Showing papers on "Murashige and Skoog medium published in 1996"

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Abstract: Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N6 benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 μM BA and 4.9 μM IBA. Although the same BA concentration but with reduced IBA concentration (0.49 μM) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 μM BA and 0.49 μM IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.

In vitro propagation of the medicinal herbs Ocimum americanum L. syn. O. canum Sims. (hoary basil) and Ocimum sanctum L. (holy basil).

Abstract: A procedure is outlined for in vitro propagation of two medicinal herbs, Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum sanctum L. (holy basil), using axillary shoot buds. Multiple shoot formation was induced from shoot bud explants of both species on Murashige and Skoog medium (MS) supplemented with benzyladenine (BA). The optimum BA concentrations for shoot proliferation were 0.25 mg/l for O. americanum and 1.0 mg/l for O. sanctum. Incorporation of 0.5 mg/l gibberellic acid (GA3) along with BA in the culture medium resulted in a marked increase in the frequency of axillary branching as well as multiple shoot formation. Shoot buds collected between September through December were most responsive in culture. Shoots of O. americanum were rooted on half-strength MS supplemented with 1.0 mg/l indole-3-butyric acid (IBA), whereas O. sanctum rooted best on medium with 1.0 mg/l naphthaleneacetic acid (NAA). The plantlets were hardened off and successfully established in natural soil, where they grew and matured normally.

High frequency somatic embryogenesis from coffee leaves : Factors influencing embryogenesis, and subsequent proliferation and regeneration in liquid medium

Abstract: An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called “high frequency” embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1-1 in a modified MS medium containing 4.5 μM 2,4-dichlorophenoxyacetic acid, under 3 μmol m-2 s-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 μm in diameter) at an inoculum density of 5 g 1-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×105 somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.

In vitro regeneration of chickpea (Cicer arietinum L.): Stimulation of direct organogenesis and somatic embryogenesis by thidiazuron

Abstract: In vitro regeneration in chickpea (Cicer arietinum L.) was achieved by direct culture of mature seeds on Murashige and Skoog (MS) medium supplemented with either N-phenyl-N′(-1,2,3-thidiazol-5-yl) urea (thidiazuron, TDZ) or N6-benzylaminopurine (BAP). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch region of the seedlings within 2 to 3 weeks of culture initiation. TDZ was found to be more effective compared to BAP as an inductive signal of regeneration. The former induced multiple shoot formation at all the concentrations tested (1 μM to 100 μM), although, maximum morphogenic response was observed at 10 μM concentration. Addition of naphthaleneacetic acid (NAA) alone or in combination with BAP to the MS medium failed to invoke a similar response. When the TDZ supplemented medium was amended with L-proline, the resultant regenerants were mostly somatic embryos. Histological investigations confirmed the switch in the regeneration pathway from directly formed adventitious shoots to embryogenesis. For obtaining plantlets, adventitious shoots were rooted on MS medium supplemented with 2.5 μM NAA; somatic embryos were germinated and established on MS medium. Normal plants were regenerated from both adventitious shoots and somatic embryos and transferred to soil.

Plant regeneration from immature embryos of 48 elite CIMMYT bread wheats.

Abstract: Forty-eight bread wheat (Triticum aestivum L.) released cultivars and elite advanced lines were evaluated for their ability to produce embryogenic callus using three different media. Basal N6 medium supplemented with dicamba (E1), MS medium containing 2,4-D (E3) or MS medium containing 2,4-D plus different amino acids (E5) were used for callus initiation and maintenance. Plant regeneration was achieved on basal MS medium with indole-3-acetic acid (IAA) and 6-benzylamino purine (BAP) and rooting on MS with 1-naphthaleneacetic acid (NAA). Percentage regeneration varied widely with both genotype and initiation medium, with values ranging from 2% to 94%. The number of plantlets produced per embryo ranged from 6 to 42. Thirteen genotypes showed at least 50% regeneration after culture on E5 medium; 3 genotypes after culture on E3 initiation medium and 1 after initiation on E1. After four subcultures, over a 16-week period, 41 genotypes (85%) lost their ability to regenerate plants while the remaining 7 lines (15%) retained plant regeneration potential but at reduced levels. E3 medium was found to be the best for maintaining regeneration potential after four subcultures.

