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Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow for 3D X-ray μCT and associated xylem staining and visualisation techniques, in particular secondary xylem programmed-cell-death (PCD) delay during gelatinous fibre (g-fibre) development. Many of the staining techniques here are transferable to other woody species such as poplar and Eucalyptus.

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Sample Preparation for X-ray Micro-computed Tomography of Woody Plant Material and Associated Xylem Visualisation Techniques
木本植物材料样本的制备用于X射线显微计算机断层扫描和木质部可视化技术

植物科学 > 植物细胞生物学 > 组织分析
作者: Nicholas J. B. Brereton
Nicholas J. B. BreretonAffiliation: Institut de recherche en biologie végétale, Université de Montréal, Montreal, Canada
For correspondence: Nicholas.brereton@umontreal.ca
Bio-protocol author page: a2999
Vol 6, Iss 6, 3/20/2016, 1766 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1767

[Abstract] Variation in the tissue structure of short rotation coppice (SRC) willow is a principle factor driving differences in lignocellulosic sugar yield yet much of the physiology and development of this tissue is unknown. Traditional sectioning can be both difficult and destructive in woody tissue; however, technology such as three dimensional X-ray micro-computational tomography (μCT) scanning can be used to move biological researchers beyond traditional two dimensional assessment of tissue variation without having to destructively cut cells. This technology does not replace classical microscopic techniques but rather can be carefully integrated with traditional methods to improve exploration of the world of plant biology in three dimensions. The procedures below outline preparation of willow for 3D X-ray μCT and associated xylem staining and visualisation techniques, in particular secondary xylem programmed-cell-death (PCD) delay during gelatinous fibre (g-fibre) development. Many of the staining techniques here are transferable to other woody species such as poplar and Eucalyptus.
Keywords: Micro-computed Tomography(微型计算机断层扫描), Tension Wood(张力Wood), Cell wall staining(细胞壁染色), Xylem visualisation(木质部的可视化), Plant Histology(植物组织学)

[Abstract]

Materials and Reagents

  1. Glass slides
  2. Cover slips
  3. 48 well plates
  4. Razor blades
  5. Short rotation coppice (SRC) willow trees [cultivar Resolution-pedigree: {S. viminalis. x (S. viminalis. x S. schwerinii SW930812)] x [S. viminalis. x (S. viminalis. x S. schwerinii ‘Quest’)]}
  6. 37% Formaldehyde (36.5-38% in H2O) (Sigma-Aldrich, catalog number: F8775 )
  7. Acetic acid (≥ 99.7%) (Sigma-Aldrich, catalog number: 320099 )
  8. Ethanol
  9. FAA: 3.7% formaldehyde, 5% acetic acid and 47.5% ethanol
  10. Oasis® floral foam (OASIS Floral Products) (http://oasisfloralproducts.com/)
  11. Safranin-O solution (Sigma-Aldrich, catalog number: HT90432 )
  12. DPX Mountant for histology (Sigma-Aldrich, catalog number: 06522 )
    Note: This product is named “Slide mounting medium (p-Xylene-bis(N-pyridinium bromide))” at Sigma-Aldrich.
  13. Distilled water
  14. β-D-glucosyl Yariv reagent (Biosupplies Australia Pty Ltd., catalog number: 100-2 )
  15. Histoclear clearing agent (National Diagnostics, catalog number: HS-200 )
  16. Chlorazol black E/direct black 38 (Sigma-Aldrich, catalog number: C1144 )

Equipment

  1. Reichert Sliding Microtome
  2. Tweezers (for handling section)
  3. Light microscope

Procedure

X-ray μCT can also potentially be used for a broader range of crops, and a more diverse set of biological questions. In most cases, care should be taken to air-dry material prior to scanning; water saturation will reduce cell wall contrast while forced drying beyond fibre saturation point runs the risk of inducing cell collapse, altering the plant’s tissue architecture. An example of this wider potential for the technology in plant research is provided in Figure 1 and Video 1.


