FACTORS AFFECTING AGROBACTERIUM- MEDIATED GENETIC TRANSFORMATION IN GROUNDNUT (ARACHIS HYPOGAEA L.)RAJINIKANTH MARKA AND RAMA SWAMY NANNA
The development of Agrobacterium- mediated transformation protocol for a recalcitrant species like groundnut requires the identification and optimisation of the factors affecting T-DNA delivery and plant transformation. During the present investigation, we have used de-embryonated cotyledon (DC), leaflet (LL) and cotyledonary node (CN) explants of groundnut cv ICG 7827 and the Agrobacterium strain LBA 4404 harbouring the binary vector pBAL2 containing nptII and uidA (Gus) genes. Factors that affect significant differences in T-DNA delivery and transformation included explant type, duration of pre-culture of explant, incubation period in bacterial suspension, co-cultivation, antibiotics used for selection and the presence of Acetosyringone (AS) in the medium. Three days precultured explants (DC, LL and CN) were incubated in Agrobacterium suspension for 20 min. Optimized the cocultivation period in the shoot induction medium with 100 mg/L AS at 25 Â°C in the dark for 4 days. A concentration of 75 mg/L (LL) and 100 mg/L (DC, CN) Kan was added to shoot induction medium (SIM)) to select transformed shoots. The KanR shoots were excised and cultured on modified MS medium (MMS) +50 mg/L Kan+0.5 mg/L BAP for shoot elongation. These elongated micro shoots were rooted on root induction medium (RIM) supplemented with 1 mg/L NAA+50 mg/L Kan. The in vitro rooted plantlets were acclimatized and maintained in the green house. The expression of GUS gene (uidA gene) in the regenerated plants (T0) was verified by histochemical GUS assay. Stable integration and expression of the transgenes (uidA and nptII) in T0 plants were confirmed by using PCR analysis. Showing the transformation efficiency as, 16.29% for DC, 13.15% for LL and 10.59% for CN explants of groundnut cv ICG 7827. Thus, the optimized factors of Agrobacterium mediated genetic transformation showed the enhanced transformation frequencies to transfer gene of interest into groundnut.