Enzymolysis and Isolation Conditions of Protoplasts from Cotyledons and Calli in Lotus corniculatus

doi:10.11733/j.issn.1007-0435.2010.06.009

›› 2010, Vol. 18 ›› Issue (6): 798-804.DOI: 10.11733/j.issn.1007-0435.2010.06.009

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Enzymolysis and Isolation Conditions of Protoplasts from Cotyledons and Calli in Lotus corniculatus

LI Yu-zhu¹, TAO Rong¹, WANG Juan¹, SHI Shang-li¹, MA Hui-ling¹, ZHAO Xiao-qiang²

1. Pratacultural College, Gansu Agricultural University, Key Laboratory of Grassland Ecosystem, Ministry of Education, Sino-US Centers for Grazingland Ecosystem Sustainability, Lanzhou, Gansu Province 730070, China;
2. College of Biological Sciences, China Agricultural University, Beijing 100193, China

Received:2010-06-01 Revised:2010-11-11 Online:2010-12-15 Published:2010-12-15

百脉根原生质体的分离和酶解条件的研究

李玉珠¹, 陶茸¹, 王娟¹, 师尚礼¹, 马晖玲¹, 赵小强²

1. 甘肃农业大学草业学院, 草业生态系统教育部重点实验室, 中美草地畜牧业可持续发展研究中心, 甘肃, 兰州, 730070;
2. 中国农业大学生物学院, 北京, 100193

通讯作者: 师尚礼,E-mail:shishl@gsau.edu.cn
作者简介:李玉珠(1979),女,陕西宁强人,博士研究生,主要从事牧草和草坪草种质资源保护与育种的研究,E-mail:liyz@gsau.edu.cn
基金资助:
国家“十一五”科技支撑计划(2007BAD52B06);现代农业产业技术体系建设专项资金;人工草地优质牧草生产技术研究与示范(nyhyzx07-022)资助

Abstract

Abstract: In vitro-grown hypocotyls and cotyledons of Lotus corniculatus served as explant sources for studies reported here.Protoplasts were isolated from cotyledons,and embryogenic calli induced from hypocotyls and cotyledons.The effects of different enzymolysis conditions on protoplast isolation of Lotus corniculatus were investigated to get foundation data of somatic cell hybridization and breeding.Results show that the best enzyme combinations for both cotyledons and calli are 2% cellulase onozuka R-10+0.5% pectinase Y-23+0.3% hemicellulose and 0.55 mol·L^-1 mannitol.The optimum enzymolysis time is 6 h for cotyledons and 12 h for calli.The yield and viability of protoplasts are 4.13×10⁶/g,65% and 3.8×10⁶/g,67.4%,respectively.Results indicate that both yield and viability of calli protoplasts significantly increase(P<0.01)through pre-culture for 8~10 d,preplasmolyzing with CPW-10 for 1 h,or under darkness for 1 d.

Key words: Lotus corniculatus, Cotyledons, Calli, Enzymolysis conditions, Protoplasts

摘要： 分别以百脉根里奥(Lotus corniculatus L.cv.Leon)无菌苗子叶及由下胚轴和子叶形成的胚性愈伤组织为供体材料,进行原生质体游离,研究不同酶解条件对原生质体分离的影响,以期为进一步开展属间体细胞杂交及培育新品种奠定研究基础。结果表明:子叶和愈伤组织原生质体分离最适的酶液组合均为2%纤维素酶+0.5%果胶酶+0.3%半纤维素酶、以及0.55 mol·L^-1甘露醇,产量及活力分别可达4.13×10⁶个·g^-1,65%和3.8×10⁶个·g^-1,67.4%;最佳酶解时间分别为6 h和12 h。预培养8~10 d,CPW-10溶液中预质壁分离1 h或预暗培养1 d等预处理措施可显著提高愈伤组织原生质体的产量及活力(P<0.01)。

关键词: 百脉根, 子叶, 愈伤组织, 酶解条件, 原生质体

CLC Number:

LI Yu-zhu, TAO Rong, WANG Juan, SHI Shang-li, MA Hui-ling, ZHAO Xiao-qiang. Enzymolysis and Isolation Conditions of Protoplasts from Cotyledons and Calli in Lotus corniculatus[J]. , 2010, 18(6): 798-804.

李玉珠, 陶茸, 王娟, 师尚礼, 马晖玲, 赵小强. 百脉根原生质体的分离和酶解条件的研究[J]. , 2010, 18(6): 798-804.

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Enzymolysis and Isolation Conditions of Protoplasts from Cotyledons and Calli in Lotus corniculatus

百脉根原生质体的分离和酶解条件的研究

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