【作者】 郭余龙；

【导师】 李名扬； 裴炎；

【作者基本信息】 西南农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 棉花是世界上第一位的天然纤维作物和第二大油料作物。棉花的生产和消费是全球经济的支柱之一，在我国国民经济中也占有非常重要的地位。由于棉花的种质基础非常狭窄，且由于长期以来通过品种间杂交进行育种，品种间的遗传变异越来越小，常规育种方法在棉花遗传改良上已不象过去那么有效，我们需要新的手段来培育棉花新品种，以提高单产，改进纤维品质，降低生产成本，提高棉农收益。转基因技术是实现作物品种定向改良的新手段，它一经出现就在棉花上得到了成功的运用。转基因抗虫棉和抗除草剂棉在生产上得到了广泛应用，带来了可观的效益。但是，棉花仍然是一个遗传转化比较困难的作物，存在转化周期长，基因型限制和再生植株变异频率高等不足，需要对转化技术进行改进。病害也是棉花生产中的严重问题，虽有转基因抗虫棉和抗除草剂棉的成功，但尚未有关于转基因抗病棉花的报告。因此，有必要将抗病基因转入棉花，评价其效果。棉花再生植株的体细胞无性系变异的频率比较高，这对转基因工作的开展是很不利的。另外，植物体细胞无性系变异现象本身也是很值得研究的，因此，有必要对棉花体细胞无性系变异的规律和机理进行分析。本文对棉花的遗传转化方法进行了比较，建立了高效遗传转化体系，然后将多个抗病基因导入棉花，对部分转基因植株的形态特征进行了分析；对棉花体细胞无性系变异的几个特征进行了分析，并定位研究了棉花再生植株的一个花变异体chv1的形态特征和基因表达。主要研究结果如下：1．高频再生材料的筛选在无激素但KNO₃加倍的MSB培养基上，冀棉14和川棉239体胚苗种子的下胚轴切段诱导愈伤组织形成的频率为70％左右，这些愈伤组织90％以上都能形成体胚，最快能在3个月内实现再生。在用冀棉14体胚苗种子的下胚轴切段进行培养时，观察到典型的直接体胚发生现象，发生频率为25.5％。体胚苗种子田间繁衍后代保持较强的体胚发生能力，但单株间有差异。冀棉14体胚苗种子分别在含IAA0.5mg／L+KT0.1mg／L、2,4-D0.1mg／L+KT0.1mg／L和ZT2.0mg／L的MSB培养基上培养，体胚发生频率分别为87.3％、63.9％和90.0％，是高频再生材料。2．遗传转化方法研究胚性愈伤组织基因枪转化：以川棉239胚性愈伤组织为受体，用基因枪法导入外源基因，获得了经GUS组织染色鉴定的转基因植株。以GUS基因瞬时表达蓝点数为评价指标，考察了影响基因枪转化的几个因素，结果表明：使用碱裂解法提取PEG纯化的质粒DNA优于试剂盒大量提取的。当轰击压力为1550PSI时，轰击距离设为6cm或9cm较好，轰前经1～2周培养的愈伤组织较佳。实验还表明：玉米Ubi启动子在棉花胚性愈伤组织中有活性。胚性愈伤组织农杆菌转化：以冀棉14胚性愈伤组织为受体材料，农杆菌介导法导入外源基因，获得了经GUS染色鉴定的转基因植株。优化后的转化程序为：农杆菌用YEB液体培养基培养36h后，离心收集菌体，用含100μmol／L乙酰丁香酮的液体MSB培养基重悬菌体， 西南农业大学博士学位论文早早卑早攀甲钾巴早毕粤.早早里里理卑娜甲卑攀早只匆卑甲娜峨甲脚甲即甲毕早毕早碑早毕毕卑早早碑理调OD6。。到0.4左右，感染20min，24佗5℃暗培养3d，直接用含Kmloomg/L的N畜sB+认AO.sln创L+ KTO，!m岁L的培养基筛选抗性愈伤组织，体胚诱导和成熟阶段也加入Kl们00In留L。再生植株的GUS染色率在90%以上。 下胚轴农杆菌转化:以有G US基因瞬时表达的外植体百分数为评价指标，研究不同材料对农杆菌的敏感性，发现子叶外植体最敏感，下胚轴次之，胚根较差。供试的6个品种都能被农杆菌感染，但敏感性有差异.愈伤组织诱导培养基用MsB+2，4一DO.lm留L+KTO.!mgL优于MSB+l AAO.sm留L十KTO.lm留L。只有当使用冀棉14体胚苗种子作外植体时，获得了转基因植株。萌发前的培养阶段可以一直加入Km 1 oom留L。抗性植株叶片Gus染色呈阳性。二杭病器因导入栩花 以川棉239胚性愈伤组织为受体，用基因枪法导入osB基因，获得了4个经PCR验证的转基因株系;以川棉239茎尖为受体，用基因枪法导入cEMA基因，获得3株转基因嵌合植株;应用胚性愈伤组织农杆菌转化法，导入A FP一SPCEMA双价基因，共获得6个抗性细胞系，再生抗性植株28株，Southem杂交鉴定表明，AFP基因已经整合进棉花基因组;用下胚轴农杆菌介导法导入AFP一CHI双价基因，共获得抗性细胞系10个、再生抗性植株100余株，CHI基因的整合经PCR扩增证实:以下胚轴和胚性愈伤组织为受体，用农杆菌介导法导入SPCEMA一CHI外源基因，获得抗性系29个，抗性植株200余株，C川基因的整合经PCR扩增证实。4.转cEMA基因棉花的表型 转C EMA基因棉花呈嵌合表型。在表型不正常的枝条上，叶片扭曲崎形，出现灰绿色与绿色相间的花叶，花器官发育异常，无花粉，人工授粉不能结实。花叶区的叶绿素含量降低20%，叶肉组织细胞局部缺损，维管束系统退化。叶片灰绿区叶肉细胞的次生壁明显增厚，_局部区域吞噬现象明显，叶绿体内基粒片?更多还原

