冰冻切片技术在铁皮石斛显微结构上的应用
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摘要
为了快速高效的观察兰科植物铁皮石斛的显微结构,利用光镜和改进的蔗糖保护——液氮速冻冰冻切片法,观察铁皮石斛根、茎、叶的显微结构。该技术方法是将铁皮石斛器官经过蔗糖磷酸缓冲液保护液处理后抽真空,再经过液氮速冻、包埋、切片、展片观察、染色以及拍照等步骤,制作出铁皮石斛根、茎和叶较完整的显微结构切片。研究结果表明,适合铁皮石斛根的最适条件为:蔗糖质量体积分数为8%、冷凝温度-25℃、切片厚度20μm;茎的最适条件为:蔗糖质量体积分数为16%、冷凝温度-20℃、切片厚度15μm;叶的最适条件为:蔗糖质量体积分数为4%、冷凝温度-20℃、切片厚度10μm。该研究在兰科植物显微结构观察和组织化学研究中将具有广阔的应用前景。
In order to observe the microscopic structure of Dendrobium officinale quickly and efficiently. This study used the light microscope and the improved sucrose protection-liquid nitrogen quick-freezing freezing section method for orchid tissues and organs,Which improved sucrose protection-liquid nitrogen frozen method,observed root,stem and leaf of microscopic structure on D. officinale. The technology and methods treated D. officinale with sucrose phosphate buffer protection fluid,and then Vacuum processing. By liquid nitrogen frozen,embedding,sectioning,exhibition slice observation,staining and photo produced D. officinale roots,stems and leaves a complete microscopic section. The results show that the optimal condition for root was 8%sucrose mass volume fraction,condensation temperature -25℃,slice thickness 20 μm; the optimal condition for stem was 16% sucrose mass volume fraction,frozen temperature-20℃ and slice thickness 15 μm; the optimal condition for leaf was 4% sucrose mass volume fraction,frozen temperature -20℃ and slice thickness 10 μm. The study on the Orchidaceae microstructure observation and histochemical study will have broad prospect of application.
In order to observe the microscopic structure of Dendrobium officinale quickly and efficiently. This study used the light microscope and the improved sucrose protection-liquid nitrogen quick-freezing freezing section method for orchid tissues and organs,Which improved sucrose protection-liquid nitrogen frozen method,observed root,stem and leaf of microscopic structure on D. officinale. The technology and methods treated D. officinale with sucrose phosphate buffer protection fluid,and then Vacuum processing. By liquid nitrogen frozen,embedding,sectioning,exhibition slice observation,staining and photo produced D. officinale roots,stems and leaves a complete microscopic section. The results show that the optimal condition for root was 8%sucrose mass volume fraction,condensation temperature -25℃,slice thickness 20 μm; the optimal condition for stem was 16% sucrose mass volume fraction,frozen temperature-20℃ and slice thickness 15 μm; the optimal condition for leaf was 4% sucrose mass volume fraction,frozen temperature -20℃ and slice thickness 10 μm. The study on the Orchidaceae microstructure observation and histochemical study will have broad prospect of application.
引文
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8 .张新成,李志刚,李素丽,等.冷冻切片在植物微管骨架研究中的应用[J].广西植物,2008,28(2):164-166,178.Zhang X C,Li Z G,Li S L,et al. Cryosectioning method for the observation of microtubule cytoskeleton in plant cells[J]. Guihaia,2008,28(2):164-166,178.
9 .李娜,郭学民,孟阳.沙棘雌雄株叶片解剖结构比较研究[J].植物研究,2017,37(1):1-7.Li N,Guo X M,Meng Y. Comparison on leaf anatomical structures betweenthe female and the male Plants of Eippophae rhamnoides L.[J]. Bulletin of Botanical Research,2017,37(1):1-7.
10 .李贺,刘松梅,孙艳,等.冰冻切片技术在海滨锦葵细胞学研究中的应用[J].广西植物,2010,30(2):170-173,184.Li H,Liu S H,Sun Y,et al. Cryo-sectioning technique for cytological studies in Kosteletzkya virginica[J]. Guihaia,2010,30(2):170-173,184.
