提高甘草原生质体游离产量及分裂频率的研究

为探讨影响胀果甘草(Glycyrrhiza inflata Batal.)原生质体游离的因素,提高原生质体游离产量和分裂频率,试验采用纤维素酶、果胶酶、离析酶不同浓度组合的酶解液,在3种酶解方式下对甘草叶片、下胚轴、愈伤组织进行了原生质体游离。结果表明:苗龄11 d的甘草叶片、下胚轴及继代5次的愈伤组织是原生质体游离的良好材料,三者均在2.0%纤维素酶+0.5%果胶酶和2.0%纤维素酶+0.25%离析酶+0.5%果胶酶的酶解液中原生质体游离频率最高,其产量和活力分别为1.25×10⁵~1.26×10⁵个·g^-1,65.7%~70.5%;1.43×10⁵~1.44×10⁵个·g^-1,69.5%~72.8%;1.10×10⁵~1.16×10⁵个·g^-1,61.9%~69.4%;纵向切割的下胚轴平均原生质体产量和活力高于叶片和愈伤组织,分别为1.19×10⁵个·g^-1,65.2%。此外,在40 r·min^-1摇床上处理7 h的酶解混合物中原生质体产量最高;而先静置后缓慢震荡(40 r·min^-1)的酶解方式可得到最佳原生质体产量和活力。将原生质体密度调整到1×10⁵个·g^-1并培养于KM8P培养基中,能获得最高频率的原生质体分裂频率(2.34%)。试验获得了较好的甘草原生质体游离及分裂体系。

Improving Protoplast Dissociation Yield and Division Frequency of Glycyrrhizia inflata Batal

This study reports influencing factors of protoplast isolation,protoplast yield and division frequency of Glycyrrhizia inflata Batal..Five enzyme combinations with different concentrations of cellulase,pectolase,macerozyme and three enzymolysis methods were used to dissociate the protoplast of leaves,hypocotyl and calli from Glycyrrhizia inflata Batal..Results show that the best materials for protoplast isolation are leaves and hypocotyls from 11-day seedlings as well as calli subcultured five times.The highest frequency of protoplast isolation is obtained by using either 2.0% cellulase+0.5% pectolase,or 2% cellulase+0.25% macerozyme+0.5% pectolase.Protoplast yield and viability for each of three methods are 1.25×105～1.26×105 individual·g-1 and 65.7%～70.5%;1.43×105～1.44×105 individual·g-1 and 69.5%～72.8%;1.10×105～1.16×105 individual·g-1 and 61.9%～69.4%,respectively.The average yield and viability of protoplast from hypocotyls cut longitudinally(1.19×105 individual·g-1 and 65.2%) was higher than from leaves and calli.Furthermore,the highest protoplast yield was obtained by shaking(40 r·min-1) 7h after enzymolysis.However,both optimizing protoplast yield and viability were reached after standing a while then shaking(40 r·min-1).The highest rate of protoplast division(2.34%) was obtained by adjusting the protoplast density to 1×105individual·g-1 then cultured in KM8P medium.