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.

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.

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.

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.

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.

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

PubMed

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

1991-11-01

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

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

NASA Technical Reports Server (NTRS)

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

1991-01-01

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

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

PubMed

Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

2012-03-01

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

Effect of 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.

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.

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 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.

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.

Effect of various factors on shoot regeneration from citrus epicotyl explants

USDA-ARS?s Scientific Manuscript database

The effect of various treatments on shoot organogenesis from seedling epicotyl explants from various scion and rootstock polyembryonic citrus types was determined. Treatments included water source, gelling agent, explant insertion, seed size, light intensity, malachite green, nonionic surfactants, a...

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.

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

PubMed

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

2014-01-01

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

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

PubMed Central

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

2014-01-01

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

In vitro propagation, 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.

[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.

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

PubMed

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

2016-09-01

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

In Vitro 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.

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.

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

In Vitro Plant Regeneration from Commercial Cultivars of Soybean

PubMed Central

Raza, Ghulam; Singh, Mohan B.

2017-01-01

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

In Vitro Plant Regeneration from Commercial Cultivars of Soybean.

PubMed

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

2017-01-01

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

Micropropagation 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.

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

Carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2006-01-01

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

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).

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

PubMed

Kumar, Pankaj; Srivastava, D K

2015-04-01

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

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.

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.

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.

Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

PubMed

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

2018-04-28

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

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.

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.

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.

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 ...

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.

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.

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.

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...

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

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

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.

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.

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.

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.

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.

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

PubMed

Fortes, A M; Pais, M S

2000-07-01

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

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.

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.

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.

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

Induction of somatic embryogenesis in explants of shoot cultures established from adult Eucalyptus globulus and E. saligna × E. maidenii trees.

PubMed

Corredoira, E; Ballester, A; Ibarra, M; Vieitez, A M

2015-06-01

A reproducible procedure for induction of somatic embryogenesis (SE) from adult trees of Eucalyptus globulus Labill. and the hybrid E. saligna Smith × E. maidenii has been developed for the first time. Somatic embryos were obtained from both shoot apex and leaf explants of all three genotypes evaluated, although embryogenic frequencies were significantly influenced by the species/genotype, auxin and explant type. Picloram was more efficient for somatic embryo induction than naphthaleneacetic acid (NAA), with the highest frequency of induction being obtained in Murashige and Skoog medium containing 40 µM picloram and 40 mg l(-1) gum Arabic, in which 64% of the shoot apex explants and 68.8% of the leaf explants yielded somatic embryos. The embryogenic response of the hybrid was higher than that of the E. globulus, especially when NAA was used. The cultures initiated on picloram-containing medium consisted of nodular embryogenic structures surrounded by a mucilaginous coating layer that emerged from a watery callus developed from the initial explants. Cotyledonary somatic embryos were differentiated after subculture of these nodular embryogenic structures on a medium lacking plant growth regulators. Histological analysis confirmed the bipolar organization of the somatic embryos, with shoot and root meristems and closed procambial tissue that bifurcated into small cotyledons. The root pole was more differentiated than the shoot pole, which appeared to be formed by a few meristematic layers. Maintenance of the embryogenic lines by secondary SE was attained by subculturing individual cotyledonary embryos or small clusters of globular and torpedo embryos on medium with 16.11 µM NAA at 4- to 5-week intervals. Somatic embryos converted into plantlets after being transferred to liquid germination medium although plant regeneration remained poor. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Germination and Plantlet Regeneration of Encapsulated Microshoots of Aromatic Rice (Oryza sativa L. Cv. MRQ 74)

PubMed Central

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. PMID:22919338

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

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

Mokhtarzedeh, A.; Constantin, M.J.

1978-01-01

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

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.

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.

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.

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

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...

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.

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.

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

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

PubMed

Wang, Qiaochun; Valkonen, Jari P T

2009-01-01

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

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.

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

PubMed

Wang, Y-H; Bhalla, P L

2004-01-01

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

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.

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.

Isolation of Temperature-Sensitive Mutants of Arabidopsis thaliana That Are Defective in the Redifferentiation of Shoots.

PubMed Central

Yasutani, I.; Ozawa, S.; Nishida, T.; Sugiyama, M.; Komamine, A.

1994-01-01

Three temperature-sensitive mutants of Arabidopsis thaliana that were defective in the redifferentiation of shoots were isolated as tools for the study of organogenesis. M3 lines were constructed by harvesting M3 seeds separately from each M2 plant. Comparative examination of shoot redifferentiation in root explants of 2700 M3 lines at 22[deg]C (permissive temperature) and at 27[deg]C (restrictive temperature) led to the identification of seven temperature-sensitive mutant lines. Genetic tests of three of the seven mutant lines indicated that temperature-sensitive redifferentiation of shoots in these three lines resulted from single, nuclear, recessive mutations in three different genes, designated SRD1, SRD2, and SRD3. The morphology of root explants of srd mutants cultured at the restrictive temperature suggests that the products of these SRD genes function at different stages of the redifferentiation of shoots. PMID:12232244

Micropropagation of an exotic ornamental plant, Calathea crotalifera, for production of high quality plantlets.

PubMed

Rozali, Shahril Efzueni; Rashid, Kamaludin A; Taha, Rosna Mat

2014-01-01

A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.

Genetic stability assessment of wWasabi plants regenerated from long-term cryopreserved shoot tips using morphological, biochemical and molecular analysis.

PubMed

Matsumoto, Toshikazu; Akihiro, Takashi; Maki, Shinya; Mochida, Kouhei; Kitagawa, Masaru; Tanaka, Daisuke; Yamamoto, Shin-Ichi; Niino, Takao

2013-01-01

This study compared the effect of cryopreserved storage duration of wasabi shoot tips, which derived from the same in vitro mother-plant. We compared the survival of shoot tips and the genetic stability of regenerated plants originating from four experimental groups: shoot tips stored in a -150°C deep-freezer for 10 years; shoot tips stored in liquid nitrogen for 2 h; shoot tips treated with PVS2 vitrification solution; and untreated controls. No significant difference in survival was observed between the four experimental groups. Survival ranged between 93 and 100%. Genetic stability of plants regenerated from cryopreserved shoot tips was assessed over a period of 24 months using morphological, biochemical and molecular markers. While glucose, fructose and glutamic acid concentrations differed slightly between experimental groups after 16 months, these differences disappeared after 24 months. No significant differences were noted for the morphological markers studied (petiole length, shoot number and leaf index). No differences were observed in RAPD profiles obtained with the six primers tested.

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

PubMed

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

2012-07-01

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

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

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

PubMed

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

1992-07-01

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

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

PubMed

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

2015-09-01

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

Micropropagation of an Exotic Ornamental Plant, Calathea crotalifera, for Production of High Quality Plantlets

PubMed Central

Efzueni Rozali, Shahril; Rashid, Kamaludin A.; Mat Taha, Rosna

2014-01-01

A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera. PMID:25136669

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.

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.

Combining thermotherapy with cryotherapy for efficient eradication of Apple stem grooving virus from infected in vitro-cultured apple shoots

USDA-ARS?s Scientific Manuscript database

Almost all of the literature reporting on the cryopreservation of shoot tips does not specify the sanitary status of the stock material from which shoot tips were taken. The effect of the presence of viruses in plant tissues on the recovery of shoot tips after cryopreservation has not been previousl...

Genetic transformation of carnation (Dianthus caryophylus L.).

PubMed

Nontaswatsri, Chalermsri; Fukai, Seiichi

2010-01-01

This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

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...

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.

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.

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

PubMed

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

2010-01-01

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

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

PubMed

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

2004-08-01

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

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

NASA Technical Reports Server (NTRS)

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

1994-01-01

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

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.

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.

Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing; Sink, K C

2004-12-01

Transient expression studies using blueberry leaf explants and monitored by beta-glucuronidase (GUS) assays indicated Agrobacterium tumefaciens strain EHA105 was more effective than LBA4404 or GV3101; and the use of acetosyringone (AS) at 100 microM for inoculation and 6 days co-cultivation was optimum compared to 2, 4, 8, 10 or 12 days. Subsequently, explants of the cultivars Aurora, Bluecrop, Brigitta, and Legacy were inoculated with strain EHA105 containing the binary vector pBISN1 with the neomycin phosphotransferase gene (nptII) and an intron-interrupted GUS gene directed by the chimeric super promoter (Aocs)3AmasPmas. Co-cultivation was for 6 days on modified woody plant medium (WPM) plus 100 microM AS. Explants were then placed on modified WPM supplemented with 1.0 mg l(-1) thidiazuron, 0.5 mg l(-1) alpha-naphthaleneacetic, 10 mg l(-1) kanamycin (Km), and 250 mg l(-1) cefotaxime. Selection for Km-resistant shoots was carried out in the dark for 2 weeks followed by culture in the light at 30 microE m(-2) s(-1) at 25 degrees C. After 12 weeks, selected shoots that were both Km resistant and GUS positive were obtained from 15.3% of the inoculated leaf explants of cultivar Aurora. Sixty-eight independent clones derived from such shoots all tested positive by the polymerase chain reaction using a nptII primer. Eight of eight among these 68 clones tested positive by Southern hybridization using a gusA gene derived probe. The transformation protocol also yielded Km-resistant, GUS-positive shoots that were also PCR positive at frequencies of 5.0% for Bluecrop, 10.0% for Brigitta and 5.6% for Legacy.

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

PubMed

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

2007-01-01

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

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

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...

Regeneration of Cuphea tolucana Peyr. in in vitro culture.

PubMed

Przybecki, Z; Olejniczak, J; Adamska, E

2001-01-01

In order to regenerate Cuphea tolucana from hypocotyl, cotyledon and root explants, a solid culture and 8 hormone combinations were used. Only the root explants did not react to any of the media. On most of the media, the other explants formed shoots, roots or callus, or their reaction was more complex. The regeneration probably occurred via direct organogenesis. The regenerants displayed a wide variety of morphological characteristics. However, their offspring did not show any differences from plants, which had not undergone culture.

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.

Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique.

PubMed

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

2014-04-01

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

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

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.

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.

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.

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

PubMed

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

2013-01-01

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

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.

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.

Improved regeneration and transformation protocols for three strawberry cultivars

PubMed Central

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

2014-01-01

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

A filter paper-based liquid culture system for citrus shoot organogenesis - a mixture-amount experiment

USDA-ARS?s Scientific Manuscript database

The objective of this study was to determine the effects of a static liquid culture system on shoot regeneration from citrus epicotyl explants. Two citrus types were used, Carrizo citrange and Ridge Pineapple sweet orange. A liquid culture system comprised of a Petri dish, cellulose filter paper dis...

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.

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.

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.

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.

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.

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.

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.

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.

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.

Cloning higher plants from aseptically cultured tissues and cells

NASA Technical Reports Server (NTRS)

Krikorian, A. D.

1982-01-01

A review of aseptic culture methods for higher plants is presented, which focuses on the existing problems that limit or prevent the full realization of cloning plants from free cells. It is shown that substantial progress in clonal multiplication has been made with explanted stem tips or lateral buds which can be stimulated to produce numerous precocious axillary branches. These branches can then be separated or subdivided and induced to root in order to yield populations of genetically and phenotypically uniorm plantlets. Similarly, undifferentiated calluses can sometimes be induced to form shoots and/or roots adventitiously. Although the cell culture techniques required to produce somatic embryos are presently rudimentary, steady advances are being made in learning how to stimulate formation of somatic or adventive embryos from totipotent cells grown in suspension cultures. It is concluded that many problems exist in the producing and growing of totipotent or morphogenetically competent cell suspensions, but the potential benefits are great.

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.

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

PubMed

Arockiasamy, S; Ignacimuthu, S; Melchias, G

2000-12-01

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

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

PubMed

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

2013-10-15

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

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

Somaclonal variation in hybrid poplars for resistance to Septoria leaf spot

Treesearch

M.E. Ostry; D. D. Skilling

1987-01-01

Tissue culture techniques have been used to obtain hybrid poplars with putative resistance to leaf spot caused by Septoria musiva from clones previously susceptible to the disease. Stem internode explants were used to obtain proliferating callus cultures. Adventitious bud formation and shoot proliferation were then induced. Elongated shoots were excised and rooted in a...

Acceleration of adventitious shoots by interaction between exogenous hormone and adenine sulphate in Althaea officinalis L.

PubMed

Naz, Ruphi; Anis, M

2012-11-01

In the current study attempts were made to investigate the effects of three different phases of callus induction followed by adventitious regeneration from leaf segments (central and lateral vein). Callus induction was observed in Murashige and Skoog's (MS) medium supplemented with 15.0 μM 2,4-dichloro phenoxy acetic acid (2,4-D). Adventitious shoot buds formation was achieved on MS medium supplemented with 7.5 μM 2,4-D and 20.0 μM AdS in liquid medium as it induced 19.2 ± 0.58 buds in central vein explants. Addition of different growth regulators (cytokinins-6-benzyladenine, kinetin and 2-isopentenyl adenine alone or in combination with auxins-indole-3-acetic acid, indole-3-butyric acid and α-naphthalene acetic acid, improved the shoot regeneration efficiency, in which 5.0 μM 6-benzyl adenine along with 0.25 μM α-naphthalene acetic acid was shown to be the most effective medium for maximum shoot regeneration (81.3 %) with 24.6 number of shoots and 4.4 ± 0.08 cm shoot length per explant. Leaf culture of central veins led to better shoot formation capacity in comparison to lateral vein. Rooting was readily achieved on the differentiated shoots on 1/2 MS medium augmented with 20.0 μM indole-3-butyric acid. The plants were successfully hardened off in sterile soilrite followed by their establishment in garden soil with 80 % survival rate.

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

PubMed

Danso, K E; Ford-Lloyd, B V

2003-04-01

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

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.

An Efficient Plant Regeneration and Transformation System of Ma Bamboo (Dendrocalamus latiflorus Munro) Started from Young Shoot as Explant

PubMed Central

Ye, Shanwen; Cai, Changyang; Ren, Huibo; Wang, Wenjia; Xiang, Mengqi; Tang, Xiaoshan; Zhu, Caiping; Yin, Tengfei; Zhang, Li; Zhu, Qiang

2017-01-01

Genetic engineering technology has been successfully used in many plant species, but is limited in woody plants, especially in bamboos. Ma bamboo (Dendrocalamus latiflorus Munro) is one of the most important bamboo species in Asia, and its genetic improvement was largely restricted by the lack of an efficient regeneration and transformation method. Here we reported a plantlet regeneration and Agrobacterium-mediated transformation protocol by using Ma bamboo young shoots as explants. Under our optimized conditions, embryogenic calluses were successfully induced from the excised young shoots on callus induction medium and rapidly grew on callus multiplication medium. Shoots and roots were regenerated on shoot induction medium and root induction medium, respectively, with high efficiency. An Agrobacterium-mediated genetic transformation protocol of Ma bamboo was established, verified by PCR and GUS staining. Furthermore, the maize Lc gene under the control of the ubiquitin promoter was successfully introduced into Ma bamboo genome and generated an anthocyanin over-accumulation phenotype. Our methods established here will facilitate the basic research as well as genetic breeding of this important bamboo species. Key achievements: A stable and high efficiency regeneration and Agrobacterium-mediated transformation protocol for Ma bamboo from vegetative organ is established. PMID:28798758

Organogenesis from transformed tomato explants.

PubMed

Frary, Anne; Van Eck, Joyce

2005-01-01

Tomato was one of the first crops for which a genetic transformation system was reported involving regeneration by organogenesis from Agrobacterium-transformed explants. Since the initial reports, various factors have been studied that affect the efficiency of tomato transformation and the technique has been useful for the isolation and identification of many genes involved in plant disease resistance, morphology and development. In this method, cotyledon explants from in vitro-grown seedlings are precultured overnight on a tobacco suspension feeder layer. The explants are then inoculated with Agrobacterium and returned to the feeder layer for a 2-d period of cocultivation. After cocultivation, the explants are transferred to an MS-based selective regeneration medium containing zeatin. Regenerated shoots are then rooted on a separate selective medium. This protocol has been used with several tomato cultivars and routinely yields transformation efficiencies of 10-15%.

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.

Stable integration and expression of wasabi defensin gene in "Egusi" melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot.

PubMed

Ntui, Valentine Otang; Thirukkumaran, Gunaratnam; Azadi, Pejman; Khan, Raham Sher; Nakamura, Ikuo; Mii, Masahiro

2010-09-01

Production of "Egusi" melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer "Egusi" resistant to these diseases, cotyledonary explants of two "Egusi" genotypes, 'Ejagham' and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mg l(-l) kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and 'Ejagham', respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype 'Ejagham' were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1-5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.

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.

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

PubMed

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

2011-01-01

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

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.

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.

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.

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

PubMed

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

2017-12-01

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

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.

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

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.

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

PubMed

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

1995-05-01

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

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.

The antioxidant melatonin boosts recovery of cryopreserved shoot tips

USDA-ARS?s Scientific Manuscript database

Many useful plant species found in Canada are of conservation concern. In vitro storage and cryopreservation techniques guarantee 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 Wor...

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...

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.

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.

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.

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

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.

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 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.

In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants.

PubMed

Gnanaraj, Wesely Edward; Antonisamy, Johnson Marimuthu; R B, Mohanamathi; Subramanian, Kavitha Marappampalyam

2012-01-01

To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants. Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3-4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration. Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field. The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing.

PubMed

Liu, Juan; Zhang, XueJiao; Zhang, FangPeng; Hong, Ni; Wang, GuoPing; Wang, Aiming; Wang, LiPing

2015-11-16

MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures. Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots. This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

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.

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.

