京亚和克瑞森葡萄高效再生体系建立
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摘要
转基因技术已发展成为植物育种和分子设计的一种重要手段,当前在葡萄育种中应用的限制因子是再生体系。虽然过去有较多葡萄组织培养的报道,但大多采用侧芽培养的方法,不适合转基因再生要求。本试验以鲜食葡萄品种京亚和克瑞森无核的成年态为试材,经消毒、继代扩繁后,进一步以无菌试管苗的叶片为外植体,建立了叶盘法再生的技术体系,并为转基因技术应用做了部分前期探索。得出以下结论:
1.对单芽茎段采用0.1%升汞和2%NaClO进行消毒,设计了不同消毒时间的组合,实验表明用2%NaClO消毒20 min灭菌效果最好。取材在4月中旬至5月中旬污染最低。
2.初代培养基:京亚采用MS+6-BA 1.0mg/L,克瑞森采用MS+6-BA 2.0mg/L都能有效的诱导腋芽的萌发。
继代增殖培养基:采用MS+6-BA 1.5mg/L+IBA 0.1mg/L可以有效的诱导腋芽的增殖,增殖倍数在5倍以上。
3.克服玻璃化:降低温度、增加琼脂含量都能够缓解组培苗的玻璃化,但都不能完全抑制玻璃化,随着温度的升高及继代次数的增加,玻璃化现象仍然严重。在继代增殖培养基中加入0.4mg/L的活性炭,虽然增殖倍数有所降低,但却可以完全抑制玻璃化,同时还可以为叶盘再生提供稳定而健壮的外植体材料。
4.葡萄叶盘再生体系:克瑞森无核的叶盘再生最佳培养基为MS+6-BA5.0mg/L+IBA0.05mg/L,再生率为11.2%;京亚叶盘再生的最佳培养基为MS+6-BA3.0mg/L+IBA0.05mg/L,再生率为65.2%。前2片展开叶是最理想的外植体,只切断叶片主脉而不将叶片完全切断、将叶片背面接触培养基也可以提高再生率。
5.叶片再生芽生根的适宜培养基为1/2MS+IBA 1.0mg/L+7g/L Agar+30g/LSuc,处理须根多,茎杆粗壮,生根效果最为理想。
6.遗传转化摸索发现:卡那霉素不适合京亚叶片再生筛选,只适合于生根筛选,浓度为10mg/L;Cef抑菌浓度为300 mg/L。
Transgene technology has been developed a kind of most important instrument of plant breeding and molecular design.However,too low regeneration frequency restricted transgene project developing of grape now.Although there were many reports in grape tissue culture,they used axillary bud as explants,these is not fit to transgene regeneration. In this study,Jingya and Crimson Seedless were used as the materials,after sterilize、subculture and multiplication,leaves of plants in vitro were used as explants,established leaf disc regeneration system,a portion prophase research for transgene was done also, and get following conclusion:
1.To sterilize single bud stem with 0.1%HgCl and 2%NaClO solution,and a variety of combinations with unequal sterilization time were designed.The test shows that the 2%NaClO sterilize 20 min has the best effect for eliminating bacterium.The lowest contamination was obtained at April and May.
2.The first generation culture medium:MS + 6-BA 1.0 mg/L is the most suitable for Jingya;MS + 6-BA 2.0 mg/L is the most suitable for Crimson Seedless.
The subculture medium:MS + 6-BA 1.5 mg/L + IBA 0.1 mg/L is the most suitable medium for both of them,and the Multiplication times are up to 5.
3.Inhibit Vitrification:Reduce the temperature and increase agar content can delay the occurrence of vitrification,but it cann't stop the occurrence of vitrification as rising of the temperature and the times of the subculture.With 0.4 g/L active carbon in subculture medium cann't get more multiplication,but it can totally stop vitrification, and can provide stabilization and strong explants for leaf disc regeneration.
4.Leaf regeneration system:The leaf best hormone match of Crimson Seedless was MS + 6-BA 6.0 mg/L + IBA 0.05 mg/L regeneration rate was 11.2%;Jingya was:MS + 6-BA 3.0 mg/L + IBA0.05 mg/L,regeneration rate was 65.2%.we also found that the top 2 leaves is the best explants,Leaf abaxial to medium and partially cut in the leaf vein can improve regeneration rate too.
5.Best of the radication medium of adventitious bud was 1/2MS + IBA 0.5 mg/L+ sucrose 30 g/L + agar 7 g/L.The taproot distributes radially,with strong stem and developed lateral root.It is the ideal result for rhizogenesis.
