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Plant cell wall biomass is an abundant and renewable organic resource. Of the polymers it encloses, cellulose and hemicellulose are regarded as a raw material for the production of fuels and other products (Klemm et al., 2005; Slavov et al., 2013). Nonetheless, current usage of lignocellulosic biomass is still below its full potential due to a series of limiting factors mainly related to the cell wall recalcitrance to saccharification, a severe constraint to maximum biomass usability in downstream processing (Pauly and Keegstra, 2008).
As a strategy to optimise bio-energy and bio-refining applications, an increasing amount of effort is being put into the advancement of our knowledge concerning the cell wall compositional roots of recalcitrance. Fourier transform mid-infrared spectroscopy (FTIR) represents a very useful tool on this enterprise, as it allows for a high-throughput, non-destructive and low unit cost procedure for the examination of cell wall biomass (Allison et al., 2009; Carpita and McCann, 2015). Furthermore, the use of Attenuated Total Reflection (ATR) in conjunction with infrared spectroscopy (IR) enables cell wall biomass samples to be examined in solid state without extensive preparation. Nonetheless, the analysis of purified cell wall preparations instead of the intact plant biomass is highly recommended, as it minimises or even eradicates interference from biomass components which are not part of the cell wall. Further information regarding the fundamentals of FTIR may be found elsewhere (Smith, 2011).
Datasets generated from FTIR spectroscopy can be extensive and complex. In these situations, data-driven modelling techniques are often used as exploratory approaches to identify the most distinctive features of the collected spectra. Here we suggest the use of Principal Component Analysis (PCA), a frequently employed method to transform a large set of variables into a smaller set of new variables (principal components), effectively reducing dataset dimensionality.
When the aim is a complete and detailed biomass characterisation, the FTIR-PCA method here described does not exclude the need for parallel wet gravimetric and analytical procedures. However, it does lead to a rapid identification of the major compositional shifts across large sets of samples; thus contributing to steer research pathways, minimise time-draining analytical procedures and reduce overall research costs.

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Cell Wall Biomass Preparation and Fourier Transform Mid-infrared (FTIR) Spectroscopy to Study Cell Wall Composition
细胞壁生物质的制备和傅里叶变换中红外(FTIR)光谱法研究细胞壁组成

植物科学 > 植物生物化学 > 糖类
作者: Ricardo M. F. da Costa
Ricardo M. F. da CostaAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
For correspondence: dacosta.rmf@gmail.com
Bio-protocol author page: a2279
Gordon G. Allison
Gordon G. AllisonAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
For correspondence: goa@aber.ac.uk
Bio-protocol author page: a2280
 and Maurice Bosch
Maurice BoschAffiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Ceredigion, UK
Bio-protocol author page: a2281
Vol 5, Iss 11, 6/5/2015, 1870 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1494

[Abstract] Plant cell wall biomass is an abundant and renewable organic resource. Of the polymers it encloses, cellulose and hemicellulose are regarded as a raw material for the production of fuels and other products (Klemm et al., 2005; Slavov et al., 2013). Nonetheless, current usage of lignocellulosic biomass is still below its full potential due to a series of limiting factors mainly related to the cell wall recalcitrance to saccharification, a severe constraint to maximum biomass usability in downstream processing (Pauly and Keegstra, 2008).
As a strategy to optimise bio-energy and bio-refining applications, an increasing amount of effort is being put into the advancement of our knowledge concerning the cell wall compositional roots of recalcitrance. Fourier transform mid-infrared spectroscopy (FTIR) represents a very useful tool on this enterprise, as it allows for a high-throughput, non-destructive and low unit cost procedure for the examination of cell wall biomass (Allison et al., 2009; Carpita and McCann, 2015). Furthermore, the use of Attenuated Total Reflection (ATR) in conjunction with infrared spectroscopy (IR) enables cell wall biomass samples to be examined in solid state without extensive preparation. Nonetheless, the analysis of purified cell wall preparations instead of the intact plant biomass is highly recommended, as it minimises or even eradicates interference from biomass components which are not part of the cell wall. Further information regarding the fundamentals of FTIR may be found elsewhere (Smith, 2011).
Datasets generated from FTIR spectroscopy can be extensive and complex. In these situations, data-driven modelling techniques are often used as exploratory approaches to identify the most distinctive features of the collected spectra. Here we suggest the use of Principal Component Analysis (PCA), a frequently employed method to transform a large set of variables into a smaller set of new variables (principal components), effectively reducing dataset dimensionality.
When the aim is a complete and detailed biomass characterisation, the FTIR-PCA method here described does not exclude the need for parallel wet gravimetric and analytical procedures. However, it does lead to a rapid identification of the major compositional shifts across large sets of samples; thus contributing to steer research pathways, minimise time-draining analytical procedures and reduce overall research costs.

