玉米、小麦、水稻原生质体制备条件优化

Optimized condition for protoplast isolation from maize, wheat and rice leaves

摘要:

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％。

Abstract:

Maize (Zea mays L.), wheat (Triticum aestivum L.) and rice (Oryza sativa L.) are three staple crops and accordingly it is very meaningful to optimize the condition of their protoplasts isolation. The concentration of the enzyme, the time of isolation and centrifugal force in protoplast isolation were investigated to find their effects on protoplast yield and viability using leaves of maize (Zong 3), wheat (Chinese Spring) and rice (Nipponbare). The results show that the concentration of the enzyme and the time of isolation affected the protoplast yield significantly. Although the yield of protoplast was increased with high concentration of enzyme and long incubated time, it led to too much cells breakdown. The orthogonal experimental design results show that the best condition of maize protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 7 h, 100×g 2 min and the protoplasts yield was 7×106 cells/g fresh weight (FW); the best condition of wheat protoplast isolation was Cellulase R-10 1.5%, Macerozyme R-10 0.5%, 50 r/min 5 h, 100×g 2?min and the protoplasts yield was 6×106 cells/g FW; the best condition of rice protoplast isolation was Cellulase R-10 2.0%, Macerozyme R-10 0.7%, 50 r/min 7 h, 1 000×g 2 min and the protoplasts yield was 6×106 cells/g FW. The vitalities were more than 90% using fluorescein diacetate staining method. 50%?80% transformation efficiency was obtained when protoplasts were transformed by green fluorescent protein using PEG-Ca2+ method.