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玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativa L.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义。以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响。结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多。水稻经真空处理后,原生质体产量大幅度提高。通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5%,离析酶0.5%,50 r/min酶解7 h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5%,离析酶0.5%,50 r/min酶解5 h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0%,离析酶0.7%,50 r/min酶解7 h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW。通过二乙酸荧光素染色发现原生质体活力均在90%以上。用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50%～80%。
Zea mays L., wheat Triticum aestivum L., and Oryza sativa L. are three important food crops and are important for the optimization of protoplast preparation conditions. The 10-day-old seedlings of maize (Zong3), wheat (Chinese Spring) and rice (Nipponbare) were used to study the effect of enzyme concentration, enzymolysis time and centrifugal force on the yield and vigor of mesophyll cell protoplasts influences. The results showed that the enzyme concentration and enzymolysis time had significant effects on the protoplast yield. With the increase of enzymolysis time and concentration, the protoplast yield increased but the cell debris increased at the same time. After vacuum treatment of rice, protoplast yield increased significantly. The optimal conditions for the isolation of maize mesophyll protoplasts were: 1.5% cellulase, 0.5% isozyme, enzymatic hydrolysis at 50 r / min for 7 h, centrifugation at 100 × g for 2 min, The protoplast yield was 7 × 106 / g FW. The optimal conditions for the isolation of wheat mesophyll protoplasts were: cellulase 1.5%, isozyme 0.5%, enzymatic hydrolysis 50 r / min 5 h, centrifugation at 100 × g for 2 min , And the protoplast yield was 6 × 106 / g FW. The optimum conditions for protoplast isolation of mesophyll cells were: cellulase 2.0%, isozyme 0.7%, enzymatic hydrolysis at 50 r / min for 7 h, centrifugation at 1000 × g min and the resulting protoplast yield was 6 × 10 6 / g FW. The viability of protoplasts was above 90% by fluorescein diacetate staining. The plasmid containing green fluorescent protein was transformed into protoplast by PEG-Ca2 + mediated method, the conversion rate was up to 50% -80%.