11 .丁晖,韩素芬,王光萍,等,卡特兰与丝核菌共培养体系的建立及卡特兰菌根显微结构的研究[J].菌物系统,2002,21(3):425-429.Ding H,Han S F,Wang G P,et al. Symbiosis between Cattleya sp. and Rhizoctonia sp. and mycorrhizal microstructure of Cattleya sp.[J]. Mycosystema,2002,21(3):425-429.
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13 . Dickson S,Kolesik P. Visualisation of mycorrhizal fungal structures and quantification of their surface area and volume using laser scanning confocal microscopy[J]. Mycorrhiza 1999,9(4):205-213.
14 .刘剑锋,程云清,阎秀峰,等.植物冰冻切片技术的改进[J].南京林业大学学报:自然科学版,2006,30(3):128-130.Liu J F,Cheng Y Q,Yan X F,et al. Improvement of plant cryo-sectioning technique[J]. Journal of Nanjing Forestry University:Natural Science Edition,2006,30(3):128-130.
15 .刘剑锋,阎秀峰,程云清,等.冰冻切片技术在高山红景天细胞学研究中的应用[J].东北林业大学学报:自然科学版,2006,34(4):118-119.Liu J F,Yan X F,Cheng Y Q,et al. Application of cryosectioning techique to cytology studies of Rhodiola sachalinensis[J]. Journal of Northeast Forestry University:Natural Science Edition,2006,34(4):118-119.
16 . Solísramos L Y,Nahuathdzib S,Andradetorres A,et al. Indirect somatic embryogenesis and morphohistological analysis in Capsicum chinense[J]. Biologia,2010,65(3):504-511.
17 . Scheen A C,Bendiksby M,Ryding O,et al. Molecular phylogenetics,character evolution,and suprageneric classification of Lamioideae(Lamiaceae)[J]. Annals of the Missouri Botanical Garden,2010,97(2):191-217.
18 . Kobae Y,Fujiwara T. Earliest colonization events of Rhizophagus irregularis in rice roots occur preferentially in previously uncolonized cells[J]. Plant and Cell Physiology,2014,55(8):1497-1510.
19 .陈丹,赵洁.适合于植物花器官的冰冻切片技术[J].武汉植物学研究,2005,23(3):285-290.Chen D,Zhao J. Suitable cryo-sectioning technique in floral organs of plants[J]. Journal of Wuhan Botanical Research,2005,23(3):285-290.
2 . Annie J,Nathalie D,Walter D,et al. In situ localisation of cytokinins in the shoot apical meristem of Sinapisalba at floral transition[J]. Planta,2002,214:970-973.
3 .林月惠,李寒冰,贺新强.高度木质化材料的冰冻切片技术[J].植物学通报,2001,18(1):118-120.Lin Y H,Li H B,He X Q. Application of cryo-sectioning technique in highly lignified tissues[J]. Chinese Bulletin of Botany,2001,18(1):118-120.
4 .谢佩松,徐中根,韦存虚.冰冻切片技术在植物显微结构和组织化学中的应用[J].生物学杂志,2009,26(3):72-74.Xie P S,Xu Z G,Wei C X. Cryo-sectioning technique for the observation of microstructure and histochemistry in plant[J]. Journal of Biology,2009,26(3):72-74.
5 .范黎,郭顺星,肖培根.密花石斛等6种兰科植物菌根的显微结构研究[J].植物学报,2000,17(1):73-79.Fan L,Guo S X,Xiao P G. A study on the mycorrhizal microstructure of six orchids[J]. Chinese Bulletin of Botany,2000,17(1):73-79.
6 .伍建榕,吕梅,刘婷婷,等. 6种兰科植物菌根的显微及超显微结构研究[J].农林科技大学学报:自然科学版,2009,37(7):199-207.Wu J R,LüM,Liu T T,et al. Study on observation of the mycorrhiza under optical and electronic microscope of six orchids[J]. Journal of Northwest A&F University:Natural Science Edition,2009,37(7):199-207.
