【目的】建立高效的‘冬枣’(Ziziphus jujuba ‘Dongzao’)果实原生质体分离技术体系,系统分析不同因素对‘冬枣’果实原生质体提取的影响,为后续基因功能研究及果实发育机制解析提供技术支撑。【方法】采用半红期‘冬枣’果实为试验材料,以65 mmol/L 氯化钙(CaCl₂)、10 mmol/L 2-吗啉乙磺酸(MES)、1 mmol/L 二硫苏糖醇(DTT)混合溶液(pH为5.8)为基础,通过分析不同质量浓度酶液(纤维素酶、离析酶、果胶酶)、氯化钾浓度(150、205、255、305、355、405、430、455、505 mmol/L)、酶解时间(2.0、2.5、3.0、3.5、4.0 h)、真空(200 Pa)处理时间(10、20、30 min)4个条件组合,经过多因素试验筛选出纤维素酶、果胶酶、离析酶、氯化钾的最适浓度,最后确定分离‘冬枣’果实原生质体的最优酶解方案。【结果】以酶解液为0.02 g/mL纤维素酶、0.01 g/mL果胶酶、0.01 g/mL离析酶、430 mmol/L氯化钾(KCl),采用200 Pa真空处理20 min,于水平摇床上30 r/min避光酶解3.0 h,原生质体产量可达1.82×10⁶个/mL,且原生质体细胞形态完整、活性最佳。【结论】通过对酶浓度、酶解时间、氯化钾浓度、真空处理时间4个酶解影响因素的筛选,得到可以提取出细胞形态饱满、活力较高的原生质体的酶解液组合,建立了高效的‘冬枣’果实原生质体分离技术体系,为后续‘冬枣’基因功能验证及果实发育的分子机制研究提供技术支持。

【Objective】This study aims to establish an efficient protoplast isolation system for Ziziphus jujuba ‘Dongzao’ fruit. By systematically analyzing the effects of various factors on protoplast extraction, it seeks to provide technical support for subsequent research on gene function and the molecular mechanisms underlying fruit development.【Method】Using semi-red fruits of Z. jujuba ‘Dongzao’ as experimental material, a multi-factor experimental design was applied to optimize key parameters. The factors investigated included: enzyme combination (cellulase, macerozyme, pectinase), KCl concentration (150, 205, 255, 305, 355, 405, 430, 455, 505 mmol/L), enzymatic hydrolysis duration (2.0, 2.5, 3.0, 3.5, 4.0 h), and vacuum (200 Pa) infiltration time (10, 20, 30 min). Through systematic screening, the optimal concentrations of cellulase, pectinase, macerozyme, and KCl were determined, leading to the identification of the most effective enzyme solution for protoplast isolation.【Result】The optimal enzymatic solution consisted of 0.02 g/mL cellulase, 0.01 g/mL pectinase, 0.01 g/mL macerozyme, 430 mmol/L KCl, 65 mmol/L CaCl₂, 10 mmol/L MES (2-morpholinoethanesulfonic acid), and 1 mmol/L DTT (dithiothreitol), adjusted to pH 5.8. The protoplast yield reached 1.82 × 10⁶ /mL when the tissues were subjected to vacuum infiltration at 200 Pa for 20 min, followed by 3.0 h of enzymatic digestion in the dark on a horizontal shaker. Under these conditions, the isolated protoplasts exhibited intact morphology and high viability.【Conclusion】By screening four key factors—enzyme concentration, hydrolysis time, KCl concentration, and vacuum treatment duration—an efficient enzymatic protocol was established for isolating protoplasts with intact cellular structure and high viability from Z. jujuba ‘Dongzao’ fruit. This study successfully develops a robust protoplast separation system, which provides a reliable technical foundation for future genetic functional studies and the molecular analysis of fruit development in Z. jujuba ‘Dongzao’.