Figure 1. Miscanthus internode 3D render. X-ray 3D Computational tomography example images of mature Miscantus x giganteus stem.

Video 1. 3D render of X-Ray μCT scans of willow (cultivar Resolution). 2 cm stem section (debarked). Vessel lumens are coloured blue in silico.

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  1. Stem sampling
    The research where 3D X-ray μCT scanning was used to assess xylem fibre PCD (Brereton et al., 2015) used short rotation coppice (SRC) willow trees (cultivar Resolution-pedigree: [S. viminalis. x (S. viminalis. x S. schwerinii SW930812)] x [S. viminalis. x (S. viminalis. x S. schwerinii ‘Quest’)] grown in pots for 12 weeks. At week six the trees were tipped at a 45° angle to the horizontal and held in place for a further 6 weeks before harvesting (with the stem tied to a straight bamboo stem every two days to maintain the 45° angle). This alteration to the vector of gravitational stimulus elicits a unique change in tissue development known as the reaction wood response (Andersson-Gunneras et al., 2003; Brereton et al., 2011; Pitre et al., 2007; Wardrop and Dadswell, 1955).
    1. Harvest a sample whole stem segment of 6-8 cm from the midpoint of the stem.
      Note: This is deliberately chosen in preference to the traditional set breast height of 1.3 meters in order to allow developmentally comparable analysis between genotypes of different morphologies.
    2. Cut two small triangular notches into the wood 3-4 cm along the segment using a razor blade (making sure to reach through the bark to the secondary xylem), both notches should be at asymmetric positions from a transverse perspective (see Figure 2).


      Figure 2. Chlorazol black and Safranin O staining. Classical sectioning and staining with light microscopy can be used to help navigate and orientate tissue architecture of the stem (Panel A). In this context, chlorazol black E (counter-stained with safranin O) is used to stain the g-layer of g-fibres in tension wood of a tipped stem sample of SRC willow (cultivar resolution)-this can then be aligned with images from the μCT scan (Panel B). Note, there is variation throughout the entirety of the 3D stem (longitudinal variation in tissue as well as transverse) so multiple notches in the stem biomass being scanned (one is visible here as a single triangle cut in panel A) are an important tool to help navigate the 3D volume data from the scan alongside natural asymmetry of the tissue. Scale bar = 4 mm.

    3. Cut the segment in half to create two 3-4 cm segments. One 3-4 cm stem segment (stem A) will be used for X-ray μCT scanning while the other (stem B) will be used for associated staining and light or confocal microscopy. The notches will assist in navigation of the segments (allowing overlay and alignment) and can be removed in silico for 3D µCT render (stem A) or during sectioning (stem B).
    4. To make sectioning, staining and X-ray μCT scan differentiation simpler, bark can be removed at this point from both stem segments by manual peeling or with a scalpel.
    5. After harvesting stem segments should be “fixed” in FAA-formaldehyde, acetic acid, alcohol solution (3.7% formaldehyde, 5% acetic acid and 47.5% ethanol).

  2. Drying and pre-μCT scan prep
    1. After fixation of Stem A for > 3 days, the segment should be removed from FAA solution and briefly washed in H2O 4 days prior to 3D X-ray µCT scanning.
    2. Air-drying of stem A should be performed at room temperature (no greater than 30 °C) for over 4-5 days in a clean environment with relatively good air turnover. Forced drying at higher temperatures can pull H2O from the cell wall (beyond fibre saturation point) resulting in cell wall collapse. In very dry climates (low humidity) this can also occur during air-drying.
      Note: Air-drying of stem samples seems an essential step in creating contrast between the three elements within the biological system under scrutiny: the cell wall, the protoplast and void space. The important factor of air-dried samples being that “full” cells, tubular in shape, are easily distinguished from intracellular void space (which was not the case when “wet”, as intracellular void space is full of water (Figure 3).


