CN116636533B - Method for improving cold resistance of rubber sapling - Google Patents
Method for improving cold resistance of rubber sapling Download PDFInfo
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- CN116636533B CN116636533B CN202310930639.9A CN202310930639A CN116636533B CN 116636533 B CN116636533 B CN 116636533B CN 202310930639 A CN202310930639 A CN 202310930639A CN 116636533 B CN116636533 B CN 116636533B
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- 244000043261 Hevea brasiliensis Species 0.000 claims abstract description 69
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
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- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/22—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom rings with more than six members
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/14—Measures for saving energy, e.g. in green houses
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Botany (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Cultivation Of Plants (AREA)
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
Technical Field
The invention relates to the technical field of agricultural crop planting, in particular to a method for improving cold resistance of rubber seedlings.
Background
Climate is an important factor affecting the production of rubber trees, while cold damage is one of the main natural disasters of rubber trees, directly affecting yield and survival. Although rubber trees have been introduced into china, cold damage remains a bottleneck limiting the development of the gum planting industry. Therefore, the research on the cold-resistant mechanism of the rubber tree and the cultivation and screening of the cold-resistant planting material have important significance. Research has reported that plant hormones can improve the cold tolerance of plants. Brassinosteroids (BRs) are a plant hormone which can regulate various physiological processes of plants, and have been shown to improve cold resistance of plants such as arabidopsis, however, whether cold resistance of rubber trees can be improved has not been reported yet. Moreover, the problems of long conventional breeding period, less filial generation, high yield, difficult aggregation of cold-resistant characters and the like of the rubber tree result in deficient high-yield cold-resistant varieties, and the effective cold-resistant method for the rubber tree seedlings has important significance for the economic benefit of the gum planting industry. Of brassinosteroids, brassinolide (BL) is the most active compound, but the prior art has rarely been applied to improve the cold resistance of rubber seedlings.
The invention patent application of Chinese patent publication number CN105693836A, publication date of 2016, 6-month and 22-day and patent name of "a cold-resistant gene and protein of rubber tree and application thereof" discovers that HbICE1 gene responds to low-temperature stress to up-regulate expression, and the expression level in the rubber tree variety with strong cold resistance is obviously higher than that of the variety with weak cold resistance, and the transgenic verification of HbICE1 can enhance cold resistance, drought resistance, salt resistance and the like of transgenic plants.
The invention patent application with the Chinese patent publication number of CN112961229A, the publication date of 2021, 6 and 15 days, and the patent name of rubber tree transcription factor HbICE4, encoding gene and application thereof discovers that HbICE4 gene responds to low-temperature stress to up-regulate expression, the expression quantity of the gene is obviously higher than that of a variety with low cold resistance in a rubber tree variety with high cold resistance, and the transgenic verification of HbICE4 can enhance cold resistance, drought resistance, salt resistance and the like of transgenic plants. The method is used for finding a cold-resistant gene in a rubber tree, and over-expressing cold resistance found in model organisms, and can only be used as a cold-resistant gene resource, so far, the method cannot be directly applied to the rubber tree and can improve the cold resistance of the rubber tree.
The invention discloses a cold-proof method for rubber tree seedlings, which is disclosed in the patent application of Chinese patent publication No. CN102511344A, publication No. 2011, publication No. 6 and 27, and patent name of "a cold-proof method for rubber tree seedlings". The method is a physical cold-proof method, and the liquid cold-proof heat-insulating agent can lose effect once being washed away or disappeared with the lapse of time when meeting rain, and has short cold-proof time.
The invention discloses a cold-resistant agent for rubber trees, which has the main functions of spraying leaves and branches of bud grafting seedlings and sprouts of the rubber trees in advance and improving the cold resistance of the bud grafting seedlings and sprouts of the rubber trees, and discloses the cold-resistant agent for the rubber trees, wherein the Chinese patent publication number is CN106900728A, the publication date is 22 of 2017 and the publication date is 22 of 2017; and can enhance the recovery capability of cold damage of the cutting surface. The method is mainly applied to improving the cold resistance of the bud grafting, the bud strips and the rubber cutting surface of the rubber tree, and the cold resistance of the whole rubber tree seedling is unknown.
