Test method for improving drought resistance of kiwi fruits based on trans-zeatin

Abstract

The invention belongs to the technical field of gardening, and discloses a test method for improving drought resistance of kiwi fruit based on trans-zeatin, which comprises the following steps: collecting germplasm resources of kiwi fruit, and breeding seedlings; carrying out natural drought treatment on the kiwi fruit rootstock seedlings, and carrying out trans-zeatin leaf spraying on the control group and the treatment group for three days before drought treatment; according to the method, morphological indexes and physiological and biochemical indexes after drought treatment are analyzed, drought resistance related index determination covers several main aspects of plant drought resistance characterization such as photosynthetic physiology, morphological adaptation, osmotic substance adjustment and antioxidant enzyme adjustment, the indexes are extremely representative, and the drought resistance of the kiwi fruit rootstock can be comprehensively and reliably represented; the external application of 50 [mu] M of trans-zeatin can significantly influence the photosynthesis, morphological growth, osmotic substance regulation and active oxygen scavenging ability of the macaque stock, thereby promoting the improvement of drought resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of horticulture, and particularly relates to an experimental method for improving the drought resistance of kiwifruit based on trans-zeatin. Background Art

[0002] Kiwifruit is a fruit crop with high added value in the world, and China is the world's largest kiwifruit producer. Kiwifruit is very sensitive to drought stress. It has low axial xylem resistance, high plant hydraulic conductivity, and high transpiration rate. It is a drought-intolerant fruit tree that is sensitive to water. Drought occurs in kiwifruit producing areas around the world, and is more prominent in the high-quality kiwifruit producing areas in northwest my country. It is one of the most important abiotic factors restricting the sustainable development of the global kiwifruit industry. Fruit tree rootstocks have a great effect on early fruiting, high yield, quality improvement and stress resistance of scion varieties. Cytokinins control the growth and development of plant organs, regulate the response of plants to the external environment, such as photosynthesis, osmotic regulation and active oxygen scavenging, and play an important role in responding to biotic and abiotic stresses. The research on exogenous cytokinins in crops such as wheat, rice, soybeans, and apples is relatively in-depth, and their application in production is also relatively common. However, there is little work on the effect of exogenous cytokinins on the drought resistance of kiwifruit rootstocks at home and abroad. Cytokinins are N6-adenine derivatives, which can be divided into isoprenoid cytokinins and aromatic cytokinins according to their side chain structures. In previous studies, it was found that the isoprenoid cytokinin trans-zeatin plays an important role in improving the drought resistance of kiwifruit rootstocks. Therefore, it is urgent to screen the concentration of exogenous zeatin suitable for improving the drought resistance of kiwifruit. Therefore, an experimental method based on trans-zeatin to improve the drought resistance of kiwifruit is proposed. Summary of the invention

[0003] The object of the present invention is to provide a test method for improving the drought resistance of kiwifruit based on trans-zeatin, so as to solve the problems raised in the above background technology.

[0004] To achieve the above object, the present invention provides the following technical solution: an experimental method for improving the drought resistance of kiwifruit based on trans-zeatin, comprising the following steps:

[0005] S1: Collect kiwifruit germplasm resources and breed seedlings;

[0006] S2: The kiwifruit rootstock seedlings were subjected to natural drought treatment, and the control group and the treatment group were sprayed with trans-zeatin leaves for three days before the drought treatment;

[0007] S3: Analysis of morphological and physiological and biochemical indices after drought treatment.

[0008] Preferably, in S1, the treatment duration of natural drought is 14 days, and the field water holding capacity is 25% to 30%.

[0009] Preferably, in said S1, the concentration of exogenously applied trans-zeatin includes 0uM, 25uM, 50uM and 100uM.

[0010] Preferably, the morphological indicators include: plant height, number of leaves, leaf area, longest root length, and number of roots.

[0011] Preferably, the physiological and biochemical indicators include: leaf photosynthesis related indicators net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO 2 concentration, chlorophyll a content, chlorophyll b content, carotenoid content, total chlorophyll content, ratio of chlorophyll a content to chlorophyll b content, leaf and root osmotic regulation factors and active oxygen related indicators active oxygen histochemical staining, hydrogen peroxide content, superoxide anion content, catalase content, superoxide dismutase content, peroxidase content, and malondialdehyde content.

[0012] Preferably, the determination of plant height, number of leaves, leaf area, longest root length, and number of roots comprises the steps of:

[0013] The height of the plants and the length of the longest root were counted by measuring tape and vernier caliper, the number of roots with a length ≥ 5 cm was counted, and the leaf area was measured by LI-3100 leaf area meter. Ten plants were randomly selected from each group of 30 plants and the average value was taken.

