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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.

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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

植物科学 > 植物发育生物学 > 形态建成
作者: Min-Jung Kim
Min-Jung KimAffiliation: Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea
Bio-protocol author page: a2952
 and Jungmook Kim
Jungmook KimAffiliation: Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, Korea
For correspondence: jungmkim@jnu.ac.kr
Bio-protocol author page: a2953
Vol 6, Iss 5, 3/5/2016, 1747 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1752

[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)

[Abstract]

Materials and Reagents

  1. 0.2 μm sterile syringe filter (Corning, catalog number: 431219 )
  2. Microscope slide and coverslip
  3. Needle (30 gauge)
  4. 3 mm Whatman filter paper (GE Healthcare, Dharmacon, catalog number: 1006090 )
    Note: Currently, it is “Sigma-Aldrich, catalog number: 1006090”.
  5. Mineral oil (Sigma-Aldrich, catalog number: M5904 )
  6. Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
  7. 25% glutaraldehyde (Junsei, catalog number: 273721250 )
  8. Sodium carcodylate (Ted Pella, catalog number: 18851 )
  9. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5379 )
  10. Ethanol (Merck Millipore Corporation, Calbiochem®, catalog number: 1.00983.1011 )
  11. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 30620 )
  12. Osmium tetroxide (OsO4) (Ted Pella, catalog number: 96049 )
  13. Spurr resin (Ted Pella, catalog number: 183004221 )
  14. Double end mold (Ted Pella, catalog number: 10590 )
  15. Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
  16. EDTA (BioShop, catalog number: EDT001.500 )
  17. Triton X-100 (Sigma-Aldrich, catalog number: T8532 )
    Note: This product has been discontinued.
  18. 4’, 6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: 32670 )
  19. Acetic acid (Merck Millipore Corporation, Calbiochem®, catalog number: 1.00063.1011 )
  20. 1/2 strength Karnovsky’s fixative solution (see Recipes)
  21. Toluidine blue O solution (see Recipes)
  22. Fixative solution (see Recipes)
  23. DAPI solution for developing pollen (see Recipes)
  24. DAPI solution for mature pollen grain (see Recipes)

Equipment

  1. Ultramicrotome (Boeckeler Instruments, model: MT990 motorized precision microtome )
  2. Hot plate
  3. Water bath
  4. Incubator
  5. Fume hood
  6. Light and fluorescence microscope (Leica Microsystems, model: DM2500 )
  7. Vacuum pump and vacuum jar
  8. Glass pipette

Procedure

  1. Semi-thin sectioning of Arabidopsis floral buds for light microscopy
    1. Embedding of Arabidopsis floral buds
      1. Collect the whole inflorescence in a 2 ml tube.
      2. Add 1 ml of 1/2 strength Karnovsky’s fixative solution (Karnovsky, 1965) into the tube and cover it with 3MM Whatman filter paper beneath the surface of the solution.
      3. Incubate the tube in a vacuum jar under 400 mmHg for 30 min.
      4. Incubate the tube at 4 °C for 4 h to overnight.
      5. Wash the samples three times with 1 ml of 0.1 M sodium carcodylate (pH 7.4) for every 5 min on the rocker with gentle agitation.
      6. Carry out a post-fixation treatment for overnight at 4 °C with 1 ml of 1% osmium tetroxide using a glass pipette.
        Note: If you want to do immuno-staining, do not perform post-fixation with osmium tetroxide. This can inhibit the binding of antibody to antigen.
      7. Dehydrate the sample with 10, 20, 30, 40, 50, 60, 70, 80, and 90% ethanol for 30 min for each treatment sequentially and with 100% ethanol for overnight.
      8. Prepare medium spurr resin according to manufacturer’s protocol in the hood and then incubate the spurr resin in a vacuum jar for 30 min to eliminate air bubbles.
      9. Infiltrate the spurr resin prepared at step A1-h to the dehydrated sample at step A1-g and remove the residual ethanol of the sample by the following procedures.

        Step
        100 % Ethanol :  Pure resin
        Time (h)

        3                     : 1
        4

        1                     : 1
        4

        1                     : 3
        4

        −                    : 4
        Overnight

      10. Fill the bottom of the double end mold tray with spurr resin, and then incubate it at 58 °C for overnight to make drop panel.
      11. Immerse the sample in the mold tray with spurr resin, and then incubate it at 58 °C for 3 days.
      12. Take out the block from the mold tray and keep the sample at room temperature.

