【摘要】 【目的】探究百合原生质体分离纯化和培养条件，为百合原生质体融合、再生体系建立、转基因育种等研究奠定基础。【方法】以‘白天堂’百合无菌苗叶片为材料，采用纤维素酶、果胶酶和离析酶组合分离原生质体，采取单因素试验法，对影响原生质体的制备及再生条件的主要因素即渗透压、酶解组合、细胞筛和酶解时间进行筛选与优化。【结果】以百合无菌苗叶片在1.0%纤维素酶+0.5%离析酶+0.1%果胶酶+0.6 mol/L甘露醇+10.0 mmol/L CaCl₂·2H₂O+20.0 mmol/L MES+20.0 mmol/L KCl混合酶解液为最优条件，25℃,25 r/min, pH为5.6黑暗酶解6 h;孔径75μm细胞筛过滤，600 r/min离心5 min收集细胞，原生质体产量可达6.4×10⁵个/mL,原生质体活力为78.0%;将纯化后密度为1×10⁵个/mL的原生质体溶液接种在葡萄糖作为唯一碳源的培养基中可以存活，并生长产生细胞壁，但没有启动细胞分裂和诱导形成愈伤组织。【结论】初步建立‘白天堂’百合的高效原生质体分离与纯化体系，为对原生质体进一步操作研究奠定基础。更多还原

【Abstract】 [Objective] In order to explore the isolation, purification and culture conditions of protoplasts establish an efficient means of preparing and regenerating the fungal protoplasts of Lilium ‘White heaven’, the protoplast fusion, establishment of regeneration system and research on transgenic breeding and so on were studied. [Methods] Driselase at cellulase、pectinase and dissociating enzyme were combined and used for the protoplast preparations. A single-factor experiment was conducted to analyze the effects of osmotic pressure, enzyme concentration, cell sieve and time of the protoplasts. Conditions for the subsequent protoplast regeneration in medium were also screened and optimized. [Results] The results show that the optimal condition is the enzymatic hydrolysate composed of 1.0 % cellulase + 0.5% macerozyme + 0. 6 mol/L mannitol +10.0 mmol/L CaCl₂·2H₂O+20.0 mmol/L MES + 20.0 mmol/L KCl. The cells are collected by centrifugation at 600 r/min for 5 mins after 6 h of enzymatic hydrolysis at 25 ℃,25 r/min in dark and 75μm cell sieve coarse filtration. Then the protoplast yield reaches 6.4×10⁵/mL with the viability of about 78.0%. Purified protoplasts can grow in a medium with a density of 1×10⁵/mL and glucose as the only carbon source, with cells surviving and producing cell walls, but no cell division was initiated and no callus was formed. [Conclusion]These results would have laid a foundation for the research of protoplast of Lilium ‘White heaven’.更多还原