农杆菌介导转化棉花过程中畸形苗发生机理的研究
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摘要
农杆菌介导法转化棉花是目前棉花转基因的最主要的方法，但用此法转化棉花后诱导成苗的过程中90％以上为畸形苗，这已成为棉花转基因的瓶颈，严重制约着转基因效率。
本研究采用农杆菌介导法转化棉花纤维改良基因，得到抗卡那霉素植株，PCR验证纤维改良基因已成功转入棉花。优化农杆菌介导法转化棉花的主要技术参数，拓宽了棉花基因型范围，建立了四个棉花品种的高效遗传转化体系。研究发现采用嫁接技术，可使转基因植株成活率达到90％以上，可有效提高转化成效。
对棉花胚状体染色体制片技术进行了大量研究，摸索出了适合棉花胚状体染色体制片的流程。染色体观察发现不同品种的胚状体均存在染色体变异，且以染色体数目介于27～52条的非整倍变异细胞为最多，不同品种的染色体变异率差别很大，胚胎发生能力弱的品种染色体变异率高。
转化后采用平行实验得到不同品种的不同培养体系的愈伤组织、胚性愈伤组织、胚状体、类胚状体、畸形苗和正常苗，以最终正常苗诱导率高的体系的材料作为正常苗的发生过程，以诱导率低的作为畸形苗的发生过程，即分为正常苗的发生过程，愈伤组织——球型胚状体——心型胚状体——鱼雷型胚状体阶段——子叶型胚状体——正常苗：畸形茁的发生过程，愈伤组织——类球型胚状体——类心型胚状体——类鱼雷型胚状体阶段——类子叶型胚状体——畸形苗。以测量大田种子形成过程和种子萌发过程中内源激素的动态变化做参照系，测量两再生苗发生过程的不同发育时期的内源激素的动态变化，结果表明，黄绿色的胚性愈伤组织与球型胚为棉花体细胞胚胎发生的两个关键时期，亦是两个转折点；畸形苗发生过程中的内源激素含量在球型胚之后比正常苗高；分化过程中球型胚状体阶段的IAA和ABA的低含量和细胞分裂素含量下降慢是导致畸形苗最终发生的原因之一，IAA／ABA值的高峰的推迟也是造成使畸形胚状体及畸形苗的原因之一；在心型胚状体阶段适当降低IAA／CKs值，以来诱导正常胚状体和正常苗。
The agrobacterium-mediated transformation is a main useful method in cotton transformation, in which induce the abnormal plantlet would be more than 90% during the regeneration of plantlet, which becomes the bottleneck of cotton transformation and restricts the efficiency of the cotton transformation.
Fiber-improvement transgenic cotton plants were obtained by Agrobacteriwn-mediated transformation and were identified by PCR amplification. The main technical parameters were improved, the genotypes of cotton acceptors were widened, and a high efficient system of transformation of four varieties was established. The results showed the surviving rates of the regeneration transgenic plants of different varieties exceeded 90% by grafting.
The chromosome aberrance of cotton embryoid was observed. All of the four varieties cotton occurred Chromosome aberrance, and cells with 27~52 chromosomes were the majority in the aberrant cells. Different varieties had different rates of aberrance; the varieties with poor capacity of somatic embryogenesis had a higher rate of aberrance chromosome.
Callus, embryogenetic callus, embryoid, abnormal plantlet and normal plantlet of different varieties were obtained from a same media in the induction stage. The normal and abnormal embryoid were distinguished by the rate of normal plantlets induced and the regeneration modes of the cottons varieties were compared to two ways, one with the highest rate of normal inducement was the normal way of regeneration plantlet from callus, embryogenetic callus, global embryoid, heart-shape embryoid, torpedo-shape embryoid, cotyledon embryoid to normal plantlet, etc, the other with the lowest rate of normal inducement was the abnormal way from callus, embryoid callus, global embryoid-like, heart-shape embryoid-like, fish torpedo-shape embryoid-like, cotyledon embryoid-like, abnormal plantlet to normal plantlet, etc. The procedure of seed development and the seed germination as the controls were compared to two ways to measure the changes of the endogenous hormones contents during the induction stage of cotton abnormal pl
antlets. The results showed that the stage of olivine embryogenic callus and the global embryoid were the key stages during induction stage of the plantlet.
The contents of Endo-hormones in abnormal induction plants were higher than that in the normal induction plants after the stage of global-embryoid; abnormal induction plantlets maybe resulted from the lower contents of IAA and ABA in the stage of global-embryoid and a little slowingdown of CKs, thereafter, delaying of the peak ratio of IAA/ABA possibly caused the induction of abnormal plants as a factor. To induce the normal plant and embryoid, reducing the content of lAA/CKs after heart-shape embryoid was suggested.
