Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues
Lynn Doran, Amanda P. De Souza
Abstract
Ethanolic extraction of total soluble sugars (liquid fraction) and starch (precipitated fraction) from leaf tissue.
Before start
Freeze-dry and grind leaf tissue into a fine powder.
- Freeze-dry according to equipment manufacturer's instructions.
- Freeze-dried leaf tissue can be ground using the tissuelyzer WITHOUT THE USE OF LIQUID NITROGEN.
- Improperly freeze-dried tissue will experience total sugar degradation and significant starch degradation. Improperly freeze-dried leaf tissue will look oxidized and more brown.
Steps
Ethanolic Extraction
Weigh 10-15 mg of freeze-dried pulverized material into a labeled, 2 mL screw-cap tube. Record the weight.
Add 1mL of 80% ethanol to each sample. A repeat pipettor is useful for large sample numbers.
Vortex to mix the samples until all powder dissolved.
Incubate the samples at 80°C in water bath for 0h 20m 0s.
Centrifuge the tubes at max speed (>15,000 g) for 0h 3m 0s to precipitate solids.
Decant the supernatant into alabeled 8 mL tube.
Repeat steps 2-6 four to six times depending on the tissue/plant. All the supernatants from the same sample should be combined into the correctly labeled 8 mL tube after each centrifugation.
Store 8 mL supernatant tubes 80On ice or at 4°C while tissue is incubating at 80°C.
Total Sugar Resuspension
After combining the last ethanolic supernatant, remove the ethanol using a Speed Vac Concencentrator, following manufacturers recommendations.
Dry samples on Speed Vac Concentrator just until dry and all traces of ethanol have evaporated.
Add 1mL of distilled or MilliQ water to the dried sugars in the tube.
If using polystyrene 8 mL tubes, transfer the aqueous sugar solution to a polypropylene, polyethylene, or other organic solvent resistant tube.
Vortex the tube for 30 seconds. Use a pasteur pipette to pipette the liquid vigourously ensuring all sugars in the tube have dissolved in the water.
Leave the tubes in an ice bath on a plate shaker for 1h 0m 0s to help dissolve all the sugar.
Transfer the entire aqueous sugar solution to a new labeled chemical resistant tube such as a 2 mL polypropylene or polyethylene microcentrifuge tube.
Add 500uL of chloroform.
Vortex.
Centrifuge the tubes at max speed (>15,000 g) for 0h 1m 0s to separate the solvent phases.
Transfer only the aqueous phase (upper phase) to a new labeled 2 mL tube.
Store at -20°C up to several months.
Starch Precipitate
After removing the last ethanolic supernatant, leave the lids off and dry the remaining pellet at 40°Covernight
After the pellet is dry, replace the lids. The dried starch pellet can be stored at room temperature.
