大豆幼苗根和叶片原生质的分离与纯化

研究了大豆幼苗根和叶片原生质体的分离、纯化方法及其影响因素.结果表明:适宜大豆根和叶片原生质体分离的酶种类、浓度分别为CPW-13M{CPW(细胞清洗液)+13%(W/V)甘露醇}+3%纤维素酶(cellulose R10)+1.1%果胶酶(macerozyme R-10)+1.0%半纤维素酶(hemicellulase)和0.15%CaCl2·2H2O+9%甘露醇+1%cellulase R-10+0.20%pectolase Y-23,pH 5.8,酶解温度为28℃.在根酶解时间为16 h时,原生质体产量可高达1.46×105个·g-1FW,活力达57.8%;叶片酶解时间为4 h时,原生质体产量可高达1.74×106个·g-1FW,活力达70.3%.对于根而言,从产量和活力两方面考虑,其原生质体用23%蔗糖和CPW-18M混合后的下沉法纯化效果较好,而叶片用25%蔗糖的上浮法纯化效果较好.

Isolation and Purification of Protoplasts in Roots and Leaves of Soybean Seedlings

The methods for isolation and purification of protoplast in roots and leaves of soybean seedling were investigated together in this study. The results showed that, the suitable enzyme species and concentrations for isolation of root protoplasts were CPW-13M + 13% (W/V) mannotol+3% cellulose R-10 + 1.1% macerozyme R- 10 + 1.0% hemicellulase, pH5.8 ; those for leaf were 0. 15% CaCl_22H_2O+9% mannitol + 1% celhilase R-10 +0.20% pectolase Y-23, pH 5.8, and the temperature was 28℃. The reasonable enzymatic times for isolation of root and leaf protoplasts were 16 and 4 h, respectively; and the higher protoplast yield and viability were accordingly 1. 46 × 10~5 protoplasts · g~(-1) FW and 57. 8% ,1. 74 × 10~6 protoplasts ? g FW and 70. 3% . The better purification for root protoplasts was the sinking method of 23% sucrose mixed with CPW- 18M ,that for leaf was the rising method of 25% sucrose.