【作者】 谢德意；

【导师】 张献龙；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2007， 博士

【摘要】 棉花体细胞培养和植株再生技术是进行原生质体培养、遗传转化、体细胞杂种创造等技术的必要条件，利用遗传工程改良棉花品种是目前棉花遗传改良研究的一个热门领域，它能有效打破物种间的界限，实现动物、植物和微生物间基因资源的相互利用，并可以实现对目标性状的定向变异，同时能大大缩短育种年限、加快育种进程。但当前棉花生物技术研究仍然存在着很多障碍，如棉花体细胞胚胎发生的基因型依赖性强、棉花组织培养周期长、体胚发生率低、基因遗传转化效率低、再生植株结实困难等等，这些问题已经成为棉花组织培养、棉花生物技术、棉花功能基因的验证研究领域的障碍。本研究内容主要涉及棉花体细胞胚胎发生的调控，不同生态区棉花品种体细胞胚胎发生及再生能力比较；不同基因型海岛棉体细胞胚胎发生能力研究；高效棉花遗传转化体系的建立；ipt基因的遗传转化及转基因植株的获得；同时对转基因引起的棉花性状变异的规律进行初步研究，为转基因棉花育种提供理论指导。取得的主要研究结果如下：1．在陆地棉愈伤组织诱导阶段，适宜浓度的2，4-D+KT和IBA+KT激素组合均能高效诱导愈伤组织产生，其最优激素配比为0.1 mg／L 2，4-D+0.15 mg／L KT和0.5 mg／L IBA+．0.15 mg／L KT。2．在陆地棉诱导愈伤组织时，诱导培养基中NH₄NO₃的水平对愈伤组织以后能否发生体细胞胚胎分化至关重要，在分化培养基中撤去NH₄NO₃，并增加KNO₃，可以明显提高愈伤组织胚分化率，使珂字201、YZ1的愈伤组织胚分化率达到80％以上。铁盐对愈伤组织诱导和胚分化起着十分重要的作用。当愈伤组织诱导培养基中不含铁盐时，不同基因型的外植体均未见膨大，更没有诱导出愈伤组织。硫酸亚铁的浓度以56 mg／L为佳，此时愈伤组织诱导率达95-100％，将愈伤组织转入分化培养基上时，添加硫酸亚铁的浓度为28 mg／L、56 mg／L时，不同基因型的愈伤组织生长都比较迅速，胚分化的时间缩短。3．利用IBA和KT激素组合，结合不同阶段的无机盐调控，使供试的22个陆地棉品种中有15个品种实现体细胞胚胎分化和植株再生，其中鄂抗棉3号、5号、鄂棉20、23号、豫棉9号、豫早73、豫棉12号、豫棉1221等8个品种是首次获得体细胞胚胎发生的品种。考察以上8个品种的系谱发现，鄂抗棉3、5号主要含有岱字棉15号的血统，鄂棉20含有金字棉的血统，豫棉9、12号则含有福字棉的血统，豫早73含有乌干达棉的血统，其他的材料遗传背景比较复杂。许多研究表明，珂字棉的再生能力最强，岱字棉较易再生，而含有金字棉、福字棉、乌干达棉血统的品种则较难获得体细胞胚和再生植株。本试验中，首次实现了5个含有金字棉、福字棉、乌干达棉血统的品种的体细胞胚胎发生和植株再生。比较长江和黄河流域棉区棉花品种的胚胎发生和植株再生能力发现，长江流域的10个品种再生6个（分别是鄂抗棉3、5号、鄂抗棉9号、鄂荆1号、鄂棉20、23号）、黄河流域的10个品种再生7个（分别是中棉27号、晋棉18、20号、豫棉9、12号、豫早73和豫棉1221），表明长江和黄河流域棉区品种的胚胎发生和植株再生潜力没有明显差别，这与以往研究结果棉花体细胞胚胎发生有严格地域性限制有所不同。但两大棉区品种体细胞胚胎发生和植株再生时间有明显差异，长江流域棉区的品种分化时间平均比黄河流域的品种晚近20d。4．研究发现海岛棉愈伤组织诱导困难、出愈时间长，与相同诱导条件下诱导陆地棉品种相比，出愈时间延迟10—15天，出愈率偏低，主要原因是海岛棉外植体易褐化导致生长缓慢，且所有供试海岛棉品种刚开始诱导的愈伤组织质地均为硬块状且增殖缓慢，由这种硬块状愈伤组织进一步诱导、继代，形成质地疏松的愈伤组织需要一个漫长的过程。最长达236天，这也是海岛棉品种难以进行组织培养的主要原因之一。5．2，4-D的加入对海岛棉愈伤组织诱导和胚分化有显著影响，加入2，4-D不仅能提高出愈率、缩短出愈时间，而且还能使获得质地疏松愈伤组织的时间大大提前，平均提前达40天以上。在海岛棉愈伤组织诱导阶段，最佳激素配比为0.2 mg／L 2，4-D+0.4 mg／L KT+1.0 mg／L IBA，而在愈伤组织分化阶段以0.05 mg／L 2，4-D+0.2 mg／L KT+0.5 mg／L IBA为最好。悬浮培养有利于海岛棉愈伤组织分化，与相同的固体培养相比，愈伤组织分化率平均提高5％以上，分化时间提前20天以上，但利用悬浮培养未能使在固体培养条件下没有发生胚分化的海岛棉基因型产生胚分化。6．对遗传转化的影响因子优化研究表明，以预培养12天的棉花胚性愈伤组织为转化受体，用OD₆₀₀值为0.4的农杆菌菌液侵染，加入50mg／L的乙酰丁香酮（AS）活化，能够使YZ1、珂字201的抗性愈伤率分别达25.1％和23.8％。7．利用农杆菌介导棉花胚性愈伤组织为转化受体的遗传转化体系，将ipt基因导入到Coker 201、YZ1、Ym4和Em23棉花品种中，获得的再生植株经卡那霉素涂抹、PCR扩增、Southern杂交检验，证明ipt基因已整合到部分再生的棉花植株的基因组中。通过对再生植株田间初步观察发现，转基因植株表现出生长旺盛、果枝数增多、叶功能期延长等特征。8．研究了不同基因型的遗传转化效率并建立了转基因再生植株的有效成苗技术体系。结果表明：在相同的转化条件下，不同基因型的转化效率差异较大，豫早1号和珂字201的遗传转化效率及植株再生能力显著高于豫棉4号和鄂棉23。降低培养基无机盐浓度，用Phytagel固化，能够提高胚萌发成苗率，降低畸形苗频率，在生根培养中添加少量活性碳提高了根系活力。再生植株长到3～5片真叶时，出现的植株根系发褐，可以切除，转移到新鲜的低盐培养基中，重新萌发生根。在试管苗移栽过程中，采用逐步揭开封口膜，炼苗的措施，可以提高试管苗的环境适应能力，显著地提高移栽成活效率。不同的培养基生根能力比较，以1／4MS+NAA（0.1 mg．／L）和1／4MS+IBA（1.2 mg．／L）较好，1／4MS+NAA（0.1 mg．／L）+IBA（1.2 mg．／L）较差。不同品种间比较来看，Coker 201和YZ1的生根能力较强，Em23生根能力较弱。更多还原

