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Scanning Electron Microscope (SEM) Imaging to Determine Inflorescence Initiation and Development in Olive
扫描电子显微镜(SEM)观测橄榄花序的起始和发育   

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Abstract

Here we present a protocol that describes how to image the structure of the olive axillary bud meristem with a scanning electron microscope (SEM) in order to characterize its identity and developmental stage. Briefly, the specimen is fixed with glutaraldehyde, saturated with ethanol, dried in a critical point dryer (CPD) system, dissected, coated with a conducting material and imaged with a scanning electron microscopy (SEM).

Keywords: SEM(SEM), Scanning Electron Microscopy(扫描电子显微镜), Olive(橄榄), Flowering(开花), Inflorescence(花序), Meristem(分生组织), Bud(芽)

Background

The exact timing of flowering induction and inflorescence initiation in olive (Olea europaea L.) is in controversy (Haberman et al., 2017). In olive, inflorescences emerge from lateral buds at the end of winter and flower in the spring. We have developed a protocol to better characterize the timing of inflorescence initiation in olive by imaging the meristem in the olive bud with a SEM at different times during the year. In these SEM images the meristem structure can be identified unambiguously, and the definition level of the meristem can be much higher than images of bud meristem sections presented in previous studies.

Materials and Reagents

  1. Scalpel blade No. 11 (Sigma-Aldrich, catalog number: S2771 )
  2. Double-sided adhesive tape
  3. Glass scintillation vials with screw caps, volume 20 ml (Sigma-Aldrich, catalog number: Z190535 )
  4. Pipette (BRAND, catalog number: 747760 ) or a similar instrument
  5. Gold annular target for the sputter coater (Agar scientific, catalog number: AGB7370 )
  6. Olive (Olea europaea L.) buds
  7. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S3264 )
  8. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S3139 )
  9. 25% glutaraldehyde (Sigma-Aldrich, catalog number: G5882 )
  10. Ethanol absolute (Sigma-Aldrich, catalog number: 24102 )
  11. Optional: Technical grade ethanol (Sigma-Aldrich, catalog number: V0T0042 )
  12. 0.1 M phosphate buffer pH 7.2 (sodium phosphate buffer; see Recipes)
  13. 5% glutaraldehyde solution (in 0.1 M phosphate buffer pH 7.2; see Recipes)

Equipment

  1. Scalpel handle No. 3 (Sigma-Aldrich, catalog number: S2896 )
  2. Tweezers style #5 (Sigma-Aldrich, catalog number: T4537 )
  3. Critical point dryer system (BAL-TEC, model: CPD 030 )
  4. Stereo-microscope (Olympus, model: SZX12 )
  5. Sputter coater (E510 scanning electron microscope coating unit) (Polaron Instruments, model: E510 )
  6. Scanning electron microscope (JEOL, model: JSM-5410 LV )

Procedure

  1. Using a scalpel, separate the axillary buds from the shoot. Leave a portion from the stem connected to the bud for gripping the bud with tweezers. Cut the opposite side to the bud of the stem piece vertically so that you can place the sample with the bud facing up (see Figure 1B).
  2. Add 5-10 ml (enough to cover the buds) of the 5% glutaraldehyde solution (see Recipes) to a scintillation vial. Immerse the buds in the glutaraldehyde solution for 24 h at room temperature (fixative solution [Sabatini et al., 1963]; see Figure 1C).
  3. Use a pipette to remove the glutaraldehyde solution from the vial.
  4. Wash the buds by adding phosphate buffer (see Recipes) to the buds. After 10 min, remove the buffer. Repeat this wash step 5 times.
  5. Add 25% ethanol solution, suspend for 1 h and remove the solution.
  6. Add 50% ethanol solution, suspend for 1 h and remove the solution.
  7. Add 75% ethanol solution, suspend for 1 h (break point) and remove the solution.
    Note: If desired the fixing procedure can be suspended at this step (75% ethanol solution). Buds can be stored in a 75% ethanol solution at 4 °C over-night or up to several weeks.
  8. Add 95% ethanol solution, suspend for 1 h and remove the solution.
  9. Add 100% ethanol solution, suspend for 1 h and remove the solution.
  10. Add 100% ethanol solution.
  11. Dry the buds in a Critical point dryer (CPD) instrument according to manufacturer's instructions (the drying procedure should take about 2 h).
  12. Use tweezers to hold the dry bud by the stem piece. Using a stereo microscope, carefully remove the leaf primordia from the bud with a scalpel or tweezers (Style #5). Normally, after 4-5 pairs of leaf primordia are removed, the meristem is exposed.
    Note: This is a crucial step, take the time to gently remove the leaf primordia without damaging the meristem.
  13. Bond a piece of double-sided adhesive tape to a metal stub (the stub goes into the SEM; see Figures 1E and1F). Use tweezers to bind the stem piece of the dissected bud to the metal stub, with the exposed meristem facing upwards (see Figure 1G).
  14. Coat the specimen with gold (Au) or gold/palladium (Au/Pd) in a sputter coater instrument according to manufacturer's instructions (see Figures 1H and 1I).
    *Coating parameters we used: vacuum 0.02 Mbar, sputtering voltage 2.4 KV, current 20 mA, coating time 150 sec.


