【作者】 蒋建雄；

【导师】 李宗道；

【作者基本信息】 湖南农业大学 ， 作物栽培学与耕作学， 2000， 博士

【摘要】 为了提高苎麻育种效率，创造高产优质的苎麻新种质材料，本研究探索了将植物基因工程技术和外源总DNA导入技术应用于苎麻品种改良的可行性，并获得了如下结果。1．建立了高效的苎麻转基因植株再生体系 对“湘苎3号”苎麻的茎段、子叶、带柄子叶、下胚轴等外植体的不定芽分化能力进行了比较，结果认为下胚轴分化频率高，材料制备容易，适宜用作苎麻遗传转化的受体，但从下胚轴上产生丛生芽的频率比较低。不同的组织培养条件下，下胚轴上不定芽分化频率高低依次为昼夜变温光照培养（日温26-28℃，夜温18-20℃）＞恒温暗培养（26-28℃）＞恒温光照培养（26-28℃），说明下胚轴再分化对温度变化的敏感性高于光照。研究还表明，苎麻外植体极易分化出根，但这个过程仍需要外源生长素的诱导。在仅添加了BA的分化培养基上，下胚轴脱分化和再分化都比较困难，而BA和IAA配合使用时却能很好地促进下胚轴的脱分化和再分化。不同BA和IAA配比组合中下胚轴的不定芽分化率存在差异，其中以BA2.0-3.0mg/l＋IAA0.5mg/l组合的不定芽分化率最高，均达到了70％左右。此外，分化培养基中添加1mg/lAgNO₃在一定程度上也可以促进下胚轴上不定芽的分化。2．实现了农杆菌介导抗除草剂基因转化苎麻 通过对影响根癌农杆菌转化效率的因素进行优化，建立了农杆菌介导外源抗除草剂Bar基因转化苎麻的遗传转化体系。结果表明，苎麻是一种对除草剂很敏感的植物，其耐除草剂“Basta”的临界浓度约为0.3mg/l，而且Basta对不定芽分化的抑制尤为明显。羧苄青霉素可以促进下胚轴上丛生芽的发生，但对不定芽生根的抑制作用不明显。同时苎麻下胚轴对根癌农杆菌菌株的敏感性高，容易被感染和转化，下胚轴与农杆菌共培养1-2d后其GUS瞬间表达率均接近100％。EHA105菌株比较适宜用于苎麻下胚轴的遗传转化，其合适的感染方式是将下胚轴外植体在用1/2MS液体培养基重新悬浮并稀释至浓度为OD₆₀₀=0.1-0.2的菌液中感染5min。下胚轴的预培养时间以及下胚轴与农杆菌的共培养时间对转化效率的促进作用都比较大，其中以预培养6-9d和共培养4d的转化效果达到最佳。试验还表明GUS瞬间表达率和GUS稳定表达率以及抗性愈伤组织中阳性转化体的百分率三者之间并不总是呈紧密的平行关系，因此综合考虑这三个参数来优化苎麻遗传转化体系是很有必要的。此外，本研究发现通过在X-Gluc缓冲液中加入6％PVP，可以有效地防止GUS组织化学显色过程中苎麻材料的严重褐化，而对其显色效果无不良影响。通过筛选培养，最后获得了21株抗性植株，经GUS组 织化学显色和DNA点杂交鉴定出8株含有抗除草剂草丁磷的Bar基因同源片段的转基 因芒麻植株。 3．棉花总 DNA导入芒麻引起了性状变异 应用超干胚浸渍法，成功地将棉花总DNA导入到芒麻细胞内，在D；代获得了两 株叶型发生奇特变异的新材料97－24和97－25。其中97－24的D。代发生分离，大部分子 代植株恢复了受体性状，只有少数子代植株叶型的变异比D；代植株更加明显。而由 97．24地下茎发生的分株上则呈现自下而上叶型变异逐渐恢复的趋势。在另一植株 97－28的D。代中又筛选到一株叶背无茸毛的变异株，其生长势弱，叶色黄绿，易受虫 害。它在D。代也发生了分离，只有约l／4的子代植株仍保持了上一代的变异。但从97－28 的D。代植株地下茎发生的分株却均能稳定地保持这些变异性状，说明外源总DNA导 入引起的变异在低世代即可通过无性繁殖方式固定下来，这非常有利于缩短芒麻育种 周期。在试管苗快速繁殖中，大多数变异材料在H基本培养基上生根良好，但在MS 基本培养基上则生根困难，这从细胞学的角度证明变异后代在遗传基础上确实发生了 改变。IEInPD分析结果也证明部分棉花DNA片段已经整合入芒麻基因组，并导致芒麻 基因组序列发生了一定的变化。更多还原

