CN116636533B - Method for improving cold resistance of rubber sapling - Google Patents

Abstract

The invention relates to the technical field of agricultural crop planting, in particular to a method for improving cold resistance of rubber seedlings. The method specifically comprises the following steps: taking rubber seedlings with 4-5 months old and strong tree vigor, spraying cold-resistant regulator on the front and back surfaces of the leaves and the stem tip positions of the rubber seedlings, and spraying a layer of water mist on the surfaces of the leaves of the rubber seedlings; the cold-resistant regulator is a solution containing brassinolide; after the cold-resistant regulator volatilizes until no water mist is seen, culturing the rubber tree seedlings under the conditions that the temperature is 0-10 ℃, the relative humidity is 50-70%, the illumination intensity is 80-150 mu mol/m2.S and the photoperiod is 16h/8 h; spraying the cold-resistant regulator for 1 time every 1 day for 2-3 times, and co-culturing for 5-9 days to obtain the rubber tree seedlings with improved cold resistance. The advantages are that: the cold-resistant regulator is simple to prepare, safe and nontoxic, and can enhance the cold resistance of the rubber tree seedlings in a short time and reduce the freezing injury rate.

Description

A method to improve the cold resistance of rubber saplings

Technical field

The invention relates to the technical field of agricultural crop planting, and in particular to a method for improving the cold resistance of rubber saplings.

Background technique

Climate is an important factor affecting rubber tree production, and cold damage is one of the main natural disasters for rubber trees, directly affecting yield and survival. Although rubber trees have been introduced into China, cold damage is still a bottleneck restricting the development of the rubber planting industry. Therefore, it is of great significance to study the cold-resistant mechanism of rubber trees and cultivate and screen cold-resistant planting materials. Studies have reported that plant hormones can improve the cold tolerance of plants. Brassinosteroids (BRs) are a type of plant hormone that can regulate various physiological processes of plants. They have been shown to improve the cold resistance of plants such as Arabidopsis thaliana. However, it has not been shown whether it can improve the cold resistance of rubber trees. Report. Moreover, problems such as long conventional breeding cycles of rubber trees, few hybrid generations, and difficulty in aggregating high-yielding and cold-resistant traits have led to a shortage of high-yield and cold-resistant varieties. Effective cold-resistant methods for rubber tree seedlings are of great significance to the economic benefits of the rubber planting industry. Among the brassinosteroids, brassinolide (BL) is the most active compound, but it is rarely used in existing technology to improve the cold resistance of rubber saplings.

The Chinese patent publication number is CN105693836A, the publication date is June 22, 2016, and the invention patent application titled "A rubber tree cold-resistant gene and protein and its application" found that the HbICE1 gene up-regulates expression in response to low temperature stress, and in cold tolerance The expression level in rubber tree varieties with strong resistance is significantly higher than that in varieties with weak cold tolerance. Through transgene verification, HbICE1 can enhance the cold tolerance, drought tolerance and salt tolerance of transgenic plants.

The Chinese patent publication number is CN112961229A, the publication date is June 15, 2021, and the invention patent application titled "Rubber tree transcription factor HbICE4 and its encoding gene and application" found that the HbICE4 gene up-regulates expression in response to low temperature stress, and plays a role in cold tolerance. The expression level in strong rubber tree varieties is significantly higher than that in weak cold-tolerant varieties. Through transgene verification, HbICE4 can enhance the cold tolerance, drought tolerance and salt tolerance of transgenic plants. This method is the discovery of a cold-resistant gene in a rubber tree, and it overexpresses the cold-resistant gene found in model organisms. It can only be used as a cold-resistant genetic resource. So far, it has not been directly applied to rubber trees to improve it. of cold resistance.

The Chinese patent publication number is CN102511344A, the publication date is June 27, 2011, and the invention patent application titled "A Method for Cold-proofing Rubber Tree Seedlings" discloses a method for cold-proofing rubber tree seedlings, which uses liquid cold-proof insulation agent to be evenly sprayed A cold insulation layer is formed on the leaves and branches of the rubber tree seedlings to block the impact of low temperature on the rubber tree seedlings and reduce cold damage to the rubber tree seedlings. This method is a physical method of cold protection. Once the liquid cold-proof insulation agent is washed away by rain or disappears over time, it will lose its effect and the cold-resistant effect will be short.

The Chinese patent publication number is CN106900728A, the publication date is February 22, 2017, and the invention patent application titled "A rubber tree cold-resistant agent and its application" discloses a rubber tree cold-resistant agent whose main function is to Spraying the leaves and branches of rubber tree bud grafts and sprouts can improve the cold resistance of rubber tree sprouts and sprouts; and can enhance the recovery ability of the cut surface from cold damage. The modified method is mainly used to improve the cold resistance of rubber tree budding seedlings, sprouting strips and rubber tapping surfaces. The overall cold resistance of rubber tree seedlings is unknown.

