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  Section: Biotechnology Methods » Tissue Culture Techniques
 
 
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Preparation of Protoplasts

 
     
 
Content
Tissue Culture Techniques
  Tissue Culture Methods
  Plant Tissue Culture
  Plant Tissue Culture (Cont.)
  Many Dimensions of Plant Tissue Culture Research
  What is Plant Tissue Culture?
  Uses of Plant Tissue Culture
  Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance
  Demonstration of Tissue Culture for Teaching
  Preparation of Plant Tissue Culture Media
  Plant Tissue Culture Media
  Preparation of Protoplasts
  Protoplast Isolation, Culture, and Fusion
  Agrobacterium Culture and Agrobacterium — Mediated transformation
  Isolation of Chloroplasts from Spinach Leaves
  Preparation of Plant DNA using
  Suspension Culture and Production of Secondary Metabolites
  Protocols for Plant Tissue Culture
  Sterile Methods in Plant Tissue Culture
  Media for Plant Tissue Culture
  Safety in Plant Tissue Culture
  Preparation of Media for Animal Cell Culture
  Aseptic Technique
  Culture and Maintenance of Cell Lines
  Trypsinizing and Subculturing Cells from a Monolayer
  Cellular Biology Techniques
  In Vitro Methods
  Human Cell Culture Methods

Method for isolating large numbers of metabolically competent protoplasts from leaves of monocotyledons (grasses), dicotyledons (such as spinach and sunflower), or from hypocotyl tissue (e.g., Brassica napus).
  1. Leaf slices of monocots and dicots are prepared by cutting the leaves with a sharp razor blade into segments 0.5–1 mm in size. In the case of dicots, the epidermis can be scraped off before cutting by rubbing with fine carborundum powder or with a fine nylon brush.
  2. Set up 50 mL of digestion medium (for 10–15 g of plant tissue) according to the recipe to Solution A.
  3. Incubate the leaf slices or pieces in a 19-cm-diameter dish containing the digestion medium for 3 hours at 25°C, covered with a plastic film. It may be advantageous to replace the digestion medium at intervals of 1 hour, as the enzymes might become inactivated by substances released from broken cells.
  4. After completion of the incubation the digestion medium is carefully removed and discarded. It normally contains very few protoplasts. The plant tissue is then washed 3 times by shaking gently with 20-mL wash medium (Solution B).
  5. After each wash, the tissue is collected by pouring through a tea strainer (0.5- to 1-mm pore size) and the combined washes are then filtered through nylon mesh (100–200 mm pore size) to remove vascular tissue and undigested material.
  6. The protoplasts are collected by centrifugation of the combined filtered washes for 3 minutes at 50–100 xg and the supernatant is aspirated and discarded.
  7. This crude protoplast preparation also contains some cells and chloroplasts and it is important to purify the protoplasts to remove these contaminants. This can be done with solutions of sucrose and sorbitol of different densities.
  8. The protoplast pellet is gently resuspended in 40 mL of Solution C, and this suspension is divided among two 100-mL centrifuge tubes.
  9. To each tube, slowly add 5 mL of Solution D and then overlay this with 5 mL of wash medium (Solution B) to make a 3-step gradient.
  10. Centrifuge at 300 g for 5 minutes.
  11. The protoplasts now collect as a band at the interface between the 2 top layers. Carefully remove them with a Pasteur pipette.
  12. The protoplasts should be examined with a light microscope to ensure that the preparation is free of cells and chloroplasts.
  13. When a large portion of the protoplasts is pelleted in this sucrose/sorbitol gradient, the density of the 2 layers can be increased by adding 5%–10% Dextran (15000–20000 Mr) or 10%–20% Ficoll to increase the percentage of the floating protoplasts.
  14. The purified protoplasts can be concentrated by diluting with 10 mL of Solution B, cenrifuging at 100 g for 3 minutes and then resuspending the pellet in a small amount of medium by gently shaking the tubes.
  15. The protoplasts are stable for up to 24 hrs when stored on ice. Photosynthetic activity of the protoplasts can be determined by measuring codependent evolution with an oxygen electrode, provided rapid stirring is avoided, as this will break some of the protoplasts. A suitable medium is listed as Solution E.
    Protoplasts exhibit a relatively broad pH optimum, but at more acidic pH values the bicarbonate concentration should be lowered.

Solutions
Solution A (Digestion medium)   Solution B (Wash medium)
Composition
Composition
For 50 mL use:
For 100 mL use:
500 mM
500 mM
D-Sorbitol
D-Sorbitol
4.56 g
9.11 g
1 mM
1 mM
CaCl2
CaCl2
7.35 mg 14.70 mg
5 mM
5 mM
MES-KOH, pH 5.5
MES-KOH, pH 6.0
49.00 mg 98.00 mg
2% The pH of the solution must be
Cellulase Onozuka R10
adjusted to 6.0 with KOH.
1.00 g  
0.3%
 
Macerozyme R10
 
0.15 g  
The pH must be adjusted to 5.5 with  
KOH before adding the enzymes  
   
Solution C Solution D
Composition Composition
For 100 mL use: For 100 mL use:
500 mM 400 mM
Sucrose Sucrose
8.56 g 6.80 g
1 mM 100 mM
CaCl2 D-Sorbitol
7.40 mg 0.90 g
5 mM 1 mM
MES-KOH, pH 6.0 CaCl2
49.00 mg 7.40 mg
The pH of the solution must be 5 mM
adjusted to 6.0 by adding KOH.
MES-KOH, pH 6.0
  49.00 mg
  The pH of the solution must be
  adjusted to 6.0 by adding KOH.
   
Solution E  
Composition
 
For 100 mL use:
 
500 mM
 
D-Sorbitol
 
9.10 g    
1 MM  
CaCl2  
15.00 mg  
30 mM  
Tricine-KOH
 
538.00 mg  
5 mM  
NaHCO3  
42.0 mg

 
     
 
 
     




     
 
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