Susan Eapen

Plant regeneration from leaf base callus of turmeric and random amplified polymorphic DNA analysis of regenerated plants

Callus cultures were initiated from leaf bases of turmeric on Murashige and Skoogs basal medium (MS) supplemented with dicamba, picloram (2 mg l−1) or 1-naphthaleneacetic acid (NAA) (5 mg l−1) in combination with benzyladenine (BA) (0.5 mg l−1). On transfer of callus cultures to medium supplemented with benzyladenine (BA) (5 mg l−1) in combination with triiodebenzoic acid (TIBA) or 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1 mg l−1), green shoot primordia were seen to differentiate from the surface of the callus. On transfer of regenerating cultures to half MS media supplemented with Kn, shoot primordia developed into well developed shoots. When shoots were transferred to medium devoid of phytohormones, complete rooted plants were obtained. Ninety percent of the plants survived to maturity on transfer to soil. Random Amplified Polymorphic DNA (RAPD) analysis of eight regenerated plants using 14 primers when separated on non-denaturing polyacrylamide gels showed 38 novel bands. About 51 bands present in the control were absent in the regenerants. The result indicates that variation at DNA level has occurred during in vitro culture.

Potential for rhizofiltration of uranium using hairy root cultures of Brassica juncea and Chenopodium amaranticolor.

Hairy root cultures of Brassica juncea and Chenopodium amaranticolor were developed by genetic transformation using Agrobacterium rhizogenes. The stable, transformed root systems demonstrated a high growth rate of 1.5-3.0 g/g dry weight/day in Murashige and Skoog medium. In the present study, hairy root system was used for removal of uranium from the solution of concentration up to 5,000 microM. The results indicated that the hairy roots could remove uranium from the aqueous solution within a short period of incubation. B. juncea could take up 20-23% of uranium from the solution containing up to 5,000 microM, when calculated on g/g dry weight basis. C. amaranticolor showed a slow and steady trend in taking up uranium, with 13% uptake from the solution of 5,000 microM concentration. Root growth was not affected up to 500 microM of uranium nitrate over a period of 10 days.

Influence of phytohormones, carbohydrates, aminoacids, growth supplements and antibiotics on somatic embryogenesis and plant differentiation in finger millet

Cultured caryopses of finger millet (Eleusine coracana GAERTN) produced callus from shoot apices or mesocotyls depending upon the concentration of picloram and combination of cytokinins in MS basal medium. On subsequent subcultures, numerous somatic embryos differentiated from the callus on MS medium supplemented with picloram and kinetin. The embryos germinated into complete plants on medium devoid of phytohormones. When different carbohydrates were tested, basal medium containing glucose and sucrose produced the highest frequency of germinating somatic embryos. Supplementation of MS basal medium with a variety of aminoacids, osmotic agents and growth supplements had an adverse effect on the germination of embryos. Incorporation of different antibiotics such as carbenicillin, cefotaxime and streptomycin sulfate enhanced plant differentiation from somatic embryos. Cytological analysis of regenerated plants showed normal diploid chromosome number in their root tips.

Plant regeneration from peduncle segments of oil seed Brassica species: Influence of silver nitrate and silver thiosulfate

Cultured peduncle segments of B. juncea, B. campestris, B. napus, B. nigra and B. carinata produced shoot buds on Murashige and Skoog medium supplemented with benzyladenine and 1-naphthalene acetic acid. Supplementation of the media with 30 µm silver nitrate or silver thiosulfate enhanced the frequency of shoot regeneration. The regenerated shoots could be rooted at a frequency of 95% and transferred to soil where 75% survived and set seed.

Enhanced plant regeneration in pearl millet (Pennisetum americanum) by ethylene inhibitors and cefotaxime

Cefotaxime, a cephalosporin antibiotic, and different ethylene inhibitors, such as silver nitrate, cobalt chloride, nickel chloride and O-acetyl salicylic acid, significantly delayed the loss of regeneration potential in embryogenic cultures of Pennisetum americanum. In the presence of these chemicals, ethylene content in the atmosphere of the culture vessel was less than that of the control. Cefotaxime, silver nitrate and O-acetyl salicyclic acid did not have any effect on callus growth based on fresh weight, while growth based on dry weight was enhanced by O-acetyl salicyclic acid.

Glutathione transferase from Trichoderma virens enhances cadmium tolerance without enhancing its accumulation in transgenic Nicotiana tabacum.