【Abstract】 Cotton tissure culture and plant regeneration was the precondition of protoplast fusion, genetical transformation and creation of hybrids; Genetic improving of cotton using the genetic engineering was one of the hot regions of cotton breeding, which can overcome sexual-crossing barriers and share all genes existing in the plant, animal, and animals. Especially, the genetic engineering can impove the given characters and significantly shorten the breeding periods. However, many hinder still exisist in cotton biotechnology. The main reason is that cultivars of transgenic cotton, which requires transformation of appropriate tissue followed by regeneration, remains extremely difficult in somatic embryogenesis and regeneration. Recalcitrant cotton cultivars, long tissue culture duration, the unpredictability of tissue culture, and a high degree of genotype dependence are more troublesome in regeneration of cotton. In this study, we have optimized some factors that can significantly affect the efficiency of regeneration in cotton, and compared the regeneability of different genotypes of Seaisland cotton.（Gossypium barbadense L. ） The regeneration of transgenic plants with ipt gene is developed through transformation mediated by Agrobacterium tumefaciens, and some variations of transgenic plants are examined and analyzed. The main results obtained are listed below:1. Two kinds of hormone combinations IBA + KT and 2, 4-D+KT were all efficient for callus induction of cotton （Gossypium hirsutum L.）, and the optional combinations were IBA 1.0 mg/L + KT 0.5 mg/L and 0.1 mg/L 2,4-D+0.15 mg/L KT.2. Somatic embryogenesis from calli derived from hypocotyls segments are observed only on inducing original calli medium containing NH₄NO₃. Calli do not form somatic embryos in the original calli inducing medium without NH₄NO₃. Doubled KNO₃ （3.8 g/L） but free of NH₄NO₃ can significantly improve the efficiency of somatic embryogenesis and regeneration. The percentage of embryogenic calli from all cultures reached more than 80% in Coker 201 through doubling KNO₃ and omitting NH₄NO₃. Medium supplemented with MgSO₄·7H₂O is necessary for callus induction and proliferation. And the medium supplemented with 50mg/L FeSO₄·7H₂O could promote the growth of calli, which can obtained 90-100 % callus induction rate. 28 mg/L、56 mg/L MgSO₄·7H₂O in the subculture medium can shorten the priod of somatic embryogenesis.3. To optimize the somatic embryogenesis of upland cotton, totally 22 cultivars including 10 cultivars from Yellow River valley, 10 cultivars from Yangtze River valley and two highly responsible genotypes Coker 201 and YZ-1, were used for studying the factors affecting the callus induction and differentiation. Most of the genotypes showed high regenerability under the IBA/KT hormone regime.Under the optimized culture procedure, most of the genotypes collected from the two valleys showed the similar regeneable abilities, except a relative longer time taken during the somatic embryogenesis of cultivars of the Yangtze River valley.In this study, somatic embryogenesis and plant regeneration had happened in 8 genotypes, namely, Ek3, Ek5, Em20, Em23, Ym9, Ym12, Yz73, and Ym1221 for the first time.4. Comparing to Upland cotton, the callus induction and proliferation of G. babardence was difficult, which would take 10-15 more days and obtained relatively low rate in callus induction, mainly because of the the death of the brownish calli. At the beginning of callus induction, all the calli of five different genotypes grew slowly and the texture was hardish. After several rounds of subculture, these hardish calli grew rapidly and became loosen, so this process would cost much time and lead to the delay of somatic embryogenesis of G. babardence, which would take about 8 months before somatic embrogenesis.5. 