be phosphorylated. We present several lines of evidence that the activity of this aquaporin is regulated by phosphorylation. First, mutation of the putative phosphorylation sites in alpha-TIP (Ser7Ala, Ser23Ala and Ser99Ala) reduced the apparent water transport activity of alpha-TIP in oocytes, suggesting that phosphorylation of alpha-TIP occurs in the oocytes and participates in the control of water channel activity. Second, exposure of oocytes to the cAMP agonists 8-bromoadenosine 3',5'-cyclic monophosphate, forskolin and 3-isobutyl-1-methylxanthine, which stimulate endogenous protein kinase A (PKA), increased the water transport activity of alpha-TIP by 80-100% after 60 min. That the protein can be phosphorylated by PKA was demonstrated by phosphorylating alpha-TIP in isolated oocyte membranes with the bovine PKA catalytic subunit. Third, the integrity of the three sites at positions 7, 23 and 99 was necessary for the cAMP-dependent increase in the Pf of oocytes expressing alpha-TIP, as well as for in vitro phosphorylation of alpha-TIP. These findings demonstrate that the alpha-TIP water channel can be modulated via phosphorylation of Ser7, Ser23 and Ser99.(ABSTRACT TRUNCATED AT 250 WORDS)

Difference in in vitro response and esculin content in two populations of Taraxacum officinale Weber.

PubMed

Jamshieed, Sumiya; Das, Sandip; Sharma, M P; Srivastava, P S

2010-12-01

In vitro micropropagation has been achieved in medicinally important plant, Taraxacum officinale collected from two different regions, Kashmir (J & K) and Garhwal (Uttarakhand). Leaf segments inoculated on MS supplemented with different combinations of Indole-3-acetic acid (IAA) and Benzyladenine (BA) produced indirect regeneration. For root induction MS fortified with Indole-3-butyric acid (IBA) was used. Taraxacum officinale collected from Garhwal responded two weeks earlier and showed shoot regeneration whereas in Kashmir population only callus proliferation occurred. Esculin content was also higher in the samples from Garhwal. The content was affected by both, the hormone concentration as well as age of the cultures. RAPD of the in vitro raised regenerants confirmed genetic stability.

Plant regeneration via direct somatic embryogenesis from leaf explants of Tolumnia Louise Elmore 'Elsa'.

PubMed

Shen, Hui-Ju; Chen, Jen-Tsung; Chung, Hsiao-Hang; Chang, Wei-Chin

2018-01-22

Tolumnia genus (equitant Oncidium) is a group of small orchids with vivid flower color. Thousands of hybrids have been registered on Royal Horticulture Society and showed great potential for ornamental plant market. The aim of this study is to establish an efficient method for in vitro propagation. Leaf explants taken from in vitro-grown plants were used to induce direct somatic embryogenesis on a modified 1/2 MS medium supplemented with five kinds of cytokinins, 2iP, BA, kinetin, TDZ and zeatin at 0.3, 1 and 3 mg l -1 in darkness. TDZ at 3 mg l -1 gave the highest percentage of explants with somatic globular embryos after 90 days of culture. It was found that 2,4-D and light regime highly retarded direct somatic embryogenesis and showed 95-100% of explant browning. Histological observations revealed that the leaf cells divided into meristematic cells firstly, followed by somatic proembryos, and then somatic globular embryos. Eventually, somatic embryos developed a bipolar structure with the shoot apical meristem and the root meristem. Scanning electron microscopy observations showed that the direct somatic embryogenesis from leaf explants was asynchronously. The somatic embryos were found on the leaf tip, the adaxial surface and also the mesophyll through a cleft, and it reflected the heterogeneity of the explant. The 90-day-old globular embryos were detached from the parent explants and transferred onto a hormone-free 1/2 MS medium in light condition for about 1 month to obtain 1-cm-height plantlets. After another 3 months for growth, the plantlets were potted with Sphagnum moss and were acclimatized in a shaded greenhouse. After 1 month of culture, the survival rate was 100%. In this report, a protocol for efficient regenerating a Tolumnia orchid, Louise Elmore 'Elsa', was established via direct somatic embryogenesis and might reveal an alternative approach for mass propagation of Tolumnia genus in orchid industry.

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

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.

Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers.

PubMed

Sreedhar, Reddampalli V; Venkatachalam, Lakshmanan; Bhagyalakshmi, Neelwarne

2007-08-01

Occurrence of genetic variants during micropropagation is occasionally encountered when the cultures are maintained in vitro for long period. Therefore, the micropropagated multiple shoots of Vanilla planifolia Andrews developed from axillary bud explants established 10 years ago were used to determine somaclonal variation using random amplified polymorphic DNA (RAPD) and intersimple sequence repeats markers (ISSR). One thousand micro-plants were established in soil of which 95 plantlets (consisting of four phenotypes) along with the mother plant were subjected to genetic analyses using RAPD and ISSR markers. Out of the 45 RAPD and 20 ISSR primers screened, 30 RAPD and 7 ISSR primers showed 317 clear, distinct and reproducible band classes resulting in a total of 30 115 bands. However, no difference was observed in banding patterns of any of the samples for a particular primer, indicating the absence of variation among the micropropagated plants. Our results allow us to conclude that the micropropagation protocol that we have used for in vitro proliferation of vanilla plantlets for the last 10 years might be applicable for the production of clonal plants over a considerable period of time.

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

Differential accumulation of hyperforin and secohyperforin in Hypericum perforatum tissue cultures.

PubMed

Charchoglyan, Armen; Abrahamyan, Arusyak; Fujii, Isao; Boubakir, Zakia; Gulder, Tobias A M; Kutchan, Toni M; Vardapetyan, Hrachik; Bringmann, Gerhard; Ebizuka, Yutaka; Beerhues, Ludger

2007-11-01

Hyperforin is a pharmacologically active constituent of Hypericum perforatum (St. John's wort). In vitro cultures of this medicinal plant were found to contain hyperforin and three related polyprenylated acylphloroglucinol derivatives. The accumulation of these compounds was coupled to shoot regeneration, with secohyperforin being the major constituent in morphogenic cultures. The structure of secohyperforin was elucidated online by LC-DAD, -MS, and -NMR. In multiple shoot cultures, the ratio of hyperforin to secohyperforin was strongly influenced by the phytohormones N6-benzylaminopurine (BAP) and naphthalene-1-acetic acid (NAA). While increasing concentrations of BAP stimulated the formation of hyperforin, increasing concentrations of NAA elevated the level of secohyperforin. No differential stimulation was observed after elicitor treatment. Hyperforin and secohyperforin are proposed to arise from a branch point in the biosynthetic pathway.

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.

Thermal study of bare tips with various system parameters and incision sizes.

PubMed

Osher, Robert H; Injev, Valentine P

2006-05-01

To identify major and minor surgeon-controlled parameters that affect incision temperature when performing microincision lens removal using the Alcon Infiniti Vision System. In vitro research and development laboratory, Alcon Research, Irvine, California, USA. Phacoemulsification was performed in eye-bank cadaver eyes and the following parameters evaluated: incision, duty cycle, ultrasound (US) power, aspiration flow rate (AFR), vacuum, pulse, bottle height and balanced salt solution temperature, and tip design/size. Each parameter was varied while the others remained constant. The resulting temperature of the incision and US tip was measured using a thermal camera. Major contributors to elevated incision temperature included incision size, US power, duty cycle, AFR, vacuum setting, tip design, and presence of an ophthalmic viscosurgical device (OVD). Minor contributors included pulse frequency, bottle height, and temperature of the infusate. Microincision lens removal can be performed at safe temperatures with the knowledgeable selection of surgeon-controlled parameters.

