甘薯叶片原生质体快速制备方法的优化

Optimization of Rapid Protoplast Preparation Method for Sweet Potato Leaves

原生质体由于没有细胞壁,在植物育种、遗传转化以及基础研究中都有极为广泛的用途。常用的酶解法制备原生质体需要的时间较长,缩短原生质体的制备时间对后续的瞬时表达、植物育种等相关实验具有重要意义。本文以甘薯[Dioscorea esculenta(Lour.)Burkill]叶片为材料,通过调整叶片处理方式、酶浓度、酶解时间、酶解温度等条件对原生质体的制备方法进行了优化,旨在建立一种快速制备甘薯原生质体的方法。结果表明:利用优化的原生质体制备方法可将20 h的酶解时间缩短到2 h;优化的条件为撕去甘薯叶片下表皮后再进行酶解,使用含1.8%纤维素酶R-10+0.9%离析酶R-10酶溶液,在28℃条件下酶解,350×g离心力离心。

Protoplasts are widely used in plant breeding,genetic transformation and other basic researches since protoplats have no cell wall.It takes a long time to prepare protoplasts by routine enzymolysis method.Shortening the preparation time of protoplasts is important for subsequent transient expression,plant breeding and other related experiments.It was aimed to establish a method for rapid preparation of sweet potato protoplasts,sweet potato[Dioscorea esculenta(Lour.)Burkill]leaves were used as materials in this study,and experimental conditions were optimized by adjusting conditions such as leaf treatment,enzyme concentration,enzymatic hydrolysis time and enzymatic hydrolysis temperature.The results showed that the enzymatic hydrolysis time could be shortened from 20 hours to 2 hours by tearing off the lower epidermis of sweet potato leaves,using the enzyme solution containing 1.8%Cellulase Onozuke R-10 and 0.9%Macerozyme R-10 at 28℃,centrifuging with 350×g centrifugal force.