Fluorescence Measurement of Postharvest Physiological Deterioration (PPD) in Cassava Storage Roots

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.