    Figure 1. Preparation of the specimen for imaging. A. Picture of the olive shoot; B. Separated bud samples with attached stem pieces; C. Bud samples immersed in the fixative solution in a scintillation vial; D. Dried bud samples after drying in the CPD instrument; E. Metal stub used for the SEM imaging; F. Metal stub with a piece of double-sided adhesive tape; G. Dissected dried bud sample mounted on a metal stub; H. Dissected dried bud sample coated with gold; I. Dissected dried bud sample coated with gold-palladium.

  15. Place the stub in the SEM and operate the SEM according to manufacturer's instructions to produce images of the meristem (see Figure 2).


    Figure 2. SEM image of a whole specimen. Taken under high vacuum condition at accelerating voltage of 20 kV. Bud was sampled on 23 February 2011. Scale bar = 1 mm.

Data analysis

Crop the images to zoom and center the area of interest in the image, make sure you retain an accurate scale bar. Create a figure from the images you produced, using the software of your choice (see examples for figures created with PowerPoint [Microsoft] in Figures 3 and 4).


Figure 3. Development of inflorescence in Barnea olive. SEM images showing olive inflorescence at different stages of development. Images were produced from lateral buds sampled on 9 February 2011 (A), 16 February 2011 (B), 23 February 2011 (C-E) and 7 February 2010 (F). A-C. Initial stage of inflorescence development. The apical meristem (Am) do not initiate new leaf primordia, enlarge and bulge (‘dome’) subsequently becoming a terminal flower meristem. Lateral flower meristems (Lm) surrounding the Am begin bulging. Leaf primordia develop into bracts (Br). D-E. Subsequent stages of inflorescence meristem development. Apical flower meristem (Am) develops into apical flower (Af). Lateral flower meristems (Lm) differentiate into lateral flowers (Lf). F. Inflorescence before anthesis. Scale bar = 0.25 mm.


Figure 4. Development of Olive flowers. SEM images showing olive flowers at different stages of development. Images proudest from Barnea olive lateral buds sampled on 23 February 2011. A. Initial bulging (‘doming’) and enlargement of the meristem; B. The periphery of the meristem differentiates into the calyx (Ca; sepals); C-E. Subsequently, the central floral meristem develops into the corolla (Co) and two stamens (St), the pistil is not seen. F. Flower bud before anthesis. Scale bar = 0.1 mm.

Notes

  1. The diluted ethanol solutions can be prepared from cheaper technical grade ethanol (See Materials and Reagents #11).
  2. This protocol can be implemented in other plant species as demonstrated in apple (Malus domestica Borkh.; Haberman et al., 2016) and passion fruit (Passiflora edulis Sims; Nave et al., 2010).