Rejuvenation of Sequoia sempervirens by Repeated Grafting of Shoot Tips onto Juvenile Rootstocks in Vitro 1

PubMed Central

Huang, Li-Chun; Lius, Suwenza; Huang, Bau-Lian; Murashige, Toshio; Mahdi, El Fatih M.; Van Gundy, Richard

1992-01-01

Repeated grafting of 1.5-centimeter long shoot tips from an adult Sequoia sempervirens tree onto fresh, rooted juvenile stem cuttings in vitro resulted in progressive restoration of juvenile traits. After four successive grafts, stem cuttings of previously adult shoots rooted as well, branched as profusely, and grew with as much or more vigor as those of seedling shoots. Reassays disclosed retention for 3 years of rooting competence at similar levels as originally restored. Adventitious shoot formation was remanifested and callus development was depressed in stem segments from the repeatedly grafted adult. The reversion was associated with appearance and disappearance of distinctive leaf proteins. Neither gibberellic acid nor N6-beneyladenine as nutrient supplements duplicated the graft effects. ImagesFigure 2Figure 5Figure 8 PMID:16668609

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

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.

Effect of environmental and cultural conditions on medium pH and explant growth performance of Douglas-fir ( Pseudotsuga menziesii) shoot cultures

PubMed Central

Chen, Chien-Chih; Bates, Rick; Carlson, John

2015-01-01

The medium pH level of plant tissue cultures has been shown to be essential to many aspects of explant development and growth. Sensitivity or tolerance of medium pH change in vitro varies according to specific requirements of individual species. The objectives of this study are to 1) determine medium pH change over time in storage conditions and with presence of explants, 2) evaluate the effects of medium pH change on explant growth performance and 3) assess the effects of adding a pH stabilizer, 2-(N-morpholino)ethanesulfonic acid (MES) that is commonly used in Douglas-fir micropropagation medium. Vegetative buds were collected in the spring before breaking dormancy from juvenile and mature donor trees for conducting these evaluations. Medium, with or without MES, was pre-adjusted to five pH levels before adding MES, agar and autoclaving. Medium pH changes and explant growth parameters were measured at eight different incubation times. Overall, MES provided a more stable medium pH, relative to starting pH values, under both light and dark storage conditions as well as with presence of explants. A general trend of decreasing medium pH over time was found comparing explants from juvenile and mature donor genotypes. Explant height and weight growth increased over time, but differ among explants from juvenile and mature donor genotypes. Our findings suggest that a 21-day subculture practice may best sustain medium freshness, medium pH level and desirable explant growth. PMID:26535110

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

PubMed

Ceasar, S Antony; Ignacimuthu, S

2011-09-01

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

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.

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...

[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%.

Low light and low ammonium are key factors for guayule leaf tissue shoot organogenesis and transformation.

PubMed

Dong, Niu; Montanez, Belen; Creelman, Robert A; Cornish, Katrina

2006-02-01

A new method has been developed for guayule tissue culture and transformation. Guayule leaf explants have a poor survival rate when placed on normal MS medium and under normal culture room light conditions. Low light and low ammonium treatment greatly improved shoot organogenesis and transformation from leaf tissues. Using this method, a 35S promoter driven BAR gene and an ubiquitin-3 promoter driven GUS gene (with intron) have been successfully introduced into guayule. These transgenic guayule plants were resistant to the herbicide ammonium-glufosinate and were positive to GUS staining. Molecular analysis showed the expected band and signal in all GUS positive transformants. The transformation efficiency with glufosinate selection ranged from 3 to 6%. Transformation with a pBIN19-based plasmid containing a NPTII gene and then selection with kanamycin also works well using this method. The ratio of kanamycin-resistant calli to total starting explants reached 50% in some experiments.

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.

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.

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.

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.

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

PubMed

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

2008-01-01

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

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.

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.

Efficient propagation of citrus rootstocks by stem cuttings

USDA-ARS?s Scientific Manuscript database

A simple multicomponent system is described that is effective for rapid propagation of a diversity of citrus rootstock genotypes by single node cuttings, including new hybrids and those that are most commonly used as rootstocks. Efficiency of this system for rooting shoot explants of six important r...

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...

Uneven drying of zygotic embryos and embryonic axes of recalcitrant seeds: challenges and considerations for cryopreservation.

PubMed

Ballesteros, Daniel; Sershen; Varghese, Boby; Berjak, Patricia; Pammenter, Norman W

2014-08-01

Cryopreservation is the most promising option for the long-term germplasm conservation of recalcitrant-seeded species. However, the variable post-cryo success achieved with the excised zygotic explants traditionally used for cryopreservation has been a concern for some time. Differential drying rates amongst explants of different species, uneven drying amongst explants within a batch of seeds and uneven drying across tissues within individual embryos could be contributory factors to this variable success and these phenomena form the foci of the present study. Using zygotic explants from a range of recalcitrant-seeded species, which included sub-tropical dicotyledonous trees and sub-tropical monocotyledonous geophytes, the study showed that embryo morphology and anatomy are critical determinants of the drying characteristics of the different tissues composing the explant and hence, post-cryo survival. The results suggest that the rates of drying of explants to water contents (WCs) in the theoretically optimal range for successful cryopreservation are species-specific, and that more rapid drying rates may promote post-cryo survival. However, the large variation in WC amongst individual explants in bulk samples challenges the selection of the theoretically optimum WC for cryopreservation. As a consequence of differential drying rates across the different tissues composing explants, either lethal ice crystal damage or desiccation damage may sometimes be likely in tissues responsible for the onwards development of the embryo. Drying times for cryopreservation of such explants should, therefore, be selected on the basis of WC of segments containing root or shoot meristem, rather than embryo bulk WC. Drying intensity and duration also interact with explant morphology and embryo/axis size and anatomy to bring about - or preclude - post-cryo survival. Copyright © 2014 Elsevier Inc. All rights reserved.

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.

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

PubMed

Radice, Silvia

2010-01-01

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

Transgene expression in pear (Pyrus communis L.) driven by a phloem-specific promoter

USDA-ARS?s Scientific Manuscript database

A gene expression cassette carrying ß-glucuronidase (uidA) reporter gene under the control of the promoter of the Arabidopsis sucrose-H+ symporter gene (AtSUC2) was introduced to pear plants via an Agrobacterium-mediated leaf-explant transformation procedure. Transgenic shoots were regenerated from...

Micropropagation of pear (Pyrus sp.).

PubMed

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

2013-01-01

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

In vitro 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.

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 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 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.

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.

Apricot (Prunus armeniaca L.).

PubMed

Petri, César; Alburquerque, Nuria; Burgos, Lorenzo

2015-01-01

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

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

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.

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.

Regeneration of plants from Fraxinus pennsylvanica hypocotyls and cotyledons

Treesearch

Ningxia Du; Paula M. Pijut

2008-01-01

An adventitious shoot regeneration and rooting protocol was developed for green ash (Fraxinus pennsylvanica) seedling explants. The best regeneration medium for freshly isolated hypocotyls and cotyledons was Murashige and Skoog (MS) supplemented with 13.3 µM6-benzylaminopurine (BA) plus 4.5 µM thidiazuron (TDZ), and 22.2 µM...

A dark incubation period is important for Agrobacterium-mediated transformation of mature internode explants of sweet orange, grapefruit, citron, and a citrange rootstock.

PubMed

Marutani-Hert, Mizuri; Bowman, Kim D; McCollum, Greg T; Mirkov, T Erik; Evens, Terence J; Niedz, Randall P

2012-01-01

Citrus has an extended juvenile phase and trees can take 2-20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.

A Dark Incubation Period Is Important for Agrobacterium-Mediated Transformation of Mature Internode Explants of Sweet Orange, Grapefruit, Citron, and a Citrange Rootstock

PubMed Central

Marutani-Hert, Mizuri; Bowman, Kim D.; McCollum, Greg T.; Mirkov, T. Erik; Evens, Terence J.; Niedz, Randall P.

2012-01-01

Background Citrus has an extended juvenile phase and trees can take 2–20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees. Methodology/Principal Findings Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings. Significance The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid. PMID:23082165

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.

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...

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.

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

PubMed

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

2017-01-01

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

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

PubMed

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

2012-01-01

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

A highly efficient protocol for micropropagation of Begonia tuberous.

PubMed

Duong, Tan Nhut; Nguyen, Thanh Hai; Mai, Xuan Phan

2010-01-01

A protocol for micropropagation of begonia was established utilizing a thin cell layer (TCL) system. This system has been employed to produce several thousand shoots per sample. Explant size and position, and plant growth regulators (PGRs) contribute to the tissue morphogenesis. By optimizing the size of the tissue and applying an improved selection procedure, shoots were elongated in 8 weeks of culture, with an average number of 210 +/- 9.7 shoots per segment. This system has facilitated a number of studies using TCL as a model for micropropagation and will enable the large-scale production of begonia. On an average, the best treatment would allow production of about 10,000 plantlets by the micropropagation of the axillary buds of one plant with five petioles, within a period of 8 months.

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...

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.

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...

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.

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.

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

PubMed

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

2014-03-01

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

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...

Finger millet [Eleusine coracana (L.) Gaertn].

PubMed

Ceasar, Stanislaus Antony; Ignacimuthu, Savarimuthu

2015-01-01

Millets are the primary food source for millions of people in tropical regions of the world supplying mineral nutrition and protein. In this chapter, we describe an optimized protocol for the Agrobacterium-mediated transformation of finger millet variety GPU 45. Agrobacterium strain LBA4404 harboring plasmid pCAMBIA1301 which contains hygromycin phosphotransferase (hph) as selectable marker gene and β-glucuronidase (GUS) as reporter gene has been used. This protocol utilizes the shoot apex explants for the somatic embryogenesis and regeneration of finger millet after the transformation by Agrobacterium. Desiccation of explants during cocultivation helps for the better recovery of transgenic plants. This protocol is very useful for the efficient production of transgenic plants in finger millet through Agrobacterium-mediated transformation.

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.

High taxonomic diversity of cultivation-recalcitrant endophytic bacteria in grapevine field shoots, their in vitro introduction, and unsuspected persistence.

PubMed

Thomas, Pious; Sekhar, Aparna C; Shaik, Sadiq Pasha

2017-11-01

Molecular and microscopic analyses reveal enormous non-cultivable endophytic bacteria in grapevine field shoots with functional significance. Diverse bacteria enter tissue cultures through surface-sterilized tissues and survive surreptitiously with varying taxonomic realignments. The study was envisaged to assess the extent of endophytic bacterial association with field shoot tissues of grapevine and the likelihood of introduction of such internally colonizing bacteria in vitro adopting molecular techniques targeting the non-cultivable bacterial community. PowerFood ® -kit derived DNA from surface-sterilized field shoot tips of grapevine Flame Seedless was employed in a preliminary bacterial class-specific PCR screening proving positive for major prokaryotic taxa including Archaea. Taxonomic and functional diversity were analyzed through whole metagenome profiling (WMG) which revealed predominantly phylum Actinobacteria, Proteobacteria, and minor shares of Firmicutes, Bacteroidetes, and Deinococcus-Thermus with varying functional roles ascribable to the whole bacterial community. Field shoot tip tissues and callus derived from stem segments were further employed in 16S rRNA V3-V4 amplicon taxonomic profiling. This revealed elevated taxonomic diversity in field shoots over WMG, predominantly Proteobacteria succeeded by Actinobacteria, Firmicutes, Bacteroidetes, and 15 other phyla including several candidate phyla (135 families, 179 genera). Callus stocks also displayed broad bacterial diversity (16 phyla; 96 families; 141 genera) bearing resemblance to field tissues with Proteobacterial dominance but a reduction in its share, enrichment of Actinobacteria and Firmicutes, disappearance of some field-associated phyla and detection of a few additional taxonomic groups over field community. Similar results were documented during 16S V3-V4 amplicon taxonomic profiling on Thompson Seedless field shoot tip and callus tissues. Video microscopy on tissue homogenates corroborated enormous endophytic bacteria. This study elucidates a vast diversity of cultivation-recalcitrant endophytic bacteria prevailing in grapevine field shoots, their in vitro introduction, and unsuspecting sustenance with possible silent participation in tissue culture processes.

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

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.

Elimination of viruses through thermotherapy and meristem culture in apple cultivar 'Oregon Spur-II'.

PubMed

Vivek, Manu; Modgil, Manju

2018-03-01

In the present study, an attempt was made to eliminate apple chlorotic leaf spot virus, apple mosaic virus, apple stem grooving virus and apple stem pitting virus from apple cultivar 'Oregon Spur-II'. Thermotherapy was carried out at 37-40 °C for 4 weeks followed by culturing of meristems of different sizes. During establishment of explants, highest survival percentage (62.35%) and proliferation (30.68%) was recorded during summer season. However, size of meristems and position of buds from where meristems were excised also influenced their survival. The meristems of size 0.6-0.7 mm were found to be the most appropriate for maximum establishment. Meristems excised from buds positioned on distil portions of actively growing shoots showed better results. MS medium supplemented with BA (1.0 mg/l), IBA (0.05 mg/l) and GA 3 (0.1 mg/l) resulted in 56.62% establishment of explants, while maximum number of meristems proliferated with low BA (0.5 mg/l), IBA (0.08 mg/l) and same GA 3 concentration. Two to fourfold multiplication was observed. Virus indexing of shoots raised from different sizes of meristems was carried out and found that 0.3-0.6 mm size was able to eliminate ACLSV, ApMV, ASGV and ASPV. However, some of 0.5-0.6 mm sized shoots were found infected with ACLSV. Larger meristems could not completely eliminate the viruses under study.

Comparison of somatic embryogenesis in Medicago sativa and Medicago truncatula.

PubMed

Hoori, F; Ehsanpour, A A; Mostajeran, A

2007-02-01

In this study, the regeneration through embryogenesis of two species of Medicago were studied. Seeds of Medicago sativa cv. Rehnani and M. truncatula line A17 were grown on MS medium. After 4-6 weeks, segments of leaf and stem from two species were transferred to MS medium containing 2 mg L(-1) NAA, 2,4-D and Kinetin. The results indicated that callus formation from leaf explants of M. sativa was higher than M. trancatula. In the next stage, media with different combinations of auxin, cytokinin or ethinyl estradiol were provided for regeneration. Then in two stages, explants of leaf and stem of two species were transferred on these media. Results after 3-6 weeks showed that in medium containing NAA and TDZ, stem pieces ofM. sativa produced shoots while leaf pieces on NAA and ethinyl estradiol formed roots. Leaf explants of M. truncatula in the medium containing NAA and BAP, produced somatic embryos. Also in media with auxin and ethinyl estradiol, somatic embryos were formed on calli of two species. Ethinyl estradiol and auxin together can induce somatic embryogenesis and root production on calli and stem or leaf explants.

Endophytic Penicillium citrinum Thom. from Scoparia dulcis Linn.

PubMed

Mathew, Annie J; Jayachandran, K; Mathew, Jyothis

2010-10-01

Scoparia dulcis of Scrophulariaceae is an annual herb distributed through out the tropics. Penicillium citrinum was obtained from apparently healthy roots, stem, leaves and fruits of this plant. Callus and multiple shoots produced during micropropagation from various explants were also symptomless but showed occurrence of Penicillium citrinum when cultured in Murashige & Skoog liquid medium for the production of secondary metabolites.

Shoot regeneration and plantlet formation by cascade huckleberry, mountain huckleberry, and in oval-leaf bilberry on a zeatin-containing nutrient medium

USDA-ARS?s Scientific Manuscript database

A plant regeneration protocol was developed for Cascade huckleberry (Vaccinium deliciosum Piper), mountain huckleberry (V. membranaceum Douglas ex Hooker) and for oval-leaf bilberry (V. ovalifolium Smith) clones. The effects of zeatin concentrations (0, 4.6, 9.1 and 13.7 µM) and explant type (leaf a...

Direct and indirect organogenesis of Alpinia galanga and the phytochemical analysis.

PubMed

Rao, Kiranmayee; Chodisetti, Bhuvaneswari; Gandi, Suryakala; Mangamoori, Lakshmi Narasu; Giri, Archana

2011-11-01

Alpinia galanga is a rhizomatous herb rich in essential oils and various other significant phytoconstituents. Rapid direct regeneration was obtained from the rhizome explants (15.66 ± 0.57 shoots) on MS media supplemented with zeatin at a concentration of 2 mg/l. The callus cultures of A. galanga were initiated from the rhizome explants on MS media supplemented with 2 mg/l each of BAP, 2,4-D, and NAA. The callus was analyzed for the presence of a vital phytoconstituent--acetoxychavicol acetate (ACA) associated with various biological properties. ACA was detected in the young friable callus as well as the stationary phase callus. Moreover, the induction of morphogenetic response in callus resulted in higher accumulation of ACA. The phytohormone withdrawal from the propagation media and the subsequent transfer of callus to BAP (2 mg/l) containing MS media has resulted in multiple shoot induction. The regenerated (indirect) plants have shown 1.6-fold higher ACA content (1.253%) when compared to the control plant (0.783%). Micropropagation of such conventionally propagated plants is very essential to meet the commercial demand as well as to ensure easy storage and transportation of disease free stocks.

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.

Micropropagation of African violet (Saintpaulia ionantha Wendl.).

PubMed

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

2013-01-01

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

Potato leaf explants as a spaceflight plant test system

NASA Technical Reports Server (NTRS)

Wheeler, R. M.

1986-01-01

The use of explant tissues or organs may circumvent limitations facing whole-plant experimentation during spaceflight. In the case of potato, a crop currently being studied for application to bioregenerative life support systems, excised leaves and their subtended axillary buds can be used to test a variety of stem growth and development phases ranging from tubers through stolons (horizontal stems) to upright leafy shoots. The leaves can be fit well into small-volume test packages and sustained under relatively low irradiance levels using light-weight growing media. Tubers formed on potato leaf cuttings can yield up from 0.5 to 1.0 g fresh mass 10 days after excision and up to 2.0 g or more, 14 days from excision.

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

PubMed

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

1996-11-01

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

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

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

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

PubMed

Soriano, Thelma L; Cangao, Evangelina C

2016-01-01

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

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.

Petunia (Petunia hybrida).

