蕨麻愈伤组织原生质体制备条件的优化

Preparation and Optimization of Callus Protoplast of Potentilla anserina

以青海‘蕨麻4号’诱导培养的愈伤组织为材料,采用4因素3水平L9(34)正交实验,研究酶类组合、酶解时间、甘露醇浓度及离心速度等主要因素对蕨麻原生质体分离的影响,建立高效、稳定的蕨麻原生质体分离体系,为进一步通过原生质体融合、基因工程等方法对蕨麻进行品种改良奠定基础。结果表明:各因素对蕨麻原生质体产量的影响顺序为:酶类组合>酶解时间>甘露醇浓度>离心速度;青海‘蕨麻4号’愈伤组织原生质体的最适酶解条件为:2.0%纤维素酶+0.75%果胶酶,40r/min振荡酶解10h,甘露醇浓度为0.5mol/L,离心转速为1 000r/min时原生质体的产量达最大(8.96×10~5 cells/g),活力为92.77%。

The present study aimed to establish an efficient and stable separation method of Potentilla anserina callus protoplast,which were induced from root tuber of Qinghai Juema No.4.The design method of L9(34)orthogonal experimental was adopted to optimize the combination conditions.The effects of some factors on the separation of Potentilla anserinacallus protoplast were investigated,including enzyme combination,digestiontime,mannitol concentration and centrifugal speeds.The results indicated that the sequential order of every factor for protoplast yield was enzyme combination,digestion time,mannitol concentration and centrifugal speeds.The optimal enzyme solution for protoplast isolation was 2.0% cellulase R-10 + 0.75% Pectinase Y-23 + 0.5mol/L mannitol.The digestion was conducted on constant-temperature shaker with 40r/min at 25℃ for 10 hin dark and centrifugation with 1 000r/min for 6min for protoplast collection.The protoplast yield amounted to 8.96×10~5 cells/g and the vitality was up to 92.77%.