Recipes

  1. 0.1 M sodium phosphate buffer pH 7.2 (1 L)
    1. First produce 1 M stock solutions of Na2HPO4 (dibasic) and NaH2PO4 (monobasic).
      Dissolve 141.96 g of Na2HPO4 in distilled H2O and complete the final volume to 1 L. Do the same for 119.98 g of NaH2PO4.
    2. Mix 68.4 ml of 1 M Na2HPO4 with 31.6 ml of 1 M NaH2PO4 and dilute to a final volume of 1 L. You get 1 L of 0.1 M sodium phosphate buffer pH 7.2.
    Note: If you are using a hydrated sodium phosphate, make sure you adjust the amount of sodium phosphate for the production of the 1 M solutions.
  2. 5% glutaraldehyde solution in phosphate buffer
    Dilute your glutaraldehyde solution according to its initial dilution in your 0.1 M phosphate buffer (Recipe 1). If you are using a stock solution of 25% glutaraldehyde, for a final volume of 100 ml, mix 20 ml of the 25% glutaraldehyde solution with 80 ml of the phosphate buffer.
    Note: Glutaraldehyde is toxic and a strong irritant, always wear gloves, work in a chemical hood, dispose of it properly.

Acknowledgments

The fixation method in the protocol was composed according to the book, Fixation for electron microscopy (Hayat, 1981).

References

  1. Haberman, A., Ackerman, M., Crane, O., Kelner, J. J., Costes, E. and Samach, A. (2016). Different flowering response to various fruit loads in apple cultivars correlates with degree of transcript reaccumulation of a TFL1-encoding gene. Plant J 87(2): 161-173.
  2. Haberman, A., Bakhshian, O., Cerezo-Medina, S., Paltiel, J., Adler, C., Ben-Ari, G., Mercado, J. A., Pliego-Alfaro, F., Lavee, S. and Samach, A. (2017). A possible role for flowering locus T-encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction. Plant Cell Environ.
  3. Hayat, M. A. (1981). Fixation for electron microscopy. Academic Press.
  4. Nave, N., Katz, E., Chayut, N., Gazit, S. and Samach, A. (2010). Flower development in the passion fruit Passiflora edulis requires a photoperiod-induced systemic graft-transmissible signal. Plant Cell Environ 33(12): 2065-2083.
  5. Sabatini, D. D., Bensch, K. and Barrnett, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17: 19-58.

简介

在这里,我们提出一个协议,描述如何使用扫描电子显微镜(SEM)图像橄榄腋芽分生组织的结构,以表征其身份和发育阶段。 简言之,将样品用戊二醛固定,用乙醇饱和,在临界点干燥器(CPD)系统中干燥,解剖,用导电材料涂覆并用扫描电子显微镜(SEM)成像。
【背景】橄榄(Olea europaea)中开花诱导和花序开始的确切时间是争议的(Haberman等人,2017)。 在橄榄中,花序在冬季结束时从侧芽出现,春季开花。 我们制定了一个协议,通过在一年中的不同时间用SEM对橄榄芽中的分生组织进行成像,更好地表征橄榄花序开始的时间。 在这些SEM图像中,可以明确地确定分生组织结构,并且分生组织的定义水平可以高于以前研究中呈现的芽分生组织部分的图像。

关键字:SEM, 扫描电子显微镜, 橄榄, 开花, 花序, 分生组织, 芽

材料和试剂

  1. Scalpel刀片11号(Sigma-Aldrich,目录号:S2771)
  2. 双面胶带
  3. 带螺旋盖的玻璃闪烁瓶,体积为20ml(Sigma-Aldrich,目录号:Z190535)
  4. 移液器(BRAND,目录号:747760)或类似仪器
  5. 溅射涂层机的金环形靶(Agar科学,目录号:AGB7370)
  6. 橄榄(Olea europaea L.)芽
  7. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S3264)
  8. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S3139)
  9. 25%戊二醛(Sigma-Aldrich,目录号:G5882)
  10. 乙醇绝对(Sigma-Aldrich,目录号:24102)
  11. 可选:技术级乙醇(Sigma-Aldrich,目录号:V0T0042)
  12. 0.1M磷酸盐缓冲液pH 7.2(磷酸钠缓冲液;参见食谱)
  13. 5%戊二醛溶液(0.1M磷酸缓冲液pH7.2;参见食谱)

