【作者】 申书兴；

【导师】 侯喜林；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2007， 博士

【摘要】 创建大白菜三体和单体是开展基因染色体定位等细胞分子遗传学研究的基础。大白菜二、四倍体杂交不育,不能利用三倍体获得三体和单体。本研究开展利用同源四倍体大白菜游离小孢子培养获得三体和单体的研究,并对该途径获得三体和单体的细胞学依据进行研究;鉴定出系列三体和部分单体;对其传递率进行了测定,并分析了各非整倍体传递率不同的组织细胞学原因;开展了25S rDNA和5S rDNA在大白菜二倍体和三体染色体上的FISH定位比较研究;并以随体染色体的单体、双体、三体为试材,开展了染色体的附加和减少对光合作用影响的研究。研究结果如下:1四倍体大白菜小孢子植株获得研究以同源四倍体大白菜为试材,进行了游离小孢子培养的胚胎诱导条件及植株再生研究。结果表明:单核靠边期小孢子是诱导胚胎发生的最佳时期;不同基因型的四倍体大白菜小孢子胚胎发生能力不同,其中9405品系产胚率达81胚/蕾;适于小孢子胚胎发生的培养基为NLN附加15%蔗糖、0.05～0.2mg/L BA和0-0.5mg/L NAA;NLN中添加0.05～0.1mg/mL活性炭可有效提高小孢子胚胎产率及发育同步性;33℃、24h热激处理是诱导小孢子胚胎发生的必要条件;子叶型胚适宜植株再生,在B₅或MS附加1.2%琼脂的培养基上,子叶型胚植株再生可达62%～65%。2四倍体大白菜小孢子培养获得三体和单体的细胞学研究通过对四倍体大白菜雄配子形成过程观察表明:由于同源四倍体大白菜的花粉母细胞内,Ⅰ、Ⅲ、Ⅳ型配对的同源染色体发生不规则分离,后期Ⅰ、Ⅱ部分染色体落后和向两极分配不均衡,致使后期Ⅱ形成染色体数目变化于15～24的各类细胞,以染色体数为20的细胞为主,其次是19、21、18和22条染色体的细胞;具有21条染色体的小孢子（2x+1）再生植株能力较强,部分具有19或22条染色体的小孢子能再生成植株,其余小孢子不能再生植株;减数分裂的各种异常频率和三体株获得率在品系间存在差异。从四倍体大白菜9405自交系游离小孢子培养获得的植株中,筛选出79个2n+1植株、7个2n+2植株、9个2n-1植株。通过根尖中期染色体核型分析,并结合减数分裂和植株外部形态特征观察,从中鉴定筛选出了一整套初级三体、2个双三体、4个单体。各初级三体、双三体和单体在植株的形态特征上表现出不同程度的差异。四倍体大白菜游离小孢子培养是创建初级三体的快捷简便的有效途径。3大白菜初级三体、单体的配子形成及传递率研究以大白菜初级三体系、单体为亲本,分别与二倍体正反交,测定结籽率和雌雄配子传递率;并对各三体和单体的减数分裂过程中的染色体行为,雌雄配子发育的组织结构及授粉受精和胚胎发育进行了详细研究。研究结果表明:4个单体结籽率为零,无传递力;大白菜初级三体系除10号三体（Tri-10）传递率为零外,其它三体均能通过n+1雌雄配子传递,但传递率在不同三体间以及雌雄配子间有差异。减数分裂过程观察表明:各三体和单体的减数分裂过程中,从三价体的形成到后期Ⅰ、后期Ⅱ染色体分离等方面都存在异常,这些共同影响n-1或n+1雄配子的传递率。雄配子发育的组织结构观察表明:2号、5号、7号、9号三体的雄配子发育过程与二倍体基本相似;3号、5号、10号单体和4号三体、10号三体的雄配子发育过程中,绒毡层细胞的异常普遍存在,而且均延迟解体,小孢子都出现了严重的败育现象,最终形成大量的败育花粉,是影响这些非整倍体雄配子传递率的主要因素,另外,花药维管组织发育异常和药室合并等异常也是造成其花粉败育的原因之一。胚胎学研究表明:胚囊败育是Tri-10结籽率为零,雌配子没有传递率的主要原因;胚珠受精率低是Tri-6的n+1雌配子传递率低的主要原因:幼胚败育率高是Tri-3和Tri-9的n+1雌配子传递率低的主要原因。以上研究结果对这些珍贵材料的繁殖和保存,利用其进行遗传分析,开展基因定位、分子细胞遗传学等研究都有重要参考价值。4 25S和5S rDNA在大白菜二倍体和三体染色体上的FISH定位为了确定rDNA在中国大白菜基因组中的位点数目和分布位置,并建立识别大白菜不同染色体的特异标记,在建立大白菜FISH技术体系基础上,对25S rDNA和5S rDNA在大白菜二倍体和不同三体中期染色体上进行了FISH定位研究。结果表明,（1）通过对FISH的相关技术参数的研究,建立了大白菜染色体制备和制片预处理、探针杂交、杂交后洗脱以及信号检测的技术体系。（2）25S rDNA在二倍体和不同三体（Tri-3除外）的中期染色体上,均在1号、2号、3号、4号染色体的长臂和10号染色体的随体上检出了杂交信号;但杂交信号在不同三体同一编号染色体上检出的位置并不完全相同,不同染色体的荧光信号强度也有变化,最弱的信号出现在1号染色体,最强的信号出现在10号染色体的随体。（3）5SrDNA在二倍体和3个三体的中期染色体上检出的杂交信号均位于2号染色体的长臂和9号、10号染色体的短臂,检出的信号个数分别为6个（二倍体、Tri-3）和7个（Tri-2、Tri-9）。通过两类rDNA的FISH定位,为大白菜1-4号、9号和10号染色体提供了准确的识别标记。5大白菜随体染色体的单体、双体、三体的光合生理特性研究以大白菜随体单体、三体和二倍体为试材,在自然条件下,对生殖生长期的功能叶的净光合速率、光合速率日变化、叶绿素含量、希尔反应活性、Rubisco活性、可溶性糖含量及气孔密度等生理生化指标进行了测定。结果表明:当光照强度低于750μmol·m^-2·s^-1时,随体染色体的减少或附加对大白菜的光合速率有正效应,但高于900μmol·m^-2·s^-1有负效应;随体三体和二倍体净光合速率日变化均呈典型的双峰曲线,有明显的“午休”现象,随体单体“午休”现象不明显:随体染色体的减少或附加对叶绿素含量有正效应,对叶绿素a/b的比值有负效应;随体染色体的减少或附加对希尔反应和Rubisco活性均有负效应;随体染色体的减少或附加对可溶性糖含量有负效应;希尔反应活性和Rubisco活性的降低是引起随体单体、三体光合速率降低的直接原因。本研究对了解大白菜随体染色体的功能和开展染色体工程育种有重要参考价值。更多还原

【Abstract】 In Chinese cabbage,development of trisomics and monosomics provides a useful cytogenetic tool to associate genes and molecular markers with their respective chromosomes.It is very difficult to obtain trisomics and monosomics using triploid,because the crossed of diploid and tetraploid Chinese cabbage could not obtain triploid.In the present study we applied the isolated microspore culture of autotetraploid Chinese cabbage to identify trisomics and monosomics,explore the cytological basis of this approach.A complete set of trisomics and some monosomics were identified,and their gamete transmission rate was observed.The tissue cytological cause differentiated gamete transmission rates among various aneuploids were further discussed.Fluorescene in Situ Hybridization（FISH） technology was used to detect the positions of 25S rDNA and 5S rDNA on chromosome of Chinese cabbage diploid and trisomics.Furthermore,the monosomic,disomic and trisomic of satellite chromosome were used to examine the effect of addition and reduction of chromosome on photosynthesis.The research results are listed as follows:1 Plant Regeneration from Isolated Microspore Culture of