Efficient transgenic plant regeneration through Agrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Abstract: Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 260C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.

Micropropagation of Hemidesmus indicus (L.) R. Br. through axillary bud culture

Abstract: A procedure for rapid in vitro propagation of the aromatic and medicinal plant Hemidesmus indicus (L.) R.Br. (Family Asclepiadaceae) from nodal explants is described. The highest shoot multiplication rate of 8.2 ± 0.4 shoots/explant with a 95% frequency was achieved in S weeks culture period on Murashige and Skoog medium supplemented with 1.15 μM kinetin and 0.054 μM α-naphthaleneacetic acid. Excised shoots were rooted on the same basal medium supplemented with 1.15 μM kinetin and 7.35 μM indole-3-butyric acid. Shoots derived from subcultures exhibited better rooting response than those from primary cultures. After a hardening phase of 2 weeks, there was a 70% transplantation success in the field.

Effect of genotype, explant and medium onin vitro regeneration of red pepper

Abstract: In vitro plant regeneration was achieved inCapsicum praetermissum, C. baccatum andC. annuum cvs. G4, Bhiwapuri Sweet pepper, Cayenne pepper and Hybrid pepper. Shoots were induced from hypocotyl, cotyledon and leaf explants on Murashige and Skoog medium supplemented with 5.7 μM indoleacetic acid (IAA)+13.3 μM benzyladenine (BA); 22 μM BA; and 44 μM BA. Analysis of variance revealed that the most significant effect on shoot regeneration was due to the explant and it accounted for 56.3% of total variation observed. The genotype x explant effect on regeneration was minor relative to all other 2- and 3-way interactions because leaf explants consistently regenerated more shoots than hypocotyls or cotyledons in all the genotypes and thereby reduced the variation among the genotypes. Explant x medium interaction revealed that 22 μM BA was the best growth regulator supplement in regeneration medium for optimal shoot regeneration from leaf explants. Rooting of regenerated shoots was achieved on 5.7 μM IAA-containing medium, and the rooting response was better from shoots induced on medium fortified with 5.7 μM IAA plus 13.3 μM BA. Complete plantlets with diploid chromosome number (2n=2x=24) were transferred to soil and 60–70% of these plantlets survived and grew well.

Callus induction and plant regeneration from immature and mature embryos of winter durum wheat genotypes.

Abstract: Seven genotypes of winter durum wheat (Triticum durum Desf.) were cultured to establish an efficient method of callus formation and plant regeneration from mature embryo culture, and to compare the responses of immature and mature embryo cultures. Immature embryos were aseptically dissected from seeds and placed, with the scutellum upwards, in dishes containing Murashige and Skoog's (MS) mineral salts and 2mg 2,4- dichlorophenoxyacetic acid (2,4-D) per litre. Calli and regenerated plants were maintained on 2,4-D-free medium. Mature embryos were moved slightly on the imbibed seeds. For callus formation, the seeds with moved embryos were placed, furrow downwards, in dishes containing 8 mg 2,4-D per litre. The developed calli and regenerated plants were maintained on the MS medium. Plants regenerated from both embryo cultures were vernalized and grown to maturity in soil. Variability was observed among the wheat genotypes tested for various culture responses in both explant cultures. Callus induction rate and regeneration capacity of callus were independent of each other. Mature embryos have a low frequency of callus induction but a high regeneration capacity. Considering availability, rapidity and reliability, this form of mature embryo culture can be used as an alternative method for immature embryo culture.

Induction of direct somatic embryogenesis and plant regeneration in pepper (Capsicum annuum L.).

Abstract: Pepper (cv. New Mexico — 6 and Rajur Hirapur) plants were regenerated from immature zygotic embryos via direct somatic embryogenesis. Somatic embryos were formed directly, without any intervening callus, on the zygotic embryo apex, embryo axis and cotyledons on Murashige and Skoog's (MS) medium containing 2,4-D (418 μM), thidiazuron (10 μM) and a high concentration of sucrose (6–10%). The best response was observed on MS medium containing 2,4-D (9 μM), coconut water (10%) and high sucrose (8%). The entire process of induction and maturation of the embryos was completed on the same medium. Histological examination indicated that secondary embryogenesis also occurred directly from the primary somatic embryos. Differentiation of embryos was nonsynchronous, and some embryos were swollen and distorted with fasciation. More than 70% of the mature normal somatic embryos germinated readily on MS medium containing GA3 or TDZ, alone and in combination, and following transfer to pots developed into normal plants.