Keywords: FTIR(FTIR), Cell wall(细胞壁), Lignocellulose(木质纤维素), Biomass(生物量), Biofuel(生物燃料)

[Abstract]

Materials and Reagents

  1. Lignocellulosic biomass
    Note: Depending on the aims of the researcher, lignocellulosic biomass from different species, organs or tissues may be used, providing it is conveniently prepared as indicated in the Procedure section below. For an example of the application of this protocol, please refer to da Costa et al. (2014).
  2. Deionised H2O
  3. 70% (v/v) aqueous ethanol (molecular biology grade)
  4. Chloroform/methanol (1:1 v/v) (molecular biology grade)
  5. 100% acetone (molecular biology grade)
  6. Type-I porcine α-amylase (Sigma-Aldrich, catalog number: A6255 ) (saline suspension; 29 mg protein/ml; 1714 units/mg protein)
  7. 0.1 M sodium acetate buffer (see Recipes)
  8. 0.1 M ammonium formate buffer (see Recipes)
  9. 0.001 M sodium azide (see Recipes)

Equipment

  1. Laboratory safety equipment (gloves, eye protection and safety mask are recommended particularly during sodium azide handling)
  2. Freezer
  3. pH meter
  4. Freeze dryer (Edwards Pirani 501 Super Modulyo, Edwards Ltd.)
  5. Analytical mill (Ika, A11 basic)
  6. Sieves (pore sizes: 0.18 and 0.85 mm)
  7. Plastic centrifuge tubes (50 ml, with screw cap) (Greiner Bio-One GmbH)
  8. Vortex mixer
  9. Shaking incubator
  10. Centrifuge
  11. Fume hood
  12. Block heater
  13. FTIR spectrometer (Equinox 55, Bruker Optik) equipped with a Golden Gate ATR accessory (Specac)

Software

  1. Bruker OPUS IR spectroscopy software (version 5.0; Bruker Optik)

Procedure

  1. Biomass preparation-organic solvent wash
    1. Pre-freeze and freeze-dry lignocellulosic biomass (time may vary depending on sample source (for whole Miscanthus spp. tillers the samples were freeze-dried for an excess of 7 days to ensure complete dryness).
    2. Grind tissues to a particle size in the range 0.18-0.85 mm (suggested equipment: IKA A11 Handheld Analytical Mill; sieves with mesh sizes of 80 and 20 µm).
    3. Weigh approximately 1 g of the ground plant biomass into a 50 ml plastic centrifuge tube.
    4. Add 30 ml 70% (v/v) aqueous ethanol, mix thoroughly using a vortex mixer and leave in a shaking incubator set at 40 °C/150 rpm for 12 h.
    5. Centrifuge at 900 x g for 10 min and discard the supernatant by decantation or aspiration.
    6. Add 30 ml 70% (v/v) aqueous ethanol, mix using a vortex mixer, but this time incubate the samples for 30 min at 40 °C/150 rpm.
    7. Centrifuge at 900 x g for 10 min and discard the supernatant.
    8. Repeat steps A6-7.
    9. Add 20 ml of the chloroform/methanol (1:1 v/v) solution, mix to re-suspend the pellet and leave in a shaking incubator for 30 min at 25 °C and 150 rpm.
    10. Centrifuge at 900 x g for 10 min and discard the supernatant after the chloroform/methanol wash.
    11. Repeat steps A9-10 twice.
    12. Add 15 ml of acetone, mix to re-suspend the pellet and leave in a shaking incubator for 30 min at 25 °C and 150 rpm.
    13. Centrifuge at 900 x g for 10 min and discard the supernatant after the acetone wash.
    14. Repeat steps A12-13 twice.
    15. Let the organic-washed biomass samples dry overnight in a fume hood (alternatively they can be left in an oven set at 35 °C for approximately 16 h).

