Morphological And Genetic Analysis Of Somatic Embryogenesis And Cloning And Expressional Analysis Of Related Genes During Initial Cellular Dedifferentiation In Cotton

Cotton is not only one of the most important fiber crops, but also is the most important sources for oils and proteins industry, and is one of the most important economic crops in the world. The steady production of cotton directly affects the whole agricultural production, farmer income and textile industry. Therefore, durative improvement and innovation of cotton variety, including high yield, quanlity and strong anti-adversity competence, were the persistent objective of cotton breeding. Cotton tissue culture is common and effective method for cotton breeding, which accelerates the breeding procedure with various biotechnologies, and improves breeding efficiency, and provides an effective tool for modern molecular breeding and gene engineering. As we know, cotton tissue culture began lately than other crops, and it is accredited that cotton is one of the most diffcult crop for plant regeneration via somatic embryogenesis. Based on above, broadening materials for cotton gene engineering, investigating the genetic controlling and origin of embryogenic cells, and disclosing the moleculr mechanism of cellular dedifferentiation are important to further improve cotton gene engineering. Besides, those studies also help us realize somatic embryogenesis in various aspects. And the results are as follows:1 Callus induction and embryogenic callus differentiation of 40 asian cotton (Gossypium arboreum L.) were investigated, which laid a solid foundation for establishment of regeneration system in asian cotton. Calli were easily induced under 2, 4-D + KT, NAA + KT, IBA + KT treatments of all varieties, and the rates of callus induction were 100%. The configuration of callus in different treatments were remarkably different, the subculture of calli in different treatments indicated NK treatment was more suitable for durative subculture, and calli could maintain a advantaged state. Subsequently, alteration of culture mode, medium component and other treatments, or combinations of above treatments were used for embryogenic callus differentiation, almost 300 different mediums were used and the somatic embryo still did not emerge, and only morphology of some cells like embryogenic cell were observed.2 Somatic embryogenesis of YZ₁ in different phytohormone combinations were investigated, a more effective and rapid regeneration system was established; procedure of somatic embryogenesis with histological observation was observed, which suggested the patterns of somatic embryogenesis were distinct in different treatments. Plant regenerations via somatic embryogenesis were represented in the eight treatments. Somatic embryogenesis competence were distinct in different treatments, and the highest ratio of embryogenic calli presented in IBA+KT treatment was 97.6%, which was three times the ratio of embryogenic calli in 2, 4-D+KT treatment. Furthermore, the whole procedure of somatice embryogenesis (from explant inoculated on medium to emergence of somatic embryo) only took 33 days, it was the most short time used for somatic embryogenesis in cotton. Somatic embryogenesis was also represented in MSB without phytohormone. Besides, histological observation suggested the patterns of somatic embryogenesis in DK and IK treatment were different, embryogenic cells were originated from primary meristem and cortex, which partly explained somatic embryogenesis could be rapidly induced in IK treatment.3 To study the genetic controll of embryogenic competence, cross between upland cotton and island cotton, and corresponding F₂ individuals, complete diallel cross with 8 different regenerative competence varities were constructed. Embryogenic variety Coker201(upland cotton) and non-embryogenic variety Pima90(island cotton) were selected for crossing, the somatic embryogenesis of the parents and F₁ were investigated in different phytohormone treatments, Pima90 and F₁ cloud not take somatic embryogenesis in those treatments. Next year, corresponding F₂ seeds was got, none of them took somatic embryogenesis after callus induction and differentiation treatment for more than six months. Subsequently, we designed complete diallel cross with embryogenic varieties, including Coker201, Coker312, Y668, YZ₁ and non-embryogenic varieties, including upland cotton TM-1, EK4 and island cotton Pima90, Pima3-79, and 47 F₁ seeds were acquired, callus induction and differentiation treatment were performed for somatic embryogenesis as above, only two hybrids between upland cotton regenetated plantlet, but the correponding anti-cross hybrids did not. Subsequently, cross and anti-cross hybrids of nonembryogenic F₁ (YZ₁ and EK4) was selected for selfcrossing, F₂ family was used to genetic analysis for somatic embryogenesis competence. Only 14 individuals from F₂ seeds with YZ₁ as male parent represented somatic embryogenesis, and the characterization of embryogenic calli from differnt individuals were distinct, the embryogenic competence were also remarkable. Therefore, somatic embryogenesis competence in cotton was a complicated characterization, which might be controlled by multi-genes from cytoplasm and nucleous.4 SSH library related cellular dedifferentiation was constructed, expression analysis were preformed with partly important genes, and provided a putative molecular mechanism for cellular dedifferentiation with SAM-dependent transmethylation. Functional validations of some related genes were also performed. cDNA of explants of three different treatments sampled at 6 h, 12 h, 24 h, 48 h, 72 h and 168 h as tester and 0 h(hypocotyl) as driver, subtract especially expressed genes and twice PCR, SSH library were constructed. Especially expressed clones were picked out and sequenced, a total of 286 differential cDNA clones were sequenced.. Among these clones, 112 unique ESTs were isolated and identified, and 40.2% of the ESTs were first identified, which might mean new genes related to cellular dedifferentiation. Expression analysis of related genes was performed, GST was highly expressed from 6 to 24 h after induction with phytohormone treatment. PRPs were predominantly expressed and exhibited distinct expression patterns in different treatments, suggested they are closely related to cellular dedifferentiation in cotton. Besides, Putative GhSAMS, GhSAMDC, GhSAHH and GhACO3 involvement in SAM metabolism was identified in this library. The analysis of realtime-PCR showed that two remarkable increased expressions of the four SAM-related genes happened during the early phase of phytohormone induction, and that a highly positive correlation existed between GhSAMS and GhSAHH, which further indicated the highest expression level of GhSAMS might be associated with reentry into the cell cycle. The histological observations further showed that some cells accomplished cellular dedifferentiation and division within 72 h in 2, 4-D treatment, and cellular dedifferentiation might be regulated through two alterations in SAM-dependent transmethylation activity in cotton. In addition, the expression patterns of differential genes in different treatments disclosed the complicated interaction between 2, 4-D and kinetin.Furthermore, we constructed RNAi vector of Extensin and PRPL, and overexpression vector of SAMS, EF1A4 and PRPL with Gateway technology, and then infected hypocotyls of YZ₁. The differentiation rate of PRPL RNAi infected hypocotyls was remarkablely lower than CK, which might be associated with interference of cellular dedifferentiation, and the normal procedure of somatic embryogenesis was affected subsequently. Besides, embryogenic calli and regenerated plantlets were acquired with infected hypocotyls with three overexpression vectors, positive rate of transformation was 72.73% with PCR detection, exact functions needed further certification.