      Figure 3. Wet and air-dry biomass 2D computational tomography scan examples. 3D X-Ray µCT models are rendered from >1,000 2D images, such as those from a tipped stem sample of SRC willow (cultivar resolution) shown above. A represents a 2D slice of a sample scanned “wet” whereas B represents a 2D slice of the same sample after having been air-dried for 3-4 days. Greater contrast between cell walls, cells with a protoplast (having not completed programmed cell death) and void volume is visible in the air-dried sample.

    3. Once stem A is air-dried the sample can be secured in a low-density media, usually standard Oasis® floral foam (a phenolic based polymer, care should be taken if specifically studying cellulose, as such low densities are hard to differentiate) and positioned for scan with the structure/s of interest in the centre of the scan volume.

  3. Infiltration
    1. As X-ray absorption increases with atomic number of atoms within a voxel, stem samples can potentially be vacuum infiltrated with heavy metals before 3D X-ray µCT scanning. Such as with phosphotungstate (Staedler et al., 2013).

  4. Post-μCT scan
    1. Once stem A has been scanned (Brereton et al., 2015) a destructive density measurement can be taken. Basic density (Biermann, 1996; Jourez et al., 2001) is best suited for biomass density quantification as it incorporates tissue structural variation often meaningful in terms of physiology.
    2. Vacuum infiltrate stem A with water before green volume is measured via water displacement (Biermann, 1996). Vacuum infiltration can be performed by pulling a vacuum over submerged wood samples for around 24 h depending on the material used. Wood oven dry weight is then measured after drying overnight at 105 °C.
    3. Calculated as (Biermann, 1996; Jourez et al., 2001): Basic density = oven dry mass / volume.

  5. Sectioning and staining
    Staining of the transverse sections from Stem B allows for different tissue types to be visualised and quantified within the stem. By clearly following each stem segment and navigating natural asymmetry (as well as notches cut into the stems), the in silico 3D X-ray µCT render, g-layers of g-fibres [chlorazol black E (Brereton et al., 2015; Brereton et al., 2011; Brereton et al., 2012; Robards and Purvis, 1964)], β-galactosyl Yariv reagent (Tryfona et al., 2012; Kitazawa et al., 2013), secondary cell wall (safranin O) and remnants of cell contents (Coomassie and β-galactosyl Yariv) can be aligned and compared in situ. The in situ β-galactosyl Yariv reagent and Coomassie staining seem to be without published precedence but are simple and effective. Coomassie staining is particularly effective for macro navigation (across a whole transverse section) of cell content remnants using light microscopy, as classical viability staining can be ineffective in woody tissue (such as: Tunnel and NBT) due to cell disruption during the sectioning process. Staining duration and concentrations are only suggestions here as cell wall structure will vary depending on the plant studied and the growth conditions.
    1. Once Stem B is secured in the Reichert sledge microtome, adjust angle of blade to the correct plane (this may require some trial and error depending on the material), dampen the sample with water and cut the thickness of < 30 µm.
    2. A 48 well plate is useful to perform multiple staining pipelines simultaneously on a common set of sections.
    3. Using tweezers throughout, first place a section in H2O to hydrate for 2 min before using section for either chlorazol black E staining, β-galactosyl Yarive reagent in situ staining or Coomassie in situ staining (cell wall antibodies can also be integrated with analysis here).

    Chlorazol black E staining (example Figure 2)
    1. Counter-stain by placing sections in a well containing 1% safranin O (aq) for 2-3 min.
    2. Wash in wells containing H2O x2 for 30 sec.
    3. Place section in progressive dehydration series: 50% ethanol for 30 sec, 75% ethanol for 30 sec, 100% ethanol x2 for 1 min.
    4. Place section in well containing 1% chlorazol black E (in methoxyethanol) for 4 min to stain g-layers.
    5. Wash in well containing 100% ethanol x2 for 1 min.