The Chinese patent publication number is CN112674123A, the publication date is April 20, 2021, and the invention patent application is titled "A cold-resistant regulator that effectively inhibits the gum flow of rubber trees and its application", which discloses a method that effectively inhibits the gum flow of rubber trees. The method provides a cold-resistant regulator and its application. The method provides a cold-resistant regulator whose active ingredients include magnesium sulfate, calcium chloride, chitosan, sodium thiram, abscisic acid, basil extract and palm oil. Under low temperature stress conditions, it can effectively slow down the decline in chlorophyll content, net photosynthetic rate, intercellular carbon dioxide concentration, etc. of rubber tree leaves, extremely significantly slow down the increase in phloem relative conductivity, malondialdehyde content, etc., and can extremely significantly increase phloem soluble sugar content, leaf PSII maximum photochemical efficiency, leaf actual photochemical efficiency, phloem soluble protein content, phloem superoxide dismutase content, phloem peroxidase activity, etc., to reduce the damage to the phloem cell membrane and leaf photosynthetic system of rubber trees caused by low temperature stress, etc. Inhibit rubber tree gum flow and reduce gum flow. The cold-resistant regulator preparation process of this method is cumbersome and complex, and it needs to be evenly coated on the stems of the rubber tree, making the operation complicated.

Relevant prior art shows that there are very few cold-resistant methods for rubber tree seedlings, and the existing cold-resistant regulators have short cold-resistant effects, complicated preparation processes, and complex application operations, which are not conducive to promoting the continued health of the rubber industry. develop.

Contents of the invention

In order to solve the above problems, the present invention provides a method for improving the cold resistance of rubber saplings.

The object of the present invention is to provide a method for improving the cold resistance of rubber saplings, which specifically includes the following steps:

S1. Take a 4-5 month old rubber sapling with strong vigor, spray the cold-resistant regulator on the front and back surfaces of the leaves and the stem tips of the rubber sapling, and spray a layer of water mist on the surface of the leaves of the rubber sapling. ;

S2. After the cold-resistant regulator evaporates until no water mist is visible, place the rubber sapling at a temperature of 0 to 10°C, a relative humidity of 50 to 70%, a light intensity of 80 to 150 μmol/m2·S, and a photoperiod of Cultivate under the conditions of 16h/8h;

S3. Spray the cold-resistant regulator once every other day, spray 2 to 3 times, and culture for a total of 5 to 9 days to obtain rubber saplings with improved cold resistance.

Preferably, the cold resistance regulator is a solution containing brassinosteroid.

Preferably, the preparation method of the cold-resistant regulator is as follows: adding brassinosteroid to 95% ethanol solution to prepare a mother solution; diluting with water before spraying to prepare a solution containing brassinosteroid.

Preferably, the concentration of brassinosteroid in the cold resistance regulator is 50 to 200 nM/L.

Preferably, the concentration of brassinosteroid in the cold resistance regulator is 50 nM/L.

Preferably, the molecular weight of brassinosteroid is 480.68g/M.

Preferably, the temperature in step S2 is 4~10°C.

Preferably, the humidity in step S2 is 60% and the light intensity is 100 μmol/m2·S.

Preferably, the cold resistance regulator described in step S3 is sprayed three times and cultured for a total of 7 days.

Compared with the existing technology, the present invention can achieve the following beneficial effects:

(1) In the method of improving the cold resistance of rubber saplings of the present invention, the cold resistance regulator is simple to prepare, safe and non-toxic, and has the functions of promoting the elongation of rubber tree cells and increasing the growth rate and yield of rubber saplings;

(2) Use the method of spraying the cold-resistant regulator so that the cold-resistant regulator can be evenly spread on the surface of the leaves and stem tips without falling off and causing waste;

(3) The cold resistance of rubber tree seedlings can be enhanced in a short period of time, the frost damage rate can be reduced, and the growth rate and yield of rubber tree seedlings can be increased;

(4) Under low temperature stress, it can quickly activate the cold resistance adaptation mechanism of plants, increase the expression of cold stress genes of plants, and then increase the content of protective enzymes, which can significantly increase the superoxide dismutase of rubber leaves. content and catalase activity, etc., extremely significantly slow down the increase in relative conductivity, malondialdehyde, proline, hydrogen peroxide content, etc. of rubber tree leaves, and effectively slow down the decrease in chlorophyll content of rubber tree leaves, thereby reducing the impact of low temperature stress on rubber trees. It has good social benefits and considerable economic benefits, which in turn helps to promote the sustainable and healthy development of the rubber industry.

Description of the drawings

Figure 1 is a phenotypic diagram of the changes in saplings under low temperature stress after being treated with different concentrations of cold resistance regulators according to embodiments of the present invention; wherein, BL represents brassinosteroid.