[0014] Preferably, the net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO 2 The method for determining the concentration comprises the steps of:

[0015] The CI RAS-3 portable photosynthesis / fluorescence measurement system was used to measure the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO from 9:00 to 11:00 on sunny days. 2 For concentration parameters, 10 plants were randomly selected from each group of 30 plants and the average value was taken.

[0016] Preferably, the calculation formula for the chlorophyll a content is:

[0017] Chlorophyll a content (mg / g) = (13.95*A665-6.88*A649)*V*D / m;

[0018] The calculation formula of the chlorophyll b content is:

[0019] Chlorophyll b content (mg / g) = (24.96*A649-7.32*A665)*V*D / m;

[0020] The calculation formula of the carotenoid content is:

[0021] Carotenoid content (mg / g) = ((1000D470-2.05*a-114.8*b) / 245)*V*D / m;

[0022] The calculation formula of the total chlorophyll content is:

[0023] Total chlorophyll content (mg / g) = chlorophyll a content + chlorophyll b content;

[0024] The calculation formula for the ratio of chlorophyll a content to chlorophyll b content is:

[0025] Chlorophyll a / b ratio = chlorophyll a content / chlorophyll b content;

[0026] Among them, A663, A645 and A470 represent the absorbance values ​​at 663nm, 645nm and 470nm respectively;

[0027] V is the volume of the extract, D is the dilution factor, m is the sample mass, and each treatment was repeated 3 times.

[0028] Preferably, the method for determining the hydrogen peroxide content is based on diaminobenzidine staining, and specifically includes:

[0029] Use leaves with the same growth status, soak them in diaminobenzidine dye solution, evacuate with a vacuum pump for 20 minutes, dye them overnight for 12 hours in the dark at room temperature, and then decolorize them. Pick leaves from the upper, middle and lower parts of the plant, freeze them with liquid nitrogen, and grind them into powder with a grinder. Weigh 0.20g of powder in each measurement tube, and determine the hydrogen peroxide content by visible spectrophotometry. Repeat each treatment step 3 times.

[0030] The method for determining the superoxide anion content is based on the nitro blue tetrazolium chloride method, which specifically includes:

[0031] Use leaves with the same growth state and soak them in nitro tetrazolium chloride blue dye solution. Use a vacuum pump to evacuate for 20 minutes, decolorize immediately, rinse the leaves with clean water for 3 times, decolorize them in 75% ethanol at 60°C for 2 hours, change the decolorizing solution until the decolorization is complete, and take pictures after the samples are soaked in 10% glycerol.

[0032] Leaves were picked from the upper, middle and lower parts of the plants, frozen with liquid nitrogen, and ground into powder using a sample grinder. 0.20 g of powder was weighed for each measuring tube. The superoxide anion content was determined by visible spectrophotometry. Each treatment step was repeated 3 times.

[0033] The content of the catalase was determined based on the ultraviolet absorption method, and each treatment step was repeated 3 times.

[0034] Preferably, the content of superoxide dismutase is determined based on the hydroxylamine method, and each treatment step is repeated 3 times;

[0035] The content of the peroxidase was determined based on the guaiacol method, the content of the malondialdehyde was determined based on the thiobarbituric acid method, and each treatment step was repeated 3 times.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The determination of drought resistance-related indicators in the present invention covers several main aspects of plant drought resistance characterization, such as photosynthetic physiology, morphological adaptation, osmotic substance regulation and antioxidant enzyme regulation. The indicators are highly representative and can represent the drought resistance of kiwifruit rootstocks in a relatively comprehensive and reliable manner. The external application of 50uM trans-zeatin can significantly affect the photosynthesis, morphological growth, osmotic substance regulation and active oxygen scavenging ability of kiwifruit rootstocks, thereby promoting the improvement of drought resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is a schematic diagram of a control group and a comparison diagram of an embodiment of the present invention;

[0039] Figure 2 Schematic diagram of the comparison of morphology and physiological and biochemical indicators of an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] See also Figure 1-Figure 2 As shown, the present invention provides the following technical solutions:

[0042] Two-year-old 'Meixian No. 1' kiwifruit cuttings were selected as materials. When selecting materials, attention should be paid to ensuring that the growth potential of the identified materials is basically consistent to ensure the accuracy of the measurement results.

[0043] The application of exogenous trans-zeatin includes the following steps:

[0044] S1: Before natural drought treatment, all groups of kiwifruit rootstocks were sprayed with trans-zeatin for three days at concentrations of 0uM, 25uM, 50uM, and 100uM.

[0045] S2: In order to facilitate comparative experiments, the kiwifruit rootstock control group was watered normally, and the treatment group was subjected to natural drought for 14 days. Figure 1 shown.

[0046] S3: Determination of morphological and physiological and biochemical indicators.

