山丹原生质体的分离条件探究

Separation Conditions of Protoplasts of Lilium pumilum

本研究旨在建立山丹高效原生质体分离体系,为山丹种质资源的有效利用提供科学依据。以山丹鳞茎胚性愈伤组织为材料,通过酶解法获得山丹原生质体,进一步采用3因素4水平L16(34)正交试验,对原生质体分离条件进行优化研究。结果表明:(1)在预处理条件下(0.015 g/mL纤维素酶+0.005 g/mL果胶酶+0.65 mol/L甘露醇)得出最佳酶解时间为6 h,产量为3.47×10^6个/g。(2)对正交试验结果进行极差分析,确定原生质体产量主次因素为纤维素酶浓度>果胶酶浓度>甘露醇浓度,同时得到最佳酶组合为0.02 g/mL纤维素酶、0.008 g/mL果胶酶,最佳渗透压浓度为0.6 mol/L甘露醇,原生质体产量最高为3.47×10^6个/g,活性为60.32%。(3)在最佳酶组合处理下,采用暗处理可将原生质体活性提高到76.15%;在15 min、1000 r/min时,原生质体产量最高,为3.51×10^6个/g;在10 min、800 r/min时,原生质体活性最高,为61.2%。试验获得了山丹原生质体最佳分离条件,建立原生质体分离体系,可为山丹种质资源的有效利用提供参考。

The present study aims to establish an efficient and stable separation method of Lilium pumilum callus protoplast, to provide a basis for L. pumilum breeding research. The design method of L16(34) orthogonal experiment was adopted to optimize the combination conditions. The effects of some factors on the separation of L. pumilum callus protoplast were investigated, including digestion time, enzyme combination, mannitol concentration, methods of pretreatment, and centrifugal speeds. The results indicated that:(1) under the pretreatment conditions(0.015 g/mL cellulase+0.005 g/mL pectinase+ 0.65 mol/L mannitol), the optimal enzymolysis time was 6 h and the yield was 3.47×10^6 cells/g;(2) the range analysis of orthogonal test results showed that the primary and secondary factors of protoplast yield were cellulase>pectinase concentration>mannitol concentration;at the same time, the optimal enzyme combination was 0.02 g/mL cellulase+0.008 g/mL pectinase, the optimal osmotic pressure concentration was 0.6 mol/L mannitol, the highest protoplast yield was3.47×10^6 cells/g, and the activity was 60.32%;(3) under the optimal enzyme combination treatment, the protoplast activity could be increased to 76.15% by dark treatment;at 15 min and 1000 r/min, the highest protoplast yield was 3.51×10^6 cells/g;at 10 min and 800 r/min, the highest protoplast activity was 61.2%. The experiment obtained the best conditions for protoplasts isolation of L. pumilum, it could provide necessary help for protoplasts culture, cellfusion and genetic transformation of L. pumilum.