欢迎访问作物学报,今天是

作物学报 ›› 2012, Vol. 38 ›› Issue (09): 1688-1697.doi: 10.3724/SP.J.1006.2012.01688

• 耕作栽培·生理生化 • 上一篇    下一篇

固定和染色方法对小麦胚乳细胞结构显示的影响

王玲玲1,**,刘智1,**,熊飞1,李栋梁1,周卫东2,陈义芳2,王忠1,*   

  1. 1扬州大学 / 江苏省作物遗传生理重点实验室 / 农业部长江中下游作物生理生态与栽培重点开放实验室,江苏扬州 225009;2扬州大学分析测试中心,江苏扬州 225009
  • 收稿日期:2012-02-10 修回日期:2012-04-20 出版日期:2012-09-12 网络出版日期:2012-07-03
  • 通讯作者: 王忠, E-mail: wangzhong@yzu.edu.cn
  • 基金资助:

    本研究由国家自然科学基金项目(31071341, 31171482)和江苏省自然科学基金项目(BK2011445)资助。

Effect of Fixation and Staining Methods on Structure Observation of Endosperm Cell of Wheat

WANG Ling-Ling1,**,LIU Zhi1,**,XIONG Fei1,LI Dong-Liang1,ZHOU Wei-Dong2,CHEN Yi-Fang2,WANG Zhong1,*   

  1. 1Key Laboratory of Crop Genetics and Physiology of Jiangsu Province / Key Laboratory of Crop Physiology, Ecology and Cultivation in Middle and Lower Reaches of Yangtze River of Ministry of Agriculture, Yangzhou 225009, China; 2Testing Center, Yangzhou University, Yangzhou 225009, China?
  • Received:2012-02-10 Revised:2012-04-20 Published:2012-09-12 Published online:2012-07-03
  • Contact: 王忠, E-mail: wangzhong@yzu.edu.cn

摘要: 以扬麦16小麦颖果为材料,采用戊二醛-四氧化锇(GA-OsO4)或高锰酸钾固定,并分别用5种染色方法[甲苯胺蓝-O (TBO)、多色性染液、高碘酸–希夫试剂(PAS)、考马斯亮蓝和PAS-TBO]进行半薄切片染色,对超薄切片进行醋酸双氧铀和柠檬酸铅染色,在光学显微镜和透射电镜下观察比较了小麦胚乳细胞结构显示的差异。结果表明,GA-OsO4固定的半薄切片染色效果整体上优于高锰酸钾固定,但是在PAS-TBO复染中,高锰酸钾固定制样效果最好。在超薄切片观察中,GA-OsO4固定制样能较好地保存胚乳细胞超微结构,但对膜性结构显示不够清晰,而高锰酸钾固定制样则能较好地固定细胞的膜性结构,并且能够增强膜结构反差,只是对多糖和蛋白质的固定和显色较差。因此,应根据观察对象来选择适当的固定及染色方法。

关键词: 小麦, 胚乳细胞, 固定方法, 染色方法, 半薄切片, 超薄切片

Abstract: Two fixing methods and five staining agents were used to compare which one is best for optimal observation of endosperm cell of wheat under optical and transmission electron microscopes. Caryopsis samples of Yangmai 16 were fixed with glutaraldehyde-osmium tetroxide (GA-OsO4) or potassium permanganate and stained withtoluidine blue (TBO), polychromatic dye, periodic acid schiff (PAS), coomassie brilliant blue, or PAS-TBO during the preparation of semi-thin sections. The GA-OsO4 fixation showed better overall performance than the potassium permanganate fixation, but the best observation was got by potassium permanganate fixationand PAS-TBO counterstaining. The GA-OsO4 fixation had the advantage of preserving endosperm cell intrinsic feature in ultra-thin section but the disadvantage of unclear display of the membranous structure. The potassium permanganate fixation was able to show the structure of the cell endomembranevividly with satisfactory contrast between background and membranous structure; however, the color stained in polysaccharide and protein was poor. Therefore, appropriate fixation and staining methods should be based on the target and objective of experiment.