设备

  1. Scalpel手柄3号(Sigma-Aldrich,目录号:S2896)
  2. 镊子式#5(Sigma-Aldrich,目录号:T4537)
  3. 临界点干燥器系统(BAL-TEC,型号:CPD 030)
  4. 立体显微镜(Olympus,型号:SZX12)
  5. 溅射涂布机(E510扫描电子显微镜涂布单元)(Polaron Instruments,型号:E510)
  6. 扫描电子显微镜(JEOL,型号:JSM-5410 LV)

程序

  1. 使用手术刀,将腋芽与射击分开。留下一个与茎连接的部分,用镊子夹住芽。垂直切割茎杆的相对侧,使您可以将样品放在芽边朝上(参见图1B)。
  2. 将5-10毫升(足以覆盖芽)的5%戊二醛溶液(见食谱)加入到闪烁瓶中。在室温下将芽浸入戊二醛溶液中24小时(固定溶液[Sabatini等人,1963];参见图1C)。
  3. 使用移液管从小瓶中除去戊二醛溶液。
  4. 通过向芽中加入磷酸盐缓冲液(参见食谱)清洗芽。 10分钟后,取出缓冲液。重复洗涤步骤5次。
  5. 加入25%乙醇溶液,悬浮1 h,取出溶液
  6. 加入50%乙醇溶液,悬浮1 h,取出溶液
  7. 加入75%乙醇溶液,悬浮1小时(断点)并除去溶液 注意:如果需要,可以在该步骤(75%乙醇溶液)下暂停定影程序。芽可以在4℃过夜或几周内在75%乙醇溶液中储存。
  8. 加入95%乙醇溶液,悬浮1 h,取出溶液
  9. 加入100%乙醇溶液,悬浮1 h,取出溶液
  10. 加入100%乙醇溶液
  11. 根据制造商的说明,在临界点干燥器(CPD)仪器中干燥芽(干燥程序大约需要2小时)。
  12. 用镊子将茎干放在干茎上。使用立体显微镜,用手术刀或镊子小心地从芽中除去叶原基(Style#5)。通常,除去4-5对叶原基后,分生组织暴露。
    注意:这是一个关键的步骤,花时间轻轻地除去叶原基而不损害分生组织。
  13. 将一块双面胶带粘合到金属短截线上(短截线进入SEM;参见图1E和1F)。使用镊子将解剖的芽的茎片与金属菌根结合,暴露的分生组织面朝上(参见图1G)。
  14. 在溅射涂布仪器中,按照制造商的说明书,用金(Au)或金/钯(Au / Pd)涂覆样品(见图1H和1I)。
    我们使用的涂层参数:真空0.02 Mbar,溅射电压2.4 KV,电流20 mA,涂布时间150秒。


    图1.成像样本的准备。 A.橄榄枝的图片B.分离的芽样品,附有茎片; C.浸泡在闪烁瓶中的固定溶液中的芽样品; D.在CPD仪器中干燥后的干芽样品; E.用于SEM成像的金属短棒; F.带有一块双面胶带的金属短棒; G.安装在金属桩上的解剖干芽样品; H.用金覆盖的解剖干芽样品I.用金钯覆盖的解剖干芽样品。

  15. 将存根放置在扫描电镜中,并根据制造商的说明操作SEM,以生成分生组织的图像(见图2)。


    图2.整个样品的SEM图像在高真空条件下在20 kV的加速电压下进行。 Bud于2011年2月23日采样。比例尺= 1 mm。

数据分析

裁剪图像以缩小图像中感兴趣区域的中心位置,确保保留精确的比例尺。使用您选择的软件从您生成的图像创建一个数字(请参见图3和图4中使用PowerPoint [Microsoft]创建的图形示例)。