Bacillus pumilus ES4: candidate plant growth-promoting bacterium to enhance establishment of plants in mine tailings

PubMed Central

de-Bashan, Luz E.; Hernandez, Juan-Pablo; Bashan, Yoav; Maier, Raina

2014-01-01

Three plant growth-promoting bacteria (PGPB; Bacillus pumilus ES4, B. pumilus RIZO1, and Azospirillum brasilense Cd) were tested for their ability to enhance plant growth and development of the native Sonoran Desert shrub quailbush (Atriplex lentiformis) and for their effect on the native bacterial community in moderately acidic, high-metal content (AHMT) and in neutral, low metal content natural tailings (NLMT) in controlled greenhouse experiments. Inoculation of quailbush with all three PGPB significantly enhanced plant growth parameters, such as germination, root length, dry weight of shoots and roots, and root/shoot ratio in both types of tailings. The effect of inoculation on the indigenous bacterial community by the most successful PGPB Bacillus pumilus ES4 was evaluated by denaturating gradient gel electrophoresis (PCR-DGGE) fingerprinting and root colonization was followed by specific fluorescent in situ hybridization (FISH). Inoculation with this strain significantly changed the bacterial community over a period of 60 days. FISH analysis showed that the preferred site of colonization was the root tips and root elongation area. This study shows that inoculation of native perennial plants with PGPB can be used for developing technologies for phytostabilizing mine tailings. PMID:25009362

Artificially induced polyploidization in Humulus lupulus L. and its effect on morphological and chemical traits.

PubMed

Trojak-Goluch, Anna; Skomra, Urszula

2013-12-01

Chemically induced polyploids were obtained by the colchicine treatment of shoot tips of Humulus lupulus L. 'Sybilla'. Flow cytometry revealed that most of the treatments resulted in the production of tetraploids. The highest number of tetraploids was obtained when explants were immersed in 0.05% colchicine for 48 h. A field experiment was conducted to compare diploid and tetraploid plants and assess the effect of genome polyploidization on the morphological and chemical characteristics. Tetraploids showed significant differences in relation to diploids. They had thinner and shorter shoots. The influence of chromosome doubling was also reflected in the length, width and area of leaves. The length of female flowers in the tetraploids was significantly shorter than that observed in diploids. Tetraploids produced a diverse number of lupuline glands that were almost twice as large as those observed in diploids. The most distinct effect of genome polyploidization was a significant increase in the weight of cones and spindles. Contents of major chemical constituents of hop cones was little affected by ploidy level. Total essential oils were significantly lower than those in diploids. However there was a significant increase in the proportion of humulene, caryophyllene and farnesene, oils desired by the brewing industry.

Iron deficiency regulated OsOPT7 is essential for iron homeostasis in rice.

PubMed

Bashir, Khurram; Ishimaru, Yasuhiro; Itai, Reiko Nakanishi; Senoura, Takeshi; Takahashi, Michiko; An, Gynheung; Oikawa, Takaya; Ueda, Minoru; Sato, Aiko; Uozumi, Nobuyuki; Nakanishi, Hiromi; Nishizawa, Naoko K

2015-05-01

The molecular mechanism of iron (Fe) uptake and transport in plants are well-characterized; however, many components of Fe homeostasis remain unclear. We cloned iron-deficiency-regulated oligopeptide transporter 7 (OsOPT7) from rice. OsOPT7 localized to the plasma membrane and did not transport Fe(III)-DMA or Fe(II)-NA and GSH in Xenopus laevis oocytes. Furthermore OsOPT7 did not complement the growth of yeast fet3fet4 mutant. OsOPT7 was specifically upregulated in response to Fe-deficiency. Promoter GUS analysis revealed that OsOPT7 expresses in root tips, root vascular tissue and shoots as well as during seed development. Microarray analysis of OsOPT7 knockout 1 (opt7-1) revealed the upregulation of Fe-deficiency-responsive genes in plants grown under Fe-sufficient conditions, despite the high Fe and ferritin concentrations in shoot tissue indicating that Fe may not be available for physiological functions. Plants overexpressing OsOPT7 do not exhibit any phenotype and do not accumulate more Fe compared to wild type plants. These results indicate that OsOPT7 may be involved in Fe transport in rice.

Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia.

PubMed

Suwińska, Anna; Wasąg, Piotr; Zakrzewski, Przemysław; Lenartowska, Marta; Lenartowski, Robert

2017-05-01

Calreticulin is involved in stabilization of the tip-focused Ca 2+ gradient and the actin cytoskeleton arrangement and function that is required for several key processes driving Petunia pollen tube tip growth. Although the precise mechanism is unclear, stabilization of a tip-focused calcium (Ca 2+ ) gradient seems to be critical for pollen germination and pollen tube growth. We hypothesize that calreticulin (CRT), a Ca 2+ -binding/buffering chaperone typically residing in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, is an excellent candidate to fulfill this role. We previously showed that in Petunia pollen tubes growing in vitro, CRT is translated on ER membrane-bound ribosomes that are abundant in the subapical zone of the tube, where CRT's Ca 2+ -buffering and chaperone activities might be particularly required. Here, we sought to determine the function of CRT using small interfering RNA (siRNA) to, for the first time in pollen tubes growing in vitro, knockdown expression of a gene. We demonstrate that siRNA-mediated post-transcriptional silencing of Petunia hybrida CRT gene (PhCRT) expression strongly impairs pollen tube growth, cytoplasmic zonation, actin cytoskeleton organization, and the tip-focused Ca 2+ gradient. Moreover, reduction of CRT alters the localization and disturbs the structure of the ER in abnormally elongating pollen tubes. Finally, cytoplasmic streaming is inhibited, and most of the pollen tubes rupture. Our data clearly show an interplay between CRT, Ca 2+ gradient, actin-dependent cytoplasmic streaming, organelle positioning, and vesicle trafficking during pollen tube elongation. Thus, we suggest that CRT functions in Petunia pollen tube growth by stabilizing Ca 2+ homeostasis and acting as a chaperone to assure quality control of glycoproteins passing through the ER.

Rational design and validation of a Tip60 histone acetyltransferase inhibitor

NASA Astrophysics Data System (ADS)

Gao, Chunxia; Bourke, Emer; Scobie, Martin; Famme, Melina Arcos; Koolmeister, Tobias; Helleday, Thomas; Eriksson, Leif A.; Lowndes, Noel F.; Brown, James A. L.

2014-06-01

Histone acetylation is required for many aspects of gene regulation, genome maintenance and metabolism and dysfunctional acetylation is implicated in numerous diseases, including cancer. Acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases and currently, few general HAT inhibitors have been described. We identified the HAT Tip60 as an excellent candidate for targeted drug development, as Tip60 is a key mediator of the DNA damage response and transcriptional co-activator. Our modeling of Tip60 indicated that the active binding pocket possesses opposite charges at each end, with the positive charges attributed to two specific side chains. We used structure based drug design to develop a novel Tip60 inhibitor, TH1834, to fit this specific pocket. We demonstrate that TH1834 significantly inhibits Tip60 activity in vitro and treating cells with TH1834 results in apoptosis and increased unrepaired DNA damage (following ionizing radiation treatment) in breast cancer but not control cell lines. Furthermore, TH1834 did not affect the activity of related HAT MOF, as indicated by H4K16Ac, demonstrating specificity. The modeling and validation of the small molecule inhibitor TH1834 represents a first step towards developing additional specific, targeted inhibitors of Tip60 that may lead to further improvements in the treatment of breast cancer.

Dilute-and-shoot coupled to nanoflow liquid chromatography high resolution mass spectrometry for the determination of drugs of abuse and sport drugs in human urine.

PubMed

Alcántara-Durán, Jaime; Moreno-González, David; Beneito-Cambra, Miriam; García-Reyes, Juan F

2018-05-15

In this work, a sensitive nanoflow liquid chromatography high-resolution mass spectrometry screening method has been developed for the determination of multiclass drugs of abuse and sport drugs in human urine. 81 drugs belonging to different multiclass pharmaceuticals were targeted. The method is based on the use of a nanoLC column (75 µm × 150 mm, 3 µm particle size and 100 Å pore) with the nanospray emitter tip integrated so that dead volumes are significantly minimized. Data acquisition method included both full-scan and all ion fragmentation experiments using an Orbitrap analyser (Q-Exactive) operated in the positive ionization mode. To increase laboratory throughput, a dilute-and-shoot methodology has been tested and proposed, based solely on direct urine dilution without further sample workup. Matrix effects were evaluated, showing a negligible effect for all studied compounds when a dilution 1:50 was implemented. Despite this high-dilution factor, limits of quantification were still satisfactory, with values below 5 µg L -1 in most cases, being lower than their minimum required performance limits correspond established by the World Anti-Doping Agency. Therefore, the use of the dilute-and-shoot method with the enhanced sensitivity provided by nanoflow LC setup could be useful tool for the determination of studied compounds in drug testing, thus increasing laboratory performance, because a minimum sample treatment steps are required. Copyright © 2018 Elsevier B.V. All rights reserved.

