早熟棉‘中棉所16’再生纯合系的创制及遗传转化
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摘要
为拓展可再生陆地棉的基因型,利用转基因技术直接改良已有的优良早熟棉品种,设置不同浓度的2,4-D和激动素(KT)4种组合进行诱导,成功获得体细胞胚和再生植株,构建早熟棉品种‘中棉所16’再生体系。研究表明:诱导初生愈伤组织以添加0.1 mg/L 2,4-D、0.1 mg/L KT的组合诱导培养基效果最佳,愈伤组织形态较好,有利于撤除激素的胚胎发生和植株再生;‘中棉所16’胚胎发生状态只有1种,在去掉2,4-D和KT的第一次继代便开始分化出白-灰色粗颗粒胚性愈伤组织,继代1～2次后有胚状体出现和植株再生,整个再生过程需要6～8个月。经过2次组培获得再生系,其不同株系胚胎发生率均有不同程度的明显提高。随后获得后代种子100%可再生纯合系材料,经过进一步自交扩繁,其后代种子用于转化受体,提高了转化和再生效率,并利用该体系将EDT1基因成功导入‘中棉所16’基因组。以9个月为限统计,其遗传转化胚胎发生率高达40%以上。
In order to expand the regenerative genotype of Gossypium hirsutum,a genetic transformation system of early maturing cotton cultivar ‘Zhongmiansuo 16' was constructed by using four different combinations of hormones.The results showed that the primary calluses were effectively induced and produced on the combined media of 0.1 mg/L KT and 0.1 mg/L 2,4-D,and well-formed calluses was beneficial to embryogenesis and plant regeneration after withdrawing KT and 2,4-D.Moreover,only one kind of embryo in ‘Zhongmiansuo 16' was observed,and the embryogenic callus of white-gray graininess were induced and differentiated after the first subculture of withdrawing the hormone.The appearance of embryos occurred after subcultured for 1 to 2 times.The embryos further developed into plantlets.Under this protocol,normal plantlets with strong roots well developed from these cotton cultivars after 6 to 8 months.Different regeneration lines were obtained by two rounds of tissue culture,and the ratio of embryogenesis in different lines was significantly increased.Subsequently,100% regeneration homozygous line seeds were obtained,and the homozygous receptors for transformation were obtained by further expanding propagation,which improved the efficiency of transformation and regeneration.EDT1 gene was successfully introduced into ‘Zhongmiansuo 16'genome by using the genetic transformation system in this study.The statistical analysis data,which was obtained in this study over a period of 9 months,indicated that the ratio of embryogenesis of genetic transformation was as high as 40%.
In order to expand the regenerative genotype of Gossypium hirsutum,a genetic transformation system of early maturing cotton cultivar ‘Zhongmiansuo 16' was constructed by using four different combinations of hormones.The results showed that the primary calluses were effectively induced and produced on the combined media of 0.1 mg/L KT and 0.1 mg/L 2,4-D,and well-formed calluses was beneficial to embryogenesis and plant regeneration after withdrawing KT and 2,4-D.Moreover,only one kind of embryo in ‘Zhongmiansuo 16' was observed,and the embryogenic callus of white-gray graininess were induced and differentiated after the first subculture of withdrawing the hormone.The appearance of embryos occurred after subcultured for 1 to 2 times.The embryos further developed into plantlets.Under this protocol,normal plantlets with strong roots well developed from these cotton cultivars after 6 to 8 months.Different regeneration lines were obtained by two rounds of tissue culture,and the ratio of embryogenesis in different lines was significantly increased.Subsequently,100% regeneration homozygous line seeds were obtained,and the homozygous receptors for transformation were obtained by further expanding propagation,which improved the efficiency of transformation and regeneration.EDT1 gene was successfully introduced into ‘Zhongmiansuo 16'genome by using the genetic transformation system in this study.The statistical analysis data,which was obtained in this study over a period of 9 months,indicated that the ratio of embryogenesis of genetic transformation was as high as 40%.
引文
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[14] 朱华国,张献龙,金双侠,刘冠泽.两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察[J].棉花学报,2012,24(2):159-166Zhu H G,Zhang X L,Jin S X,Liu G Z.Histological observation of the processes during somatic embryogenesis in the two most commonly used PGR regimes in cotton[J].Cotton Science,2012,24(2):159-166 (in Chinese)
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[18] 陈天子,吴慎杰,李飞飞,郭旺珍,张天真.新疆棉花4个主栽品种的体细胞胚胎发生及植株再生[J].作物学报,2008,34(8):1374-1380Chen T Z,Wu S J,Li F F,Guo W Z,Zhang T Z.In vitro regeneration of four commercial cotton (Gossypium hirsutum L) cultivars grown in Xinjiang,China[J].Acta Agronomica Sinica,2008,34(8):1374-1380 (in Chinese)
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