MSB Medium Research Articles

Optimization of Agrobacterium-mediated genetic transformation of shoot tip explants of green gram (Vigna radiata (L.) Wilczek)

Shoot tip explants prepared from seedlings of ML-267 genotype of green gram were inoculated on MSB5 medium supplemented with BAP (0–20 μM) individually or in combination with minimal concentration of auxins (NAA/IAA/IBA) for adventitious shoots formation. BAP alone without auxins was observed to be efficient in multiple shoot induction and optimum shoot proliferation was achieved on MSB5 medium containing 10 μM BAP with 100 % shoot induction frequency. 3-day-old explants gave best shoot multiplication response and the mean shoot number decreased significantly in 4-day and 5-day-old explants. The induced shoots rooted profusely on ½ MSB5 + 2.46 µM IBA and about 90 % of the plantlets survived after acclimatization and set seed normally. Shoot tip explants infected with A.tumefaciens (LBA4404) harboring pCAMBIA 2301 + AnnBj1 recombinant vector. Various factors which influence the competence of transformation were optimized based on the frequency of transient GUS expression in shoot tip explants. Optimum levels of transient GUS expression were recorded at pre-culture of explants for 2 days, infection for 10 min with Agro-culture of 0.8 OD and co-cultivation for 3 days on co-cultivation medium containing 100 µM acetosyringone in dark at 23 °C. Putative transformed shoots were produced on selection medium (shoot inductionmedium with100 mg/l kanamycin and 250 mg/l cefotaxim). PCR analysis confirmed the presence of AnnBj1, nptII, and uidA genes in T0 plants. Stable GUS activity was detected in flowers of T0 plants and leaves of T1 plants. PCR analysis of T1 progeny revealed AnnBj1 gene segregated following a Mendelian segregation pattern.

High frequency induction of multiple shoots and plant regeneration from zygotic embryo axis explants of cotton cultivar L-604 (Gossypium hirsutum L.).

An efficient, robust regeneration protocol has been developed using zygotic embryo axis explants of an elite cultivar L-604 (Gossypium hirsutum L.). Explants regenerated on MSB5 medium supplemented with 0.5 mg-l Thidiazuron, 0.1 mg-l Naphthalene acetic acid, 5.0 mg-l Silver nitrate and 1.0 g-l activated charcoal, was identified as the best combination for producing the maximum number (12.67) of multiple shoots per explant. In addition, the highest shoot elongation (5.30 cm) and maximum regeneration frequency (89.0%) was also recorded in the same hormonal combinations. In vitro shoots were successfully rooted on ½ MSB5 medium containing 1.5 mg-l IBA. Well rooted plants were subsequently transferred to polyhouse and greenhouse conditions for primary and secondary hardenings respectively and about 95% of the plants were established successfully. The conditions optimized in this study for inducing multiple shoots could be best suitable for development of transgenic L-604 variety for agronomically important traits.

Silver nitrate promotes high-frequency multiple shoot regeneration in cotton (Gossypium hirsutum L.) by inhibiting ethylene production and phenolic secretion

The regeneration frequency of cotton (Gossypium spp.) is greatly influenced by its genetic makeup and recalcitrant nature. In particular, phenolic secretion is a major problem in cotton tissue culture. The present study was carried out to develop a rapid and efficient in vitro regeneration method, without phenolic secretion, from cotyledonary node explants of cotton. Multiple shoot induction was achieved after 3 wk of culture on MSB5 medium supplemented with N6-benzyl adenine (BA), kinetin (KN), thidiazuron (TDZ) (each tested at 0.5–3.0 mg L−1), Pluronic F-68 (0.1–0.6% [w/v]), or silver nitrate (AgNO3; 0.5–3.0 mg L−1). AgNO3 at 2 mg L−1 produced the greatest number of shoots (a mean of 22.2 ± 0.21 shoots per cotyledonary node explant), with no phenolic secretion. The induced shoots were healthy with intense green color and multiplied rapidly on the same medium. BA at 2.5 mg L−1 produced 10.3 ± 0.37 shoots/explant with moderate phenolic secretion. Pluronic F-68 at 0.3% produced 7.6 ± 0.28 shoots/explant along with high phenolic secretion. Individual shoots were elongated on MSB5 medium + gibberellic acid (GA3; 0.2 mg L−1; shoot length 7.8 ± 0.31 cm) and rooted on MSB5 medium + α-naphthalene acetic acid (NAA; 0.3 mg L−1; 8.3 ± 0.32 roots/shoot). Rooted plantlets were acclimatized in paper cups inside a plant growth chamber at 25°C and 70% relative humidity with a 70% survival rate. From the present study, AgNO3 was found to be suitable for obtaining a high frequency of multiple shoot production from cotyledonary node explants of cotton without phenolic secretion. The current protocol can be adopted for developing transgenic plants of commercial cotton cultivars through Agrobacterium-mediated genetic transformation.

