Effects Of LEC1 On Cotton Somatic Embryogenesis

Objective: As we all know, cotton has important economic value in textile processing and oil production, but many stress factors influence its yield,quality and stress resistance to a large degree. At present, molecular breeding by gene engineering is a hot topic of cotton germplasm innovation. Besides, cotton somatic embryogenesis and plant regeneration are an esssential choke point of cotton genetic engineering. However, most of the cotton somatic embryogenesis system has low incidence rate of somatic embryo, low germination rate, high deformity rate, low regeneration rate, and strong dependence in genotype. Thus, some embryogenesis related genes have been cloned and isolated. LEC1 gene is an indispensable regulatory factor which is necessary for standarding cotyledon features and completing development and maturity of embryo. For one thing, LEC1 can induce the formation of somatic embryo by activating related specific genes that participate embryonic morphogenesis and cell differentiation in early development phase, for another thing, LEC1 can induce some seed maturation course, such as acquiring embryonic dry tolerance and the accumulation of stock. Agrobacterium-mediated genetic transformation can induces cotton inherent defense reaction, reducing its genetic transformation rate. In monocot ryegrass, the lack of inositol, glutamine and cold shock treatment can reduce its defense reaction, improving its genetic transformation rate.Therefore, this study, on the one hand, expects to obtain transgenic plants by the LEC1 genetic transformation in cotton and somatic embryogenesis, then exploring the effects of LEC1 on cotton somatic embryogenesis. On the other hand, through the optimization of lack of inositol, glutamine and cold shock, this study explore the effects of the treatment on cotton hypocotyl defense reaction and genetic transformation rate. The study is aimed at providing theoretical guidance for cotton germplasm innovation by means of genetic engineering.Methods: This research use dexamethasone(DEX) inducible expression vectors p IGb LEC1 A, p IGb LEC1 B, p IKd LEC1 to perform agrobacterium-mediated Kd LEC1 genetic transformation on Xinhai 15(Gossypium barbadense L.)hypocotyl and Gb LEC1 A,Gb LEC1 B,Kd LEC1 genetic transformation on Xinluzao 33(Gossypium hirsutum L.) embryogenic callus, respectively. Kd LEC1 transgenic Xinhai15 embryogenic callus lines and regeneration plants as well as Gb LEC1 A,Gb LEC1 B, Kd LEC1 transgenic Xinluzao33 regeneration plants are identified by PCR analysis respectively after hygromycin selection and somatic embryogenesis. Moreover, three transgenic Xinhai15 embryogenic callus lines which have been identified correctly are treated respectively by 30 uM DEX and no DEX suspension induction for 6h. The Kd LEC1 relative expression level was analyzed by real-time quantitative PCR. Besides, the T-DNA insertion site of genome in Kd LEC1-4 transgenic Xinhai15 regeneration plant was analyzed by high-efficiency thermal asymmetric interlaced PCR(hi TAIL PCR),sequencing and blast sequence alignment.The study is intended to explore the effects of LEC1 on embryogenic conversion, somatic embryogenesis and plant regeneration by F1 generation of transgenic regeneration plants latterly. Furthermore, the study performs different combined treatment of inositol, glutamine and cold shock before and in the process of the agrobacterium-mediated p CAMBIA1301 genetic transformation, which is intended to explore the effects of different treatment on cotton defense reaction and its genetic transformation rate by p CAMBIA1301 genetic transformation, the DAB staining and GUS staining after cocultivation.Results and Conclusions:(1) The study obtains two Kd LEC1 transgenic Xinhai15 regeneration plants by somatic embryogenesis as long as 13 months and PCR identification. Besides, The study also obtains one Gb LEC1 A, Gb LEC1 B and Kd LEC1 transgenic Xinluzao33 regeneration plants, respectively, which regeneration period is about 10 months. Real-time quantitative PCR analysis shows that Kd LEC1 relative expression level fluctuates between 2.5 and 23 times in three transgenic Xinhai15 embryogenic callus lines, demonstrating that dexamethasone(DEX) inducible expression vector p INDEX3 can induce exogenous gene expression at different levels. Hi TAIL PCR and blast sequence alignment show that T-DNA of Kd LEC1 really insert into the genome of Kd LEC1-4 transgenic Xinhai15 regeneration plant, but its exact insertion site is unknown duo to little formation of cotton gene which has been sequenced alignment.(2)Compared to other treatments, no inositol, glutamine and cold shock physiologic treatment reduce the content of H2O2 of cotton which have been infected by agrobacterium and enhance its genetic transformation rate, illustrating that no inositol, glutamine and cold shock physiologic optimization treatment can improve the genetic transformation rate of cotton hypocotyl by attenuating defense response to some extent.