板栗非胚性愈伤组织DsRed基因瞬时转化方法建立和优化

Establishment and optimization of transient transformation method of non-embryonic callus with DsRed gene in Chinese chestnut (Castanea mollissima Blume)

【目的】为建立并优化板栗非胚性愈伤组织瞬时转化技术体系。【方法】以‘燕山红栗’幼胚根尖诱导的非胚性愈伤组织为材料,参照美洲栗胚性愈伤组织转化方法,转化DsRed红色荧光蛋白基因,建立板栗非胚性愈伤组织瞬时转化技术体系。针对影响转化率的重要因子,设计菌液浓度、超声处理时间、共培养时间、干燥时间四因素五水平正交试验优化该方法。【结果】成功诱导板栗非胚性愈伤组织并观察到红色荧光,表明成功建立并优化了板栗非胚性愈伤组织瞬时转化方法:菌液浓度OD600=1.50,超声处理时间90s,共培养时间1h,干燥时间3d。【结论】该方法对于在细胞水平上研究糖类和淀粉代谢的分子机理具有重要意义。

【Objective】 The experiment is to establish and optimize the transient transformation technology system of non-embryonic callus in Chinese chestnut ( Castanea mollissima Blume).【Methods】The Chinese chestnut non-embryonic callus was induced by the young embryonic root tip of Chinese chestnut cultivar 'Yanshanhongli', and then the DsRed gene was transferred into non-embryonic callus based on the transient transformation method of American chestnut embryogenic callus. The Chinese chestnut non-embryonic callus transformation efficiency was determined by four factors: Agrobacterium tumefaciens concentration, sonication duration, co-culture duration and desiccation duration, therefore, the experiment was optimized by designing a four-factor and five-level orthogonal experiments.【Results】We successfully observed red fluorescence from Chinese chestnut non-embryonic callus transferred DsRed gene . The results showed that the optimal transient transformation technology system of non-embryonic callus in Chinese chestnut was as the following: the concentration of culture was OD 600 =1.50, the sonication time was 90 s , and the co-culture time was 1 h , the drying time was 3 d .【Conclusion】This method is significant for studying the molecular mechanism of carbohydrate and starch metabolism at the cellular level.