【作者】 朱雪云；

【导师】 陈利萍； 张明方；

【作者基本信息】 浙江大学 ， 蔬菜学， 2009， 博士

【摘要】 榨菜（Brassica juncea Coss．var．tumida Tsen et Lee）和紫甘蓝（B．oleracea var．capitata L．）属于十字花科芸薹属植物，是重要的蔬菜作物。以榨菜和紫甘蓝为材料通过试管离体嫁接可以获得种间嵌合体。本论文以榨菜和紫甘蓝种间嵌合体为研究材料，通过植物学、细胞学、生物化学、分子生物学等方法，研究了嵌合体营养时期和生殖时期生长发育特性；通过嵌合体光合特性研究，探讨嫁接“优势”’与两种遗传型的细胞系之间的相互关系；通过嵌合体不同部位的不定器官再生研究，揭示了植物不定器官的起源和发生规律，以及不同细胞层间的相互影响。本研究为植物育种学和植物发育学研究提供了新思路和新方法，为细胞互作机制研究提供了良好的试验体系，也为利用嵌合体改良植物性状以及生产实践应用提供理论依据，主要研究结果如下：1在形态学、细胞水平、蛋白质水平和分子水平上对榨菜和紫甘蓝的种间嵌合体进行了分析。研究结果发现嵌合体的形态特征在具有了两个亲本特点的基础上，其形态发生了一定变化。利用扫描和透射电镜观察叶片表皮和叶肉细胞亚显微结构，发现嵌合体的气孔密度超过两个亲本，叶绿体、淀粉粒等的形态指标基本处于两个亲本之间。通过可溶性蛋白质的SDS-PAGE电泳图谱观察到嵌合体产生了特异性条带。分子水平上的RAPD和特异引物PCR扩增结果显示，嵌合体没有产生其特异性条带。这些研究结果证明嵌合体中两种基因型不同的细胞系之间存在着相互作用，使嵌合体在形态学、细胞学和生物化学特性发生了改变。另外，嵌合体采用离体保存的方法，用1／2MS培养基可以长期保存。利用腋芽繁殖途径对嵌合体进行无性繁殖，这种方法可以使后代植株保持与母株一样的嵌合状态。用1／2MS+1mg／L BA培养基扩繁时，周缘嵌合体转化为扇形嵌合体和混合型嵌合体的比例分别是8．2％和2．4％。而用1／2MS+0．1mg／L BA时周缘嵌合体基本不会产生其它类型的嵌合体，但腋芽生长速度较慢。田间植株腋芽离体繁殖所用的诱导培养基为1／2 MS+1mg／LBA，但腋芽的诱导率比试管苗低。2从光合作用、叶绿素荧光、叶绿素含量、Rubisco的活性以及Rubisco大亚基和小亚基基因的转录水平等进行了测定分析。研究结果发现，周缘嵌合体TCC（LⅠ-LⅡ-LⅢ=TCC，LⅠ-茎尖分生组织层最外层；LⅡ-中间层；LⅢ-最内层．T表示榨菜，C表示紫甘蓝）的净光合速率为18.09μmol CO2·m^-2·s^-1，与亲本榨菜相当，但比亲本紫甘蓝高出24％。而嵌合体的气孔导度和胞间CO₂浓度显著高于两个亲本。叶绿素荧光参数中光系统Ⅱ实际的量子效率（φPSⅡ）和光化学猝灭系数（qP）在榨菜中最高，而嵌合体和紫甘蓝的这两个参数基本一致。叶绿素含量测定后发现，嵌合体的叶绿素a和b的总含量与榨菜的比较接近，比紫甘蓝高97％。TCC嵌合体Rubisco酶的初始活性和总活性处于榨菜（最高）和紫甘蓝（最低）之间，为1.76和3.75μmolCO₂·g^-1·min^-1。而TCC嵌合体的Rubisco酶大亚基和小亚基基因的相对表达量与榨菜和紫甘蓝相比明显增高。以上结果说明，与TCC嵌合体光合机构的层源亲本——紫甘蓝相比，其叶绿素含量升高、Rubisco酶的活性以及其大小亚基基因表达的增强导致其净光合速率的提高。可见，TCC嵌合体的异源表皮（来自榨菜）对其内部光合组织光合能力的改善有很大的促进作用。3对榨菜和紫甘蓝种间周缘嵌合体TCC的生殖器官和生殖特性等方面进行了研究。研究结果发现，TCC嵌合体的有性生长发育与紫甘蓝相近，具有严格的绿体春花特性。其抽薹开花习性和花序、花冠形态也与紫甘蓝类似，但在异源表皮——榨菜的影响下发生了改变，很多形态指标，如花蕾长、短轴长度，花瓣的长、宽，雄蕊、雌蕊的长度以及花粉粒的大小等都基本介于榨菜和紫甘蓝相应指标的中间。此外，对嵌合体进行蕾期自交、杂交后，嵌合体可以产生果实但不能产生种子。从以上的研究可以说明，在TCC嵌合体的有性生殖生长发育过程中LⅠ（榨菜）与LⅡ和LⅢ（紫甘蓝）之间的相互作用使TCC嵌合体的生殖器官和生殖特性相对于亲本发生了较大变化。从嵌合体花冠的大小和形状改变以及产生无籽果实的角度来看，嵌合体在花卉和瓜类等作物育种上有很大的应用潜力。4以榨菜和紫甘蓝种间周缘嵌合体TCC的茎段和叶片为外植体诱导了不定芽的再生。利用形态标记和分子生物学方法鉴定分析不定芽的起源。经统计分析结果显示，茎段上叶腋处不定芽的诱导频率随着MS培养基中BA浓度的增加而升高，而茎段基部不定芽的诱导频率在含有不同浓度BA的培养基中没有差异。叶腋处产生的不定芽绝大多数是榨菜（TTT），只有4个嵌合体的产生，由此可知：与LⅡ和LⅢ相比，这些不定芽更多的起源于LⅠ层。从茎段基部产生的不定芽全部为紫甘蓝（CCC），说明这些不定芽起源于LⅡ或LⅢ，或者是两层共同参与的结果。叶片外植体上不定芽的再生频率在三种不同的NAA和BA组合的培养基之间存在着显著的差异。叶片切块边缘再生的不定芽绝大多数是紫甘蓝（CCC），70个不定芽中只有2个是嵌合体。从这一结果可以推出：叶片上再生的不定芽绝大多数是由LⅡ或（和）LⅢ参与形成的，LⅠ参与的几率较低。另外由试验发现，再生的嵌合体类型与其亲本TCC的类型相比完全不同。从上述的研究结果可以推出，不定芽的起源会因为外植体从供体植株上的来源不同而发生改变，而且嵌合体是多细胞甚至是多组织参与起源的。5同样以周缘嵌合体TCC的叶片和茎段为外植体诱导不定根。TCC嵌合体叶片和茎段上的诱导频率分别为94％和86％，显著高于它的亲本。其中榨菜叶片和茎段不定根的诱导频率分别为25％和38％，而紫甘蓝分别为73％和47％。另外，每株TCC嵌合体不定根的数目平均是13．11，平均重量为0．274g，这些指标也显著高于榨菜和紫甘蓝。这些结果说明嵌合体异源LⅠ（表皮）与内部LⅡ和LⅢ之间可能存在着正向互作从而改善了嵌合体不定根的再生能力。利用PCR技术（聚合酶链式反应）和组织解剖学观察来鉴定不定根的起源部位。鉴定结果显示，不定根最终起源于LⅢ。通过本论文的研究，可以发现种间嵌合体的不同发育时期和各个器官的特性都受到两种基因型不同的细胞层相互作用的影响，特别是数量性状基本介于两个亲本的中间。嵌合体的不定根的再生能力、营养生长时期的光合能力相比其层源亲本有很大改善，同时嵌合体的生殖器官和生殖特性也有较大的变化。嵌合体中不同遗传型细胞之间的相互作用所带来的性状改变在植物育种中有较大的应用潜力。另外，利用嵌合体茎尖细胞层组成的特殊性揭示了不定器官的起源规律，为植物的无性繁殖和组织培养提供了一定的理论基础。更多还原

