Experiments on the greening effect of cytokinins on isolated cotyledons
Experiments on the greening effect of cytokinins on isolated cotyledons
To understand the greening effect of cytokinins on isolated cotyledons and the basic principles and methods.
Operation method
Experiments on the greening effect of cytokinins on isolated cotyledons
Principle
Cytokinins can hinder the production of nucleic acid hydrolases and protein hydrolases, so they can delay organ senescence and keep isolated leaves green. The strength of the greening effect can be measured by determining the change in chlorophyll content, or by the time of yellowing and dying of the leaf color.
Materials and Instruments
Cucumber cotyledons Radish cotyledons Move I. Materials, instrumentation and reagents For more product details, please visit Aladdin Scientific website.
16-BA solution 95 % ethanol CaCO3 powder
Electronic analytical balance Spectrophotometer Constant temperature incubator Constant temperature water bath 25 ml volumetric flasks 25 ml graduated test tube racks Test tube racks Pipettes Petri dishes for cucumber cotyledons Porcelain plates Blades Pointed tweezers Filter paper Pipette ear balls
1. Materials: cucumber cotyledons, radish cotyledons.
2. Instrumentation: electronic analytical balance, spectrophotometer, constant temperature incubator, constant temperature water bath, 25 ml volumetric flasks, 25 ml graduated test tubes, test tube racks, pipettes, Petri dishes of cucumber cotyledon leaves (diameter 5 cm), porcelain plates, razor blades, pointed tweezers, filter paper, absorbing earballs and so on.
3. Reagents and preparations: 100 μg- ml-16-BA solution, 95 % ethanol, CaCO3 powder.
Preparation of 100μg- ml-16-BA mother liquor: accurately weigh 10mg of 6-BA, add a few drops of 1mol-L-1HC1 to dissolve (can be slightly heated to dissolve), and then slowly add distilled water to dilute the volume to 100 ml.
Experimental steps
1. Cultivation of cucumber seedlings
Selected cucumber seeds, sown into a ceramic plate lined with filter paper, add distilled water to make it moist (note that the seeds should not be impregnated), wait for the seeds to sprout, and then cultivate them under the light for about 2-3d until the cotyledons are green. Spare.
2. Series of concentration 6-BA solution preparation: absorb 100 μg - ml - 16-BA mother solution, diluted into 10, 5, 0.5
μg- ml-1 of series concentration solution.
3. Culture of isolated cucumber cotyledons
3.1 Four sets of Petri dishes were taken, numbered, and each set of Petri dishes was padded with a piece of filter paper for four treatments of 0.5, 5, and 10 μg- ml-1 of 6-BA solution and distilled water (CK), i.e., the first set.
3.2 Twenty cucumber seedlings with uniform cotyledon size and growth were selected, 5 plants (10 cotyledons) per treatment, and the cotyledons without petioles were cut off with a razor blade, weighed freshly separately, and the data were recorded in the table below. The cotyledons were put into the first group of petri dishes with different concentrations of 6-BA solution (3 ml) and the control (CK) with distilled water (3 ml), then covered with petri dish lids and placed in a dark place at a temperature of 25-28°C for 3 d. The cotyledon was then incubated in a dark place at a temperature of 25-28°C for 3 days.
In addition, 20 cucumber seedlings of the same type as the first group were taken, and the cotyledons without petioles were cut off with a razor blade for the second group, which was used to determine the chlorophyll content directly.
4. Determination of chlorophyll content of cucumber cotyledons in the second group
4.1 Extraction of chlorophyll
Take three 25 ml graduated test tubes and number them. Weigh the cucumber cotyledons of the second group in three portions (three replicates), 10 cotyledons per portion, and then put them into the corresponding numbered graduated test tubes, each adding a small amount of CaCO3 powder and 95% ethanol 3-4 ml, and then carefully crush the cotyledons with a glass rod and put the test tubes into the 45 ℃ water bath to extract the chlorophyll for about 30 min until the cotyledons are colorless, and then filter it through a filter paper and put it into a 25 ml volumetric flask (if the residue still has a greenish color). Filter through filter paper into a 25 ml volumetric flask (if the residue is still green, add a few more ml of ethanol to continue extraction until the residue is not green). The pigment on the filter paper was washed into the volumetric flask with ethanol slowly and dropwise, and then finally it was fixed to the scale, shaken well, to be measured.
4.2 Determination of chlorophyll content
The absorbance (A value) of the chlorophyll extract was measured at 652 nm with 95 % ethanol as blank control. And the chlorophyll content was calculated according to the formula. It is the original chlorophyll content of cotyledons before culture.
5. Determination of chlorophyll residue in cucumber cotyledons of the first group
After the first group of cucumber cotyledons were cultured in the dark for 3d, the chlorophyll content of cotyledons of each treatment was determined according to the method of step (4) above, and the chlorophyll content of each treatment was calculated according to the formula, which was the chlorophyll content of cotyledons after dark culture.
Calculation of results
Chlorophyll content was calculated by the formula: 
IV. Recording results
Record the experimental data and analyze the results according to the table below 