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Fluorescence Measurement of Postharvest Physiological Deterioration (PPD) in Cassava Storage Roots
荧光测定木薯储藏根采后的生理劣变(PPD)   

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Abstract

Cassava (Manihot esculenta Crantz) is a perennial root crop in the tropics. Within 24-72 hours of harvest the storage roots deteriorate rapidly, thereby necessitating their prompt processing or consumption. Postharvest physiological deterioration (PPD) of cassava storage roots is the result of a rapid oxidative burst, which leads to discoloration of the vascular tissues. The various fluorogenic probes available for in vivo reactive oxygen species (ROS) imaging could reveal complex spatial and temporal dynamics in plant tissues. Fluorescence measurement of PPD became widely used assay for ROS. Most of the ROS probes passively diffuse across cell membranes localize in the mitochondria, and exhibit fluorescence. Due to its high sensitivity to ROS and ease of loading and detection, the Dihydrorhodamine123 probe has been widely used in plants to monitor ROS accumulation in response to various stimuli and range of developmental processes.

Keywords: Cassava(木薯), Storage root(贮藏根), Postharvest physiological deterioration(采后生理恶化), Fluorescence(荧光)

Materials and Reagents

  1. DMSO (Sangon Biotech, catalog number: D0231 )
  2. Dihydrorhodamine123 (Invitrogen, Molecular Probes®, catalog number: D632 ) (see Recipes)
  3. MitoTracker Deep Red FM (Invitrogen, Molecular Probes®, catalog number: M22426 ) (see Recipes)
  4. 0.1 M sodium phosphate buffer (pH 7.0) (see Recipes)

Equipment

  1. 48-well plate
  2. Razor blade
  3. Slides
  4. Centrifuge
  5. Confocal laser scanning microscope (Zeiss, model: LSM 510 META )

Procedure

  1. Cut fresh cassava storage roots into smaller segments about 5 x 5 x 0.5 mm in length, width and height by razor blade. The areas (Figure 1):
    1. The center: vascular tissue found within the center of the root cross-section.
    2. The middle: the tissue filled with storage cells with streaks of xylem throughout.


      Figure 1. Cassava storage root cross-section (Rickard, 1985). 1: periderm; 2: sclerenchyma; 3: parenchyma; 4: latex tubes; 5: cambium; 6: parenchyma (The middle); 7: xylem vessels (The middle); 8:  xylem bundles (The center).

  2. Immersed in the sodium phosphate buffer with Dihydrorhodamine123 or MitoTracker Deep Red FM, stain for 10 and 20 min, respectively in the dark at room temperature.
  3. Afterwards, wash with sodium phosphate buffer once before viewing under the microscope. Use sodium phosphate buffer to mount the sample on slides.
  4. Use a Zeiss LSM (Laser Scanning Microscope) 510 META Confocal with the capturing program LSM 510 equipped with a10x and 20x objective.
  5. Allow the microscope and lasers to warm up for about 20 minutes before use.
  6. The settings used for each stain are as follows:
    1. Dihydrorhodamine123 excitation/emission 488/515 nm.
    2. MitoTracker Deep Red FM excitation/emission 635/680 nm.
  7. First use the visible spectrum to find the sample, after that change to fluorescence spectrum (Figure 2).


    Figure 2. Fluorescence determination of PPD in cassava storage roots. a, xylem vessel; b, bundle sheath; Scale bar = 20 μm.

Recipes

  1. 0.1 M sodium phosphate buffer, pH 7.0 (100 ml)
    Mix 61.5 ml 1 M K2HPO4 and 38.5 ml 1 M KH2PO4
    Filter sterilize (0.45 μm)
    Store at 4 °C
  2. Dihydrorhodamine123 working solution
    1. 50 mM Storage solution
      10 mg Dihydrorhodamine123 with 577.4 μl DMSO  
    2. 50 μM Working solution 
      Sodium phosphate buffer dilution 
      Store at -70 °C
  3. MitoTracker Deep Red FM
    1. 1 mM Storage solution 
      50 μg MitoTracker Deep Red FM with 91.98 μl DMSO 
    2. 250 nM Working solution
      Sodium phosphate buffer dilution 
      Store at -70 °C

Acknowledgments

The protocol was mainly adapted from the publication Xu et al. (2013). This work was supported by grants from the National Natural Science Foundation of China (31271775), the National Basic Research Program (2010CB126605), the National High Technology Research and Development Program of China (2012AA101204), the Earmarked Fund for China Agriculture Research System (CARS-12-shzp) and the BioCassava Plus Program from the Bill & Melinda Gates Foundation.

References

  1. Rickard J. E. (1985). Physiological deterioration in cassava roots. J Sci Food Agric 36: 167-176.
  2. Xu, J., Duan, X., Yang, J., Beeching, J. R. and Zhang, P. (2013). Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol 161(3): 1517-1528.

