【作者】 武自念；

【导师】 魏臻武；

【作者基本信息】 扬州大学 ， 草业科学， 2013， 博士

【摘要】 我国当前苜蓿青干草产量低,品质较低,育成品种数量少,远远不能满足苜蓿生产多方面的需求,因此迫切需要加强苜蓿种质资源的研究和利用,加快优良品种选育,提高苜蓿的产量和质量,以满足市场的需要。多叶苜蓿具有提高苜蓿产量和品质的潜力。(1)本文首先通过两年的田间试验对多叶苜蓿产草量相关性状及遗传多样性进行了分析研究。利用相关性分析研究叶茎比与产草量相关性状之间的关系,进而采用SSR分子标记的方法研究多叶苜蓿的遗传多样性。(2)通过田间调查,对多叶苜蓿的性状表达特征进行了分析；研究了多叶苜蓿各复叶节位叶面积,叶茎比、多叶率及叶面积之间关系。(3)利用光合仪研究多叶苜蓿和三叶型苜蓿的光合效率,在此基础上,利用光合仪和叶绿体超微结构研究不同复叶类型的苜蓿光合效率。(4)采用扫描电镜研究多叶苜蓿的顶端分生组织形态发育特性。比较研究了多叶苜蓿和三叶型苜蓿的复叶形态发生及苜蓿花的形态发育特征等。(5)进行多叶苜蓿花药培养的研究,筛选适合多叶苜蓿花药培养的最佳培养基,对花药培养再生植株进行倍性鉴定等工作。本文的主要研究结果如下：(1)多叶苜蓿WL323干草产量两年平均最高,为19551.45kg/hm2;8920MF和皇冠的平均干草产量分别为15395.23kg/hm2和15245.64kg/hm2,干草产量高于Super7、巨人、牧歌和品系GL0601等4份材料,在所有参试材料中处于较高水平。多叶苜蓿的皇冠和8920MF各茬次的叶茎比均高于三叶型苜蓿。苜蓿叶茎比与干草产量正相关,与分枝数、节间数显著负相关。利用SSR遗传多样性分析发现,具有多叶性状的各苜蓿材料间的遗传关系较近,单独聚为一类。(2)多叶苜蓿主茎中4+5叶复叶类型的最多,各材料平均为13.06%；只含4片复叶的主茎最少,各材料平均为0.01%。苜蓿4+5型多叶型复叶越多,苜蓿的多叶率越高：WL343HQ的多叶率最高,为25.71%,其4+5型复叶多叶率为13.88%。多叶苜蓿各复叶节位的叶面积均大于对照淮阴苜蓿；各节叶面积平均后发现多叶苜蓿复叶的叶面积在8.65-9.10cm2之间,而对照淮阴苜蓿仅为6.22cm2。通过多叶率、叶面积和叶茎比的相关性分析发现,多叶率与叶茎比呈显著正相关,多叶率越高,叶茎比越大,多叶率可以显著提高苜蓿的叶茎比：多叶率与苜蓿的叶面积也显著正相关,多叶率越高,叶面积也越大。叶面积和叶茎比正相关,但没有显著性。(3)通过对多叶型和三叶型苜蓿品种的光合参数分析发现,多叶苜蓿的Pn平均在25.79μmol·m-2·s-1左右,光合效率显著大于3叶型苜蓿。不同复叶类型的光合效率发现,5叶型复叶Pn最大,Pn的排列顺序是5叶型>4叶型>3叶型>6叶型>7叶型>8叶型>9叶型。5叶型复叶的叶肉中,每细胞的叶绿体数目最多,叶绿体中基粒多,基质浓厚,基粒片层堆积致密,嗜锇体数目较少、体积小,每叶绿体中的淀粉粒数目多而饱满。因此,在苜蓿多叶性状育种中,以5片复叶类型为目标,可以提高苜蓿的光合效率。(4)通过对3叶型和5叶型的复叶形态发生分析发现,苜蓿的复叶发育模式为向顶式,3叶型和5叶型茎顶端分生组织形态特征均为不规则方形结构,之后可按其形态发生分为明显的7个阶段。S0和S1阶段形态发生特征相似,形成条状共同原基；S2阶段,出现叶原基,隆起的数目不同；S3阶段,3叶型出现2个侧叶原基,而5叶型出现4个；之后的形态发育特征基本相似,由S2-S3阶段是关键阶段,决定了叶片的数目和叶片的着生方式。对苜蓿花的形态发育过程研究发现,苜蓿的花为两侧对称,为典型的21个器官。其形态发育特征与其他蝶形花亚科类似,出现初级花序分生组织和次级花序分生组织的分化,次级花序分生组织再发育成花分生组织,花分生组织发育成其他各器官,在发育过程中又有四个共同原基的划分。我们根据发育的特征不同,将其分为8个阶段,这对于研究雄性不育、花药培养花蕾的精确选择、苜蓿花发育的分子生物学机制等具有重要的意义。(5)通过对整个花器内,苜蓿的花药培养能力研究发现苜蓿现蕾后15-28d,花药培养愈伤组织诱导效果最好。愈伤组织分化的最佳培养基为改良Blaydes培养基,即Blaydes培养基添加肌醇100mg·L-1、水解酪蛋白200mg·L-1、2-IP0.5mg·L-1、NAA0.2mg·L-1、KT1.0mg·L-1时愈伤组织分化效率较高：1/2B5培养基诱导生根的效果优于1/2MS。对再生植株进行流式细胞术检测发现,花药培养后的植株,倍性比较复杂,会产生混倍体和非整倍体等,该研究对苜蓿单倍体和双单倍体育种具有重要的意义。更多还原

【Abstract】 At present, the yield and quality of alfalfa hey are low, and the number of improved varieties is not rich. The same reason,it is far from adequate to meet the needs of alfalfa production. So, there is an urgent need to strengthen alfalfa germplasm research and utilization, speed up the breeding of superior varieties, improving yield and quality of alfalfa, so as to meet the needs of the market. Some study suggests that multifoliate alfalfa has the potential to improve the yield and quality, but there is highly controversial on if the multifoliate alfalfa has the ability to increase yield and photosynthetic efficiency.(1) Firstly, this study spent a two-year field experiment on alfalfa forage yield-related traits and genetic diversity analysis; and study the relationship between stem/leaf ratio and yield-related traits by correlation analysis, then multifoliate alfalfa genetic diversity can be done by SSR molecular marker methods.