Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of groundnut (Arachis hypogaea L.) via somatic embryogenesis

Abstract

An efficient transformation protocol was developed for groundnut (Arachis hypogaea L.) plants. Precultured cotyledons were co-cultured with Agrobacterium tumefaciens strain LBA 4404 harbouring the binary vector pBI121 containing the uidA (GUS) and nptII genes for 2 days and cultured on an embryo induction medium containing 0.5 mg/l NAA, 5.0 mg/l BAP, 75 μg/ml kanamycin and 300 μg/ml cefotaxime. The putatively transformed embryos were transferred to the medium with reduced kanamycin (50 μg/ml) for further development. Prolific shoots developed from these embryos on a MS medium containing 0.5 mg/l BAP and 50 μg/ml kanamycin with a transformation efficiency of 47%. The elongated kanamycin-resistant shoots were subsequently rooted on the MS medium supplemented with 1.0 mg/l IBA. The transgenic plants were later established in plastic cups. A strong GUS activity was detected in the putatively transformed plants by histochemical assay. Transformation was confirmed by PCR analyses. Integration of T-DNA into nuclear genome of transgenic plants was further confirmed by Southern hybridization with nptII gene probe. A large number of transgenic plants were obtained in this study. This protocol allows effective transformation and quick regeneration via embryogenesis.