Studies On Donor-Recipient Fusion And Genetic Transformation In Cotton

Cotton genetics and breeding is on the basis of the development of new germplasm. However, using the existing germplasm is very difficult to improve cotton cultivars, mostly because that many important characters (pest and disease resistance, herbicide resistance, and so on) were lack in cultivars. And the favorable genes were present in the wild and related species. It is very difficult to make wide cross between cultivars and wild species for the distant relationship or very low fertility. Application of biotechnology is an effective way for new germplasm development in cotton. Somatic culture, protoplast fusion and genetic transformation are of great use in cotton breeding, and have the broad application prospects in genetic improvement in cotton. Our studies involved somatic embryogenesis of Ekang cultivars, organogenesis in wild cotton (Gossypium bickii Prokhanov, G Genome), donor-recipient fusion between cultivars and wild species and genetic transformation of protoplasts. The main results of this research were as follows.1. Somatic embryogenesis of Ekang genotypes was carried out. The effect of different plant growth regulators on callus induction, embryogenic callus induction and somatic embryos germination of Ekang spectrum was compared. The results indicated that 2, 4-D was benefit for callus induction and proliferation at the early stage. Embryogenic callus was not obtained on the medium with 2, 4-D, but the yellow and gray granulated embryogenic calli of EKang 5 and EKang 8 were seen on the medium with NAA, and developed to plantlet finally. 0.5 mg/L NAA + 0.1 mg/L KT and 0.5 mg/L IBA + 0.1 mg/L KT were beneficial to embryogenic calli maintenance and somatic embryos germination.2. Organogenesis of wild cotton (Gossypium bickii Prokhanov, G Genome) was carried out. The effect of main factors on organogenesis in wild cotton (Gossypium bickii Prokhanov, G Genome) (the types of explants, cytokinin types and concentrations) was investigated. Among the explants (cotyledonary nodes, hypocotyls and cotyledons) used, only the cotyledonary nodes explants produced multiple shoots on the MSB medium supplemented with BAP or TDZ alone or in combination, and more than one shoot per explant were induced in these medium (>2). The better medium for organogenesis for G. bickii was the combination of 4 mg/L BAP and 0.1 mg/L TDZ. In addition, Flow cytometric analysis and chromosome counting indicated that no obvious change in the ploidy of the regenerants and their offsprings happened to the control diploid parent. The RAPD analysis showed that those plants have genetic homogeneity.3. donor-recipient fusion between cultivars and wild species was performed. (1) Before fusion, iodoacetamide (IOA) was used to treat recipient protoplasts of cultivars and ultraviolet (UV) was used to treat donor protoplasts of wild species. The different IOA solution, treat temperature, treat duration and IOA concentration were studied. As a result, protoplasts treated with 0.5 mM IOA which was diluted with CPW9M solution for 15 min at room temperature was chosen as the best condition of IOA treatment (treated protoplasts can't divid continually, but can survive several days, have a few division and low breakage). On the other hand, five doses were managed on G. davidsonii protoplasts. And the dose of 38.7 J/cm~2 was chosen for UV irradiation (treated protoplasts can't divid continually, but can survive several days, have a few division and low breakage). The condition was used in protoplast fusion later. (2) The effect of electrofusion parameters (Alternate current field strength and duration, direct current pulse strength and duration, direct pulse number) on protoplast fusion was analyzed. The best electrofusion parameters (short bunchiness, low breakage, high fusion efficiency) were as follow: Alternate current field strength of 100 V/cm, AC duration of 15s; direct current pulse strength 1250V/cm, DC pulse duration of 30μs; direct pulse number of 3. (3) In this research, donor-recipient fusion mediated by electricity was carried out, including 4 combinations. Plants regenerated from YZ-1 + G. davidsonii. Most fused regenerants displayed different Morphological characteristics compared to the two parents. Some were intermediate morphologically to the two parents, and a few was similar to the recipient. And the chromosome number of the regenerants was between 40 and 73. Analysis of RAPD and SSR verified the hybridity of the regenerated plantlets, and Analysis of cpSSR and CAPS indicated that rearrangements or recombination might have occurred in some regions of the mtDNA and cpDNA in the somatic hybrids. This was a novel case in hybrid production following the symmetric fusion and asymmetric fusion based on UV irradiation in cotton.4. Genetic transformation of YZ-1 and G. davidsonii protoplasts was carried out. Protoplasts isolated from embryogenic callus suspension as explants and GFP gene as marker gene were used in this experiment. And the foreign plasmid (pBIN m-gfp5-ER) DNA was transformed to protoplasts via electroporation transformation to establish a protoplasts transformation system in cotton. In this study, the factors affecting the transformation efficiency were investigated. The transformation efficiency was the highest (5.36 %) when the direct current pulse strength of 1000 V/cm, pulse duration of 20 us and the plasmid concentration of 100 ng/μl. As a result, no transformed calli of YZ-1 was obtained, but 5 transformed calli and 3 plants of G. davidsonii were produced.