[0047] In addition, in the present invention, the above-mentioned morphological and physiological and biochemical index determinations specifically include the following steps:

[0048] After 14 days, the samples of the control group and the treatment group were tested for morphological and physiological and biochemical indicators. The height of the plant and the length of the longest root were counted with a tape measure and a vernier caliper, and only the roots with a length of ≥5 cm were counted. The leaf area was measured using an LI-3100 leaf area meter, and 10 plants were randomly selected from each group of 30 plants to take the average value.

[0049] In addition, in the present invention, it is necessary to measure the net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO 2 concentration, chlorophyll a content, chlorophyll b content, carotenoid content, total chlorophyll content, ratio of chlorophyll a content to chlorophyll b content, marked active oxygen histochemical staining, hydrogen peroxide content, superoxide anion content, catalase content, superoxide dismutase content, peroxidase content, and malondialdehyde content.

[0050] The specific measurement methods for the above indicators are as follows:

[0051] The CI RAS-3 portable photosynthesis / fluorescence measurement system was used to measure the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO from 9:00 to 11:00 on sunny days. 2 For concentration parameters, 10 plants were randomly selected from each group of 30 plants and the average value was taken.

[0052] The chlorophyll concentration was determined by acetone extraction. Take 0.5g of fresh leaves, cut them into pieces, and extract them with 5ml of 80% acetone overnight in the dark. Using 80% acetone solution as a control, measure the absorbance of the extract solution at 663nm, 645nm and 470nm respectively.

[0053] In addition, in the present invention, the determination formulas for the above-mentioned chlorophyll a, chlorophyll b content, carotenoid content, total chlorophyll content, and chlorophyll a / b ratio are as follows:

[0054] The formula used is chlorophyll a content (mg / g) = (13.95*A665-6.88*A649)*V*D / m;

[0055] Chlorophyll b content (mg / g) = (24.96*A649-7.32*A665)*V*D / m;

[0056] Carotenoid content (mg / g) = ((1000D470-2.05*a-114.8*b) / 245)*V*D / m; total chlorophyll content (mg / g) = chlorophyll a content + chlorophyll b content, chlorophyll a / b ratio = chlorophyll a content / chlorophyll b content, calculate chlorophyll content and other indicators. Among them, A663, A645 and A470 represent the absorbance values ​​at 663nm, 645nm and 470nm respectively. V is the volume of the extract, D is the dilution factor, m is the sample mass, and each treatment is repeated 3 times.

[0057] The diaminobenzidine staining method is used to detect the content of hydrogen peroxide in plants. Leaves with the same growth state are used, immersed in the diaminobenzidine dye solution, vacuumed for 20 minutes with a vacuum pump, and dyed overnight for 12 hours at room temperature in the dark, and then decolorized. The nitro tetrazolium chloride blue method is used to detect the content of superoxide anions in plants. Leaves with the same growth state are used, immersed in the nitro tetrazolium chloride blue dye solution, vacuumed for 20 minutes with a vacuum pump, and decolorized immediately. Decolorization: Rinse the leaf discs with clean water 3 times, decolorize them in 75% ethanol at 60°C for 2 hours, replace the decolorization solution until the decolorization is complete, and take pictures after the samples are infiltrated with 10% glycerol.

[0058] Leaves were picked from the upper, middle and lower parts of the plant respectively, frozen with liquid nitrogen, and then ground into powder with a sample grinder. 0.20 g of powder was weighed in each measuring tube. The hydrogen peroxide content and superoxide anion content were determined by visible spectrophotometry; the catalase content was determined by ultraviolet absorption method; the superoxide dismutase content was determined by hydroxylamine method; the peroxidase content was determined by guaiacol method; the malondialdehyde content was determined by thiobarbituric acid method; each treatment was repeated 3 times.

[0059] The data in S3 above were statistically analyzed using Microsoft Excel 2007 and SPSS 22, and the average values ​​were visualized using a heat map.

[0060] like Figure 2 As shown in the results, under drought stress, exogenous application of 50uM trans-zeatin can significantly increase the plant height, leaf number, longest root length, chlorophyll a content, carotenoid content, total chlorophyll content, ratio of chlorophyll a content to chlorophyll b content, net photosynthetic rate, catalase content and peroxidase content in leaves of macaque rootstock, and reduce the hydrogen peroxide content, superoxide anion content and malondialdehyde content in leaves and roots, thereby affecting the photosynthesis, morphological growth, osmotic substance regulation and active oxygen scavenging ability of macaque rootstock, and promoting the improvement of the overall drought resistance of the plant.

