Establishment of High Efficient Regeneration System of Perennial Ryegrass and PEPC Gene Transformation Mediated by Agrobacterium tumefaciens

doi:10.11733/j.issn.1007-0435.2012.04.024

›› 2012, Vol. 20 ›› Issue (4): 747-752.DOI: 10.11733/j.issn.1007-0435.2012.04.024

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Establishment of High Efficient Regeneration System of Perennial Ryegrass and PEPC Gene Transformation Mediated by Agrobacterium tumefaciens

LI Wei, ZHANG Na, LI Ren, ZHAO Bing, GUO Yang-dong

College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China

Received:2012-02-14 Revised:2012-03-27 Online:2012-08-15 Published:2012-08-28

多年生黑麦草高频再生体系的建立及农杆菌介导PEPC基因的转化

李蔚, 张娜, 李仁, 赵冰, 郭仰东

中国农业大学农学与生物技术学院, 北京 100193

通讯作者: 郭仰东
作者简介:李蔚(1987-),女,吉林白山人,硕士研究生,研究方向为植物遗传育种与生物技术,E-mail:liwei2010cau@sina.cn
基金资助:
"十二五"国家科技支撑计划"优质多抗牧草新品种选育与良种繁育关键技术研究与示范"(2011BAD17B01);国家自然科学基金项目(31171989);中央高校基本科研业务专项(2009-2-06)资助

Abstract

Abstract: In order to establish a highly efficient regeneration and transformation system of perennial ryegrass (Lolium perenne L.), its mature seeds were used as explants. Result showed that the best induction rate was 61.33%. Callus differentiated after 2~3 generation. The optimum medium for callus differentiation was MS+6-BA at 1.0 mg稬^-1 with 61.33% regeneration rate. The best medium for rooting was 1/2 MS+NAA at 0.5 mg稬^-1 and the ratio of rooting was 100%. Based on this regeneration system, the embryonic callus cells were used for Agrobacterium-mediated transformation. Transformed tissues were regenerated with media containing hyg as a selective solvent. PCR and Real-Time PCR analysis of transformants showed that PEPC gene was successfully integrated into the genome of perennial ryegrass and transformation rate was 8.67%.

Key words: Perennial ryegrass (Lolium perenne L.), Embryogenic callus, Plant regeneration, PEPC gene, Transformation

摘要： 以多年生黑麦草(Lolium perenne L.)成熟种子作为外植体,建立其高频再生体系,通过农杆菌介导法将磷酸烯醇式丙酮酸羧化酶(PEPC)基因转入,为多年生黑麦草的抗逆转基因工作奠定基础。结果表明:种子充分灭菌后在附加6.0 mg·L^-1 2,4-D的培养基中诱导愈伤组织,诱导率可达61.33%。愈伤组织在2,4-D浓度减半的培养基上继代培养长势良好,分化培养基为MS培养基附加1.0 mg·L^-16-BA,分化率最高达到61.33%,在1/2 MS+0.5 mg·L^-1 NAA培养基中进行生根培养,生根率达100%。以该再生体系为基础,将获得的愈伤组织作为外植体进行遗传转化,经筛选培养后得到再生植株,通过PCR及实时荧光定量(Real-Time)PCR验证表明PEPC基因已成功转入,转化率为8.67%。试验结果将为多年生黑麦草抗性品种的研究奠定基础。

关键词: 多年生黑麦草, 胚性愈伤组织, 植株再生, 磷酸烯醇式丙酮酸羧化酶(PEPC)基因, 遗传转化

CLC Number:

Q943.2

LI Wei, ZHANG Na, LI Ren, ZHAO Bing, GUO Yang-dong. Establishment of High Efficient Regeneration System of Perennial Ryegrass and PEPC Gene Transformation Mediated by Agrobacterium tumefaciens[J]. , 2012, 20(4): 747-752.

李蔚, 张娜, 李仁, 赵冰, 郭仰东. 多年生黑麦草高频再生体系的建立及农杆菌介导PEPC基因的转化[J]. , 2012, 20(4): 747-752.

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Establishment of High Efficient Regeneration System of Perennial Ryegrass and PEPC Gene Transformation Mediated by Agrobacterium tumefaciens

多年生黑麦草高频再生体系的建立及农杆菌介导PEPC基因的转化

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