图3.在巴尼亚橄榄中开花花序 。在不同发育阶段显示橄榄花序的SEM图像。 2011年2月9日(A),2011年2月16日(B),2011年2月23日(C-E)和2010年2月7日(F))采集的侧芽产生图像。 A-C。花序发育的初期阶段顶端分生组织(Am)不会启动新的叶原基,扩大和膨胀(“圆顶”),随后成为末端花分生组织。围绕Am的侧花分生组织(Lm)开始膨胀。叶原基发育成苞片(Br)。 D-E。花序分生组织发育的后续阶段。顶端花分生组织(Am)发育成根尖花(Af)。侧花分生组织(Lm)分化为侧花(Lf)。 F.开花前花序。比例尺= 0.25 mm。


图4.橄榄花的开发。在不同发育阶段显示橄榄花的SEM图像。从2011年2月23日采集的巴尼亚橄榄侧芽最骄傲的图像。A.初始膨胀(“垄断”)和分生组织的扩大;分生组织的外围区分成花萼(Ca;萼片); C-即随后,中心花分生组织发育成花冠(Co)和雄蕊(St),雌蕊未见。 F.花芽在开花前。刻度棒= 0.1 mm。

笔记

  1. 稀释的乙醇溶液可以由廉价的工业级乙醇制备(参见材料和试剂#11)。
  2. 如苹果(Malus domestica,Borkh; Haberman等人,2016)和西番莲(西番莲)所示,该方案可以在其它植物物种中实现, Sims; Nave等人,2010)。

食谱

  1. 0.1M磷酸钠缓冲液pH 7.2(1L)
    1. 首先生产Na 2 HPO 4(二元)和NaH 2 PO 4(一元)的1M储备溶液, 。
      在蒸馏的H 2 O 3中溶解141.96g的Na 2 HPO 4,并将最终体积完全溶解至1L。对119.98g NaH 2 PO 4
    2. 将68.4ml 1M Na 2 HPO 4与31.6ml 1M NaH 2 PO 4混合并稀释最终体积为1L。得到1L 0.1M磷酸钠缓冲液pH7.2。
    注意:如果您正在使用水合磷酸钠,请确保调整磷酸钠的用量以生产1M溶液。
  2. 磷酸盐缓冲液中的5%戊二醛溶液
    根据其0.1M磷酸盐缓冲液(配方1)中的初始稀释度稀释戊二醛溶液。如果您使用25%戊二醛的储备溶液,最终体积为100ml,将20 ml 25%戊二醛溶液与80 ml磷酸盐缓冲液混合。
    注意:戊二醛是有毒的和强烈的刺激性,总是戴着手套,在化学防护罩上工作,妥善处理。

致谢

方案中的固定方法是根据“电子显微镜固定”(Hayat,1981)所着的。

参考

  1. Haberman,A.,Ackerman,M.,Crane,O.,Kelner,JJ,Costes,E.and Samach,A。(2016)。  对苹果品种的各种果实负荷的不同开花反应与TFL1编码基因的转录本再累积程度相关。植物J 87(2):161-173。
  2. Haberman,A.,Bakhshian,O.,Cerezo-Medina,S.,Paltiel,J.,Adler,C.,Ben-Ari,G.,Mercado,JA,Pliego-Alfaro,F.,Lavee,S和Samach,A.(2017)。 开花基因座T编码基因在解释影响橄榄的环境和内部提示中的可能作用( Olea europaea L.)花诱导。植物细胞环境。
  3. Hayat,M.A。(1981)。 电子显微镜的固定。 a> 学术出版社。
  4. Nave,N.,Katz,E.,Chayut,N.,Gazit,S。和Samach,A。(2010)。 番茄果实中的花卉发育西番莲需要光周期诱发的系统性移植物传播信号。植物细胞环境33(12):2065-2083。
  5. Sabatini,D.D.,Bensch,K。和Barrnett,R.J。(1963)。 细胞化学和电子显微镜。通过醛固定保存细胞超微结构和酶活性。细胞生物学17:19-58。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Haberman, A., Zelinger, E. and Samach, A. (2017). Scanning Electron Microscope (SEM) Imaging to Determine Inflorescence Initiation and Development in Olive. Bio-protocol 7(19): e2575. DOI: 10.21769/BioProtoc.2575.
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