Shu Yingjie, Huang Liyan, Chen Ming, Tao Yuan, Wang Zhankui, Ma Hao
College of Agronomy, Anhui Science and Technology University, Fengyang 233100, Anhui, China.
State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2017 Jun 25;33(6):976-985. doi: 10.13345/j.cjb.170086.
Young leaves of Kabuli chickpea as well as soybean Xiangdou No.3, which are the current plants that studied in our laboratory were selected as materials. Effects on protoplasts yield and survival rate of different enzyme combination, concentration of D-Mannitol in enzyme combinations, pH of enzyme combinations and enzymolysis time are detected. The results showed that, the best condition for Xiangdou No.3 leaf protoplasts isolation is to rotate the cut materials for 6 hours in enyzme solution under temperature of 27 ℃ and rotate speed of 45 r/min for 6 h. Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%) in combination with Pectolyase Y-23 (0.4%) dissolving in CPW solution with MES (0.1%) and Mannitol (10%), pH 6.0 was found best for protoplasts isolation of Xiangdou No.3 leaves.The best condition for protoplasts isolation of Kabuli chickpea is to put the cut materials into enzymatic hydrolysate enzymolyse for 7 to 8 hours under temperature of 27 ℃ and rotate speed of 45 r/min on water bath shaker, the optimum combination of enzyme consists of Onozuka R-10 (0.5%), Hemicellulase (0.8%), Macerozyme R-10 (0.8%), MES (0.1%) and Mannitol (10%) dissolved in CPW solution with pH 4.8. The protoplasts prepared with the methods above are used in subcellular location and the effects show well.
选用本实验室目前研究的卡布利鹰嘴豆幼叶以及大豆湘豆3号作为材料。检测了不同酶组合、酶组合中D-甘露醇浓度、酶组合pH值和酶解时间对原生质体产量和存活率的影响。结果表明，湘豆3号叶片原生质体分离的最佳条件是将切割后的材料在27℃、转速45 r/min的条件下于酶液中旋转6 h。发现将Onozuka R-10（0.5%）、半纤维素酶（0.8%）、离析酶R-10（0.8%）与果胶酶Y-23（0.4%）溶解于含有MES（0.1%）和甘露醇（10%）的CPW溶液中，pH值为6.0时，最适合湘豆3号叶片原生质体的分离。卡布利鹰嘴豆原生质体分离的最佳条件是将切割后的材料置于酶解液中，在27℃、转速45 r/min的条件下于水浴振荡器上酶解7至8 h，最佳酶组合为将Onozuka R-10（0.5%）、半纤维素酶（0.8%）、离析酶R-10（0.8%）、MES（0.1%）和甘露醇（10%）溶解于pH值为4.8的CPW溶液中。用上述方法制备的原生质体用于亚细胞定位，效果良好。
