CN110089430B - Eucommia ulmoides seedling culture method - Google Patents
Eucommia ulmoides seedling culture method Download PDFInfo
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- CN110089430B CN110089430B CN201910349791.1A CN201910349791A CN110089430B CN 110089430 B CN110089430 B CN 110089430B CN 201910349791 A CN201910349791 A CN 201910349791A CN 110089430 B CN110089430 B CN 110089430B
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- 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
- A01G7/00—Botany in general
- A01G7/02—Treatment of plants with carbon dioxide
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- 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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
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- 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
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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- 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
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- 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
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention relates to a eucommia ulmoides seedling raising method, which comprises the following steps: pretreatment: taking new shoots of eucommia containing axillary buds, sterilizing, and cutting into stem segments containing axillary buds for later use; inoculation: inoculating the axillary bud-containing stem segments to a germination culture medium, and inducing germination; and (3) proliferating cluster buds: inoculating the germinated tissue to a subculture medium, and performing cluster bud proliferation to obtain a rootless bud section; strengthening and rooting seedlings: transferring the rootless bud segments into a container filled with mixed matrix of vermiculite and river sand, adding culture solution for culturing, and introducing CO during the culture process 2 Keeping illumination and controlling humidity to enable the rootless bud segments to grow roots to obtain eucommia ulmoides seedlings; transplanting: transplanting the seedling with root system. The method makes Eucommiae cortex seedling adapt to natural humidity from early stage, and improves survival rate of the seedling. Solves the problems of low survival rate of transplanted seedlings, easy water shortage and wilting of the traditional bottle seedling method due to long-term adaptation to high-humidity environment.
Description
Technical Field
The invention relates to the technical field of plant vegetative propagation, in particular to a eucommia seedling raising method.
Background
Eucommia ulmoides is the dried bark of eucommia ulmoides oliv of the family eucommia ulmoides and is a famous and precious Chinese medicinal material for nourishing. It is sweet in taste and warm in nature. Has effects in tonifying liver and kidney, strengthening muscle and bone, regulating Chong and ren meridians, and preventing miscarriage. Can be used for treating lumbago, leg pain or soreness and weakness due to kidney yang deficiency, and leukorrhagia and scrotum pruritus due to liver qi deficiency. It is listed as the superior in Shen nong Ben Cao Jing.
The modern research on eucommia bark has the effects of clearing away in-vivo garbage, strengthening the metabolism of human cell substances, preventing the aging of musculoskeletal tissues, balancing the blood pressure of a human body, decomposing in-vivo cholesterol, reducing in-vivo fat, restoring the elasticity of blood vessels, inducing diuresis, clearing heat, resisting bacteria in a broad spectrum, exciting central nerves and improving the pharmacological action of white blood cells.
With the increase of the demand on eucommia, the yield of the eucommia is difficult to continue, and the eucommia is used for asexual propagation seedling raising, so that the seedlings can be quickly grown and strong seedlings can be cultivated, and the effect of quickly improving the yield of the eucommia is achieved. However, in the conventional eucommia ulmoides seedling raising method, the problem of bacterial contamination often occurs, and a large amount of vermiculite and river sand are required to be used, which has the problem of high cost.
Disclosure of Invention
Therefore, it is necessary to provide a method for culturing eucommia ulmoides seedlings, which can greatly reduce the bacterial contamination rate of the traditional culture medium and improve the survival rate of the seedlings.
A eucommia ulmoides seedling raising method comprises the following steps:
pretreatment: taking new shoots of eucommia ulmoides containing axillary buds, sterilizing, and cutting into stem segments containing the axillary buds for later use;
inoculation: inoculating the axillary bud-containing stem segments to a germination culture medium, and inducing germination;
and (3) cluster bud multiplication: inoculating the germinated tissue to a subculture medium, and performing cluster bud proliferation to obtain a rootless bud section;
strengthening seedlings and rooting: transferring the rootless bud segments into a container filled with mixed matrix of vermiculite and river sand, adding culture solution for culturing, and introducing CO during the culture process 2 Keeping illumination and controlling humidity to enable the rootless bud segments to grow roots to obtain eucommia ulmoides seedlings;
transplanting: transplanting the seedling with root system.
The eucommia ulmoides seedling culture method is characterized in that CO is introduced in the process of strong seedling and rooting according to the growth characteristics of eucommia ulmoides 2 The method avoids adding sugar, can inhibit the growth of harmful bacteria, reduces the humidity of the container, enables the eucommia ulmoides seedlings to adapt to natural humidity from the early stage, and improves the survival rate of the seedlings. The traditional bottle seedling method is suitable for a high-humidity environment for a long time, so that the survival rate of transplanted seedlings out of bottles is low, and the seedlings are easy to lack water and wilting.
In one embodiment, in the pre-treatment step, the sterilization process is: soaking young shoots containing axillary buds of eucommia ulmoides in 10 + -2% hydrogen peroxide solution for 5 + -2 min, washing with water for 30 + -5 min, soaking and sterilizing in mixed solution of 1 + -0.2% sodium hypochlorite and 10 + -2 mg/L Vc (vitamin C) in a super clean bench for 20 + -5 min, and washing with sterile water.
In one embodiment, in the pretreatment step, the length of the axillary bud-containing stem is 0.5-1cm.