  2. Biomass preparation-starch removal
    1. Re-suspend the dry, organic solvent-extracted biomass in 15 ml of 0.1 M sodium acetate buffer (pH 5.0).
    2. Heat for 20 min at 80 °C in a heating block to induce starch gelatinisation.
    3. Cool suspensions on ice for 15 min.
    4. Centrifuge at 900 x g for 10 min and discard the supernatant.
    5. Wash the pellet twice with 30 ml H2O, with centrifugation (900 x g for 10 min) and supernatant removal after each wash.
    6. Add to the pellet the following reagents: 10 ml 0.1 M ammonium formate buffer (pH 6.0), 10 μl type-I porcine α-amylase (47 units per 100 mg cell wall) and 500 µl 0.001 M sodium azide solution.
    7. Leave in a shaking incubator for 48 h at 25 °C/110 rpm.
    8. Terminate the digestion by heating to 95 °C/15 min.
    9. Cool samples on ice for 15 min.
    10. Centrifuge at 900 x g for 10 min and discard the supernatant.
    11. Wash the pellet three times with 30 ml H2O and twice with 20 ml acetone, with centrifugation (900 x g for 10 min) and supernatant removal after each wash.
    12. Let the prepared cell wall material samples dry overnight in a fume hood (alternatively they can be left in an oven set at 35 °C for approximately 16 h; the samples are stable for months at room temperature if kept in a sealed container and protected from direct light).

  3. Biomass examination-Fourier transform mid-infrared spectroscopy
    1. Without further preparation, place approximately 10 mg of the dry cell wall powder onto the Golden Gate ATR crystal.
    2. Press the sample into optimal contact with the ATR crystal using the anvil of the Golden Gate ATR accessory.
    3. Collect spectra in duplicate for each biomass sample
      Note: Each spectrum is collected by ATR in a mid-infrared range of 4,000-600 cm-1 and consists of the average over 32 scans at a resolution of 4 cm-1. Spectra were corrected for background absorbance by subtraction of the spectrum of the empty ATR crystal.
    4. Recover the sample using a spatula or a razor blade (Video 1).
    5. Clean the contact areas with a laboratory tissue wipe and ethanol between samples.
    6. Repeat steps C1-5 for all samples.
    7. Convert each collected spectrum into an individual text file containing the spectral two dimensional Cartesian coordinates (x, y) (Figure 1) in two separate columns (we used the Bruker OPUS IR spectroscopy software). Subsequently, matrices containing the raw data may be created and the underlying relationships between the spectra may be investigated using numerical or statistical computing software (such as R or MATLAB). As an example, we used PCA to identify the features most distinctive of spectra collected from Miscanthus spp. leaf and stem cell wall biomass (Figure 2).

      Video 1. Collection of FTIR spectra from cell wall biomass

      To play the video, you need to install a newer version of Adobe Flash Player.

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      Figure 1. FTIR spectra of cell wall biomass (Miscanthus spp.), plotted using Opus IR spectroscopy software (A), and after being converted into text files containing their Cartesian coordinates in two separate columns (B)


      Figure 2. Plot of principal component one (PC1) and principal component two (PC2) scores for Miscanthus spp. leaf and stem cell wall spectra generated by FTIR (da Costa et al., 2014)

Recipes

  1. 0.1 M sodium acetate buffer (pH 5.0)
    Prepare 0.2 M acetic acid (A) by mixing 11.55 ml glacial acetic acid in 500 ml H2O and adjusting to 1 L with H2O
    Prepare 0.2 M sodium acetate solution (B) by dissolving 27.21 g sodium acetate trihydrate in 800 ml H2O and adjust to 1 L with H2O
    Mix 14.8 ml of A, 35.2 ml of B and 50 ml of H2O
    Confirm pH=5.0 with a pH meter (if needed, the pH may be adjusted with 10 M sodium acetate or with glacial acetic acid)
  2. 0.1 M ammonium formate buffer (pH 6.0)
    Dissolve 6,306 mg ammonium formate in 500 ml H2O and adjust pH to 6.0 with formic acid
    Make up to 1 L with H2O
    Confirm pH=6.0 with a pH meter (if needed, the pH may be adjusted with formic acid)
  3. 0.001 M sodium azide
    Dissolve 10 mg sodium azide in 145 ml H2O

