Optimization of factors affecting Agrobacterium-mediated genetic transformation in groundnut (Arachis hypogaea L.)

Abstract

The efficiency of Agrobacterium-mediated genetic transformation of groundnut is dependent on various factors such as type and age of explants, antibiotics used for selection, Acetosyringone (AS), duration of co cultivation and infection period. Hence, we have attempted to standardize these parameters to improve the Agrobacterium mediated genetic transformation efficiency in groundnut cvICG 13942. To evaluate the optimization of factors affecting Agrobacterium mediated genetic transformation in which we used three different explants (Deembryonated cotyledon-DC, Leaflet-LL, Cotyledonary node-CN) and studied, the effect of inoculums concentration (0.5, 1.0 and 1.5 OD600 of A. tumefaciens), pre culture period (1-11 days), co cultivation period (1-7 days), Acetosyringone concentration (25-200mg/L) and antibiotic (Kan) concentration (25-150mg/L) on the efficiency of genetic transformation. The transformation was verified by GUS staining and by means of PCR amplification of the uidA and nptII genes. The optimized factors for A. tumefaciens LBA 4404 harboring pBAL2 binary vector mediated transformation in groundnut cvICG 13942 are: Pre culture period-3 days, Concentration of A. tumefaciens-1.0 at OD600, Co-cultivation period-4 days, Kan concentration-100mg/L for DC &CN, 75mg/L for LL explants, Concentration of AS-100mg/L during infection and co cultivation and Cefotaxime concentration-250mg/L. Thus, the optimized protocol showed the enhanced transient transformation frequencies such as 18.13% for DC, 15.33% for LL and 12.32% for CN explants.