The invention discloses a cold-resistant regulator for effectively inhibiting rubber tree gumming and application thereof, and provides the cold-resistant regulator, wherein the active components of the cold-resistant regulator comprise magnesium sulfate, calcium chloride, chitosan, sodium fermet, abscisic acid, basil extract and palm oil. Under the condition of low-temperature stress, the reduction of chlorophyll content, net photosynthetic rate, intercellular carbon dioxide concentration and the like of the rubber tree leaves can be effectively slowed down, the rise of relative conductivity, malondialdehyde content and the like of phloem can be obviously slowed down, the content of soluble sugar of the phloem, the maximum photochemical efficiency of the leaf PSII, the actual photochemical efficiency of the leaf, the content of soluble protein of the phloem, the content of superoxide dismutase of the phloem, the activity of peroxidase of the phloem and the like of the phloem can be obviously improved, the damage of the low-temperature stress to cell membranes of the phloem of the rubber tree, the photosynthetic system of the leaf and the like can be obviously reduced, the gumming of the rubber tree can be inhibited, and the gumming can be reduced. The preparation process of the cold-resistant regulator is complex, and the cold-resistant regulator needs to be uniformly coated on the stalks of the rubber trees, so that the operation is complex.
The related prior art shows that the existing method for resisting cold of the rubber tree seedlings is very few, has the defects of short cold-resisting time effect of the cold-resisting regulator, complex preparation process, complex operation during application and the like, and is not beneficial to promoting the continuous healthy development of the rubber industry.
Disclosure of Invention
The invention provides a method for improving cold resistance of rubber seedlings in order to solve the problems.
The invention aims to provide a method for improving cold resistance of rubber sapling, which comprises 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;
s2, when 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;
and S3, spraying the cold-resistant regulator for 1 time every 1 day, spraying for 2-3 times, and co-culturing for 5-9 days to obtain the rubber tree seedlings with improved cold resistance.
Preferably, the cold resistance modifier is a solution containing brassinolide.
Preferably, the preparation method of the cold-resistant regulator comprises the following steps: adding brassinolide into 95% ethanol solution to prepare mother solution; diluting with water before spraying to obtain solution containing brassinolide.
Preferably, the concentration of brassinolide in the cold-resistant regulator is 50-200 nM/L.
Preferably, the concentration of brassinolide in the cold resistance regulator is 50nM/L.
Preferably, the molecular weight of brassinolide is 480.68g/M.
Preferably, the temperature in step S2 is 4-10 ℃.
Preferably, the humidity in step S2 is 60% and the light intensity is 100. Mu. Mol/m2.multidot.S.
Preferably, the cold-resistance regulator in step S3 is sprayed 3 times and cultured for 7 days.
Compared with the prior art, the invention has the following beneficial effects:
(1) In the method for improving the cold resistance of the rubber tree seedlings, the cold resistance regulator is simple to prepare, is safe and nontoxic, and has the functions of promoting the elongation of the rubber tree cells, improving the growth speed and the yield of the rubber tree seedlings and the like;
(2) The method of spraying the cold-resistant regulator is adopted, so that the cold-resistant regulator can be uniformly paved on the surfaces of the leaves and the stem tips, and waste caused by falling can be avoided;
(3) The cold resistance of the rubber tree seedlings can be enhanced in a short time, the freezing injury rate is reduced, and the growth speed and the yield of the rubber tree seedlings are improved;
(4) Under the condition of low temperature stress, the cold-resistant adaptation mechanism of the plant can be activated rapidly, the expression of the cold-resistant gene of the plant is increased, the content of protective enzyme is increased, the superoxide dismutase content, the catalase activity and the like of the rubber tree leaves can be remarkably improved, the rise of the relative conductivity, malondialdehyde, proline, hydrogen peroxide content and the like of the rubber tree leaves can be remarkably slowed down, the drop of the chlorophyll content of the rubber tree leaves is effectively slowed down, the damage of the low temperature stress to the rubber tree can be reduced, good social benefit and considerable economic benefit are achieved, and the continuous healthy development of the rubber industry is promoted.