Ethyl methane sulphonate (EMS) mediated changes in callus growth of clusterbean (Cyamopsis tetragonoloba L.) raised under saline conditions

Soil salinity is the major biotic stress, which restricts the distribution and productivity of the crops. Agricultural production in the future will increasingly rely on our ability to grow plants on salt affected and marginal lands using saline water. Ethyl methane sulphonate is the most commonly used chemical mutagen to increase genetic variability in crop plants and could be useful in increased plant tolerance to salinity. Clusterbean is one of the most important summer annual legume. In the present study, aseptically grown7-day old seedling explants of clusterbean viz. cotyledon, cotyledonary node, hypocotyl and the embryo axis (cut on radical side) were cultured on MS medium + B5 vitamins (MSB5 medium) supplemented with various growth regulators. Among various explant tried, cotyledonary node gave good response in terms of callus growth which was further selected for future experimentation. The best medium for callus growth was MSB5 with 2 mgl'1 2, 4-D and 1 mgl'1 BAP. All the calli produced were compact and their color changed with increase of salt concentration from greenish to brownish green, dark brownish and finally blackish at 200 mM NaCl. Further, calli pieces dipped in sterile liquid MS medium adjuncted with aqueous filter sterilized solution of 0.5% EMS for a range of time duration (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 h) were raised on MSB medium without NaCl. These calli were subcultured on the above medium with or without 200 mM NaCl. EMS treatment of 2.5, 3.0 and 3.5h duration improved callus growth on salt amended medium; growth being maximum after 3.0h EMS treatment. 11 seems that exposing mutagen treated calli to salinity/salt stress, forces these to face stress doubly-ionic toxicity and toxic effects of mutagen. The survival and regeneration of the putative variant calli is suppressed under such circumstances.

Evaluation of different carbon sources for high frequency callus culture with reduced phenolic secretion in cotton (Gossypium hirsutum L.) cv. SVPR-2

An efficient protocol was developed to control excessive phenolic compound secretion during callus culture of cotton. As cotton is naturally rich in phenolic compounds factors influencing the phenolic compound secretion, callus induction and proliferation were optimized for getting high frequency callus culture. Different carbon sources such as fructose, glucose, sucrose and maltose were tested at various concentrations to control phenolic secretion in callus culture. Among them, 3% maltose was found to be the best carbon source for effectively controlling phenolic secretion in callus induction medium. High frequency of callus induction was obtained on MSB5 medium supplemented with 3% Maltose, 2,4-D (0.90μM) and Kinetin (4.60μM) from both cotyledon and hypocotyl explants. The best result of callus induction was obtained with hypocotyl explant (94.90%) followed by cotyledon explant (85.20%). MSB5 medium supplemented with 2,4-D (0.45μM) along with 2iP (2.95μM) gave tremendous proliferation of callus with high percentage of response. Varying degrees of colors and textures of callus were observed under different hormone treatments. The present study offers a solution for controlling phenolic secretion in cotton callus culture by adjusting carbon sources without adding any additives and evaluates the manipulation of plant growth regulators for efficient callus culture of SVPR-2 cotton cultivar.

