大丽花花瓣高效的原生质体分离体系研究

Study on Efficient Protoplast Isolation System for Dahlia pinnata Petals

为解决大丽花常规育种进程缓慢、现代生物技术育种体系不完善、大丽花遗传转化和基因功能验证体系不成熟等问题,以大丽花花瓣为试验材料,研究分析了原生质体制备过程中的材料处理方式、甘露醇浓度、酶解时间、酶解组合、纯化方式等因素对大丽花原生质体产量和活力的影响。结果表明,大丽花花瓣原生质体分离的最佳组合为:采用切丝法将花瓣组织切成1 mm条带状,置于1.00%纤维素酶+0.50%离析酶+0.40%果胶酶+1.00 mol/L甘露醇的酶解液组合酶解10 h,得到的大丽花花瓣原生质体产量最高,为5.46×10^(6)个/mL,活力最大,为88.83%;建立了高效、稳定、快速的大丽花花瓣原生质体分离体系,可为后续大丽花体细胞融合和基因功能分析研究提供良好的试验平台和技术支持。

In order to solve the problems of slow conventional breeding process,imperfect breeding system of modern biotechnology,and immature system of genetic transformation and gene function verification in Dahlia pinnata,Dahlia pinnata petals were used as experimental materials,and the effects of material treatment,mannitol concentration,enzymolysis time,enzymolysis combination and purification method on the yield and viability of Dahlia pinnata protoplasts were studied.The results showed that the most effective method for isolating Dahlia pinnata petal protoplasts involved cutting the petal tissue into 1 mm strips using the filament-cutting technique.The strips were then placed in an enzyme solution consisting of 1.00% cellulase,0.50% macerozyme,0.40% pectinase,and 1.00 M mannitol for 10 hours.The highest yield of dahlia petal protoplasts was 5.46×10^(6)/mL,and the highest viability was 88.83%.An efficient,stable and rapid system for isolating dahlia petal protoplasts has been established,in order to provide a good experimental platform and technical support for subsequent analysis of Dahlia pinnata somatic cell fusion and gene function.