多年生黑麦草成熟胚再生体系的建立及基因枪转化  

Production of Transgenic Perennial Ryegrass(Lolium perenne L.) Plants by Particle Bombardment of Mature Seed-deried Highly Regenerative Tissues

作　　者：张永彦[1] 徐子勤[1] 高丽美[1] 黄萱[1] 王健[1] 

机构地区：[1]西北大学生命科学学院,西安710069

出　　处：《中国生物工程杂志》2005年第3期53-57,59,共6页China Biotechnology

基　　金：陕西高等学校重点实验室项目资助

摘　　要：目的 :建立以多年生黑麦草成熟胚为起始材料的再生体系 ,用于基因枪转化。方法 :多年生黑麦草成熟种子在附加 5mg L 2 ,4 D的MS培养基上诱导愈伤组织 ,转至新继代培养基上产生胚性愈伤组织。分化培养基为无激素MS培养基。再生植株在培养基成分减半的无激素MS培养基生根 ,之后移栽至土壤。基于这一再生体系 ,用含有水稻几丁质酶基因RC2 4的质粒pARN6和含有草丁膦乙酰转移酶基因Bar的质粒pDB1 ,通过基因枪轰击胚性愈伤组织。用附加PPT的继代培养基进行转化植株的抗性筛选。结果 :共获得 2 4 3株再生植株。通过PCR进行检测 ,获得1 8株整合有RC2 4基因的植株 ,1 5株整合有Bar基因的植株 ,同时转入 2个基因的植株 2株。An efficient perennial ryegrass regeneration system was developed using mature embryos. Mature seeds were surface-sterilized , they were used as explants to initiate callus on MS induction medium supplemented with 5.0mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). The induction frequency of primary calli was 24% for HUNTER inbred line. The primary calli were then subcultured on callus induction medium. Following three week subculture process, embryoenic calli were formed. The embryogenic calli were transferred to hormone-free MS medium to regenerate.The regeneratants were transferred to 1/2 MS medium without hormone to produce roots The regenerated plantlets were successfully transplant to soil using this system, plantlets were regenerated from two perennial ryegrass inbred lines.Based on this regeneration system, embryonic callus tissue were used for particle bombardment.Two plasmids,pARN6 containing rice basic chitinase(RC24) and pDB1 containing Bar gene were used for co-transformation.Transformed tissues were regenerate at Medium containing low concentration PPT as selective solvent.Up to 243 plants regenerated,through PCR analysis ,18 plants contained RC24 gene,15 plants cotained bar gene, two plants harboured RC24 gene and bar gene simultaneously.

关 键 词：再生植株 再生体系 MS培养基 多年生黑麦草 成熟胚 继代培养基 胚性愈伤组织 激素 基因 转移酶 

分 类 号：Q943.1[生物学—植物学] S511[农业科学—作物学]