Efficient plant regeneration of Malaysian aromatic rice (Oryza sativa L.) via improved somatic embryogenesis pathway

Aromatic rice MRQ 74, MRQ 80 and MRQ 50 are the most popular rice in Malaysia. Establishing an efficient regeneration protocols via somatic embryogenesis is required for varietal improvements. Somatic embryogenesis system was established in three aromatic rice varieties by using mature seeds. Optimised MS-B5 medium containing 1 mg/L 2, 4-D (2, 4-dichlorophenoxyacetic acid) and 10 mg/L NAA (α-naphthaleneacetic acid) produced highest production of embryogenic callus (89%) without any browning effect. The highest whitish somatic embryos frequency (75 %) was initiated by incubating embryogenic calli on same media containing 10 mg/L ABA and 9 g/L Gelrite agar for 8 weeks. However, the numbers of regenerated plantlets on medium containing NAA and 2,4-D which was previously pre-treated with 10 mg/L ABA, 9 g/L Gelrite agar and incubation at 8 weeks was the best method for shoots induction (14 plantlets per 3 g of somatic embryos) of three rice varieties.

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO3 on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO3 for multiple shoot induction. AgNO3 influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO3 in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA3 (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.

High frequency somatic embryogenesis and plant regeneration from hypocotyl and leaf explants of gherkin (Cucumis anguria L.)

Gherkin (Cucumis anguria L.) is an important commercial vegetable crop. An efficient protocol for plantlet regeneration from hypocotyl and leaf explants through direct somatic embryogenesis was developed. High frequency of somatic embryos (21.6 and 34.0) were obtained from hypocotyl and leaf explants when cultured on Murashige and Skoog (MS) salts plus B5 vitamins (MSB5) supplemented with 5% sucrose, 1.5mgL−1 2,4-D, 0.5mgL−1 BAP and 150μM l-glutamine. Initial culturing of embryos in the dark conditions for two weeks, followed by four weeks under light resulted in a higher frequency of embryo formation when compared to continuous light conditions. Histological observation showed that the somatic embryos originated from single cells of the epidermal layer. Histological evidence on formation of shoot and root poles during conversion of the embryos confirmed that these structures were true somatic embryos. Maturation of somatic embryos occurred on plant growth regulator-free MSB5 semi-solid medium containing 3% sucrose and 0.5% (w/v) activated charcoal (AC). Conversion of embryos into plantlets was achieved on MSB5 medium supplemented with 3% sucrose, 0.3% AC and 0.5mgL−1 gibberellic acid (GA3). Sucrose was found to be the best carbon source for SE induction, maturation and germination. Ninety percent of embryos were converted into normal plantlets. The plantlets were successfully acclimatized in the greenhouse with 95% survival rate and transferred to ex vitro conditions which developed with normal phenotypes. This regeneration protocol assured successful embryo induction and plantlet conversion. This is the first report for the induction of direct somatic embryogenesis from hypocotyl and leaf explants of C. anguria. The result of this study is beneficial for genetic transformation and mass clonal propagation.

Optimization of factors influencingin vitroflowering of gherkin (Cucumis anguriaL.)

This study investigated the factors influencing in vitro flowering of gherkin (Cucumis anguria L.). Multiple shoots were efficiently regenerated from cotyledonary node and axillary bud explants of C. anguria within 15 days on MSB5 medium containing 3% sucrose and supplemented with 1.5 mg l-1 6-benzyladinine (BA). The elongated shoots were excised and transferred to MSB5 medium containing 4% sucrose supplemented with 0.5 mg l(-1) gibberellic acid (GA(3)) and 1.0 mg l(-1) indole-3-butyric acid (IBA) induced maximum number of flowers (9.5 flowers/plant) and root induction (16.5 roots/plant). Factors that influence the in vitro flowering were optimizing pH, photoperiod and temperature. In vitro flowering was significantly early and higher number of flowers produced at pH (5.8), photoperiod (12/12 h) and room temperature (28 °C). In vitro developed flowers were less viable (80 ± 1.0%) compared to control plants (90 ± 2.0%). Our in vitro flower induction procedures provide an extremely effective method for further research on flowering regulation mechanisms in C. anguria. These plantlets were successfully transferred to the soil where they grew well for 3 to 5 weeks with 90% survivability. Plants grew normally and produced flowers with viable pollen and fertile seeds.

