Quantitative determination of erythromycin-like substances have many methods: such as barley dextrin layer α-amylase-induced formation method, acid mold leaf greening method, wheat yellowing seedling first leaf base cut off elongation method, the second leaf leaf sheath elongation of the rice seedling, "drip method" and so on. Among them, the rice seedling method is better. This method utilizes the important physiological property of gibberellin to stimulate the elongation of internodes in young plants. Within a certain concentration range (0.1-100 pp M), the elongation of leaf sheaths is proportional to the concentration.
Operation method
Measurement of gibberellin concentration
Principle
Quantitative determination of erythromycin-like substances have many methods: such as barley dextrin layer α-amylase-induced formation method, acid mold leaf greening method, wheat yellowing seedling first leaf base cut off elongation method, the second leaf leaf sheath elongation of the rice seedling, "drip method" and so on. Among them, the rice seedling method is better. This method utilizes the important physiological property of gibberellin to stimulate the elongation of internodes in young plants. Within a certain concentration range (0.1-100 pp M), the elongation of leaf sheaths is proportional to the concentration.
Materials and Instruments
Rice Seeds Move I. Materials and equipment Fig. 1 Placement of selected seeds with bud sheaths up to 2 mm long Fig. 2 Carefully place 1 µl of acetone solution of GA3 Figure 3 Length of the second leaf sheath measured at the end of the experiment For more product details, please visit Aladdin Scientific website.
Beakers Pressure cookers Glass cases Glass rulers
Rice seeds
Glass cup or beaker of about 6 cm height and 4 cm diameter, autoclave, square glass box of about 30 cm side length and 15 cm height, 1 MM 3 micro syringe, glass ruler capable of differentiating 1 MM.
Medicines:
100pp M GA3:10 mg GA3 dissolved in 100 ml 50% acetone.
1% Agar: Cut up weighed agar, immerse in water at 1:100 (W/W) and sterilize at 15 lbs autoclave for 20 minutes. Pour the agar lysate into a glass before it solidifies, until the lysate reaches a height of 5 cm.
Saturated Bleach Solution
II. Experimental Procedure
1. Take rice seeds with full grains, sterilize them with bleach solution for 30 minutes, rinse them and then add appropriate amount of water so that it can cover the seeds and germinate them in the dark at 30°C for 2 days.
2. After the seeds are white, select seeds with a bud length of 2 M M, with the germ facing upwards, and arrange them on the agar gel in a small cup. Discharge about 10 seeds per cup (see Figure 6). The small cups were placed in a glass box with a lid and placed in a thermostatic incubator for 2 days at 30°C under 2000-6000 lux light.
on agar in small cups incubated under continuous light Drops between the bud sheath and the first leaf
3. Select seedlings with the tip of the second leaf just protruding from the first leaf and with a height of 0.9-1 cm, and remove those that do not conform.
4. Dilute 100 pp M of GA3 into 0.1, 1.0, 10, and 100 pp M of GA3 solution with 50% acetone.
5. Remove the small beakers from the thermostat incubator, number them, and treat them with 0.1, 1.0, 10, and 100 pp M of GA3, respectively, and the control with 50% acetone solution. The treatments were performed by carefully dropping the 3 M M 3GA3 solution between the germinal sheath and the leaf axil of the first leaf of the seedling with a microsyringe (see Fig. 7), without letting it slip. If the droplet slips, this seedling should be pulled out immediately. Repeat at least three cups for each concentration.
6. All the spotted cups should be incubated in a thermostatic incubator for another 3 days.
7. Determine the length of the leaf sheath of the second leaf (as in Figure 8).
Results
Plotting the logarithm of the GA3 concentration as the horizontal coordinate and the length of the second leaf sheath as the vertical coordinate, this is the standard curve, from which it is possible to find out how many concentrations of GA3 are equivalent to the activity of the gibberellins in the samples to be tested.