棉花组织培养与雪花莲凝集素基因转化
摘要
棉花是世界性的重要经济作物。但是棉花生产由于长期受到病虫害的危害，棉花的产量和品质受到严重影响。基因工程技术的出现为棉花品质改良和病虫害防治提供了一个有力手段。外源苏云金芽孢杆菌(Bacillus thuringicnsis)晶体蛋白基因(Bt基因)的表达对于鳞翅目害虫有明显的抑制作用，而雪花莲凝集素(GNA)对同翅目害虫有较好的毒杀作用，能与Bt基因的抗虫谱形成互补，在抗虫转基因棉花研究中具有良好的应用前景。
转基因棉花的研制必须以成熟的组织培养技术为基础。本试验选用转Bt基因棉33B、99B和中棉29为研究材料，从不同培养基、基因型、外植体、激素配比与浓度、碳源、氮源和外源添加物等影响棉花组织培养的因素进行探讨，结果如下：以33B下胚轴为外植体，接种于附加2，4-D 0.1mg／L、KT 0.1mg/L、IAA 0.1 mg／L，葡萄糖3％的MSB培养基上能够高频率的诱导愈伤组织。在愈伤组织继代培养过程中，去掉2，4-D并将琼脂浓度降低到6g／L能够获得较高的胚性愈伤组织诱导频率。以33B的茎尖为材料，建立起茎尖培养体系：以3d苗龄，下胚轴长度为5-7mm的茎尖为外植体，接入不加任何激素的MSB培养基中培养四周，成苗率能达到88.9％。
与棉花体细胞胚胎再生途径相比，棉花茎尖分生组织培养植株成苗的时间短，培养方法简单，也不受基因型的限制，适合作为基因转化的受体系统。因此，本试验利用基因枪法将雪花莲凝集素(GNA)基因导入33B茎尖分生组织，恢复培养7d后，在含有Bialaphos10mg/L的筛选培养基上进行直接筛选，最终得到抗性植株。
Cotton is a kind of economic crop. Every year virus and pests cause serious damage to cotton production. Gene engineering has provided a useful tool for improvement in cotton quality and biological protection from pests. For some insect pests, the expression of Bacillus thuringiensis (Bt) endotoxin genes in transgenic plants has been shown to be an effective means of control, although the long-term use of trans-Bt gene may depend on devising suitable management strategies to delay the buildup of Bt-resistant insect populations. Since no reported strain of Bt is effective against homopterans, GNA lectin expressed in the transgenic plants decreased survival and overall fecundity (production of offspring) of the insects. The two anti-pest spectra can be complementaty, which underlie the research in transgenic cotton with resistance
to pests.
The development of transgenic cotton must base on successful tissue culture. In this research, we chose transgenic cotton strains 33B, 99B and Zhongmian 29 as materials, studied the factors that affected cotton tissue culture from media, genotypes, explant types, hormones, carbon sources, nitrogen sources and exogenous materials. The results are as followed: incubating hypocotyls from 33B as explants on MSB medium containing 0.lmg/L 2,4-D, 0.lmg/L KT, 0.lmg/L IAA, 3% glucose, we got high-rate induction of calli; we reduced the concentration of agar to 6g/L in the medium free of 2,4-D in the subculture, and got high inducing rate of embryogenic calli; in the meantime, we used shoot apical meristems of 33B isolated from 3-day-old seedlings as explants, then incubated them on MSB medium free of hormones for 4 weeks. The rate of plantlet from shoot apical meristem is 88.9%.
In comparison with embryogenesis, it is easy to get plantlets from shoot apical meristems, and it is suitable and convenience to be used as transformation system. We used particle bombardment method to transfer GNA gene into shoot apical meristems and finally got plantlets with resistance to l0mg/L Bialaphos after primary selection.
转基因棉花的研制必须以成熟的组织培养技术为基础。本试验选用转Bt基因棉33B、99B和中棉29为研究材料，从不同培养基、基因型、外植体、激素配比与浓度、碳源、氮源和外源添加物等影响棉花组织培养的因素进行探讨，结果如下：以33B下胚轴为外植体，接种于附加2，4-D 0.1mg／L、KT 0.1mg/L、IAA 0.1 mg／L，葡萄糖3％的MSB培养基上能够高频率的诱导愈伤组织。在愈伤组织继代培养过程中，去掉2，4-D并将琼脂浓度降低到6g／L能够获得较高的胚性愈伤组织诱导频率。以33B的茎尖为材料，建立起茎尖培养体系：以3d苗龄，下胚轴长度为5-7mm的茎尖为外植体，接入不加任何激素的MSB培养基中培养四周，成苗率能达到88.9％。
与棉花体细胞胚胎再生途径相比，棉花茎尖分生组织培养植株成苗的时间短，培养方法简单，也不受基因型的限制，适合作为基因转化的受体系统。因此，本试验利用基因枪法将雪花莲凝集素(GNA)基因导入33B茎尖分生组织，恢复培养7d后，在含有Bialaphos10mg/L的筛选培养基上进行直接筛选，最终得到抗性植株。
Cotton is a kind of economic crop. Every year virus and pests cause serious damage to cotton production. Gene engineering has provided a useful tool for improvement in cotton quality and biological protection from pests. For some insect pests, the expression of Bacillus thuringiensis (Bt) endotoxin genes in transgenic plants has been shown to be an effective means of control, although the long-term use of trans-Bt gene may depend on devising suitable management strategies to delay the buildup of Bt-resistant insect populations. Since no reported strain of Bt is effective against homopterans, GNA lectin expressed in the transgenic plants decreased survival and overall fecundity (production of offspring) of the insects. The two anti-pest spectra can be complementaty, which underlie the research in transgenic cotton with resistance
to pests.
The development of transgenic cotton must base on successful tissue culture. In this research, we chose transgenic cotton strains 33B, 99B and Zhongmian 29 as materials, studied the factors that affected cotton tissue culture from media, genotypes, explant types, hormones, carbon sources, nitrogen sources and exogenous materials. The results are as followed: incubating hypocotyls from 33B as explants on MSB medium containing 0.lmg/L 2,4-D, 0.lmg/L KT, 0.lmg/L IAA, 3% glucose, we got high-rate induction of calli; we reduced the concentration of agar to 6g/L in the medium free of 2,4-D in the subculture, and got high inducing rate of embryogenic calli; in the meantime, we used shoot apical meristems of 33B isolated from 3-day-old seedlings as explants, then incubated them on MSB medium free of hormones for 4 weeks. The rate of plantlet from shoot apical meristem is 88.9%.
In comparison with embryogenesis, it is easy to get plantlets from shoot apical meristems, and it is suitable and convenience to be used as transformation system. We used particle bombardment method to transfer GNA gene into shoot apical meristems and finally got plantlets with resistance to l0mg/L Bialaphos after primary selection.
引文
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