Somatic Embryogenesis and Genetic Engineering of Banana with Elongation Factor Receptor Gene for Resistance to Banana Xanthomonas Wilt
Ochieng, Mark Adero
MetadataShow full item record
Banana is among the top ten most important food crops worldwide in production and consumption. However, its production is threatened by several phytopathogens. Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pathovar (pv.) musacearum (Xcm) is one of the biggest threats to banana production in the African Great Lakes region. Crop breeding via conventional means is key to overcoming yield losses to phytopathogens. However, conventional breeding of clonally propagated crops like banana is limited by the lack of diversity and important traits in the gene pool. At present, only Musa balbisiana (banana progenitor species) is resistant to BXW, but breeders do not prefer it because it harbors banana streak virus (BSV) sequences in its genome, which get activated during abiotic stress, leading banana streak disease (BSD). Thus, genetic engineering serves as a viable alternative and complement to conventional breeding for banana improvement. Interfamily transfer of pattern recognition receptor genes, such as elongation factor receptor (EFR), through transgenic approach can enhance plant resistance to bacterial diseases of diverse genera. An efficient regeneration system based on embryogenic cell suspensions (ECS) is a prerequisite for efficient genetic transformation of bananas. Till now, limited progress has been made on establishing protocols for somatic embryogenesis and transformation of most East African highland banana (EAHB) cultivars. This study aimed to establish somatic embryogenesis, cell suspension cultures, and transformation protocol for EAHB cultivars ‘Ngombe’ and ‘Kisansa’ and transform banana with elongation factor receptor (AtEFR) gene from Arabidopsis thaliana for resistance to BXW. Various media and plant growth regulators (PGRs) were tested for their ability to induce somatic embryogenesis and plant regeneration or generation of cell suspension cultures. Embryogenic calli were transformed with Agrobacterium strain EHA 105 containing a binary vector pCAMBIA2301 harboring a reporter gene (β-glucuronidase, gusA), followed by selection and plant regeneration. Putative transformants were subjected to GUS assay and molecular characterization. For the development of transgenic bananas expressing AtEFR, ECS of two banana cultivars, ‘Sukari Ndizi’ and ‘Dwarf Cavendish’ were transformed with Agrobacterium strain EHA 105 containing a binary vector pBIN19g-35S:: EFR and the generated transgenic events were evaluated for BXW resistance in the greenhouse. Among the medium and PGRs tested for callus induction and somatic embryogenesis, half-strength Murashige and Skoog (MS) medium containing 2 mg/l picloram or dicamba was optimum for both varieties. The highest frequency of plant regeneration from embryogenic calli (35%) was achieved in MS medium containing 1 mg/l 6-benzylaminopurine (BAP). In total, 257 putatively transformed events expressing AtEFR were regenerated and characterized using polymerase chain reaction (PCR), Southern blot, reverse transcription (RT)-PCR, and quantitative RT-PCR analyses. Among the 48 transgenic events evaluated for BXW resistance in the greenhouse, three showed complete resistance, 23 partial resistance, and 22 were susceptible. The transgenic events also showed responsiveness to the N-terminal acetylated peptide elf18 of bacterial elongation factor thermal unstable (EFTu), suggesting that the function of the AtEFR gene was retained in banana. Overall, this study indicates that EAHBs are amenable to somatic embryogenesis and genetic transformation and further confirms that adaptors of EFR are conserved across a diverse range of plant families and can potentially enhance the resistance of bananas to BXW under field conditions.