Agrobacterium-mediated transformation of embryogenic cell suspensions of the banana cultivar Rasthali (AAB)

A protocol was developed for establishing embryogenic suspension cultures f rom in vitro-grown, thin shoot-tip sections of the banana cultivar Rasthali . The best medium for callus induction was an MS-based medium supplemented with 2 mg/l 2,4-D and 0.2 mg/l zeatin. The callus was transferred to liquid medium to establish embryogenic cell suspensions. These cultures were subs equently used for Agrobacterium-mediated transformation. The Agrobacterium tumefaciens strain EHA105 containing the binary vector pVGSUN with the als gene as a selectable marker and an intron-containing the gusA gene as a rep orter gene was used for transformations. The herbicide Glean was used as a selection agent. Two hundred putative transformants were recovered, of whic h a set of 16 was tested by histochemical analysis for GUS expression and b y Southern blot analysis with a probe for the gusA gene. The plants were po sitive for GUS expression and integration of the gusA gene. Two of the tran sformants were grown to maturity under greenhouse conditions. Bananas were harvested to test GUS expression by histochemical analysis. The fruit from both transgenics tested positive for GUS expression.