PubMed

Lutke, W Kevin

2006-01-01

Petunia hybrida genetic transformation continues to be a valuable tool for genetic research into biochemical pathways and gene expression, as well as generating commercial products with varying floral colors. In this chapter, we describe a simple and reproducible genetic transformation protocol for generating transgenic petunia plants harboring a gene of interest and selectable marker. The system utilizes Agrobacterium tumefaciens for transgene integration with plant recovery via shoot organogenesis from leaf explant material. Selection for transgenic plants is achieved using the bar gene conferring resistance to glufosinate or nptII gene for resistance to kanamycin. Transformation efficiencies of around 10% are achievable with shoots being recovered about 8 wk after transgene insertion and rooted plants transferred to the greenhouse about twelve weeks after inoculation.

An Efficient Method for Adventitious Root Induction from Stem Segments of Brassica Species

PubMed Central

Srikanth, Sandhya; Choong, Tsui Wei; Yan, An; He, Jie; Chen, Zhong

2016-01-01

Plant propagation via in vitro culture is a very laborious and time-consuming process. The growth cycle of some of the crop species is slow even in the field and the consistent commercial production is hard to maintain. Enhanced methods of reduced cost, materials and labor significantly impact the research and commercial production of field crops. In our studies, stem-segment explants of Brassica species were found to generate adventitious roots (AR) in aeroponic systems in less than a week. As such, the efficiency of rooting from stem explants of six cultivar varieties of Brassica spp was tested without using any plant hormones. New roots and shoots were developed from Brassica alboglabra (Kai Lan), B. oleracea var. acephala (purple kale), B. rapa L. ssp. chinensis L (Pai Tsai, Nai Bai C, and Nai Bai T) explants after 3 to 5 days of growing under 20 ± 2°C cool root zone temperature (C-RZT) and 4 to 7 days in 30 ± 2°C ambient root zone temperature (A-RZT). At the base of cut end, anticlinal and periclinal divisions of the cambial cells resulted in secondary xylem toward pith and secondary phloem toward cortex. The continuing mitotic activity of phloem parenchyma cells led to a ring of conspicuous white callus. Root initials formed from the callus which in turn developed into ARs. However, B. rapa var. nipposinica (Mizuna) explants were only able to root in C-RZT. All rooted explants were able to develop into whole plants, with higher biomass obtained from plants that grown in C-RZT. Moreover, explants from both RZTs produced higher biomass than plants grown from seeds (control plants). Rooting efficiency was affected by RZTs and explant cuttings of donor plants. Photosynthetic CO2 assimilation rate (Asat) and stomatal conductance (gssat) were significantly differentiated between plants derived from seeds and explants at both RZTs. All plants in A-RZT had highest transpiration rates. PMID:27446170

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 tulip: production of virus-free stock plants.

PubMed

Podwyszyńska, Małgorzata; Sochacki, Dariusz

2010-01-01

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

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.

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.

Micropropagation and genetic transformation of Tylophora indica (Burm. f.) Merr.: a review.

PubMed

Teixeira da Silva, Jaime A; Jha, Sumita

2016-11-01

This review provides an in-depth and comprehensive overview of the in vitro culture of Tylophora species, which have medicinal properties. Tylophora indica (Burm. f.) Merr. is a climbing perennial vine with medicinal properties. The tissue culture and genetic transformation of T. indica, which has been extensively studied, is reviewed. Micropropagation using nodal explants has been reported in 25 % of all publications. Leaf explants from field-grown plants has been the explant of choice of independent research groups, which reported direct and callus-mediated organogenesis as well as callus-mediated somatic embryogenesis. Protoplast-mediated regeneration and callus-mediated shoot organogenesis has also been reported from stem explants, and to a lesser degree from root explants of micropropagated plants in vitro. Recent studies that used HPLC confirmed the potential of micropropagated plants to synthesize the major T. indica alkaloid tylophorine prior to and after transfer to field conditions. The genetic integrity of callus-regenerated plants was confirmed by RAPD in a few reports. Tissue culture is an essential base for genetic transformation studies. Hairy roots and transgenic T. indica plants have been shown to accumulate tylophorine suggesting that in vitro biology and transgenic methods are viable ways of clonally producing valuable germplasm and mass producing compounds of commercial value. Further studies that investigate the factors affecting the biosynthesis of Tylophora alkaloids and other secondary metabolites need to be conducted using non-transformed as well as transformed cell and organ cultures.

Citrus transformation using juvenile tissue explants.

PubMed

Orbović, Vladimir; Grosser, Jude W

2015-01-01

The most frequently used method for production of citrus transgenic plants is via Agrobacterium-mediated transformation of tissues found on explants obtained from juvenile seedlings. Within the last decade and especially within the last 5-6 years, this robust method was employed to produce thousands of transgenic plants. With the newly applied screening methods that allow easier and faster detection of transgenic shoots, estimates of transformation rate for some cultivars have gone up making this approach even more attractive. Although adjustments have to be made regarding the (varietal) source of the starting material and Agrobacterium strain used in each experiment preformed, the major steps of this procedure have not changed significantly if at all. Transgenic citrus plants produced this way belong to cultivars of rootstocks, sweet oranges, grapefruits, mandarins, limes, and lemons.

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.

Pineapple [Ananas comosus (L.) Merr].

PubMed

Gangopadhyay, Gaurab; Mukherjee, Kalyan K

2015-01-01

The efficacy of Agrobacterium-mediated pineapple transformation technique has been improved (mean percentage of transgenic micro-shoots regenerated from initial callus explants up to 20.6%) using a novel encapsulation-based, antibiotic selection procedure. The detailed protocol using a standard plant transformation vector (pCAMBIA1304) as reported in an 'elite' Indian variety (Queen) of pineapple [Ananas comosus (L.) Merr] can be applied to other varieties of pineapple for introgression of target genes.

Micropropagation of Calophyllum brasiliense (Cambess.) from nodal segments.

PubMed

Silveira, S S; Cordeiro-Silva, R; Degenhardt-Goldbach, J; Quoirin, M

2016-05-03

Micropropagation of Calophyllum brasiliense Cambess. (Clusiaceae) is a way to overcome difficulties in achieving large-scale plant production, given the recalcitrant nature of the seeds, irregular fructification and absence of natural vegetative propagation of the species. Cultures were established using nodal segments 2 cm in length, obtained from 1-2 year old seedlings, maintained in a greenhouse. Mercury chloride and Plant Preservative Mixture™ were used in the surface sterilizing stage, better results being achieved with Plant Preservative Mixture™ incorporation in culture medium, at any concentration. Polyvinylpyrrolidone, activated charcoal, cysteine, ascorbic acid or citric acid were added to the culture medium to avoid oxidation. After 30 days of culture, polyvinylpirrolidone and ascorbic acid gave better results, eliminating oxidation in most explants. For shoot multiplication, benzylaminopurine was used in concentrations of 4.4 and 8.8 µM in Woody Plant Medium, resulting in an average of 4.43 and 4.68 shoots per explant, respectively, after 90 days. Indole-3-butyric acid and α-naphthalene acetic acid were used to induce root formation, reaching a maximum rooting rate of 24% with 20µM α-naphthalene acetic acid. For acclimatization. the rooted plants were transferred to Plantmax® substrate and cultured in a greenhouse, reaching 79% of survival after 30 days and 60% after one year.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects.

PubMed

Cipriano, Jamile L D; Cruz, Ana Cláudia F; Mancini, Karina C; Schmildt, Edilson R; Lopes, José Carlos; Otoni, Wagner C; Alexandre, Rodrigo S

2018-01-01

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

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.

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.

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.

Enzyme-Linked Immunosorbent Assay Testing of Shoots Grown In Vitro and the Use of Immunocapture-Reverse Transcription-Polymerase Chain Reaction Improve the Detection of Prunus necrotic ringspot virus in Rose.

PubMed

Moury, B; Cardin, L; Onesto, J P; Candresse, T; Poupet, A

2000-05-01

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 10(4) to 10(5) increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

Study on suitable for regeneration system of genetic transformation of kiwifruit

NASA Astrophysics Data System (ADS)

Yuan, Yun Xiang

2011-02-01

The stems of Actinidia Qinmei were taken as explants and induced callus formation after having gained the aseptic seedling in primary culture successfully, and then the calli were placed on different combinations regeneration medium. The results showed that the induction medium added 1 mg/L 6-BA and 0.1 mg/L NAA was beneficial to the callus induction in dark culture condition. The suitable regeneration medium was MS containing 2.0 mg/L 6BA, 0.1 mg/L NAA and 3% (W/V) sucrose, it could improve obviously the frequency of regenerated shoots. This method separated the callus induction from shoot regeneration and obtained more callus to optimize the regenerated medium, and also was advantageous to Kiwifruit genetic transformation.

Optimization of Agrobacterium-Mediated Transformation in Soybean.

PubMed

Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

2017-01-01

High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens -mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium -mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD 650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA 3 ) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA 3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA 3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA 3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide (glufosinate) painting, and QuickStix Kit for Liberty Link ( bar ) were used to verify the positive transgenic plants, and absolute quantification PCR confirmed the exogenous gene existed as one to three copies in the soybean genome. This study provides an improved protocol for Agrobacterium -mediated transformation in soybean and a useful reference to improve the transformation efficiency in other plant species.

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

PubMed

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

2010-02-01

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

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...

Development of an Agrobacterium-Mediated Stable Transformation Method for the Sensitive Plant Mimosa pudica

PubMed Central

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements. PMID:24533121

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

PubMed

Mano, Hiroaki; Fujii, Tomomi; Sumikawa, Naomi; Hiwatashi, Yuji; Hasebe, Mitsuyasu

2014-01-01

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2013-05-01

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

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

PubMed Central

Dohling, Stadwelson; Kumaria, Suman; Tandon, Pramod

2012-01-01

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

In Vitro Propagation and Conservation of Withania somnifera (Dunal) L.

PubMed

Fatima, Nigar; Ahmad, Naseem; Anis, Mohammad

2016-01-01

Plant tissue culture offers several techniques for rapid clonal propagation, germplasm conservation, regeneration of genetically manipulated superior clones, production of phyto-constituents, and ex vitro conservation of valuable phytodiversity. An improved and efficient micropropagation protocol for Withania somnifera (L.), a drug-producing medicinal plant, using juvenile explants (nodal explants) has been developed. Highest multiplication and subsequent elongation of shoots is observed on MS medium containing BA and NAA. The regenerated microshoots roots best on ½ MS medium containing NAA, established in earthen pots containing garden soil and are maintained in the greenhouse with 95 % survival rate. Genetic uniformity of micropropagated plants is confirmed by PCR-based DNA fingerprinting techniques, viz., RAPD and ISSR. No variation is observed in DNA fingerprinting patterns among the micropropagated plants, which are similar to that of the donor plant illustrating their genetic uniformity.

Development of guayule (Parthenium argentatum) research in cell culture

NASA Technical Reports Server (NTRS)

Ball, E. A.

1981-01-01

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

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 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.

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

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

NASA Astrophysics Data System (ADS)

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

2018-04-01

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

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

Plant regeneration through protocorm-like bodies induced from rhizoids using leaf explants of Rosa spp.

PubMed

Tian, Chuanwei; Chen, Ying; Zhao, Xiaolan; Zhao, Liangjun

2008-05-01

A new protocol for plant regeneration via protocorm-like bodies (PLBs) induced from rhizoids that developed from leaf explants of Rosa spp. (R. canina L., R. multiflora var. cathayensis Rehd. et Wils., and R. multiflora f. carnea Thory.) has been established. Rhizoids were induced from calli of leaf explants incubated under dark conditions on Murashige and Skoog (MS) medium containing 1.5 mg/l 2, 4-D. PLBs developed from the tip of rhizoids cultured under light conditions on (1/2) MS medium containing 20 mg/l TDZ. About 90, 17 and 93% of rhizoid formation were achieved for the above-mentioned Rosa spp., respectively using this protocol. The frequency of PLB clusters formation and the number of PLB clusters per explant reached 50% and 5.1 for R. canina, 46.7% and 0.8 for R. multifolra var. cathayensis, 46.7% and 4.2 for R. multiflora f. carnea, respectively. PLB clusters regenerated on MS medium supplemented with 2 mg/l 6-BA, 0.1 mg/l IBA, and 0.1 mg/l GA(3). The best result of regenerated plantlets per leaf explant achieved via PLBs for the three Rosa spp. mentioned above was 3.6, 0.1, and 1.2, respectively. Environmental scanning electron microscope and histological studies revealed that rhizoids were structurally different from roots grown in vitro, and PLBs developed from proembryos.

Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

PubMed Central

Han, Xue; Ma, Shurong; Kong, Xianghui; Takano, Tetsuo; Liu, Shenkui

2013-01-01

Poplar is a model organism for high in vitro regeneration in woody plants. We have chosen a hybrid poplar Populus davidiana Dode × Populus bollena Lauche. By optimizing the Murashige and Skoog medium with (0.3 mg/L) 6-benzylaminopurine and (0.08 mg/L) naphthaleneacetic acid, we have achieved the highest frequency (90%) for shoot regeneration from poplar leaves. It was also important to improve the transformation efficiency of poplar for genetic breeding and other applications. In this study, we found a significant improvement of the transformation frequency by controlling the leaf age. Transformation efficiency was enhanced by optimizing the Agrobacterium concentration (OD600 = 0.8–1.0) and an infection time (20–30 min). According to transmission electron microscopy observations, there were more Agrobacterium invasions in the 30-day-old leaf explants than in 60-day-old and 90-day-old explants. Using the green fluorescent protein (GFP) marker, the expression of MD–GFP fusion proteins in the leaf, shoot, and root of hybrid poplar P. davidiana Dode × P. bollena Lauche was visualized for confirmation of transgene integration. Southern and Northern blot analysis also showed the integration of T-DNA into the genome and gene expression of transgenic plants. Our results suggest that younger leaves had higher transformation efficiency (~30%) than older leaves (10%). PMID:23354481

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

PubMed

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

2014-08-01

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

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.

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.

Biological activities of indoleacetylamino acids and their use as auxins in tissue culture

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

Hangarter, R.P.; Peterson, M.D.; Good, N.E.

1980-05-01

The auxin activities of a number of indoleacetylamino acid conjugates have been determined in three test systems: growth of tomato hypocotyl explants (Lycopersicon esculentum Mill. cv. Marglobe); growth of tobacco callus cultures (Nicotiana tabacum L. cv. Wisconsin 38); and ethylene production from pea stems (Pisum sativum L. cv. Alaska). The activities of the conjugates differ greatly depending on the amino acid moiety. Indoleacetyl-L-alanine supports rapid callus growth from the tomato hypocotyls while inhibiting growth of shoots and roots. Indoleacetlyglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting growth of shoots andmore » roots. Indoleacetylglycine behaves in a similar manner but is somewhat less effective in supporting callus growth and in inhibiting shoot formation. The other amino acid conjugates tested (valine, leucine, aspartic acid, threonine, methionine, phenylalanine, and proline) support shoot formation without supporting root formation or much callus growth. The tobacco callus system, which forms abundant shoots in the presence or absence of free indoleacetic acid, produces only rapid undifferentiated growth in the presence of indoleacetyl-L-alanine and indoleacetylglycine. The other conjugates inhibit shoot formatin weakly if at all. Most of the conjugates induce sustained ethylene production from the pea stems but at rates well below the initial rates observed with free indoleacetic acid. Many, but not all of the effects of conjugates such as indoleacetyl-L-alanine can be mimicked by frequent renewals of the supply of free indoleacetic acid.« less

Blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing

2015-01-01

Vaccinium consists of approximately 450 species, of which highbush blueberry (Vaccinium corymbosum) is one of the three major Vaccinium fruit crops (i.e., blueberry, cranberry, and lingonberry) domesticated in the twentieth century. In blueberry the adventitious shoot regeneration using leaf explants has been the most desirable regeneration system to date; Agrobacterium tumefaciens-mediated transformation is the major gene delivery method and effective selection has been reported using either the neomycin phosphotransferase II gene (nptII) or the bialaphos resistance (bar) gene as selectable markers. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining the optimal conditions for efficient plant regeneration, reliable gene delivery, and effective selection. The protocol has led to successful regeneration of transgenic plants from leaf explants of four commercially important highbush blueberry cultivars for multiple purposes, providing a powerful approach to supplement conventional breeding methods for blueberry by introducing genes of interest.

Cryopreservation of in vitro grown nodal segments of Rauvolfia serpentina by PVS2 vitrification.

PubMed

Ray, Avik; Bhattacharya, Sabita

2008-01-01

This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant. The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested. Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2. When optimizing the PVS2 vitrification of nodal segments, those of 0.31 - 0.39 cm in size were better than other nodal sizes and or apices. Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants. Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments. PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66 percent. Plantlets derived after cryopreservation resumed growth and regenerated normally.

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.

Factors enhancing Agrobacterium tumefaciens-mediated gene transfer in peanut (Arachis hypogaea L.)

NASA Technical Reports Server (NTRS)

Egnin, M.; Mora, A.; Prakash, C. S.; Mortley, D. G. (Principal Investigator)

1998-01-01

Parameters enhancing Agrobacterium-mediated transfer of foreign genes to peanut (Arachis hypogaea L.) cells were investigated. An intron-containing beta-glucuronidase uidA (gusA) gene under the transcriptional control of CaMV 35S promoter served as a reporter. Transformation frequency was evaluated by scoring the number of sectors expressing GUS activity on leaf and epicotyl explants. The 'Valencia Select' market type cv. New Mexico was more amenable to Agrobacterium transformation than the 'runner' market type cultivars tested (Florunner, Georgia Runner, Sunrunner, or South Runner). The disarmed Agrobacterium tumefaciens strain EHA101 was superior in facilitating the transfer of uidA gene to peanut cells compared to the disarmed strain C58. Rinsing of explants in half-strength Murashige-Skoog (MS) media prior to infection by Agrobacterium significantly increased the transformation efficiency. The use of cocultivation media containing high auxin [1.0 or 2.5 mg/l (4.53 micromolar or 11.31 micromolar) 2,4-D] and low cytokinin [0.25 or 0.5 mg/l (1.0 micromolar or 2.0 micromolar) BA] promoted higher transformation than either hormone-free or thidiazuron-containing medium. The polarity of the epicotyl during cocultivation was important; explants incubated in an inverted (vertically) manner followed by a vertically upright position resulted in improved transformation and shoot regeneration frequencies. Preculture of explants in MS basal medium or with 2.5 mg thidiazuron per l prior to infection drastically decreased the number of transformed zones. The optimized protocol was used to obtain transient transformation frequencies ranging from 12% to 36% for leaf explants, 15% to 42% for epicotyls. Initial evidence of transformation was obtained by polymerase chain reaction and subsequently confirmed by Southern analysis of regenerated plants.