Acknowledgments

The methods here described were employed in da Costa et al. (2014) for the analysis of Miscanthus spp. cell wall samples. Portions of this procedure were modified from various sources: organic solvent washing and starch gelatinization from Foster et al. (2010); starch removal from Persson et al. (2007) and Kong et al. (2011); and Fourier transform mid-infrared spectroscopy from Allison et al. (2009). The original work was supported by the European Regional Development Funding through the Welsh Government for BEACON Grant number 8056 to R. M. F. da Costa, G. G. Allison and M. Bosch.

References

  1. Allison, G. G., Thain, S. C., Morris, P., Morris, C., Hawkins, S., Hauck, B., Barraclough, T., Yates, N., Shield, I., Bridgwater, A. V. and Donnison, I. S. (2009). Quantification of hydroxycinnamic acids and lignin in perennial forage and energy grasses by Fourier-transform infrared spectroscopy and partial least squares regression. Bioresour Technol 100(3): 1252-1261.
  2. Carpita, N. C. and McCann, M. C. (2015). Characterizing visible and invisible cell wall mutant phenotypes. J Exp Bot. (Epub ahead of print)
  3. da Costa, R. M., Lee, S. J., Allison, G. G., Hazen, S. P., Winters, A. and Bosch, M. (2014). Genotype, development and tissue-derived variation of cell-wall properties in the lignocellulosic energy crop Miscanthus. Ann Bot 114(6): 1265-1277.
  4. Foster, C. E., Martin, T. M. and Pauly, M. (2010). Comprehensive compositional analysis of plant cell walls (Lignocellulosic biomass) part I: lignin. J Vis Exp (37): e1745.
  5. Klemm, D., Heublein, B., Fink, H. P. and Bohn, A. (2005). Cellulose: fascinating biopolymer and sustainable raw material. Angew Chem Int Ed Engl 44(22): 3358-3393.
  6. Kong, Y., Zhou, G., Yin, Y., Xu, Y., Pattathil, S. and Hahn, M. G. (2011). Molecular analysis of a family of Arabidopsis genes related to galacturonosyltransferases. Plant Physiol 155(4): 1791-1805.
  7. Pauly, M. and Keegstra, K. (2008). Cell‐wall carbohydrates and their modification as a resource for biofuels. Plant J 54(4): 559-568.
  8. Persson, S., Caffall, K. H., Freshour, G., Hilley, M. T., Bauer, S., Poindexter, P., Hahn, M. G., Mohnen, D. and Somerville, C. (2007). The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity. Plant Cell 19(1): 237-255.
  9. Slavov, G., Allison, G. and Bosch, M. (2013). Advances in the genetic dissection of plant cell walls: tools and resources available in Miscanthus. Front Plant Sci 4: 217.
  10. Smith, B. (2011). Fundamentals of Fourier Transform Infrared Spectroscopy, Second Edition. CRC Press.

材料和试剂

  1. 木质纤维素生物质
    注:根据研究者的目的,可以使用来自不同物种,器官或组织的木质纤维素生物质,只要其如以下程序部分所示方便地制备即可。 有关此协议的应用示例,请参阅da Costa等。 (2014)。
  2. 去离子的H 2 O 2 /
  3. 70%(v/v)含水乙醇(分子生物学级)
  4. 氯仿/甲醇(1:1 v/v)(分子生物学级)
  5. 100%丙酮(分子生物学级)
  6. I型猪α-淀粉酶(Sigma-Aldrich,目录号:A6255)(盐悬浮液; 29mg蛋白质/ml; 1714单位/mg蛋白质)
  7. 0.1 M乙酸钠缓冲液(见配方)
  8. 0.1 M甲酸铵缓冲液(见配方)
  9. 0.001 M叠氮化钠(参见配方)

