ZHANG Li, YANG Dongxu, KONG Xiaoping, LI Xuaner, HAN Nuo, LIU Yumei, HAN Fengqing, YAN Xiangping, ZHANG Xiaomei, LI Zhansheng, XU Jin
To establish and optimize an efficient protoplast differentiation and regeneration system for different genotypes of broccoli（Brassica oleracea L. var. italic），thereby providing technical support for subsequent biotechnological breeding approaches such as gene editing and somatic hybridization，this study primarily used cotyledons，true leaves，and hypocotyls of two broccoli genotypes（B1 and B16）as experimental materials．It systematically investigated the effects of enzymatic hydrolysis systems，pretreatment duration，and osmotic pressure conditions on protoplast isolation efficiency，and optimized the key parameters governing protoplast differentiation and regeneration．The main methodologies included the following：a single-factor experimental design was adopted to screen the optimal concentration combination of cellulase and pectinase；gradient pretreatment durations ranging from 12 h to 48 h were set to analyze the suitability of different tissue materials；mannitol was used as the osmotic stabilizer to optimize the appropriate concentrations of both the enzymatic hydrolysis solution and the washing buffer（W5）；a complete regeneration protocol was constructed through sequential processes of protoplast culture，callus induction，and differentiation culture．The results demonstrated that the optimal enzymatic hydrolysis system for broccoli protoplast isolation consisted of 0.8%（m/V）celluase R-10 + 0.1%（m/V）pectolyase Y-23，with a 13 h hydrolysis period sufficient to achieve complete enzymatic digestion of leaf tissues．Significant variations were observed in the optimal pretreatment durations among different tissue types：24-48 h for cotyledons，12-24 h for true leaves，and 48 h for hypocotyls．Under these optimized conditions，the protoplast yield reached 144 × 105-195 × 105 cells · g-1（fresh weight，FW），with viability maintained at 91.5%-96.9%．Osmotic pressure adaptability analysis revealed that the optimal mannitol concentration in the enzymatic hydrolysis solution for cotyledon protoplasts
was 0.5 mol · L-1，with the corresponding washing buffer being W5 solution containing 0 mol · L-1 mannitol．In contrast，true leaf protoplasts required a higher osmotic pressure environment，with the optimal mannitol concentrations in the enzymatic hydrolysis solution and washing buffer being 0.7 mol · L-1 and 0.2 mol · L-1，respectively．The optimized system enabled efficient acquisition of high-yield and high-viability broccoli protoplasts，which could be successfully induced to form calli and complete differentiation and regeneration upon culture．Although the regeneration efficiency exhibited certain variations among different genotypes，all genotypes maintained stable performance．The broccoli protoplast differentiation and regeneration system established in this study exhibits strong practicability and reliability，thus providing critical technical support for molecular breeding and gene function research of broccoli．
