GENETIC TRANSFORMATION FOR POD BORER RESISTANCE USING cry1A (b) IN PIGEONPEA [Cajanus cajan ( L ) Millsp.] cv. ICPL-8863 (MARUTI)
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Date
2002
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University of Agricultural Sciences, Dharwad
Abstract
A study was undertaken to standardize in vitro plant regeneration and Agrobacterium mediated transformation procedure for pigeonpea (Cajunus cajan) cv. ICPL-8863 (Maruti). For regeneration direct organogenesis was attempted using different explants viz., shoottip (ST), cotyledonary node (CN), half cotyledon with cotyledonary node (V2 CNC) and CNC. These were cultured on various levels of benzyl amino purine (BAP) (1, 2, 3, 4 mg W) and thidiazuron (TDZ) (0.01, 0.05, 0.1, 0.5 mg l1). CNC found to produce average of 1.69 shoots/explant and was better among all explants used. Among different levels of BAP and TDZ tried, BAP 2 mg l1 was found to be better for multiple shoot and shootbud induction. Shootbuds were cultured on MS with reduced levels of cytokinins and TDZ 0.05 mg l 1 gave better elongation compared to other levels, elongated shoots were rooted on MS with IBA (0.1-0.5 mg W). Among all the levels tried 0.2 mg l1 IBA gave good healthy roots. For transformation Agrobacterium strains EHA 105 harboring pBinBtl plasmid [cryl A{b)} and GV2260 harboring pCAMBIA1301 plasmid (gus) with nptll as selectable marker, which confers kanamycin resistance were used. Initially kanamycin sensitivity of control explants was tested at different growth stages. Inhibitory levels at different stages were used for selection of transformants. Precultivation of explants on MS with 2 mg l 1 BAP for two days prior to cocultivation resulted in increased survival. Explnats were cocultured for two days in dark and transferred to selection medium (with kanamycin and cefotaxime). Approximately 1.4 per cent shoots obtained were cry positive. In in planta approach plants were treated with Agrobacterium inoculum at different growth stages. Germinating seeds were injected with GV2260 strain and shoots were histochemically assayed and 0.9% of shoots were gus positive. In seedling dip and flower injection methods cryl A (b) gene was transferred and confirmed through PCR analysis (9/54, 11/26 plants were cry positive respectively). Thus efficient regeneration and transformation protocol has been standardized for pigeonpea cv. ICPL-8863 (Maruti).
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