Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.
A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.
Huang
Xia
X
The Key Laboratory of Gene Engineering of the Ministry of Education, School of Life Sciences, Zhongshan (Sun Yat-sen) University, Guangzhou 510275, China.
Huang
Xue-Lin
XL
Xiao
Wang
W
Zhao
Jie-Tang
JT
Dai
Xue-Mei
XM
Chen
Yun-Feng
YF
Li
Xiao-Ju
XJ
D018014:Gene Transfer Techniques; D028521:Musa / Q000166:cytology* / Q000235:genetics*; D030821:Plants, Genetically Modified; D012231:Rhizobium / Q000235:genetics*; D014170:Transformation, Genetic