    β-galactosyl Yariv reagent staining (example Figure 4)
    1. Place section in 0.1 mg/ml dilute β-galactosyl Yariv reagent in 0.15 M NaCl and incubate at room temperature for 4 h on gently shaking platform.
    2. Wash as necessary in wells containing H2O.
    3. Move onto step E9.




      Figure 4. In situ β-galactosyl Yariv reagent staining. β-galactosyl Yariv reagent has long been used to condense arabinogalactan proteins (AGPs). As the g-layer of g-fibres in tension wood are rich in Fasciclin-like arabinogalactan protein (Lafarguette et al., 2004; Andersson-Gunneras et al., 2006), β-galactosyl Yariv reagent works well as an in situ stain for g-layers in tension wood of SRC willow stem as well as a protoplast stain (live cells will also include AGPs). Light microscopy of a tipped stem sample of SRC willow (cultivar resolution) shows A patterning of tensions wood g-layers and delayed PCD well aligned those of Figures 2. The same sample magnified using light microscopy to x63 illustrates: B. Tension wood (TW), C Opposite wood (OW) and D TW/OW interface show how clear the in situ β-galactosyl Yariv reagent staining can differentiate these cellular components. Scale bar = 4 mm.

    Coomassie staining (example Figure 5)
    1. Place section in 1% Coomassie solution and incubate at room temperature for 4 h on gently shaking platform.
    2. Wash as necessary in wells containing H2O.
    3. Move onto step E9.

    Slide construction
    1. Rinse staining section in Histoclear clearing agent for 1 min. At this stage apply DPX to slide to slightly set (1 min or so).
    2. Place section on to the DPX and apply cover slip, lowering gently. Apply pressure to force out air bubbles, place some weight (a few grams) on top and leave to set for 24 h.



      Figure 5. In situ Coomassie staining. A. Stitched 25 μm transverse sections stained with 1% Coomassie solution and counter stained with safranin O. B. Stitched 25 μm transverse sections without counterstain, remnants of cell contents is visible if present. While z-stacked images are difficult to assemble clearly using Coomassie staining, the process was invaluable to assess remnants of cell content using light microscopy (by eye, as one can move slightly through 3D space using light microscopes) and led to the identification of the same PCD patterning using 3D X-ray μCT. Scale bar = 4 mm.

Acknowledgments

The author was financially supported by BioFuelNet Canada. X-ray μCT scanning was performed in collaboration with Farah Ahmed and Dan Sykes at the Natural History Museum with funding from BBSRC Sustainable Bioenergy Centre (BSBEC), working within the BSBEC BioMASS (http://www.bsbec-biomass.org.uk/) Programme. In situ staining techniques were developed in collaboration with Dr Michael Jasmine Ray.