6.Kanamycin was used in the rooting select but not the leaf select because of the sensitivity,and the selecting concentrate was 10 mg/L,and the concentrate of Cef was 300 mg/L which can inhibit the Agrobacterium effectively.
1.对单芽茎段采用0.1%升汞和2%NaClO进行消毒,设计了不同消毒时间的组合,实验表明用2%NaClO消毒20 min灭菌效果最好。取材在4月中旬至5月中旬污染最低。
2.初代培养基:京亚采用MS+6-BA 1.0mg/L,克瑞森采用MS+6-BA 2.0mg/L都能有效的诱导腋芽的萌发。
继代增殖培养基:采用MS+6-BA 1.5mg/L+IBA 0.1mg/L可以有效的诱导腋芽的增殖,增殖倍数在5倍以上。
3.克服玻璃化:降低温度、增加琼脂含量都能够缓解组培苗的玻璃化,但都不能完全抑制玻璃化,随着温度的升高及继代次数的增加,玻璃化现象仍然严重。在继代增殖培养基中加入0.4mg/L的活性炭,虽然增殖倍数有所降低,但却可以完全抑制玻璃化,同时还可以为叶盘再生提供稳定而健壮的外植体材料。
4.葡萄叶盘再生体系:克瑞森无核的叶盘再生最佳培养基为MS+6-BA5.0mg/L+IBA0.05mg/L,再生率为11.2%;京亚叶盘再生的最佳培养基为MS+6-BA3.0mg/L+IBA0.05mg/L,再生率为65.2%。前2片展开叶是最理想的外植体,只切断叶片主脉而不将叶片完全切断、将叶片背面接触培养基也可以提高再生率。
5.叶片再生芽生根的适宜培养基为1/2MS+IBA 1.0mg/L+7g/L Agar+30g/LSuc,处理须根多,茎杆粗壮,生根效果最为理想。
6.遗传转化摸索发现:卡那霉素不适合京亚叶片再生筛选,只适合于生根筛选,浓度为10mg/L;Cef抑菌浓度为300 mg/L。
Transgene technology has been developed a kind of most important instrument of plant breeding and molecular design.However,too low regeneration frequency restricted transgene project developing of grape now.Although there were many reports in grape tissue culture,they used axillary bud as explants,these is not fit to transgene regeneration. In this study,Jingya and Crimson Seedless were used as the materials,after sterilize、subculture and multiplication,leaves of plants in vitro were used as explants,established leaf disc regeneration system,a portion prophase research for transgene was done also, and get following conclusion:
1.To sterilize single bud stem with 0.1%HgCl and 2%NaClO solution,and a variety of combinations with unequal sterilization time were designed.The test shows that the 2%NaClO sterilize 20 min has the best effect for eliminating bacterium.The lowest contamination was obtained at April and May.
2.The first generation culture medium:MS + 6-BA 1.0 mg/L is the most suitable for Jingya;MS + 6-BA 2.0 mg/L is the most suitable for Crimson Seedless.
The subculture medium:MS + 6-BA 1.5 mg/L + IBA 0.1 mg/L is the most suitable medium for both of them,and the Multiplication times are up to 5.
3.Inhibit Vitrification:Reduce the temperature and increase agar content can delay the occurrence of vitrification,but it cann't stop the occurrence of vitrification as rising of the temperature and the times of the subculture.With 0.4 g/L active carbon in subculture medium cann't get more multiplication,but it can totally stop vitrification, and can provide stabilization and strong explants for leaf disc regeneration.
4.Leaf regeneration system:The leaf best hormone match of Crimson Seedless was MS + 6-BA 6.0 mg/L + IBA 0.05 mg/L regeneration rate was 11.2%;Jingya was:MS + 6-BA 3.0 mg/L + IBA0.05 mg/L,regeneration rate was 65.2%.we also found that the top 2 leaves is the best explants,Leaf abaxial to medium and partially cut in the leaf vein can improve regeneration rate too.
5.Best of the radication medium of adventitious bud was 1/2MS + IBA 0.5 mg/L+ sucrose 30 g/L + agar 7 g/L.The taproot distributes radially,with strong stem and developed lateral root.It is the ideal result for rhizogenesis.
6.Kanamycin was used in the rooting select but not the leaf select because of the sensitivity,and the selecting concentrate was 10 mg/L,and the concentrate of Cef was 300 mg/L which can inhibit the Agrobacterium effectively.
引文
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