【作者】 司家钢；

【导师】 朱德蔚；

【作者基本信息】 中国农业科学院 ， 蔬菜学， 2001， 博士

【摘要】 胡萝卜瓣化型细胞质雄性不育性常规回交转育一般至少需要5-6年的时间，原生质体非对称融合方式转移这种雄性不育性可使时间大大缩短，从而加快育种的进程。本试验在Tanno Suenaga等的研究基础上，对胡萝卜原生质体非对称融合的基础体系进行了较广泛的研究；进行了61次胡萝卜种内原生质体非对称融合，从19次融合中获得愈伤和胚状体，从6个组合获得了植株再生；对再生植株进行了形态学、细胞学和分子生物学鉴定。主要研究结果如下： 1.基因型是影响胡萝卜固体培养和细胞悬浮培养中愈伤组织状态的重要因子，并由此影响到分离原生质体的产量和质量，进而影响原生质体的愈伤再生和植株再生，因此，在进行胡萝卜原生质体非对称融合前首先对基因型进行筛选是必要的。 2.通过麦芽提取物和谷氨酰胺两种物质的添加，可以起到抑制非胚性愈伤生长的作用，使培养物主要以胚性愈伤为主；通过液体培养基2，4-D浓度的改变，可调节胚性愈伤处于合适的发育时期，便于原生质体的分离。 3.通过胡萝卜原生质体培养愈伤和胚状体发生过程的观察，认为胡萝卜原生质体培养胚状体的直接来源是愈伤组织，而不是原生质体细胞本身。 4.UV和X-射线的作用机制相似，原生质体对UV射线更为敏感。UV辐射可以很好地起到抑制原生质体再生和愈伤组织生长的作用，可以用作胡萝卜的供体处理；UV和X-射线辐射处理供体原生质体都存在处理效果不稳定的情况，因此在融合杂种筛选中应着重注意淘汰供体基因型；IOA抑制胡萝卜原生质体细胞分裂的适宜剂量是15mM浓度处理10min，其处理效果稳定。 5.Ca²⁺离子浓度过高，使原生质体在交流电场下不能串珠状连接，从而影响电融合，胡萝卜原生质体电融合Ca²⁺离子的适宜浓度为0.5mM；优化的胡萝卜原生质体电融合参数是：交流电场（AC）70V/cm，作用时间60s；直流电场（DC）1500V／。m，5次脉冲，间隔卜，直流脉冲印加时间50 K 6．STS。标记与胡萝卜瓣化型胞质雄性不育性密切相关，可以用作胡萝卜体细胞杂种瓣化型胞质雄性不育性的分子鉴定，并可用于胡萝卜一代杂种的纯度鉴定。 7．愈伤组织的植株再生是制约胡萝一卜对称融合转移胡萝卜雄性不育性最终能否成功的一个重要环节，内源激素的调控与植株再生相关。此外，原生质体预处理的后效应也是值得考虑的因素。影响胡萝卜融合体愈伤植株再生的因子是今后研究的重要内容。 8．温度是影响胡萝卜再生植株移栽成活的主要因素。胡萝卜组培苗较耐低温，10度左右的平均温度下仍能移栽成活，但是对于高温忍耐性较差，平均温度为30度以上的高温情况下，难于移栽成活。 9．通过形态学、细胞学和分子生物学鉴定，确认组合7－0－8－4＋66－3的再生植株为胞质杂种，成功通过非对称融合方式将供体7．0．8．4的瓣化型胞质雄性不育性转移到受体66－3中，认为原生质体非对称融合可以成为转育胡萝卜瓣化型胞质雄性不育性的补充手段。 论文对影响胡萝卜原生质体非对称融合的若干因子、组培茵的先期抽墓、利用非对称融合转移胡萝卜瓣化型雄性不育性的可行性、本研究存在的问题及改进方案以及非对称原生质体融合在胡萝卜资源创新中的应用价值等进行了讨论。更多还原

【Abstract】 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更多还原