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

PubMed

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

2011-06-01

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

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

PubMed Central

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

2014-01-01

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

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.

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)

Optimization of Agrobacterium-Mediated Transformation in Soybean

PubMed Central

Li, Shuxuan; Cong, Yahui; Liu, Yaping; Wang, Tingting; Shuai, Qin; Chen, Nana; Gai, Junyi; Li, Yan

2017-01-01

High transformation efficiency is a prerequisite for study of gene function and molecular breeding. Agrobacterium tumefaciens-mediated transformation is a preferred method in many plants. However, the transformation efficiency in soybean is still low. The objective of this study is to optimize Agrobacterium-mediated transformation in soybean by improving the infection efficiency of Agrobacterium and regeneration efficiency of explants. Firstly, four factors affecting Agrobacterium infection efficiency were investigated by estimation of the rate of GUS transient expression in soybean cotyledonary explants, including Agrobacterium concentrations, soybean explants, Agrobacterium suspension medium, and co-cultivation time. The results showed that an infection efficiency of over 96% was achieved by collecting the Agrobacterium at a concentration of OD650 = 0.6, then using an Agrobacterium suspension medium containing 154.2 mg/L dithiothreitol to infect the half-seed cotyledonary explants (from mature seeds imbibed for 1 day), and co-cultured them for 5 days. The Agrobacterium infection efficiencies for soybean varieties Jack Purple and Tianlong 1 were higher than the other six varieties. Secondly, the rates of shoot elongation were compared among six different concentration combinations of gibberellic acid (GA3) and indole-3-acetic acid (IAA). The shoot elongation rate of 34 and 26% was achieved when using the combination of 1.0 mg/L GA3 and 0.1 mg/L IAA for Jack Purple and Tianlong 1, respectively. This rate was higher than the other five concentration combinations of GA3 and IAA, with an 18 and 11% increase over the original laboratory protocol (a combination of 0.5 mg/L GA3 and 0.1 mg/L IAA), respectively. The transformation efficiency was 7 and 10% for Jack Purple and Tianlong 1 at this optimized hormone concentration combination, respectively, which was 2 and 6% higher than the original protocol, respectively. Finally, GUS histochemical staining, PCR, herbicide (glufosinate) painting, and QuickStix Kit for Liberty Link (bar) were used to verify the positive transgenic plants, and absolute quantification PCR confirmed the exogenous gene existed as one to three copies in the soybean genome. This study provides an improved protocol for Agrobacterium-mediated transformation in soybean and a useful reference to improve the transformation efficiency in other plant species. PMID:28286512

Genetic transformation of Begonia tuberhybrida by Ri rol genes.

PubMed

Kiyokawa, S; Kikuchi, Y; Kamada, H; Harada, H

1996-04-01

We have developed an Agrobacterium -mediated transformation system for commercial Begonia species. The leaf explants of Begonia semperflorens, Begonia x hiemalis and B. tuberhybrida were inoculated with Agrobacterium tumefaciens LBA4404 harboring a binary vector pBI121 which contains rolA, B and C genes of an agropine type Ri plasmid (pRiA4b). Kanamycin resistant shoots of B. tuberhybrida were obtained on MS agar medium supplemented with 0.1 mg/l NAA, 0.5 mg/l BA, 500 mg/l claforan and 100 mg/l kanamycin. These shoots exhibited GUS activity and Southern analysis showed a single copy insertion into the genome. When the transgenic plants were transferred to soil, they displayed the phenotype specific to the transgenic plants by A. rhizogenes such as dwarfness, delay of flowering, and wrinkled leaves and petals.

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.

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

PubMed Central

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

2017-01-01

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

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.

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

Apical Correlative Effects in Leaf Epinasty of Tomato 1

PubMed Central

Kazemi, Saidollah; Kefford, Noel P.

1974-01-01

The influence of the stem apex on leaf curvature was investigated using debudded tomato (Lycopersicon esculentum Mill. cv Anahu) plants and petiole explants, consisting of a section of petiole attached to a section of stem. Decapitation of the main shoot of tomato plants induced hyponasty of petioles in young leaves. Application of auxin in place of the removed apex or fumigation of intact tomato plants with ethylene produced epinastic curvature at the base of the petiole. Simultaneous carbon dioxide treatments prevented the development of petiolar epinasty due to auxin and ethylene treatments. Application of ethylene gas to the decapitated shoot or injection into the stem, induced petiolar epinasty. In a saturating level of ethylene gas, tomato petioles did not respond to indole-3-acetic acid applied to the cut apex. Auxin-induced ethylene production in petiole explants preceded the development of epinasty. Application of indoleacetic acid in lanolin to the entire lower side of the petioles of leaves in situ produced petiole epinasty. Petiolar epinasty due to apically applied indoleacetic acid resulted from differential cell elongation. The auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, and naphthalene-1-acetic acid induced epinasty when applied apically to decapitated tomato plants, while gibberellic acid, kinetin, abscisic acid, and auxin or gibberellin antagonists had no effect. When such compounds were applied to petiole explants, only indole-3-acetic acid and kinetin caused an increase in ethylene production and the effect of kinetin was relatively weak. Application of 2,3,5-triiodobenzoic acid around the stem did not change the effect on petiolar epinasty of auxin applied to the decapitated shoot or around the stem. Radioautography showed that the label from 14C-indoleacetic acid applied apically entered the petiole and midrib tissue; however, extraction showed that only a fraction of the label in these tissues was in the form of indoleacetic acid. Removal of leaflets from leaves induced hyponasty in the midrib region, and application of auxin to the leaflet stubs produced midrib epinasty; carbon dioxide did not block the action of auxin in this type of epinasty. Removal of leaflets from leaves did not alter the effect of apically applied auxin on petiolar epinasty. The data are consistent with the hypothesis that the oblique orientation of leaves in tomato plants is influenced by two epinastic responses. Petiolar epinasty is controlled by the apical region on the stem and is due to the action of auxin-induced ethylene; and midrib epinasty is due to an action of auxin other than through ethylene. PMID:16658919

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

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.

Increased Agrobacterium-mediated transformation and rooting efficiencies in canola (Brassica napus L.) from hypocotyl segment explants

NASA Technical Reports Server (NTRS)

Cardoza, V.; Stewart, C. N.

2003-01-01

An efficient protocol for the production of transgenic Brassica napus cv. Westar plants was developed by optimizing two important parameters: preconditioning time and co-cultivation time. Agrobacterium tumefaciens-mediated transformation was performed using hypocotyls as explant tissue. Two variants of a green fluorescent protein (GFP)-encoding gene--mGFP5-ER and eGFP--both under the constitutive expression of the cauliflower mosaic virus 35S promoter, were used for the experiments. Optimizing the preconditioning time to 72 h and co-cultivation time with Agrobacterium to 48 h provided the increase in the transformation efficiency from a baseline of 4% to 25%. With mGFP5-ER, the transformation rate was 17% and with eGFP it was 25%. Transgenic shoots were selected on 200 mg/l kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 10 g/l sucrose and 0.5 mg/l indole butyric acid in the presence of kanamycin.

Histological characterization of Passiflora pohlii Mast. root tips cryopreserved using the V-Cryo-plate technique.

PubMed

Simão, Mariela J; Collin, Myriam; Garcia, Renata O; Mansur, Elisabeth; Pacheco, Georgia; Engelmann, Florent

2018-05-01

Cryopreservation stands out as the main strategy to ensure safe and cost efficient long-term conservation of plant germplasm, especially for biotechnological materials. However, the injuries associated with the procedure may result in structural damage and low recovery rates after cooling. Histological analysis provides useful information on the effects of osmotic dehydration, LN exposure, and recovery conditions on cellular integrity and tissue organization, allowing the determination of the critical steps of the cryopreservation protocol and, thus, the use of optimized treatments. Passiflora pohlii Mast. (Passifloraceae) is a native species from Brazil with potential agronomic interest. Recent studies showed the presence of saponins in its roots, which presented antioxidant activity. The goal of this work was to develop a cryopreservation technique for root tips of in vitro-derived plants of P. pohlii using the V-Cryo-plate technique and to characterize the anatomical alterations that occurred during the successive steps of the protocol. Root tips were excised from in vitro plants and precultured before adhesion to cryo-plates and then treated for different periods with the plant vitrification solutions PVS2 or PVS3. Treatment with PVS2 for 45 min resulted in higher recovery (79%) when compared with PVS3 (43%). The greatest number of adventitious roots per cryopreserved explant was also observed after a 45-min exposure to PVS2. Plasmolysis levels were higher in cortical cells of cryopreserved explants treated with PVS2, while pericycle and central cylinder cells were not damaged after this treatment. Thirty days after rewarming, no plasmolysis could be detected, regardless of the experimental conditions.

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.

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.

Establishment of the regeneration system for Vicia faba L.

PubMed

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

2009-01-01

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

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.

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.

The effect of low- and high-power microwave irradiation on in vitro grown Sequoia plants and their recovery after cryostorage.

PubMed

Halmagyi, A; Surducan, E; Surducan, V

2017-09-01

Two distinct microwave power levels and techniques have been studied in two cases: low-power microwave (LPM) irradiation on in vitro Sequoia plants and high-power microwave (HPM) exposure on recovery rates of cryostored (-196°C) Sequoia shoot apices. Experimental variants for LPM exposure included: (a) in vitro plants grown in regular conditions (at 24 ± 1°C during a 16-h light photoperiod with a light intensity of 39.06 μEm -2 s -1 photosynthetically active radiation), (b) in vitro plants grown in the anechoic chamber with controlled environment without microwave irradiation, and (c) in vitro plants grown in the anechoic chamber with LPM irradiation for various times (5, 15, 30, 40 days). In comparison to control plants, significant differences in shoot multiplication and growth parameters (length of shoots and roots) were observed after 40 days of LPM exposure. An opposite effect was achieved regarding the content of total soluble proteins, which decreased with increasing exposure time to LPM. HPM irradiation was tested as a novel rewarming method following storage in liquid nitrogen. To our knowledge, this is the first report using this type of rewarming method. Although, shoot tips subjected to HPM exposure showed 28% recovery following cryostorage compared to 44% for shoot tips rewarmed in liquid medium at 22 ± 1 °C, we consider that the method represent a basis and can be further improved. The results lead to the overall conclusion that LPM had a stimulating effect on growth and multiplication of in vitro Sequoia plants, while the HPM used for rewarming of cryopreserved apices was not effective to achieve high rates of regrowth after liquid nitrogen exposure.

Artificial Neural Network Genetic Algorithm As Powerful Tool to Predict and Optimize In vitro Proliferation Mineral Medium for G × N15 Rootstock

PubMed Central

Arab, Mohammad M.; Yadollahi, Abbas; Shojaeiyan, Abdolali; Ahmadi, Hamed

2016-01-01

One of the major obstacles to the micropropagation of Prunus rootstocks has, up until now, been the lack of a suitable tissue culture medium. Therefore, reformulation of culture media or modification of the mineral content might be a breakthrough to improve in vitro multiplication of G × N15 (garnem). We found artificial neural network in combination of genetic algorithm (ANN-GA) as a very precise and powerful modeling system for optimizing the culture medium, So that modeling the effects of MS mineral salts (NH4+, NO3-, PO42-, Ca2+, K+, SO42-, Mg2+, and Cl−) on in vitro multiplication parameters (the number of microshoots per explant, average length of microshoots, weight of calluses derived from the base of stem explants, and quality index of plantlets) of G × N15. Showed high R2 correlation values of 87, 91, 87, and 74 between observed and predicted values were found for these four growth parameters, respectively. According to the ANN-GA results, among the input variables, NH4+ and NO3- had the highest values of VSR in data set for the parameters studied. The ANN-GA showed that the best proliferation rate was obtained from medium containing (mM) 27.5 NO3-, 14 NH4+, 5 Ca2+, 25.9 K+, 0.7 Mg2+, 1.1 PO42-, 4.7 SO42-, and 0.96 Cl−. The performance of the medium optimized by ANN-GA, denoted as YAS (Yadollahi, Arab and Shojaeiyan), was compared to that of standard growth media for all Prunus rootstock, including the Murashige and Skoog (MS) medium, (specific media) EM, Quoirin and Lepoivre (QL) medium, and woody plant medium (WPM) Prunus. With respect to shoot length, shoot number per cultured explant and productivity (number of microshoots × length of microshoots), YAS was found to be superior to other media for in vitro multiplication of G × N15 rootstocks. In addition, our results indicated that by using ANN-GA, we were able to determine a suitable culture medium formulation to achieve the best in vitro productivity. PMID:27807436

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.

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...

Experience With Cardiac Implantable Electrical Device Explantation After Cardiac Transplantation: A Report of 16 Cases From a Single Center in a Period of 5 Years.

PubMed

Çiftci, Orçun; Yılmaz, Kerem Can; Sezgin, Atilla; Özin, Mehmet Bülent; Müderrisoğlu, İbrahim Haldun; Haberal, Mehmet

2018-03-01

Cardiac implantable electrical devices are widely used for patients with advanced heart failure and are usually explanted during orthotopic heart transplant. However, lead fragments and the pulse generator are sometimes left after the procedure. Given the concerns of infectious and thromboembolic complications, their removal is recommended. Herein, we report our experience with cardiac implantable electrical device explantation after orthotopic heart transplant. We included recipients of heart transplants performed at Başkent University Faculty of Medicine, Department of Cardiovascular Surgery, who underwent lead and pulse generator explantation by manual traction between January 2012 and June 2017. We analyzed patient demographic, clinical, biochemical, and treatment properties. Sixteen patients (11 males, 5 females) with a median age of 45 years (range, 18-52 y) were included. Two patients (12.5%) died during follow-up but not secondary to device explantation. All patients were using immunosuppressives and 50% were receiving antiplatelet/anticoagulant agents. All pulse generators were located at the left prepectoral area, with tips of lead fragments in the superior vena cava or left subclavian vein. No procedural complications were observed. Aspirin was continued uninterrupted perioperatively, warfarin was stopped 2 days before the procedure, and low-molecular-weight heparins were skipped on the morning and evening of the procedure. One patient (6.3%) complained of postoperative pain, and another (6.3%) developed a pocket hematoma, which was treated conservatively. No patient developed fever, clinical infection, or major bleeding. Preoperative and postoperative levels of hemoglobin, white blood cells, and C-reactive protein were similar. No demographic, procedural, or biochemical variable was significantly correlated with postprocedural complications. In our cohort, explantation of lead fragments and pulse generators of cardiac implantable electrical devices was safe after heart transplant. It appears that neither antiplatelet/anticoagulant agents nor immunosuppressives seem to put patients at increased risk of postoperative complications.

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.

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

PubMed

Peña, Leandro; Pérez, Rosa M; Cervera, Magdalena; Juárez, José A; Navarro, Luis

2004-07-01

Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

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.

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

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).

Micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin production.

PubMed

Ya-ut, Pornwilai; Chareonsap, Piyarat; Sukrong, Suchada

2011-12-01

An efficient system was developed for the in vitro micropropagation and hairy root culture of Ophiorrhiza alata Craib for camptothecin (CPT) production. Shoot multiplication on leaf and node explants from germinated seeds of O. alata was successful on half-strength Murashige and Skoog medium supplemented with varying amounts of kinetin and α-naphthaleneacetic acid. Node explants grown in vitro were successfully infected by Agrobacterium rhizogenes TISTR 1450 for the establishment of hairy root culture. The amount of CPT in various parts of O. alata was analyzed by HPLC. The accumulation of CPT in transformed hairy roots was twice that in soil-grown plants (785 ± 52 and 388 ± 32 μg/g dry wt, respectively). In the presence of a polystyrene resin (Diaion HP-20) that absorbed CPT, the CPT content in the culture media increased sevenfold compared with controls (1,036 and 151 μg per 250 ml medium, respectively). These results enable the feasible production of CPT of O. alata by means of a cell culture strategy. These measures can help safeguard the plant from extinction. © Springer Science+Business Media B.V. 2011

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

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.

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.

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.

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...

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

PubMed

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

2012-06-01

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

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.

Melatonin enhances root regeneration, photosynthetic pigments, biomass, total carbohydrates and proline content in the cherry rootstock PHL-C (Prunus avium × Prunus cerasus).

PubMed

Sarropoulou, Virginia; Dimassi-Theriou, Kortessa; Therios, Ioannis; Koukourikou-Petridou, Magdalene

2012-12-01

The present study, investigates the effects of melatonin (0, 0.05, 0.1, 0.5, 1, 5 and 10 μM) on the morphogenic and biochemical responses in the cherry rootstock PHL-C (Prunus avium L. × Prunus cerasus L.), from shoot tip explants. The incorporation of melatonin (0-10 μM) in the Murashige and Skoog (MS) medium, greatly influenced rooting either positively or negatively. Melatonin, irrespective of its concentration, had a negative effect concerning the number of roots. However, application of 0.5 μM melatonin significantly increased the root length; while 1 μM melatonin increased the root length by 2.5 times, and the fresh weight of the roots by 4 times, in comparison to the control. Although 0.05 μM melatonin increased rooting by 11.11%, 5 μM melatonin had a significant reduction on the number, the fresh weight of roots, and the rooting percentage. Melatonin concentration of 0.1 μM resulted in the greatest chlorophyll (a + b) content, and 5-10 μM reduced the chlorophyll concentration by 2 times, compared to the control. The high melatonin concentrations (5 and 10 μM), increased the levels of proline and carbohydrates in leaves by 3-4 times. In the roots, 0.5 μM of melatonin concentration increased the carbohydrate levels by 1.5 times, while 0.05, 0.1 and 1 μM melatonin concentration significantly reduced the proline content. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

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 (-...

