银白杨再生体系建立及rolB-pttGA20ox双价基因遗传转化促进生根和高生长的研究
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摘要
银白杨(Populus alba)是我国西北地区重要的速生造林树种,其材质优良,抗逆性强,但扦插繁殖不易生根,生长速度也比其他白杨慢,在很大程度上限制了这一优良树种的推广与应用。本研究的主要目标是通过农杆菌介导的遗传转化方法导入rolB-pttGA20ox双价基因,利用基因工程技术开展改良生根性状的转基因研究,从而培育扦插生根能力强、生长快的银白杨新品种。
围绕这一目标,本研究以银白杨当年生枝条的带芽茎段为外植体,首先建立了该树种的离体快繁及植株再生体系,并就培养条件和植物生长调节剂等因素对银白杨叶片直接再生体系的影响进行了详细的研究;然后利用NPTⅡ筛选基因对影响根癌农杆菌介导银白杨遗传转化的条件做了深入探索,通过优化转化条件,建立了银白杨遗传转化体系;最后在此基础上,进行rolB-pttGA20ox双价基因的转化,并由PCR、Southern杂交和形态特征差异检测,以证明是否为转化植株。主要研究结果如下:
1.茎段再生不定芽体系的建立。消毒的最佳方法为:70%的酒精浸泡30s+1%NaClO灭菌15min～35min。启动培养基最佳组合为MS + 6-BA0.5mg/L +NAA0.1mg/L ,启动率达到了74.1 %。MS + 6-BA1.0mg/L + NAA0.3mg/L +KT0.2mg/L+IBA0.4mg/L为增殖培养的最佳配方,增殖系数可达8.3以上。生根培养的最适培养基为1/2MS+IBA0.2mg/L+蔗糖15g/L+琼脂6g/L,生根率达到100%。移栽炼苗以蛭石作为最佳基质,移栽成活率达90.2%以上。
2.叶片再生不定芽的研究。叶片不定芽诱导的最适培养基是MS+6-BA0.5mg/L+NAA0.1mg/L+蔗糖2.5%+琼脂0.55%,pH5.8,不定芽再生率达95%以上;叶片不定芽再生能力强的最佳取材位置是自试管苗顶端向下伸展叶的第1~3片叶;生根试管植株叶片的不定芽再生能力强于无根试管植株。
3.玻璃苗产生机理的研究。与正常苗相比,玻璃苗的叶片和茎段横切面结构发生了很大变化;玻璃苗组织含水量增加;叶绿素含量、可溶性蛋白含量、相对电导率、MDA和RNA含量降低;SOD、CAT活性和DNA含量变化不大;采用聚丙烯酰胺凝胶电泳法对蛋白质进行分析,发现电泳谱带均出现增加、缺失等异常现象。
The major objective of this study was to breed new Populus alba varieties with improved rooting ability and apical dominance through transformation of the rolB-pttGA20ox two genes by using Agrobacterium tumefaciens-mediate transformation technology. For this purpose, first, an efficient in vitro propagation system and plant regeneration system were developed for Populus alba. Factors that affecting shoots regeneration via direct organogenesis, including cultivation conditions and plant growth regulators, etc., were also investigated in details. Subsequently, parameters that affecting the efficiency of Agrobacterium tumefaciens-mediate transformation of Populus alba were comprehensively studied and optimized by accessing NPTⅡexpression, and a transformation protocol was established. Finally, on the basis of the established transformation protocol, rolB-pttGA20ox genes were stably transferred to the Populus alba by PCR and Southern blotting tests.
The main results of this study were introduced separately as following:
1. Regeneration system of adventitious buds from stem segments
Stem segments with axillary buds were sterilized in 70% alcohol about 30s and then in 1%NaClO about 15min～35min. Survival percentage of explants was 94%. The most optimal induction medium was MS+6-BA0.5mg/L+NAA0.1mg/L,in which the differentiation rate of explants could be up to 74.1%. The most optimal proliferation medium was MS+6-BA1.0mg/L+NAA0.3mg/L+KT0.2mg/L+IBA0.4mg/L, the proliferation coefficient of buds being 8.3. The most optimal rooting medium was 1/2MS+IBA0.2mg/L, rooting rate being up to 100%.
2. Regeneration System of adventitious bud from leaves
The optimum medium for adventitious bud regeneration was MS medium supplementing with 6-BA and NAA(ratio was 5:1) (MS+6-BA0.5mg/L+NAA0.1mg/L+Sugar2.5%+agar0.55%),and the frequency of adventitious bud regeneration is more 95%;The best leaves for adventitious bud regeneration is the first three top leaves of a shoot.The leaf explants of rooted tube plants is more propitious to induce adventitious buds than those of non-rooted tube plants.The differentiation rate of adventitious bud is higher under 14h illuminations per day than others.
