【作者】 孙保娟；

【导师】 曹家树；

【作者基本信息】 浙江大学 ， 蔬菜学， 2005， 博士

【摘要】 白菜是十字花科芸薹属的一种重要蔬菜,目前其优良品种选育借助的主要手段还是杂种优势利用。本文针对雄性不育亲本保存和繁殖上存在的一些问题,采用带柄及子房的花托、带子房的花托、花柄和花序轴等4种不同外植体,进行了再生体系的研究筛选,并对再生体系建立中出现的一些现象进行了观察分析,进而以不定芽为材料,研究了不定芽离体春化反应特性、抗氧化酶活性及可溶性蛋白含量变化与春化作用的关系,借助cDNA-AFLP技术分离离体条件下与春化反应相关的EST。主要研究结果如下: （1） 核雄性不育两用系‘ZUB97-01AB’不育株和可育株,以带柄及子房的花托为外植体,再生频率因育性的不同而出现了差异。在BAP浓度固定为2 mg·L^-1的条件下,可育在NAA浓度0.1rag-L^-1时获得最高的再生频率72.6%,不育在NAA浓度0.2 mg·L^-1时获得最高的再生频率77.1%,在各自相对最适的NAA浓度下,可育株对BAP浓度要求严格,不育株相对宽泛。带柄及子房的花托,带子房的花托,花柄和花序轴这几种外植体都能再生不定芽,但就诱导率和芽的发生速度而言,以带柄及子房的花托为最理想。总体来说,可育外植体再生表现要优于不育外植体。 （2） 不育株和可育株外植体内源激素含量有所差异,前者GA₃,IAA和ZT含量均比后者高。这能用来解释不育和可育外植体再生的差异。 （3） 来源于花组织的再生芽以有丝分裂的形式传承了母体的春化态,所以导致高频试管开花;这种生殖生长趋势会随着继代扩繁时间的增加逐渐减弱。这种不定芽生长状态的改变是多种因素综合作用的结果,培养基的基本成分,外源激素,细胞的分化形式都可能与此有关。 （4） 经多次继代后得到的营养芽在5℃低温下处理14 d时就能够重新获得春化态,在生根培养过程中抽薹开花,5℃条件下,14 d的处理是本材料可以通过春化的临界时间,要保证获得较高水平的春化态,春化处理时间应在21 d以上。低温处理21 d后再进行脱春化处理,不能完全实现脱春化。 （5） GA₃能够部分代替低温的作用,添加到生根培养基中,能够诱导不定芽开花,但同时造成生根困难。赤霉素诱导与低温春化诱导后开花表现有所不同,GA₃诱导产生花臺的速度及高度不及后者。它们是有交叉作用,但又是有本质不同的两种开花途径。 （6） 再生可育株和不育株小孢子发育进程与母体基本一致,不育小孢子败育发生在减数分裂Ⅱ这一阶段。在离体成花条件下,可育株小孢子不能正常发育,最终导致败育。 （7） 在超长时间的春化处理下,不定芽在感受低温作用通过春化发育后,在低温无光的条件下会继续进行开始花芽分化,形成花原基;即使在花芽分化程度比较高的情况下,因BAP的作更多还原

【Abstract】 Chinese cabbage-pak-choi {Brassica campestris L. ssp. chinensis Makino) is an important vegetable of Brassica. The main methods of obtaining better quality cultivars depend on heterosis. As far as the maintenance and propagation of parents is concerned, there are some problems. In our studies receptacle with ovary and pedicel, receptacle with ovary, pedicel and rhachis were used as explants to construct regeneration system. At the same time, some phenomena were observed. The characteristics of vernalization were studied with adventitious shoots. In addition, the relationships between vernalization and antioxidant enzymes, soluble protein content were probed into. The ESTs related to vernalization were screened by the means of cDNA-AFLP. The main results as followers:(1) There was a difference of regeneration frequency between fertile and sterile resulting from fertility, as the receptacle with ovary and pedicel was used as explants. As BAP concentration was 2 mg·L-1, the better concentration for fertile was 0.1 mg·L-1, but for sterile is 0.2 mg·L-1. Under this condition the shoot induction rate were 72.6%, 77.1%, respectively. Fertile explants required for a strict BAP concentration, but the sterile didn’t. Receptacle with ovary and pedicel, receptacle with ovary, pedicel and the cuttings of rhachis all could regenerate adventitious shoots, but among of them, the receptacle with ovary and pedicel was the best. On the whole, the regeneration of fertile explants was better than that of the sterile.(2) The endogenous hormones are different in the explants of the fertile and sterile. It was found that IAA, GA3, ZT contents in fertile were all higher than that of in sterile.(3) The vernalized state was transfered to the adventitious shoots through the cell mitosis, which maybe the main reason of high frequency flowering in vitro. However, the regenerative tendency declined with the increasing of subculture. It was many factors, such as the component of the medium, exogenous hormones, and so on, that resulted in this change.(4) Vegetative shoots were vernalized again at 5°C for more than 14 days (the critical period of vernalization). It can’t be devernalize completely at 5°C for 21 days, subsequently 33 °C for 5 days.(5) GA3 partly substituted for the role of low temperature to induce to flower. However, to some extent, it repressed the origination of roots. The flowering speed and the length of rachides were not a patch on vernalization.(6) It was observed there was no difference for microsporegenesis between regeneration plants andtheir parents.(7) After vernalization with an over long time, the adventitious initiated flower differentiation, and formed floral primordial.(8) After vernalization, the soluble content increased in all the tissue (shoot apical meristem, young leafs, callus) tested. At 5℃ for 7 days, except for SOD increase, the activities of APX, CAT and POD declined significantly; for 14 days. The activities of the three enzymes had an increasing process. The activity of SOD and POD in SAM showed the tendency of increase with the treating duration increasing. Devernalization treatment decreased all the enzymes tested in all tissues. Theses results seemed to suggest that changes of antioxidant enzymes, especially SOD and POD, probably play a vital role in SAM vernalization regulation in vitro of B. campestris ssp. chinensis. After cold treatment, the product of lipid peroxidation MDA decreased at the start, and then increased gradually in all tissues tested. Furthermore, under the treating period of inducing vernalization the content of soluble protein increased significantly in all parts researched, and it was 28 d > 21 d > 14 d. The increase of the soluble protein possibly offers the base of the transition of flowering induced by vernalization.(9) After vernalization treatment the expressions of some genes are repressed, but of some are induced. Genes repressed are possible the enzymes involved in the disassimilation, in which including genes probably encoding hydrolytic enzyme associated with更多还原