Semi-thin Sectioning, Light and Fluorescence Microscopy of Floral Bud to Study Microspore Development in Arabidopsis
通过花芽半薄切片、光学和荧光显微镜研究拟南芥中的花粉粒发育
发布: 2016年03月05日第6卷第5期 DOI: 10.21769/BioProtoc.1752 浏览次数: 9775
评审: Samik BhattacharyaVinay PanwarAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Mar 2015
Abstract
Pollen grains are male gametophytes produced within the pollen sacs of the anthers of the flower. Recent genetic studies have revealed several components involved in microspore development (Borg et al., 2009; Berger and Twell, 2011), and yet many components controlling microspore development remain to be identified. Semi-thin sectioning of anthers and light and fluorescence microscopy of floral bud (Kim et al., 2015) are the initial key experiments to characterize Arabidopsis mutants and transgenic plants for understanding the roles of new genetic components during microspore development. Herein, we describe a protocol for semi-thin sectioning of anthers and light and fluorescence microscopy of floral bud in Arabidopsis.
Keywords: Pollen (花粉)
Anther (花药)
Nuclei (核)
Bicellular (二)
Tricellular (三)
Materials and Reagents
- 0.2 μm sterile syringe filter (Corning, catalog number: 431219 )
- Microscope slide and coverslip
- Needle (30 gauge)
- 3 mm Whatman filter paper (GE Healthcare, Dharmacon, catalog number: 1006090 )
Note: Currently, it is “Sigma-Aldrich, catalog number: 1006090”. - Mineral oil (Sigma-Aldrich, catalog number: M5904 )
- Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
- 25% glutaraldehyde (Junsei, catalog number: 273721250 )
- Sodium carcodylate (Ted Pella, catalog number: 18851 )
- Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
- Ethanol (Merck Millipore Corporation, Calbiochem®, catalog number: 1.00983.1011 )
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 30620 )
- Osmium tetroxide (OsO4) (Ted Pella, catalog number: 96049 )
- Spurr resin (Ted Pella, catalog number: 183004221 )
- Double end mold (Ted Pella, catalog number: 10590 )
- Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
- EDTA (BioShop, catalog number: EDT001.500 )
- Triton X-100 (Sigma-Aldrich, catalog number: T8532 )
Note: This product has been discontinued. - 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: 32670 )
- Acetic acid (Merck Millipore Corporation, Calbiochem®, catalog number: 1.00063.1011 )
- 1/2 strength Karnovsky’s fixative solution (see Recipes)
- Toluidine blue O solution (see Recipes)
- Fixative solution (see Recipes)
- DAPI solution for developing pollen (see Recipes)
- DAPI solution for mature pollen grain (see Recipes)
Equipment
- Ultramicrotome (Boeckeler Instruments, model: MT990 motorized precision microtome )
- Hot plate
- Water bath
- Incubator
- Fume hood
- Light and fluorescence microscope (Leica Microsystems, model: DM2500 )
- Vacuum pump and vacuum jar
- Glass pipette
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kim, M. and Kim, J. (2016). Semi-thin Sectioning, Light and Fluorescence Microscopy of Floral Bud to Study Microspore Development in Arabidopsis. Bio-protocol 6(5): e1752. DOI: 10.21769/BioProtoc.1752.
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分类
植物科学 > 植物发育生物学 > 形态建成
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