二穗短柄草幼胚再生体系及农杆菌介导转化的初步研究

草业学报 ›› 2010, Vol. 19 ›› Issue (5): 9-16.

二穗短柄草幼胚再生体系及农杆菌介导转化的初步研究

吴雪莉¹,刘金星²,Klaus K Nielsen³,杨志民^1*,高彩霞^2,3*

1.南京农业大学园艺学院,江苏南京 210095;
2.中国科学院遗传与发育生物学研究所, 植物细胞与染色体工程
国家重点实验室,北京 100101;
3.Research Division,DLF-TRIFOLIUM A/S,丹麦 466060

收稿日期:2009-10-10 出版日期:2010-05-25 发布日期:2010-10-20
作者简介:吴雪莉(1982-),女,山东青岛人,在读硕士。E-mail:xueli19821201@live.cn
基金资助:
欧共体(EU)第七框架课题Renewall资助

Agrobacterium-mediated transformation of Brachypodium distachyon through embryogenic calli derived from immature embryos

WU Xue-li¹, LIU Jin-xing², Klaus K Nielsen³,YANG Zhi-min¹,GAO Cai-xia^2,3

1.College of Horticulture, Nanjing Agricultural University, Nanjing 210095,China;
2.Institute of
Genetics and Developmental Biology Chinese Academy of Sciences, State Key Laboratory of
Plant Cell and Chromosome Engineering, Beijing 100101,China;
3.Research Division,
DLF-TRIFOLIU M A/S 466060, Denmark

Received:2009-10-10 Online:2010-05-25 Published:2010-10-20

摘要/Abstract

摘要： 以二穗短柄草3个品系BDR018,BD21,BD21-3的幼胚为外植体,研究了幼胚大小对胚性愈伤组织诱导和再生的影响。采用携带gus和bar基因双元表达载体(pDM805)的根癌农杆菌菌株AGL1对BDR018品系的幼胚愈伤组织进行了遗传转化。本实验探讨了影响幼胚愈伤组织诱导再生及遗传转化的几个因素。结果发现,幼胚大小介于0.5～1.0mm之间的愈伤组织诱导率最高。随愈伤组织年龄的增加,BDR018,BD21,BD21-3愈伤组织的再生率下降,白化率上升。愈伤组织年龄在5～8周范围内转化效率较高,平均转化效率为38.5%。真空处理5min和0.01%的SilwetL-77处理均可提高转化效率。对转化植株进行GUS基因化学组织检测和PCR鉴定,初步证明外源基因已整合再生植株基因组中。

Abstract: A very efficient transformation system, using Agrobacterium-mediated transformation, has been developed for the production of transgenic plants of Brachypodium distachyon. Embryogenic calli initiated from immature embryos were transformed with Agrobacterium tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. Transformation efficiency was strongly influenced by genotype, size of immature embryos and age of embryogenic callus. An average transformation efficiency of 38.5% was obtained using 5 to 8 week-old embryogenic callus as transformation targets and the highest transformation efficiency achieved was 43.1%. The existence and integration of the bar gene in transgenic plants was confirmed by PCR (polymerase chain reaction).

中图分类号:

吴雪莉,刘金星,Klaus K Nielsen,杨志民,高彩霞. 二穗短柄草幼胚再生体系及农杆菌介导转化的初步研究[J]. 草业学报, 2010, 19(5): 9-16.

WU Xue-li, LIU Jin-xing, Klaus K Nielsen,YANG Zhi-min,GAO Cai-xia. Agrobacterium-mediated transformation of Brachypodium distachyon through embryogenic calli derived from immature embryos[J]. Acta Prataculturae Sinica, 2010, 19(5): 9-16.

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