Efficient plant regeneration from petiole explants of West Indian gherkin (Cucumis anguria L.) via indirect organogenesis

An efficient protocol for in vitro organogenesis was achieved from callus-derived immature and mature petiole explants of West Indian gherkin (Cucumis anguria L.). Calluses were induced from immature petiole explants excised on 7-day-old in vitro seedlings and mature petiole explants of 40-day-old in vivo plants. The maximum frequency of immature petiole explants (98.0 %) and mature petiole (91.5 %) produced green, compact organogenic callus in Murashige and Skoog (MS) medium with Gamborg (B5) vitamins containing 30 g l−1 sucrose, 8.0 g l−1 agar and 4.0 μM naphthalene acetic acid (NAA) with 2.0 μM benzyl amino purine (BAP) after two successive subculture at 11 days interval. Adventitious shoots were produced from the organogenic callus when it was transferred to MSB5 medium supplemented with 3.0 μM TDZ, 1.0 μM NAA and 0.05 mM L-glutamine with shoot induction frequency of immature petiole 45 shoots and mature petiole 40 shoots per explant. The shoots were excised from callus and elongated in MSB5 medium fortified with 3.0 μM gibberellic acid (GA3). Then elongated shoots were rooted in half strength MSB5 medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Histological analyses of the regeneration process confirmed the indirect organogenesis pattern. Plantlets with well-developed shoot and root systems were successfully acclimatized (95 %) in winter season and exhibited normal morphology and growth characteristics. The survival percentage differed with seasonal variations.

Effect of 6-BA on the Plant Regeneration via Organogenesis from Cotyledonary Node of Cowpea (Vigna unguiculata L. Walp)

The present study compares effects of different concentrations of 6-BA on regeneration from cotyledonary node explants of cowpea (Vigna unguiculata L. Walp). The seeds were inoculated on MSB5 medium [Murashige and Skoog (1962) salts and Gamborg B5 vitamins (1968)] containing different concentrations (0, 1, 2, 3, 4, 5 mg/L) of 6-BA for 4 days. The cotyledonary node explants with one cotyledon excised from 4-day-old seedlings, placed in vitro on MSB5 medium containing 6-BA at different dose (0, 0.5, 1.0, 1.5 mg/L) for shoot induction and elongation. Best response in terms of shoot number and shoot length were obtained with explants derived from seedling preconditioning with 3mg/L 6-BA followed by the induction and elongation stage pretreated with 0.5mg/L 6-BA. The elongated shoots were rooted on MSB5 medium without hormone.

Efficient in vitro plant regeneration and generation of transgenic plants in barley (Hordeum vulgare L.) using particle bombardment

Plant regeneration from immature embryos of 15 Indian barley genotypes (Hordeum vulgare L.) was examined. Highest percent callus induction and number of regenerated plantlets were obtained in cultivars BL 2 (94.4 %; 12.1), RD 2668 (92.6 %; 9.1) and RD 2552 (90.8 %; 7.8). The highly responding cultivar BL 2 was selected for further development of transformation protocol. The plasmid DNA isolated from pCAMBIA1381 harbouring hptII gene as selectable marker and gusA gene as reporter was used. Particle bombardment was used for transformation of immature embryos and regeneration of transgenic plants in Indian barley genotype for the first time. Transformation experiments were carried out using different parameters and optimum conditions for DNA delivery was standardized. The transient expression of gusA gene was investigated as a preliminary test of optimum DNA delivery and for selecting the most appropriate bombardment parameters. The optimum conditions were: gold microparticles (diameter 1.0 μm) shot with 1,100 psi rupture disc pressure. The 3 cm distance between rupture disk and macrocarrier and 9 cm target tissue distance yielded high transient GUS expression. The immature embryos were bombarded twice to increase area for efficient gene delivery. Osmotic medium optimization with 0.4 M sorbitol and preculture of immature embryos for 5 days prior to bombardment resulted into efficient gene transfer in barley. Selection of transformed tissue was performed after 7 days resting step on selection medium containing 50 mg l−1 hygromycin. After two more selection steps, green shoots were rooted on MSB5 medium with 50 mg l−1 hygromycin. PCR analysis using primers specific for hptII and gusA genes and Southern blot analysis with hptII probes confirmed the stable integration of transgene in barley genome. Molecular analysis of T1 generation plantlets revealed the amplification of selectable marker hptII gene in the progeny.

