| Transfer of petaloid cytoplasmic male sterility (CMS) in carrot with the routine backcross method needs at least 5-6 years, while the time could be saved largely by using the asymmetric protoplast fusion method to transfer this male sterility, and breeding progress could be speeded up. Based on the study of Tanno Suenaga et al, much research work was conducted on the carrot asymmetric protoplast fusion basic systems; 61 times of asymmetric protoplast fusion in carrot had been made, callus and embryoid were obtained in 19 times of fusion, and the plantlets were regenerated from 7 combinations; identification was done on the regenerated plants by using morphological, cytological and molecular methods. The main results of this research were as follows:1.Genotype was an important factor affecting the callus status in solid culture and suspension cell culture, which could further affect the quality and yield of the isolated protoplast, and in the last, the regeneration of calli and plantlets from protoplast culture would be influenced, so the selection of genotype before fusion is necessary.2.The addition of malt extract and glutamine could inhibit non-embryonic calli, and make the culture be mainly composed of embryonic calli; change of the 2,4-D concentration in liquid culture medium could adjust the status of embryonic calli to the suitable developmental stage for protoplast isolation.3.According to the observation of the genesis of calli and embryoid in protoplast culture, the direct source of embryoid was considered to be callus, rather than the protoplast cell.4.The modes of the action of UV (Ultra Violet rays) and X-rays were similar, but the protoplast was more sensitive to UV irradiation. The protoplast regeneration and callus growth could be inhibited well by UV irradiation, so it could be used to pre-treat the donor protoplast in asymmetric protoplast fusion. The treatment effect on donor protoplast of UV and X-ray irradiation was not stable, so more attention should be paid to the elimination of the donor genotypes in the selection of the somatic hybrid. 10 mm treatment with 15 mM IOA was suitable to inhibit recipient protoplast cell division, and the effect of IOA treatment was stable.5.Extremely high concentration of Ca2~ would prevent the protoplast from connection with each other under AC field, and affect the electronic fusion, 0.5 mM Ca2~ was the favorite concentration in the carrot protoplast electronic fusion. The optimum parameters for carrot3electronic protoplast fusion was as follows: Alternative current fleld(AC), 7OVIcm, exerting time, 60s; Direct current field(DC), 1 500V/cm,S pulses, is interval, DC pulse exerting time, 50~s.6.STS4 marker was closely related to petaloid cytoplasmic male sterility in carrot, it could be used as molecular marker in the identification of the carrot somatic hybrids on the petaloid cytoplasmic male sterility, and also could be used in the purity identification of F1 hybrid variety.7.Plantlet regeneration from callus was an important step in the transfer of carrot male sterility by asymmetric protoplast fusion, the regulation of endogenous hormone was related to plantlet regeneration. In addition, the post-effect of the pretreatment of protoplast also should be considered. The influencing factors on the regeneration of fusion product were the important content in the future research work.8.Temperature was the main factor affecting the transplanting of the regenerated plantlets in carrot. The carrot plantlets in vitro was tolerant to lower temperature, and could be successfully transplanted into the soil and survived under the average temperature about bC, however they were intolerable to high temperature conditions, difficult to survive when the average temperature is above 30C.9.According to the results of morphological, cytological and molecular identification, it was proved that cybrids were obtained from 7-0-8-4+66-3 fusion combination, and the petaloid cytoplasmic male st...
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