二倍体野生种马铃薯Solanum pinnatisectmum原生质体分离纯化与培养的研究

Studies on Isolation,Purification and Culture of Protoplasts from Diploid Wild Potato(S.pinnatisectmum)

为获得较好的原生质体分离和原生质体纯度,试验以野生种马铃薯(S.pinnatisectmum)试管苗叶片为材料,进行了原生质体分离和纯化的研究。结果表明:培养3周左右的试管苗叶片均在含有0.4%纤维素酶+0.7%果胶酶+0.1%离析酶,渗透压为0.3 mol/L的酶解液中解离效果最好,原生质体产量为1.93×106/g FW;在(25±1)℃酶解温度条件下静置14 h,可促进叶片原生质体的大量释放。而且,外源激素组合0.5 mg/L IAA+2.5 mg/L Zeatin有利于S.pinnatisectmum叶片原生质体的分裂,原生质体一次分裂频率达到6.32%。研究获得了稳定的原生质体分离体系以及较好的原生质体培养条件,为马铃薯野生种Solanum pinnatisectmum的体细胞融合提供了依据。

In order to obtain better protoplast isolation and protoplast purity,the leaves from test-tube plantlet of wild potato（S.pinnatisectmum） were uesd as material to study on protoplast isolation and purification.The result showed that the optimum combination of enzymes to separate protoplast from culture leaves of plantlets in vitro for about 3 weeks was 0.4% cellulase＋0.1% pectinase＋0.7% macerozym,.and the optimum osmotic pressure was 0.30 mol/L.Under the above condition,the protoplast yield of S.pinnatisectmum was 1.93 ×106/g FM.Resting under the condition of（25 ±1）℃ digesting temperature for 14 h could promote the release of protoplasts in leaves.In addition,exogenous hormone combination 0.5 mg/L IAA＋2.5 mg/L Zeatin was helpful to the division of protoplast in leaves of S.pinnatisectmum,and frequency of protoplast division could reach 6.32%.Stable protoplast isolation system and better protoplast culture conditions were obtained in this study,which could provide reference for the somatic cell fusion of S.pinnatisectmum.