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

In Vitro Conservation of Sweet Potato Genotypes

PubMed Central

Arrigoni-Blank, Maria de Fátima; Tavares, Fernanda Ferreira; dos Santos, Maria Clézia; Menezes, Thays Saynara Alves; de Santana, Aléa Dayane Dantas

2014-01-01

The aim of this study was to develop a protocol for the in vitro conservation of sweet potato genotypes using the slow growth technique. The first experiment was conducted in a 4 × 5 × 2 factorial scheme, testing four genotypes (IPB-007, IPB-052, IPB-072, and IPB-137), five concentrations of abscisic acid (ABA) (0.0, 1.0, 2.0, 4.0, and 8.0 mg·L−1), and two temperatures (18 and 25°C). The second experiment was conducted in a 4 × 3 × 3 factorial scheme at 18°C, testing four genotypes (IPB-007, IPB-052, IPB-072, and IPB-137), three variations of MS salts (50, 75, and 100%), and three concentrations of sucrose (10, 20, and 30 g·L−1). Every three months, we evaluated the survival (%), shoot height, and shoot viability. In vitro conservation of the sweet potato genotypes IPB-052 and IPB-007 was obtained over three and six months, respectively, using MS medium plus 2.0 mg·L−1 of ABA at either 18 or 25°C. Genotypes IPB-072 and IPB-137 can be kept for three and six months, respectively, in MS medium without ABA at 18°C. It is possible to store IPB-052 and IPB-072 for six months and IPB-007 and IPB-137 for nine months using 30 g·L−1 of sucrose and 50% MS salts. PMID:24563627

Somatic embryogenesis in cell cultures of Glycine species.

PubMed

Gamborg, O L; Davis, B P; Stahlhut, R W

1983-08-01

This report describes the development of procedures for the production of somatic embryos in cell cultures of Glycine species including soybean. The conditions for callus induction and initiation of rapidly growing cell suspension cultures were defined. Methods for inducing embryogenesis were tested on 16 lines of several Glycine species and cultivars of soybean. The SB-26 Culture of a G. soja gave the best results and was used in the experiments. Embryogenesis required the presence of picloram or 2,4-D. AMO 1618, CCC, PP-333 and Ancymidol enhanced the embryogenesis frequency. Plants of the G. soja (SB-26) were grown to maturity from seed-derived shoot tips. Characteristics of the plants are discussed.

Flowering in Vitis: Conversion of tendrils into inflorescences and bunches of grapes.

PubMed

Srinivasan, C; Mullins, M G

1979-01-01

Inflorescences and fruits with viable seeds were produced in place of tendrils in plants of Vitis vinifera L. cv. "Muscat of Alexandria" and in a staminate hybrid grapevine (Vitis vinifera x V. rupestris Scheele) following repeated applications of 10-20 μl of 50-200 μM 6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-purine (PBA) to apices. Young leaves, shoot tips and axillary buds were removed before the PBA treatments were commenced. The number and weight of berries produced by inflorescences derived from tendrils was closely correlated with the number and area of leaves retained. When application of PBA was continued after floral initiation there was formation of fused flowers and cleistogamous pollination.

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.

Ion transport: Tipping a cell's ionic balance

NASA Astrophysics Data System (ADS)

Davis, Jeffery T.

2014-10-01

A synthetic compound that transports chloride across membranes can kill both normal cells and cancer cells in vitro. The transporter works together with sodium channels to move NaCl into the cells, which triggers cell death.

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.

Novel histone deacetylase inhibitor N25 exerts anti-tumor effects and induces autophagy in human glioma cells by inhibiting HDAC3

PubMed Central

Sun, Xin-Yuan; Qu, Yue; Ni, An-Ran; Wang, Gui-Xiang; Huang, Wei-Bin; Chen, Zhong-Ping; Lv, Zhu-Fen; Zhang, Song; Lindsay, Holly; Zhao, Sibo; Li, Xiao-Nan; Feng, Bing-Hong

2017-01-01

N25, a novel histone deacetylase inhibitor, was created through structural modification of suberoylanilide hydroxamic acid. To evaluate the anti-tumor activity of N25 and clarify its molecular mechanism of inducing autophagy in glioma cells, we investigated its in vitro anti-proliferative effect and in vivo anticancer effect. Moreover, we detected whether N25 induces autophagy in glioma cells by transmission electron microscope and analyzed the protein expression level of HDAC3, Tip60, LC3 in glioma samples by western blot. We additionally analyzed the protein expression level of HDAC3, Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment with N25 in glioma cells. Our results showed that the anti-tumor activity of N25 in glioma cells is slightly stronger than SAHA both in vitro and in vivo. We found that N25 induced autophagy, and HDAC3 was significantly elevated and Tip60 and LC3 significantly decreased in glioma samples compared with normal brain tissues. Nevertheless, N25 inhibited HDAC3 and up-regulated the protein expression of Tip60, ULK1 (Atg1), and Beclin-1 (Atg6) after treatment of glioma cells with N25. In conclusion, these data suggest that N25 has striking anti-tumor activity in part due to inhibition of HDAC3. Additionally, N25 may induce autophagy through inhibiting HDAC3. PMID:29088860

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo.

PubMed

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-10-28

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

PubMed Central

Stenzel, Denise; Franco, Claudio A; Estrach, Soline; Mettouchi, Amel; Sauvaget, Dominique; Rosewell, Ian; Schertel, Andreas; Armer, Hannah; Domogatskaya, Anna; Rodin, Sergey; Tryggvason, Karl; Collinson, Lucy; Sorokin, Lydia; Gerhardt, Holger

2011-01-01

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo. PMID:21979816

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.

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.

Effect of benzyl amino purine and indole-3-acetic acid on propagation of Sterculia foetida in vitro

NASA Astrophysics Data System (ADS)

Yuniastuti, E.; Widodo, C. E.; Samanhudi; Delfianti, M. N. I.

2018-03-01

Sterculia foetida is an oval seed plants that can be used as biofuel, which is one of the environmental friendly fuels. This plant is quite hard to find because not many peoples cultivate the plants. An in vitro propagation is one way to preserve the plant. This research aimed to determine optimum concentration of benzyl amino purine (BAP) and indole-3-acetic acid (IAA) to propagate S. foetida in vitro. The results showed that woody plant medium (WPM) added by 4 mg L BAP-1 and 0.5 mg L IAA-1 was able to produce complete plantlet, whereas those added by 4 mg L BAP-1 and 1 mg L IAA-1 generated the best growth of shoot and leaves.

Development of efficient plant regeneration and transformation system for impatiens using Agrobacterium tumefaciens and multiple bud cultures as explants.