Drawings
FIG. 1 is a phenotype diagram of a change in seedlings under low temperature stress after treatment with different concentrations of a cold resistance regulator provided according to an embodiment of the present invention; wherein BL represents brassinolide.
FIG. 2 is a graph showing the change of the content of physiological indexes related to cold stress before and after cold treatment according to an embodiment of the present invention; A. conductivity changes; B. malondialdehyde content; C. proline content; D. chlorophyll changes; E. hydrogen peroxide content.
FIG. 3 is a graph showing the relative expression level of cold stress genes after cold treatment according to an embodiment of the present invention.
FIG. 4 is a graph showing the effect of cold stress on the content of peroxidase SOD (A) and CAT (B) in rubber seedlings according to an embodiment of the present invention.
Detailed Description
Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the following description, like modules are denoted by like reference numerals. In the case of the same reference numerals, their names and functions are also the same. Therefore, a detailed description thereof will not be repeated.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention.
Example 1
A method for improving cold resistance of rubber seedlings specifically comprises the following steps:
s1, taking a single rubber tree tissue culture seedling with 4-5 months old and strong tree vigor as a test material, and spraying a cold-resistant regulator on the front and back surfaces of the leaves and the positions of the stem tips of the single rubber tree tissue culture seedling by using a spray bottle until a layer of water mist on the surfaces of the leaves is obviously observed; the cold resistance regulator is a working solution containing 50nM/L Brassinosteroids (BL);
s2, when the cold-resistant regulator volatilizes until no water mist is seen, placing the rubber tree seedlings in a 10 ℃ illumination incubator, setting the relative air humidity to be 60%, the illumination intensity to be 100 mu mol/(m2.S), and the photoperiod to be 16h/8h (day/night);
and S3, spraying the cold-resistant regulator for 1 time every 1 day for 3 times, and co-culturing for 7 days to obtain the rubber tree seedlings with improved cold resistance.
The preparation method of the cold-resistant regulator comprises the following steps: 2mg of brassinolide (molecular weight 480.68 g/M) was added to 1mL of a 95% ethanol solution to prepare 4.16077. Mu.M/L of a mother liquor, which was diluted with water before spraying to prepare a working solution containing 50nM/L of brassinolide.
Example 2
The influence of cold-resistant regulators with different concentrations on the post-rubber sapling under the condition of low-temperature stress is researched, and the specific method comprises the following steps:
the cold resistance regulator was formulated into 3 concentration gradients (50 nM/L, 100nM/L and 200 nM/L) and sprayed on the rubber seedling leaves and stem tips using the method of example 1 with no cold treatment as a control group (control); culturing rubber tree seedlings in an illumination incubator at 10 ℃, setting the relative air humidity to be 60%, and culturing the rubber tree seedlings in a light period of 16h/8h (day/night) with the illumination intensity of 100 mu mol/(m2.S), wherein photographing is carried out after 5 days;
as a result, as shown in FIG. 1, it was found from the graph that the rubber seedlings sprayed with the cold resistance regulator were significantly superior to the rubber seedlings not sprayed (control group), and that the optimum concentration was 50nM/L brassinolide-containing working fluid.
Example 3
The specific method and the specific result of the cold stress related physiological index content detection are as follows:
according to the apparent observation result in the example 2, the most obvious cold-resistant regulator concentration of 50nM/L is selected, the tissue culture seedling of the rubber tree with the age of four five months is taken as a material, the leaf of the rubber tree is sprayed on the stem tip by the cold-resistant regulator with the concentration of 50nM/L, the rubber tree seedling is placed in a 10 ℃ illumination incubator, the relative humidity of air is set to 60%, the illumination intensity is 100 mu mol/(m2.S), and the photoperiod is 16h/8h (day/night); taking 0d, 1d, 3d, 5d, 7d and 9d rubber tree leaves respectively, and measuring the contents of conductivity, malondialdehyde, proline, chlorophyll and hydrogen peroxide, wherein the results are shown in figure 2; the conductivity, malondialdehyde, proline and hydrogen peroxide contents of the sprayed cold-resistant regulator group are obviously lower than those of the non-sprayed cold-resistant regulator group (fig. 2E is a physiological and biochemical index measurement related to cold stress, and the hydrogen peroxide is taken as 4d and 6d according to the general DAB method), so that the chlorophyll content is obviously higher than that of the non-sprayed cold-resistant regulator group.