    2. Semi-thin sectioning of the sample embedded in the spurr resin.
      1. Trim the resin block ends with a razor blade to make the shape of trapezoid centering around a specimen (Figure 1).


        Figure 1. Resin blocks embedded specimens. A. Arabidopsis inflorescences embedded in solidified resin. B. Trimmed resin by razer blade.

      2. Fix the resin block to the head of the microtome.
      3. Cut 1-10 μm transverse sections of the resin-embedded specimens using a MT-990 motorized precision microtome with the glass knives. Four μm transverse sections were made in the case of Borg et al. (2009).
      4. Collect the floating samples in water on to a glass slide with a platinum wire and put the glass slide with the samples on a hot plate at 50 °C for 5 min.
      5. Drop 100 μl of the toluidine blue solution on the surface of the sections by using a syringe, and then dry it on the hot plate at 50 °C for 5 min.
      6. Wash the samples with sprinkled water using a syringe, and then dry the excess moistures on the slide put on the hot plate at 50 °C.
      7. Drop 20 μl of the mineral oil and cover it with a coverslip, then seal the edges of a coverslip with nail varnish (Figure 2).


        Figure 2. Sections stained with toluidine blue solution on a slide with a coverslip

  2. Fluorescence microscopic analysis of developing microspore and mature pollen with 4', 6-diamidino-2-phenylindole (DAPI)
    1. Preparation of mature pollen grains with DAPI staining.
      1. Fix the whole inflorescence with opened flowers in a fixative solution (Oh et al., 2010) for 5 min.
        Note: If you want to detect fluorescent proteins or GUS staining, skip this procedure.
      2. Collect 3-4 opened flowers at flower stage 13-14 from Arabidopsis (Sanders et al., 1999) in a 1.5 ml tube containing 200 μl of DAPI solution (Park et al., 1998).
      3. Vortex the tube briefly at maximum speed for 10 sec to separate pollen grains from the dehiscent anther wall.
      4. Incubate the tube in the dark at room temperature for 30 min.
      5. Transfer the supernatant to a new 1.5 ml tube with a 200 μl pipette man.
        Note: Do not centrifuge. Do not transfer any other floral organs except pollen grains in the supernatant.
      6. Concentrate the pollen grains by centrifuging at 13,000 rpm for 30 sec at room temperature, and then discard the supernatant.
      7. Resuspend the pollen pellets in 5-10 μl of DAPI solution.
      8. Mount this sample on the microscope slide under the coverslip.
    2. Preparation of developing microspores from anthers with DAPI staining
      1. Fix the whole inflorescence with opened flowers in a fixative solution (Oh et al., 2010) for 5 min.
        Note: If you want to detect fluorescent proteins or GUS staining, skip this procedure.
      2. Dissect the single anther from inflorescence on a microscope slide with a needle while adding 4-5 μl of 0.3 M mannitol or DAPI solution (Park et al., 1998).
      3. Collect free microspores on a slide in a 1.5 ml tube and incubate them in the dark for 30 min.
      4. Mount this sample on the microscope slide under the coverslip.
    3. Acquire images with a camera affixed to a Leica DM2500 microscope under the epifluorescence channel (15% of 405 nm/430-470 nm for DAPI, 470 nm/530 nm for GFP and 561 nm/570-610 nm for DsRed) (Figure 3) (Kim et al., 2015).


      Figure 3. Image of transverse sections of Arabidopsis anther stained with toluidine blue solution by light microscopy according to the protocol

Recipes

  1. 1/2 strength Karnovsky’s fixative solution
    2% paraformaldehyde
    2.5% glutaraldehyde
    0.1 M sodium cacodylate (pH 7.4)
    Prepare this solution freshly prior to use
  2. Toluidin blue O solution
    Dissolve powder in 1 or 2 ml of water to make final 0.05% solution, and filter this solution through a 0.2 μm sterile syringe filter. Store this solution at 4 °C protected from light.
  3. Fixative solution
    Ethanol: acetic acid=3:1
    Prepare this solution freshly prior to use.
  4. DAPI solution for developing pollen
    0.1 M sodium phosphate (pH 7.0)
    1 mM EDTA (pH 8.0)
    0.1% Triton X-100
    1 μg/ml DAPI
    Store this solution at 4 °C protected from light
  5. DAPI solution for mature pollen grain
    0.1 M sodium phosphate (pH 7.0)
    1 mM EDTA, pH 8.0
    0.1% Triton X-100
    0.4 μg/ml DAPI
    Store this solution at 4 °C protected from light