设备

  1. 实验室安全设备(特别是在叠氮化钠处理期间,建议戴手套,护目镜和安全面罩)
  2. 冰柜
  3. pH计
  4. 冷冻干燥机(Edwards Pirani 501 Super Modulyo,Edwards Ltd.)
  5. 分析磨(Ika,A11基本)
  6. 筛(孔径:0.18和0.85mm)
  7. 塑料离心管(50ml,带螺旋盖)(Greiner Bio-One GmbH)
  8. 涡流搅拌器
  9. 摇动培养箱
  10. 离心机
  11. 通风橱
  12. 阻止加热器
  13. 装备有Golden Gate ATR附件(Specac)的FTIR光谱仪(Equinox 55,Bruker Optik)

软件

  1. Bruker OPUS IR光谱软件(版本5.0; Bruker Optik)

程序

  1. 生物质制备 - 有机溶剂洗涤
    1. 预冷冻和冷冻干燥木质纤维素生物质(时间可变化 取决于样品来源(对于整个芒草 spp 将样品冷冻干燥超过7天以确保完成 干燥)。
    2. 研磨组织至粒度在0.18-0.85范围内   mm(建议设备:IKA A11手持分析磨;筛 筛目尺寸为80和20μm)。
    3. 称取约1g地面植物生物质放入50ml塑料离心管中
    4. 加入30ml 70%(v/v)乙醇水溶液,使用涡旋充分混合 混合器中,并置于设定在40℃/150rpm的摇动培养箱中12小时
    5. 在900×g离心10分钟,通过倾析或吸出弃去上清液。
    6. 加入30ml 70%(v/v)乙醇水溶液,使用涡旋混合器混合 这一次在40℃/150rpm下孵育样品30分钟
    7. 在900×g离心10分钟并弃去上清液
    8. 重复步骤A6-7。
    9. 加入20ml氯仿/甲醇(1:1 v/v)溶液,混合 重悬浮沉淀并在25℃下在振荡培养箱中放置30分钟 ℃和150rpm
    10. 在900×g离心10分钟,并在氯仿/甲醇洗涤后弃去上清液。
    11. 重复步骤A9-10两次。
    12. 加入15ml丙酮,混合以重悬浮沉淀,并在25℃和150rpm下在振荡培养箱中放置30分钟。
    13. 在900×g离心10分钟,并在丙酮洗涤后弃去上清液。
    14. 重复步骤A12-13两次。
    15. 使有机洗涤的生物质样品在通风橱中干燥过夜   (或者它们可以放置在设定在35℃的烘箱中 约16小时)。

  2. 生物质制剂 - 淀粉去除
    1. 将干燥的,有机溶剂萃取的生物质再悬浮于15ml的0.1M乙酸钠缓冲液(pH5.0)中
    2. 在加热块中在80℃下加热20分钟以诱导淀粉糊化
    3. 在冰上冷却悬浮液15分钟。
    4. 在900×g离心10分钟并弃去上清液
    5. 用30ml H 2 O 2洗涤沉淀两次,离心(900×g 10分钟),每次洗涤后除去上清液。
    6. 向沉淀中加入以下试剂:10ml 0.1M铵 甲酸缓冲液(pH6.0),10μlI型猪α-淀粉酶(47单位/ 100mg细胞壁)和500μl0.001M叠氮化钠溶液
    7. 在25℃/110rpm下,在振荡孵育器中保持48小时
    8. 加热至95℃/15分钟终止消化。
    9. 在冰上冷却样品15分钟
    10. 在900×g离心10分钟并弃去上清液
    11. 用30ml H 2 O洗涤沉淀三次,用20ml洗涤两次 丙酮,离心(900×g,10分钟)和上清液 每次洗涤后除去
    12. 让制备的细胞壁材料 样品在通风橱中干燥过夜(或者可以留在其中 设定在35℃的烘箱约16小时; 样品稳定 个月,如果保存在密封容器中并保护 从直接光)。