References

  1. Andersson-Gunneras, S., Hellgren, J. M., Bjorklund, S., Regan, S., Moritz, T. and Sundberg, B. (2003). Asymmetric expression of a poplar ACC oxidase controls ethylene production during gravitational induction of tension wood. Plant J 34(3): 339-349.
  2. Andersson-Gunneras, S., Mellerowicz, E. J., Love, J., Segerman, B., Ohmiya, Y., Coutinho, P. M., Nilsson, P., Henrissat, B., Moritz, T. and Sundberg, B. (2006). Biosynthesis of cellulose-enriched tension wood in Populus: global analysis of transcripts and metabolites identifies biochemical and developmental regulators in secondary wall biosynthesis. Plant J 45(2): 144-165.
  3. Biermann, C. J. (1996). Handbook of pulping and papermaking, 2nd edn. Academic Press.
  4. Brereton, N. J., Ahmed, F., Sykes, D., Ray, M. J., Shield, I., Karp, A. and Murphy, R. J. (2015). X-ray micro-computed tomography in willow reveals tissue patterning of reaction wood and delay in programmed cell death. BMC Plant Biol 15: 83.
  5. Brereton, N. J., Pitre, F. E., Ray, M. J., Karp, A. and Murphy, R. J. (2011). Investigation of tension wood formation and 2,6-dichlorbenzonitrile application in short rotation coppice willow composition and enzymatic saccharification. Biotechnol Biofuels 4: 13.
  6. Brereton, N. J., Ray, M. J., Shield, I., Martin, P., Karp, A. and Murphy, R. J. (2012). Reaction wood - a key cause of variation in cell wall recalcitrance in willow. Biotechnol Biofuels 5(1): 83.
  7. Jourez, B., Riboux, A. and Leclercq, A. (2001). Comparison of basic density and longitudinal shrinkage in tension wood and opposite wood in young stems of Populus euramericana cv. Ghoy when subjected to a gravitational stimulus. Canadian Journal of Forest Research-Revue Canadienne De Recherche Forestiere 31, 1676-1683.
  8. Kitazawa, K., Tryfona, T., Yoshimi, Y., Hayashi, Y., Kawauchi, S., Antonov, L., Tanaka, H., Takahashi, T., Kaneko, S., Dupree, P., Tsumuraya, Y. and Kotake, T. (2013). beta-galactosyl Yariv reagent binds to the beta-1, 3-galactan of arabinogalactan proteins. Plant Physiol 161(3): 1117-1126.
  9. Lafarguette, F. et al. (2004). Poplar genes encoding fasciclin-like arabinogalactan proteins are highly expressed in tension wood. New Phytologist 164, 107-121.
  10. Pitre, F. E., Cooke, J. E. K. and Mackay, J. J. (2007). Short-term effects of nitrogen availability on wood formation and fibre properties in hybrid poplar. Trees-Structure and Function 21, 249-259.
  11. Robards, A. W. and Purvis, M. J. (1964). Chlorazol black E as a stain for tension wood. Biotechnic and Histochemistry 39, 309-315.
  12. Staedler, Y. M., Masson, D. and Schonenberger, J. (2013). Plant tissues in 3D via X-ray tomography: simple contrasting methods allow high resolution imaging. PLoS One 8(9): e75295.
  13. Timell, T. E. (1969). The chemical composition of tension wood. Comptes Rendus Biologies 72, 173-181.
  14. Tryfona, T., Liang, H. C., Kotake, T., Tsumuraya, Y., Stephens, E. and Dupree, P. (2012). Structural characterization of Arabidopsis leaf arabinogalactan polysaccharides. Plant Physiol 160(2): 653-666.
  15. Wardrop, A. and Dadswell, H. (1955). The nature of reaction wood. IV. Variations in cell wall organization of tension wood fibres. Australian Journal of Botany 3, 177-189.

材料和试剂

  1. 玻璃滑槽
  2. 盖玻片
  3. 48孔板
  4. 剃刀片
  5. 短旋转枝(SRC)柳树[栽培品种分辨率 - 系谱: viminalis。 x( S。viminalis。 x S。schwerinii SW930812)] x [ viminalis。 x( s。viminalis。 x S。schwerinii 'Quest')]}
  6. 37%甲醛(36.5-38%,在H 2 O中)(Sigma-Aldrich,目录号:F8775)
  7. 乙酸(≥99.7%)(Sigma-Aldrich,目录号:320099)
  8. 乙醇
  9. FAA:3.7%甲醛,5%乙酸和47.5%乙醇
  10. Oasis ?花卉泡沫(OASIS Floral Products)( http://oasisfloralproducts.com/
  11. Safranin-O溶液(Sigma-Aldrich,目录号:HT90432)
  12. 用于组织学的DPX Mountant(Sigma-Aldrich,目录号:06522)
    注意:本产品在Sigma-Aldrich公司名为"载玻片(对二甲苯 - 双(N-吡啶鎓溴化物))"。
  13. 蒸馏水
  14. β-D-葡萄糖基Yariv试剂(Biosupplies Australia Pty Ltd.,目录号:100-2)
  15. Histoclear清除剂(National Diagnostics,目录号:HS-200)
  16. 氯唑黑E /直接黑38(Sigma-Aldrich,目录号:C1144)

