蝴蝶兰组织培养褐变机理的研究
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摘要
蝴蝶兰是一种深受人们喜爱的名贵花卉，被称为热带兰中的“兰花皇后”，组织培养为其主要的繁殖方式。褐变在植物组织培养快速繁殖过程中普遍存在，严重影响外植体分化，也是制约蝴蝶兰工厂化繁殖的难题之一，研究蝴蝶兰组织培养褐变机理具有理论与实践意义。
本研究以蝴蝶兰无菌苗为材料，通过对其组培褐变过程中外植体细胞形态及生理生化变化进行初步研究，试图探明蝴蝶兰组织培养褐变机理。研究结果如下：
1．光学显微镜观察表明：与未褐变的外植体相比，褐变的外植体细胞形态结构发生不规则变化，细胞壁边缘堆积黑色颗粒，且褐变越严重，黑色颗粒物质堆积越多；透射电镜观察发现：未褐变的外植体细胞形态结构规则正常，细胞质壁不分离，叶绿体结构完整；褐变的外植体细胞严重皱缩变形，细胞质壁分离，褐变严重的外植体细胞壁断裂，叶绿体片层结构严重破坏，细胞核解体；
2．研究了培养基中五种矿质元素(Fe、Cu、K、Ca、Zn)含量对蝴蝶兰褐变的影响。发现初代培养时，培养基中Fe、Cu浓度越高，褐变越严重；一定范围内，培养基中K、Ca、Zn浓度越高，褐变越轻；
3．通过对外植体不同处理的9种酚酸高效液相色谱定性定量分析，初步证明绿原酸、邻苯二酚、儿茶酚、咖啡酸及没食子酸、对羟基苯甲酸、香豆酸可能与蝴蝶兰褐变相关，苯甲酸对蝴蝶兰褐变只有很小影响；
4．蝴蝶兰在褐变过程中，总酚含量呈先降后升再降的趋势，与褐变程度呈正相关；褐变过程中PAL和PPO活性与褐变程度呈正相关，POD参与了褐变的过程；总酚含量与PAL活性呈正相关，与PPO和POD活性呈负相关。
Phalaenopsis. Spp is one of beloved rare flowers that regarded as "The Queen of Tropical Orchid ", tissue culture is its main progenitive measure. Tissue browning which hamper cell differentiation inhabits the whole process of tissue culture, and also restricts the industrial product of Phalaenopsis Spp. Studies on the mechanism of of tissue browning in Phalaenopsis Spp is with great significance. Using Phalaenopsis Spp as experimental material, changes in cytology, physiology and biochemistry of explants when browning have been investigated. The main results are as follows:
1. There were black substances deposited in parenchyma cell of explants, and more browning more black substances deposition under microscopic observation. TEM pictures showed that, compared with controls, browning cells were not regular-shaped, cell wall break, chloroplast structures seriously damaged and nuclus decomposed.
2. In experiment of mineral element (Fe, Cu, K, Ca and Zn) influence on tissue browning of Phalaenopsis Spp, Fe and Cu concentration in culture medium enhanced browning, however, K、 Ca、 Zn in media showed a negative correlation with browning.
3. High Performance Liquid Chromatography (HPLC) was used to analyze
本研究以蝴蝶兰无菌苗为材料，通过对其组培褐变过程中外植体细胞形态及生理生化变化进行初步研究，试图探明蝴蝶兰组织培养褐变机理。研究结果如下：
1．光学显微镜观察表明：与未褐变的外植体相比，褐变的外植体细胞形态结构发生不规则变化，细胞壁边缘堆积黑色颗粒，且褐变越严重，黑色颗粒物质堆积越多；透射电镜观察发现：未褐变的外植体细胞形态结构规则正常，细胞质壁不分离，叶绿体结构完整；褐变的外植体细胞严重皱缩变形，细胞质壁分离，褐变严重的外植体细胞壁断裂，叶绿体片层结构严重破坏，细胞核解体；
2．研究了培养基中五种矿质元素(Fe、Cu、K、Ca、Zn)含量对蝴蝶兰褐变的影响。发现初代培养时，培养基中Fe、Cu浓度越高，褐变越严重；一定范围内，培养基中K、Ca、Zn浓度越高，褐变越轻；
3．通过对外植体不同处理的9种酚酸高效液相色谱定性定量分析，初步证明绿原酸、邻苯二酚、儿茶酚、咖啡酸及没食子酸、对羟基苯甲酸、香豆酸可能与蝴蝶兰褐变相关，苯甲酸对蝴蝶兰褐变只有很小影响；
4．蝴蝶兰在褐变过程中，总酚含量呈先降后升再降的趋势，与褐变程度呈正相关；褐变过程中PAL和PPO活性与褐变程度呈正相关，POD参与了褐变的过程；总酚含量与PAL活性呈正相关，与PPO和POD活性呈负相关。
Phalaenopsis. Spp is one of beloved rare flowers that regarded as "The Queen of Tropical Orchid ", tissue culture is its main progenitive measure. Tissue browning which hamper cell differentiation inhabits the whole process of tissue culture, and also restricts the industrial product of Phalaenopsis Spp. Studies on the mechanism of of tissue browning in Phalaenopsis Spp is with great significance. Using Phalaenopsis Spp as experimental material, changes in cytology, physiology and biochemistry of explants when browning have been investigated. The main results are as follows:
1. There were black substances deposited in parenchyma cell of explants, and more browning more black substances deposition under microscopic observation. TEM pictures showed that, compared with controls, browning cells were not regular-shaped, cell wall break, chloroplast structures seriously damaged and nuclus decomposed.
2. In experiment of mineral element (Fe, Cu, K, Ca and Zn) influence on tissue browning of Phalaenopsis Spp, Fe and Cu concentration in culture medium enhanced browning, however, K、 Ca、 Zn in media showed a negative correlation with browning.
3. High Performance Liquid Chromatography (HPLC) was used to analyze
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