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中国农学通报

中国农学通报 ›› 2019, Vol. 35 ›› Issue (34): 64-71.doi: 10.11924/j.issn.1000-6850.casb20190700384

• 林学 园艺 园林 • 上一篇    下一篇

山丹丹原生质体的分离条件探究

王珺华,王凯琪,王凯,孙志宏,曹园,齐向英   

  1. 1.延安大学生命科学学院;2.延安大学;3.延安大学(陕西省区域生物资源保育与利用工程技术研究中心）;4.延安大学(陕西省区域生物资源保育与利用工程技术研究中心)
  • 收稿日期:2019-07-04 修回日期:2019-09-16 接受日期:2019-09-20 出版日期:2019-12-09 发布日期:2019-12-09
  • 通讯作者: 齐向英
  • 基金资助:
    国家自然科学基金审批项目“延安地区野生山丹丹种质资源收集、保护与筛选研究”(3165007)；延安大学校级科研项目“山丹丹种球室内 标准化生产工艺研究”(YDQ2014-41)；延安大学校级大学生创新创业训练计划项目“山丹丹多花亭新品种培育”(D2017080)。

Separation Conditions of Protoplasts of Lilium pumilum

  • Received:2019-07-04 Revised:2019-09-16 Accepted:2019-09-20 Online:2019-12-09 Published:2019-12-09

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摘要： [目的]本研究旨在建立山丹丹高效原生质体分离体系,为山丹丹种质资源的有效利用提供科学依据。[方法]以山丹丹鳞茎胚性愈伤组织为材料,通过酶解法获得山丹丹原生质体,进一步采用3因素4水平L16(34)正交试验 。[结果]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3.47×106个/ mL；(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>果胶酶浓度>甘露醇浓度,同时得到最佳酶组合为0.02 g/mL的纤维素酶浓度、0.008g/mL的果胶酶浓度和最佳渗透压浓度为0.6 mol/L甘露醇,原生质体产量最高为3.47×106个/ mL,活力为60.32%；(3)在最佳酶组合处理下,采用暗处理可将原生质体存活率提高到76.15 %；在15 min、1000 r/min时,原生质体产量最高,为3.51×106/mL；在10 min、800 r/min时,原生质体活力最高,为61.2 %。 [结论] 该试验获得了山丹丹原生质体最佳分离条件,建立原生质体分离体系,为山丹丹种质资源的有效利用提供研究奠定基础。

关键词: 迎春花, 迎春花, 组织培养, 快速繁殖技术, 试管苗, 生根率

Abstract: [Objective] The present study aim to establish an efficient and stable separation method of Lilium pumilum DC. callus protoplast, to provide scientific basis for Lilium pumilum DC. breeding research. [Method]The design method of L16(34) orthogonal experimental was adopted to optimize the combination conditions. The effects of some factors on the separation of Lilium pumilum DC. callus protoplast was investigated, including enzymolysis time, enzyme combination, osmotic pressure, pretreatment,and centrifugal speeds.[Result] (1)Pretreatment was carried out under the conditions of 0.015 g/mL cellulase + 0.005 g/mL pectinase + 0.65 mol/L mannitol. The results showed that the optimal hydrolysis time was 6 h and the yield was 3.47×106 cells/mL.(2) According to the results of the orthogonal test ,the optimum condition for protoplast isolation from Lilium pumilum DC. included a mixed enzyme solution of 0.02 g/mL cellulase、0.008 g/mL pectolyase、0.6 mol/L mannitol. High activity and pure protoplasts could be prepared by using the optimum condition, protoplast yield up to 3.465×106 cells/mL, viability of 60.32%;(3) Under the optimal enzyme combination treatment,Under these conditions, the dark matter treatment could increase the protoplast survival rate to 76.15%; At 15 min and 1000 r/min, the protoplast yield was the highest at 3.51×106/mL; at 10 min and 800 r/min, the protoplast activity was the highest at 61.2%.[Conclusion]The experiment obtained the best separation conditions of protoplasts of Lilium pumilum DC. and established a protoplast isolation system, which laid a foundation for further research on Lilium pumilum DC. breeding.