【作者】 柳霖坡；

【导师】 张成合；

【作者基本信息】 河北农业大学 ， 蔬菜学， 2004， 硕士

【摘要】 本文以二、四倍体结球甘蓝为试材，采用雌蕊整体压片荧光显微观察和石蜡切片等技术，对二、四倍体正反交的授粉受精和胚胎发育过程进行了较系统地观察比较，并对其杂交败育的原因及解决方法进行了研究探讨。同时，对所获得三倍体的生长发育特性、生理生化指标以及减数分裂行为、花粉败育途径和配子传递率等进行了观察测定。此外，我们还发现了能自然产生2n配子的甘蓝材料，并通过对其减数分裂中期Ⅱ纺锤体的动态观察，探讨了其产生2n雄配子细胞学机制。所得结果主要有以下几点： 1 甘蓝2x×4x、4x×2x授粉受精过程均能正常进行，但胚胎发育均不正常，存在不同程度的胚胎败育现象，且败育均是由胚胎发育过程中胚乳异常退化、内珠被细胞严重坏死，从而断绝了胚胎发育的营养供给所致，但两者发生败育的时间不同，2x×4x主要发生在小球胚阶段，胚胎尚未出现组织分化，因此败育十分彻底，最终形不成种子；而4x×2x主要发生在心形胚或晚心形胚阶段，胚胎已有了明显的组织分化，最终能形成少量具幼胚的小粒种子。 2 甘蓝4x×2x和2x×4x结荚率都比较高，无明显差异，但结实性差异很大，2x×4x仅结有极少量的大粒种子(0.57％)，而4x×2x除结有少量的大粒种子(0.54％)外，还结有一些小粒种子(3.25％)。经倍性鉴定，4x×2x所结小粒种子中绝大部分为3x=27的三倍体，还有极少量为2n=26亚倍体和2n=28的超倍体，4x×2x和2x×4x所结的大粒种子均为四倍体。 3 激素处理膨大过程中的子房对杂种幼胚的发育有一定的促进作用，其作用大小因激素种类、浓度和配比而异，其中200ppmIAA+200ppmBA的处理效果最好，结籽率较对照提高了3.87倍。 4 胚珠离体培养的效果受胚胎发育程度的影响很大。在MS基本培养基上，心形胚以前的胚珠均未能培养成功，心形胚以后的部分胚珠能够完成后熟作用，并直接萌发成苗。 5 杂交获得的小粒种子在MS基本培养基上或在铺有湿滤纸的培养皿中均能萌发，前者的萌发率为45.56％，后者为31.10％，但后者的畸形苗相对较少，且操作简便。 6 三倍体结球甘蓝生长健壮，耐热性增强，结球紧实，叶型指数变小，平均叶球重和部分营养品质指标高于其二倍体。 7 甘蓝三倍体减数分裂行为较二倍体复杂，终变期多以三价体存在，单价体和二价体占少数；中期Ⅰ、中期Ⅱ均含有游离染色体；后期Ⅰ染色体分离不均衡有9／18、10／17、11／16、12／15、13／14等方式，频率分别为0.77％，3.87％，8.76％，31.19％和48.45％，其中10／17分离是创建三体系的细胞学基础。 8 三倍体甘蓝的花粉败育主要是由于减数分裂行为异常，产生的小孢子本身染色体组成不均衡，导致其遗传生理不协调所致，而与绒毡层细胞的发育关系不大。 9 甘蓝3x×2x的结籽率为10.36％，2x×3x的结籽率为0.83％，但前者的2n+1株率为26.47%，后者为72.70%。表明甘蓝三倍体产生的各种非整倍性配子中，通过花粉一般只能传递的n+1的配子，而通过胚囊，不仅能传递n+1的配子，还能传递n+2、n+3等染色体组成更加不均衡的配子。 10二倍体甘蓝中存在能自然产生2n配子的植株，其形成2n配子的细胞学机制主要是由于减数分裂中期11纺锤体定向发生改变，形成三分体和二分体所致，并以形成二分体为主。更多还原

【Abstract】 The process of pollination, fertilization and embryo development of 4x × 2x and 2x× 4x in head cabbage was observed by the ways of fluorescence microscopy and paraffin section . Then the characters of quality, growth and development, meiosis behavior and gamates transmitting rate of the triploid plants obtained by 4x×2x were studied ,the methods of saving young embryo collapse and improving the seed setting were researched in this paper.The results were showed as follows:1 The process of pollination and fertilization was normal in 4x× 2x and 2x × 4x which is similar to 2x and 4x .The cause of embryo collapse in both combinations lied in cutting off supply of nutrition necessary to embryo development due to endosperm degeneration and inner integument cells dying off in early embryo development phase.But the time of the embryo collapse was different in the both combinations, in 4x×2x the embryo collapse generally took place at heart-shaped or late heart-shaped embryo stage and finally formed small seeds,while in 2x ×4x,the embryos collapsed at small globe-shaped embryo stage and could not formed seeds.2 The pod setting in both 4x ×2x and 2x ×4x were very high and without marked difference ,but their fecundities were different remarkablely,some small seeds and a few of big seeds were obtained in 4x×2x which the former included triploid,hyperploid and hypoploid ,the later were all tetraploid, but only a few big seeds were observed in 2x X 4x which were all tetraploid .3 The effect on accelerating young embryo development was based on kind, concentration and proportion of IAA and BA which were treated to swelling ovaries. It showed that the treatment of 200 ppmIAA +200ppmBA was most effective which the seed setting 3.87 times CK.4 About 50% of the isolated ovales with late heart-shaped embryo could accomplish afterripening and germinate into seedlings directly when cultivated in MS medium, while those with early stage embryo failed completely.5 The small seeds obtained in 4x ×2x could germinate in MS medium as well in wet filter paper, and the rates of germination and abnormal seedling were higher in the former than in the later.6 The triploid head cabbage showed the general characters of polyploid plant, such as,having stronger resistance to higher temperature, weight and some minion indexes of plant being higher, leaf type index becoming smaller than diploid plants. The anthesis was similar to tetraploid but later than diploid plants.7 The meiosis behavior of triploid head cabbage was much more complex than that of diploid.It showed as foilowsrat diakinesis,trivalent was major while divalent and univalent was minor.Some free monoverient chromosomes were observed at metaphase I , II. The separate types showed 9/18,10/17,11/16,12/15,13/14 whose rates were 0.77%, 3.87%, 8.76%, 31.19% and 48.45% respectively at anaphase I , of which the 10/17 was the most useful in selecting the trisomics.8 That abnomal meiosis behavior produced many microspores with unbalanced composing of chromosomes was the main cause of pollen collapse.9 Seed sets and rate of 2n+1 plants were 10.46% and 26.47% respectively in 3x X 2x while 0.83% and 72.7% respectively in 2xX3x. It showed that pollen could only transfer n+lgametes,whereas megaspore even could transfer n+2,n+3 gametes besides n+1 gamete.10 Diploid cabbage plants which could naturally produce unreduced 2n gamete were discovered for the first time.Similar to in Chinese cabbage .the cytological mechanism of unreduced 2n gamete formation in cabbage was mainly due to the abmormal orientation of spindles at metaphase II which led to formation of dyad and triad, dyads were the direct souce of 2n gamete in cabbage unlike Triads in Chinese cabbage.更多还原