设备

  1. Reichert滑动切片机
  2. 镊子(用于处理部分)
  3. 光学显微镜

程序

X射线μCT也可以潜在地用于更广泛的作物,以及更多样化的生物问题集。在大多数情况下,在扫描前应注意空气干燥材料;水饱和度将降低细胞壁对比度,而强制干燥超过纤维饱和点会带来诱导细胞萎缩的风险,从而改变植物的组织结构。图1和视频1中提供了植物研究技术更广泛潜力的一个例子

图1. Miscanthus 节间3D渲染。X/3D 3D计算层析成像 miscantus x giganteus
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视频1.杨柳X射线μCT扫描的3D渲染(品种分辨率)。 2厘米茎段(剥皮)。血管内腔 。
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  1. 干抽样
    使用3D X射线μCT扫描的研究 评估木质部纤维PCD(Brereton等人,2015)使用短旋转 杨树(SRC)柳树(栽培品种:分辨率谱系: x(
    1. 收获从茎的中点6-8厘米的样品整茎段。
      注意:这是故意选择的,优先于传统的集合 乳房高度为1.3米,以允许发育相当 ?分析不同形态的基因型。
    2. 切两个 小三角形切口插入木材3-4厘米沿线段使用a 剃刀刀片(确保通过树皮到达次要 木质部),两个凹口应当位于与横向不对称的位置 ?透视(见图2)。


      图2.氯唑黑和番红 ?O染色。 经典切片和光学显微镜染色可以 ?用于帮助导航和定向茎的组织结构 (图A)。在这种情况下,氯唑黑E(抗褪色 safranin O)用于在a的张力木材中染色g-纤维的g-层 ?然后可以得到SRC柳树的尖端茎样品(品种分辨率) ?与来自μCT扫描的图像(图B)对准。注意,有 整个3D茎的变化(纵向变化 ?在组织中以及横向),因此在茎中有多个缺口 生物质被扫描(一个在这里可以看作一个单一的三角形切入 面板A)是帮助导航3D体数据的重要工具 扫描与组织的自然不对称一起。比例尺= 4 mm。

    3. 将片段切成两半,以创建两个3-4厘米的片段。一个3-4厘米 茎段(茎A)将用于X射线μCT扫描 其他(茎B)将用于相关染色和光或 共聚焦显微镜。凹口将有助于导航 片段(允许重叠和对齐),并且可以在3DμCT渲染(茎A)或切片期间(茎B)在计算机中移除。
    4. 至 使切片,染色和X射线μCT扫描分化更简单, 树皮可以在这一点从两个茎段手动除去 剥皮或用手术刀
    5. 收获后茎段应 在FAA - 甲醛,乙酸,酒精溶液(3.7% 甲醛,5%乙酸和47.5%乙醇)。

  2. 干燥和pre-μCT扫描准备
    1. 在将茎A固定到> 3天,分段应该 从FAA溶液中移出,并在3D前4天在H 2 O中简单洗涤 X射线μCT扫描。
    2. 茎A的空气干燥应在 室温(不大于30℃)下干燥4-5天以上 环境空气流通相对较好。强制干燥较高 温度可以从细胞壁吸出H 2 O 2(超过纤维饱和 点)导致细胞壁塌陷。在非常干燥的气候(低 湿度),这也可以在空气干燥期间发生。
      注意:风干 的茎样本似乎是在创造对比之间的必要步骤 ?生物系统中的三个要素正在仔细检查:细胞 壁,原生质体和空隙空间。空气干燥的重要因素 样品是"全"细胞,管状形状,容易 区别于胞内空隙空间(这不是这种情况 ?"湿",因为胞内空隙空间充满水(图3)。