PubMed

Dan, Yinghui; Baxter, Aaron; Zhang, Song; Pantazis, Christopher J; Veilleux, Richard E

2010-08-09

Impatiens (Impatiens walleriana) is a top selling floriculture crop. The potential for genetic transformation of Impatiens to introduce novel flower colors or virus resistance has been limited by its general recalcitrance to tissue culture and transformation manipulations. We have established a regeneration and transformation system for Impatiens that provides new alternatives to genetic improvement of this crop. In a first step towards the development of transgenic INSV-resistant Impatiens, we developed an efficient plant regeneration system using hypocotyl segments containing cotyledonary nodes as explants. With this regeneration system, 80% of explants produced an average of 32.3 elongated shoots per initial explant plated, with up to 167 elongated shoots produced per explant. Rooting efficiency was high, and 100% of shoots produced roots within 12 days under optimal conditions, allowing plant regeneration within approximately 8 weeks. Using this regeneration system, we developed an efficient Agrobacterium-mediated Impatiens transformation method using in vitro multiple bud cultures as explants and a binary plasmid (pHB2892) bearing gfp and nptII genes. Transgenic Impatiens plants, with a frequency up to 58.9%, were obtained within 12 to 16 weeks from inoculation to transfer of transgenic plants to soil. Transgenic plants were confirmed by Southern blot, phenotypic assays and T1 segregation analysis. Transgene expression was observed in leaves, stems, roots, flowers, and fruit. The transgenic plants were fertile and phenotypically normal. We report the development of a simple and efficient Agrobacterium-mediated transformation system for Impatiens. To the best of our knowledge, there have been no reports of Agrobacterium-mediated transformation of Impatiens with experimental evidence of stable integration of T-DNA and of Agrobacterium-mediated transformation method for plants using in vitro maintained multiple bud cultures as explants. This transformation system has the advantages of 1) efficient, simple and rapid regeneration and transformation (with no need for sterilization or a greenhouse to grow stock plants), 2) flexibility (available all the time) for in vitro manipulation, 3) uniform and desirable green tissue explants for both nuclear and plastid transformation using Agrobacterium-mediated and biolistics methods, 4) no somaclonal variation and 5) resolution of necrosis of Agrobacterium-inoculated tissues.

A novel RNA binding surface of the TAM domain of TIP5/BAZ2A mediates epigenetic regulation of rRNA genes.

PubMed

Anosova, Irina; Melnik, Svitlana; Tripsianes, Konstantinos; Kateb, Fatiha; Grummt, Ingrid; Sattler, Michael

2015-05-26

The chromatin remodeling complex NoRC, comprising the subunits SNF2h and TIP5/BAZ2A, mediates heterochromatin formation at major clusters of repetitive elements, including rRNA genes, centromeres and telomeres. Association with chromatin requires the interaction of the TAM (TIP5/ARBP/MBD) domain of TIP5 with noncoding RNA, which targets NoRC to specific genomic loci. Here, we show that the NMR structure of the TAM domain of TIP5 resembles the fold of the MBD domain, found in methyl-CpG binding proteins. However, the TAM domain exhibits an extended MBD fold with unique C-terminal extensions that constitute a novel surface for RNA binding. Mutation of critical amino acids within this surface abolishes RNA binding in vitro and in vivo. Our results explain the distinct binding specificities of TAM and MBD domains to RNA and methylated DNA, respectively, and reveal structural features for the interaction of NoRC with non-coding RNA. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

[In vitro regeneration and applications using vegetable cell and tissue culture].

PubMed

Jordán, M

1990-10-01

Plant cells by means of their totipotency and aided by in vitro culture techniques can be induced to perform morphogenesis leading to somatic embryoids and massive clonal multiplication; microspores or pollen can be triggered to recover haploid plants, then characters expressed via haploidy can be selected and fixed. Protoplasts from different species can lead to recombinations. We report here work done on Carica pubescens, where somatic embryoids were obtained from cells; in Prunus avium androgenesis leading to pollen calli was triggered, while plants were recovered from Nicotiana tabacum anthers. Fusion products were obtained using C. pubescens and C. papaya protoplasts, leading up to calli and shoots.

Sunflower (Helianthus annuus L.).

PubMed

Radonic, Laura M; Lewi, Dalia M; López, Nilda E; Hopp, H Esteban; Escandón, Alejandro S; Bilbao, Marisa López

2015-01-01

Sunflower (Helianthus annuus L.) is still considered as a recalcitrant species to in vitro culture and transformation in spite of the publication of different protocols. Here we describe a routine transformation system of this crop which requires mature HA89 genotype seeds and Agrobacterium tumefaciens EHA105 strain for gene delivery, being both easily available. Selection of transformed shoots depends on root development in kanamycin-selective media, instead of shoot color, avoiding selection of escapes. The establishment of this protocol proved successful for the incorporation of both reporter and agronomic important genes and also for the evaluation of the specific expression patterns of different promoters in transgenic sunflower plants. Stable expression of the incorporated transgenes was confirmed by RT-PCR and GUS reporter gene visualization. Stable inheritance of transgenes was successfully followed until T2 generation in several independent lines.

Anti-freezing-protein type III strongly influences the expression of relevant genes in cryopreserved potato shoot tips.

PubMed

Seo, Ji Hyang; Naing, Aung Htay; Jeon, Su Min; Kim, Chang Kil

2018-06-04

AFP improved cryopreservation efficiency of potato (Solanum tuberosum cv. Superior) by regulating transcript levels of CBF1 and DHN1. However, the optimal AFP concentration required for strong induction of the genes was dependent on the type of vitrification solution to which the AFP was added: This finding suggests that AFP increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Despite the availability of many studies reporting the benefits of anti-freeze protein III (AFP III) as a cryoprotectant, the role of AFP III in this process has not been well demonstrated using molecular analysis. In addition, AFP III has not been exploited in the cryopreservation of potato thus far. Therefore, we studied the effects of AFP III on the cryopreservation of potato (Solanum tuberosum cv. Superior). We found that CBF1 and DHN1 genes are low temperature-inducible in potato leaves (S. tuberosum cv. Superior). Transcript levels of these genes expressed in shoot tips cryopreserved with AFP III were higher than those of the controls. However, the optimal AFP III concentration required for strong induction of the genes was dependent on the type of cryoprotection solution to which the AFP III was added: 500 ng/mL worked best for PVS2, while 1500 ng/mL was optimal for LS. Interestingly, the involvement of AFP III in the cryoprotection solutions improved cryopreservation efficiency as compared to the control, and expression levels of the detected genes were associated with cryopreservation efficiency. This finding suggests that AFP III increased cryopreservation efficiency by transcriptional regulation of these genes, which might protect plant cell membranes from cold stress during cryopreservation. Therefore, we expect that our findings will lead to the successful application of AFP III as a potent cryoprotectant in the cryopreservation of rare and commercially important plant germplasms.

CLIMATE CONDITIONS AFFECTING THE WITHIN-PLANT SPREAD OF BROAD MITES ON AZALEA.

PubMed

Mechant, E; Pauwels, E; Gobin, B

2014-01-01

The broad mite Polyphagotarsonemus latus (Banks) is considered a major pest in potted azalea, Flanders' flagship ornamental crop of Rhododendron simsii hybrids. In addition to severe economic damage, the broad mite is dreaded for its increasing resistance to acaricides. Due to restrictions in the use of broad spectrum acaricides, Belgian azalea growers are left with only three compounds, belonging to two mode of action groups and restricted in their number of applications, for broad mite control: abamectin, milbemectin and pyrethrin. Although P. latus can be controlled with predatory mites, the high cost of this system makes it (not yet) feasible for integration into standard azalea pest management systems. Hence, a maximum efficacy of treatments with available compounds is essential. Because abamectin, milbemectin and pyrethrin are contact acaricides with limited trans laminar flow, only broad mites located on shoot tips of azalea plants will be controlled after spraying. Consequently, the efficacy of chemical treatments is influenced by the location and spread of P. latus on the plant. Unfortunately, little is known on broad mites' within-plant spread or how it is affected by climatic conditions like temperature and relative humidity. Therefore, experiments were set up to verify whether climate conditions have an effect on the location and migration of broad mites on azalea. Broad mite infected azalea plants were placed in standard growth chambers under different temperature (T:2.5-25°C) and relative humidity (RH:55-80%) treatments. Within-plant spread was determined by counting mites on the shoot tips and inner leaves of azalea plants. Results indicate that temperature and relative humidity have no significant effect on the within-plant spread of P. latus. To formulate recommendations for optimal spray conditions to maximize the efficacy of broad mite control with acaricides, further experiments on the effect of light intensity and rain are scheduled.

