In light of the critical need to increase genetic diversity in the gene pool,cotton improvement programs are increasingly turning to the application of molecular approaches to breeding and germplasm utilizations.The abundant species of wild cotton (Gossypium spp.) are an important renewable resources and have been the valuable genetic germplasms for cotton genentic 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 developing new germplasm in cotton,and we aim 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 follows:1.Calli were induced from 9 wild cotton species.Among them,the normally 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 calli 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 and germination,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 useful for callus induction in all tested wild species.Different combinations of PGR,sugar sources,suspension culture and environmental stress etc improved the formation of embryogenic callus,the maturation and germination of somatic embryos,and plant regeneration to some degree.Embryogenic calli of wild species subcultured and conserved on MSB semi-solid medium supplementing with IBA 0.984 μmol/L,KT 0.232 mol/L for 4 years still have the capability of differentiation and provide a mass of materials.It 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 YZ1) 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,embryogenic callus-and somatic embryo-protoplast,hypocotyl-young root-and leaf-protoplast was 10%,6%,less than 2%.The plating frequencies of plants regenerated form protoplast cultures isolated from embryogenic suspension cultures,somatic embryos and embryogenic callus in YZ1 were lower (1%-2%) than that of plants regenerated 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%.RAPD analysis demonstrated that the regenerated plants were genetically homogeneous.This study emphasized on enzyme combinations for protoplast isolation,the influences of culture density and PGR combinations etc for protoplasts sustained division,callus formation,and then a practical protocol for protoplast culture in cotton is established.3.In this research,symmetric fusion including 8 combinations mediated by electricity was carried out.Plants regenerated from Coker 201+G.klotzschianum，Coker 201+G.davidsonii，Coker 201+G.bickii,Coker 201+G.stockii,which were morphologically intermediated 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,the sum of that of parents,and were somatic hybrids.Analysis of RAPD and other molecular markers demonstrated that the majority of regenerated plants had its 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 at fall in the field and at 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.It 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 aspects of somatic cell culture,protoplast culture in wild cotton species and regeneration of somatic hybrids between wild speciesand cultivars,in order to improve the efficiency of regeneration in wild cotton species and broarden the range of wild species in which plant regenerated via somatic embryogenesis.