【作者】 邹建；

【导师】 宋明；

【作者基本信息】 西南农业大学 ， 蔬菜学， 2004， 硕士

【摘要】 本试验以3个茄子品种(渝研一号、三月茄、萍早秋一号)为材料，通过花药愈伤组织的诱导、增殖和分化培养，获得了一定数量的单倍体植株，并对小孢子的发育动态、愈伤组织的分化做了组织细胞学和生理生化方面的研究。主要的工作和结果分述如下： 1 材料的选取和预处理 取不同发育期的花蕾，用碘和碘化钾染色液染色压片镜检，选花粉发育为单核中期或单核中晚期的花药进行离体培养。花蕾的发育形态与花粉发育时期的相关性因品种与外界条件而异，一般以花萼刚刚裂开，还未开放，花冠包含在萼片之中、呈绿白色为宜。此时花药长7厘米左右。将采回来的花蕾大部分用湿纱布包裹放于温度为5～6℃冰箱中进行低温预处理，其余部分于接种后进行常规培养(对照)或热激处理。 2 茄子花蕾适宜消毒方法的建立 茄子花蕾外被大量的绒毛，故花蕾的消毒是花药培养中最基础的一环。试验结果表明：次氯酸钠的消毒效果差，单纯氯化汞的消毒效果也不好，最佳的消毒方法为70％酒精预处理30秒后，用0.1％的氯化汞滴加一滴吐温消毒10～12分钟。 3 花药愈伤组织的诱导 将茄子花药接种到培养基上对其培养的最适激素、有机物质组合、低温预处理时间、热激处理时间、蔗糖浓度进行筛选。试验结果表明，茄子花药愈伤组织的诱导受多种因素的影响。其中茄子花药愈伤组织培养的最适激素、有机物质组合为1升培养基中添加2，4-二氯苯氧乙酸1.0毫克，激动素0.5毫克，水解乳蛋白1克和谷氨酰胺500毫克；5～6℃下最适低温预处理时间为48～72小时；33℃下最适热激处理时间为48～60小时，热激处理和低温预处理的效应没有叠加作用；最适蔗糖浓度为1升培养基中添加蔗糖30～40克。在各种最适条件下，渝研一号、三月茄、萍早秋一号的愈伤诱导率分别达到了12.00％、7.00％、25.00％左右。 4 花药愈伤组织的分化和不定芽的生根 将诱导和增殖培养产生的具形态建成愈伤组织转到不同分化培养基上诱导不定芽的产生。结果表明最适的分化培养基为1升MS培养基中添加玉米素1.0毫克，α-萘乙酸0.001～0.005毫克，赤霉素0.5毫克以及水解乳蛋白1克。在最适分化培养基上，渝研一号、三月茄、萍早秋一号的分化率分别达到了33.33％、24.00％、68.89％。随后将产生的不定芽转到1/2MS附加α-萘乙酸0～0.005毫克/升、蔗糖20克/升的生根培养基中生根良好，移栽存活率达90％以上。 5 单倍体植株的鉴定和倍性变化 将培养获得的再生植株取其根尖用去壁低渗法进行染色体的倍性鉴定。结果显示，在鉴定的95株花药培养植株中，单倍体、二倍体、三倍体、四倍体及非整倍、混倍体的比例分别为16.84％、54.74％、4.21％、9.47％和14.74％，但3个基因型的花培苗群体的倍性比例分布有西南农业大学硕士学位论文所不同 6小拘子发育动态观察 在花药接种后的第2、5、l()、15天各取样一次观察花药离体培养过程中小泡子的发育动态(以带丰早秋一号为研究对象)，用卡宝品红染色法分别制成花粉粒涂片，用显微镜进行细胞学观察和显微摄影。结果显示，雄核发育的启动及多细胞花粉的形成并不要求培养基中必须有外源激素存在，但外源激素在茄子花药离体培养前期几天内对雄核发育的启动和多细胞花粉的形成有促进作用，即促使有更多的花粉由配子体发育转向抱子体发育而形成多细胞花粉。同时试验中观察到了茄子.花药离体培养中花粉细胞分裂的两种途径:营养核分裂途径和花粉均等分裂途径。7具形态建成愈伤组织分化的组织细胞学观察 在不定芽发生过程中(以萍早秋一号为研究对象)，从接入具形态建成愈伤组织块起在O、l、3、6、9、12、15、18天时取材，用纳瓦兴氏液m固定，爱氏苏木精染色，石蜡包埋切片，切片厚度10微米，显微观察照相。具形态建成愈伤组织转接到分化培养基上后，表面和内部的具形态建成细胞逐渐形成两个分生中心，即分生细胞团和分生组织结节，以后前者发育成了芽原基，后者发育成了根原基。8愈伤组织分化的生理生化指标测定 本试验分别测定了具形态建成和不具形态建成愈伤组织分化时的多项生理生化指标(以萍早秋一号为研究对象):过氧化物酶同工酶谱带变化、过氧化物酶活性、可溶性糖、淀粉、蛋白质和核酸含量，从而较系统地从多角度对两类愈伤组织分化时的生理生化变化进行了研究。结果表明，在转入分化培养基前具形态建成与不具形态建成愈伤组织的酶谱基本相同，都有B、E、G、H、I带，但前者酶带相对要浓，同时前者比后者多了一条谱带C，C带在具形态建成愈伤组织分化的过程中始终存在;其他生理生化指标也是前者高于后者。在分化过程中，具形态建成愈伤组织要比不具形态建成愈伤组织多产生3条谱带，即谱带A、D、J，其它生理生化指标的变化前者也要比后者剧烈得多。与此同时，在具形态建成愈伤组织的3个处理中，处理I、n的各生理生化指标变化趋势基本相似，只是处理n的变化要比处理I大些，酶谱多产生了谱带D;处理111各生理生化指标的前期变化趋势与处理I、n相似，后期与二者表现出较大差异，结果未能产生不定芽;整个分化过程中更多还原

