Agrobacterium -mediated transformation of banana cultivar 'Rastali' ( Musa , AAB genome) with chitinase gene.

摘要

A rice chitinase gene (RCC2), multiplied in Agrobacterium strain (EHA 101), was simultaneously introduced into single buds of in vitro grown in the banana (Musa spp.) cultivar 'Rastali' (AAB genome). Plasmid pBI333-EN4-RCC2 contained a hygromycin phosphotransferase gene (hptII) as the selectable marker and gusA gene as a reporter marker to identify the transformants. Treatment A contained hygromycin at 25 mg L-1 and treatment B contained hygromycin at 50 mg L-1 in both Murashige and Skoog (MS) medium, supplemented with 5 mg L-1 of 6-Benylaminoupurine (BAP) and 2.7 g of gelrite agar. Single buds, derived from multiple bud clumps (Mbcs), were the target explants for transformation. An assay was performed to identify the minimum concentration required for two antibiotics (carbenicillin and cefotaxime) that is most effective against the Agrobacterium strain, EHA 101 and the effect on tissue regeneration capacity. Even though the transformation frequency based on the hygromycin selection medium (treatment A) was higher, no transformant could be confirmed based on PCR and southern blot analyses, as compared to the 50 mg L-1 hygromycin selection medium. Stable gusA gene expression was detectable in transformed single buds, Mbcs, shoots, leaves and roots derived from treatment B. The assay of protein extract from the transgenic plantlets showed an increased in chitinase enzyme activity over the untransformed plantlets. The presentation of Agrobacterium-mediated transformation reported here is suitable for using tiny meristem tissues to obtain fungal disease tolerant or resistant banana through genetic engineering.