【Abstract】 Cotton is the world’s leading natural fiber crop and the second largest oil crop. The production and consumption of cotton fiber is a mainstay of the world’s economy, and is also very important for the economy of china. Because of the narrow germplasm base of today’s cultivated cottons , the variation among today’s cultivated varieties is very small. Traditional breeding methods are not so effective today as in the past. New breeding methods are demanded to create new cotton varieties for the purpose of increasing fiber yield per acre, improving fiber quality, lowering production cost and increasing the income of cotton growers.Biotechnology is a new approach of plant breeding. Its goal is to manipulate gene expression in ways that produce desired phenotypic effect. Cotton biotechnology have achieved complete success in the past decade, the total acres committed to transgenic cotton engineered for insect or herbicide resistance have been increasing fast from its commercial release. However, the development of transgenic cotton plants is still very difficult with barriers such as long transformation cycle, highly genotype dependent and fertile of regenerated plants in the way. The cotton transformation methods should be improved.Cotton disease causes enormous loss of potential production every year. Although, the insect and herbicide transgenic cotton have reached great success, disease-resistant transgenic cottons have not reached the market yet. It is needed to deliver disease-resistant genes into cotton to evaluate their effects for cotton disease resistance.The high frequent somaclonal variation of regenerated cotton plants is an obstacle to the cotton transformation, and the plant somaclonal variation itself is worth exploring. So we decided to study the rules and mechanism of cotton somclonal variation.In this dissertation, gene delivery systems used to generate transgenic cotton were compared, effective cotton transformation procedure was set up, then we delivered a few disease genes intocotton genome, and morphological changes of transgenic cotton plants \\ith disease gene were analysed. A few characters of cotton somalclonal variation were analysed, the morphological and gene expression difference between the chvl, a homeotic somalclonal variani of conon. and the wild type were researched. The main results are as following:1. Selection of lines with high regeneration abilityWhen hypocotyls segments of seeds from regenerated Chuanmian 239 and Jimian14 plants were cultured on hormone free MSB+KNO31.9g/1hMSBK) medium, callus induction frequency was 70%, plant regeneration can achieved within 3 months in some cases. Typical direct embryogenesis was found when hypocotyls segments of seeds from regenerated Jiminn14 plants were cultured on MSBK medium. The second generation seeds reproduced by regenerated plants remained high regeneration ability but the ability was different among seeds collected from different individual plants.Seeds from regenerated Jimian14 plants are our selected material with high regeneration ability, the embryogenetic frequency of the material was 87.3%. 63.9% or 90.0% on MSB medium with IA A 0.5mg/L+KT0.1 mg/L, 2.4-D0. 1 mg/L+KT0. 1 mg/L or ZT2.0mg/L respectively.2. Cotton transformation approachesTransformation of embryogenetic callus by particle bombardment: Embryogenic callus of ’Chuanmian 239’ were bombarded with gold particle coated with plasmids coding B-glucuronidase (GUS) and neomycin phosphatransferase (NPTII). Regenerated plants were verified to be transgenic by histologicai staining for GUS expression and PCR amplification of the NPTII. Factors that affect biolistic transformation efficiency were studied. Plasmids isolated by alkaline lysis and purified by PEG precipitation produced more blue foci, when bombarded into cotton callus, than plasmids isolated with Roche DNA maxprep kit; When the helium was 1550PSI, the suitable target distance were 6cm and 9cm; Embryogentic callus cultured for l-2week before bombardment were found to be the best t更多还原