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...

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

Robust genetic transformation of sorghum (Sorghum bicolor L.) using differentiating embryogenic callus induced from immature embryos.

PubMed

Belide, Srinivas; Vanhercke, Thomas; Petrie, James Robertson; Singh, Surinder Pal

2017-01-01

Sorghum ( Sorghum bicolor L.) is one of the world's most important cereal crops grown for multiple applications and has been identified as a potential biofuel crop. Despite several decades of study, sorghum has been widely considered as a recalcitrant major crop for transformation due to accumulation of phenolic compounds, lack of model genotypes, low regeneration frequency and loss of regeneration potential through sub-cultures. Among different explants used for genetic transformation of sorghum, immature embryos are ideal over other explants. However, the continuous supply of quality immature embryos for transformation is labour intensive and expensive. In addition, transformation efficiencies are also influenced by environmental conditions (light and temperature). Despite these challenges, immature embryos remain the predominant choice because of their success rate and also due to non-availability of other dependable explants without compromising the transformation efficiency. We report here a robust genetic transformation method for sorghum (Tx430) using differentiating embryogenic calli (DEC) with nodular structures induced from immature embryos and maintained for more than a year without losing regeneration potential on modified MS media. The addition of lipoic acid (LA) to callus induction media along with optimized growth regulators increased callus induction frequency from 61.3 ± 3.2 to 79 ± 6.5% from immature embryos (1.5-2.0 mm in length) isolated 12-15 days after pollination. Similarly, the regeneration efficiency and the number of shoots from DEC tissue was enhanced by LA. The optimized regeneration system in combination with particle bombardment resulted in an average transformation efficiency (TE) of 27.2 or 46.6% based on the selection strategy, 25% to twofold higher TE than published reports in Tx430. Up to 100% putative transgenic shoots were positive for npt - II by PCR and 48% of events had < 3 copies of transgenes as determined by digital droplet PCR. Reproducibility of this method was demonstrated by generating ~ 800 transgenic plants using 10 different gene constructs. This protocol demonstrates significant improvements in both efficiency and ease of use over existing sorghum transformation methods using PDS, also enables quick hypothesis testing in the production of various high value products in sorghum.

Artificial Neural Network Genetic Algorithm As Powerful Tool to Predict and Optimize In vitro Proliferation Mineral Medium for G × N15 Rootstock.

PubMed

Arab, Mohammad M; Yadollahi, Abbas; Shojaeiyan, Abdolali; Ahmadi, Hamed

2016-01-01

One of the major obstacles to the micropropagation of Prunus rootstocks has, up until now, been the lack of a suitable tissue culture medium. Therefore, reformulation of culture media or modification of the mineral content might be a breakthrough to improve in vitro multiplication of G × N15 (garnem). We found artificial neural network in combination of genetic algorithm (ANN-GA) as a very precise and powerful modeling system for optimizing the culture medium, So that modeling the effects of MS mineral salts ([Formula: see text], [Formula: see text], [Formula: see text], Ca 2+ , K + , [Formula: see text], Mg 2+ , and Cl - ) on in vitro multiplication parameters (the number of microshoots per explant, average length of microshoots, weight of calluses derived from the base of stem explants, and quality index of plantlets) of G × N15. Showed high R 2 correlation values of 87, 91, 87, and 74 between observed and predicted values were found for these four growth parameters, respectively. According to the ANN-GA results, among the input variables, [Formula: see text] and [Formula: see text] had the highest values of VSR in data set for the parameters studied. The ANN-GA showed that the best proliferation rate was obtained from medium containing (mM) 27.5 [Formula: see text], 14 [Formula: see text], 5 Ca 2+ , 25.9 K + , 0.7 Mg 2+ , 1.1 [Formula: see text], 4.7 [Formula: see text], and 0.96 Cl - . The performance of the medium optimized by ANN-GA, denoted as YAS (Yadollahi, Arab and Shojaeiyan), was compared to that of standard growth media for all Prunus rootstock, including the Murashige and Skoog (MS) medium, (specific media) EM, Quoirin and Lepoivre (QL) medium, and woody plant medium (WPM) Prunus . With respect to shoot length, shoot number per cultured explant and productivity (number of microshoots × length of microshoots), YAS was found to be superior to other media for in vitro multiplication of G × N15 rootstocks. In addition, our results indicated that by using ANN-GA, we were able to determine a suitable culture medium formulation to achieve the best in vitro productivity.

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...

[Introduction of hexaploid of Chinese narcissus and analysis of its chromosome change].

PubMed

Wang, Rui; Zhang, Ya Nan; Wang, Ya Ying; Tian, Hui Qiao

2007-06-01

Anthers of Chinese narcissus (Narcissus tazetta L. var chinesis Roem) were used as explants for callus induction and plant regeneration. About 80% anthers produced callus and 28% of the callus differentiated out bulbs, making a good experiment system of tissue culture of Chinese narcissus for further cellular and gene engineering. The 700 callus were treated by 0.5% colchicin for 5-6 days and then transformed into a MS medium containing 3 mg/L 6-BA to induce differentiation. 90 bulbs were obtained and 55 bulbs among them were checked the chromosome number from their root tips for three times. 29 bulbs (53%, 29/55) still kept triploidy and the most cells of root tips contained 30 chromosomes. 22 bulbs (40%, 22/55) displayed aneuploidy and the most cells of its root tips contained 10-50 chromosomes. 4 bulbs displayed hexaploidy and contained 60 chromosomes. After three months growing, the cells of root tips containing aneuploidy chromosomes disappeared, and the bulbs became triploidy. The chromosomes of 4 hexaploidy bulbs did not changed during three checks. The origin and disappearance of aneuploidy cells of Chinese narcissus after treated by colchicin were discussed.

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.

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...

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...

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...

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...

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.

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.

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

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.

Temporary Immersion System for Date Palm Micropropagation.

PubMed

Othmani, Ahmed; Bayoudh, Chokri; Sellemi, Amel; Drira, Noureddine

2017-01-01

The temporary immersion system (TIS) is being used with tremendous success for automation of micropropagation of many plant species. TIS usually consists of a culture vessel comprising two compartments, an upper one with the plant material and a lower one with the liquid culture medium and an automated air pump. The latter enables contact between all parts of the explants and the liquid medium by setting overpressure to the lower part of the container. These systems are providing the most satisfactory conditions for date palm regeneration via shoot organogenesis and allow a significant increase of multiplication rate (5.5-fold in comparison with that regenerated on agar-solidified medium) and plant material quality, thereby reducing production cost.

Synthesis of deuterium-labelled substrates for the study of oleuropein biosynthesis in Olea europaea callus cultures.

PubMed

Serrilli, Anna Maria; Maggi, Agnese; Casagrande, Valentina; Bianco, Armandodoriano

2016-01-01

We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-(2)H2]-7-deoxy-8-epi-loganin (2(D)), [8,10-(2)H2]-8-epi-loganin (4(D)) and [7,8-(2)H2]-7-deoxy-loganin (3(D)).

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

PubMed

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

2015-08-01

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

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

PubMed

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

2017-07-01

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

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.

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...

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

Limb salvage following iatrogenic arterial injury: complications of cardiac support using intra-aortic balloon pumps.

PubMed

Hunt, Adam; Waldin, Owen; Slim, Hani; Brar, Ranjeet

2016-06-06

This is a unique case report describing complex limb salvage in a patient who experienced acute limb ischaemia due to a complication of intra-aortic balloon pump (IABP) counterpulsation. This case focuses on a patient who had an IABP counterpulsation device inserted following myocardial infarction, requiring urgent coronary artery bypass grafting for acute coronary syndrome. Postoperatively, the IABP could not be removed, with consequent iliac thrombosis and acute limb ischaemia. Emergency femoral-to-femoral crossover bypass was performed using a polytetrafluoroethylene graft. A portion of the IABP balloon tip was entrapped and retained, however, and became the focus of delayed septicaemia, resulting in graft infection and wound breakdown 6 weeks later. Explantation of the residual balloon tip, ligation of the right external iliac artery and redo femoral-femoral crossover using the great saphenous vein were successfully performed. 2016 BMJ Publishing Group Ltd.

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

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.

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.

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.

Blueberry (Vaccinium corymbosum L.).

PubMed

Song, Guo-Qing; Sink, Kenneth C

2006-01-01

Recent advances in plant biotechnology have led to a reliable and reproductive method for genetic transformation of blueberry. These efforts built on previous attempts at transient and stable transformation of blueberry that demonstrated the potential of Agrobacterium tumefaciens-mediated transformation, and as well, the difficulties of selecting and regenerating transgenic plants. As a prerequisite for successful stable transformation, efficient regeneration systems were required despite many reports on factors controlling shoot regeneration from leaf explants. The A. tumefaciens-mediated transformation protocol described in this chapter is based on combining efficient regeneration methods and the results of A. tumefaciens-mediated transient transformation studies to optimize selected parameters for gene transfer. The protocol has led to successful regeneration of transgenic plants of four commercially important highbush blueberry cultivars.

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.

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.

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…

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 ...

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

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

PubMed

Gomez, M P; Segura, J

1996-08-01

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

In vitro regeneration of Basella alba L

NASA Technical Reports Server (NTRS)

Edney, Norris Allen; Rizvi, Muhammad A.; Rizvi, Narjis F.

1989-01-01

Basella alba L. is a tropical vine used as a vegetable in some Asian and African countries. It has potential as a nontraditional crop for small family farms. A short day plant, it blooms during the fall, provided the temperatures are mild. In the southeastern U.S., the short days of fall are associated with subfreezing temperatures, and plants are killed before blooming. Attempts were made to regenerate the plant using tissue culture techniques. Several trials were conducted with different media, hormones, and explants. It was found that nodal segments on Gamborg medium regenerated shoots. Interaction studies of auxins and cytokinins indicated that its endogeneous auxin content might be high because callus proliferated in almost all treatments and roots initiated even when the medium was not supplemented with an auxin.

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.

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.

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

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

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.

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.

The combination effect of auxin and cytokinin on in vitro callus formation of Physalis angulata L. - A medicinal plant

NASA Astrophysics Data System (ADS)

Mastuti, Retno; Munawarti, Aminatun; Firdiana, Elok Rifqi

2017-11-01

Physalis angulata L. (Ciplukan) is one member of Solanaceae that has a potential as herbal medicine. This plant grows wild in the crop fields, forest edges, etc. However, ciplukan is increasingly difficult to find recently. In vitro callus is an alternative source to produce secondary metabolite production as well as to regenerate plants through indirect organogenesis. This study aims to identify the response of hypocotyl explants on in vitro callus formation induced by a combination of auxin and cytokinins. Two types of cytokinins, Kinetin and BAP (0.5 ppm) were combined with three types of auxin, i.e. 2.4-D, IBA and IAA, at three concentrations 0.5, 1.0 and 1.5 ppm. In all combinations of cytokinin and auxin, 50-100% of hypocotyl explants derived from in vitro seedling were able to produce callus either in a compact or watery friable texture. In MS medium supplemented with 2.4-D, callus FW (fresh weight) began to decline in the fourth week after culture. Callus FW that increased until 5 weeks of culture was obtained in medium IAA 0.5 + Kin 0.5, IBA 1.0 + Kin 0.5 and IBA 1 + BA 0.5. Almost all calli induced on a medium + Kinetin also produced roots. While medium + BAP was able to induce shoots regeneration.

An effective protocol for micropropagation of edible bamboo species (Bambusa tulda and Melocanna baccifera) through nodal culture.

PubMed

Waikhom, Sayanika Devi; Louis, Bengyella

2014-01-01

High demand for edible bamboo shoots of Bambusa tulda and Melocanna baccifera in many Asian ethnic groups has led to the need for developing intensive bamboo farming. To achieve this, in vitro regeneration of bamboo plantlets is needed due to the long and irregular bamboo flowering cycle and scarcity of bamboo seeds. An effective protocol for plantlets regeneration in B. tulda and M. baccifera from nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is described. Effective axillary bud breaking was achieved at 3 mg/L of 6-benzylaminopurine (BAP) in MS medium. Importantly, combining 2 mg/L of kinetin (Kn) with 3 mg/L of BAP produced a synergistic effect for shoot multiplication in B. tulda and M. baccifera. Under optimized conditions in half-strength MS medium supplemented with 3 mg/L of indole-3-butyric acid (IBA), 10 mg/L of coumarin, and 3% sucrose, profuse production of dark-brown rhizome in B. tulda and abundant rooting (81.67%, P < 0.05, F = 15.46) for M. baccifera within 30 days were achieved. The established protocol and the validation of the reported species at the molecular level will be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity.

Micropropagation of onion (Allium cepa L.) from immature inflorescences.

PubMed

Marinangeli, Pablo

2013-01-01

In vitro plant production by direct organogenesis from immature flower heads is an ideal approach for clonal propagation of onions (Allium cepa L.). This technique ensures genetic stability, high propagation rate, and maintains donor plant of explants with an advantage over other means of in vitro regeneration. Onion micropropagation is usually applied in breeding programs, maintenance, and multiplication of cytoplasmic-male sterile lines for hybrid production, germplasm conservation, and as a tool for the application of other biotechnologies. For in vitro culture, mature onion bulbs are induced to reproductive phase by vernalization and forced to inflorescence initiation. Immature umbels are dissected from bulbs or cut directly when they appear from the pseudostem among the leaves. Disinfected inflorescences are cultivated in BDS basal medium supplemented with 30 g/L sucrose, 0.1 mg/L naphthalene acetic acid, 1 mg/L N (6)-benzyladenine, and 8 g/L agar, pH 5.5, under 16 h photoperiod white fluorescent light (PPD: 50-70 μmol/m(2)s) for 35 days. The regenerated shoot clumps are divided and subculture under the same conditions. For bulbification phase, the individual shoots are cultured in BDS basal medium containing 90 g/L sucrose, without plant growth regulators, pH 5.5, under 16 h photoperiod. Microbulbs can be directly cultivated ex vitro without acclimation.

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...

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

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.

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.

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.

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.

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.

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato1

PubMed Central

Coenen, Catharina; Lomax, Terri L.

1998-01-01

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways. PMID:9576775

High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode.

PubMed

Cho, H J; Farrand, S K; Noel, G R; Widholm, J M

2000-01-01

Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and beta-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54-95% of the cotyledon explants on MXB selective medium containing 200 microg ml(-1) kanamycin and 500 microg ml(-1) carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode (Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4-5 weeks after inoculation. Thus the soybean cyst nematode could complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for testing genes that might impart resistance to soybean cyst nematode.

Optimization of factors influencing microinjection method for Agrobacterium tumefaciens-mediated transformation of tomato.

PubMed

Vinoth, S; Gurusaravanan, P; Jayabalan, N

2013-02-01

A simple and efficient protocol for Agrobacterium-mediated genetic transformation of tomato was developed using combination of non-tissue culture and micropropagation systems. Initially, ESAM region of 1-day-old germinated tomato seeds were microinjected for one to five times with Agrobacterium inoculums (OD(600) = 0.2-1.0). The germinated seeds were cocultivated in the MS medium fortified with (0-200 mM) acetosyringone and minimal concentrations of (0-20 mg L(-1)) kanamycin, and the antibiotic concentration was doubled during the second round of selection. Bacterial concentration of OD(600) = 0.6 served as an optimal concentration for infection and the transformation efficiency was significantly higher of about 46.28 %. In another set of experiment, an improved and stable regeneration system was adapted for the explants from the selection medium. Four-day-old double cotyledonary nodal explants were excised from the microinjected seedlings and cultured onto the MS medium supplemented with 1.5 mg L(-1) thidiazuron, 1.5 mg L(-1) indole-3-butyric acid, 30 mg L(-1) kanamycin, and 0-1.5 mg L(-1) adenine sulphate. Maximum of 9 out of 13 micropropagated shoots were shown positive to GUS assay. By this technique, the transformation efficiency was increased from 46.28 to 65.90 %. Thus, this paper reports the successful protocol for the mass production of transformants using microinjection and micropropagation techniques.

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea.

PubMed

Prabhudesai, V; Bhaskaran, S

1993-03-01

An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

An Effective Protocol for Micropropagation of Edible Bamboo Species (Bambusa tulda and Melocanna baccifera) through Nodal Culture

PubMed Central

Waikhom, Sayanika Devi; Louis, Bengyella

2014-01-01

High demand for edible bamboo shoots of Bambusa tulda and Melocanna baccifera in many Asian ethnic groups has led to the need for developing intensive bamboo farming. To achieve this, in vitro regeneration of bamboo plantlets is needed due to the long and irregular bamboo flowering cycle and scarcity of bamboo seeds. An effective protocol for plantlets regeneration in B. tulda and M. baccifera from nodal explants following validation of the species using the sequence of trnL-F intergenic spacer region is described. Effective axillary bud breaking was achieved at 3 mg/L of 6-benzylaminopurine (BAP) in MS medium. Importantly, combining 2 mg/L of kinetin (Kn) with 3 mg/L of BAP produced a synergistic effect for shoot multiplication in B. tulda and M. baccifera. Under optimized conditions in half-strength MS medium supplemented with 3 mg/L of indole-3-butyric acid (IBA), 10 mg/L of coumarin, and 3% sucrose, profuse production of dark-brown rhizome in B. tulda and abundant rooting (81.67%, P < 0.05, F = 15.46) for M. baccifera within 30 days were achieved. The established protocol and the validation of the reported species at the molecular level will be of help to stakeholders in edible bamboo trade to conserve gene-pool and increase productivity. PMID:24967429

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 .