Autotetraploid Chinese Cabbage（Brassica campestris ssp.pekinensis）Autotetraploid Chinese cabbage was used to study microspores culture response and plant regeneration.The results showed that the late uninucleate stage was the best period to induce embryos.Embryogenesis was related to different genotypes,of which 9405 line was the highest yield with 81 embryos per bud.Medium NLN supplemented with 15%sucrose,BA 0.05-0.2mg/L and NAA 0-0.5mg/L induced high frequency embryogenesis,which is suggested a basic medium for microspore culture.And addition of 0.05-0.1g/L activated charcoal to microspore suspension had significant effect upon the yield and developmental uniform of embryos and percentage of cotyledary embryos.Furthermore,the optimal response was obtained at 33℃for 24h at initial culture period,which the frequency of embryogenesis was the highest. Cotyledary embryos had a higher rate of plant regeneration,reaching 62%-65%when embryos germination was conducted on B₅ or MS medium with 1.2%agar.2 Cytological Research on Trisomics and Monosomies Obtained by Microspore Culture in Autotetraploid Chinese CabbageThe observation of male gametophyte formation indicated that the chromosome pairing in PMC forms at diakinesis wereⅠ、Ⅱ、Ⅲ、Ⅳ.The irregular separation ofⅠ、Ⅲ、Ⅳ, straggling chromosomes at metaphaseⅠ,resulted in the chromosome number of PMC at anaphaseⅡwere varied from 15-24,the type with 20 chromosomes was the most,and then they were 19 and 21 chromosomes.The plant regeneration rate of microspore（2x+1） with 21 chromosomes was similar to that of the normal microspore（2x） with 20 chromosomes. The irregular behaviour of chromosome and developmental rate of trisomics were different among the strains.Seventy nine aneuploid plants with one extra chromosome（2x+1）,7 with two extra chromosome（2x+2） and 9 with loss one chromosome（2x-1） were obtained from the isolated microspore cultured plantlets of the tetraploid Chinese cabbage line 9405. Through karyotype analysis of the root tip metaphase chromosome,meiosis observation and plant characters investigation,a complete set of primary trisomics（Tri-1,Tri-2,Tri-3, Tri-4,Tri-5,Tri-6,Tri-7,Tri-8,Tri-9 and Tri-10）,two double trisomics（Double tri-3,5 and Double tri-3,6） and four monosomics（Mono-3,Mono-5,Mono-6,Mono-10） were identified, each of which had some unique characters on plant morphological traits.Thus,the isolated microspore culture of tetraploid is a convenient and effective way to select trisomics in Chinese cabbage.3 Gamete Formation and Transmission Rate of a Set of Primary Trisomics and Several Monosomies in Chinese CabbageA set of primary trisomics and several monosomes in Chinese cabbage was used as parents to make reciprocal crosses with diploid,the seeding set and the male and female gamete transmission rate were investigated,and chromosome behaviours of each trisomic and monosomic during meiotic divisions,gamete development structure,pollination, fertilization and embryo development were systematically studied.These results indicated that the seeding set of four monosomes were zero without transmissibility,n+1 gametes of the Chinese cabbage trisomics transmitted through female and male parents,except for Tri-10,with different transmission rate among various trisomics.The main influencing factors of n+1 or n-1 male gamete transmission rate were the trivalent formation and abnormal chromosome segregation at anaphaseⅠandⅡduring meiotic divisions of each trisomics and monosome.The observation of male gamete development tissue anatomy showed:male gamete development of Tri-2、Tri-5、Tri-7 and Tri-9 was similar to diploid, whereas abnormal tapetum cells were commonly found in Mono-3,Mono-5,Mono-10, Tri-4 and Tri-10 with delayed degeneration of the tapetum cells.It was the main factor influenced the transmission rate