Oxygen Electrode Determination of Wheat Embryo Respiratory Activity
Experiment preparation:
Equipment: Oxytherm oxygen electrode system; scissors, double-sided knife (or single-sided cutter or scalpel), 1ml pipette, tweezers, pipette, absorbent paper, 3 beakers
Reagents: Determination medium 50mmol / L Tris-HCl buffer solution pH7.5. Weigh out 6.05 grams of Tris (trishydroxymethylaminomethane) 800-900ml of water, dropwise add 1mol / L HCl, adjust the pH to 7.5, and add 1000ml of water. In addition, the measurement medium can also use phosphoric acid, Hepes, Tricine and other buffers with pH7.5 50mmol / L.
Experimental steps:
1 Prepare the required instruments and reagents, install the aerobic electrode, connect it to the computer, and calibrate (see the instruction manual or Chinese manual provided by the manufacturer for detailed operation steps), add 2mL buffer solution after calibration, turn on the rotor, and go to the baseline (see Whether the baseline is stable, the baseline must be stable before measurement).
2 Evenly select ten wheat seeds and cut the embryo with a scalpel
3 Aspirate the buffer solution from the reaction cup, re-add 2mL of buffer solution, put the cut embryo into the reaction cup, the measurement curve is as follows:
4 Ten minutes after the measurement, the respiration rate decreased steadily, and the slope of the curve (respiration rate) was calculated using software. Suppose the slope of the curve is A.
5 After the measurement is completed, suck the sample out, wash the reaction cup, re-add the wheat germ, and repeat step 3
6 Calculation of the respiration rate of wheat seed embryos:
Respiratory rate Sr (umol O2 / Per * s) = curve slope A (umol O2 / ml * s) * 2ml = A / 300 (umol O2 / Per * s)
Note: Suppose the slope of the curve is A; Per represents each embryo
Precautions:
1 First refer to the precautions for the use of oxygen electrodes
2 When measuring the activity of the embryo, only the embryo needs to be cut off, and the whole seed need not be measured. Only the embryo in the seed is active
3 Select the wheat seeds to be uniform and full, the amount of each measurement is consistent
The instruments that can complete the experiment are: Oxygraph; Oxytherm; Chlorolab-2
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