Hormetic Response by Silver Nanoparticles on In Vitro Multiplication of Sugarcane (Saccharum spp. Cv. Mex 69-290) Using a Temporary Immersion System.

PubMed

Bello-Bello, Jericó J; Chavez-Santoscoy, Rocío A; Lecona-Guzmán, Carlos A; Bogdanchikova, Nina; Salinas-Ruíz, Josafhat; Gómez-Merino, Fernando Carlos; Pestryakov, Alexey

2017-01-01

Hormesis is considered a dose-response phenomenon characterized by growth stimulation at low doses and inhibition at high doses. The hormetic response by silver nanoparticles (AgNPs) on in vitro multiplication of sugarcane was evaluated using a temporary immersion system. Sugarcane shoots were used as explants cultured in Murashige and Skoog medium with AgNPs at concentrations of 0, 25, 50, 100, and 200 mg/L. Shoot multiplication rate and length were used to determine hormetic response. Total content of phenolic compounds of sugarcane, mineral nutrition, and reactive oxygen species (ROS) was determined. Results were presented as a dose-response curve. Stimulation phase growth was observed at 50 mg/L AgNPs, whereas inhibition phase was detected at 200 mg/L AgNPs. Mineral nutrient analysis showed changes in macronutrient and micronutrient contents due to the effect of AgNPs. Moreover, AgNPs induced ROS production and increased total phenolic content, with a dose-dependent effect. Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNP-induced hormesis and that phenolic accumulation was obtained as a response of the plant to stress produced by high doses of AgNPs. Therefore, small doses of AgNPs in the culture medium could be an efficient strategy for commercial micropropagation.

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

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

PubMed

Mhatre, Minal

2013-01-01

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

Hormetic Response by Silver Nanoparticles on In Vitro Multiplication of Sugarcane (Saccharum spp. Cv. Mex 69-290) Using a Temporary Immersion System

PubMed Central

Chavez-Santoscoy, Rocío A.; Lecona-Guzmán, Carlos A.; Bogdanchikova, Nina; Salinas-Ruíz, Josafhat; Gómez-Merino, Fernando Carlos; Pestryakov, Alexey

2017-01-01

Background: Hormesis is considered a dose–response phenomenon characterized by growth stimulation at low doses and inhibition at high doses. The hormetic response by silver nanoparticles (AgNPs) on in vitro multiplication of sugarcane was evaluated using a temporary immersion system. Methods: Sugarcane shoots were used as explants cultured in Murashige and Skoog medium with AgNPs at concentrations of 0, 25, 50, 100, and 200 mg/L. Shoot multiplication rate and length were used to determine hormetic response. Total content of phenolic compounds of sugarcane, mineral nutrition, and reactive oxygen species (ROS) was determined. Results: Results were presented as a dose–response curve. Stimulation phase growth was observed at 50 mg/L AgNPs, whereas inhibition phase was detected at 200 mg/L AgNPs. Mineral nutrient analysis showed changes in macronutrient and micronutrient contents due to the effect of AgNPs. Moreover, AgNPs induced ROS production and increased total phenolic content, with a dose-dependent effect. Conclusion: Results suggested that the production of ROS and mineral nutrition are key mechanisms of AgNP-induced hormesis and that phenolic accumulation was obtained as a response of the plant to stress produced by high doses of AgNPs. Therefore, small doses of AgNPs in the culture medium could be an efficient strategy for commercial micropropagation. PMID:29238274

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.

Somatic embryo-like structures of strawberry regenerated in vitro on media supplemented with 2,4-D and BAP.

PubMed

Omar, Genesia F; Mohamed, Fouad H; Haensch, Klaus-Thomas; Sarg, Sawsan H; Morsey, Mohamed M

2013-09-01

Somatic embryo-like structures (SELS) were produced in vitro from leaf disk and petiole explants of two cultivars of strawberry (Fragaria x ananassa Duch) on Murashige and Skoog medium with different concentrations and combinations of 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP) and sucrose to check the embryonic nature of these structures histologically. A large number of SELS could be regenerated in both cultivars on media with 2-4 mg L(-1) 2,4-D in combination with 0.5 -1 mg L(-1) BAP and 50 g x L(-1) sucrose. Histological examination of SELS revealed the absence of a root pole. Therefore these structures cannot be strictly classified as somatic embryos. The SELS formed under the tested culture conditions represent malformed shoot-like and leaf-like structures. The importance of these results for the propagation of strawberries via somatic embryogenesis is discussed.

Cryopreservation of banana's cv Grand Naine in vitro rhizomes.

PubMed

Londe, Luciana C N; Vendrame, Wagner A; Sanaei, Massy; Oliveira, Alexandre B DE

2018-01-01

The preservation of banana genetic material is usually performed through seedlings. However, most banana cultivars do not produce seed and are propagated vegetatively. Therefore, cryopreservation is a feasible technique that allows the preservation of banana genotypes indefinitely. For the success of cryopreservation protocols, the selection of cryoprotectants and pre-freezing techniques are important factor. Therefore, the objective of this study was to verify the effects of different cryoprotectants with and without 1% phloroglucinol and pre-cooling periods on the development of a protocol for cryopreservation of in vitro rhizomes ofMusa accuminata(AAA) cv Grand Naine banana. The addition of 1% phloroglucinol to the cryoprotective solutions, such as PVS2 enhanced recovery of cryopreserved banana rhizomes. In addition, pre-cooling of explants in ice for 3 hours in PVS2 + 1% of phloroglucinol allowed efficient cryopreservation of banana rhizomes, followed by successful recovery and regeneration of in vitro shoots of banana cv Grand Naine.

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

PubMed

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

2013-04-01

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

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.

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.

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.

Embryogenic callus proliferation and regeneration conditions for genetic transformation of diverse sugarcane cultivars.

PubMed

Basnayake, Shiromani W V; Moyle, Richard; Birch, Robert G

2011-03-01

Amenability to tissue culture stages required for gene transfer, selection and plant regeneration are the main determinants of genetic transformation efficiency via particle bombardment into sugarcane. The technique is moving from the experimental phase, where it is sufficient to work in a few amenable genotypes, to practical application in a diverse and changing set of elite cultivars. Therefore, we investigated the response to callus initiation, proliferation, regeneration and selection steps required for microprojectile-mediated transformation, in a diverse set of Australian sugarcane cultivars. 12 of 16 tested cultivars were sufficiently amenable to existing routine tissue-culture conditions for practical genetic transformation. Three cultivars required adjustments to 2,4-D levels during callus proliferation, geneticin concentration during selection, and/or light intensity during regeneration. One cultivar gave an extreme necrotic response in leaf spindle explants and produced no callus tissue under the tested culture conditions. It was helpful to obtain spindle explants for tissue culture from plants with good water supply for growth, especially for genotypes that were harder to culture. It was generally possible to obtain several independent transgenic plants per bombardment, with time in callus culture limited to 11-15 weeks. A caution with this efficient transformation system is that separate shoots arose from different primary transformed cells in more than half of tested calli after selection for geneticin resistance. The results across this diverse cultivar set are likely to be a useful guide to key variables for rapid optimisation of tissue culture conditions for efficient genetic transformation of other sugarcane cultivars.

In vitro seed germination of economically important edible bamboo Dendrocalamus membranaceus Munro.

PubMed

Brar, Jasmine; Anand, Manju; Sood, Anil

2013-01-01

An in vitro propagation protocol using mature seeds of D. membranaceus was successfully established. Scarcity of seeds in bamboos because of their long flowering periods and irregular seed set resulting in low viability and germination potential, motivated us to undertake the present study. The effects of sterilants, light conditions, exogenous application of plant growth regulators and temperature in overcoming germination barriers in ageing seeds of bamboo were studied. It was found that HgCl2 (0.1%) along with bleach (15%) was more effective in raising aseptic cultures. Dark conditions, high temperatures around 30 degrees C and soaking of seeds in GA3 solution (50 ppm) overnight stimulated high percent of seed germination with corresponding increase in shoot length (2.7 +/- 0.7 mm) and number of sprouts (2.1 +/- 0.7) per explants during culture initiation. 6-benzylaminopurine acted synergistically with kinetin to give optimum germination rate of 70 +/- 13.9% as compared to 63.13% when used individually. For prolonged maintenance of cultures, 2% sucrose was found to be suitable for promoting photomixotrophic micropropagation. Following this procedure, about 65% survival of plantlets could be achieved during hardening. Biochemically seeds consume starchy endosperm for emergence of radicle which is taken as a sign of germination as also evident from the present study. Loss of viability and vigour after a year was confirmed by Tetrazolium chloride test. Micropropagation protocol developed here will ensure regeneration of large number of plants in a relatively short time. Conclusively, in vitro propagation protocol developed in D. membranaceus using mature seeds as an explants is reported for the first time.

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

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

Bacterial stimulation of adventitious rooting on in vitro cultured slash pine (Pinus elliottii Engelm.) seedling explants.

PubMed

Burns, J A; Schwarz, O J

1996-02-01

A bacterium has been isolated that initiates adventitious rooting when co-cultured under in vitro conditions with seedling-produced hypocotylary explants of slash pine (Pinus elliottii). Rooting efficiencies produced through bacterial-explant co-culture range from approximately 15% to greater than 90% over non-treated controls. Explant exposure to the root inducing bacterium has produced no obvious pathology in the regenerated plantlets. Seedling explants rooted by bacterial-explant co-culture have been successfully transitioned to ambient greenhouse conditions.

Development of ecotoxicology procedures for use in assessing health of coral reefs

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

Davies, P.S.; Marubini, F.

1995-12-31

There is widespread concern over the apparent worldwide decline in the health of coral reefs. However, assessment methods, other than long-term monitoring, have not yet been attempted. To remedy this the authors are developing ecotoxicology procedures to assess the effects of water quality factors on the corals themselves. Because of the problems of working with large, attached organisms, the authors have concentrated on developing small clonal preparations from corals with both a branching and a massive growth-form. For branching corals, tips (`nubbins`) are removed, and the cut surface ground smooth before cementing to 30mm x 30mm acrylic squares. Cores, ormore » `explants`, 25mm in diameter are removed from massive corals and cemented into injection-moulded plastic cups, to protect the cut surfaces of the skeleton. Trays of up to 18 nubbins and explants may then be transferred to the reef, where they are affixed to previously installed concrete breeze blocks. They may then be retrieved as required to assess the effects of water quality upon them. For laboratory ecotoxicology experiments, the authors have devised a system of artificial lighting, using halide lamps, to grow coral nubbins and explants under controlled conditions. To test the effects of pollutants, the authors use measurements of respiration, photosynthesis and skeletal growth rate. Growth is measured by a very simple buoyant weighing procedure, which requires only an analytical laboratory balance, and an easily-constructed plastic box-shaped chamber. The method is sufficiently sensitive to measure growth over a 24 hour period in some fast-growing corals. The authors will describe the results of initial experiments, carried out at the Bellairs Research Institute, Barbados, on the effects of different levels of phosphate and nitrate in the seawater. For the first time, they are able to demonstrate the concentration-related decrease in growth rate associated with nitrate eutrophication.« less

Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus.

PubMed

Alam, Pravej; Khan, Zainul Abdeen; Abdin, Malik Zainul; Khan, Jawaid A; Ahmad, Parvaiz; Elkholy, Shereen F; Sharaf-Eldin, Mahmoud A

2017-05-01

Catharanthus roseus is an important medicinal plant known for its pharmacological qualities such as antimicrobial, anticancerous, antifeedant, antisterility, antidiabetic activities. More than 130 bioactive compounds like vinblastine, vindoline and vincristine have been synthesized in this plant. Extensive studies have been carried out for optimization regeneration and transformation protocols. Most of the protocol described are laborious and time-consuming. Due to sophisticated protocol of regeneration and genetic transformation, the production of these bioactive molecules is less and not feasible to be commercialized worldwide. Here we have optimized the efficient protocol for regeneration and transformation to minimize the time scale and enhance the transformation frequency through Agrobacterium and sonication-assisted transformation (SAAT) method. In this study, hypocotyl explants responded best for maximal production of transformed shoots. The callus percentage were recorded 52% with 1.0 mg L -1 (BAP) and 0.5 mg L -1 (NAA) while 80% shoot percentage obtained with 4.0 mg L -1 (BAP) and 0.05 mg L -1 (NAA). The microscopic studies revealed that the expression of GFP was clearly localized in leaf tissue of the C. roseus after transformation of pRepGFP0029 construct. Consequently, transformation efficiency was revealed on the basis of GFP localization. The transformation efficiency of SAAT method was 6.0% comparable to 3.5% as conventional method. Further, PCR analysis confirmed the integration of the nptII gene in the transformed plantlets of C. roseus.

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

PubMed

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

2010-10-01

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

Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number.

PubMed

Khuong, Thi Thu Huong; Crété, Patrice; Robaglia, Christophe; Caffarri, Stefano

2013-09-01

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions. Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.

Validation of murine and human placental explant cultures for use in sex steroid and phase II conjugation toxicology studies

PubMed Central

Sato, Brittany L.; Ward, Monika A.; Astern, Joshua M.; Kendal-Wright, Claire E.; Collier, Abby C.

2014-01-01

Human primary placental explant culture is well established for cytokine signaling and toxicity, but has not been validated for steroidogenic or metabolic toxicology. The technique has never been investigated in the mouse. We characterized human and mouse placental explants for up to 96hr in culture. Explant viability (Lactate dehydrogenase) and sex steroid levels were measured in media using spectrophotometry and ELISA, respectively. Expression and activities of the steroidogenic (3β-hydroxysteroid dehydrogenase, Cytochrome P45017A1, Cytochrome P45019), conjugation (UDP-glucuronosyltransferase, sulfotransferase (SULT)), and regeneration (β-glucuronidase, arylsulfatase C (ASC)) enzymes were determined biochemically in tissues with fluorimetric and spectrophotometric assays, and western blot. Explants were viable up to 96hr, but progesterone, estrone, and 17β-estradiol secretion decreased. Steroidogenic enzyme expression and activities were stable in mouse explants and similar to levels in freshly isolated tissues, but were lower in human explants than in fresh tissue (P<0.01). Human and mouse explants exhibited significantly less conjugation after 96hr, SULT was not detected in the mouse, and neither explants had active ASC, although proteins were expressed. Mouse explants may be useful for steroid biochemistry and endocrine disruption studies, but not metabolic conjugation. In contrast, human explants may be useful for studying conjugation for <48hr, but not for steroid/endocrine studies. PMID:25283089

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

Plantlet regeneration potential from seedling explants of vitegnus (Vitex agnus castus).

PubMed

Chamandoosti, F

2007-11-15

In this research a simple and repeatable method for regeneration of a important medicinal plant (Vitex agnus castus) described. Different seedling explants such as hypocotyl, cotyledon, root and apical meristem were cultured in MS basal media with different kinds and concentrations of PGRs. Root and apical meristem explants were the only explants that have regeneration whole plantlets potential. It was interesting that regeneration whole plantlets from root and apical meristem explants have different developmental pathways. Whole plantlets from apical meristem explants regenerated by passing phase callusing whereas regeneration whole plantlets from root was direct and without phase callusing. This subject implies that we can have many manipulation possibilities in order to different objects of tissue culture by selecting different explants in vitegnus.

Activity and Accumulation of Cell Division-Promoting Phenolics in Tobacco Tissue Cultures 1

PubMed Central

Teutonico, Rita A.; Dudley, Matthew W.; Orr, John D.; Lynn, David G.; Binns, Andrew N.

1991-01-01

Dehydrodiconiferyl alcohol glucosides (DCGs) are derivatives of the phenylpropanoid pathway that have been isolated from Catharansus roseus L. (Vinca rosea) crown gall tumors. Fractions containing purified DCGs have been shown previously to promote the growth of cytokinin-requiring tissues of tobacco in the absence of exogenous cytokinins. In this study, we utilized synthetic DCG isomers to confirm the cell division-promoting activity of DCG isomers A and B and show that they neither promote shoot meristem initiation on Nicotiana tabacum L., cv Havana 425, leaf explants nor induce betacyanin synthesis in amaranth seedlings. Analysis of cultured tobacco pith tissue demonstrated that DCG accumulation was stimulated by cytokinin treatment and correlated with cytokinin-induced cell division. Thus, the accumulation of metabolites that could replace cytokinin in cell division bioassays is stimulated by cytokinins. These data support the model that DCGs are a component of a cytokinin-mediated regulatory circuit controlling cell division. ImagesFigure 2 PMID:16668384

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

PubMed Central

2013-01-01

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

Causes of Phakic Implantable Collamer Lens Explantation/Exchange at King Khaled Eye Specialist Hospital

PubMed Central

AlSabaani, Nasser A.; Behrens, Ashley; Jastanieah, Sabah; Al Malki, Salem; Al Jindan, Mohanna; Al Motowa, Saeed

2016-01-01

PURPOSE: The purpose of this study is to evaluate the causes of phakic implantable collamer lens (ICL) explantation/exchange at an eye hospital in Saudi Arabia. MATERIALS AND METHODS: A retrospective chart review was performed for patients who underwent ICL implantation from 2007 to March 2014 and data were collected on cases that underwent ICL explantation. RESULTS: Of the 787 ICL implants, 30 implants (3.8% [95% confidence interval 2.6%; 5.3%]) were explanted. The causes of explantation included incorrect lens size (22), cataract (4), high residual astigmatism (2), rhegmatogenous retinal detachment (1), and intolerable glare (1). Corrective measures mainly included an exchange with an appropriately sized lens (9), ICL explantation (11), with phacoemulsification and posterior chamber intraocular lens implantation (6), or replacement with an ICL of correct power (2). CONCLUSION: Incorrect ICL size was the most common cause of ICL explantation. More accurate sizing methods for ICL are required to reduce the explantation/exchange rate. PMID:27994391

The effect of in vitro tracheal occlusion on branching morphogenesis in fetal lung explants from the rat nitrofen model of congenital diaphragmatic hernia.