of these aneuploid,thereafter microspores were serious abortion generally,and finally produced vast abortive pollen.Additionally abnormal vascular tissue of the anther and the anther chambers unified also caused pollen abortion. Embryological observation results showed that the embryo sac abortion was main factor that led to the Tri-10 seeding set with zero and female gametes without transmission rate. However,the low transmission rate of Tri-6 through n+1 female gametes was due to the low fertilization rate of the ovule,and the main factor of the low transmission rate of Tri-3 and Tri-9 through n+1 female gametes was young embryo abortion.The experimental results described above were a valuable reference for the rarity material propagation and preservation and for carrying out gene orientation and molecular and cytogenetical study using aneuploid of Chinese cabbage.4 FISH Mapping of 25S rDNA and 5S rDNA in Chromosome of Chinese Cabbage Diploid and TrisomicsIn order to clarify the number and location of rDNA sites in Chinese cabbage genome, and identify special molecular markers related to different chromosomes,fluorescene in situ hybridization（FISH） technique system was established.25S rDNA and 5S rDNA were further mapped in the mitotic metaphase chromosomes of Chinese cabbage diploid and different trisomics based on this method.The results were showed that:（1） With the study of correlative parameters during FISH,the technique system on Chinese cabbage was established,including chromosome preparation,chromosome pre-treatment,probe hybridization,washing and signal surveying.（2） The hybridization signals of 25S rDNA were observed in metaphase chromosomes of Chinese cabbage diploid and trisomics （except for Tri-3）,which were located on the long arms of chromosomes1-4 and the satellite of chromosome 10.But the hybridization signal position was different in the same chromosome of each trisomics,and the hybridization signal intension was various in different chromosomes,the weakest signal appeared on chromosome 1 and the strongest signal appeared on the satellite of chromosome 10.（3） The hybridization signals of 5S rDNA were observed in the metaphase chromosomes of Chinese cabbage diploid and three trisomics,in which they were located on the long arms of chromosome 2 and the short arms of chromosome 9 and 10.The number of hybridization signals was 6 for diploid,7 for Tri-2, 6 for Tri-3,and 7 for Tri-9 respectively.This study provides accurate identified marks for chromosome 1-4,9 and 10 of Chinese cabbage by two rDNA-FISH location.5 Photosynthetic characteristics of satellite chromosome monosome,trisome and diploid in Chinese cabbageIn order to research the effect of addition or reduction of satellite chromosome on the photosynthetic characteristics of Chinese cabbage,the satellite chromosome monosome, diploid and trisome of Chinese cabbage were used to examine the photosynthetic rate（Pn）, diurnal variation and chlorophyll content,Hill reaction activity,Rubisco activity so on.The result showed that the addition or reduction of sat-chromosome had a negative effect on the photosynthetic rate（Pn）,when PAR was over 900μmol·m^-2.s^-1.The curves of diurnal change of Pn of trisome and diploid were quite similar,displaying typical two peaks and conspicuous "noon sleep".The addition or reduction of sat-chromosome had a positive effect on chlorophyll content and a negative effect on chla/b and boosted the degree of thylakoid stacking.The decline of Hill reaction activity and Rubisco activity were direct factors resulting in low photosynthetic rate of sat-monosome and sat-trisome.These results could offer valuable information to realize the function of sat-chromosome of Chinese cabbage and high-photosynthetic efficiency breeding using the chromosome engineering.更多还原