Optimization of in vitro regeneration and genetic transformation protocol for selected banana and plantain (musa spp.) Cultivars and generation of transgenic banana resistant to bacterial xan

ABSTRACT

Banana and plantain (Musa spp.) are important staple food crops for rural and urban consumers and provide a source of income for resource poor farmers in the humid tropics of sub-Saharan Africa. However, banana production is severely limited by both biotic and abiotic factors. Application of conventional breeding for both disease and pest resistance has resulted only in limited success due to the long generation times and the high sterility and triploidy of most cultivated bananas and plantains. Genetic transformation offers an alternative and viable means for crop improvement. However, to be successful, these applications require a rapid and efficient plant regeneration and transformation protocol for both banana and plantain. Currently, most transformation protocols for banana use cell suspension cultures. Establishing cell suspension cultures is a lengthy process, highly cultivar-dependent and most farmer-preferred banana and plantain cultivars are recalcitrant to generation of embryogenic cell suspensions. The objective of this study was to optimize a genetic transformation protocol for banana and plantain cultivars using meristematic tissues and to develop transgenic plants tolerant to BXW. Multiple bud clumps and intercalary meristematic tissues of 10 cultivars (Grande naine, Gross Michel, Gonja Manjaya, Nusu Ngombe, Ngombe, Mpologoma, Uganda green, Kayinja, Zebrina and Calcutta 4) were cocultivated with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 or modified pCAMBIA2300-GFP, followed by selection and regeneration of kanamycin-resistant plantlets. Results of this study indicated 5mg/L as the optimal concentration of cytokinin for multiple bud induction. Several parameters affecting transformation efficiency were explored in this study. The optimal acetosyringone concentration was 200 µM and 100 µM for multiple bud clumps and intercalary meristems respectively), optimal infection time (30 minutes and 10 minutes for multiple bud clumps and intercalary meristems respectively), optimal vacuum infiltration time (2 minutes and 5 minutes for multiple bud clumps and intercalary meristems respectively), effect of explant type ( multiple bud clumps had a higher transformation efficiency, 10%, compared to intercalary meristems, 7%), optimal sonication time (2 seconds for multiple bud clumps) and combined optimal sonication time and vacuum infiltration time was reported to give a higher transformation efficiency (12%) compared to sonication and vacuum infiltration a lone. Uniform GFP expression was observed after the fifth sub culture. The presence and integration of the nptII and gusA genes in the progenies were confirmed by PCR and Southern blot analysis, respectively. Transformation efficiency of banana cultivar Mpologoma with hypersensitive response assisting protein (Hrap) gene was 8%. Out of the twenty lines expressing Hrap gene screened for Xanthomonas wilt resistance resulted in four resistant lines, five partial resistant and eleven susceptible lines. This study augments the ongoing genetic improvement of banana and plantain and contributes to the food security of communities living in Africa and relying on banana as a staple food.