Callus induction and plant regeneration from different explant types of Miscanthus x ogiformis Honda ‘Giganteus’

Abstract: Different explants of Miscanthus x ogiformis Honda ‘Giganteus’ were tested in order to develop an efficient tissue culture system. Shoot apices, leaf and root sections from in vitro-propagated plants, and leaf and immature inflorescence sections from 6-month-old greenhouse-grown plants were used. The explants were cultured on Murashige and Skoog medium supplemented with 4.5, 13.6, 22.6 or 31.7 μM 2,4-dichlorophenoxyacetic acid. Three types of callus were formed but only one was embryogenic and regenerated plants. Callus induction and formation of embryogenic callus depended on the type and developmental stage of the explants. Shoot apices formed the highest percentage of embryogenic callus. There was a difference in the formation of embryogenic callus between leaf explants from in vitro-propagated shoots and greenhouse-grown plants. The best results were obtained from newly formed leaves of in vitro-propagated shoots and older leaves of greenhouse-grown plants. Immature inflorescences smaller than 2.5 cm produced a higher percentage of embryogenic callus than larger more mature inflorescences. Embryogenic callus derived from immature inflorescences had the highest regeneration capacity. Differences in 2,4-dichlorophenoxyacetic acid concentrations had no significant effect on callus induction, embryogenic callus formation and plant regeneration.

Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana.

Abstract: Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv. WSP-3 was very superior on MS medium, compared to B5 medium, supplemented with four cytokinins (TDZ, 4PU-30, BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D. Optimum conditions for regeneration from explants (leaf, stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5–10 mg/l and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30–100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil.

Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica.

Abstract: Immature endosperm of Acacia nilotica formed a nodular callus on MS medium supplemented with 2,4-D, BAP and CH. In the third passage on this medium, in the dark, the callus differentiated somatic embryos. The embryos germinated on MS only after 15 d pre-treatment on modified MS medium in which major salts were replaced by those of major salts of B5 medium and supplemented with glutamine, CH and CW. Triploid nature of the somatic embryos was confirmed by Feulgen cytophotometry.

An improved and reliable chili pepper (Capsicum annuum L.) plant regeneration method.

Abstract: In this work we report a new method forin vitro chili pepper (Capsicum annuum L.) plant regeneration based on shoot formation from wounded hypocotyls. Chili pepper seeds were surface sterilized and germinated on agar (0.8%) at 25 ± 2°C in the dark. Five factors that may influence shoot regeneration were studied: age of seedlings, hypocotyl wounding site, time elapsed between wounding the hypocotyls and decapitation of seedlings, culture media and cultivars. In order to study the influence of the first three factors on shoot regeneration, the apical, middle or basal hypocotyl regions of seedlings of cv. Mulato Bajio at different stages of development (9, 15, 16, 21 and 28 d old) were wounded with a syringe needle, and the seedlings were cultured on MS semisolid medium without growth regulators at 25 ± 2°C under a 16/8 h light/dark photoperiod (daylight fluorescent lamps; 35 μmol m-2 s--1) until decapitation. The seedlings were decapitated (3 mm below the cotyledons) at different times after wounding (0, 2, 4, 10, 12 and 14 d), and each explant was evaluated for bud and shoot formation (≥ 5 mm in length) at the wounded site after 30 d of incubation. In general, seedlings at the stage of curved hypocotyl (9 d old) wounded in the apical region of hypocotyl were the best explants for shoot regeneration when inoculated on culture medium without growth regulators. Decapitation after wounding also influenced the shoot regeneration efficiency, with 10–14 d being the best period. Up to 90% shoot regeneration in cv. Mulato Bajio was obtained under these conditions. Statistically significant differences were observed for shoot formation among 21 cultivars tested. Regeneration of whole plants was achieved by rooting the shoots with indole-3-butyric acid pulses of 60 mg L−1 for 3 h and then subculturing on MS medium without growth regulators.