7 .陆叶,席梦利,郑佳,等.杨树和杉木茎段组织的冰冻切片技术研究[J].南京林业大学学报:自然科学版,2009,33(6):44-48.Lu Y,Xi M L,Zheng J,et al. Cryo-section techniquedeveloped for stem tissue gene expression research on poplar and Chinese fir[J]. Journal of Nanjing Forestry University:Natural Science Edition,2009,33(6):44-48.
8 .张新成,李志刚,李素丽,等.冷冻切片在植物微管骨架研究中的应用[J].广西植物,2008,28(2):164-166,178.Zhang X C,Li Z G,Li S L,et al. Cryosectioning method for the observation of microtubule cytoskeleton in plant cells[J]. Guihaia,2008,28(2):164-166,178.
9 .李娜,郭学民,孟阳.沙棘雌雄株叶片解剖结构比较研究[J].植物研究,2017,37(1):1-7.Li N,Guo X M,Meng Y. Comparison on leaf anatomical structures betweenthe female and the male Plants of Eippophae rhamnoides L.[J]. Bulletin of Botanical Research,2017,37(1):1-7.
10 .李贺,刘松梅,孙艳,等.冰冻切片技术在海滨锦葵细胞学研究中的应用[J].广西植物,2010,30(2):170-173,184.Li H,Liu S H,Sun Y,et al. Cryo-sectioning technique for cytological studies in Kosteletzkya virginica[J]. Guihaia,2010,30(2):170-173,184.
11 .丁晖,韩素芬,王光萍,等,卡特兰与丝核菌共培养体系的建立及卡特兰菌根显微结构的研究[J].菌物系统,2002,21(3):425-429.Ding H,Han S F,Wang G P,et al. Symbiosis between Cattleya sp. and Rhizoctonia sp. and mycorrhizal microstructure of Cattleya sp.[J]. Mycosystema,2002,21(3):425-429.
12 .丁松爽,孙坤,苏雪,等.栒子属(Cotoneaster Medikus)植物叶表皮微形态特征及其分类学意义[J].植物研究,2008,28(2):187-194.Ding S S,Sun K,Su X,et al. Characters of Leaf Epidermis and Their Taxonomic Significance in Cotoneaster Medikus[J]. Bulletin of Botanical Resarch,2008,28(2):187-194.
13 . Dickson S,Kolesik P. Visualisation of mycorrhizal fungal structures and quantification of their surface area and volume using laser scanning confocal microscopy[J]. Mycorrhiza 1999,9(4):205-213.
14 .刘剑锋,程云清,阎秀峰,等.植物冰冻切片技术的改进[J].南京林业大学学报:自然科学版,2006,30(3):128-130.Liu J F,Cheng Y Q,Yan X F,et al. Improvement of plant cryo-sectioning technique[J]. Journal of Nanjing Forestry University:Natural Science Edition,2006,30(3):128-130.
15 .刘剑锋,阎秀峰,程云清,等.冰冻切片技术在高山红景天细胞学研究中的应用[J].东北林业大学学报:自然科学版,2006,34(4):118-119.Liu J F,Yan X F,Cheng Y Q,et al. Application of cryosectioning techique to cytology studies of Rhodiola sachalinensis[J]. Journal of Northeast Forestry University:Natural Science Edition,2006,34(4):118-119.
16 . Solísramos L Y,Nahuathdzib S,Andradetorres A,et al. Indirect somatic embryogenesis and morphohistological analysis in Capsicum chinense[J]. Biologia,2010,65(3):504-511.
17 . Scheen A C,Bendiksby M,Ryding O,et al. Molecular phylogenetics,character evolution,and suprageneric classification of Lamioideae(Lamiaceae)[J]. Annals of the Missouri Botanical Garden,2010,97(2):191-217.
18 . Kobae Y,Fujiwara T. Earliest colonization events of Rhizophagus irregularis in rice roots occur preferentially in previously uncolonized cells[J]. Plant and Cell Physiology,2014,55(8):1497-1510.
19 .陈丹,赵洁.适合于植物花器官的冰冻切片技术[J].武汉植物学研究,2005,23(3):285-290.Chen D,Zhao J. Suitable cryo-sectioning technique in floral organs of plants[J]. Journal of Wuhan Botanical Research,2005,23(3):285-290.