(2) This study analyse the trait expression characteristics of multifoliate alfalfa through field investigation;besides,the relationship between Compound leaf area, stem/leaf ratio, leafy rate and leaf area were also analysed.(3) Using a photosynthetic apparatus to study photosynthetic efficiency of multifoliate alfalfa and three-leaf alfalfa, on this basis, photosynthetic apparatus and chloroplast ultra-structure can be used to study the photosynthetic efficiency in different types of compound leaves of alfalfa.(4) Using scanning electron microscopic to study the apical meristem development characteristics of multifoliate alfalfa,and compared the morphogenesis of compound leaves and the development characteristics of flowers of multifoliate alfalfa and three-leaf alfalfa.(5) Study on anther culture of multifoliate alfalfa, filtering the best medium for anther culture of multifoliate alfalfa, doing identification of the ploidys of regenerated plants from anther culture.This article’s main findings are as follows:(1) The average hay yield of multifoliate alfalfa WL323in two years is the highest,19551.45kg/hm2; the average hay yield of8920MF and Phabulous is15395.23kg/hm2and15245.64kg/hm2, at a high level in all participants, better than Super7, AmeriStand. GL0601and AmeriGraze. All stem/leaf ratio of multifoliate alfalfa in every stubble are higher than three-leaf alfalfa. Leaf-stem ratio of alfalfa has a positive correlation with hey yield.but significant negative correlation with branches and section number. SSR analysis of genetic diversity found that alfalfa materials with leafy traits are in the close genetic relationship,can be separately gathered as one.(2) In the main stem of multifoliate alfalfa, type of4+5compound leaves is the most, the average of each material is about13.06%; type of4compound leaves is the least, the average of each material is about0.01%; the more4+5compound leaves, the higher leafy rate; WL343HQ has the highest leafy rate,25.71%, and its’ leafy rate of4+5compound leaves is13.88%.Each compound leaves’ leaf area of multifoliate alfalfa is larger than the control of huaiyin; compound leaves’ leaf area of multifoliate alfalfa is between8.65cm2and9.10cm2,the control material, huaiyin,is only6.22cm2.By the correlation analysis of leafy rate, leaf area and stem/leaf ratio, this study found that leafy rate had a positive correlation with stem/leaf ratio, the higher leafy rate, the higher stem/leaf ratio; as well as stem/leaf ratio, leafy rate also had a significantly positively correlation with leaf area, the higher leafy rate, the larger leaf area; Leaf area and stem/leaf ratio are related, but not significantly.