【Abstract】 Tuber mustard （Brassica juncea ） and red cabbage （B. oleracea） are importantvegetable crops of the Brassica in the Cruciferae. The plant chimeras obtained by invitro grafting between tuber mustard and red cabbage were used as the materials forthis study. The characteristics of vegetative and reproductive stages at the aspects ofbotany, cytology, biochemistry and molecular biology, and the photosyntheticcharacteristics of the chimeras were studied for clarifying the interactions betweengenetically different apical cell layers.The regeneration of adventitious organs indifferent explants of the chimeras was investigated in quest of the origin and ontogeny.The interactions in the chimeras can result in changes of phenotypes and improvemany characters, which can be useful for plant breeding. In addition, these studieswill provide academic supports for applying chimeras to plant improvement and putforward some novel ideas and viewpoints at the aspect of plant development. Themain results as follows:1 The characterization of chimera was observed at the level of morphology,cytology, molecular biology and so on. The results were that the chimeras not onlycombined the morphological characters from both donor plants, tuber mustard and redcabbage, but also they undergo some changes in phenotypes. The stomata density ofthe leaf epidermis in the chimeras surpassed the tuber mustard and red cabbage, andthe morphology of chloroplast and starch grains were altered in the chimeras. Inaddition, there were novel bands in the soluble proteins of the chimeras bySDS-PAGE analysis. However, no novel band sepsific for chimeras was detected inRAPD and PCR analysis of the chimeras compared with their donor plants. Thesestudies suggested that there were interactions at the level of morphology, cytology andbiochemistry in the interspecific chimeras of tuber mustard and red cabbage. Theoptimal medium and method for the chimera vegetative proliferation was selected.The chimeras could be conserved in half strength MS medium and propagated in half strength MS medium with 0.1 mg/L BA in which periclinal chimeras would notchange to sectorial and mericlinal chimeras by culturing the nodes with axillary buds.2 In this study, the photosynthesis, chlorophyll fluorescence and chlorophyllcontent, Rubisco activities and the large and small subunits of Rubisco were assayedin the periclinal chimera TCC （LⅠ-LⅡ-LⅢ=T-C-C, LⅠ-the outmost layer of shootapical meristem; LⅡ-the middle layer; LⅢ-the innermost layer T=tuber mustard, C=red cabbage） synthesized by grafting in vitro between tuber mustard （Brasscia juncea）and red cabbage （B. oleracea）. The net photosynthesis