S. K. Sen

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

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.

Somatic embryogenesis from immature cotyledons of an elite Darjeeling tea clone

Abstract Cotyledons from immature embryos of Camellia sinensis O. Kuntze var. T-78, an elite Darjeeling tea clone, were exposed to various levels of 2,4-D, NAA, IBA, BAP and Kin (1–10 mg/1). While no embryos were formed in MS basal medium without growth regulators, the auxins tried were also inefficient in stimulating primary embryogenesis. Somatic embryos were induced directly on the cotyledonary explants in MS medium with 10 mg/1 BAP alone. Somatic embryo formation could be enhanced by addition of 0.5 mg/1 IBA and 80 mg/1 adenine to 10 mg/1 BAP. Embryo conversion was 2–3% in MS medium with or without growth regulators. Almost 20% of somatic embryos converted into whole plants following transfer to B5 medium containing 3 mg/1 BAP and 2 mg/1 IBA. Embryogenic potential was maintained for over 30 months by secondary embryogenesis through successive generation of embryos. Somatic embryo derived plants were successfully transferred to potted soil at 70% frequency under green house conditions. Morphologically and cytologically (2n = 30) plants are true to type.

Micropropagation of an elite Darjeeling tea clone

Shoot cultures of Camellia sinensis (L.) O. Kuntz var. T-78, an elite Darjeeling tea clone, were established from cotyledonary nodes and shoot tips of germinated seedlings as well as from nodal explants of field grown plants. Shoot multiplication rate ranged from 4x in nodal explants to 35x in cotyledonary nodes after 18 weeks of culture. Rooting was achieved in 80–90% micro-shoots by either placing them on an inductive medium for 10 d and then transferring shoots to hormone-free medium, or by treating micro-shoots with a chronic dose of IBA (500 mg/l) for 30–40 min. Rooted plants were established in soil under glasshouse condition at 60% frequency after hardening phase of 4–6 weeks. The regenerated plants show a constant chromosome number of 2n=30 and are morphologically true to type. This procedure can be applied for conservation and utilisation of an elite clone of Darjeeling tea.

Plant regeneration from stem-derived callus of the seed legume Lathyrus sativus L.

Callus cultures from stem explants of six Lathyrus sativus L. cultivars were tested for their morphogenic capacity. Shoot-buds were formed in calli of only one cultivar. Maximum response was observed in the medium containing 10-8 M picloram and 10-6 M benzylaminopurin. Supplementation with adenine sulphate was required for shoot-bud formation. The greatest frequency of shoot-bud formation was detected at the second passage and complete lack of regeneration capacity was observed after the 8th passage. Cell regenerates were diploid.

Homologous promoter derived constitutive and chloroplast targeted expression of synthetic cry1Ac in transgenic chickpea confers resistance against Helicoverpa armigera

The insecticidal crystal protein derived from gram positive soil bacterium Bacillus thuringiensis plays an important role in controlling lepidopteran infestation. The present study seeks to protect chickpea plants from Helicoverpa armigera infestation by over expressing cry1Ac. Homologous Ubiquitin and RuBisCO small subunit (rbcS) promoters were used to transcribe cry1Ac in transgenic chickpea both constitutively and in a tissue specific manner through Agrobacterium mediated transformation of chickpea var. ICCV89314. Expressed Cry1Ac was specifically targeted to the chloroplast rich tissues using transit peptide sequence. After monitoring transgene integration by Southern hybridization, transgenic chickpea lines were further analyzed by western blot, ELISA and insect bioassay. Expression of cry1Ac in chickpea under the control of above two promoters conferred a high level of protection against pod borer infestation, where chloroplast targeting system was found to be more efficient in controlling this particular devastating lepidopteran pest.

Interspecific Somatic Protoplast Fusion Products in Cultivated Jute Species

Accounts of success on isolation and culture of plant protoplasts and various genetic manipulation studies including fusion of plant protoplasts from distantly related species have been reported in the past (1, 2, 3). However, records of success amongst major crop plants are not many. Jute is an important fiber crop plant extensively grown in the eastern part of India and Bangladesh. The two cultivated species (Corchorus capsularis and C. olitorius) complement the needs of each other. Production of sexual hybrids is prevented by barriers of sexual incompatibility between the two species. We reported recently (4) that interspecific somatic hybrids may be possible to evolve. On further improving our technical approach, we are convinced that somatic hybrid cell lines have been produced which, however, failed to differentiate into complete plants till date.

