Rohini Sreevathsa

In planta transformation of pigeon pea: a method to overcome recalcitrancy of the crop to regeneration in vitro

Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans’ AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22–25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.

Overexpression of AtNAC2 (ANAC092) in groundnut (Arachis hypogaea L.) improves abiotic stress tolerance

Groundnut (Arachis hypogaea L.) is an important oilseed crop grown in semi-arid tropics where it experiences moisture stress at different stages of growth resulting in reduced growth and productivity. In this study, we report that the stress tolerance of groundnut can be improved by overexpression of stress-specific transcription factor through transgenic approach. In silico electronic-northern analysis of AtNAC2 showed increased expression under different abiotic stresses. The transcript levels of a homolog of AtNAC2 gene were upregulated under different drought regimes in groundnut. Groundnut transgenics overexpressing AtNAC2 showed enhanced tolerance to drought and salinity with improved yield under water-limited conditions. The study demonstrates that AtNAC2 is a potential candidate gene to improve stress tolerance by transgenic approach.

Agrobacterium-Mediated In Planta Transformation of Field Bean (Lablab purpureus L.) and Recovery of Stable Transgenic Plants Expressing the cry1AcF Gene

The amenability and reproducibility of a tissue culture-independent Agrobacterium tumefaciens-mediated transformation strategy was analyzed in field bean and the stability of the transgenes was examined. The protocol involves in planta inoculation of embryo axes of germinating seeds and allowing them to grow into seedlings ex vitro. Transformants were raised using a chimeric Bt gene, cry1AcF, and putative transformants were analyzed by PCR for both cry1AcF as well as the nptII genes. Bioassays against Helicoverpa armigera, the major pod borer, showed that several T1 plants performed well with 17% of T1 plants harboring the transgene. Further, enzyme-linked immunosorbent assay (ELISA) and quick dip strip test confirmed the expression of the chimeric Bt toxin. The stability of the transgenes was checked in three generations for integration, expression, and efficacy against the two insects, H. armigera and Spodoptera litura. Southern blot analysis of 10 high expressing plants confirmed the integration of the transgene, whereas single copy integration of the T-DNA in 5 events was also evident. Transcript accumulation of the cry1AcF gene by Northern analysis supported the expression analysis by ELISA. Likewise, Western blot analysis for the NPTII protein further confirmed the transgenic nature of the plants. At the end of the analysis in the T3 generation, five plants from five T1 events were selected as promising. Therefore, the study proved not only the amenability of the field bean to the transformation protocol but also the stability of the introduced genes through three generations.

Overexpression of phytochelatin synthase (AtPCS) in rice for tolerance to cadmium stress

Phytoremediation is an important strategy adapted by plants to sequester and/or detoxify pollutants. Phytochelatins, a family of cysteine-rich thiol-reactive peptides, bind to various heavy metals and metalloids making them good candidates for phytoremediation. Phytochelatin synthase catalyses the final step in the biosynthesis of phytochelatins and can be used as a strategy to improve tolerance against heavy metals. In the present study, an AtPCS gene was overexpressed in rice following the in planta transformation approach. Stringent screening strategies were standardized to select putative transformants under a Cd stress of 125 μM at both seedling and plant levels. Molecular analysis by PCR in 18 tolerant plants confirmed the transgene integration and absence of Agrobacterium. Genomic Southern analysis further confirmed the integration of the T-DNA as a single copy. The stability of the T-DNA in the progeny of 5 selected T1 generation plants was confirmed by tolerance assay, molecular characterization and biochemical analysis for the reduced glutathione, phytochelatin content and lipid peroxidation. This strategy is discussed as a potential mechanism to enhance the tolerance of rice plants to Cd stress.

Amenability of castor to an Agrobacterium -mediated in planta transformation strategy using a cry1AcF gene for insect tolerance

Agrobacterium tumefaciens mediated in planta transformation protocol was developed for castor, Ricinus communis. Two-day-old seedlings were infected with Agrobacterium strain EHA105/pBinBt8 harboring cry1AcF and established in the greenhouse. Screening the T1 generation seedlings on 300 mg L−1 kanamycin identified the putative transformants. Molecular and expression analysis confirmed the transgenic nature and identified high-expressing plants. Western blot analysis confirmed the co-integration of the nptII gene in the selected transgenic plants. Bioassay against Spodoptera litura corroborated with high expression and identified five promising effective lines. Analysis of the T2 generation plants proved the stability of the transgene indicating the feasibility of the method.

Simple yet stringent screening methodologies for evaluation of putative transformants for abiotic stress tolerance: salt and cadmium stress as a paradigm.

Rigorous and stringent screening methodologies to select transformants at both seedling and plant level under cadmium or NaCl stress were developed. At seedling level, two screening strategies were standardized. One involved germination on filter paper/agar in the presence of either CdCl2 (125 μM) or NaCl (350–450 mM) for 9 days and selection of tolerant putative transformants. The other involved germination of the seedlings on soilrite by irrigation of 450 mM NaCl. Further, at plant level, in vitro evaluation for stress tolerance involved a simple leaf senescence bioassay. Combination of the seedling and plant level screening strategies would result in the initial identification of promising transformants for further analysis.