Development of in vitro plant regeneration system and agrobacterium–mediated genetic transformation in Mungbean (Vigna radiata (L.) wilczeck)

Abstract

An efficient protocol has been developed for Agrobacterium-mediated genetic transformation of two important mungbean (Vigna radiata (L.) Wilczeck) varieties cultivated in Bangladesh, namely, Binamoog-5 and BARI mung-6. Prior to transformation an efficient in vitro regeneration protocol was developed for these two mungbean varieties. Cotyledonary node, shoot tip and cotyledone attached decapitated embryo were used as explants for direct regeneration of shoot. MS medium with various combinations and concentrations of BAP, Kn and NAA were used for direct regeneration of shoots. MS medium supplemented with 10.0 μM BAP and 0.5 μM NAA was found to be the best towards the development of highest number of multiple shoots. Full strength of MS medium supplemented with 2.0 μM IBA was found to be the most effective for healthy root induction from the regenerated excised shoots. Following root induction the in vitro regenerated plantlets were successfully transplanted to soil. Transformation experiments with cotyledone attached decapitated embryo, cotyledonary node and decapitated embryo explants were conducted using Agrobacterium tumefaciens strain, namely, LBA4404 harboring the binary plasmid pBI121 conferring GUS (β-glucuronidase) and nptII (neomycin phosphotransferase II) genes. Transformation experiments were monitored through GUS histochemical assay since the strain of Agrobacterium contained GUS gene. Different factors influencing successful transformation such as, density of bacterial suspension, incubation and co-cultivation periods were optimized. Maximum responses towards transformation were obtained with bacterial suspension having an optical density of 0.56 at 600 nm. Moreover, 30 minutes of incubation period followed by 72 hours of co-cultivation period were found to be the most effective towards transformation as determined by transient GUS assay. Selection of transformed shoots was carried out using different concentrations of kanamycin as the Agrobacterium strain contained nptII gene. Transformed shoots were selected using 150 mg/l kanamycin. The survival of green shoots on the optimum selection medium indicated the production of transformed shoots. Stable expression of the GUS gene was detected in various parts of transformed plantlets. Putatively transformed shoots were rooted on full strength of MS medium containing 2.0 μM IBA and 100 mg/l ticrecilin. Rooted transformed plantlets were successfully transferred to soil. Genomic DNA was isolated from the putatively transformed mungbean shoots and stable integration of GUS and nptII genes was confirmed by polymerase chain reaction (PCR) analysis.