Micropropagation of switchgrass by node culture.

Abstract: Switchgrass (Panicum virgatum L.) is naturally outcrossing; therefore, maintenance of genotype is difficult through sexual propagation. The objective of this study was to develop a micropropagation procedure for the multiplication of desired or selected genotypes. Nodal segments were surface sterilized, split longitudinally, and placed with the cut surface on solid MS medium containing 30 g L-1 maltose and different concentrations of 6-benzylaminopurine (BAP) as the only growth regulator. Best shoot proliferation was obtained with 12.5 micromoles BAP and culture at 29 degrees C. Shoots were easily rooted on MS medium without BAP. Under optimum conditions, it is possible to produce approximately 500 plantlets from one parent plant in 12 wk.

Callus induction and plantlet formation through culture of isolated microspores of eggplant (Solanum melongena L.)

Abstract: Morphogenic calli were obtained efficiently from ab initio cultures of isolated microspores in eggplant. Initial culture of freshly isolated microspores in sucrose-free medium at high temperature (35°C) for 3 d was a prerequisite for callus induction. The microspores were re-cultured in modified NLN medium containing 2% sucrose and phytohormones (NAA 0.5 mg l(-1), BA 0.5 mg l(-1)) in the dark. After 4 weeks of re-culture, small calli derived from microspores were transferred to MS medium containing 4 mg l(-1) zeatin and 0.2 mg l(-1) IAA for shoot regeneration. The ploidy of 12 randomly selected regenerants was assessed by chromosome counts in root tips. Only one of the regenerants was haploid, 7 were diploid, 3 were triploid and one was tetraploid. The diploids set seeds after self-pollination and showed no segregation for morphological traits in the progeny, suggesting that they were spontaneously doubled haploids.

High frequency shoot regeneration from leaf explants of muskmelon

Abstract: Efficient in vitro plant regeneration systems are critical for many purposes including plant transformation. Current regeneration systems for melon (Cucumis melo L.) plants generally utilize cotyledon explants; regeneration from melon leaves has received limited attention. We investigated several factors that influence regeneration from melon leaves including: genotype growth conditions and age of the source plant, leaf age, explant orientation, gelling agent, and the addition of silver nitrate and sulfonylurea herbicide to the culture media. Critical factors that influenced regeneration were preculture conditions of the donor plants, leaf size, and the use of silver nitrate and Phytagel in the medium. The best results were obtained with 3–4 cm diam leaves excised from pot grown greenhouse or growth chamber plants cultured on MS medium with 5 μM IAA, 5 μM BA, 1 μM ABA, 30 μM silver nitrate and 2.6 g l-1 Phytagel. Low concentratons of sulfonylurea herbicide (0.25 mg l-1 DPX-M 6316) also enhanced regeneration. Under optimized conditions 80–100% of the explants regenerated, with 10–100 shoots per explant

Interaction of hyperhydricity-preventing Pseudomonas sp. with oregano (Origanum vulgare) and selection of high phenolics and rosmarinic acid-producing clonal lines

Abstract: Phenolic metabolites from oregano and related species in the family Lamiaceae are important sources of antimicrobials and antioxidants. The content of phenolic metabolites in oregano and related species are highly variable due to genetic heterogeneity. This genetic heterogeneity is due to the breeding character being influenced by natural cross‐pollination. In order to develop gene pools to improve ingredient quality and quantity genetically uniform shoot‐based clonal lines were isolated using plant tissue culture techniques. Clonal lines were generated from multiple shoots induced by 1 mg/1 benzylaminopurine in standard Murashige and Skoog medium with 3 % sucrose. Under these optimum conditions 7–10 shoots per explant were generated for further clonal propagation or regeneration of plants. Shoot‐inducing hormones like thidiazuron and adenine sulfate did not improve multiple shoot‐forming ability. Individual shoots of several individual clonal lines with each originating from a single heterozygou...

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

Abstract: An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.