Key words: Wheat, Endosperm cell, Fixation method, Stain method, Semi-thin section, Ultra-thin section

[1]Buttrose M S. Ultrastructure of the developing wheat endosperm. Aust J Biol Sci, 1963, 16: 305–317

[2]Bechtel D B, Gains R L, Pomeranz Y. Early stages in wheat endosperm formation and protein body initiation. Annu Bot, 1982, 50: 507–518

[3]Parker M L. The relationship between A-type and B-type starch granules in the developing endosperm of wheat. J Cereal Sci, 1985, 3: 271–278

[4]Shewry P R, Napier J A, Tatham A S. Seed storage proteins: structures and biosynthesis. Plant Cell, 1995, 7: 945–956

[5]Langeveld S M J, van Wijk R, Stuurman N, Kijne J W, de Pater S. B-type granule containing protrusions and interconnection between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression. J Exp Bot, 2000, 51: 1357–1361

[6]Shewry P R, Halford N G. Cereal seed storage proteins: structures, properties and role in grain utilization. J Exp Bot, 2002, 53: 947–958

[7]Kumamaru T, Ogawa M, Satoh H, Okita T W. Protein body biogenesis in cereal endosperms. In: Olsen O A ed. Plant Cell Monogry: Endosperm. Heidelberg: Springer-Verlag Berlin, 2007. pp 141–158

[8]Sheng J(盛婧), Guo W-S(郭文善), Zhu X-K(朱新开), Feng C-N(封超年), Peng Y-X(彭永欣). Starch granules development in grain endosperm of wheat for different end uses. Acta Agron Sin (作物学报), 2004, 30(9): 953–954 (in Chinese with English abstract)

[9]Wei C-X(韦存虚), Zhang J(张军), Zhou W-D(周卫东), Chen Y-F(陈义芳), Xu R-G(许如根). Accumulation of storage protein and formation of protein body during wheat endosperm development. J Triticeae Crops (麦类作物学报), 2009, 29(1): 73–78 (in Chinese with English abstract)

[10]Yin Y-A(银永安), Qi J-C(齐军仓), Li W-H(李卫华), Cao L-P(曹连莆), Wang Z-B(王自布). Physico- chemical characteristics of A, B type starch granule in wheat endosperm. Sci Agric Sin (中国农业科学), 2010, 43(11): 2372–2379 (in Chinese with English abstract)

[11]Liu Z(刘智), Wang L-L(王玲玲), Zhou W-D(周卫东), Chen Y-F(陈义芳), Wang Z(王忠). The effect of fixation and staining methods on observed the endosperm cell in rice. Chin J Rice Sci (中国水稻科学), 2011, 25(6): 667–671 (in Chinese with English abstract)

[12]Bechtel D B, Zayas I, Kaleikau L, Pomeranz Y. Size distribution of wheat starch granules during endosperm development. Cereal Chem, 1990, 67: 59–63

[13]Liu D-J(刘冬娟), Zhu S-W(祝素文), Li Y-J(李艳杰). The application of semi-section staining with borax toluidine blue. J Chin Med Univ (中国医科大学学报), 2004, 33(1): 33 (in Chinese)

[14]Wang S(王爽), Wang C-M(王春梅), Chen-D(陈丹), Huang X-F(黄晓峰). Polychromatic dying method on semithin resin sections resin. J Fourth Mil Med Univ (第四军医大学学报), 2002; 23(9): 775 (in Chinese)

[15]Lyon H O, Schulte E K, Prento P, Barer M R, Béné M C. Standardized staining methods: feulgen-rossenbeck reaction for desoxyribonucleic acid and periodic acid -schiff (PAS) procedure. Biotech Histochem, 2002, 77: 121–125

[16]Hu S-Y(胡适宜), Xu L-Y(徐丽云). A cytochemical technique for demonstration of lipids, polysaccharides and protein bodies in thick resin sections. Aata Bot Sin (植物学报), 1990, 32(11): 841–846 (in Chinese with English abstract)

[17]Dang J M, Copeland L. Studies of the fracture surface of rice grains using environmental scanning electron microscopy. J Sci Food Agric, 2004, 84: 707–713

[18]Buttrose M S. Submicroscopic development and structure of starch granules in cereal endosperms. J Ultrastructure Res, 1960, 4: 231–257

[19]Fu H-L(付洪兰). Practical Electronic Microscope Technology (实用电子显微镜技术). Beijing: Higher Education Press, 2004. pp 12–13 (in Chinese)

[20]Song J-D(宋今丹). The membrane structure of cultured cell by potassium permanganate fixed. J Chin Electron Microscopy Soc (电子显微学报), 1990, 9(3): 1 (in Chinese)

[21]Loussert C, Popineau Y, Mangavel C. Protein bodies ontogeny and localization of prolamin components in the developing endosperm of wheat caryopses. J Cereal Sci, 2008, 47: 445–456

[22]Hughes C E. The Developing Endosperm of Triticum aestivum (L.): an Ultrastructural and Morphometric Study. PhD Dissertation of University of Nottingham, 1976