Figure 2 is a diagram showing the changes in the content of physiological indicators related to cold stress before and after cold treatment according to the embodiment of the present invention; A. Changes in electrical conductivity; B. Malondialdehyde content; C. Proline content; D. Changes in chlorophyll; E. Process Hydrogen oxide content.

Figure 3 is a diagram of relative expression levels of cold stress genes after cold treatment provided according to an embodiment of the present invention.

Figure 4 shows the effect of cold stress on the contents of peroxidase SOD (A) and CAT (B) in rubber saplings according to an embodiment of the present invention.

Detailed ways

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, the same modules are designated with the same reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, its detailed description will not be repeated.

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention and do not constitute limitations of the present invention.

Example 1

A method for improving the cold resistance of rubber saplings, specifically including the following steps:

S1. Use a 4-5-month-old, strong rubber tree single plant tissue culture seedling as the test material. Use a spray bottle to spray the cold-resistant regulator on the front and back surfaces of the leaves and the stem tip until the surface of the leaves is clearly visible. until there is a layer of water mist; the cold-resistant regulator is a working solution containing 50nM/L brassinosteroid (BL);

S2. After the cold-resistant regulator evaporates until no water mist is visible, place the rubber sapling in a 10°C light incubator, set the relative air humidity to 60%, the light intensity to 100 μmol/(m2·S), and the photoperiod to 16h/8h. (day and night);

S3. Spray the cold-resistant regulator once every other day, spray three times, and culture for a total of 7 days to obtain rubber saplings with improved cold resistance.

The preparation method of the cold-resistant regulator is: add 2 mg of brassinosteroid (molecular weight: 480.68 g/M) into 1 mL of 95% ethanol solution to prepare a 4.16077 uM/L mother solution, and dilute it with water before spraying. into a working solution containing 50nM/L brassinosteroid.

Example 2

To study the effects of different concentrations of cold resistance regulators on rubber saplings under low temperature stress, the specific method is:

The cold-resistant regulator was prepared into three concentration gradients (50nM/L, 100nM/L and 200nM/L), and the non-cold treatment was used as the control group. The method of Example 1 was used to spray the cold-resistant regulator on the rubber. Seedling leaves and stem tips; place the rubber tree seedlings in a 10°C light incubator for culture, set the relative air humidity to 60%, the light intensity to 100 μmol/(m2·S), and the photoperiod to 16h/8h (day/night) for culture, 5 The diva takes photos;

The results are shown in Figure 1. From the figure, it can be found that the rubber saplings sprayed with cold-resistant regulators are significantly better than the rubber saplings not sprayed (control group), and the concentration with the best effect is containing 50nM/L brassinosteroids. Ester working fluid.

Example 3

To detect the content of physiological indicators related to cold stress, the specific methods and results are as follows:

According to the phenotypic observation results in Example 2, the most obvious cold resistance regulator concentration of 50nM/L was screened. Four or five-year-old rubber tree tissue culture seedlings were used as materials, and the cold resistance regulator at a concentration of 50nM/L was sprayed. Rubber seedling leaves are placed at the stem tips, and the rubber seedlings are placed in a 10°C light incubator with a relative air humidity of 60%, a light intensity of 100 μmol/(m2·S), and a photoperiod of 16h/8h (day/night); respectively The conductivity, malondialdehyde, proline, chlorophyll, and hydrogen peroxide content of the rubber tree leaves at 0d, 1d, 3d, 5d, 7d and 9d were measured. The results are shown in Figure 2; it was found that the conductivity of the group sprayed with cold-resistant regulator rate, malondialdehyde, proline, and hydrogen peroxide contents were significantly lower than those in the group that was not sprayed with cold-resistant regulators (Figure 2E is the measurement of physiological and biochemical indicators related to cold stress. Generally, the DAB method is used to measure hydrogen peroxide for 4 days. and 6d), the chlorophyll content was significantly higher than that of the group without cold-resistant regulator spraying.

Example 4

To study the effect of cold stress on the expression of cold stress genes in rubber saplings, the specific methods and results are as follows:

According to the phenotypic observation results in Example 2, the most obvious cold resistance regulator concentration of 50nM/L was screened. Four or five-year-old rubber tree tissue culture seedlings were used as materials, and the cold resistance regulator at a concentration of 50nM/L was sprayed. Rubber seedling leaves are placed at the stem tips. Place the rubber seedlings at 4°C. Because RNA expression is faster and shorter, it requires lower temperature stimulation and the results are more obvious. Take the rubber tree leaves at 0 hours, 3 hours, 6 hours and 12 hours. RNA was extracted by grinding in liquid nitrogen, and after reverse transcribing cDNA, fluorescence quantitative PCR was used to identify the expression levels of cold stress genes HbCBF1 , HbCBF2 , HbCBF3 , HbCOR , HbICE1 and HbICE2 .