PubMed

Grushka, Jeremy R; Al-Abbad, Saleh; Baird, Robert; Puligandla, Pramod; Kaplan, Feige; Laberge, Jean-Martin

2010-05-01

Fetal tracheal occlusion (TO) has been investigated as a treatment option for lung hypoplasia secondary to congenital diaphragmatic hernia. Tracheal occlusion has been shown to accelerate lung growth, but its effect on bronchial branching is unknown. In this study, we characterize the effects of in vitro TO on bronchial branch development in fetal lung explants derived from the nitrofen rat model of congenital diaphragmatic hernia. Rat dams were gavaged nitrofen on gestational day 9.5, and fetal lungs were harvested for explant culture on gestational day 14 (term, 22 days). Four experimental groups were investigated, with TO performed ex vivo using cautery: control, control + TO, nitrofen, and nitrofen + TO. Explants were incubated for 72 hours. Representative photographs were taken at 0, 24, 48, and 72 hours from the time of culture, and the number of distal branches was counted for each explant. The Student t test was used to compare distal branch measurements. A minimum of 12 fetal lung explants were cultured for each group. By 24 hours, all explants undergoing TO had more branch iterations than explants that did not. Moreover, TO in nitrofen-exposed explants increased bronchial branching to control levels by 24 hours in culture. Our results suggest that TO at day 14 increases branching in normal and nitrofen-exposed lung explants. In addition, TO increases airway branching in nitrofen-exposed explants to control levels suggesting that early TO reverses the lung hypoplasia seen in this model. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Localization of QTLs for in vitro plant regeneration in tomato

PubMed Central

2011-01-01

Background Low regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato. Results We developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration. Conclusions In this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation. PMID:22014149

Localization of QTLs for in vitro plant regeneration in tomato.

PubMed

Trujillo-Moya, Carlos; Gisbert, Carmina; Vilanova, Santiago; Nuez, Fernando

2011-10-20

Low regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato. We developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration. In this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation.

Feasibility of real-time magnetic resonance imaging-guided endomyocardial biopsies: An in-vitro study.

PubMed

Lossnitzer, Dirk; Seitz, Sebastian A; Krautz, Birgit; Schnackenburg, Bernhard; André, Florian; Korosoglou, Grigorios; Katus, Hugo A; Steen, Henning

2015-07-26

To investigate if magnetic resonance (MR)-guided biopsy can improve the performance and safety of such procedures. A novel MR-compatible bioptome was evaluated in a series of in-vitro experiments in a 1.5T magnetic resonance imaging (MRI) system. The bioptome was inserted into explanted porcine and bovine hearts under real-time MR-guidance employing a steady state free precession sequence. The artifact produced by the metal element at the tip and the signal voids caused by the bioptome were visually tracked for navigation and allowed its constant and precise localization. Cardiac structural elements and the target regions for the biopsy were clearly visible. Our method allowed a significantly better spatial visualization of the bioptoms tip compared to conventional X-ray guidance. The specific device design of the bioptome avoided inducible currents and therefore subsequent heating. The novel MR-compatible bioptome provided a superior cardiovascular magnetic resonance (imaging) soft-tissue visualization for MR-guided myocardial biopsies. Not at least the use of MRI guidance for endomyocardial biopsies completely avoided radiation exposure for both patients and interventionalists. MRI-guided endomyocardial biopsies provide a better than conventional X-ray guided navigation and could therefore improve the specificity and reproducibility of cardiac biopsies in future studies.

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.

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.

Trehalose 6-phosphate is involved in triggering axillary bud outgrowth in garden pea (Pisum sativum L.).

PubMed

Fichtner, Franziska; Barbier, Francois F; Feil, Regina; Watanabe, Mutsumi; Annunziata, Maria Grazia; Chabikwa, Tinashe G; Höfgen, Rainer; Stitt, Mark; Beveridge, Christine A; Lunn, John E

2017-11-01

Trehalose 6-phosphate (Tre6P) is a signal of sucrose availability in plants, and has been implicated in the regulation of shoot branching by the abnormal branching phenotypes of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays) mutants with altered Tre6P metabolism. Decapitation of garden pea (Pisum sativum) plants has been proposed to release the dormancy of axillary buds lower down the stem due to changes in sucrose supply, and we hypothesized that this response is mediated by Tre6P. Decapitation led to a rapid and sustained rise in Tre6P levels in axillary buds, coinciding with the onset of bud outgrowth. This response was suppressed by simultaneous defoliation that restricts the supply of sucrose to axillary buds in decapitated plants. Decapitation also led to a rise in amino acid levels in buds, but a fall in phosphoenolpyruvate and 2-oxoglutarate. Supplying sucrose to stem node explants in vitro triggered a concentration-dependent increase in the Tre6P content of the buds that was highly correlated with their rate of outgrowth. These data show that changes in bud Tre6P levels are correlated with initiation of bud outgrowth following decapitation, suggesting that Tre6P is involved in the release of bud dormancy by sucrose. Tre6P might also be linked to a reconfiguration of carbon and nitrogen metabolism to support the subsequent growth of the bud into a new shoot. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Multiple bud cultures of 'Barhee' date palm (Phoenix dactylifera) and physiological status of regenerated plants.

PubMed

Fki, Lotfi; Bouaziz, Neila; Kriaa, Walid; Benjemaa-Masmoudi, Raja; Gargouri-Bouzid, Radhia; Rival, Alain; Drira, Noureddine

2011-09-15

Adventitious bud clusters of date palm 'Barhee' were successfully established from juvenile leaves (<1cm) using reduced amounts of 2,4-D (0.2mgL(-1)) to limit the risk of somaclonal variation. An average of 8.4 adventitious buds per explant were obtained. Histological examination showed that the superficial cell layers of leaves had the highest caulogenic capacity. High sucrose concentration (70gL(-1)) was used for the conversion of initial buds to multiple bud clusters. The promoting effect of temporary immersion on shoot proliferation was found to be significant when compared to cultivation on solid media. Elongation of shoots was also better using a thin film of PGR-free liquid medium instead of a solid medium. Anatomical observations indicated that roots from vitroplants were potentially functional at various developmental stages. However, only 12-month-old vitroplants were found to be physiologically able to control transpirational vapor loss. Additionally, the photochemical activity of photosystem II in these vitroplants was close to that measured in plants that were already acclimatized. As a result, 83.3% of regenerated plants were successfully acclimatized. No phenotypic variation was observed among more than 500 adventitious bud-derived plants. All regenerants survived after field transplantation. We found that the production of adventitious bud clusters in small bioreactors was able to provide an efficient micropropagation system for date palm cv. 'Barhee'. An in vitro hardening step was a prerequisite for the successful transfer of vitroplants in soil. Copyright © 2011 Elsevier GmbH. All rights reserved.

Optic nerve compression as a late complication of a hydrogel explant with silicone encircling band.

PubMed

Crama, Niels; Kluijtmans, Leo; Klevering, B Jeroen

2018-06-01

To present a complication of compressive optic neuropathy caused by a swollen hydrogel explant and posteriorly displaced silicone encircling band. A 72-year-old female patient presented with progressive visual loss and a tilted optic disc. Her medical history included a retinal detachment in 1993 that was treated with a hydrogel explant under a solid silicone encircling band. Visual acuity had decreased from 6/10 to 6/20 and perimetry showed a scotoma in the temporal superior quadrant. On Magnetic Resonance Imaging (MRI), compression of the optic nerve by a displaced silicone encircling band inferior nasally in combination with a swollen episcleral hydrogel explant was observed. Surgical removal of the hydrogel explant and silicone encircling band was uneventful and resulted in improvement of visual acuity and visual field loss. This is the first report on compressive optic neuropathy caused by swelling of a hydrogel explant resulting in a dislocated silicone encircling band. The loss of visual function resolved upon removal of the explant and encircling band.

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.

Genome-wide transcriptome and expression profile analysis of Phalaenopsis during explant browning.

PubMed

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning.

Fibrin glue inhibits migration of ocular surface epithelial cells

PubMed Central

Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

2016-01-01

Purpose Fibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro. Methods Corneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed. Results Explants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14–16 for explants with fibrin glue. Conclusions Fibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy. PMID:27367746

Genome-Wide Transcriptome and Expression Profile Analysis of Phalaenopsis during Explant Browning

PubMed Central

Xu, Chuanjun; Zeng, Biyu; Huang, Junmei; Huang, Wen; Liu, Yumei

2015-01-01

Background Explant browning presents a major problem for in vitro culture, and can lead to the death of the explant and failure of regeneration. Considerable work has examined the physiological mechanisms underlying Phalaenopsis leaf explant browning, but the molecular mechanisms of browning remain elusive. In this study, we used whole genome RNA sequencing to examine Phalaenopsis leaf explant browning at genome-wide level. Methodology/Principal Findings We first used Illumina high-throughput technology to sequence the transcriptome of Phalaenopsis and then performed de novo transcriptome assembly. We assembled 79,434,350 clean reads into 31,708 isogenes and generated 26,565 annotated unigenes. We assigned Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) annotations, and potential Pfam domains to each transcript. Using the transcriptome data as a reference, we next analyzed the differential gene expression of explants cultured for 0, 3, and 6 d, respectively. We then identified differentially expressed genes (DEGs) before and after Phalaenopsis explant browning. We also performed GO, KEGG functional enrichment and Pfam analysis of all DEGs. Finally, we selected 11 genes for quantitative real-time PCR (qPCR) analysis to confirm the expression profile analysis. Conclusions/Significance Here, we report the first comprehensive analysis of transcriptome and expression profiles during Phalaenopsis explant browning. Our results suggest that Phalaenopsis explant browning may be due in part to gene expression changes that affect the secondary metabolism, such as: phenylpropanoid pathway and flavonoid biosynthesis. Genes involved in photosynthesis and ATPase activity have been found to be changed at transcription level; these changes may perturb energy metabolism and thus lead to the decay of plant cells and tissues. This study provides comprehensive gene expression data for Phalaenopsis browning. Our data constitute an important resource for further functional studies to prevent explant browning. PMID:25874455

Induction of bulb organogenesis in in vitro cultures of tarda tulip (Tulipa tarda Stapf.) from seed-derived explants.

PubMed

Maślanka, Małgorzata; Bach, Anna

2014-01-01

A protocol for obtaining bulbs via in vitro organogenesis was developed for tarda tulip ( Tulipa tarda Stapf). Scale explants were obtained from bulbs formed at the base of seedlings or from adventitious bulbs that developed from callus tissue forming on stolons or on germinating seeds. Some explants were subjected to chilling at 5°C for 12 wk. The culture media contained 3 or 6% sucrose and was supplemented with either no growth regulators, either 0.5 μM 6-benzyl-aminopurine (BAP) or 18.9 or 94.6 μM abscisic acid (ABA). Cultures were maintained in the dark at 20°C. Callus tissue developed mainly on media without growth regulators or with BAP. Callus was formed from up to 96% of explants derived from non-chilled adventitious bulbs that were treated with 3% sucrose and 0.5 μM BAP. Less callus was formed from chilled explants compared with non-chilled explants. Newly formed adventitious bulbs appeared on the explants via direct and indirect organogenesis. The media with BAP promoted the formation of adventitious bulbs at a rate of 56-92% from non-chilled explants, whereas a maximum rate of 36% was observed from chilled explants. ABA inhibited the induction of adventitious bulbs and callus. The adventitious bulbs obtained in these experiments contained a meristem, which was evidence that they had developed properly.

Does the Implantation Technique for Totally Implantable Venous Access Ports (TIVAPs) Influence Long-Term Outcome?

PubMed

Biacchi, Daniele; Sammartino, Paolo; Sibio, Simone; Accarpio, Fabio; Cardi, Maurizio; Sapienza, Paolo; De Cesare, Alessandro; Atta, Joseph Maher Fouad; Impagnatiello, Alessio; Di Giorgio, Angelo

2016-02-01

Totally implantable venous access ports (TIVAP) are eventually explanted for various reasons, related or unrelated to the implantation technique used. Having more information on long-term explantation would help improve placement techniques. From a series of 1572 cancer patients who had TIVAPs implanted in our center with the cutdown technique or Seldinger technique, we studied the 542 patients who returned to us to have their TIVAP explanted after 70 days or more. As outcome measures we distinguished between TIVAPs explanted for long-term complications (infection, catheter-, reservoir-, and patient-related complications) and TIVAPs no longer needed. Univariate and multivariate analyses were run to investigate the reasons for explantation and their possible correlation with implantation techniques. The most common reason for explantation was infection (47.6 %), followed by catheter-related (20.8 %), patient-related (14.7 %), and reservoir-related complications (4.7 %). In the remaining 12.2 % of cases, the TIVAP was explanted complication free after the planned treatments ended. Infection correlated closely with longer TIVAP use. Univariate and multivariate analyses identified the Seldinger technique as a major risk factor for venous thrombosis and catheter dislocation. The need for long-term TIVAP explantation in about one-third of cancer patients is related to the implantation techniques used.

Sweet Potato [Ipomoea batatas (L.) Lam].

PubMed

Song, Guo-qing; Yamaguchi, Ken-ichi

2006-01-01

Among the available transformation methods reported on sweet potato, Agrobacterium tumefaciens-mediated transformation is more successful and desirable. Stem explants have shown to be ideal for the transformation of sweet potato because of their ready availability as explants, the simple transformation process, and high-frequency-regeneration via somatic embryogenesis. Under the two-step kanamycin-hygromycin selection method and using the appropriate explants type (stem explants), the efficiency of transformation can be considerably improved in cv. Beniazuma. The high efficiency in the transformation of stem explants suggests that the transformation protocol described in this chapter warrants testing for routine stable transformation of diverse varieties of sweet potato.

Effects of laser irradiation on immature olfactory neuroepithelial explants from the rat

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

Mester, A.F.; Snow, J.B. Jr.

1988-07-01

The photobiological effect of low-output laser irradiation on the maturation and regeneration of immature olfactory bipolar receptor cells of the rat was studied. The maturation and regeneration of the receptor cells of rat fetuses were quantified in neuroepithelial explants with morphometric analysis. The number of explants with outgrowth and the number and length of neuritic outgrowths were determined on a regular basis for 12 days. Explants in the experimental group were irradiated with a helium-neon laser using different incident energy densities (IED). Explants in the fluorescent light control group were exposed to fluorescent light for the same periods of timemore » as those in the experimental group were exposed to laser irradiation. Explants in another control group were not exposed to laser or fluorescent light irradiation. The IED of 0.5 J/cm2 laser irradiation has been found to increase significantly the number of explants with outgrowth and the number and length of the outgrowths. Other laser IEDs or fluorescent light irradiation did not influence maturation or regeneration.« less

Mesenchymally-derived insulin-like growth factor 1 provides a paracrine stimulus for trophoblast migration.

PubMed

Lacey, Helen; Haigh, Teresa; Westwood, Melissa; Aplin, John D

2002-04-24

Trophoblast migration into maternal decidua is essential for normal pregnancy. It occurs in a defined time window, is spatially highly restricted, and is aberrant in some pathological pregnancies, but the control mechanisms are as yet ill-defined. At the periphery of the placenta, chorionic villi make contact with decidua to form specialised anchoring sites that feed interstitially migrating cytotrophoblast into the placental bed. Explants of first trimester mesenchymal villi on collagen type I developed cytotrophoblast outgrowths from the villous tips. However, in medium changed daily, cells did not progress to a migratory phenotype, remaining instead as a contiguous multi-layered sheet. This suggested the need for another migration stimulus. To test the possibility that this might arise from mesenchymal cells, serum-free conditioned medium from first trimester placental fibroblasts was added to explant cultures. Cytotrophoblasts were stimulated to migrate in streams across the gel. Affinity depletion of Insulin-like growth factor from fibroblast medium reduced streaming activity, while the addition of exogenous IGF-I (10 ng/ml) to serum-free medium produced a streaming phenotype. IGF receptor type 1 (IGFR1) was present on cells in the columns, and streaming could be inhibited by antibody to this receptor. IGF-II and activin, known stimulators of cytotrophoblast migration, were also active in this model. These data suggest a paracrine interaction between villous mesenchyme and the cytotrophoblast in anchoring sites that stimulates trophoblast infiltration of decidua. Such a signal would be self-limiting since it diminishes with distance from the placenta. This is a novel mechanism in placental development.

Rooting and acclimatization of micropropagated marubakaido apple rootstock using Adesmia latifolia rhizobia.

PubMed

Muniz, Aleksander Westphal; de Sá, Enilson Luiz; Dalagnol, Gilberto Luíz; Filho, João Américo

2013-01-01

In vitro rooting and the acclimatization of micropropagated rootstocks of apple trees is essential for plant development in the field. The aim of this work was to assess the use of rhizobia of Adesmia latifolia to promote rooting and acclimatization in micropropagated Marubakaido apple rootstock. An experiment involving in vitro rooting and acclimatization was performed with four strains of rhizobium and two controls, one with and the other without the addition of synthetic indoleacetic acid. The inoculated treatments involved the use of sterile inoculum and inoculum containing live rhizobia. The most significant effects on the rooting rate, primary-root length, number of roots, root length, fresh-shoot biomass, and fresh-root biomass were obtained by inoculation with strain EEL16010B and with synthetic indole acetic acid. However, there was no difference in the growth of apple explants in the acclimatization experiments. Strain EEL16010B can be used to induce in vitro rooting of the Marubakaido rootstock and can replace the use of synthetic indoleacetic acid in the rooting of this cultivar.

Cell and tissue culture of Miscanthus Sacchariflorus

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

Godovikova, V.A.; Moiseyeva, E.A.; Shumny, V.K.