      图3.湿和空气干燥生物量2D计算层析成像扫描 3D X-RayμCT模型是从> 1,000张2D图片中渲染, 例如来自SRC柳树(栽培品种)的倾斜茎样品的那些 分辨率)。 A表示扫描的样本的2D切片 "湿",而B表示在具有之后相同样品的2D切片 空气干燥3-4天。细胞壁,细胞之间的对比度更大 与原生质体(尚未完成程序性细胞死亡)和空白 体积在空气干燥的样品中可见
    3. 一旦茎A 空气干燥的样品通常可以固定在低密度介质中 标准Oasis ?花卉泡沫(酚醛基聚合物,应小心 如果具体研究纤维素,因为这样低的密度是硬的 ?以区分)并且被定位用于具有一个或多个结构的扫描 兴趣在扫描体积的中心。

  3. 渗透
    1. 随着X射线吸收随a中的原子数的增加而增加 体素,茎样品可能被真空渗透重 金属前3D X射线μCT扫描。如用磷钨酸 (Staedler等人,2013)。

  4. 后μCT扫描
    1. 一旦茎A 已经被扫描(Brereton等人,2015)破坏性密度 可以进行测量。基本密度(Biermann,1996; Jourez等人, 2001)最适合于生物质密度定量 并入组织结构变异通常有意义 生理学
    2. 绿色之前,真空渗透茎A与水 体积通过水置换测量(Biermann,1996)。真空 可以通过在浸没木材上抽真空来进行渗透 样品约24小时,取决于所使用的材料。木烤箱干燥 然后在105℃下干燥过夜后测量重量
    3. 计算为(Biermann,1996; Jourez等人,2001):基本密度=烘干质量/体积。

  5. 切片和染色
    来自茎B的横截面的染色允许不同的 组织类型在茎内可视化和定量。清楚 ?跟随每个茎段和导航自然不对称(以及 作为凹口切入茎),in silico 3D X射线μCT渲染, g-纤维的g-层[chlorazol black E(Brereton等人,2015; Brereton等人,2011; Brereton等人, >,2012; Robards and Purvis,1964)], β-半乳糖基Yariv试剂(Tryfona等人,2012; Kitazawa等人, 2013),继发性细胞壁(safranin O)和细胞内容物的残留物 (考马斯和β-半乳糖基Yariv)可以比对并原位比较。 原位β-半乳糖基Yariv试剂和考马斯染色似乎是 ?没有公布的优先顺序,但简单有效。考马斯 染色对于宏观导航(在整个过程中)特别有效 横断面)的细胞内容物残留使用光学显微镜,as 经典的活力染色在木质组织中是无效的(例如 作为:隧道和NBT)由于在切片期间的细胞破坏 处理。染色时间和浓度只是建议 因为细胞壁结构将取决于所研究的植物和 生长条件。
    1. 一旦茎B固定在Reichert雪橇上 切片机,将刀片角度调整到正确的平面(这可能需要 一些试验和错误取决于材料),用样品缓冲 水切割, 30μm。
    2. 48孔板对于在同一组部分上同时进行多个染色管道是有用的
    3. 使用镊子在整个过程中,首先在H 2 O中放置一段以水合 2分钟,然后使用切片进行氯唑黑E染色, β-半乳糖基Yarive试剂原位染色或考马斯原位染色(细胞壁抗体也可以与分析 这里)。

    Chlorazol black E染色(示例图2)
    1. 通过将切片置于含有1%番红O(aq)的孔中2-3分钟进行逆染色。
    2. 在含有H 2 O x2的孔中洗涤30秒。
    3. 放置部分进行脱水系列:50%乙醇30秒,75%乙醇30秒,100%乙醇x2 1分钟。
    4. 将部分置于含有1%氯唑黑E(在甲氧基乙醇中)的孔中4分钟以染色g层。
    5. 在含有100%乙醇x2的孔中洗涤1分钟。

