Studies On Protoplast Culture And Somatic Hybridization In Diploid Potato

Potato (Solanum tuberosum L.) is one of the most important food crops in the world. Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of potato. The worldwide control strategy has consisted of plant breeding for resistance. Tuber chip color is an important trait in determining the quality and acceptability of special varity for processing, which is demonded now. Some wild and related species of potato are known to be resistant to bacterial wilt and to be of good chip color. However it is difficult to incorporate these traits to cultivated potato in conventional breeding due to crossing barriers between cultivated potato and the species. In this thesis, diploid potato protoplast culture and somatic hybridization were studied to obtain new germplasm. The results were as follows:1. Ethylene accumulation often occurs in plant tissue culture owing to limited gas exchange. The effect of silver thiosulfate (STS), an inhibitor of ethylene activity, on reducing ethylene-induced plant abnormalities was investigated with different STS concentrations in culture medium. The results showed that supplement of 1 mg/L STS in medium could alleviate ethylene stress and improve plantlet growth by increasing the leaf areas and total chlorophyll contents and eliminating aerial root. Toxic symptoms like black purple pigment accumulation and callus occurring on leaf abaxial surface were observed on plantlets when the STS concentration was over 4 mg/L. Though no morphological abnormality was observed in plantlets, some physiological parameters showed STS toxicity to plantlets of HS66 and DH401 in the medium containing 2 mg/L STS. It was suggested that the STS concentration toxic to plantlets in vitro be judged not only by morphological abnormality but also physiological parameters changes. The 1mg/L STS was added into medium in different concentration sucrose (1%,2%,3%, and 4%) and under different closed-type (closed and difussive) in order to investigate the effect of STS on plantlets. The results showed that the leaf areas, fresh weight and the total chlorophyll content of plantlets increased in the medium containing 1mg/L STS under 2～3% sucrose compared with control (0 mg/L STS). Shoot height and MDA content of plantlets were the lowest whereas leaf areas and total chlorophyll content were the biggest in medium containing 1mg/L STS in diffusion. Therefore, the 1mg/L STS was added into medium with 2～3% sucrose under diffusion benefited diploid potato growth for protoplast donor.2. Reactive oxygen species plays a positive role in plant growth and development such as cell wall polymerizing, cell cycle, cell wall loosening, and signal transduction. Overproduction of reactive oxygen species results in oxidative stress and cellular damage. In the process of protoplast isolation, it is inevitable for plant cell to be under oxidative stress and the balance of reactive oxygen species will be broken. The H2O2 contents, CAT activities and SOD activities were investigated during the protoplast isolation for ED13 with excellent division capability and DH405 with no division capability. The results showed that the H2O2 content of ED13 was higer than that of DH405 at the early stage of isolation (before 8h). The SOD activities of ED13 were lower than that of DH405 at the late stage of isolation (after 8h) but CAT activities of ED13 were higher than that of DH405 in the early stage of isolation (before 8h). It could be concluded that the difference of division capability between ED13 and DH405 was probably related to reactive oxygen species and antioxidative enzyme activity.3. The studies on protoplast culture for diploid potato with resistant to bacterial wilt or with good chip color (ED13, CE171, and HS66) and ones with good agricultural traits (DH401 and DH405) showed that there were no significant difference of protoplast yield and viability between leaves in suspension medium in dark and complete plantlets in dark for pretreatment. When 0.5mol/L mannitol was used as an osmosis regulator, the suitable cellulose concentration (0.4%) for protoplast isolation of CE171 and DH401 was higher than those of ED13, HS66 and DH401(0.3%) under 25℃for 12h. The protoplast of ED13, DH401, CE171, and HS66 could be induced into callus and the protoplast of ED13, DH401could regenerate into complete plantlets after 4 month culture, but protoplast of DH405 had no division capability.4. The studies on electric parameters in somatic hybridization indicated that better fusion effect was in the condition of 156V/cm for 10s or 50 V/cm for 30s in alternate current and 1250V/cm in direct current for 45μs. 242 callus derived from fusion of ED13 and DH401 regenerated 23 plantlets and SSR maker showed that 11 plantlets were somatic hybrid.