1995-11-01

Since recent time search and introduction of new species of plants have paid attention. More perspective are perennial low maintenance landscape plants from genera Phragmites L. and Miscanthus Anderss. known as high speed growing and great amount of cellulose`s containing. Absence of seeds production and limited distribution area prevent from immediately introduction the plants of this species. The main goal of our investigation is the scientific development of the cell and tissue culture methods to get changing clones, salt and cold tolerant plants and their micropogation. At present there are collection of biovariety represented by subspecies, ecotypes and plant regenerantsmore » of two species - Miscanthus purpurascens (Anders.) and Miscanthus sacchariflorus (Maxim.). Successful results have been achieved in screening of culture media, prepared on MS base medium and contained a row of tropic components to protect the explant and callus tissue from oxidation and necrosis. Initially the callus was induced from stem segments, apical and nodular meristem of vegetative shoots of elulalia, growing in hydroponic greenhouse. Morphological and cytologic analysis of plant-regenerants have been done.« less

Risk factors for explantation due to infection after sacral neuromodulation: a multicenter retrospective case-control study.

PubMed

Myer, Emily N B; Petrikovets, Andrey; Slocum, Paul D; Lee, Toy Gee; Carter-Brooks, Charelle M; Noor, Nabila; Carlos, Daniela M; Wu, Emily; Van Eck, Kathryn; Fashokun, Tola B; Yurteri-Kaplan, Ladin; Chen, Chi Chiung Grace

2018-04-07

Sacral neuromodulation is an effective therapy for overactive bladder, urinary retention, and fecal incontinence. Infection after sacral neurostimulation is costly and burdensome. Determining optimal perioperative management strategies to reduce the risk of infection is important to reduce this burden. We sought to identify risk factors associated with sacral neurostimulator infection requiring explantation, to estimate the incidence of infection requiring explantation, and identify associated microbial pathogens. This is a multicenter retrospective case-control study of sacral neuromodulation procedures completed from Jan. 1, 2004, through Dec. 31, 2014. We identified all sacral neuromodulation implantable pulse generator implants as well as explants due to infection at 8 participating institutions. Cases were patients who required implantable pulse generator explantation for infection during the review period. Cases were included if age ≥18 years old, follow-up data were available ≥30 days after implantable pulse generator implant, and the implant was performed at the institution performing the explant. Two controls were matched to each case. These controls were the patients who had an implantable pulse generator implanted by the same surgeon immediately preceding and immediately following the identified case who met inclusion criteria. Controls were included if age ≥18 years old, no infection after implantable pulse generator implant, follow-up data were available ≥180 days after implant, and no explant for any reason <180 days from implant. Controls may have had an explant for reasons other than infection at >180 days after implant. Fisher exact test (for categorical variables) and Student t test (for continuous variables) were used to test the strength of the association between infection and patient and surgery characteristics. Significant variables were then considered in a multivariable logistic regression model to determine risk factors independently associated with infection. Over a 10-year period at 8 academic institutions, 1930 sacral neuromodulator implants were performed by 17 surgeons. In all, 38 cases requiring device explant for infection and 72 corresponding controls were identified. The incidence of infection requiring explant was 1.97%. Hematoma formation (13% cases, 0% controls; P = .004) and pocket depth of ≥3 cm (21% cases, 0% controls; P = .031) were independently associated with an increased risk of infection requiring explant. On multivariable regression analysis controlling for significant variables, both hematoma formation (P = .006) and pocket depth ≥3 cm (P = .020, odds ratio 3.26; 95% confidence interval, 1.20-8.89) remained significantly associated with infection requiring explant. Of the 38 cases requiring explant, 32 had cultures collected and 24 had positive cultures. All 5 cases with a hematoma had a positive culture (100%). Of the 4 cases with a pocket depth ≥3 cm, 2 had positive cultures, 1 had negative cultures, and 1 had a missing culture result. The most common organism identified was methicillin-resistant Staphylococcus aureus (38%). Infection after sacral neuromodulation requiring device explant is low. The most common infectious pathogen identified was methicillin-resistant S aureus. Demographic and health characteristics did not predict risk of explant due to infection, however, having a postoperative hematoma or a deep pocket ≥3 cm significantly increased the risk of explant due to infection. These findings highlight the importance of meticulous hemostasis as well as ensuring the pocket depth is <3 cm at the time of device implant. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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

Role of biotechnological interventions in the improvement of castor (Ricinus communis L.) and Jatropha curcas L.

PubMed

Sujatha, M; Reddy, T P; Mahasi, M J

2008-01-01

Castor and Jatropha belong to the Euphorbiaceae family. This review highlights the role of biotechnological tools in the genetic improvement of castor and jatropha. Castor is monotypic and breeding programmes have mostly relied on the variability available in the primary gene pool. The major constraints limiting profitable cultivation are: vulnerability to insect pests and diseases, and the press cake is toxic which restrict its use as cattle feed. Conventional breeding techniques have limited scope in improvement of resistance to biotic stresses and in quality improvement owing to low genetic variability for these traits. Genetic diversity was assessed using protein based markers while use of molecular markers is at infancy. In vitro studies in castor have been successful in shoot proliferation from meristematic explants, but not callus-mediated regeneration. Genetic transformation experiments have been initiated for development of insect resistant and ricin-free transgenics with very low transformation frequency. In tropical and subtropical countries jatropha is viewed as a potential biofuel crop. The limitations in available germplasm include; lack of knowledge of the genetic base, poor yields, low genetic diversity and vulnerability to a wide array of insects and diseases. Great scope exists for genetic improvement through conventional methods, induced mutations, interspecific hybridization and genetic transformation. Reliable and highly efficient tissue culture protocols for direct and callus-mediated shoot regeneration and somatic embryogenesis are established for jatropha which indicates potential for widening the genetic base through biotechnological tools. Assessment of genetic diversity using molecular markers disclosed low interaccessional variability in local Jatropha curcas germplasm. The current status and future prospects of in vitro regeneration, genetic transformation and the role of molecular tools in the genetic enhancement of the two-oilseed crops are discussed.

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.

5-Azacytidine mediated reactivation of silenced transgenes in potato (Solanum tuberosum) at the whole plant level.

PubMed

Tyč, Dimitrij; Nocarová, Eva; Sikorová, Lenka; Fischer, Lukáš

2017-08-01

Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

Heat-shock-mediated elimination of the nptII marker gene in transgenic apple (Malus×domestica Borkh.).

PubMed

Herzog, Katja; Flachowsky, Henryk; Deising, Holger B; Hanke, Magda-Viola

2012-04-25

Production of marker-free genetically modified (GM) plants is one of the major challenges of molecular fruit breeding. Employing clean vector technologies, allowing the removal of undesired DNA sequences from GM plants, this goal can be achieved. The present study describes the establishment of a clean vector system in apple Malus×domestica Borkh., which is based on the use of the neomycin phosphotransferase II gene (nptII) as selectable marker gene and kanamycin/paramomycin as selective agent. The nptII gene can be removed after selection of GM shoots via site-specific excision mediated by heat-shock-inducible expression of the budding yeast FLP recombinase driven by the soybean Gmhsp17.5-E promoter. We created a monitoring vector containing the nptII and the flp gene as a box flanked by two direct repeats of the flp recognition target (FRT) sites. The FRT-flanked box separates the gusA reporter gene from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. Consequently, GUS expression does only occur after elimination of the FRT-flanked box. Transformation experiments using the monitoring vector resulted in a total of nine transgenic lines. These lines were investigated for transgenicity by PCR, RT-PCR and Southern hybridization. Among different temperature regimes tested, exposure to 42 °C for 3.5 to 4h led to efficient induction of FLP-mediated recombination and removal of the nptII marker gene. A second round of shoot regeneration from leaf explants led to GM apple plants completely free of the nptII gene. Copyright © 2012 Elsevier B.V. All rights reserved.

Laparoscopic Adjustable Gastric Band Explantation and Implantation at Academic Centers.

PubMed

Koh, Christina Y; Inaba, Colette S; Sujatha-Bhaskar, Sarath; Hohmann, Samuel; Ponce, Jaime; Nguyen, Ninh T

2017-10-01

The laparoscopic adjustable gastric band (LAGB) was approved for use in the US in 2001 and has been found to be a safe and effective surgical treatment for morbid obesity. However, there is a recent trend toward reduced use of LAGB nationwide. The objective of this study was to examine the prevalence and outcomes of primary LAGB implantation compared with revision and explantation at academic centers. Data were obtained from the Vizient database from 2007 through 2015. The ICD-9-Clinical Modification and ICD-10-Clinical Modification were used to select patients with a primary diagnosis of obesity who had undergone LAGB implantation, revision, or explantation. Prevalence and outcomes of primary LAGB implantation compared with revision or explantation were analyzed. Outcomes measures included length of stay, ICU admission, morbidity, mortality, and cost. From 2007 through 2015, a total of 28,202 patients underwent LAGB implantation for surgical weight loss. The annual number of LAGB implantation procedures decreased steadily after 2010. In the same time period, 12,157 patients underwent LAGB explantation. In 2013, the number of LAGB explantation procedures exceeded that of implantation. Laparoscopic adjustable gastric band revision rates remained stable throughout the study period. Mean length of stay, serious morbidity, and proportion of patients requiring ICU admission were higher for gastric band revision and explantation cases compared with primary LAGB implantation cases. There was no statistically significant difference in mortality or mean cost between the 2 groups. Since 2013, the number of gastric band explantation procedures has exceeded that of implantation procedures at academic centers. Laparoscopic adjustable gastric band revision or explantation is associated with longer length of stay, higher rate of postoperative ICU admissions, and higher overall morbidity compared with LAGB implantation. Copyright © 2017 American College of Surgeons. Published by Elsevier Inc. All rights reserved.

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

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.

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.

A novel method for coral explant culture and micropropagation.

PubMed

Vizel, Maya; Loya, Yossi; Downs, Craig A; Kramarsky-Winter, Esti

2011-06-01

We describe here a method for the micropropagation of coral that creates progeny from tissue explants derived from a single polyp or colonial corals. Coral tissue explants of various sizes (0.5-2.5 mm in diameter) were manually microdissected from the solitary coral Fungia granulosa. Explants could be maintained in an undeveloped state or induced to develop into polyps by manipulating environmental parameters such as light and temperature regimes, as well as substrate type. Fully developed polyps were able to be maintained for a long-term in a closed sea water system. Further, we demonstrate that mature explants are also amenable to this technique with the micropropagation of second-generation explants and their development into mature polyps. We thereby experimentally have established coral clonal lines that maintain their ability to differentiate without the need for chemical induction or genetic manipulation. The versatility of this method is also demonstrated through its application to two other coral species, the colonial corals Oculina patigonica and Favia favus.

Dissecting and Culturing Animal Cap Explants.

PubMed

Dingwell, Kevin S; Smith, James C

2018-05-16

The animal cap explant is a simple but adaptable tool available to developmental biologists. The use of animal cap explants in demonstrating the presence of mesoderm-inducting activity in the Xenopus embryo vegetal pole is one of many elegant examples of their worth. Animal caps respond to a range of growth factors (e.g., Wnts, FGF, TGF-β), making them especially useful for studying signal transduction pathways and gene regulatory networks. Explants are also suitable for examining cell behavior and have provided key insights into the molecular mechanisms controlling vertebrate morphogenesis. In this protocol, we outline two methods to isolate animal cap explants from Xenopus laevis , both of which can be applied easily to Xenopus tropicalis The first method is a standard manual method that can be used in any laboratory equipped with a standard dissecting microscope. For labs planning on dissecting large numbers of explants on a regular basis, a second, high throughput method is described that uses a specialized microcautery surgical instrument. © 2018 Cold Spring Harbor Laboratory Press.

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.

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.

Single-Center Experience With HeartMate II Left Ventricular Assist Device Explantation.

PubMed

Tchantchaleishvili, Vakhtang; Cheyne, Christina; Sherazi, Saadia; Melvin, Amber L; Hallinan, William; Chen, Leway; Todd Massey, Howard

2016-12-01

In patients with continuous flow left ventricular assist devices (CF-LVADs) myocardial recovery is uncommon. Given the heterogeneity of the population implanted and low incidence of recovery, the discovery of native left ventricular (LV) recovery and criteria for explantation of CF-LVAD system is not clearly determined. We sought to analyze the characteristics of the patients who underwent CF-LVAD explantation at our institution. Prospectively collected data on patients supported with CF-LVADs were reviewed retrospectively. Patients who underwent CF-LVAD explants were identified and their characteristics were analyzed with a focus on patient presentation and determinants of explantability. From November 2006 to June 2014, 223 patients (181 male, 42 female) underwent implantation of HeartMate II LVAD. Seven female (16.7%) and one male (0.6%) patients were explanted (P < 0.001). Mean age was 43 ± 9 years and etiology for cardiomyopathy was ischemic in three (37.5%) patients, nonischemic in four (50%) patients, and mixed in the one (12.5%) male patient of the cohort. Five (62.5%) patients presented acutely with significant hemolysis, and were found to have LV improvement as well as reduced, absent, or reversed diastolic flow velocities on echocardiography. Overall, mean lactate dehydrogenase level before explantation was 1709 ± 1168 U/L compared to the mean baseline level of 601 ± 316 U/L (P = 0.048). Mean LV ejection fraction (LVEF) improved from 17 ± 7% preimplant to 56 ± 11% pre-explantation (P < 0.001). Median number of days on CF-LVAD support was 870 (interquartile range, 209-975) while mean duration of follow-up after the CF-LVAD explantation was 276 ± 240 days. Mean LVEF dropped from 46 ± 19% postexplantation to 34 ± 10% during the most recent follow-up (P = 0.015). At our institution, patients who underwent LVAD explants were predominantly women with nonischemic cardiomyopathy. Clinical evidence of hemolysis and echocardiographic evidence of reduced or absent diastolic flow velocities were common findings in these patients. Over time, patient's native LV function declined in the absence of LVAD (after LVAD explantation). Significant challenges remain in predicting LV recovery and identifying those individuals who have recovered myocardial function significant enough to be explanted. Copyright © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

PubMed Central

Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

2016-01-01

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

Noninvasive assessment of articular cartilage surface damage using reflected polarized light microscopy

NASA Astrophysics Data System (ADS)

Huynh, Ruby N.; Nehmetallah, George; Raub, Christopher B.

2017-06-01

Articular surface damage occurs to cartilage during normal aging, osteoarthritis, and in trauma. A noninvasive assessment of cartilage microstructural alterations is useful for studies involving cartilage explants. This study evaluates polarized reflectance microscopy as a tool to assess surface damage to cartilage explants caused by mechanical scraping and enzymatic degradation. Adult bovine articular cartilage explants were scraped, incubated in collagenase, or underwent scrape and collagenase treatments. In an additional experiment, cartilage explants were subject to scrapes at graduated levels of severity. Polarized reflectance parameters were compared with India ink surface staining, features of histological sections, changes in explant wet weight and thickness, and chondrocyte viability. The polarized reflectance signal was sensitive to surface scrape damage and revealed individual scrape features consistent with India ink marks. Following surface treatments, the reflectance contrast parameter was elevated and correlated with image area fraction of India ink. After extensive scraping, polarized reflectance contrast and chondrocyte viability were lower than that from untreated explants. As part of this work, a mathematical model was developed and confirmed the trend in the reflectance signal due to changes in surface scattering and subsurface birefringence. These results demonstrate the effectiveness of polarized reflectance microscopy to sensitively assess surface microstructural alterations in articular cartilage explants.

Effects of pinealectomy and melatonin supplementation on endometrial explants in a rat model.

PubMed

Koc, Onder; Gunduz, Bülent; Topcuoglu, Ata; Bugdayci, Güler; Yilmaz, Fahri; Duran, Bülent

2010-11-01

To determine the effects of pinealectomy on endometrial explants in rats and evaluate the activity of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) in the rat endometriosis model. Rats with experimentally induced endometriosis were randomly divided into three groups after second-look laparotomies. Group 1 (pinealectomy, n = 8) and Group 2 (pinealectomy+melatonin, n = 8) underwent pinealectomies after the second-look laparotomies. Group 3 was presented as control group (vehicle solution+without pinealectomy (n = 6)). Melatonin was administered intraperitoneally for 4 weeks in Group 2, whereas an equal volume of vehicle solution was given to Groups 1 and 3. Evaluation of the volume of the endometrial explants, histopathological examination and preservation of explant epitheliums according to the scoring system were undertaken. There was a statistically significant increase in spherical explant volumes of Group 1 compared to Groups 2 and 3. In Group 1, the level of MDA was significantly higher and SOD and CAT activity was significantly lower compared to Groups 2 and 3. A statistically significant increase in the epithelial lining scores of explants was noted in Group 1 compared to Groups 2 and 3. The effects of pinealectomy on the progression of endometriosis explants were reversed by melatonin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

"Not all breast implants are equal: a 13-year review of implant longevity and reasons for explantation".

PubMed

Van Slyke, Aaron C; Carr, Michael; Carr, Nicholas J

2018-06-04

Augmentation mammoplasty is the most common aesthetic procedure. Textured implants control implant position and have improved capsular contracture rates; however, the impact of texturing on longevity and clinical findings at explantation is unclear. All cases of explantation between January 2005 - April 2017 from an aesthetic practice were reviewed retrospectively. Patient demographics, implant characteristics, time-to-explantation, and clinical presentation and intraoperative findings at explantation were analyzed. 539 breast implants were explanted during the study period: 249 saline, 147 smooth gel, 123 Biocell, and 20 other nonaggressively textured breast implants. Average time from placement to explantation was 7.5 years, 5.6 years, 4.9 years, and 4.0 years for saline, other textured, smooth gel, and Biocell implants, respectively (p-value = 3.25e-08). The percentage of implants removed associated with implant performance failure was 50.3%, 57.5%, 75.0%, and 85.4% for smooth gel, saline, other textured, and Biocell implants, respectively (p-value = 7.25e-09). 21.1% of Biocell implants versus 1.5% of all other implants presented with pain (p-value = 2.71e-15). 45 Biocell implants had double capsules; this phenomenon was not observed with any other implant type (p-value = 5.85e-37). Seven Biocell implants had late seromas, compared to three late seromas with any other implant type (p-value = 0.0013). Here, we provide evidence that Biocell implants have the shortest time-to-explantation and the highest proportion of implants associated with implant performance failure. This information should complement the informed consent process when selecting an appropriate implant.