Enhanced Agrobacterium-mediated transformation efficiency of banana cultivar Grand Naine by reducing oxidative stress

Abstract

Efficient protocols were developed for somatic embryogenesis (SE) and recovery of genetic transformants of banana cultivar Grand Naine. The efficiency of embryogenic callus formation was enhanced up to 14% by spraying 2, 4-dichlorophenoxyacetic acid (20 mg/l) on immature male flower buds (IMFBs) before inoculation of explants (immature male flowers) on the callus induction medium (CIM). Embryogenic cell suspension (ECS) derived from callus was used for Agrobacterium tumefaciens-mediated transformation harboring β-glucuronidase (GUS) as a reporter and hygromycin phosphotransferase (hpt II) as the selection marker genes. Four antioxidants (melatonin, quercetin, gallic acid, and α-tocopherol) were tested during and after Agrobacterium infection of ECS to reduce the oxidative stress. Melatonin boosted up nearly 1.5-fold [72.3 ± 7.5 plantlets/ml settled cell volume (SCV)] higher transformation efficiency as compared to without antioxidant treated (46.7 ± 2.5 plantlets/ml SCV) culture. Scanning electron microscopy (SEM) revealed that melatonin reinforced the bacterial attachment to the plant cell and also maintained surface morphology of the cells. The expression of the reporter gene in putatively transformed ECS and different tissues has been demonstrated by histochemical GUS assay. The transgenic plants were confirmed by PCR for the presence of hpt II and GUS amplicon. The optimized efficient protocols pave the way to generate the high number of transgenic lines with desirable traits in important commercial banana cultivar Grand Naine.