【Abstract】 The experiments have been conducted in the induction, multiplication and differentiation of anther cullus with three eggplant varieties ( Yuyan No. 1 ,Sanyueqie,Pingzaoqiu No. 1).And then a few haploid plants were obtained. In addition, further research on the triggering and growth of the microspores and the differentiation of the anther callus has been done at the cytohistology, physiology and biochemistry levels.The main results are as follows:1 Selection and pretreatment of materialAlabastrums of different development phases were dyed with I-KI, pressed with cover glass and examined with microscope so that the anthers were selected for tissue culture whose pollens were developing at mid or late uninucleate stage.The relativity between exterior shape of alabastrums and growth phases of pollens would probably differed considering different varieties and environment. In a general way, the phase with about seven cilimetre length anther was suitable when calyx was going to dehisce but not open and corolla with aqua was included in sepal.Then the majority of alabastrums picked were enwrapped with waterish pledget and pretreated with5~6℃ in refrigeratory, and the others inoculated were cultured at the room temperature (CK) or treated at the high temperature.2 Good disinfection system of eggplant alabastrumsDisinfection of alabastrums was fundmental to anther culture because eggplant alabastrums have a lot of glosses on their surface.The results indicated that the disinfection treatment of NaClO was noneffective, and the only HgCl2 was also inapparent,but pretreating30s with 70% alcohol before disinfecting 10~12min with 0.1% HgCl2 supplemented with a bolb of tween-20 was efficient and high qualitative.3 Induction of anther callusThe best condition of induction was selected about the best combination of hormone and organic compound, the optimum time of pretreatment by low temperature, the optimal time of treatment by high temperature and the suitable concentration of sucrose after the anthers were put into the induction media, the results indicated that there were many influence factors to the induction of anther callus.The optimal conbination of hormone and organic compound was 2,4-D1 .0mg/L + KT 0.5mg/L + LH1g/L + Gln500mg/L.The best concentration of sucrose was 30~40g/L.The most suitable time of low temperature pretreatment was 48-72h at 5~6℃, and the optimum time of high temperature treatment was 48~60h at 33℃.However, the influence of low temperature pretreatment couldn’t interact with that of high temperature treatment to increasing the induction rate. Under the optimal condition including forementioned factors, the callus induction rate of Yuyan No.1.Sanyueqie and Pingzaoqiu No.1 were about 12.00%,7.00%,25.00% respectively.4 Differentiation of anther callus and rhizogenesis of adventitious budMorphogenetic callus by inducing and proliferating culture was transferred into different differentiation media to induce adventitious buds. The results indicated that the optimum medium of differentiation was MS + ZT1.0mg/L +NAA0.001 -0.005mg/L+ GA30.5mg/L+ LH1g/L. Under this condition, the differentiation rate of Yuyan No.1, Yanyueqie and Pingzaoqiu No.1 attained 33.33%, 24.00% and 68.89% respectively. Then the adventitious buds were transferred into (he rhizogenesis medium of 1/2MS supplemented with 0~0.005mg/LNAA and 20g/L sucrose,and rooted well. When aftergrowthes were transplanted the livability was more than 90%.5 Identification of haploid plant and diversification of chromosome multipleThe chromosome multiple of root tip of aftergrowth was identified with the way of ridding cell wall and putting low osmosis.The results showed that the percentage of haploids, diploids, triploids, tetraploids and aneuploids-mixoploids were 16.84%, 54.74%,4.21%, 9.47% and 14.74% respectively at 95 aftergrowthes by anther culture. But the proportion of chromosome multiple was different at the anther aftergrowthes of three genotypes.6 Observation of microspore triggering and growthIn the cou更多还原