Determination of betaine content in plants
Betaine is a widely distributed cytocompatible substance, and many plants accumulate large amounts of betaine in their cells under salinity and drought stress, so betaine can be used as a biochemical indicator of plant stress tolerance. Therefore, betaine can be used as a biochemical indicator of plant stress tolerance. The chemical colorimetric method can be used to determine the content of betaine in plants. The purpose of this experiment is to learn the principle and method of determining betaine in plants by the chemical colorimetric method.
Principle
The basic principle for the determination of betaine in plants is that iodine reacts with tetravalent antimicrobial compounds (QACs) to form water-insoluble periodates, which are soluble in ethylene dichloride and have a maximum absorption at 365 nm. Depending on the pH range required for iodine precipitation of betaine and choline, the amount of glycine betaine is equal to the amount of tetravalent skeletonized compounds minus the amount of choline.
Operation method
Determination of betaine content in plants
Principle
The basic principle for the determination of betaine in plants is that iodine reacts with tetravalent antimicrobial compounds (QACs) to form water-insoluble periodates, which are soluble in ethylene dichloride and have a maximum absorption at 365 nm. Depending on the pH range required for iodine precipitation of betaine and choline, the amount of glycine betaine is equal to the amount of tetravalent skeletonized compounds minus the amount of choline.
Materials and Instruments
Material: Fresh spinach leaves or other plant leaves. Move The basic procedure for the determination of betaine content in plants can be divided into the following steps:1. Betaine extraction and purification: 1~2 g of spinach leaves were weighed and ground with 10 mL of betaine extract. The homogenate was held in a water bath at 60-70 P for 10 min. After cooling, the aqueous phase was collected by centrifugation at 1,000 g for 10 min at 20 °C. The chloroform phase was added with 10 mL of chloroform. The aqueous phase was collected by adding 10 mL of extract to the chloroform phase and centrifuging at 1,000 g for 10 min at 20 °C with repeated shaking. The lower chloroform phase was extracted with 4 mL of 50% methanol in water and centrifuged at 1,000 g for 10 min at 20 °C. The upper aqueous phases were combined, adjusted to pH 5.0-7.0, evaporated to dryness at 70 °C, and redissolved in 2 mL of water.2. Ion exchange purification: Add the sample to a Dowex 1 column (1 cmX5 cm, OH) or a mixture of Dowex 1 and Amberlite (1 + 2), wash the column with 5 times the column volume of water, and collect the effluent. The effluent was added directly to a Dowex 50 (1 cmX5 cm, FT) column and eluted with 5 times the column volume of water. The betaines were eluted with 4 mol-L'1 ammonia and the pH neutral effluent was collected, evaporated at 50-60 °C to remove the water, and dissolved in an appropriate volume of water.3. Determination： （1） Preparation of standard curve: Prepare standard curves for betaine and choline in the range of 10~400 mL'1.Standard curve for betaine: To prepare the QACs precipitation solution, 15.7 g of iodine and 20 g of KI were dissolved in 100 mL of 1 mol-L-undefined HCl solution, filtered, and stored at 4 °C for use. Add 0.5 mL of each concentration of standard solution to 0.2 mL of QACs precipitation solution and mix well. Hold at 0 °C for 90 min with intermittent shaking. Add 2 mL of pre-cooled water, quickly add 20 mL of pre-cooled dichloroethane at 10°C, shake vigorously at 4 bar for 5 min, and allow to stand at 4°C until the two phases are completely separated. Recover to room temperature, take the lower phase and measure the absorbance at 365 nm.Standard curve for choline: Same as above, but the reaction reagent can be replaced by choline precipitate solution. To prepare the choline precipitation solution, 15.7 g of iodine and 20 g of KI were dissolved in 100 mL of 0.4 mol - L_1 KH2PO4-NaOH buffer (pH 8.0) and filtered, and the filtrate was stored at 4°C for use.(2) Determination of samples: Measure the amount of tetravalent compounds and choline according to the standard curve.(3) Result: The content of glycine betaine is the difference between the amount of tetravalent compound and the amount of choline. Caveat The results would be more significant if the tested plants were pre-treated with osmotic stress. For more product details, please visit Aladdin Scientific website.
Materials: UV spectrophotometer, centrifuge, Dowex 1 column or Dowex 1 mixed with Aberlite (1 + 2), Dowex 50 column.
Reagents:
Methanol
②Chloroform
Iodine
④ Potassium iodide
⑤ Hydrochloric acid
⑥Dichloroethane
⑦KH
2
PO
4
⑧NaOH
⑨ Betaine extract was prepared in the ratio of methanol: chloroform: water = 12 : 5 : 3.