Seedling preconditioning in thidiazuron enhances axillary shoot proliferation and recovery of transgenic cowpea plants

Lack of competence of seedling explants for efficient shoot proliferation in recalcitrant grain legume cowpea restricts its genetic manipulation for crop improvement. This study aimed at establishing a protocol to increase the shoot proliferation efficiency during the regeneration of transgenic cowpea plants. Here, we describe how seedling preconditioning in thidiazuron (TDZ) could stimulate the transformation process (by 3.5-fold), shoot proliferation potential of cotyledonary node (by a factor of fourfold) and accelerate the transgenic shoot regeneration. We investigated the effect of TDZ and 6-benzyladenine (BA) at high dose (5–20 μM) in the induction phase of regeneration by preconditioning seedlings for different durations (2–6 days) with the aim of improving shoot proliferation competence from cultured explants. Cotyledonary node explants from preconditioned seedlings were cultured on MSB5 medium supplemented with 5 μM BA and 0.5 μM kinetin for 4 weeks. Best response in terms of maximum shoot proliferation (7.1 shoots per explants), and greatest shoot length (2.6 cm) were obtained with explants derived from seedlings preconditioned in 10 μM TDZ for 4 days. This enhanced shoot proliferation ability was maintained through three subsequent 4-week long regeneration passages. On comparison of the transformation rate in absence and presence of seedling preconditioning (in 10 μM TDZ for 4 days), a significant enhancement from 0.6 to 2.1% was observed. The promotive effect of seedling preconditioning had a direct beneficial effect on transgenic plant recovery time leading to a reduction of more than 2 weeks. The protocol was found applicable to seven cowpea genotypes.

Effect of culture conditions on the plant regeneration via organogenesis from cotyledonary node of cowpea (Vigna unguiculata L. Walp)

A rapid and efficient regeneration system via organogenesis from cotyledonary node explants of cowpea (Vigna unguiculata L. Walp) has been established. The cotyledonary node explants excised from 4 days old seedlings, placed in vitro on medium containing salts of Murashige and Skoog and vitamins of Gamborg’s media (MSB 5 ). Adventitious shoots occurred at the basal end of the initiated axillary buds that preexisted at the node regions. BAP at 1.25 mg/l was the optimum for shoot induction. The combination of BAP with IBA had worthless effect on shoots proliferation. The number of adventitious buds was promoted when the seeds were preconditioned with appropriate concentrations of BAP (2 to 3 mg/l), whereas was depressed with higher concentrations of BAP (5 to 15 mg/l). The regeneration system was further optimized due to the presence of cotyledons attaching to the cotyledonary node explants. Explants with two entire cotyledons from 4 days old seedlings produced the greater number of shoots (7.83) after 3 weeks on MSB5 medium supplemented with 1.25 mg/l BAP. Regenerated shoots could well elongate on regulator-free basal medium and well root with 100% of success on the half strength medium supplemented with various concentrations of IBA (0, 0.1, 0.3, and 0.5 mg/l). The regenerated plantlets were cultured on the pots containing sterilized vermiculite and soil (1:1) with 27% of survival. Key words : Cowpea, plant regeneration, cotyledonary node, organogenesis.

