玉米叶片原生质体的制备及瞬时转化体系的建立

Preparation of Maize Leaf Protoplasts and Establishment of Transient Transformation System

以玉米(Zea mays L.)自交系-郑58叶肉细胞原生质体的制备条件的研究,探讨了酶解法制备玉米叶肉细胞原生质体的分离条件和影响因素。结果表明:当沿着叶脉横切叶片时,最佳酶浓度组合为纤维素酶1.0%,离析酶0.5%;最佳酶解时间为5 h;最佳渗透压甘露醇浓度为0.4 mol/L;离心力为100×g、离心1 min时原生质体的产量最高。采用30%PEG-4000将去内毒素的p CAMBIA1302-mGFP的2种质粒制备的原生质体,在细胞核部位能清晰地看到绿色荧光。

Leaves of Zea mays L. inbred lines Zheng 58 plants were taken for protoplast isolation. The factors affecting mesophyll protoplasts isolation from leaves of maize were reported in this paper. The result showed that there were highly significant differences among enzymatic dissociation methods, time in the protoplast yield, centrifugal force in the protoplast yield. The best yield of mesophyll protoplast was obtained using young leaves incubated in the enzyme solution containing 1.0 % cellulose, 0.5 % macerozyme and the optimal concentration of 0.4 mol/L mannitol, incubating for 5 hours. Endotoxin-free plasmids were used for transformation with maize 30 % PEG-4000 and GFP fluorescence was obviously observed in cell nucleus of protoplasts.