Genetic Engineering Of Banana Using Nonexpressor Of Pathogenesis Related Gene For Resistance To Xanthomonas Wilt And Fusarium Wilt Diseases

ABSTRACT

Banana (Musa spp.) is an important fruit and staple food crop globally. It is grown in over 140 countries on more than 11 million hectares, with current production of 145 million metric tons per annum, out of which about 30 % is produced in Africa. However, its production is limited by banana Xanthomonas wilt (BXW) disease caused by bacterium Xanthomonas campestris pathovar musacearum (Xcm) and Fusarium wilt disease (FWD) caused by Fusarium oxysporum subspecies cubense race 1 (Foc). Cultivated banana varieties are sterile, triploids and have long generation period; therefore, it is difficult to develop disease resistance through conventional breeding. The use of genetic engineering is a potential option that could facilitate the development of banana cultivars resistant to a broad-spectrum of pathogens. The main objective of this study was to develop transgenic banana resistance to Xcm and Foc race 1 through over expression of non-expressor of pathogenesis related gene (NPR/NH1) from the rice. Embryogenic cells of farmer-preferred banana cultivars were generated, and Agrobacterium-mediated genetic transformation system was optemized using betaglucuronidase (gusA) reporter gene. Two assorted groups of explants, multiple buds and male flowers, were tried for their capacity to develop EC from three locally grown cultivars of banana in Africa. Embryogenic cells of ‘Gros Michel’ and ‘Cavendish Williams’ were produced from multiple buds, and EC of ‘Sukali Ndiizi’ was obtained from male flowers. The EC were regenerated with efficiency of about 20,000-50,000 plantlets per ml of settled cell volume (SCV) based on cultivars. Agrobacterium-mediated genetic transformation was established for these cultivars using gusA reporter gene. Twenty to seventy transgenic events were regenerated from one ml of SCV of EC on kanamycin (100 mg/L) containing selective medium. The presence of gusA gene in transgenic banana plants was demonstrated by PCR. The integration and expression of gusA gene was further checked by dot blot, Southern hybridization and GUS histochemical assay. Banana cultivar ‘Sukali Ndiizi’ was transformed by the nonexpressor of pathogenesis-related gene (NPR1) homolog 1 (NH1) gene from the rice. One hundred and twelve transgenic events were regenerated in selective medium containing hygromycin B (25 mg/L). The transgenic nature of the generated events was tested by PCR analysis, dot blot, and Southern hybridization. Gene expression study was performed by RTPCR and qRT-PCR for selected transgenic events. Potted transgenic events were evaluated against Xanthomonas campestris pv. musacearum and Fusarium oxysporum f. sp. cubense race 1 in the glasshouse. Out of twenty evaluated transgenic events, two events (10 %) exhibited no disease symptoms in all the three replicates and twelve events (60 %) showed reduced symptoms and five events (25 %) were susceptible. Fifteen transgenic events were tested for Fusarium wilt resistance in the glasshouse. Out of 15 transgenic events, three transgenic events were resistant (20 %) and seven events were tolerant (46 %) and five events (33 %) were susceptible with reduced symptoms in comparsion to control plant against Foc race 1. To understand the mechanism of disease resistance of the NH1 gene, pathogenesis-related genes PR1, PR2, PR3 and PR5 were analyzed by qRT-PCR after inoculation of transgenic events with Xcm at different time intervals. The results showed that the PR genes were activated more in transgenic events in contrast to control non-transgenic plants at 6, 12 and 48-hour post inoculation. This study reports an efficient and reproducible protocol for genetic transformation using embryogenic cells of banana. The results of this research also provided proof of concept that the rice NH1 can provide resistance against Xcm and Foc race 1. Overall this information will form a basis to develop Xanthomonas wilt and Fusarium wilt disease resistant transgenic banana varieties for smallholder banana farmers in Africa.