Production of Fusarium verticillioides biocontrol agents, Bacillus amyloliquefaciens and Microbacterium oleovorans, using different growth media: evaluation of biomass and viability after freeze-drying

The aims of this study were to compare the viability and biomass production of B. amyloliquefaciens and M. oleovorans in different growth media, and the efficiency of a freeze-drying method as a possible formulation process. B. amyloliquefaciens and M. oleovorans were grown in 100 ml of four different media. Media water activity was modified at 0.99, 0.98, 0.97 and 0.96. Nutrient yeast dextrose broth (NYDB) and molasses soy powder (MSB) media were selected and survival levels of cells were determined before and after the freeze-drying process. B. amyloliquefaciens showed the highest survival after freeze-drying when grown in NYDB medium at 0.99 aw, whereas, at 0.98, 0.97 and 0.96 aw, the highest survival was obtained in MSB medium. M. oleovorans showed the highest survival in MSB medium at 0.99 aw. MSB medium was select for biomass production due to high growth and survival after freeze-drying.

Hypocotyl derived in vitro regeneration of pumpkin ash (Fraxinus profunda)

Pumpkin ash (Fraxinus profunda (Bush) Bush) is at risk for extirpation by an exotic insect, the emerald ash borer (EAB). Pumpkin ash is limited to wetland areas of the Eastern United States, and has been listed as an endangered species because of EAB activity. Pumpkin ash provides many benefits to the ecosystem, and its wood is used in the manufacturing industry. In vitro regeneration provides an integral tool for the mass propagation and genetic transformation of pumpkin ash to combat EAB. Therefore, a plant regeneration protocol was developed for pumpkin ash. Aseptically extracted hypocotyls formed adventitious shoots following 4 weeks on Murashige and Skoog (MS) medium supplemented with 0-22.2 lM 6-benzyladenine (BA) and 0-6.8 lM thidiazuron (TDZ) then transferred for an additional 4 weeks on MS medium with Gamborg B5 vitamins plus 0.2 g L -1 glycine (B5G) containing 6.7 lM BA, 1 lM indole-3-butryic acid (IBA), and 0.29 lM gibberellic acid (GA3). As adventitious shoots developed, these were transferred to a MSB5G medium with 13.3 lM BA, 1 lM IBA, and 0.29 l MG A 3 for shoot elongation. Elongated shoots were successfully micropropagated using MSB5 medium with 10 l MB A and 10 lM TDZ. Adventitious root formation was as high as 94% using woody plant medium supplemented with 4.9 lM IBA with shoots cultured for 10 days in the dark followed by culture under a 16-h photoperiod. Acclimatization to the greenhouse was successful and normal plant growth was observed. This protocol will provide a means for genetic transformation for EAB resistance and mass propagation for conservation.

Hormone-Induced Indirect Regeneration Protocol for Scutellaria baicalensis Georgi (Huang-qin)

There is no report available to date on somatic embryogenesis or even callus formation and plant regeneration in Scutellaria baiclensis (Huang qin). Ours is the first report on somatic embryo development in Scutellaria baicalensis. Plant growth regulators Thidiazuron [TDZ: N-phenyl N'- (1,2,3-thidiazol-5-ylurea)], 6-benzyladenine (BA), and 2,4-dichlorophenoxyacetic acid (2,4-D) were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf, internode, nodal portion, and root explants of Scutellaria baicalensis. The TDZ (4 mg/l) showed the highest percentage of calli-induction frequency in various explants, viz., leaf (42.2%), nodal stem portion (62.9%), and root (63.4%). The TDZ-derived calli were incubated on MS salt + B5 vitamin medium containing glutamine (1.5mM) (MSE) and fortified with various combinations of TDZ, BA, and 2,4-D for induction of somatic embryos. No somatic embryogenesis was recorded in any culture at this stage. On transferring the above friable calli onto hormone-free MSB medium and after 12 days of culture, several globular and heart-shaped embryos appeared over the surface of green compact callus. The mature somatic embryos were dissected out and cultured on hormone-free MS medium for germination. Plantlets were hardened in a growth chamber and, after 30 days, transferred to greenhouse and subsequently to pots and field. There was no morphological difference in plantlets regenerated by this procedure and normal seed-grown plant.