Differential expressions of photosynthetic genes provide clues to the resistance mechanism during Fusarium oxysporum f.sp. ciceri race 1 (Foc1) infection in chickpea (Cicer arietinum L.)

Fusarium oxysporum f.sp. ciceri race 1 (Foc1), a root-invading pathogen causes vascular wilt in chickpea (Cicer arietinum L.). Foc1 is known to induce reactive oxygen species (ROS) mediated localized defense responses at the site of colonization in roots. However, the effect of this localized infection on distant shoot tissues is still unknown. In the present study, the effect of Foc1 on shoot tissues of both susceptible and resistant chickpea plants was studied. Total pigment content and fluorescence of chlorophyll was measured. Occurrence of oxidative damage in shoots was confirmed by both biochemical and lipid peroxidation assays. Expression pattern of some redox responsive transcripts were also analyzed. Additionally, transcriptional accumulations of some key genes related to light reaction, carbon reduction and photosystem II (PSII) of photosynthesis were analyzed at different time points post infection. Expressional status of stress induced sugar metabolism related genes (sucrose synthase, β amylase and invertase) were also investigated. Finally, gene networks were constructed showing interconnection of the photosynthetic genes, sugar metabolism-related genes and redox responsive transcripts with other metabolic and stress related pathways. The results demonstrate that the infection in root tissues of chickpea by Foc1 dramatically increases the ROS levels in shoot tissues of susceptible plants. The oxidative outburst in shoot tissues of susceptible plants also hampers the photosynthetic stability by down-regulating the key photosynthetic genes. On the contrary, resistant chickpea lines are grossly devoid of such instances with few behavioral irregularities at later time points.

Recovery of Virus Free Plantlets of Cultivated Jute Species

Meristem culture techniques have helped in the past to generate symptomless or virus free plants of species of over 35 genera (1). As there exists a difference between the rates of plant growth and virus multiplication, it has been possible to culture the young and fast growing tissue, i.e., the meristem of a plant under virus free conditions. Meristem culture techniques date back about three decades (2). These techniques were first used in the fifties in order to free plants of virus (3). Since then it has been used to produce virus-free plants of several crops, including cassava (4), potatoes (5), white clover (6), red clover (7, 8), and alfalfa (8). These techniques have also been adopted for rapid asexual propagation of orchids (9), pea (10) and other crops.

Embryoids from Mesophyll Protoplasts of Vinga Mungo L. Hepper, a Seed Legume Crop Plant

Isolation and culture of seed legume protoplasts of the most important group of crop plants which supply the bulk of food protein to the vegetarian population of the world, have proved difficult. There is only one report where limited success in anther derived cultured tissue protoplasts of Dolichos biflorus has been achieved (1). The present communication deals with our success of inducing embryogenesis in calli from leaf mesophyll protoplasts of Vinga mungo, a widely cultivated seed legume crop of the Indian subcontinent.

Epigenetic and transcriptional control of chickpea WRKY40 promoter activity under Fusarium stress and its heterologous expression in Arabidopsis leads to enhanced resistance against bacterial pathogen

Promoters of many defense related genes are enriched with W-box elements serving as binding sites for plant specific WRKY transcription factors. In this study, expression of WRKY40 transcription factor was analyzed in two contrasting susceptible (JG62) and resistant (WR315) genotypes of chickpea infected with Foc1. The resistant plants showed up-regulation of WRKY40 under Fusarium stress, whereas in susceptible plants WRKY40 expression was absent. Additionally, global changes in the histone modification patterns were studied in above two chickpea genotypes by immunoblotting and real-time PCR analyses under control and Fusarium infected conditions. Notably, region specific Histone 3 lysine 9 acetylation, a positive marker of transcription gets enriched at WRKY40 promoter during resistant interaction with Foc1. H3K9 Ac is less enriched at WRKY40 promoter in Foc1 infected susceptible plants. WRKY40 promoter activity was induced by jasmonic acid and pathogen treatment, while salicylic acid failed to stimulate such activity. Moreover, WRKY40 was found to bind to its own promoter and auto-regulates its activity. The present study also showed that heterologous over-expression of chickpea WRKY40 triggers defense response in Arabidopsis against Pseudomonas syringae. Overall, we present epigenetic and transcriptional control of WRKY40 in chickpea under Fusarium stress and its immunomodulatory role is tested in Arabidopsis.