3. Hyperhydricity in Populus alba.regenerated in vitro: involvement of physiological, biochemical and morphological aspects
The leaf and stem anatomy in non-hyperhydric and hyperhydric Populus alba was investigated aimed to identify structural changes associated with this phenomenon. In non-hyperhydric organs there were smaller and more organized cells, besides a more differentiated vascular system when compared
围绕这一目标,本研究以银白杨当年生枝条的带芽茎段为外植体,首先建立了该树种的离体快繁及植株再生体系,并就培养条件和植物生长调节剂等因素对银白杨叶片直接再生体系的影响进行了详细的研究;然后利用NPTⅡ筛选基因对影响根癌农杆菌介导银白杨遗传转化的条件做了深入探索,通过优化转化条件,建立了银白杨遗传转化体系;最后在此基础上,进行rolB-pttGA20ox双价基因的转化,并由PCR、Southern杂交和形态特征差异检测,以证明是否为转化植株。主要研究结果如下:
1.茎段再生不定芽体系的建立。消毒的最佳方法为:70%的酒精浸泡30s+1%NaClO灭菌15min～35min。启动培养基最佳组合为MS + 6-BA0.5mg/L +NAA0.1mg/L ,启动率达到了74.1 %。MS + 6-BA1.0mg/L + NAA0.3mg/L +KT0.2mg/L+IBA0.4mg/L为增殖培养的最佳配方,增殖系数可达8.3以上。生根培养的最适培养基为1/2MS+IBA0.2mg/L+蔗糖15g/L+琼脂6g/L,生根率达到100%。移栽炼苗以蛭石作为最佳基质,移栽成活率达90.2%以上。
2.叶片再生不定芽的研究。叶片不定芽诱导的最适培养基是MS+6-BA0.5mg/L+NAA0.1mg/L+蔗糖2.5%+琼脂0.55%,pH5.8,不定芽再生率达95%以上;叶片不定芽再生能力强的最佳取材位置是自试管苗顶端向下伸展叶的第1~3片叶;生根试管植株叶片的不定芽再生能力强于无根试管植株。
3.玻璃苗产生机理的研究。与正常苗相比,玻璃苗的叶片和茎段横切面结构发生了很大变化;玻璃苗组织含水量增加;叶绿素含量、可溶性蛋白含量、相对电导率、MDA和RNA含量降低;SOD、CAT活性和DNA含量变化不大;采用聚丙烯酰胺凝胶电泳法对蛋白质进行分析,发现电泳谱带均出现增加、缺失等异常现象。
The major objective of this study was to breed new Populus alba varieties with improved rooting ability and apical dominance through transformation of the rolB-pttGA20ox two genes by using Agrobacterium tumefaciens-mediate transformation technology. For this purpose, first, an efficient in vitro propagation system and plant regeneration system were developed for Populus alba. Factors that affecting shoots regeneration via direct organogenesis, including cultivation conditions and plant growth regulators, etc., were also investigated in details. Subsequently, parameters that affecting the efficiency of Agrobacterium tumefaciens-mediate transformation of Populus alba were comprehensively studied and optimized by accessing NPTⅡexpression, and a transformation protocol was established. Finally, on the basis of the established transformation protocol, rolB-pttGA20ox genes were stably transferred to the Populus alba by PCR and Southern blotting tests.
The main results of this study were introduced separately as following:
1. Regeneration system of adventitious buds from stem segments
Stem segments with axillary buds were sterilized in 70% alcohol about 30s and then in 1%NaClO about 15min～35min. Survival percentage of explants was 94%. The most optimal induction medium was MS+6-BA0.5mg/L+NAA0.1mg/L,in which the differentiation rate of explants could be up to 74.1%. The most optimal proliferation medium was MS+6-BA1.0mg/L+NAA0.3mg/L+KT0.2mg/L+IBA0.4mg/L, the proliferation coefficient of buds being 8.3. The most optimal rooting medium was 1/2MS+IBA0.2mg/L, rooting rate being up to 100%.
2. Regeneration System of adventitious bud from leaves
The optimum medium for adventitious bud regeneration was MS medium supplementing with 6-BA and NAA(ratio was 5:1) (MS+6-BA0.5mg/L+NAA0.1mg/L+Sugar2.5%+agar0.55%),and the frequency of adventitious bud regeneration is more 95%;The best leaves for adventitious bud regeneration is the first three top leaves of a shoot.The leaf explants of rooted tube plants is more propitious to induce adventitious buds than those of non-rooted tube plants.The differentiation rate of adventitious bud is higher under 14h illuminations per day than others.
3. Hyperhydricity in Populus alba.regenerated in vitro: involvement of physiological, biochemical and morphological aspects
The leaf and stem anatomy in non-hyperhydric and hyperhydric Populus alba was investigated aimed to identify structural changes associated with this phenomenon. In non-hyperhydric organs there were smaller and more organized cells, besides a more differentiated vascular system when compared
引文
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