2, 4-D+IBA+KT hormone combination was very efficient for callus induction and proliferation of G. babardence species, which can obtain high efficient callus induction and shorten the period of proliferation. The optimal combinations was 0.2 mg/L 2, 4-D + 0.4 mg/L KT +1.0 mg/L IBA on callus induction medium and 0.05 mg/L 2,4-D + 0.2 mg/L KT + 0.5 mg/L IBA on callus proliferation and differentiation medium. Suspension culture of calli was helpful for calli proliferation and somatical embryogenesis, which can averagely obtain 5% higher differentiation rate than calli subcultured on solidified medium6. Various aspects of transformation processes were examined in efforts to improve the efficiency of production of transgenic cotton. Embryogenic calli which were precultured for 12 days proved to be a valuable explant to cotton transformation. The effects of concentration of A. tumefaciens inoculated, time of co-cultivation, media for co-cultivation was evaluated. OD (A₆₀₀) value 0.4 proved to be significantly better than other concentrations. Concentration of acetosyringone at 50 mg 1^-1 during co-cultivation significantly increased transformation efficiency. Under the same transgeneic procedure, four genotypes obtained different transformation efficiency.7. The isopentenyl transferases gene was introduced into cotton genome via Agrobacterium tumefaciens. Transgenic plants were obtained from four cultivars, namely Coker 201、YZ1、Ym4 and Em23. The putative transgenic plants were tested by PCR and Southern blotting. The results showed that the transgenes has inserted into cotton genome. Transgenic plants with ipt gene showed many interested characters, for example, dark green leaves and flourishing branches and buds. 8. To optimize the procedure of somatic embryos germination, rooting and transplant, many factors were studied using somatic embryos of YZ-1 as target. The results showed that low concentration of inorganic salt, namely 1/2 MS inorganic salts, in the embryo germination media could increase the ratio of normal regenerated plant and help build stronger root systems. Phytagel was proved a better agent for solidifying the medium than Agar. Activated carbon added in rooting medium was proved to be helpful for root regeneration of somatic embryos. Roots of some regenerated plantlets with 3-5 true leaves would be brown due to a long culturing period in solid medium. These plantlets could rebuild strong root system after the brown roots were removed and inoculated on fresh medium with low concentration of inorganic salt. Plantlets could healthily grow after stepwise opening the lids of culture bottles, which help the regenerated plantlets accommodate to open circumstances.更多还原