[0061] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An experimental method for improving the drought resistance of kiwifruit based on trans-zeatin, characterized in that: The steps include: S1: Collect kiwifruit germplasm resources and breed seedlings; S2: The kiwifruit rootstock seedlings were subjected to natural drought treatment, and the control group and the treatment group were sprayed with trans-zeatin leaves for three days before the drought treatment; S3: Analysis of morphological and physiological and biochemical indices after drought treatment.

2. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 1, characterized in that: In S1, the natural drought treatment duration is 14 days, and the field water holding capacity is 25% to 30%.

3. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 1, characterized in that: In the S1, the concentrations of the externally applied trans-zeatin include 0uM, 25uM, 50uM and 100uM.

4. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 1, characterized in that: The morphological indices include: plant height, number of leaves, leaf area, longest root length, and number of roots.

5. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 1, characterized in that: The physiological and biochemical indicators include: leaf photosynthesis related indicators such as net photosynthetic rate, transpiration rate, stomatal conductance, intercellular CO2 concentration, chlorophyll a content, chlorophyll b content, carotenoid content, total chlorophyll content, ratio of chlorophyll a content to chlorophyll b content, leaf and root osmotic regulation factors and active oxygen related indicators such as active oxygen histochemical staining, hydrogen peroxide content, superoxide anion content, catalase content, superoxide dismutase content, peroxidase content, and malondialdehyde content.

6. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 4, characterized in that: The determination of plant height, number of leaves, leaf area, longest root length, and number of roots includes the following steps: The height of the plants and the length of the longest root were counted by measuring tape and vernier caliper, the number of roots with a length ≥ 5 cm was counted, and the leaf area was measured by LI-3100 leaf area meter. Ten plants were randomly selected from each group of 30 plants and the average value was taken.

7. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 5, characterized in that: The method for measuring the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration comprises the following steps: The CIRAS-3 portable photosynthesis / fluorescence measurement system was used to measure the net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration parameters between 9:00 and 11:00 a.m. on sunny days. Ten plants were randomly selected from each group of 30 plants, and the average value was taken.

8. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 5, characterized in that: The calculation formula of the chlorophyll a content is: Chlorophyll a content (mg / g) = (13.95*A665-6.88*A649)*V*D / m; The calculation formula of the chlorophyll b content is: Chlorophyll b content (mg / g) = (24.96*A649-7.32*A665)*V*D / m; The calculation formula of the carotenoid content is: Carotenoid content (mg / g) = ((1000D470-2.05*a-114.8*b) / 245)*V*D / m; The calculation formula of the total chlorophyll content is: Total chlorophyll content (mg / g) = chlorophyll a content + chlorophyll b content; The calculation formula for the ratio of chlorophyll a content to chlorophyll b content is: Chlorophyll a / b ratio = chlorophyll a content / chlorophyll b content; Among them, A663, A645 and A470 represent the absorbance values ​​at 663nm, 645nm and 470nm respectively; V is the volume of the extract, D is the dilution factor, m is the sample mass, and each treatment was repeated 3 times.

9. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 5, characterized in that: The method for determining the content of hydrogen peroxide is based on diaminobenzidine dyeing, and specifically includes: Use leaves with the same growth status, soak them in diaminobenzidine dye solution, vacuum pump for 20 minutes, dye them overnight for 12 hours in dark room temperature, and then decolorize them. Pick leaves from the upper, middle and lower parts of the plant, freeze them with liquid nitrogen, and grind them into powder with a grinder. Weigh 0.20g of powder in each measurement tube, and determine the hydrogen peroxide content by visible spectrophotometry. Repeat each treatment step 3 times. The method for determining the superoxide anion content is based on the nitro blue tetrazolium chloride method, which specifically includes: Use leaves with the same growth state and soak them in nitro tetrazolium chloride blue dye solution. Use a vacuum pump to evacuate for 20 minutes, decolorize immediately, rinse the leaves with clean water for 3 times, decolorize them in 75% ethanol at 60°C for 2 hours, change the decolorizing solution until the decolorization is complete, and take pictures after the samples are soaked in 10% glycerol. Leaves were picked from the upper, middle and lower parts of the plants, frozen with liquid nitrogen, and ground into powder using a sample grinder. 0.20 g of powder was weighed for each measuring tube. The superoxide anion content was determined by visible spectrophotometry. Each treatment step was repeated 3 times. The content of the catalase was determined based on the ultraviolet absorption method, and each treatment step was repeated 3 times.

10. The test method for improving drought resistance of kiwifruit based on trans-zeatin according to claim 5, characterized in that: The content of superoxide dismutase was determined based on the hydroxylamine method, and each treatment step was repeated 3 times; The content of the peroxidase was determined based on the guaiacol method, the content of the malondialdehyde was determined based on the thiobarbituric acid method, and each treatment step was repeated 3 times.

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