In vitro Regeneration and Agrobacterium-mediated Genetic Transformation of Local Varieties of Mungbean (Vigna radiata (L). Wilczek)
Keywords:Mungbean, In vitro regeneration, Genetic transformation, nptll and GUS genes
An efficient Agrobacterium-mediated transformation compatible in vitro regeneration protocol was developed for two important varieties of mungbean (Vigna radiata (L.) Wilczek) cultivated in Bangladesh, namely Binamoog-5 and BARI Mung-6. Two different zygotic embryo derived explants, such as cotyledonary node (CN) and cotyledon attached decapitated embryo (CADE) were used for direct organogenesis of shoot. MS supplemented with 4.0 μM BAP was found to be the best for the development of highest number of multiple shoots from CADE in both the varieties of mungbean. While in case CN the best shoot formation was achieved on MS containing 4.0 μM BAP and 0.5 μM NAA in both varieties. Half strength of MS with 2.0 μM IBA was found to be most effective for producing healthy root from regenerated shoots. Following root induction, the in vitro raised plantlets were successfully transplanted to soil for their establishment. Considering overall responses, genetic transformation efficiency was found to be better with CADE explant using Agrobacterium tumefaciens strain LBA4404 harboring the binary plasmid pBI121 conferring GUS and nptII genes. Different factors influencing transformation was optimized during this study. Selection of transformed shoots was carried out by gradually increasing the concentration of kanamycin and such transformed shoots were eventually selected using 200 mg/l kanamycin. Stable expression of the GUS gene was detected in various parts of regenerated transformed plantlets. Transformed shoots were rooted on half strength MS containing 2.0 μM IBA and 100 mg/l ticarcillin. Rooted transformed plantlets were successfully transferred to soil. Stable integration of GUS and nptII genes in the putative transformed shoots was confirmed through PCR analysis.
Plant Tissue Cult. & Biotech. 29(1): 81-97, 2019 (June)