“Gros Michel”香蕉胚性细胞悬浮系及遗传转化体系的建立

Establishment of Embryogenic Cell Suspension and Genetic Transformation System of "Gros Michel" Banana

本研究以"Gros Michel"香蕉未成熟雄花序为外植体,建立了其胚性细胞悬浮系及遗传转化体系。结果表明,90 d后可诱导产生浅黄色松散的胚性愈伤组织,经悬浮培养120 d后获得含有均质细胞团的胚性细胞悬浮系(ECS);将3个月龄的ECS置于胚诱导培养基30 d后有大量白色半透明状球形的体细胞胚发生,继代培养60 d后发育为成熟不透明的体细胞胚;将成熟的体胚在萌发培养基上培养10 d后有幼芽产生,转移至生根培养基30 d后得到幼苗。同时开展了含绿色荧光蛋白(GFP)的pCAMBIA0380载体的遗传转化研究,经过3次液体培养基筛选转移至半固体筛选培养基90 d后,在胚萌发培养基和生根培养基中不再添加抗生素。经对转化植株的根尖镜检及PCR鉴定,均为阳性植株。研究结果将为进一步开展"Gros Michel"优质抗病基因功能研究提供材料和技术支撑。

In the research,embryogenic cell suspension and genetic transformation of "Gros Michel"banana were established with the immature male inflorescence as explants.The results showed that yellowish and loose embryogenic.callus could be developed after 90 days.Embryogenic cell suspensions (ECS)with homogeneous cell mass were obtained after 120 days of suspension culture.3-month-old ECS were cultured in embryo-inducing medium for 30 days,followed by a large number of white translucent spherical somatic embryos,which would be developed into mature opaque somatic embryos after subculture for 60 days.Mature somatic embryos were cultured on the germination medium for 10 days,and then buds were produced and transferred to the rooting medium for 30 days to obtain seedlings.At the same time,the genetic transformation of pCAMBIA0380 vector containing green fluorescent protein (GFP)was carried out.The embryogenic cells were selected after three times of liquid screening medium and transferred to semi-solid screening culture medium for 90 days,followed by culturing in embryo germination medium and rooting medium without any antibiotic.After root tip microscopic examination and PCR identification of the transformed plants,they were found to be positive.Those results would provide materials and technical support for the further research on the function of high-quality disease resistance gene in "Gros Michel".