【作者】 丁亮；

【导师】 祝水金；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2003， 硕士

【摘要】 本研究以陆地棉（Ghirsutum）的3个品种为材料，系统研究无机盐、激素、碳源、基因型及其外植体等对陆地棉体细胞诱导、胚性愈伤组织的分化、胚状体的形成与植株再生的影响，优化棉花愈伤组织诱导、增殖和胚状体发生和植株再生培养基，以建立棉花体细胞诱导与植株再生技术体系，为棉花体细胞培养研究和棉花转基因研究奠定基础；同时以转基因棉及其遗传背景为材料，研究外源基因对棉花组织培养的影响；以陆地棉色素腺体近等基因系等为材料，研究棉花色素腺体和棉酚在棉花组织培养中的作用，主要结果如下： 1．陆地棉组织培养体系的优化： 取5日龄无菌苗的外植体，在含有0.1mg／L KT和MSB培养基中连续培养能直接诱导出胚性愈伤组织，也可在含有0.1mg／L的KT和0.05～0.1mg／L的2，4-D的MSB培养基上进行2～3次的继代培养后，转移至含有0.1mg／L KT和2g／L活性炭的MSB培养基上诱导胚性愈伤组织。胚性愈伤呈金黄色，米粒状，在水中易分散成较小的颗粒体。胚性愈伤组织的诱导往往与不定根的诱导相关，能诱导出胚性愈伤组织的外植体均有不定根的出生。此外，外植体的类型与胚性愈伤组织的诱导效率也有关系，以子叶为外植体其胚性愈伤分化率高于下胚轴。胚状体再生成植株不需要激素，但活性炭和KNO₃可以提高正常再生苗频率。再生小棉花经生根培养和壮苗培养后，再采用保湿和逐步炼苗法进行移栽可获得较高的试管苗移栽成活率。 2．外源基因对棉花组织培养的影响： 取转Bt基因抗虫棉春矮早和遗传背景亲本春矮旱进行组织培养，对下胚轴和子叶愈伤组织鲜重进行成对数据比较分析，通过t测试得下胚轴愈伤组织鲜重t=47.667＞t_0.01，P＜0.01；子叶愈伤组织鲜重t=86.711＞t_0.01，P＜0.01，表明Bt基因对棉花外植体的愈伤组织诱导有极显著影响。对转Bt基因抗虫棉春矮早与非转基因遗传背景亲本春矮早茎尖生根的成对数据进行比较分析，t测试得茎尖生根t=128.187＞t_0.01，P＜0.01，同样表明了Bt基因对棉花茎尖外植体的生根能力有极 显著影响，外源基因的导入使得转Bt基因陆地棉品种容易进行体细胞培养和茎 尖培养。对不同来源转基因品种研究发现，美国转Bt基因棉32B、33B，国产转 Bt基因棉国抗22、邯109、CPTI和Bt双价抗虫棉CK321在30d的出愈率均在‘95％以上，愈伤组织质地与生长势与坷字棉312相似，是易于进行愈伤组织诱导 的品种，而对应的遗传背景亲本多为难于培养或较难培养的推广棉花品种。可见， 外源抗虫基因的导入可使转基因棉品种容易进行体细胞培养。然而，通过花粉管 通道法转化的Bar基因对棉花组织培养无明显影响。 3．色素腺体与棉酚在棉花组织培养中的作用： 棉花色素腺体和棉酚对棉花茎尖培养不定根的再生具有较强的诱导作用， 有色素腺体棉品种的茎尖培养能力优于对应的无色素腺体近等基因系，有色素腺 体棉品种比无色素腺体近等基因系再生根平均长0．07cm～0．21cm，平均叶片数相 差0．08—0．76。此外，培养基中IBA的添加能扩大色素腺体近等基因系内的茎尖 培养效果的差异。外源棉酚的添加也能促进茎尖培养的效果，在0．ling几 IBA 和0．1刁．2吧儿外源棉酚相互作用下，对无色素腺体陆地棉茎尖培养的诱导促进 作用最为显著，其再生根率分别达到96．2％和95．8％。对于有色素腺体陆地棉茎尖 培养来讲，加有0．ling／L IBA和0．01mg儿外源棉酚的MSB培养基是茎尖培养的最 佳培养基。 不同色素腺体近等基因系的根尖培养效果也有差异，但不很有规律。中棉 所12。和中棉所16。的根尖培养效果优于对应的有色素腺体近等基因系，而中棉所 门。的根尖培养效果却差于对应的有色素腺体近等基因系。 棉花色素腺体和棉酚对于棉花体细胞培养的出愈诱导和愈伤组织生长具有 显著的抑制作用。在与 2，4干的作用下，外源棉酚达 0．2 mg几时大部分来源于 子叶的外植体很难形成愈伤组织，添加 0．01～0．1。g几外源棉酚下虽不能提高产 出量，但可以提高愈伤的质地。棉酚与KT配合使用还能大大促进外植体不定根 的诱导率，以0．1刁．Zing／L外源棉酚浓度诱导生根率较高，此外，棉酚诱导离体 叶片不定根的再生在细胞遗传学观察上具有重要的意义。更多还原

