李余润 王馨 杨丽英 杨斌 宁玲 董志渊

摘    要：原生质体是进行单细胞测序和基因编辑研究的理想材料。为了研究不同酶解时间对灯盏花原生质体分离的影响，采用纤维素酶1%+果胶酶0.3%+半纤维素酶0.5%酶液处理愈伤组织悬浮细胞2，3，4，5，6 h，纤维素酶0.5%+果胶酶0.2%+离析酶0.5%酶液处理幼叶4，6，8，10，12，14，16 h。结果表明，不同酶解时间，灯盏花悬浮细胞和幼叶的原生质体产量及活力均差异显著。悬浮细胞酶解5 h，原生质体的产量和活力达到最高，分别为3.73×105个·g-1 、75.84%;幼叶酶解12 h，原生质体产量最高，达到1.17×106个·g-1，酶解10 h，原生质体活力最高，为80.98%。叶片的原生质体产量和活力明显高于悬浮细胞。

关键词：灯盏花;原生质体;酶解时间;产量;活力

中图分类号：S567.2                文献标识码：A                DOI 编码：10.3969/j.issn.1006-6500.2020.06.004

Abstract： Protoplast is an ideal material for single-cell sequencing and gene editing. In order to optimize enzymatic hydrolysis times for the protoplast isolation of Erigeron breviscapus， suspension cells were incubated in an enzyme solution containing 1% cellulase， 0.3% pectinase and 0.5% hemicellulase for the 2，3，4，5，6 h and young leaves were incubated in an enzyme solution containing 0.5% cellulase， 0.2% pectinase and 0.5% macerozyme for the 4，6，8，10，12，14，16 h. The results showed enzymatic hydrolysis times significantly affect protoplast yield and viability. Five-hour enzyme incubation produced the maximum yield （3.73×105 protoplasts·g-1） and viability（75.84%） of protoplasts from suspension cells. Using young leaves as materials， the maximum yield（1.17×106 protoplasts·g-1） of the protoplasts was obtained at 12 h and the maximum viability（80.98%） was obtained at 10 h. The yield and viability of protoplasts from young leaves were both higher than those from suspension cells.

Key words： Erigeron breviscapus; protoplast; enzymatic hydrolysis time; yield; viability

燈盏花（Erigeron breviscapus）为菊科飞蓬属多年生草本植物，全草入药，具有祛风散寒、活血通络止痛等功效[1]。药理研究表明，灯盏花含有黄酮、咖啡酸酯、挥发油等有效成分，具有神经保护、心脑血管保护、抗氧化、抗癌以及抗炎等药理作用[2]。以灯盏花为主要原料，已生产出灯盏花素片、灯盏细辛胶囊、灯盏细辛注射液以及灯盏花素注射液等药品[3]。

灯盏花原料种植中，主要采用种子进行育苗[4]。异花授粉、自交不亲和的繁育特性造成种植群体遗传变异大，有效成分的不稳定[5-7]。生产中，急需一种高效的单株无性快繁技术，培育基因型一致、遗传稳定的种苗。目前，以灯盏花叶片为外植体，初步建立了愈伤组织诱导、不定芽分化增殖及壮苗生根的快繁体系，但不定芽诱导系数较低[8-9]。……