Studies On Protoplast Culture And Somatic Hybridization In Cotton

Cotton is not only one of important fibre crops,but also provides abundant oil and protein resource only inferior to soybean.Whether the production of cotton is stable dramatically affects the development of agricultural production,farmer income and textile industry of our country.Therefore,with the aim to stabilize cotton production and increase farmer income,improvement and innovation on cotton breeding is justified to create new germplasm with higher yield,better fiber quality and multiple resistances, satisfying the demand of market and reinforcing Chinese competitive advantage on cotton.The technology of cotton tissue culture is an effective method of cotton breeding which is able to accelerate breeding process,increase breeding efficiency,create new breeds and cultivars as well as provide an experimental platform for modem molecular breeding and gene engineering.However,work on somatic culture of cotton begin very late,and cotton has been acknowledged one of these crops which are most difficult to obtain their regenerated plants in vitro.On the basis of preceding researches,the techniques of somatic embryogenesis and plant regeneration for cotton somatic culture, including the upland cotton and wild cotton species,were systematically studied,as well as the techniques of cotton protoplast culture and protoplast fusion between upland cotton cultivars and several wild species.The main results of these researchs are as follows.1.Improving and optimizing cotton somatic culture technique system.Large embryos and regenerated plants of upload cottons and several wild cotton species were obtained via callus inducing,somatic embryogenesis and plant regeneration.This study turned out that,with hypocotyls as explants,the callus induced by the hormone combination of 0.1 mg L^-1KT+0.1 mg L^-12,4-D was loose,crisp and bright,with high differentiation potential but not benefit for somatic embryogenesis.Medium with the hormone combination of 0.2 mg L^-1KT + 0.5 mg L^-1IBA was used for embryogenic calli proliferation.High frequencies of somatic embryos were produced when the medium was supplemented with 3 mg L^-1CuSO₄ and 3 mg L^-1AgNO₃.Regenerated plants could grow normally in medium MSB₅ + 0.1 mg L^-1KT + 0.5 mg L^-1NAA+2 %active carbon +8%agorose.In addition,glucose was good for cotton callus induction,while maltose was even better than glucose for proliferation of embryogenic callus and germination of somatic embryo.The regeneration system was applied widely for different cotton species either upload cotton or wild species.The regeneration cycle of the cotton culture was short.It took 210 d to obtain regenerated plants from explants culture,and needed 90 d from embryogenic calli to regenerated plants for upload cotton2.Establishing of the regeneration system of cotton protoplast culture.Plants regenerated from protoplasts of Coker 312 and ZDM-3 via somatic embryogenesis with the influences of the isolation,purification,culture and plant regeneration of cotton protoplast isolated from embryogenic cell suspension culture of Coker 312 and ZDM-3 being studied concretely,and then many somatic embryos obtained from protoplast culture of wild cotton species G.klotzschianum and G.australe.This study indicated that plentiful protoplasts isolated with enzyme solution of 3.0%Cellulase Onzuka R-10 and 1.0%Mecerozyme R-10 or with 3.0%Cellulase,1.5%pectinase and 0.5%hemicellulose for 20-23 h were vigorous and fit well for being cultured.Purified protoplasts were cultured in thin liquid medium KMSP+0.1 mg L^-12,4-D+0.2 mg L^-1 KT+1.5%(w/v)glucose+1.5%(w/v)maltose+9.0%(w/v)mannitol.Many normal somatic embryos and plants were produced with matlose as a carbon source.The regenerated plants were transferred to the soil via an easy grafting,the highest survival rate arrived at 95%.The cotton protoplast culture system had high plating efficiency, high conversion rate of somatic embryos to plantlets and high survival probability of transplanting.RAPD analysis showed that these regenerated plants inherited stably.3.Upbuilding of cotton protoplast electro fusion technique system.The cotton protoplast fusion technique was studied with the protoplast fusion being mediated by the electricity between 2 upload cotton cultivars(ZDM-3 and Coker 201)and 3 wild cotton species(G.klotzschianum,G.trillium and G.australe).The results showed that three kinds of Somatic hybrid plants(ZDM-3+G,klotzschianum,Coker201+G.trillium, and Coker201+G.australe)were regenerated via somatic embryogenesis.Regenerated somatic hybrid plants were identified by morphological,cytological and molecular analysis.Morphologically,these regenerated plants were investigated intermediate between the fusion parents,apt to wild cotton parent,but they flowered not set bolls outside in the field.Analysis of RAPD and SSR demonstrated that some regenerated plants contained specific genomic fragments from both fusion partners.Flow cytometric analysis showed that the total nucleus DNA of these tested plants were sum of that of parents.And cytological investigation of the metaphase of root-tip cell of the regenerated plant revealed the chromosome number in the plants was close to the total of the two parents.These hexaploidy somatic hybrids obtained would be useful for breeding programs.