Plant Regeneration and Cellular Behaviour Studies in Celosia cristata Grown In Vivo and In Vitro

PubMed Central

Taha, Rosna Mat; Wafa, Sharifah Nurashikin

2012-01-01

Tissue culture studies of Celosia cristata were established from various explants and the effects of various hormones on morphogenesis of this species were examined. It was found that complete plant regeneration occurred at highest percentage on MS medium supplemented with 2.0 mg/L NAA and 1.5 mg/L BAP, with the best response showed by shoot explants. In vitro flowering was observed on MS basal medium after six weeks. The occurrence of somaclonal variation and changes in cellular behavior from in vivo and in vitro grown plants were investigated through cytological studies and image analysis. It was observed that Mitotic Index (MI), mean chromosome numbers, and mean nuclear to cell area ratio of in vitro root meristem cells were slightly higher compared to in vivo values. However, in vitro plants produced lower mean cell areas but higher nuclear areas when compared to in vivo plants. Thus, no occurrence of somaclonal variation was detected, and this was supported by morphological features of the in vitro plants. PMID:22593677

Evaluating phytoextraction efficiency of two high-biomass crops after soil amendment and inoculation with rhizobacterial strains.

PubMed

Vanessa, Álvarez-López; Ángeles, Prieto-Fernández; Sergio, Roiloa; Beatriz, Rodríguez-Garrido; Rolf, Herzig; Markus, Puschenreiter; Susan, Kidd Petra

2017-03-01

We evaluated the effect of compost amendment and/or bacterial inoculants on the growth and metal accumulation of Salix caprea (clone BOKU 01 AT-004) and Nicotiana tabacum (in vitro-bred clone NBCu10-8). Soil was collected from an abandoned Pb/Zn mine and rhizobacterial inoculants were previously isolated from plants growing at the same site. Plants were grown in untreated or compost-amended (5% w/w) soil and were inoculated with five rhizobacterial strains. Non-inoculated plants were also established as a control. Compost addition increased the shoot DW yield of N. tabacum but not S. caprea, while it decreased soil metal availability and lowered shoot Cd/Zn concentrations in tobacco plants. Compost amendment enhanced the shoot Cd/Zn removal due to the growth promotion of N. tabacum or to the increase in metal concentration in S. caprea leaves. Bacterial inoculants increased photosynthetic efficiency (particularly in N. tabacum) and sometimes modified soil metal availability, but this did not lead to a significant increase in Cd/Zn removal. Compost amendment was more effective in improving the Cd and Zn phytoextraction efficiency than bioaugmentation.

Evaluation of Clonostachys rosea for Control of Plant-Parasitic Nematodes in Soil and in Roots of Carrot and Wheat.

PubMed

Iqbal, Mudassir; Dubey, Mukesh; McEwan, Kerstin; Menzel, Uwe; Franko, Mikael Andersson; Viketoft, Maria; Jensen, Dan Funck; Karlsson, Magnus

2018-01-01

Biological control is a promising approach to reduce plant diseases caused by nematodes. We tested the effect of the fungus Clonostachys rosea strain IK726 inoculation on nematode community composition in a naturally nematode infested soil in a pot experiment, and the effect of C. rosea on plant health. The numbers of plant-parasitic nematode genera extracted from soil and plant roots decreased by 40 to 73% when C. rosea was applied, while genera of nonparasitic nematodes were not affected. Soil inoculation of C. rosea increased fresh shoot weight and shoot length of wheat plants by 20 and 24%, respectively, while only shoot dry weight increased by 48% in carrots. Light microscopy of in vitro C. rosea-nematode interactions did not reveal evidence of direct parasitism. However, culture filtrates of C. rosea growing in potato dextrose broth, malt extract broth and synthetic nutrient broth exhibited toxicity toward nematodes and immobilized 57, 62, and 100% of the nematodes, respectively, within 48 h. This study demonstrates that C. rosea can control plant-parasitic nematodes and thereby improve plant growth. The most likely mechanism responsible for the antagonism is antibiosis through production of nematicidal compounds, rather than direct parasitism.

Hydrophilic guidewires: evaluation and comparison of their properties and safety.

PubMed

Torricelli, Fabio Cesar Miranda; De, Shubha; Sarkissian, Carl; Monga, Manoj

2013-11-01

To compare physical and mechanical properties of 10 commercially available hydrophilic guidewires. In vitro testing was performed to evaluate 10 different straight hydrophilic guidewires (5 regular and 5 stiff wires): Glidewire, NiCore, EZ Glider, Hiwire, and Zipwire. The forces required for tip perforation, tip bending, shaft bending, and friction during movement were measured for all 10 wires. The tip contour was measured using high power light microscopy. The Glidewire required the greatest force to perforate our model (P = .01). The EZ Glider, Zipwire, and Glidewire had the lowest tip bending forces (P <.001). The Glidewire had the stiffest shaft (P <.001). The EZ Glider and Glidewire required the greatest forces in the friction test (P <.001). Regarding the stiff guidewires, the GlidewireS required the greatest force in the perforation test (P ≤.05). The GlidewireS and EZ GliderS required the lowest tip bending force (P ≤.004). The ZipwireS and NiCoreS had the stiffest shafts (P ≤.01). The GlidewireS required the greatest force in the friction test (P <.001). Measurement of the tip contour showed the Zipwire, HiwireS, and EZ GliderS had the roundest tips. Each wire has unique properties with advantages and disadvantages. The Glidewires (both stiff and regular) have the lowest potential for perforation, although they are less slippery. The Glidewire and EZ Glider required the least tip force to bend around a point of obstruction. Copyright © 2013 Elsevier Inc. All rights reserved.

[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%.

The PDZ protein tax-interacting protein-1 inhibits beta-catenin transcriptional activity and growth of colorectal cancer cells.

PubMed

Kanamori, Mutsumi; Sandy, Peter; Marzinotto, Stefania; Benetti, Roberta; Kai, Chikatoshi; Hayashizaki, Yoshihide; Schneider, Claudio; Suzuki, Harukazu

2003-10-03

Wnt signaling is essential during development while deregulation of this pathway frequently leads to the formation of various tumors including colorectal carcinomas. A key component of the pathway is beta-catenin that, in association with TCF-4, directly regulates the expression of Wnt-responsive genes. To identify novel binding partners of beta-catenin that may control its transcriptional activity, we performed a mammalian two-hybrid screen and isolated the Tax-interacting protein (TIP-1). The in vivo complex formation between beta-catenin and TIP-1 was verified by coimmunoprecipitation, and a direct physical association was revealed by glutathione S-transferase pull-down experiments in vitro. By using a panel of deletion mutants of both proteins, we demonstrate that the interaction is mediated by the PDZ (PSD-95/DLG/ZO-1 homology) domain of TIP-1 and requires primarily the last four amino acids of beta-catenin. TIP-1 overexpression resulted in a dose-dependent decrease in the transcriptional activity of beta-catenin when tested on the TOP/FOPFLASH reporter system. Conversely, siRNA-mediated knock-down of endogenous TIP-1 slightly increased endogenous beta-catenin transactivation function. Moreover, we show that overexpression of TIP-1 reduced the proliferation and anchorage-independent growth of colorectal cancer cells. These data suggest that TIP-1 may represent a novel regulatory element in the Wnt/beta-catenin signaling pathway.

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.

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.

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.

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.

Observations on the Feeding and Symptomatology of Xiphinema and Longidorus on Selected Host Roots

PubMed Central

Cohn, E.

1970-01-01

In vitro feeding of Xiphinema brevicolle, X. index and Longidorus africanus on roots of host seedlings is described. Both Xiphinema spp. fed mainly along roots rather than at tips and up to several days at a single site. Feeding of L. africanus was confined to root tips and lasted up to 15 min. No visible short term reaction of roots parasitized by the Xiphinema spp. could be discerned, but both swelling and cessation of growth of root tips were observed within 20 hr after feeding by L. africanus. Long-term (12-month) symptoms on roots of several host plants caused by cultured populations of X. brevicolle, X. index, X. italiae, L. africanus and L. brevicaudatus are described. All the Xiphinema spp. caused a thinning and distinct darkening of root systems and, at some sites, a breakdown of the cortex. Both species of Longidorus caused stubby and swollen root tips. Root symptom severity was in proportion to nematode population levels. PMID:19322291

Effect of utilization of tomato extract and foliar fertilizer as media on shoots multiplication of banana cv Ambon in vitro

NASA Astrophysics Data System (ADS)

Bidhari, L. A.; Purwanto, E.; Yunus, A.