Evaluation of regeneration potential of mature embryo derived callus in Indian cultivars of barley (Hordeum vulgare L.)

A protocol for plant regeneration in Indian cultivars of barley (Hordeum vulgare L.) has been developed using mature embryo culture. The influence of various auxins 2,4-D (2,4-dichlorophenoxyacetic acid), Dicamba (3,6-dichloro-o-anisic acid) and Picloram (4-amino-3,5,6-trichloropicolinic acid) on the callus induction and subsequent plant regeneration revealed highest percent of callus induction form cultivar (cv) BL 2 on MSB5 medium (MS salts + B5 vitamins) supplemented with 6 mg l−1 Picloram, but maximum number of shoot buds (6–13) were regenerated on MSB5 medium containing 0.5 mg l−1 Picloram. Regenerated shoots were rooted on half-strength MSB5 medium. Plantlets were successfully transferred to soil and grown to maturity in greenhouse. The effect of copper sulphate revealed significant improvement in callus induction and plant regeneration when the concentration of CuSO4 was increased to 3 μM (30 times higher than normal MS medium) for cv BL 2. Regeneration potential differed for different cultivars of barley used, with highest for cv BL 2 and lowest for cv BH 924. We conclude that the Indian barley genotypes exhibit plant regeneration from mature embryo cultures. The protocol has potential application in barley improvement through genetic engineering.

Regeneration of pineapple (Ananas comosus L.) plant through somatic embryogenesis

An efficient protocol for a complete plant regeneration by somatic embryogenesis was developed with Smooth Cayenne pineapple (Ananas comosus L.). Previous works showed that this species is responsive to somatic embryogenesis. In the present work the influence of components of culture medium in the induction, development and conversion of somatic embryos was investigate in order to establish a somatic embryogenesis protocol. Nodular callus (83.67%) was initiated from leaf explants of young plants on CIM3 medium. The highest frequency (37.6%) of embryogenic callus induction was obtained from 4-week-old calluses on EIM3 medium supplemented with 3.0 mg/l picloram. The highly organized callus induction and the development of somatic embryos were achieved after the transfer of callus clumps onto EIM3 medium containing 1.0 mg/l BAP + 0.1 mg/l NAA. The frequency of somatic embryo formation was of 39.5 ± 2.45 embryos per callus. Up to 97% of the somatic embryos were converted into complete plants within 4 week on MSB medium with 1.0 mg/l BAP + 0.05 mg/l GA3 + 500 mg/l glutamine. The continuation of the elongation of the shoots occurred on this medium). Shoots obtained from all the above methods were rooted in MSB medium with activated charcoal. Complete plantlets were transferred onto specially made polyethylene bags containing soil mixture and transferred to the greenhouse. Survival rate of the plantlets under ex vitro conditions was 98% and maximum average number of plantlets (80 ± 0.6). The well-developed plantlets were transferred to an open field where the plants produced normal fruits.

Impact of tryptophan and glutamine on the tissue culture of upland rice

In order to evaluate the effect of tryptophan and glutamine on the tissue culture of upland rice cultivars, serial experiments were conducted using four cultivars: Kusan, Lamsan, Selasi and Siam. Mature seeds from these cultivars were subjected to 4 levels of tryptophan and glutamine on the MSB5 (MS macro elements, B5 micro elements and B5 vitamins) medium. Callus induction results showed a positive effect of tryptophan on all cultivars except Selasi. The optimal tryptophan concentration for callus induction in cultivars Kusan and Siam was 100 µmol, while in Lamsan the optimum was 200 µmol. With the exception of the Lamsan cultivar, incorporation of glutamine generally did not result in the enhancement of callus induction response that incorporation of tryptophan did. Plantlet regeneration frequency was significantly increased when an appropriate level of tryptophan was added to culture media, the optimum being 100 µmol or Kusan, Selasi and Siam, compared to an optimum of 200 µmol for Lamsan. Glutamine did not affect regeneration frequency in any of the cultivars under the conditions tested. In summary, the results showed that tryptophan is a useful additive for upland rice tissue culture.