rate of TCC chimera was18.09μmol CO2·m^-2·s^-1, much higher by 24.8% than that of its donor plant redcabbage, and the stomatal conductance and intercellular CO2 concentration of TCCchimera markedly higher than that of both donor plants. The quantum efficiency ofphotosystemⅡ（φPSⅡ） and photochemical quenching coefficient （qP） were almostsame in TCC and red cabbage, but distinctly lower than that of tuber mustard. Thetotal content of chlorophyll a and chlorophyll b in TCC chimera was close with that oftuber mustard, but remarkably higher by 97% than that of red cabbage. The initial andtotal activities of Rubisco of TCC chimera were 1.76 and 3.75μmolCO2·g^-1·min-¹,intermediate between tuber mustard and red cabbage, while the expression of rbcLand rbcS of Rubisco in TCC chimera exceeded both donor plants. The resultssuggested that the enhancement of stomatal conductance, chlorophyll content,Rubisco activities and transcription of rbcL and rbcS genes may contribute to higherPN of TCC chimera than the donor red cabbage, and heterogenous epidermis （LⅠ） inTCC chimera exerted large effects on these physiological characteristics determinedby inner tissues （LⅡand LⅢ）.3 The characteristics of the TCC chimera reproductive organs and crossing weredetermined. It was noted that TCC chimeras required strict vernalization condition toflower, which was same with red cabbage, while tuber mustard was not so strict as redcabbage and TCC chimeras. Moreover, the flowering habit and morphologycharacteristics of inflorescence in TCC chimeras were more similar with red cabbage.However, the length and width of flower buds and petals, the size of pistils, stamensand pollens were intermediate between those of red cabbage and tuber mustard. In addition, TCC chimeras were artificially pollinated with the pollens of TCC chimera,red cabbage and tuber mustard. The capsule setting frequency of TCC chimera wasclose with both donor plants, but there was no seed in the capsule while the seedsettings of red cabbage and tuber mustard in every capsule were 5.3 and 14.6 for selfpollination, and 0.2 and 7.6 for cross pollination with TCC pollens. It suggested thatthe reproductive characteristics of TCC chimeras were changed by the effects ofinteractions between genetically different apical cell layer LⅠand LⅡ, LⅢ.4 Adventitious shoots were induced from nodal segments and leaf discs of TCC（LⅠ-LⅡ-LⅢ, LⅠ-the outmost layer of shoot apical meristem; LⅡ-the middle layer;LⅢ-the innermost layer. T=Tuber mustard, C=Red cabbage） chimeras. The origin ofshoots was analyzed by histology and molecular biology. As a result, the frequency ofadventitious shoot induction rose with the increase of BA in MS medium in the areaof nodes. However, there was no different induction