Acknowledgements

This study was supported by grants from the Next-Generation BioGreen 21 Program (PJ01104701), Rural Development Administration, Republic of Korea and Basic Science Research Programs through the National Research Foundation of Korea funded by the Ministry of Education (no. 2013R1A1A2062335). This protocol was based on Kim et al. (2015).

References

  1. Berger, F. and Twell, D. (2011). Germline specification and function in plants. Annu Rev Plant Biol 62: 461-484.
  2. Borg, M., Brownfield, L. and Twell, D. (2009). Male gametophyte development: a molecular perspective. J Exp Bot 60(5): 1465-1478.
  3. Karnovsky, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 27(2): 137A-138A.
  4. Kim, M. J., Kim, M., Lee, M. R., Park, S. K. and Kim, J. (2015). LATERAL ORGAN BOUNDARIES DOMAIN (LBD)10 interacts with SIDECAR POLLEN/LBD27 to control pollen development in Arabidopsis. Plant J 81(5): 794-809.
  5. McCormick, S. (2004). Control of male gametophyte development. Plant Cell 16 Suppl: S142-153.
  6. Oh, S. A., Park, K. S., Twell, D. and Park, S. K. (2010). The SIDECAR POLLEN gene encodes a microspore-specific LOB/AS2 domain protein required for the correct timing and orientation of asymmetric cell division. Plant J 64(5): 839-850.
  7. Park, S. K., Howden, R. and Twell, D. (1998). The Arabidopsis thaliana gametophytic mutation gemini pollen1 disrupts microspore polarity, division asymmetry and pollen cell fate. Development 125(19): 3789-3799.
  8. Sanders, P. M., Bui, A. Q., Weterings, K., McIntire, K. N., Hsu, Y. C., Lee, P. Y., Truong, M. T., Beals, T. P. and Goldberg, R. B. (1999). Another developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod 11(6): 297-322.

材料和试剂

  1. 0.2μm无菌注射器过滤器(Corning,目录号:431219)
  2. 显微镜载玻片和盖玻片
  3. 针(30号)
  4. 3mm Whatman滤纸(GE Healthcare,Dharmacon,目录号:1006090)
    注意:目前,它是"Sigma-Aldrich,目录号:1006090"。
  5. 矿物油(Sigma-Aldrich,目录号:M5904)
  6. 多聚甲醛(Sigma-Aldrich,目录号:F8775)
  7. 25%戊二醛(Junsei,目录号:273721250)
  8. sodium carcodylate(Ted Pella,目录号:18851)
  9. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5379)
  10. 乙醇(Merck Millipore Corporation,Calbiochem ,目录号:1.00983.1011)
  11. 氢氧化钠(NaOH)(Sigma-Aldrich,目录号:30620)
  12. 四氧化锇(OsO 4)(Ted Pella,目录号:96049)
  13. Spurr树脂(Ted Pella,目录号:183004221)
  14. 双端模具(Ted Pella,目录号:10590)
  15. 甲苯胺蓝O(Sigma-Aldrich,目录号:T3260)
  16. EDTA(BioShop,目录号:EDT001.500)
  17. Triton X-100(Sigma-Aldrich,目录号:T8532) 注意:此产品已停产。
  18. 4',6-二脒基-2-苯基吲哚(DAPI)(Sigma-Aldrich,目录号:32670)
  19. 乙酸(Merck Millipore Corporation,Calbiochem ,目录号:1.00063.1011)
  20. 1/2强度的Karnovsky的固定溶液(见配方)
  21. 甲苯胺蓝O溶液(见配方)
  22. 固定解决方案(参见配方)
  23. DAPI解决方案用于开发花粉(参见配方)
  24. 用于成熟花粉粒的DAPI溶液(参见配方)