In Vitro Development of Inflorescences from Switchgrass Nodal Segments

Abstract: Immature inflorescences are an important explant source for initiating in vitro cultures in gramineous species. The objective of this study with switchgrass (Panicum virgatum L.) cv. Alamo was to produce axenic inflorescences from nodes cultured in vitro. The top nodal segments from tillers in the two to four node stage of greenhouse grown plants were surface sterilized, split longitudinally, and plated with the cut surface in contact with Murashige and Skoog (MS) medium containing 30 g L-' maltose. 6-Benzylaminopurine (BAP) was added in concentrations of 0.0, 5.0, 12.5, or 25.0 μM. After 2 to 5 wk culture, panicles were produced with fully developed spikelets and perfect terminal florets. Panicles produced on medium without BAP were between 5 and 9 cm long and the number of spikelets ranged between 50 and 200. Panicles produced on medium with 5.0 to 25.0 μM BAP had a shorter rachis (2-5 cm) and branches, but contained more spikelets (200-700). Young spikelets obtained after 20- to 30-d culture of nodes, were used as explants for regeneration experiments. Calli were initiated from spikelets plated on MS solid medium with 30 g L -1 maltose, 22.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 μM BAP. They were later transferred to MS medium without growth regulators, where thousands of regenerants developed through somatic embryogenesis and organogenesis. The procedure is highly efficient for producing multiple sterile explants from a single genotype for use in various in vitro culture experiments.

Micropropagation of Lavandula latifolia through nodal bud culture of mature plants

Abstract: Cultures of Lavandula latifolia Medicus were established from axillary buds of mature field-grown plants. Explants were initially cultured on media with two different macronutrient combinations and benzyladenine or kinetin added either individually or with naphthaleneacetic acid. Subsequently, explants were subcultured in Murashige and Skoog medium supplemented with 20% coconut milk, 0.57 μM indoleacetic acid and 8.88 μM benzyladenine. Shoot proliferation from axillary buds was not affected by seasonal fluctuations in the stock plants but depended on the macronutrient composition and on the type and concentration of cytokinin tested. Best results were obtained in explants initially cultured in media with Murashige and Skoog constituents and supplemented with 5 μM benzyladenine. In vitro-grown shoots were used to induce multiple shooting by transferring them to subculture medium. Shoots were rooted on Murashige and Skoog medium with macronutrients at half-strength. Plantlets were transferred to soil and grown to maturity.

Shoot tips of wheat as an alternative source for regenerable embryogenic callus cultures

Abstract: Shoot tips of Triticum aestivum L. cvs. Turbo and Nandu, both summer wheat varieties, were excised from 4 and 10 day-old seedlings, and used for induction of embryogenic callus. A modified L3 medium, supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-d) for culture initiation, and 5 μM 2,4-d for subculturing, was optimal; 90% of 4 day-old ‘Turbo’ seedlings formed embryogenic callus. Optimal plant regeneration was achieved from callus incubated on a modified MS medium without 2,4-d, but supplemented with 2.22 μM 6-benzylaminopurine and 0.27 μM naphthaleneacetic acid. Plantlets formed via embryogenesis from all embryogenic ‘Turbo’ calli initiated from 4 day-old explants, with a mean number of 8 regenerants per explant. Regeneration occured via embryogenesis only. Results obtained using ‘Nandu’ were within the same range.

High frequency adventitious shoot regeneration from excised leaves ofPaulownia spp. cultured in vitro.

Abstract: High frequency, direct regeneration of shoots was induced in leaf cultures ofPaulownia tomentosa, P. fortunei x P. tomentosa andP. kawakamii. The optimum culture medium for the leaf explants derived from shoot cultures was Murashige-Skoog (MS) medium supplemented with 10 μM indole-3-acetic acid and 50 μM benzyladenine. Up to 40 shoots were obtained over a 4 month culture period from each leaf explant. Rooting occurred spontaneously in the shoots that were about 1 cm tall when subcultured on phytohormone-free MS medium. The plantlets could be transplanted successfully. Some of the transplantedP. tomentosa plantlets flowered in the greenhouse one year after transplanting. The protocol is suitable not only for rapid multiplication of the various species ofPaulownia, but also for analytical studies associated with adventitious shoot regeneration.

