The proline content in plants increases when they are dehydrated. The proline content in plants reflects the water situation in plants to a certain extent, and thus can be used as a reference physiological indicator of water deficiency in plants.

Using artificial zeolite (Permutit) in the pH1-7 range of oscillation solution can remove the interference of amino acids or make it not react with ninhydrin (such as glycine, glutamic acid, aspartic acid, alanine, valine, cystine, phenyl stalked ammonia, arginine, etc.), proline and ninhydrin reagent is a chromogenic reaction, the content of its color with the color of the darkness of the color is positively correlated with, can be measured by spectrophotometer. The method is specific.

Operation method

Experiments to determine the degree of water deficit in plants

Principle

The proline content in plants increases when they are dehydrated. The proline content in the plant reflects the water situation in the plant to some extent, and thus can be used as a reference physiological indicator of water deficiency in plants. Using artificial zeolite (Permutit) in the pH1-7 range of oscillation solution can remove the interference of amino acids or make it not react with ninhydrin (such as glycine, glutamic acid, aspartic acid, alanine, valine, cystine, phenyl stalked ammonia, arginine, etc.), proline and ninhydrin reagent is a chromogenic reaction, the content of its color with the color of the darkness of the color is positively correlated with, can be measured by spectrophotometer. The method is specific.

Materials and Instruments

721 spectrophotometer water bath centrifuge pipette beaker volumetric flask mortar glacial acetic acid stoppered test tube Proline 80% ethanol ninhydrin reagent

Move

II. Instrumental drugs

(1) 721 spectrophotometer; (2) water bath; (3) centrifuge; (4) pipette; (5) beaker; (6) volumetric flask; (7) mortar and pestle; (8) glacial acetic acid; (9) stoppered test tubes; (10) proline; (11) 80% ethanol; (12) ninhydrin reagent: 2.5 g of ninhydrin was dissolved in 60 ml of glacial acetic acid and 40 ml of 6M phosphoric acid, and heated (70 °C) Dissolve and the reagent was stabilized for at least 24 hours.

III. Experimental steps

1. Draw the standard curve

Take proline dissolved in 80% ethanol, and then diluted to the following concentrations: 0, 1.0, 2.5, 5.0, 7.5, 10, 15, 20, 25, 30 μg/ml. 2 ml of standard solution, 2 ml of glacial acetic acid, 2 ml of ninhydrin reagent were added to the test tube, and then tightly corked with a rubber stopper, and heated in a boiling water bath for 15 minutes. The colorimetric determination was carried out with a spectrophotometer at a wavelength of 515 nm, with concentration 0 as the blank control. The results were analyzed by using proline concentration as the horizontal coordinate and optical density as the vertical coordinate to make a standard curve.

2. Extraction of plant sample solution

Selected plant functional leaves 2g, grinding with 3ml 80% ethanol (put a little quartz sand), into a pulp, wash the mortar with 80% ethanol, the extract and washings were placed in a test tube, 80% ethanol total 10ml. cover and put in the dark at room temperature to extract for 24 hours or water bath.

The extract was filtered on filter paper placed with activated charcoal and the filtration was repeated once to remove the pigment and residue. The extract was placed in a test tube with 1/5 of its weight of artificial zeolite and strongly shaken for 5 minutes. The supernatant was centrifuged in a centrifuge for 10 minutes and the supernatant was prepared.

3. Determination of sample solution

The supernatant obtained after centrifugation, take 2 ml in a test tube, then add 2 ml glacial acetic acid and 2 ml ninhydrin reagent, sealed with a lid, and heated on a boiling water bath for 15 minutes. 3ml of 80% ethanol was used as control instead of sample solution. Colorimetric determination was carried out on a spectrophotometer at 515 nm. The optical density was read and the proline content of the sample solution was found from the coordinate curve.

4. Calculation of results:

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