设备

  1. 超微切片机(Boeckeler Instruments,型号:MT990机动精密切片机)
  2. 热板
  3. 水浴
  4. 孵化器
  5. 通风橱
  6. 光和荧光显微镜(Leica Microsystems,型号:DM2500)
  7. 真空泵和真空瓶
  8. 玻璃吸管

程序

  1. 用于光学显微镜的拟南芥花蕾的半薄切片
    1. 嵌入拟南芥花蕾
      1. 在2ml管中收集整个花序
      2. 加入1ml 1/2强度的Karnovsky's固定溶液(Karnovsky, 1965)放入管中并用3MM Whatman滤纸覆盖 溶液的表面。
      3. 将管在真空瓶中在400mmHg下孵育30分钟
      4. 将管在4℃孵育4小时至过夜
      5. 用1ml的0.1M碳酸氢钠溶液洗涤样品三次 (pH7.4),在摇动器上每5分钟轻轻搅拌
      6. 使用玻璃吸管在4℃下用1ml的1%四氧化锇进行后固定处理过夜。
        注意:如果你想做免疫染色,不要执行后固定 与四氧化锇。这可以抑制抗体的结合 抗原。
      7. 将样品用10,20,30,40,50,60,70, 80和90%乙醇,每次处理连续30分钟 100%乙醇过夜。
      8. 制备中等大小的树脂 根据制造商的协议在罩,然后孵育spurr树脂 ?在真空罐中30分钟以消除气泡
      9. 浸润 将在步骤A1-h制备的球形树脂加入到步骤中的脱水样品 A1-g,并通过以下方法除去样品的残留乙醇 程序
        步骤
        100%乙醇:纯树脂
        时间(h)

        3                    :1
        4

        1                     :1
        4

        1                    :3
        4

        -                      :4
        过夜

      10. 用spurr树脂填充双端模具托盘的底部,然后在58℃下孵育过夜以制作下拉面板。
      11. 将样品浸入模具托盘中,用spurr树脂,然后在58℃下孵育3天。
      12. 从模具托盘取出块,并将样品保持在室温。

    2. 嵌入在spurr树脂中的样品的半薄切片
      1. 用刀片修剪树脂块的端部,使梯形以试样为中心(图1)。


        图1.树脂块嵌入的标本。 A.嵌入固化树脂中的拟南芥花序。 B.用剃刀修剪的树脂 刀片。

      2. 将树脂块固定在切片机的头部。
      3. 切割1-10微米横向切片的树脂嵌入标本 使用MT-990电动精密切片机与玻璃刀。四 ?在Borg等人的(2009)的情况下制造了5μm的横截面
      4. 收集浮动样品在水中与载玻片上 铂丝并将载玻片与样品放在热板上 ?50℃5分钟
      5. 滴下100微升的甲苯胺蓝溶液 表面的部分用注射器,然后在上面干燥 热板在50℃下5分钟
      6. 洗涤样品洒 水,使用注射器,然后干燥幻灯片上的多余的水分 在50℃下放在热板上
      7. 滴20μl的矿物油和 用盖玻片覆盖,然后用指甲密封盖玻片的边缘 清漆(图2)。


