Studies On Protoplast Culture And Protoplast Symmetric Fusion In Cotton

Cotton is the world's leading natural fiber and the second largest oilseed crop in production; cotton is the number one value-added crop. Approximately 90% of cotton's value resides in the fiber, yet yield and fiber quality have declined, especially over the last 15 years. This downward trend has been attributed to general erosion in genetic diversity of cotton varieties, and an increased vulnerability of the crop to environmental stress. In light of the critical need to increase diversity in the gene pool, cotton improvement programs are increasingly turning to the application of molecular approaches to breeding and germplasm utilization. The abundant cotton wild species {Gossypium spp.) is an important renewable resource and has been the valuable genetic germplasm for cotton genenric improvement, and which has been significant in the reality and theory, potential in the application. It is very difficult to widecross between cultivars and wild species for the distant relationship, or no fertility of F₁ hybrids, or very low fertility. Application of biotechnology is an effective way for new germplasm development in cotton, and we wanted to develop new sources of cotton germplasm via somatic cell culture, protoplast culture and protoplast fusion. Our studies involved somatic embryogenesis and plant regeneration in wild cotton species, protoplast culture in Gossypium hirsutum L. and wild species, protoplast fusion between cultivars and wild species. The main results of this research were as fellows.1. Calluses were induced from 9 wild cotton species. Among them, the normal regenerated plants were obtained from G davidsonii, G. klotzschianum, G. raimondii and G stocksii via somatic embryogenesis, regenerated plants with abnormal morphology from G. aridum. Only non-embryogenic calluses were obtained from G. anomalum, G. africanum, G. thurberi and G. bickii. We studied the methods and factors for embryogenic callus induction and conservation, improving somatic embryos maturation andgermination, plant regeneration in detailed, and then a new and elementary protocol has been developed for somatic cell culture, mainly somatic embryogenesis and plant regeneration in wild cotton species. The combination of 2, 4-D/KT was very resultful for callus induction in all tested wild species. Different combinations of PGR, sugar sources, suspension culture and environmental stress et al improved embryogenic callus formation, somatic embryos maturation and germination, plant regeneration to some degree. Embryogenic calluses of wild species were subcultured and conserved on MSB semi-solid medium supplemented with IBA 0.984 (iM, KT 0.232 fiM for 4 years, they still have the capability of differentiation and then provide a mass of materials.This is the first report of regeneration of plants via somatic embryogenesis in many wild cotton species.2. Protoplasts were isolated from different explants of 2 species (Coker 201 and YZl) in Gossypium hirsutum L. (embryogenic cell suspension culture, embryogenic callus, immature somatic embryos, hypocotyls, young roots and leaves). Plants regenerated from cultured protoplasts of 6 explants in Coker 201, but the plating frequencies of protoplasts from different explants varied significantly, the plating frequency of suspension culture-protoplast was 10%, that of embryogenic callus- and somatic embryo-protoplast was 6%, that of hypocotyl-, young root- and leaf-protoplast was not over 2%. Plants regenerated form protoplast cultures isolated from embryogenic suspension cultures, somatic embryos and embryogenic callus in YZl, the plating frequencies were lower (l%-2%) than those from same explants in Coker 201.Plants regenerated from protoplasts isolated from somatic embryos and embryogenic suspension cultures in wild cotton G klotzschianum with the plating frequencies ranging 6% to 8%. Analysis of RAPD demonstrated that the regenerated plants have genetic homogeneity.This study emphasized on enzyme combinations for protoplast isolation, the influences of culture density and PGR combinations et al for protoplasts sustained division, callus formation, and then a practical protocol for protoplast culture in cotton has been developed.3. In this research, symmetric fusion mediated by electricity was carried out,including 8 combinations. Plants regenerated from Coker 201+G klotzschianum, Coker 201+G davidsonii, Coker 201+G bickii, Coker 201+G stockii, which morphologically mediated fusion parents, apt to wild cotton parent. Cytological examinations and flow cytometric analysis showed that all of the tested plants were hexaploids with chromosomes (2n=2x=78), or aneuploids nearing 78 chromosomes, sum of that of parents, and were somatic hybrids. Analysis of RAPD and other molecular markers demonstrated that the majority of regenerated plants had band patterns sum of two parents' specific bands. Somatic hybrid plants of Coker 201+G klotzschianum, Coker 201+G davidsonii flowered and set bolls in green house, but difficultly flowered outside in the field. Somatic hybrid plants of Coker 201+G bickii, Coker 201+G stockii flowered and set bolls in the fall in the field and during the winter in the green house. The fertility of four somatic hybrids was comparatively high, perhaps related with photoperiod response. Somatic hybrid callus was obtained from G arboreum +G stockii, but this callus differentiated very difficultly.This is the first report on production of somatic hybrid plants between cultivars and wild species via protoplast fusion in cotton.4. In this research, we studied various factors in efforts to improve somatic embryogenesis and plant regeneration in wild cottons through 3 aspacts of somatic cell culture, protoplast culture in wild cotton species and regeneration of somatic hybrids between wild species with cultivars, in order to improve the efficiency of regeneration in wild cotton species and broard the range of wild species in which plant regenerated via somatic embryogenesis.