甘蔗茎尖离体培养褐变影响因素及细胞区室结构分析

Cell chamber structure and influences factors analysis on sugarcane stem tips browning in vitro

关键词:  褐变, 茎尖, 甘蔗, 多酚类物质

Key words:  browning, stem tips, sugarcane, phenolic compounds

摘要:

该研究通过综合分析对甘蔗(ROC 22)茎尖离体培养褐变不同条件因素的影响以及褐变细胞区室结构的变化,探讨了甘蔗茎尖离体培养褐变的机理机制。结果表明:不同芽位茎尖诱导成活率具有明显差异,随着芽位的增加,诱导成活率不断降低; 不同季节取芽对外植体茎尖总酚类物质含量无明显影响; 但不同芽位及不同催芽天数,外植体芽的总多酚含量明显不同,随着催芽天数的增加,不同芽位的多酚含量呈现由低升高的趋势; 蔗芽在培养4周时多酚含量较低,适宜进行采芽接种培养; 从褐变甘蔗茎尖的解剖结构变化分析,褐变甘蔗茎尖细胞离体培养初期细胞核结构出现变形,线粒体有肿胀拉长,部分液泡膜开始分解; 中后期质壁分离更为严重,胞质中出现大量溶酶体,线粒体等细胞器全分解,细胞膜、液泡膜、核膜、线粒体膜的双层膜结构出现破损和缺口; 而正常发育的茎尖细胞,能基本保持细胞核的形态结构,只有少量的溶酶体出现。因此,可以推测细胞核和线粒体结构变形以及膜系统的大量破损是甘蔗茎尖培养褐变死亡的原因。

Abstract:

This research was mainly carried out to analyze the mechanism of sugarcane stem tip browning in vitro culture through investigating the influences factors which relate to phenolic browning and observing stem tip cellular compartments structure changes in the process of browning. Sugarcane variety ROC 22 which provided by sugarcane research institute of Guangxi Academy of Agricultural Sciences was selected as experiment material. The result showed that different axillary buds obtained obviously different induction survival rates, the more fresh buds resulted in the more high survival rate, the first position bud got the highest survival rate, that is to mean the more fresh bud get the more higher induction survival rate; the induction survival rate was not effected by different explants collecting seasons; pre-germination time 4 weeks resulted higher induction survival rate, and obtained significant difference compared with other pre-germination time treatments. Different buds with different pre-germination time treatments had obvious different total phenolic compounds content, the longer pre-germination time resulted in the lower phenolic compounds accumulated in stem tip cells, pre-germination time for 2 weeks and 3 weeks obtained higher total phenolic compounds in stem tip cells at different position buds, while pre-germination for 4 weeks and 5 weeks got low total phenolic compounds content, therefore, sugarcane axillary bud was pre-germinated for 4 weeks is a better period for induction tissue culture; stem tip total phenolic compounds content was not effected by explants collecting season; polyphenol oxidase acitivity changed significantly in different axillary buds with different pre-germination time treatments, lower polyphenol oxidase acitivity were obtained in 1-6 position buds with 2 weeks pre-germinated treatment and in 3-6 position buds with 4 weeks pre-germinated treatment; while higher polyphenol oxidase acitivity were obtained in 1-3 position buds with 3 weeks pre-germinated treatment and in 4-6 position buds with 5 weeks pre-germinated treatment. Sugarcane browning stem tips were observed by electron microscope, the result showed that the nucleus structure deformed, swollen mitochondria stretched, part of the vacuole membrane started to break down at the early browning stage; more serious plasmolysis appeared, a large number of lysosomes appeared in cytoplasm, organelles such as mitochondria completely decomposed, cell membrane, vacuole membrane, nuclear membrane and mitochondrial membrane broke down, cytoplasm completely dissolved, vacuoles mitochondria and other organelles completely decomposed at the later browning stage. This finding inferred that nucleus and mitochondria structural distortion and cell membrane systems break were the main reasons for sugarcane stem tip browning to death in the process of tissue culture in vitro.