To promote gene function research and somatic hybrid breeding,a protoplast isolation and purification system for Lilium davidii var. unicolor was established,and the optimal parameters for enzymatic hydrolysis of protoplasts were determined by investigating the key factors such as material selection,osmotic regulator concentration,enzyme dosage,enzymatic hydrolysis time,and purification conditions. The results demonstrated that the homogeneous mesophyll protoplasts were isolated from leaves sub-cultured for about 30 d and digested with the enzyme solution containing 15 g·L-1 cellulase and 5 g·L-1 segregation enzyme for 3 h in the presence of 0.4 mol·L-1 mannitol,and the average yield was more than 4×106 cells·g-1 with the cell viability exceeding 90％. Positive transformers were obtained successfully by 35S::GFP plasmids transfected with 0.4 g·mL-1 PEG-4000 for 15 min. At the same time,the protoplasts fused to form binucleated cells by 0.5 g·mL-1 PEG-4000 and 5 mmol·L-1 CaCl2,pH=10. This system provided high-quality cell materials for L. davidii var. unicolor protoplast fusion breeding and established a platform for gene function research.
关键词
兰州百合/原生质体/瞬时转化/化学融合
Key words
Lilium davidii var. unicolor/protoplast/transient transformation/chemical fusion
维普
万方数据