Figure 3 is a diagram of the relative expression of cold stress genes after cold treatment, where cold indicates that untreated rubber saplings have experienced cold stress, cold+BL indicates that rubber saplings treated with cold resistance regulators have experienced cold stress; as can be seen from the figure The cold stress genes in the group sprayed with cold resistance regulators were significantly higher than those in the group not sprayed with cold resistance regulators, indicating that under low temperature stress, BL can quickly activate the cold resistance adaptation mechanism of plants and increase the expression of cold stress genes in plants. The expression of cold resistance genes enhances the cold tolerance of rubber seedlings.

Example 5

To study the effect of cold stress on peroxidase content in rubber saplings, the specific methods and results are as follows:

According to the phenotypic observation results in Example 2, the most obvious cold resistance regulator concentration of 50nM/L was screened. Four or five-year-old rubber tree tissue culture seedlings were used as materials, and the cold resistance regulator at a concentration of 50nM/L was sprayed. Put the rubber sapling leaves at the stem tip, place the rubber saplings at 10°C, take the rubber tree leaves on 0d, 1d, 3d, 5d and 7d, grind them with liquid nitrogen, and use a kit to measure the SOD and CAT content.

Results: Figure 4 shows the peroxidase content diagram after cold treatment, where cold indicates that the untreated rubber saplings have experienced cold stress, and cold+BL indicates that the rubber saplings treated with cold-resistant regulators have experienced cold stress; from the figure, it can be seen It was found that the SOD and CAT contents in the group sprayed with cold-resistant regulators were significantly higher than those in the group not sprayed with cold-resistant regulators. The SOD content in the group sprayed with cold-resistant regulators was significantly higher than that in the group not sprayed with cold-resistant regulators on 1d, 3d, 5d and 7d of cold treatment. On the 1st day of cold treatment, the CAT content was significantly higher in the group sprayed with cold-resistant regulators than in the group not sprayed with cold-resistant regulators. There was no significant difference between the groups sprayed with cold-resistant regulators, but at 3d, 5d and 7d of cold treatment, the CAT content in the group sprayed with cold-resistant regulators was significantly higher than that in the group not sprayed with cold-resistant regulators.

In summary, the cold resistance of rubber trees is significantly improved after spraying cold-resistant regulators on rubber saplings, proving that the invention is highly feasible and can be widely used in the production practice of rubber saplings.

It should be understood that various forms of the process shown above may be used, with steps reordered, added or deleted. For example, each step described in the disclosure of the present invention can be executed in parallel, sequentially, or in a different order. As long as the desired results of the technical solution disclosed in the present invention can be achieved, there is no limitation here.

The above-mentioned specific embodiments do not constitute a limitation on the scope of the present invention. It will be understood by those skilled in the art that various modifications, combinations, sub-combinations and substitutions are possible depending on design requirements and other factors. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (5)

1. A method for improving cold resistance of rubber sapling by improving cold stress gene expression quantity of rubber sapling under cold stress is characterized by comprising the following steps:

s1, taking rubber tree seedlings with 4-5 months of age and strong tree vigor, spraying cold-resistant regulator on the front and back surfaces of leaves and the stem tip positions of the rubber tree seedlings, and spraying a layer of water mist on the surfaces of the leaves of the rubber tree seedlings; the cold-resistant regulator is a brassinosteroid working solution, and the concentration of brassinosteroids is 50-200 nmol/L;

s2, after the cold-resistant regulator volatilizes until no water mist is seen, placing the rubber tree seedlings at the temperature of 10 ℃ and the relative humidity of 50-70%, and enabling the illumination intensity to be 80-150 mu mol/(m) ² S), culturing under conditions of 16h/8h photoperiod day-night;

s3, spraying the cold-resistant regulator for 1 time every 1 day, spraying for 2-3 times, and co-culturing for 5-7 days to obtain rubber seedlings with improved cold resistance;

the cold stress gene isHbCBF1、HbCBF2、HbCBF3、HbICE1。

2. The method according to claim 1, wherein the cold resistance regulator is prepared by the following steps: adding brassinolide into 95% ethanol solution to prepare mother solution; before spraying, the mixture is diluted by water to prepare the brassinolide working solution.

3. The method according to claim 2, wherein the concentration of brassinosteroids in the cold resistance modifier is 50nmol/L.

4. A method according to claim 3, wherein the humidity in step S2 is 60%, the light intensity is 100 μmol/(m) ² ·S）。

5. The method for improving cold resistance of rubber tree seedlings according to claim 4, wherein said cold resistance regulator in step S3 is sprayed 3 times and cultured for 7 days.