【Abstract】 In order to improve the efficiency of ramie breeding and to create new germplasm, it was studied to improve ramie cultivars with the plant gene engineering or the total exogenous DNA introducing. The results are as follows. 1.Establishing the receptor system for ramie genetic transformation. The redifferentiation abilities of different explants of ramie were compared, and the hypocotyl was considered as a favorable receptor for genetic transformation because of its high redifferentiation potential and easy preparation. Under different tissue culture conditions, the regeneration frequency of adventitious shoots from hypocotyls varied as: that of light culture in alternating temperatures of day 26-28X2 and night 18-20C > that of dark culture in constant temperature of 26-28XD > that of light culture in constant temperature of 26-28 0C, which revealed that the differentiation of shoots was more sensitive to temperature than to illumination. The only addition of BA in medium was not benefit for dedifferentiation of hypocotyls. However, the combination of BA with IAA promoted the dedifferentiation and redifferentiation, though the regeneration frequency differed in kinds of combinations, among which the combinations of 2.0-3.0 mg/i BA + 0.5 mg/I IAA produced a highest differentiation frequency of 70 % or so. The addition of 1 mg/I AgNO3 in medium also improved the regeneration of shoots. 2. Producing herbicide-tolerant transgenic ramie via A. tumefaciens mediated transformation. A system for genetic transformation of ramie was developed by systematically optimizing the factors affecting A. tumefaciens mediated transformation efficiency. It was showed that ramie was a kind of plant susceptiable to herbicide, whose critical tolerance concentration to herbicide asta was 0.3 mg/I or so, and that the herbicide inhibited the regeneration of shoots especially. N4eanwhile, the carbecillin facilitated the emergency of clustered shoots but had no obvious repression for rooting of adventitious shoots. Hypocotyls had a high sensitivity to the strains of A. tumefaciens, with a GUS instantaneous expression frequency close to 100 % when co-cultivated 1 or 2 days with the bacteria. The strain of E1-1A105 was suitable for transformation of hypocotyl by inoculating 5 minutes in a bacterial concentration of 0D600=0.1-0.2 (the bacteria were resuspended and diluted with 1/2MS liquid medium). The preculture of hypocotyis and their co-cultivation with the bacteria improved greatly the transformation efficiency, and a preculture duration of 6-9 days and a co-cultivation duration of 4 days were the best choices for hypocotyl of ramie. In addition, the GUS instantaneous expression frequency was not closely positively correlated to the GUS stable expression frequency and to the ratio of positive transformants to herbicide-tolerant calli, which indicated it necessary to combine these three indexes for the optimization of transformation system of ramie. The oxidization of ramie materials in histochemical GUS assay could be effectively reduced by adding 6 % PVP in the X-GIuc solution. Through histochemical GUS assay and DNA dot blotting, 8 phophinothricin-tolerant transgenic plants were obtained. 3. Obtaining variants by introducing exogenous cotton DNA into ramie. The exogenous cotton DNA was successfully introduced into ramie by over-dried seed soaking. In the D1 generation, two plants 97-24 and 97-25 whose leave had extraordinary morphology, were selected out. The D2 generation of 97-24 segregated, with most of p更多还原