本研究采用农杆菌介导法转化棉花纤维改良基因，得到抗卡那霉素植株，PCR验证纤维改良基因已成功转入棉花。优化农杆菌介导法转化棉花的主要技术参数，拓宽了棉花基因型范围，建立了四个棉花品种的高效遗传转化体系。研究发现采用嫁接技术，可使转基因植株成活率达到90％以上，可有效提高转化成效。
对棉花胚状体染色体制片技术进行了大量研究，摸索出了适合棉花胚状体染色体制片的流程。染色体观察发现不同品种的胚状体均存在染色体变异，且以染色体数目介于27～52条的非整倍变异细胞为最多，不同品种的染色体变异率差别很大，胚胎发生能力弱的品种染色体变异率高。
转化后采用平行实验得到不同品种的不同培养体系的愈伤组织、胚性愈伤组织、胚状体、类胚状体、畸形苗和正常苗，以最终正常苗诱导率高的体系的材料作为正常苗的发生过程，以诱导率低的作为畸形苗的发生过程，即分为正常苗的发生过程，愈伤组织——球型胚状体——心型胚状体——鱼雷型胚状体阶段——子叶型胚状体——正常苗：畸形茁的发生过程，愈伤组织——类球型胚状体——类心型胚状体——类鱼雷型胚状体阶段——类子叶型胚状体——畸形苗。以测量大田种子形成过程和种子萌发过程中内源激素的动态变化做参照系，测量两再生苗发生过程的不同发育时期的内源激素的动态变化，结果表明，黄绿色的胚性愈伤组织与球型胚为棉花体细胞胚胎发生的两个关键时期，亦是两个转折点；畸形苗发生过程中的内源激素含量在球型胚之后比正常苗高；分化过程中球型胚状体阶段的IAA和ABA的低含量和细胞分裂素含量下降慢是导致畸形苗最终发生的原因之一，IAA／ABA值的高峰的推迟也是造成使畸形胚状体及畸形苗的原因之一；在心型胚状体阶段适当降低IAA／CKs值，以来诱导正常胚状体和正常苗。
The agrobacterium-mediated transformation is a main useful method in cotton transformation, in which induce the abnormal plantlet would be more than 90% during the regeneration of plantlet, which becomes the bottleneck of cotton transformation and restricts the efficiency of the cotton transformation.
Fiber-improvement transgenic cotton plants were obtained by Agrobacteriwn-mediated transformation and were identified by PCR amplification. The main technical parameters were improved, the genotypes of cotton acceptors were widened, and a high efficient system of transformation of four varieties was established. The results showed the surviving rates of the regeneration transgenic plants of different varieties exceeded 90% by grafting.
The chromosome aberrance of cotton embryoid was observed. All of the four varieties cotton occurred Chromosome aberrance, and cells with 27~52 chromosomes were the majority in the aberrant cells. Different varieties had different rates of aberrance; the varieties with poor capacity of somatic embryogenesis had a higher rate of aberrance chromosome.
Callus, embryogenetic callus, embryoid, abnormal plantlet and normal plantlet of different varieties were obtained from a same media in the induction stage. The normal and abnormal embryoid were distinguished by the rate of normal plantlets induced and the regeneration modes of the cottons varieties were compared to two ways, one with the highest rate of normal inducement was the normal way of regeneration plantlet from callus, embryogenetic callus, global embryoid, heart-shape embryoid, torpedo-shape embryoid, cotyledon embryoid to normal plantlet, etc, the other with the lowest rate of normal inducement was the abnormal way from callus, embryoid callus, global embryoid-like, heart-shape embryoid-like, fish torpedo-shape embryoid-like, cotyledon embryoid-like, abnormal plantlet to normal plantlet, etc. The procedure of seed development and the seed germination as the controls were compared to two ways to measure the changes of the endogenous hormones contents during the induction stage of cotton abnormal pl
antlets. The results showed that the stage of olivine embryogenic callus and the global embryoid were the key stages during induction stage of the plantlet.
The contents of Endo-hormones in abnormal induction plants were higher than that in the normal induction plants after the stage of global-embryoid; abnormal induction plantlets maybe resulted from the lower contents of IAA and ABA in the stage of global-embryoid and a little slowingdown of CKs, thereafter, delaying of the peak ratio of IAA/ABA possibly caused the induction of abnormal plants as a factor. To induce the normal plant and embryoid, reducing the content of lAA/CKs after heart-shape embryoid was suggested.
引文
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