2018-03-01

The good quality banana seeds are still difficult to obtain. There are two ways to provide seeds, namely conventional and tissue culture (in vitro). Tomato extract contains natural ZPT or phytohormone which can be utilized in modification of banana tissue culture media. The aim of this study was to determine the influence of media types and tomato extracts in various concentrations for multiplication of banana cv. Ambon in vitro. The study was conducted from October - December 2016 at the Tissue Culture Laboratory of Horticulture Seed Center, Salaman, Magelang. The experimental design used was completely randomized design with two treatment factors. The firs factor was media type with the addition of foliar fertilizer, the second factor was modification of tomato extract with 4 levels. The results showed that the different of the treated media treatment did not affect the emerge of leaf and leaf length, the number of roots and root length. The emerge of the leaves of all treatments occurred at 6 days after planting with the highest average length was obtained in MS treatment with a combination of tomato extract 50 ml/l (10.3 cm). The use of MS medium with a combination of tomato extract 50 ml/l generated the average root number 15.5 with a root lengths 7.5 cm. Substitution of MS medium with tomato extract and foliar fertilizer did not show better results compared to the use of MS media in the multiplication of banana shoots in tissue culture.

Plant stem cell niches.

PubMed

Stahl, Yvonne; Simon, Rüdiger

2005-01-01

Stem cells are required to support the indeterminate growth style of plants. Meristems are a plants stem cell niches that foster stem cell survival and the production of descendants destined for differentiation. In shoot meristems, stem cell fate is decided at the populational level. The size of the stem cell domain at the meristem tip depends on signals that are exchanged with cells of the organizing centre underneath. In root meristems, individual stem cells are controlled by direct interaction with cells of the quiescent centre that lie in the immediate neighbourhood. Analysis of the interactions and signaling processes in the stem cell niches has delivered some insights into the molecules that are involved and revealed that the two major niches for plant stem cells are more similar than anticipated.

Immunolocalization of integrin-like proteins in Arabidopsis and Chara

NASA Technical Reports Server (NTRS)

Katembe, W. J.; Swatzell, L. J.; Makaroff, C. A.; Kiss, J. Z.

1997-01-01

Integrins are a large family of integral plasma membrane proteins that link the extracellular matrix to the cytoskeleton in animal cells. As a first step in determining if integrin-like proteins are involved in gravitropic signal transduction pathways, we have used a polyclonal antibody against the chicken beta1 integrin subunit in western blot analyses and immunofluorescence microscopy to gain information on the size and location of these proteins in plants. Several different polypeptides are recognized by the anti-integrin antibody in roots and shoots of Arabidopsis and in the internodal cells and rhizoids of Chara. These cross-reactive polypeptides are associated with cellular membranes, a feature which is consistent with the known location of integrins in animal systems. In immunofluorescence studies of Arabidopsis roots, a strong signal was obtained from labeling integrin-like proteins in root cap cells, and there was little or no immunolabel in other regions of the root tip. While the antibody stained throughout Chara rhizoids, the highest density of immunolabel was at the tip. Thus, in both Arabidopsis roots and Chara rhizoids, the sites of gravity perception/transduction appear to be enriched in integrin-like molecules.

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis.

PubMed

Dudits, Dénes; Török, Katalin; Cseri, András; Paul, Kenny; Nagy, Anna V; Nagy, Bettina; Sass, László; Ferenc, Györgyi; Vankova, Radomira; Dobrev, Petre; Vass, Imre; Ayaydin, Ferhan

2016-03-01

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity. © 2016 American Society of Plant Biologists. All Rights Reserved.

Response of Organ Structure and Physiology to Autotetraploidization in Early Development of Energy Willow Salix viminalis1

PubMed Central

Dudits, Dénes; Török, Katalin; Cseri, András; Paul, Kenny; Nagy, Bettina; Sass, László; Ferenc, Györgyi; Vankova, Radomira; Dobrev, Petre; Vass, Imre; Ayaydin, Ferhan

2016-01-01

The biomass productivity of the energy willow Salix viminalis as a short-rotation woody crop depends on organ structure and functions that are under the control of genome size. Colchicine treatment of axillary buds resulted in a set of autotetraploid S. viminalis var. Energo genotypes (polyploid Energo [PP-E]; 2n = 4x = 76) with variation in the green pixel-based shoot surface area. In cases where increased shoot biomass was observed, it was primarily derived from larger leaf size and wider stem diameter. Autotetraploidy slowed primary growth and increased shoot diameter (a parameter of secondary growth). The duplicated genome size enlarged bark and wood layers in twigs sampled in the field. The PP-E plants developed wider leaves with thicker midrib and enlarged palisade parenchyma cells. Autotetraploid leaves contained significantly increased amounts of active gibberellins, cytokinins, salicylic acid, and jasmonate compared with diploid individuals. Greater net photosynthetic CO2 uptake was detected in leaves of PP-E plants with increased chlorophyll and carotenoid contents. Improved photosynthetic functions in tetraploids were also shown by more efficient electron transport rates of photosystems I and II. Autotetraploidization increased the biomass of the root system of PP-E plants relative to diploids. Sections of tetraploid roots showed thickening with enlarged cortex cells. Elevated amounts of indole acetic acid, active cytokinins, active gibberellin, and salicylic acid were detected in the root tips of these plants. The presented variation in traits of tetraploid willow genotypes provides a basis to use autopolyploidization as a chromosome engineering technique to alter the organ development of energy plants in order to improve biomass productivity. PMID:26729798

Identification of MicroRNAs and Target Genes in the Fruit and Shoot Tip of Lycium chinense: A Traditional Chinese Medicinal Plant

PubMed Central

Khaldun, A. B. M.; Huang, Wenjun; Liao, Sihong; Lv, Haiyan; Wang, Ying

2015-01-01

Although Lycium chinense (goji berry) is an important traditional Chinese medicinal plant, little genome information is available for this plant, particularly at the small-RNA level. Recent findings indicate that the evolutionary role of miRNAs is very important for a better understanding of gene regulation in different plant species. To elucidate small RNAs and their potential target genes in fruit and shoot tissues, high-throughput RNA sequencing technology was used followed by qRT-PCR and RLM 5’-RACE experiments. A total of 60 conserved miRNAs belonging to 31 families and 30 putative novel miRNAs were identified. A total of 62 significantly differentially expressed miRNAs were identified, of which 15 (14 known and 1 novel) were shoot-specific, and 12 (7 known and 5 novel) were fruit-specific. Additionally, 28 differentially expressed miRNAs were recorded as up-regulated in fruit tissues. The predicted potential targets were involved in a wide range of metabolic and regulatory pathways. GO (Gene Ontology) enrichment analysis and the KEGG (Kyoto Encyclopedia of Genes and Genomes) database revealed that “metabolic pathways” is the most significant pathway with respect to the rich factor and gene numbers. Moreover, five miRNAs were related to fruit maturation, lycopene biosynthesis and signaling pathways, which might be important for the further study of fruit molecular biology. This study is the first, to detect known and novel miRNAs, and their potential targets, of L. chinense. The data and findings that are presented here might be a good source for the functional genomic study of medicinal plants and for understanding the links among diversified biological pathways. PMID:25587984

In-Vivo Fluorescence Spectroscopy Of Normal And Atherosclerotic Arteries

NASA Astrophysics Data System (ADS)

Deckelbaum, Lawrence I.; Sarembock, Ian J.; Stetz, Mark L.; O'Brien, Kenneth M.; Cutruzzola, Francis W.; Gmitro, Arthur F.; Ezekowitz, Michael D.