Transformation of pickling cucumber with chitinase-encoding genes using Agrobacterium tumefaciens.

Abstract: Transformation of cucumber cv. Endeavor was attempted using three Agrobacterium tumefaciens strains (a supervirulent leucinopine type, an octopine type and a nopaline type), each harbouring one of three binary vectors which contained an acidic chitinase gene from petunia, and basic chitinase genes from tobacco and bean, respectively, driven by the CaMV 35S promoter. Petiole explants were inoculated with a bacterial suspension (10(8) cells·ml(-1)), cocultivated for 48-96 h and placed on Murashige and Skoog (MS) medium with 5.0 μM each of 2,4-D and BA, 50 mg·l(-1) kanamycin and 500 mg·l(-1) carbenicillin. The frequency of embryogenic callus formation ranged from 0 to 12%, depending on strains/vectors used and length of cocultivation, with the highest being obtained using the leucinopine strain with petunia acidic chitinase gene. The kanamycin-resistant embryogenic calli were used to initiate suspension cultures (in liquid MS medium with 1.0/1.0 μM 2,4-D/BA, 50 mg·l(-1) kanamycin) for multiplication of embryogenic cell aggregates. Upon plating of cell aggregates onto solid MS medium with 1.0/1.0 μM NAA/BA and 50 mg·l(-1) kanamycin, calli continued to grow and later differentiated into plantlets. Transformation by the leucinopine strain and all three vectors was confirmed by PCR amplification of the NPT II gene in transgenic calli and plants, in addition to Southern analysis. Expression of the acidic chitinase gene (from petunia) and both basic chitinase genes (from tobacco and bean) in different transgenic cucumber lines was confirmed by Western analyses.

Somatic embryogenesis and plant regeneration of pepper in liquid media

Abstract: A protocol was developed for regeneration of pepper (Capsicum annuum var. Ace) through somatic embryogenesis in liquid media. For embryogenic callus formation, mature zygotic embryo explants were used on basal Murashige and Skoog medium with 9.05 μM 2,4-dichlorophenoxyacetic acid and 3% sucrose. Embryogenic callus was transferred to liquid basal Murashige and Skoog medium with 4.52 μM 2,4-dichlorophenoxyacetic acid and 3% sucrose in order to increase the mass of the embryogenic culture. After pretreatment with potassium citrate, cells were placed into embryo initiation medium with 6 g l-1 l-proline and a decreased (10 mM) ammonium concentration. Embryos were matured in 1.89 μM abscisic acid containing half-strength Murashige and Skoog medium and converted into plants bothin vivo andin vitro at up to a 97% efficiency.

Adventitious bud formation and shoot development from in vitro leaves of Vitis x Muscadinia hybrids

Abstract: High levels of regeneration were obtained from young leaves excised from axillary shoots in proliferating nodal cultures of several Vitis x Muscadinia hybrids. Best results were obtained when the explants were cultured on solidified half-strength Murashige and Skoog medium supplemented with 8.9 μM 6-benzyladenine and 0.05 μM 1-naphthaleneacetic acid. Though variation was observed among the hybrids, the procedure used does not seem to be genotype-specific as all the hybrids and cultivars tested could regenerate. Scanning electron microscopy observations and histological studies carried out during the development of adventitious shoot organogenesis revealed that the promeristem initiation occurred in the outer cell layers near the wounded area of the petiolar stub.

Agrobacterium tumefaciens-mediated transformation of Vigna mungo (L.) Hepper

Abstract: Transformed Vigna mungo (blackgram) calli were obtained by cocultivating segments of primary leaves with Agrobacterium tumefaciens vir helper strains harbouring the binary vector pGA472 having kanamycin resistance gene as plant transformation marker. Transformed calli were selected on Murashige and Skoog medium supplemented with 50 mg/l kanamycin and 500 mg/l carbenicillin. Transformed calli were found to be resistant to kanamycin up to 900 mg/l concentration. Expression of kanamycin resistance gene in transformed calli was demonstrated by neomycin phosphotransferase assay. Stable integration of transferred DNA into V. mungo genome was confirmed by Southern blot analysis.