  3. 生物质检验 - 傅里叶变换中红外光谱仪
    1. 无需进一步制备,将约10mg的干细胞壁粉末置于金门ATR晶体上
    2. 使用金门ATR附件的砧座将样品与ATR晶体最佳接触。
    3. 对于每个生物质样品,重复收集光谱
      注意:每个光谱由ATR在中红外范围内收集 4,000-600 cm -1   4 cm -1 。 用背景吸光度校正光谱 减去空ATR晶体的光谱。
    4. 使用刮刀或刀片回收样品(视频1)。
    5. 用样品之间的实验室组织擦拭物和乙醇清洁接触区域
    6. 对所有样品重复步骤C1-5。
    7. 将每个收集的光谱转换为单个文本文件 包含光谱二维笛卡尔坐标( x , y ) (图1)在两个单独的列(我们使用Bruker OPUS IR 光谱软件)。随后,包含原始数据的矩阵 并且可以创建光谱之间的潜在关系 使用数值或统计计算软件(如 作为R或MATLAB)。例如,我们使用PCA来识别特征 最不同的是从芒草(Miscanthus)中收集的光谱。叶和茎  细胞壁生物量(图2)
      视频1.来自细胞壁生物质的FTIR光谱的收集
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      图1.细胞壁生物量( 芒草 spp。)的FTIR光谱, 使用Opus IR光谱软件(A),并转换成 文本文件包含它们在两个独立的笛卡尔坐标 栏(B)


      图2.主成分1(PC1)和 Miscanthus spp的主成分二(PC2)得分。 叶和茎 FTIR产生的细胞壁光谱(da Costa等人,2014)

食谱

  1. 0.1M乙酸钠缓冲液(pH5.0) 通过将11.55ml冰醋酸在500ml H 2 O中混合并用H 2 O调节至1L来制备0.2M乙酸(A) 通过将27.21g乙酸钠三水合物溶解在800ml H 2 O中并通过H 2 O调节至1L来制备0.2M乙酸钠溶液(B) 将14.8ml的A,35.2ml的B和50ml的H 2 O混合 用pH计确认pH = 5.0(如果需要,可以用10M乙酸钠或冰醋酸调节pH)
  2. 0.1M甲酸铵缓冲液(pH 6.0) 将6,306mg甲酸铵溶解在500ml H 2 O中并用甲酸调节pH至6.0 用H sub 2 O补充到1 L
    用pH计确认pH = 6.0(如果需要,可以用甲酸调节pH)
  3. 0.001 M叠氮化钠 将10mg叠氮化钠溶于145ml H 2 O中

致谢

这里描述的方法在da Costa等人(2014)中用于芒草属(Miscanthus)的分析。细胞壁样品。该程序的一部分改性自各种来源:Foster等人(2010)的有机溶剂洗涤和淀粉胶凝化;从Persson等人(2007)和Kong等人(2011)的淀粉去除;和来自Allison等人的傅立叶变换中红外光谱(Eur.J.Med。)(2009)。最初的工作得到欧洲区域发展基金的支持,通过威尔士政府为BEACON拨款号8056授予R. M. F. da Costa,G. G. Allison和M. Bosch。

参考文献

  1. Allison,GG,Thain,SC,Morris,P.,Morris,C.,Hawkins,S.,Hauck,B.,Barraclough,T.,Yates,N.,Shield,I.,Bridgwater,AVand Donnison, (2009)。 通过傅里叶变换红外光谱和偏最小二乘法对常年饲料和能源草中羟基肉桂酸和木质素的定量平方回归。 Bioresour Technol 100(3):1252-1261。
  2. Carpita,N. C.和McCann,M.C。(2015)。 表征可见和不可见的细胞壁突变体表型。/em>(打印前的电子版)
  3. da Costa,R.M.,Lee,S.J.,Allison,G.G.,Hazen,S.P.,Winters,A.and Bosch,M。(2014)。 木质纤维能源作物中细胞壁特性的基因型,发育和组织衍生变异Miscanthus 。 Ann Bot 114(6):1265-1277。
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How to cite this protocol: da Costa, R. M., Allison, G. G. and Bosch, M. (2015). Cell Wall Biomass Preparation and Fourier Transform Mid-infrared (FTIR) Spectroscopy to Study Cell Wall Composition. Bio-protocol 5(11): e1494. DOI: 10.21769/BioProtoc.1494; Full Text



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