【Abstract】 The inducing of callus, formation of embryo and plant regeneration, and some factors which affected the tissue culture such as the type of media, the concentration of inorganic salt, the concentration and ratio of plant hormones, the type and concentration of carbon source, the genotype and the explant type were studied systematically in this paper, using three cultivars and germplasm of Upland cotton, G. hirsutum, in order to optimize the culture technique of cotton tissue culture for callus induce, somatic embryo formation and plant regeneration, and to establish the system of cotton tissue culture, which will lie a good basis on the field of researching in somatic cell culture and transgenic research work in cotton. On the other hand, the tissue culture of transgenic cotton and its genetic background as well as three pairs of pigment gland near-isogenic lines of G. hirsutum were carried out to study the effects of extraneous genes, pigment gland and gossypol on cotton tissue culture, and the main results were as follows:1. Optimization in system of the tissue culture in upland cottonExplants from five-day asepsis seedlings were inoculated on the MSB medium containing 0.1mg/L KT to induce embryonic callus directly, or inoculated on the MSB medium with 0. 1mg/L KT and 0.01-0. 1mg/L 2, 4-D to induce callus, then transfer to the medium containing 0.1mg/L KT and 2g/L active coal to induce embryonic callus. The embryonic callus was golden-yellow and grain type, which was related to the induction of adventitious roots, and all the explants that could induce embryonic callus can produce adventitious roots. In addition, the types of the explants were also related to the induction efficiency of embryonic callus. The differentiation ratio of embryonic callus from cotyledon was higher than that of the hypocotyls. The regeneration of cotton from embryoid to plant did not need hormone, but the active coal and KNO3 could improve the frequency of the normal regeneration seedling. After the regeneration, plantlets should be planted on the rhizogenesis medium and growth medium for a few weeks, and then transplanted into the nutritive pots. By the method of keeping wet and transplanting step by step, a high survival rate for tube seedlings could be transplanted.2. The effect of foreign genes on cotton tissue cultureThe comparing tissue culture for Bt-transgenic cotton, R-CAZ, and its genetic background, CAZ, were carried out, and the fresh weight of callus that induced fromhypocotyl and cotyledon were analyzed by T- test. The results shown that the t-value of fresh weight for the hypocotyls callus was 47.667, >t0.01, P<0.01; and that of cotyledon was 86.711, >t0.01, P<0.01, which showed that the Bt gene had a very significant effects on the induction of the callus in cotton tissue culture. The root numbers that induced from stem tip culture were also analyzed by T- test, and the t-value was 128.187, >t0.01, P<0.01, it also showed the Bt gene had a very markedly effects on the ability of the adventitious root inducing in stem tip culture. The extraneous genes made the Bt-transgenic cotton into a easy material for tissue culture. American Bt-transgenic cultivars, 32B and 33B, Chinese Bt-transgenic cultivars, GK22 and HD109, and the double gene (CPTI and Bt) cotton cultivars, CK321, were also studied in this experiment, the inducing ratio for callus were all over 95%, and the callus quality and its growth potential were similar to that of Coker 312, the CK. Comparing with their genetic background, all the materials used in the experiment prove that extraneous Bt genes could change transgenic varieties to be the tissue culture easy cultivars. However, there were not significant effects on cotton tissue culture of Bar gene transgenic materials obtained through the pollen tube pathway.3. The effect of pigment gland and gossypol on cotton tissue cultureThe pigment gland and gossypol could induce the adventitious root in cotton stem tip culture dramatically. The ability of the stem tip culture with the g更多还原