        图2.用盖玻片在载玻片上用甲苯胺蓝溶液染色的切片

  2. 用4',6-二脒基-2-苯基吲哚(DAPI)对发育中的小孢子和成熟花粉的荧光显微镜分析
    1. 用DAPI染色制备成熟花粉粒
      1. 用固定溶液(Oh em et al。,2010)打开鲜花将整个花序固定5分钟。
        注意:如果您想检测荧光蛋白或GUS染色,请跳过此步骤。
      2. 在包含200μlDAPI的1.5ml管中从拟南芥(Sanders等人,1999)在花阶段13-14收集3-4个开放的花 溶液(Park等人,1998)。
      3. 以最大速度短暂涡旋管10秒,以从开裂的花药壁分离花粉粒
      4. 在黑暗中在室温下孵育试管30分钟
      5. 转移上清液到一个新的1.5毫升管与200微升移液器人 注意:不要离心。除上清中的花粉外,不得转移任何其他花器官。
      6. 通过在13,000rpm离心30分钟浓缩花粉粒 秒,然后弃去上清液
      7. 将花粉颗粒悬浮于5-10μlDAPI溶液中
      8. 将这个样品放在显微镜载玻片上的盖玻片下。
    2. 用DAPI染色从花药制备发育中的小孢子
      1. 用开放的花在固定溶液中固定整个花序(Oh等人,2010),持续5分钟。
        注意:如果您要检测荧光蛋白或GUS染色,请跳过此步骤。
      2. 在显微镜载玻片上从花序解剖单花药 用针同时加入4-5μl0.3M甘露醇或DAPI溶液 (Park等人,1998)。
      3. 在载玻片上收集1.5ml管中的游离小孢子,并在黑暗中孵育30分钟
      4. 将这个样品放在显微镜载玻片上的盖玻片下。
    3. 使用固定在Leica DM2500显微镜上的相机获取图像 在epifluorescence通道(15%的405nm/430-470nm的DAPI, 对于GFP是470nm/530nm,对于DsRed是561nm/570-610nm)(图3)(Kim等人 2015)。


      图3.拟南芥花药的横切面图像 根据光学显微镜用甲苯胺蓝溶液染色 协议

食谱

  1. 1/2强度的Karnovsky固定剂溶液
    2%多聚甲醛
    2.5%戊二醛
    0.1M二甲胂酸钠(pH7.4) 使用前请立即准备此溶液
  2. 甲苯蓝溶液
    将粉末溶解在1或2ml水中以制备最终的0.05%溶液,并将该溶液通过0.2μm无菌注射器过滤器过滤。将此溶液在4℃避光保存。
  3. 固定解决方案
    乙醇:乙酸= 3:1 在使用前请立即准备此溶液。
  4. DAPI解决方案开发花粉
    0.1M磷酸钠(pH7.0) 1mM EDTA(pH8.0) 0.1%Triton X-100 1μg/ml DAPI
    将此溶液储存在4°C避光保存
  5. 成熟花粉粒DAPI解决方案 0.1M磷酸钠(pH7.0) 1mM EDTA,pH8.0 0.1%Triton X-100 0.4μg/ml DAPI
    将此溶液储存在4°C避光保存

致谢

这项研究得到了由教育部资助的韩国国家研究基金会(no。2013R1A1A2062335)的下一代生物绿色21计划(PJ01104701),大韩民国农村发展管理局和基础科学研究计划的资助。此协议基于Kim al。(2015)。

参考文献

  1. Berger,F。和Twell,D。(2011)。 植物中的种系规范和功能 Annu Rev Plant Biol 62:461-484。
  2. Borg,M.,Brownfield,L。和Twell,D。(2009)。 男性配子体发育:分子视角 J Exp Bot 60(5):1465-1478。
  3. Karnovsky,M.J。(1965)。 高摩尔渗透压浓度的甲醛 - 戊二醛固定剂,用于电子显微镜。 J Cell Biol 27(2):137A-138A
  4. Kim,M.J.,Kim,M.,Lee,M.R.,Park,S.K.and Kim,J.(2015)。 LATERAL ORGAN BOUNDARIES DOMAIN(LBD)10 与 SIDECAR进行互动POLLEN/LBD27 以控制拟南芥中的花粉发育。 81植物J 81(5):794-809。
  5. McCormick,S。(2004)。 控制雄性配子体发育。 植物细胞 16 Suppl :S142-153。
  6. Oh,S.A.,Park,K.S.,Twell,D。和Park,S.K。(2010)。 SIDECAR POLLEN 基因编码小孢子特异性LOB/AS2结构域蛋白对于不对称细胞分裂的正确时序和方向是必需的。 64(5):839-850。
  7. Park,S.K.,Howden,R。和Twell,D。(1998)。 拟南芥配子体突变双子叶花粉 1 可破坏小孢子极性,分裂不对称和花粉细胞命运。 125(19):3789-3799。
  8. Sanders,P.M.,Bui,A.Q.,Weterings,K.,McIntire,K.N.,Hsu,Y.C.,Lee,P.Y.,Truong,M.T.,Beals,T.P.and Goldberg,R.B。(1999)。 拟南芥中的另一个发育缺陷雄性不育突变体 < em> Sex Plant Repro 11(6):297-322。
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How to cite this protocol: 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; Full Text



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