【作者】 李彤；

【导师】 宋明； 王志敏；

【作者基本信息】 西南大学 ， 蔬菜学， 2008， 硕士

【摘要】 本实验以芥菜的三个品种(圆大头菜、永胜大儿菜、容渝香芥菜)为材料,在以子叶和胚轴为外植体建立离体培养体系的基础上,对芥菜组织器官发生过程中各发育阶段的生理生化指标变化及可溶性蛋白质含量组分和过氧化物酶同工酶进行了研究,同时进行了组织细胞学观察。实验结果如下:1芥菜离体培养体系的建立不同品种的芥菜及培养基激素配比都会影响芥菜离体培养体系的建立。实验结果表明:芥菜种子消毒方法以采用5%NaClO溶液滴加2滴100%吐温-20,消毒15分钟最佳;种子无菌萌发培养基为:1/2MS+8.0g/L卡拉胶(pH5.8～6.0);最适愈伤组织诱导和分化的培养基为:MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/L NAA+3.0mg/L 6-BA,当以子叶为外植体时,圆大头菜、永胜大儿菜、容渝香芥菜的分化率均为最高,分别为:72%、84%、76%;当以胚轴为外植体时上述三个品种的分化率分别为70%、72%、70%;最适生根培养基为MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/LNAA,根系生长状态良好,生根率达到100%。2可溶性蛋白质含量及组分的研究具形态建成的愈伤组织中可溶性蛋白质平均含量为20.88mg╱g·FW,远远高于不具形态建成的愈伤组织中的平均含量12.08mg/g·FW,低水平的蛋白质表达量可能是使愈伤组织丧失分化能力,转变为不具形态建成的愈伤组织的一个重要原因。SDS-PAGE电泳结果显示,在具形态建成的愈伤组织增殖与分化过程中,有65 KDa、29 KDa、24 KDa、16 KDa的特异性蛋白质组分出现,其中16 KDa蛋白在不定芽分化后不再表达,推测是这4种特异性蛋白推动了愈伤组织的形态建成。在不具形态建成的愈伤组织中有50KDa和30KDa的蛋白质组分表达,可把这两种蛋白组分看作是芥菜子叶离体培养过程中不具形态建成的愈伤组织中的特异性蛋白。3过氧化物同工酶酶谱的研究通过过氧化物酶同工酶电泳,得到以下结果:具形态建成的愈伤组织的酶谱带比不具形态建成的愈伤组织的酶谱带颜色相对要浓,同时多出了C带和G带,其中G带是具形态建成的愈伤组织所特有的,C带是在不定芽分化后出现的,因此C带可作为不定芽分化的生化标记,D带在愈伤组织诱导期出现的,可作为脱分化开始的标志。4生理生化变化的研究两种愈伤组织随着培养时间的增加生理指标的差异越来越显著,具形态建成的愈伤组织比不具形态建成的愈伤组织内部代谢增强。外植体培养的第8天开始,具形态建成的愈伤组织中抗氧化酶(POD、SOD、CAT)活性、核酸(RNA、DNA)含量均高于不具形态建成的愈伤组织,呈明显的上升趋势,而可溶性糖和淀粉作为细胞中可利用的能源物质,在这个时期含量下降;不具形态建成的愈伤组织中以上各指标的变化则比较平缓。5组织细胞学观察利用石蜡切片技术对培养材料进行组织细胞学观察,结果表明,具形态建成的愈伤组织其表面和内部会逐渐形成两个分化中心,即分生细胞团和分生组织结节,前者将发育成为芽原基,后者将发育成为根原基。更多还原

【Abstract】 This work aimed at the study of culturing three varieties of mustard (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai), Starting with in vitro culture of cotyledons and hypocotyls of mustard established on the plant regeneration system. In the process of the organogenesis, this work mainly focused on the research of biochemical indicators, soluble proteins and peroxidase isoenzyme, and further research on the morphogenetic callus has been done at the cytohistology level. The main results are as follows:1 The establishment of in vitro culture system in mustardThe result showed that different varieties, hormone concentration played important roles in mustard organogenesis. The optimal way of seed sterilization was 5% NaClO +tween-20 2 drops for 15min. The optimal culture medium for sterilitas plants was 1/2MS+8.0g/L Carrageenan (pH5.8~ 6.0); the appropriate medium for explants differentiation was MS+30g/L sucrose+8.0g/L Carrageenan+3.0mg/L 6-BA+0.1mg/L NAA, when the explants were cotyledons, the highest differentiation rate of three varieties (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai)were 72%, 84%, 76%; and when the explants were hypocotyls, the highest differentiation rate were 70%, 72%, 70%. The best medium for rooting was MS+30g/L sucrose+8.0g/L Carrageenan+0.1mg/L NAA, with good root status and 100% rootage rate.2 The studies on components and contents of the soluble proteinsThe average content of soluble proteins in morphogenetic callus was 20.88 mg/g·FW, it was higher than the content in non-morphogenetic callus, which was 12.08 mg/g-FW. Therefore, the lower-level of the proteins might be one of the important reason that let morphogenetic callus lost the capacity of redifferentiation and turned into non-morphogenetic callus.The results of SDS-PAGE electrophoresis showed that different specific proteins found separately in different stages. There were four kinds of proteins (65KDa, 29KDa, 24KDa, 16KDa) expressed during callus differentiation. And the protein weight 16KDa didn’t express after callus differentiated. These four kinds of proteins might be related with the morphochoresis of organs. The proteins weight 50KDa and 30KDa were detected in non-morphogenetic callus and not in morphogenetic callus.So, these two kinds of proteins can be used as markers for non-morphogenetic callus during mustard in vitro culture.3 The studies on peroxidase isozymeThe peroxidase isozyme analyse was as follows: the thickness of isozyme bands of the morphogenetic callus was little stronger than those of the non-morphogenetic callus. The morphogenetic callus had C and G bands that the non-morphogenetic callus didn’t have. C band appeared after callus differentiation, so it could be used as a biochemical marker for callus differentiation. D band appeared during callus induction, this might be a sign for the begining of explants dedifferentiation.4 Studies on physiological and biochemical indexesThe physiological and biochemical indexes in two kinds of callus are obviously different during the culture process. The physiological and biochemical indexes of the morphogenetic callus had wider change range than those of the non-morphogenetic callus. When the callus had been cultivated for 8 days, the contents of nucleic acid (DNA, RNA), the antioxides enzyme (POD, SOD, CAT) activities of morphogenetic callus were higher than that of non-morphogenetic callus, and trends of those indexes were on the rise. At the same time, the contents of soluble sugars and starch were declined. While the changes of physiological and biochemical indexes of non-morphogenetic callus changed mildly.5 Histocytological analysisBy the observation of paraffin wax slice, the morphogenetic cells of exterior and interior gradually formed two kinds of meristematic centres, which were the embryonic meristematic packed cells and the meristematic tubercles.The former would develop into primordial buds, and the latter would develop into primordial roots.更多还原