frequency of adventitious shootsfrom nodal segment bases in the media with different BA concentrations. Mostadventitious shoots （clustered shoots） arose from node area were TTT （Tubermustard- Tuber mustard- Tuber mustard） but only 4 shoots were chimeras, whichindicated that more shoots originated from LⅠthan from LⅡand LⅢ. All shoots fromnodal segment bases were CCC （Red cabbage-Red cabbage- Red cabbage）, indicatingthe shoots originated from LⅡor LⅡand LⅢ. There were significant differences ofthe regeneration rate in the margin of leaf discs among the three combinations of BAand NAA. Most adventitious shoots from the margin of leaf discs were CCC but 2 of70 were chimeras, which indicated that more shoots originated from LⅡor LⅡandLⅢthan from LⅠ. All chimeras obtained by regeneration in types were different fromthe original of explants in the present study. The origin of adventitious shoots variedwith the sites of origin on the plants, and could be multicellular and multhistogenic.5 Adventitious roots were induced from stems and leaves of chimera TCC（LⅠ-LⅡ-LⅢ= TCC, T = Tuber mustard, C = Red Cabbage） synthesized by in vitrografting between tuber mustard and red cabbage previously. The induction frequencyof adventitious roots from TCC stems and leaf discs were 86% and 94%, markedlyhigher than its parents, 38% and 25% from TTT （tuber mustard）, 47% and 73% from CCC （red cabbage）, and the number and fresh weight of adventitious roots from TCCshoots, 13.11 and 0.274 respectively, was also significantly high when compared to itsparents. This investigation demonstrated that the replacement of LI in plants with adifferent genotype might improve the adventitious root regeneration ability because ofa probable positive co-operation between LI and the two inner apical cell layers.Subsequently, the origin of these adventitious roots was examined by morphology,PCR （Polymerase Chain Reaction） and histology, and it was found that adventitiousroots were originated from the LⅢ.These studies showed that the growth and development of the interspecificchimeras was affected by the interactions between the different genotypic apical celllayers. Many quantitative traits were intermediate between two donor plants. Theregeneration ability of adventitious roots and the photosynthetic characteristics ofchimeras were remarkably improved by the interactions of genetically different tissuecell layers, compared to the genotype which they derived from. The floralcharacteristics of TCC chimeras were also changed by the interactions. In conclusion,the interactions between the different genotypic apical cell layers and their derivatesresult in many characters and phenotype variation which will contribute to plantbreeding in the future.更多还原