简介

木薯( Manihot esculenta Crantz)是热带地区的多年生根作物。 在收获的24-72小时内,存储根快速劣化,从而迫使其迅速加工或消费。 木薯储存根的采后生理劣化(PPD)是快速氧化爆发的结果,其导致血管组织的变色。 可用于体内活性氧(ROS)成像的各种荧光探针可以揭示植物组织中的复杂空间和时间动力学。 PPD的荧光测量成为广泛使用的ROS测定法。 大多数ROS探针被动扩散穿过细胞膜定位在线粒体中,并且显示荧光。 由于其对ROS的高敏感性和易于加载和检测,二氢罗丹明123探针已广泛用于植物中以监测响应于各种刺激和发育过程范围的ROS积累。

关键字:木薯, 贮藏根, 采后生理恶化, 荧光

材料和试剂

  1. DMSO(Sangon Biotech,目录号:D0231)
  2. 二氢罗丹明123(Invitrogen,Molecular Probes ,目录号:D632)(参见配方)
  3. MitoTracker Deep Red FM(Invitrogen,Molecular Probes ,目录号:M22426)(参见Recipes)
  4. 0.1 M磷酸钠缓冲液(pH 7.0)(见配方)

设备

  1. 48孔板
  2. 剃刀刀片
  3. 幻灯片
  4. 离心机
  5. 共聚焦激光扫描显微镜(Zeiss,型号:LSM 510 META)

程序

  1. 将新鲜木薯储存根切成长度,宽度和高度约5×5×0.5mm的较小片段,用刀片。 区域(图1):
    1. 中心:位于根横截面中心内的血管组织
    2. 中间:组织充满储存细胞,具有整个木质部的条纹

      图1. Cassava存储根横截面(Rickard,1985)。 1:periderm; 2:sclerenchyma; 3:实质; 4:胶管; 5:形成层; 6:实质(中间); 7:木质部血管(中间); 8: 木质部束(中心)。

  2. 用二氢罗丹明123或MitoTracker Deep Red FM浸没在磷酸钠缓冲液中,在室温黑暗中分别染色10和20分钟。
  3. 然后,用磷酸钠缓冲液洗涤一次,然后在显微镜下观察。 使用磷酸钠缓冲液将样品安装在载玻片上。
  4. 使用Zeiss LSM(激光扫描显微镜)510 META共聚焦,配备10倍和20倍物镜的捕获程序LSM 510.
  5. 让显微镜和激光器在使用前预热约20分钟。
  6. 用于每种污渍的设置如下:
    1. 二氢罗丹明123激发/发射488/515nm
    2. MitoTracker深红色FM激发/发射635/680 nm。
  7. 首先使用可见光谱找到样品,然后变成荧光光谱(图2)。


    图2.木薯储存根中PPD的荧光测定。 a木质部容器; b,束鞘; 比例尺=20μm。

食谱

  1. 0.1M磷酸钠缓冲液,pH7.0(100ml) 将61.5ml 1M K 2 HPO 4和38.5ml 1M KH 2 PO 4溶液混合,得到混合物 过滤灭菌(0.45μm)
    存储在4°C
  2. 二氢罗丹明123工作液
    1. 50 mM贮存溶液
      10毫克二氢罗丹明123与577.4微升DMSO 
    2. 50μM工作解决方案 
      磷酸钠缓冲液稀释
      储存于-70°C
  3. MitoTracker深红色FM
    1. 1 mM存储解决方案
      50μgMitoTracker深红色FM与91.98μlDMSO&
    2. 250 nM工作溶液
      磷酸钠缓冲液稀释
      存储在-70°C

致谢

该方案主要从出版物Xu等人(2013)修改。 这项工作得到国家自然科学基金(31271775),国家基础研究计划(2010CB126605),国家高技术研究与发展计划(2012AA101204),中国农业研究系统专用基金 CARS-12-shzp)和BioCassava Plus Program from the Bill& 梅林达 盖茨基金会。

参考文献

  1. Rickard J.E。(1985)。 木薯根的生理恶化 。 J Sci Food Agric 36:167-176。
  2. Xu,J.,Duan,X.,Yang,J.,Beeching,J.R.and Zhang,P.(2013)。 超氧化物歧化酶和过氧化氢酶过量产生的活性氧清除增加延迟了木薯储存根的采后生理恶化。 Plant Physiol 161(3):1517-1528。
  • English
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xu, J., Fellman, J. K. and Zhang, P. (2013). Fluorescence Measurement of Postharvest Physiological Deterioration (PPD) in Cassava Storage Roots. Bio-protocol 3(18): e909. DOI: 10.21769/BioProtoc.909.
  2. Xu, J., Duan, X., Yang, J., Beeching, J. R. and Zhang, P. (2013). Enhanced reactive oxygen species scavenging by overproduction of superoxide dismutase and catalase delays postharvest physiological deterioration of cassava storage roots. Plant Physiol 161(3): 1517-1528.
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diana masika
kenya agricultural research institute
i would like to get more information on cassava post harvest physiological deterioration. can u give the links please.
4/24/2014 3:18:52 AM Reply
Peng Zhang
Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China

You can get more information about cassava PPD by:

http://www.fao.org/docrep/v4510e/v4510e00.htm
http://r4d.dfid.gov.uk/PDF/Outputs/R7550011.pdf
http://zenodo.org/record/8146/files/1319193155-Siritunga_2011AJEA784.pdf

4/24/2014 5:49:55 AM


diana masika
kenya agricultural research institute

Thank you so much.

4/24/2014 6:17:46 AM