Experiments for the determination of free proline in plants
Experiments for the determination of free proline in plants
Plants grown under normal environmental conditions have low levels of free proline, but under adversity (e.g., drought, cold, salt, etc.) the free proline content in plants can increase 10-100 times, so it has been proposed that the free proline content can be used as an indicator of plant stress tolerance.
Operation method
Experiments for the determination of free proline in plants
Principle
Plants grown under normal environmental conditions have low levels of free proline in their bodies, but under adverse conditions (such as drought, cold, salt, etc.) the free proline content in plants can increase by 10-100 times, so it has been proposed that the free proline content can be used as an indicator of plant stress tolerance. Free proline in plants is extracted with sulfosalicylic acid or alcohol. Under acidic conditions, proline and ninhydrin react to form a stable red product, and the content of proline is measured colorimetrically at 520n M wavelength.
Materials and Instruments
Wheat Move I. Instrumentation For more product details, please visit Aladdin Scientific website.
Acid ninhydrin Proline standard solution Glacial acetic acid Toluene
Spectrophotometer Analytical balance Constant temperature water bath Mason jar Volumetric flask Stoppered test tube Pipette Funnel Filter paper
Spectrophotometer, analytical balance, constant temperature water bath, mortar, volumetric flasks, stoppered test tubes, pipettes, funnels, filter paper
Plugged test tubes: 25 ml 9 pcs.
Pipettes: 4 of 2 ml, 2 of 5 ml
Reagents
1. Acid ninhydrin: weigh 2.5 grams of ninhydrin, add 60 ml of glacial acetic acid and 40 ml of 6 M phosphoric acid, heated at 70 ℃ to dissolve. Cooled and stored in a brown reagent bottle for backup. 4 ℃ in 2 ~ 3 days effective.
Proline standard solution: weigh 0.0250 g of proline, dissolved in 250 ml of distilled water, the concentration of 100 μg/ml. Then take 10 ml of this solution and dilute it to 100 ml with distilled water, i.e. 10 μg/ml of proline standard solution.
3. Glacial acetic acid
4. Toluene
If extracting with sulfosalicylic acid, you need to prepare a 3% solution of sulfosalicylic acid. (If extracted with alcohol, 80% alcohol, activated charcoal, and artificial zeolite are required.)
Third, the material
Wheat or other crops growing under irrigation and drought conditions.
IV. Methodological steps
1. Make a standard curve: Take seven 25 ml stoppered test tubes and number them. Add Proline standard solution (containing 10 micrograms of proline per milliliter) 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 ml to each tube, and then make up the volume to 2 ml with distilled water, and then shake well, then it will be formulated into a standard series containing 0, 2, 4, 8, 12, 16, 20 micrograms of proline respectively. Then 2 ml each of glacial acetic acid and acid ninhydrin were added to each tube. After shaking well, it was heated in a boiling water bath for 30 min to develop the color, removed and cooled to room temperature, and 5 ml of toluene was added to each tube with sufficient shaking to extract the red product. After extraction (leave for more than 4 hours away from light), after complete stratification, pipette the toluene layer, determine the extinction value with a spectrophotometer at 520n M wavelength, and plot the standard curve with the proline content as the horizontal coordinate and the extinction value as the vertical coordinate.
2. Free proline extraction: Free proline in plants can be extracted with 3% sulfosalicylic acid. Sulfosalicylic acid extraction: weigh 0.5 g each of fresh and wilted wheat leaves, put the weighed sample into a test tube with a stopper, add 5 ml of 3% sulfosalicylic acid solution, immerse the test tube into a boiling water bath for 15 minutes, filter, and the filtrate is to be measured.
3. Determination: Pipette 2 ml of filtrate in a test tube, and standard concentration of proline series of solutions with the same method of color determination.
4. Calculation: The result is calculated according to the following formula 
Where: C = micrograms of proline from the standard curve;
V = total volume of extract (ml);
a = number of milliliters of extract taken for the determination;
W = dry weight of the sample in grams, 106 = conversion of grams to micrograms.