Document Type : Research paper
Authors
1
Plant Genome and Disease Research Unit, Department of Agriculture and Resources, Faculty of Natural Resources and Agro-Industry, Kasetsart University, Chalermphrakiat Sakon Nakhon Province Campus, Sakon Nakhon, Thailand
2
Department of Biology, Faculty of Science, Mahasarakham University, Maha Sarakham, Thailand
3
Department of Horticulture, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom, Thailand
4
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand
5
Center of Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom, Thailand
6
Central Laboratory and Greenhouse Complex, Research and Academic Service Center, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus, Kamphaeng Saen, Nakhon Pathom, Thailand
Abstract
This study establishes a CRISPR/Cas9-based genome editing protocol for banana cultivars ‘Hin’ (ABB) and ‘Hom Thong’ (AAA), intended for future use in enhancing resistance to diseases such as banana blood disease caused by Ralstonia syzygii subsp. celebesensis. The target for genome editing was the downy mildew resistance 6 (DMR6) gene. A total of 416 potential single guide RNAs (sgRNAs) were identified, and 26 optimal sgRNAs were selected based on On-scores, minimal off-target effects, and favourable GC content. Five sgRNAs were successfully inserted into the CRISPR/Cas9 vector pRGEB32 and transformed into Agrobacterium tumefaciens EHA105. The transformation process involved co-cultivating the CRISPR/Cas9 system with banana shoot clumps under in vitro conditions. The transformation efficiency, indicated by the presence of Cas9 and hptII genes, ranged from 30% to 38% in ‘Hin’ and 13% to 25% in ‘Hom Thong’. Gene editing was confirmed by deep sequencing, which revealed base pair substitutions in the DMR6 gene, with specific mutations in both cultivars. The highest mutation frequencies were observed for sgDMR6-7, with 70.15% in ‘Hin’ and 33.24% in ‘Hom Thong’. These results demonstrate successful genome editing in bananas, offering a potential strategy to enhance disease resistance.
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