Background Cadmium (Cd) is a major heavy metal pollutant which is highly toxic to plants and animals. Vast agricultural areas worldwide are contaminated with Cd. Plants take up Cd and through the food chain it reaches humans and causes toxicity. It is ideal to develop plants tolerant to Cd, without enhanced accumulation in the edible parts for human consumption. Glutathione transferases (GST) are a family of multifunctional enzymes known to have important roles in combating oxidative stresses induced by various heavy metals including Cd. Some GSTs are also known to function as glutathione peroxidases. Overexpression/heterologous expression of GSTs is expected to result in plants tolerant to heavy metals such as Cd. Results Here, we report cloning of a glutathione transferase gene from Trichoderma virens, a biocontrol fungus and introducing it into Nicotiana tabacum plants by Agrobacterium-mediated gene transfer. Transgenic nature of the plants was confirmed by Southern blot hybridization and expression by reverse transcription PCR. Transgene (TvGST) showed single gene Mendelian inheritance. When transgenic plants expressing TvGST gene were exposed to different concentrations of Cd, they were found to be more tolerant compared to wild type plants, with transgenic plants showing lower levels of lipid peroxidation. Levels of different antioxidant enzymes such as glutathione transferase, superoxide dismutase, ascorbate peroxidase, guiacol peroxidase and catalase showed enhanced levels in transgenic plants expressing TvGST compared to control plants, when exposed to Cd. Cadmium accumulation in the plant biomass in transgenic plants were similar or lower than wild-type plants. Conclusion The results of the present study suggest that transgenic tobacco plants expressing a Trichoderma virens GST are more tolerant to Cd, without enhancing its accumulation in the plant biomass. It should be possible to extend the present results to crop plants for developing Cd tolerance and in limiting Cd availability in the food chain.

Somatic embryogenesis in peanut: Influence of growth regulators and sugars

Somatic embryogenesis and plant regeneration were induced from immature embryonal axes and immature cotyledons of peanut (Arachis hypogaea L. fastigata type cv JLM-1). Influence of different auxins, cytokinins and sugars on somatic embryogenesis from immature cotyledon explants was also investigated. Among the different auxins tested, 2,4-dichlorophenoxyacetic acid (2,4-d) was most effective, producing the highest frequency of responding cultures and highest average number of somatic embryos per responding culture, while dicamba, picloram, indolepropionic acid, α-naphthaleneacetic acid, 2,4,4-trichlorophenoxypropionic acid and α-naphthoxyacetic acid were also effective for embryogenesis. Indolebutyric acid, indoleacetic acid, p-chlorophenoxyacetic acid and trichlorophenoxyacetic acid were not beneficial. Among the four cytokinins tested, zeatin slightly enhanced the frequency of somatic embryogenesis, while kinetin, 6-γ-γ-dimethylallylaminopurine and benzyladenine were relatively inhibitory. Among the different carbon sources tested, sucrose was the best for embryo induction and at 6% sucrose the highest frequency of responding cultures and average number of somatic embryos per explant were obtained. For inducing embryogenesis from embryonal axes, 2,4-d was more effective than picloram. Highest plant conversion frequency from somatic embryos was obtained in presence of dicamba or NAA and using cotyledon explants.

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

Transgenic peanut plants were produced using Agrobacterium mediated gene transfer. Primary leaf explants of peanut were co-cultivated with Agrobacterium tumefaciens LBA 4404 harbouring the binary plasmid pBI 121 (conferring β-glucuronidase activity and resistance to kanamycin) and cultured on regeneration medium supplemented with kanamycin to select putatively transformed shoots. They were rooted and plants were transferred to soil. Stable integration and expression of the transgenes were confirmed by NPT II assay, Southern blot hybridization and GUS assay.

Organogenesis and embryogenesis from diverse explants in pigeonpea (Cajanus cajan L.)

Seed and seedling expiants of pigeonpea were evaluated for organogenesis and somatic embryogenesis. De novo plant regeneration through organogenesis was obtained from mature cotyledons, primary leaves and roots of seedlings. Production of multiple shoots from the cotyledonary node was observed in cultures of whole seeds on 6-benzylaminopurine enriched medium. Somatic embryos were induced from immature cotyledons and embryonal axes, however, well-developed plants could not be derived from these embryos. The regenerants obtained through organogenesis were transferred to the field and grown to maturity.

Plant regeneration from leaf discs of peanut and pigeonpea: Influence of benzyladenine, indoleacetic acid and indoleacetic acid-amino acid conjugates

The influence of indoleacetic acid (IAA) and some IAA-amino acid conjugates in combination with benzyladenine (BA) on in vitro shoot regeneration from leaf discs of peanut and pigeonpea was investigated. The frequency of shoot regeneration and average number of shoot buds produced was dependent on the type of auxin present in the medium. Highest frequency of plant regeneration in peanut was induced by BA in combination with IAA or IAA-l-alanine, while in pigeonpea BA in combination with IAA or IAA-l-aspartic acid produced best results. Four hundred plants of peanut and 25 plants of pigeonpea were transplanted to soil.