Example 4
The influence of cold stress on the expression quantity of the cold stress genes of the rubber seedlings is studied, and the specific method and the specific result are as follows:
based on the apparent pattern observation in example 2, the most obvious cold-resistant regulator was screened for a concentration of 50nM/L, four to fiveThe tissue culture seedling of the rubber tree of month age is used as a material, the cold-resistant regulator with the concentration of 50nM/L is sprayed on the leaf of the rubber tree to the stem tip, and the rubber tree seedling is placed at the temperature of 4 ℃, so that the RNA expression is faster, shorter, lower temperature stimulation is required, and the result is more obvious; taking 0, 3, 6 and 12 hours rubber tree leaves, grinding with liquid nitrogen to extract RNA, reverse transcribing cDNA, and identifying cold stress genes by fluorescent quantitative PCRHbCBF1、HbCBF2、HbCBF3、HbCOR、HbICE1AndHbICE2is a factor (B) of the expression level of (C).
FIG. 3 is a graph showing the relative expression level of cold stress genes after cold treatment, wherein cold represents that untreated rubber seedlings are subjected to cold stress, and cold+BL represents that rubber seedlings treated with a cold resistance regulator are subjected to cold stress; the graph shows that the cold stress genes of the sprayed cold-resistant regulator group are obviously higher than those of the non-sprayed cold-resistant regulator group, so that BL can quickly activate the cold-resistant adaptation mechanism of plants under the condition of low temperature stress, the expression of the cold-resistant genes of the cold stress genes of the plants is increased, and the cold tolerance of rubber seedlings is enhanced.
Example 5
The influence of cold stress on the content of peroxidase in rubber seedlings is studied, and the specific method and the result are as follows:
according to the apparent observation results in example 2, the most obvious cold-resistant regulator concentration of 50nM/L was selected, the tissue culture seedlings of rubber trees of the age of four five months were used as materials, the cold-resistant regulator of 50nM/L was sprayed on the leaves and the tips of the stems, the rubber seedlings were placed at 10 ℃, the leaves of the rubber trees of 0d, 1d, 3d, 5d and 7d were taken, and after grinding with liquid nitrogen, the SOD and CAT contents were measured with the kit.
Results: FIG. 4 shows a graph of peroxidase content after cold treatment, wherein cold represents untreated rubber seedlings subjected to cold stress, and cold+BL represents rubber seedlings treated with a cold resistance regulator subjected to cold stress; from the figure, it can be seen that the SOD and CAT content of the sprayed cold-resistant modifier group is significantly higher than that of the non-sprayed cold-resistant modifier group. The SOD content of the sprayed cold-resistant modifier group is obviously higher than that of the non-sprayed cold-resistant modifier group in the cold treatments 1d, 3d, 5d and 7d, and the CAT content of the cold treatment 1d is not obviously different between the sprayed cold-resistant modifier group and the non-sprayed cold-resistant modifier group, but the CAT content of the sprayed cold-resistant modifier group is obviously higher than that of the non-sprayed cold-resistant modifier group in the cold treatments 3d, 5d and 7 d.
In conclusion, after the cold-resistant regulator is sprayed on the rubber tree seedlings, the cold resistance of the rubber tree is obviously improved, and the method has high feasibility and can be widely applied to the production practice of the rubber tree seedlings.
It should be appreciated that various forms of the flows shown above may be used to reorder, add, or delete steps. For example, the steps described in the present disclosure may be performed in parallel, sequentially, or in a different order, provided that the desired results of the technical solutions of the present disclosure are achieved, and are not limited herein.
The above embodiments do not limit the scope of the present invention. It will be apparent to those skilled in the art that various modifications, combinations, sub-combinations and alternatives 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 should be included in the 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.
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