1988-06-01

Laser-induced fluorescence spectroscopy can discriminate atherosclerotic from normal arteries in-vitro and may thus potentially guide laser angioplasty. To evaluate the feasibility of laser-induced fluorescence spectroscopy in a living blood-filled arterial system we performed fiberoptic laser-induced fluorescence spectroscopy in a rabbit model of focal femoral atherosclerosis. A laser-induced fluorescence spectroscopy score was derived from stepwise linear regression analysis of in-vitro spectra to distinguish normal aorta (score>0) from atherosclerotic femoral artery (score<0). A 400 u silica fiber, coupled to a helium cadmium laser and optical multichannel analyzer, was inserted through a 5F catheter to induce and record in-vivo fluorescence from femoral and aortoiliac arteries. Arterial spectra could be recorded in all animals (n=10: 5 occlusions, 5 stenoses). Blood spectra were of low intensity and were easily distinguished from arterial spectra. The scores (mean ± SEM) for the in-vivo spectra were -0.69 +/- 0.29 for artherosclerotic femoral, and +0.54 ±. 0.15 for normal aorta (p<.01 p=NS compared to in-vitro spectra). In-vitro, a fiber tip to tissue distance <50 u was necessary for adequate arterial LIFS in blood. At larger distances low intensity blood spectra were recorded (1/20 the intensity of tissue spectra). Thus, fiberoptic laser-induced fluorescence spectroscopy can be sucessfully performed in a blood filled artery provided the fiber tip is approximated to the tissue.

phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism.

PubMed

Christie, John M; Yang, Haibing; Richter, Gregory L; Sullivan, Stuart; Thomson, Catriona E; Lin, Jinshan; Titapiwatanakun, Boosaree; Ennis, Margaret; Kaiserli, Eirini; Lee, Ok Ran; Adamec, Jiri; Peer, Wendy A; Murphy, Angus S

2011-06-01

It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.

Germination and plantlet regeneration of encapsulated microshoots of aromatic rice (Oryza sativa L. Cv. MRQ 74).

PubMed

Taha, Rosna Mat; Saleh, Azani; Mahmad, Noraini; Hasbullah, Nor Azlina; Mohajer, Sadegh

2012-01-01

Plant tissues such as somatic embryos, apical shoot tips, axillary shoot buds, embryogenic calli, and protocom-like bodies are potential micropropagules that have been considered for creating synthetic seeds. In the present study, 3-5 mm microshoots of Oryza sativa L. Cv. MRQ 74 were used as explant sources for obtaining synthetic seeds. Microshoots were induced from stem explants on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L benzylaminopurine (BAP). They were encapsulated in 3% (w/v) sodium alginate, 3% sucrose, 0.1 mg/L BAP, and 0.1 mg/L α-Naphthalene acetic acid (NAA). Germination and plantlet regeneration of the encapsulated seeds were tested by culturing them on various germination media. The effect of storage period (15-30 days) was also investigated. The maximum germination and plantlet regeneration (100.0%) were recorded on MS media containing 3% sucrose and 0.8% agar with and without 0.1 mg/L BAP. However, a low germination rate (6.67%) was obtained using top soil as a sowing substrate. The germination rate of the encapsulated microshoots decreased from 93.33% to 3.33% after 30 days of storage at 4°C in the dark. Therefore, further research is being done to improve the germination rate of the synthetic seeds.

Photosynthetic leaf area modulates tiller bud outgrowth in sorghum: Bud outgrowth is sensitive to leaf area

DOE PAGES

Kebrom, Tesfamichael H.; Mullet, John E.

2014-12-12

Shoot branches or tillers develop from axillary buds. The dormancy versus outgrowth fates of buds depends on genetic, environmental and hormonal signals. Defoliation inhibits bud outgrowth indicating the role of leaf-derived metabolic factors such as sucrose in bud outgrowth. In this study, the sensitivity of bud outgrowth to selective defoliation was investigated. At 6 d after planting (6 DAP), the first two leaves of sorghum were fully expanded and the third was partially emerged. Therefore, the leaves were selectively defoliated at 6 DAP and the length of the bud in the first leaf axil was measured at 8 DAP. Budmore » outgrowth was inhibited by defoliation of only 2 cm from the tip of the second leaf blade. The expression of dormancy and sucrose-starvation marker genes was up-regulated and cell cycle and sucrose-inducible genes was down-regulated during the first 24 h postdefoliation of the second leaf.At 48 h, the expression of these genes was similar to controls as the defoliated plant recovers. Our results demonstrate that small changes in photosynthetic leaf area affect the propensity of tiller buds for outgrowth. Therefore, variation in leaf area and photosynthetic activity should be included when integrating sucrose into models of shoot branching.« less

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

NASA Technical Reports Server (NTRS)

Rashotte, A. M.; Brady, S. R.; Reed, R. C.; Ante, S. J.; Muday, G. K.; Davies, E. (Principal Investigator)

2000-01-01

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.

Demonstration of the economic feasibility of plant tissue culture for jojoba (Simmondsia chinensis) and Euphorbia spp

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

Sluis, C.

1980-09-01

The economic feasibility of plant tissue culture was demonstrated as applied to two plants: jojoba (Simmondsia chinensis) and Euphorbia spp. The gopher weed (Euphorbia lathyris) was selected as the species of Euphorbia to research due to the interest in this plant as a potential source of hydrocarbon-like compounds. High yield female selections of jojoba were chosen from native stands and were researched to determine the economic feasibility of mass producing these plants via a tissue culture micropropagation program. The female jojoba selection was successfully mass produced through tissue culture. Modifications in initiation techniques, as well as in multiplication media andmore » rooting parameters, were necessary to apply the tissue culture system, which had been developed for juvenile seedling tissue, to mature jojobas. Since prior attempts at transfer of tissue cultured plantlets were unsuccessful, transfer research was a major part of the project and has resulted in a system for transfer of rooted jojoba plantlets to soil. Euphorbia lathyris was successfully cultured using shoot tip cultures. Media and procedures were established for culture initiation, multiplication of shoots, callus induction and growth, and root initiation. Well-developed root systems were not attained and root initiation percentages should be increased if the system is to become commercially feasible.« less

Hughmillerites vancouverensis sp. nov. and the Cretaceous diversification of Cupressaceae.

PubMed

Atkinson, Brian A; Rothwell, Gar W; Stockey, Ruth A

2014-12-01

• Two ovulate conifer cones, one of which is attached terminally to a short leafy shoot, reveal the presence of a new species of Hughmillerites in the Early Cretaceous Apple Bay flora of Vancouver Island, British Columbia, Canada. This ancient conifer expands the diversity of Cupressaceae in the Mesozoic and reveals details about the evolution of Subfamily: Cunninghamioideae.• Specimens were studied from anatomical sections prepared using the cellulose acetate peel technique.• Vegetative shoots have helically arranged leaves that are Cunninghamia-like. Seed cones have many helically arranged bract/scale complexes in which the bract is larger than the ovuliferous scale. Each ovuliferous scale has three free tips that separate from the bract immediately distal to an inverted seed. Several ovuliferous scales show interseminal ridges between seeds.• This study documents a new extinct species of cunninghamioid conifers, Hughmillerites vancouverensis, expanding the record of the genus from the Late Jurassic to the Early Cretaceous. This new extinct species emphasizes the important role that conifers from subfamily Cunninghamioideae played in the initial evolutionary radiation of Cupressaceae. In light of recent findings in conifer regulatory genetics, we use H. vancouverensis to hypothesize that variations of expression in certain gene homologues played an important role in the evolution of the cupressaceous ovuliferous scale. © 2014 Botanical Society of America, Inc.

Isolation and antioxidant activities of polysaccharides extracted from the shoots of Phyllostachys edulis (Carr.).

PubMed

Zhang, Zhongshan; Wang, Xiaomei; Yu, Shuchi; Zhao, Mingxing

2011-11-01

Polysaccharides extracted from Phyllostachys edulis (Carr.) are a group of hetero polysaccharides, and their antioxidant activities were investigated employing various established in vitro systems. Available data obtained with in vitro models suggested that among the three samples, B1 (extraction with water) showed significant inhibitory effects on superoxide radical and hydroxyl radical; its reducing power was also the strongest among the three samples. These results clearly establish the possibility that polysaccharides extracted from P. edulis could be effectively employed as ingredient in health or functional food, to alleviate oxidative stress. However, comprehensive studies need to be conducted in experimental animal models. Copyright © 2011 Elsevier B.V. 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.