(3) This study did the analysis of photosynthetic parameters of multifoliate alfalfa and three-leaf alfalfa,found that the average Pn of multifoliate alfalfa was about25.79μmol·m-2·s-1, photosynthetic efficiency was significantly greater than three-leaf alfalfa. In different types of compound leaf photosynthetic efficiency, the Pn of5-leaf leaflet was the max, the order of the Pn was quinquefoliolate alfalfa> quadrifoliate leaflets> trifoliate leaflets> six leaves type seven leaves type> eight leaves type> Nine leaves type. In the mesophyll of quinquefoliolate alfalfa, the number of chloroplasts was the most in every cell, and with many granas in chloroplasts, stroma is thick, and the grana lamella were tight; the number of Osmophilic globule was few, starch grains in chloroplast was rich and full. Therefore, in breeding of Alfalfa leafy traits, using the quinquefoliolate alfalfa as the target can improve the photosynthetic efficiency of alfalfa.(4) This study analysed the morphogenesis of3and5type of compound leaf, then found that the model of compound leaves of alfalfa was top, the apical meristem morphology characteristics of3and5type of compound leaf wre the irregular square structure, which can be classified into7stages. Morphological characteristics of SO was similar to S1, forming the common primordial; primordia appeared in stage2, the number of those uplift was different; in S3,2side leaf primordia appeared in3type of compound leaf,4side leaf primordia appeared in5type of compound leaf, the following Morphological characteristics was the similar. It was the key stage from S2to S3,it determined the number of blades and blade life. In the study of Morphological development of alfalfa, the followings were found:flower was symmetric on both sides, as typical21-organs. Its morphological characteristics were similar to other Butterfly subfamily, with the appearing of differentiation of primary inflorescence meristem and secondary inflorescence meristem, secondary inflorescence meristem then developed into floral meristem and flower meristem developed into other organs, which also has four divisions of common primordia in the development. It was divided into8stages by the different characteristics development. it was of great significance for the study of male sterile, precise selection of anther culture of flower bud,and the molecular biology mechanism of alfalfa.(5) It was found that anther culture callus induction worked the best after15-28d of alfalfa budding. The best medium for the callus differentiation was modified Blaydes medium, that was adding inositol100mg·L-1, casein hydrolysis200mg·L-1,2-IP0.5mg·L-1, NAA0.2mg·L-1and KT1.0mg·L-1to Blaydes medium, in which condition, callus differentiation was more efficient. The induction effect of1/2B5medium was better than1/2MS. Flow Cytometry was used to Detected the regenerated plants, then,it was found that plants from anther culture had a more complicated ploidy, mixoploid and aneuploid may appear. This research had a great significance to haploid and doubled haploid breeding of alfalfa.更多还原