【摘要】 为建立一套稳定高效的小麦叶肉细胞原生质体制备技术流程，以10 d苗龄的普通小麦‘济麦22号’第一片真叶为实验材料，采用正交实验确定原生质体制备的最佳条件。结果表明：酶解液中纤维素酶浓度为1.5%、离析酶浓度为0.75%、甘露醇浓度为0.8 mol/L,25℃黑暗条件下酶解3 h得到的原生质体完整性最好，原生质体产量达6.97×106个/mL，活性达93.99%，均高于其他组合。在原生质体浓度为2×105个/mL、PEG-4000浓度为25%、质粒浓度为1μg/μL及转化时间为20 min的条件下，利用PEG-Ca2+介导法将表达绿色荧光蛋白的重组质粒pAN580载体瞬时转化原生质体后，转化效率达到65.22%。该研究优化了小麦叶片原生质体的制备方法，有较好的应用价值。更多还原

【Abstract】 In order to establish a stable and efficient protocol for isolating wheat mesophyll cell protoplasts, the first true leaves of ’Jimai22’ at 7 day seedling age was used as the experimental material, and orthogonal experiment was used to determine the optimum conditions for the isolation of protoplasts. The results showed that with the cellulase concentration of 1.5%, macerozyme concentration of 0.75%, mannitol concentration of 0.8 mol/L, the protoplast damage was the least, the highest protoplast yield of 6.97×106was obtained, with little protoplast damage and activity of 93.99%. Under the conditions of protoplast concentration of 2×105cells/m L, PEG concentration of 25%, plasmid concentration of 1 μg/μL and transformation time of 20 min, the recombinant plasmid pAN580was transformed into protoplast by PEG-Ca2+mediated method, and the transformation efficiency reached to 65.22%.The study optimized the preparation method of wheat leaf protoplasts, which has good application value.更多还原