Study On The Protoplast Culture And The System Of Somatic Cell Hybridization Of Banana

Bananas (Musa spp.) are one of the most important fruit crops in the world in terms of production and consumption, which also serve as a staple diet and source of income of people worldwide. Banana plants, however, are both sterile and parthenocarpic so the fruit develops without seed, the cultivated hybrids and species are mostly triploid and most are being vegetatively propagated by tissue culture, causing low levels of genetic diversity of germplasm in Musa plants. More importantly, this crop, is vulnerable to several most devastating diseases such as Fusarium wilt. Therefore, there is compelling need to widen the knowledge base for further fundamental research and for application in future improvement of the crop, breeding cultivars with disease and pest resistance and good agronomic value, which, assure the sustainability of banana production and industry in the world.The present study attempted to establish an efficient and integrallty system for both protoplast culture and plant regeneration, and to progress in a preliminary basis research on somatic hybridization. Using a wild germplasm Musa itinerans Cheesm and a triploid variety'Guoshanxiang'(Musa AAB Silk cv. Guoshanxiang) as the materials, we established embryogenic cell suspensions cultures and developed a plant regeneration system based on that, and investigated the main affecting factors within those processing, which can provide an useful information for somatic hybridization and germplasm improvement. The main results are as follows:1. The embryogenic calli was induced using explants derived from mature seeds of Musa itinerans Cheesm. After 60 days, we obtained the loose yellow embryogenic callus. Also we obtained homogeneous embryogenic cell suspensions from embryogenic callus by suspension culture after 2 months screening and subculture.2. Using Musa itinerans Cheesm embryogenic cell suspensions as starting materials, we successfully isolated a large number of protoplast. The effects on protoplast isolation and plant regeneration were systematic study. Results showed that type and concentration of enzyme, time for enzymatic hydrolysis and osmo-regulator have a major impact on the isolated protoplast yield and viability. The optimal conditions for obtaining the Musa itinerans Cheesm protoplasts at a high yield and viability of 19.46×106 protoplasts/ml and 92.17%, were through the embryogenic cell suspensions hydrolysised for 8 hours in the enzyme solution containing 3.0% cellulase Onozuka R-10,1.0% macerozyme R-10,0.2% pectinase,0.78% CaCl2, 1.52% KCl,0.01% MES and 11% mannitol. Using nurse culture method, the microcolonies (small cell clusters) were formed from protoplast culture, afterwards they were transferred onto embryo induction medium. After 45 days induction, somatic embryos emerged, in which those of 45.97% mature embryos later germinated and 54.76% of the somatic embryos formed normal plantlets after the germinated.somatic embryos were transferred to rooting medium.3. Somatic hybridization between triploid and diploid bananas was attempted by using protoplast electrofusion on Musa itinerans Cheesm and'Guoshanxiang'. The results showed that, under the conditions of 10% Mannitol and 0.1mmol/L CaCl2 in electrofusion solution, and generated AC pulse (200 V/cm-1,30 sec) and DC pulse (1500V/cm,40μs) 2 times, the fusion frequency of protoplast was 32.4%, with the cell viability was about 81.4%.