    β-半乳糖基Yariv试剂染色(示例图4)
    1. 将切片置于0.1mg/ml稀释的β-半乳糖基Yariv试剂中,0.15 ?并在室温下温和摇动孵育4小时 平台。
    2. 根据需要在含有H 2 O的孔中洗涤
    3. 转到步骤E9。




      图4.原位β-半乳糖基Yariv试剂染色。β-半乳糖基 Yariv试剂长期以来被用于冷凝阿拉伯半乳聚糖蛋白 (AGP)。由于张力木材中的g-纤维的g-层富含 Fasciclin样阿拉伯半乳聚糖蛋白(Lafarguette等人,2004; Andersson-Gunneras等人,2006),β-半乳糖基Yariv试剂很好地起作用 作为SRC柳树茎的紧张木材中的g层的原位染色剂 以及作为原生质体染色(活细胞也将包括AGP)。光 SRC柳树尖端茎样品的显微镜检查(品种分辨率) 显示了紧张的木材g层和延迟的PCD的图案化 对齐图2的相同样品使用光放大 显微镜到x63说明:B.张力木(TW),C对立木 (OW)和D TW/OW界面显示了原位β-半乳糖基Yariv ?试剂染色可以区分这些细胞组分。比例尺 ?= 4 mm。

    考马斯染色(示例图5)
    1. 将切片置于1%考马斯溶液中,在室温下在轻轻摇动的平台上孵育4小时
    2. 根据需要在含有H 2 O的孔中洗涤
    3. 转到步骤E9。

    幻灯片构造
    1. 冲洗染色部分在Histoclear清除剂1分钟。在 此阶段应用DPX滑动到稍微设置(1分钟左右)
    2. 将部分放在DPX上,并应用盖玻片,轻轻下降。应用 ?压力迫使气泡,放一些重量(几克) 顶部并设置为24小时。



      图5.原位考马斯染色 A.用1%考马斯染色的缝合的25μm横切片 溶液并用番红O染色。B.缝合25μm 横截面无复染,细胞内含物残留 可见(如果存在)。虽然z堆叠图像难以组装 清楚地使用考马斯染色,该过程是无价的评估 使用光学显微镜检查细胞含量的残留(通过眼睛,作为一个可以移动 ?轻微通过3D空间使用光学显微镜)和导致 使用3D X射线μCT鉴定相同的PCD图案。比例尺= ?4mm。

致谢

作者在加拿大生物燃料网的资助下。 X射线μCT扫描与Farah Ahmed和Dan Sykes在自然历史博物馆合作进行,由BBSRC可持续生物能源中心(BSBEC)资助,在BSBEC BioMASS( http://www.bsbec-biomass.org.uk/)计划。 原位染色技术是与Michael Jasmine Ray博士合作开发的。

参考文献

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  2. Andersson-Gunneras,S.,Mellerowicz,EJ,Love,J.,Segerman,B.,Ohmiya,Y.,Coutinho,PM,Nilsson,P.,Henrissat,B.,Moritz,T.and Sundberg, 2006)。 山杨中富含纤维素的张力木的生物合成:转录本和代谢物的全球分析识别生化和发育调节剂在次生壁生物合成中。 植物J 45(2):144-165
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  4. Brereton,N.J.,Ahmed,F.,Sykes,D.,Ray,M.J.,Shield,I.,Karp,A.and Murphy,R.J。(2015)。 柳树中的X射线微计算机断层扫描揭示了反应木材的组织图案化和程序性细胞死亡的延迟。 BMC Plant Biol 15:83.
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  9. Lafarguette,F。等人(2004)。 编码fasciclin样阿拉伯半乳聚糖蛋白的白杨基因在张力中高表达。新植物学家 164,107-121。
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How to cite this protocol: Brereton, N. J. (2016). Sample Preparation for X-ray Micro-computed Tomography of Woody Plant Material and Associated Xylem Visualisation Techniques. Bio-protocol 6(6): e1767. DOI: 10.21769/BioProtoc.1767; Full Text



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