[23]Wei C-X(韦存虚), Zhang J(张军), Zhou W-D(周卫东), Chen Y-F(陈义芳), Xu R-G(许如根). Accumulation of storage protein and formation of protein body during barley endosperm development. Acta Agron Sin (作物学报), 2009, 35(2): 334–340 (in Chinese with English abstract)

[24]Luo Y-Y(罗玉英). Ultrastructural study on the division of amyloplast in pollen grain of Polygouatum samizui. J Chive Electr Microse Soc (电子显微学报), 1995, 3: 166–169 (in Chinese with English abstract)
[1] 胡文静, 李东升, 裔新, 张春梅, 张勇. 小麦穗部性状和株高的QTL定位及育种标记开发和验证[J]. 作物学报, 2022, 48(6): 1346-1356.
[2] 郭星宇, 刘朋召, 王瑞, 王小利, 李军. 旱地冬小麦产量、氮肥利用率及土壤氮素平衡对降水年型与施氮量的响应[J]. 作物学报, 2022, 48(5): 1262-1272.
[3] 付美玉, 熊宏春, 周春云, 郭会君, 谢永盾, 赵林姝, 古佳玉, 赵世荣, 丁玉萍, 徐延浩, 刘录祥. 小麦矮秆突变体je0098的遗传分析与其矮秆基因定位[J]. 作物学报, 2022, 48(3): 580-589.
[4] 冯健超, 许倍铭, 江薛丽, 胡海洲, 马英, 王晨阳, 王永华, 马冬云. 小麦籽粒不同层次酚类物质与抗氧化活性差异及氮肥调控效应[J]. 作物学报, 2022, 48(3): 704-715.
[5] 刘运景, 郑飞娜, 张秀, 初金鹏, 于海涛, 代兴龙, 贺明荣. 宽幅播种对强筋小麦籽粒产量、品质和氮素吸收利用的影响[J]. 作物学报, 2022, 48(3): 716-725.
[6] 马红勃, 刘东涛, 冯国华, 王静, 朱雪成, 张会云, 刘静, 刘立伟, 易媛. 黄淮麦区Fhb1基因的育种应用[J]. 作物学报, 2022, 48(3): 747-758.
[7] 王洋洋, 贺利, 任德超, 段剑钊, 胡新, 刘万代, 郭天财, 王永华, 冯伟. 基于主成分-聚类分析的不同水分冬小麦晚霜冻害评价[J]. 作物学报, 2022, 48(2): 448-462.
[8] 陈新宜, 宋宇航, 张孟寒, 李小艳, 李华, 汪月霞, 齐学礼. 干旱对不同品种小麦幼苗的生理生化胁迫以及外源5-氨基乙酰丙酸的缓解作用[J]. 作物学报, 2022, 48(2): 478-487.
[9] 徐龙龙, 殷文, 胡发龙, 范虹, 樊志龙, 赵财, 于爱忠, 柴强. 水氮减量对地膜玉米免耕轮作小麦主要光合生理参数的影响[J]. 作物学报, 2022, 48(2): 437-447.
[10] 马博闻, 李庆, 蔡剑, 周琴, 黄梅, 戴廷波, 王笑, 姜东. 花前渍水锻炼调控花后小麦耐渍性的生理机制研究[J]. 作物学报, 2022, 48(1): 151-164.
[11] 孟颖, 邢蕾蕾, 曹晓红, 郭光艳, 柴建芳, 秘彩莉. 小麦Ta4CL1基因的克隆及其在促进转基因拟南芥生长和木质素沉积中的功能[J]. 作物学报, 2022, 48(1): 63-75.
[12] 韦一昊, 于美琴, 张晓娇, 王露露, 张志勇, 马新明, 李会强, 王小纯. 小麦谷氨酰胺合成酶基因可变剪接分析[J]. 作物学报, 2022, 48(1): 40-47.
[13] 李玲红, 张哲, 陈永明, 尤明山, 倪中福, 邢界文. 普通小麦颖壳蜡质缺失突变体glossy1的转录组分析[J]. 作物学报, 2022, 48(1): 48-62.
[14] 罗江陶, 郑建敏, 蒲宗君, 范超兰, 刘登才, 郝明. 四倍体小麦与六倍体小麦杂种的染色体遗传特性[J]. 作物学报, 2021, 47(8): 1427-1436.
[15] 王艳朋, 凌磊, 张文睿, 王丹, 郭长虹. 小麦B-box基因家族全基因组鉴定与表达分析[J]. 作物学报, 2021, 47(8): 1437-1449.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!