Potential of cell cycle genes arath cycd2 and musa cycd2 for the improvement of transformation and regeneration efficiency of banana (cv “sukali ndiizi”)

Abstract

Available techniques for the genetic transformation with important genes could overcome some of the agronomic and environmental problems limiting conventional breeding of bananas. Although it is possible to transform bananas, broad application of the technology is limited because of the low overall efficiency and lack of reliability of the technique. This research reports on the potential of CycD2 genes to improve transformation and regeneration efficiency of banana (cv. “Sukali ndiizi”). Two genes Arath CycD2 and Musa CycD2 were evaluated for the cell cycle modification of the embryogenic cell suspension that is conventionally used in banana genetic engineering at the National Biotechnology Centre, Kawanda. The UidA (GUS) gene was used as reporter to establish transient transformation efficiency. The GUS reporter gene, which was used for quantification of transformants was therefore, fused with each of the CycD2 genes in the binary vector, pC1305.1. The Cauliflower mosaic virus 35S promoter was used to drive both CycD2 and the GUS reporter genes. Results indicated that cell cycle genes could significantly increase the competence of banana cells for uptake and integration of foreign genes. The Gus assay analyses of transformed cells showed a success rate of 80% to 90% for all the constructs including the control transformed with the empty vector without CycD2 gene. To assess whether the CycD2 genes could improve the regeneration efficiency of “Sukali ndiizi”, the transformed cells were cultured on selection media and the hygromycin resistant colonies developed into shoots. The gus assay of the regenerants showed that the genes were expressed in different parts of the plants (roots, corm and leaves). The PCR analysis of DNA from these shoots indicated that Musa CycD2 and Arath CycD2 genes significantly improve the regeneration of transgenic “Sukali ndiizi” cells. The regeneration effinciency of the embryogenic colonies of CycD2 genes (47%-62%) was much higher than that of the control without CycD2 (18%). The results show that “Sukali ndiizi” cells are highly competent and transformable by Agrobacterium mediated transformation system and CycD2 genes have the potential to significantly improve regeneration efficiency of “Sukali ndiizi” cells”. This study contributes to the current information about improvement of transformation and regeneration efficiency of bananas and highlights the potential of CycD2 genes in the improvement of regeneration of transgenic plants.