【作者】 黄素华；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 果树学， 2002， 硕士

【摘要】 本研究以荔枝胚性培养物为材料，采用细胞标记、生化标记、分子标记等手段，研究荔枝胚性愈伤组织培养及其体细胞胚胎发生过程中的遗传变异规律，以期为探讨荔枝体细胞胚胎发生机理和体细胞无性系变异机理提供新的线索，并为解决荔枝体胚中玻璃胚、畸形胚及体胚成苗率低等问题提供科学依据。 从“元红”、“下番枝”等荔枝品种的幼胚、花药等外植体诱导并筛选出胚性愈伤组织和非胚性愈伤组织。采用酒精-浓HCI(1:1)解离液直接解离和丙酸-铁矾-苏木精染色液染色的简化制片方法，对荔枝的非胚性愈伤组织、胚性愈伤组织、胚状体、畸形苗根尖、正常苗根尖的染色体数目检查表明：各种培养物均出现了广泛的染色体数目的变异，除正常的荔枝体细胞染色体数目2n=2X=30外，还出现2n=15～29以及2n=31～60的染色体异常细胞。对胚性愈伤组织进行筛选，很难得到染色体数目稳定在某一数目的非正常染色体数目的细胞系，但可得到染色体数目相对稳定在某一范围的细胞系。在本试验中共得到3种类型细胞系：细胞系Ⅰ，染色体数目主要为正常二倍体，2n=30；细胞系Ⅱ，染色体数目主要为少于30，2n=15～29；细胞系Ⅲ，染色体数目很不稳定，2n=15～60。激素、继代时间、外植体种类及基因型等对染色体数目的变异有一定影响。 采用不连续垂直聚丙烯酰胺凝胶电泳，对胚性愈伤组织和胚状体的POD与EST同工酶谱分析表明：3种类型细胞系(胚性愈伤组织)的POD与EST同工酶酶谱既有酶带强弱的差别，又有带数的增减；同一类型细胞系分化而来的不同胚状体之间的POD与EST同工酶酶谱也表现出一定的差异。 RAPD分析表明：荔枝胚性和非胚性愈伤组织、玻璃化胚和白色胚、筛选出的3种染色体数目不同的3种类型细胞系（胚性愈伤组织）之间的DNA分子标记存在差异，从而进一步证实荔枝胚性愈伤组织继代及再分化过程中存在遗传物质的变异。 由此可见，在染色体数口、生化与分子标记等方面，荔枝胚性愈伤组织存在广泛的遗传变异，并且这种变异可以传巡到胚状体水平；同时，在从胚性愈伤组织到胚状体的发育过程中也产生一定的遗传变异。因此，我们认为，荔枝胚性培养物的广泛遗传变异以及这种变异的传递性，是荔枝体胚萌发难的根本原因。更多还原

【Abstract】 The mechanisms of genetic variation of the in-vitro embryogenic cultures in the process of somatic embryogenesis were studied through cytological markers, biochemical markers and molecular markers in litchi [Litchi chinensis Sonn.], which could provide new clues for the study of the mechanism of somatic embryogenesis and somaclonal variation, and also provide scientific information for overcoming vitrified embryoids, abnormal embryoids and the low inversion rate of somatic embryos in litchi.Embryogenic and nonembryogenic calli were induced and screened from the culture of young embryos and anthers in the Litchi cultivars "Yuanhong"and "Xiafanzhi". The chromosome numbers of the nonembryogenic calli, embryonic calli, somatic embryos, and the root-tips of abnormal and normal plantlets were examined by the routine method combined with a simplified method using the alcohol-HCl (1:1) resolution for direct lysis of tissue and cell and using methylacetic acid-iron-haematoxylin for staining. The results showed that the variation of the chromosome numbers existed in all the above cells ,tissues or organs. Besides the normal chromosome number of 2n=30, some abnormal cells with the chromosome numbers of 2n= 15-29 or 2n=31-60 occurred. The results also showed that it was difficult to obtain the stable cell clones in which the chromosome numbers were stable in a number through the screening from embryogenic calli; however, some cell clones with relatively stable chromosome numbers in a narrow range were obtained.In this experiment, three cell lines were obtained. They were Cell Line I ( most cells with the normal chromosome number 2n=30), Cell Line II(most cells with the chromosome numbers 2n<30) and Cell Line Ill(cells with the chromosome numbers 2n=15 ~ 60). Different plant growth regulators, the time of subcultures, genotypes and explants had some effect on the numbers of the chromosomes.The POD and EST isoenzymatic patterns were analyzed by the discontinuous polyacrylamide gel electrophoresis (PAGE), The differencesof the numbers of the belts and strength of the belt activity existed in the tree types of the screened cell lines; moreover, there also existed some differences in POD and EST isoenzymatic patterns among the different embryoids which were induced from the same cell line.The results of randomly amplified polymorphic DNA (RAPD) showed there were differences between embryogenic and nonembryogenic calli, vitrified embryoids, white embryoids and the three screened cell lines, which proved that there existed the variations of genetic substance during the process of subcultures and differentiation of embryogenic calli in litchi.In a word, there existed wide genetic variations in the chromosome numbers, biochemical and molecular markers among the embryogenic calli and these variation also existed during the process of somatic embryogenesis from embryogenic calli to embryoids in litchi. Therefore, we considered that the key factor that influenced the regeneration of litchi somatic embryogenesis was the existence of the wide variations in the embryonic cells and the transfer characteristics of these variations in litchi.更多还原