In Vitro Comparison of a Novel Single Probe Dual-Energy Lithotripter to Current Devices.

PubMed

Carlos, Evan C; Wollin, Daniel A; Winship, Brenton B; Jiang, Ruiyang; Radvak, Daniela; Chew, Ben H; Gustafson, Michael R; Simmons, W Neal; Zhong, Pei; Preminger, Glenn M; Lipkin, Michael E

2018-06-01

The LithoClast Trilogy is a novel single probe, dual-energy lithotripter with ultrasonic (US) vibration and electromagnetic impact forces. ShockPulse and LithoClast Select are existing lithotripters that also use a combination of US and mechanical impact energies. We compared the efficacy and tip motion of these devices in an in vitro setting. Begostones, in the ratio 15:3, were used in all trials. Test groups were Trilogy, ShockPulse, Select ultrasound (US) only, and Select ultrasound with pneumatic (USP). For clearance testing, a single investigator facile with each lithotripter fragmented 10 stones per device. For drill testing, a hands-free apparatus with a submerged balance was used to apply 1 or 2 lbs of pressure on a stone in contact with the device tip. High-speed photography was used to assess Trilogy and ShockPulse's probe tip motion. Select-USP was slowest and Trilogy fastest on clearance testing (p < 0.01). On 1 lbs drill testing, Select-US was slowest (p = 0.001). At 2 lbs, ShockPulse was faster than Select US (p = 0.027), but did not significantly outpace Trilogy nor Select-USP. At either weight, there was no significant difference between Trilogy and ShockPulse. During its US function, Trilogy's maximum downward tip displacement was 0.041 mm relative to 0.0025 mm with ShockPulse. Trilogy had 0.25 mm of maximum downward displacement during its impactor function while ShockPulse had 0.01 mm. Single probe dual-energy devices, such as Trilogy and ShockPulse, represent the next generation of lithotripters. Trilogy more efficiently cleared stone than currently available devices, which could be explained by its larger probe diameter and greater downward tip displacement during both US and impactor functions.

Steerable catheter microcoils for interventional MRI reducing resistive heating.

PubMed

Bernhardt, Anthony; Wilson, Mark W; Settecase, Fabio; Evans, Leland; Malba, Vincent; Martin, Alastair J; Saeed, Maythem; Roberts, Timothy P L; Arenson, Ronald L; Hetts, Steven W

2011-03-01

The aims of this study were to assess resistive heating of microwires used for remote catheter steering in interventional magnetic resonance imaging and to investigate the use of alumina to facilitate heat transfer to saline flowing in the catheter lumen. A microcoil was fabricated using a laser lathe onto polyimide-tipped or alumina-tipped endovascular catheters. In vitro testing was performed on a 1.5-T magnetic resonance system using a vessel phantom, body radiofrequency coil, and steady-state pulse sequence. Resistive heating was measured with water flowing over a polyimide-tip catheter or saline flowing through the lumen of an alumina-tip catheter. Preliminary in vivo testing in porcine common carotid arteries was conducted with normal blood flow or after arterial ligation when current was applied to an alumina-tip catheter for up to 5 minutes. After application of up to 1 W of direct current power, clinically significant temperature increases were noted with the polyimide-tip catheter: 23°C/W at zero flow, 13°C/W at 0.28 cm(3)/s, and 7.9°C/W at 1 cm(3)/s. Using the alumina-tip catheter, the effluent temperature rise using the lowest flow rate (0.12 cm(3)/s) was 2.3°C/W. In vivo testing demonstrated no thermal injury to vessel walls at normal and zero arterial flow. Resistive heating in current carrying wire pairs can be dissipated by saline coolant flowing within the lumen of a catheter tip composed of material that facilitates heat transfer. Copyright © 2011 AUR. Published by Elsevier Inc. All rights reserved.

Holmium:Yttrium Aluminum Garnet Laser and Guidewires: Is There a Durability Difference Among Guidewires Against Laser Energy? An In Vitro Experimental Study.

PubMed

Bagbanci, Sahin

2017-05-01

To evaluate the durability differences between five different type of guidewires against laser energy in an in vitro experimental ureteral model. The study was performed at the Department of Urology, Medicine Faculty of Ahi Evran University. An in vitro experimental ureteral model was created for the work; a silicon ureteral model in a saline-filled container. Experiments were performed on five different type of guidewires; ZIPwire, Sensor polytetrafluoroethylene (PTFE) Nitinol guidewire, Roadrunner ® PC wire guide, Amplatz Super Stiff, and Zebra Urologic Guidewire. These guidewires were grouped from one to five, respectively. Laser fibers were contacted to the guidewire, and laser energy was fired to the premarked tip and body parts in different adjustments. The breakage of the guidewires was detected only on the flexible tip parts in group 1a, group 1b, group 2a, group 2b, group 4a, and group 4b. The body parts of the guidewires were resistant to laser energy in all groups and did not break. The breakage of the guidewires occurred after 3 J × 10 Hz (30 W) experiment. Group 1a and 1b were different from group 2a, 2b, 4a, and 4b according to Kruskal-Wallis H test. The body parts of the guidewires in all study groups were resistant to laser energy. The tip parts of Zipwire ™ , Sensor ™ PTFE Nitinol, and Amplatz Super Stiff ™ guidewire should be kept away from the surgical field when the high power settings of the laser are being used. The body parts of the guidewires can be utilized in the surgical field safely.

The human Ino80 binds to microtubule via the E-hook of tubulin: Implications for the role in spindle assembly

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

Park, Eun-Jung; Hur, Shin-Kyoung; Lee, Han-Sae

2011-12-16

Highlights: Black-Right-Pointing-Pointer The N-terminal domain of hIno80 is important for binding to the spindle. Black-Right-Pointing-Pointer The hIno80 N-terminal domain binds to tubulin and microtubule in vitro. Black-Right-Pointing-Pointer The E-hook of tubulin is critical for hIno80 binding to tubulin and microtubule. Black-Right-Pointing-Pointer Tip49a does not bind to microtubule and dispensable for spindle formation. -- Abstract: The human INO80 chromatin remodeling complex, comprising the Ino80 ATPase (hIno80) and the associated proteins such as Tip49a, has been implicated in a variety of nuclear processes other than transcription. We previously have found that hIno80 interacts with tubulin and co-localizes with the mitotic spindle andmore » is required for spindle formation. To better understand the role of hIno80 in spindle formation, we further investigated the interaction between hIno80 and microtubule. Here, we show that the N-terminal domain, dispensable for the nucleosome remodeling activity, is important for hIno80 to interact with tubulin and co-localize with the spindle. The hIno80 N-terminal domain binds to monomeric tubulin and polymerized microtubule in vitro, and the E-hook of tubulin, involved in the polymerization of microtubule, is critical for this binding. Tip49a, which has been reported to associate with the spindle, does not bind to microtubule in vitro and dispensable for spindle formation in vivo. These results suggest that hIno80 can play a direct role in the spindle assembly independent of its chromatin remodeling activity.« less

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...

Using electrical impedance to predict catheter-endocardial contact during RF cardiac ablation.

PubMed

Cao, Hong; Tungjitkusolmun, Supan; Choy, Young Bin; Tsai, Jang-Zern; Vorperian, Vicken R; Webster, John G

2002-03-01

During radio-frequency (RF) cardiac catheter ablation, there is little information to estimate the contact between the catheter tip electrode and endocardium because only the metal electrode shows up under fluoroscopy. We present a method that utilizes the electrical impedance between the catheter electrode and the dispersive electrode to predict the catheter tip electrode insertion depth into the endocardium. Since the resistivity of blood differs from the resistivity of the endocardium, the impedance increases as the catheter tip lodges deeper in the endocardium. In vitro measurements yielded the impedance-depth relations at 1, 10, 100, and 500 kHz. We predict the depth by spline curve interpolation using the obtained calibration curve. This impedance method gives reasonably accurate predicted depth. We also evaluated alternative methods, such as impedance difference and impedance ratio.