An efficient protocol for the production of chit42 transgenic Furenzhi banana (Musa spp. AA group) resistant to Fusarium oxysporum

Abstract

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a destructive fungal disease of banana. Transferring antifungal genes into banana provides a feasible way to control fungal disease, but transformation frequencies for banana are often cultivar and cell line dependent. We investigated an efficient liquid medium selection protocol for an Agrobacterium-mediated transformation system for Furenzhi (Musa spp. AA group). Embryogenic cell suspensions (ECS) of Furenzhi were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring a plasmid containing the endochitinase gene chit42 from Trichoderma harzianum. After co-cultivation, GUS-positive ECS were selected in liquid medium with antibiotics, and compared to semi-solid medium selection. In total, 186 transgenic plantlets were obtained using M2S liquid medium (based on Murashige and Skoog salts) with hygromycin, whereas no transgenic lines were obtained in parallel experiments with semi-solid selection medium. Integration of the transgene was confirmed by PCR, and Southern blots which showed a single copy of the transgene had integrated into the banana genome in three plant lines. Expression of the transgene in regenerated plants was confirmed by s-glucuronidase histochemical assays and real-time PCR. Both in vitro and ex vivo disease assays showed that the majority of transgenic lines (three of seven) expressing chit42 showed a higher level of resistance to Fusarium wilt (Foc race 4), whereas non-transgenic control plants were susceptible. These results imply a relationship between Foc4 disease resistance and the transcription levels of the transgene in the transgenic clones. This study may offer a promising approach to breed bananas resistant to the fungal disease Fusarium wilt.