In one embodiment, the germination medium is MS medium and comprises the following components:
the germination culture medium is adjusted, so that the contamination rate of the traditional culture medium can be greatly reduced, vermiculite and river sand in the subsequent steps can be recycled, and the production cost is reduced.
In one embodiment, the secondary culture medium is MS culture medium and comprises the following components:
the subculture medium is adjusted, so that the contamination rate of the traditional culture medium can be greatly reduced, vermiculite and river sand in the subsequent steps can be recycled, and the production cost is reduced. In addition, the carrageenan is used in the subculture medium instead of agar, so that the problem that the proliferation of cluster buds is influenced due to excessive callus tissues generated by using the agar is solved.
In one embodiment, in the step of strong seedling and rooting, CO is introduced 2 The time of (2) is 7 2 The concentration of the (D) is kept between 900 and 1100ppm; the illumination intensity is 3500lx, and the illumination time is 8; the temperature was maintained at 26. + -. 4 ℃.
In one embodiment, in the strong seedling rooting step, the weight part ratio of vermiculite to river sand in the mixed matrix is 3 +/-1, and the volume ratio of the culture solution to the mixed matrix is 1.5 +/-0.5.
In one embodiment, in the step of rooting strong seedlings, the culture solution comprises the following components:
in one embodiment, in the step of strong seedling and rooting, the mixed matrix is sterilized at 121 +/-5 ℃ for 30 +/-5 min before use.
In one embodiment, in the step of rooting strong seedlings, the humidity is controlled according to the following method: controlling the humidity to be 90 +/-5% in the first week, 80 +/-5% in the second week, 70 +/-5% in the third week, 60 +/-5% in the fourth week and 40% -50% in the fifth week;
and the transplanting step is carried out in the sixth week after strong seedlings are rooted.
By adopting the humidity control method, the smooth proceeding of strong seedling rooting can be ensured, the seedlings can adapt to natural humidity, and the survival rate of the seedlings is improved.
Compared with the prior art, the invention has the following beneficial effects:
according to the eucommia ulmoides seedling growing method, aiming at the growth characteristics of eucommia ulmoides, CO is introduced in the strong seedling rooting process 2 The growth of harmful bacteria can be inhibited, the humidity of the container is reduced, the eucommia ulmoides seedlings can adapt to the natural humidity from the early stage, and the survival rate of the seedlings is improved. Solves the problems of low survival rate of transplanted seedlings, easy water shortage and wilting of the traditional bottle seedling method due to long-term adaptation to high-humidity environment.
In addition, the invention also adjusts the germination culture medium and the subculture medium, so that the contamination rate of the traditional culture medium can be greatly reduced, vermiculite and river sand in the subsequent steps can be recycled, and the production cost is reduced.
And the humidity is gradually reduced and controlled in the strong seedling rooting step, so that the smooth proceeding of the strong seedling rooting can be ensured, the seedlings can adapt to the natural humidity, the survival rate of the seedlings is improved and reaches more than 90 percent.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
MS culture medium used in the following examples was SIGMA-ALORICH brand M5519 (Murashige & Skoog minimal medium)
Example 1
A eucommia ulmoides seedling raising method comprises the following steps:
1. pretreatment
Harvesting young shoots containing axillary buds for 4-5 months per year, soaking and disinfecting with 10% aqueous hydrogen peroxide solution for 5 minutes, washing with tap water for half an hour, moving to a super-clean workbench, disinfecting with 1% sodium hypochlorite and 10mg/L VC mixed solution for 20 minutes, washing with sterile water for 5 times, sucking, and cutting into 0.5-1cm axillary bud-containing stem segments for later use.
2. Inoculation of
And (3) inoculating the axillary bud-containing stem segments to a germination culture medium (wherein the germination culture medium is an MS culture medium, and 40g/L sucrose, 7g/L agar and 2g/L active carbon are added, and the rest added components are shown in the table below), and inducing germination.
TABLE 1 Germination medium composition and Germination status for each experiment
Note: , + + + + indicates that the growth state is excellent, + indicates that the growth state is good, and + indicates that the growth state is normal.
The above results show that the germination medium of test No. 4 has the best germination induction effect.
3. Multiplication of cluster buds
According to the test number, the germinated tissue is inoculated to a subculture medium (the subculture medium adopts an MS culture medium, 20g/L of sucrose and 7g/L of carrageenan are added, and the rest of added components are shown in the table below), and the cluster buds are proliferated to obtain a non-root-bud section.
TABLE 2 subculture medium components and proliferation of multiple shoots for each experiment
Note: , + + + + indicates that the growth state is excellent, + indicates that the growth state is good, and + indicates that the growth state is normal.
The above results show that the subculture medium of test No. 4 has the best effect of proliferating the cluster buds.
4. Strong seedling and root
The rootless bud segments obtained in test No. 4 were transferred to a vessel containing a mixed matrix of vermiculite and river sand (previously sterilized at 121 ℃ for 30 min), wherein the weight ratio of vermiculite to river sand was 3.
The culture solution comprises the following components:
and CO is introduced during the culture process 2 Keeping illumination and controlling humidity to enable the rootless bud segments to grow roots, thus obtaining the eucommia ulmoides seedlings.
Wherein, CO is introduced 2 The time of (2) is 7 2 The concentration of (A) is kept between 900 and 1100ppm; the illumination intensity is 3500lx, and the illumination time is 8; the temperature was kept around 26 ℃.
And controlling the humidity according to the following method: the humidity is controlled to be 90 +/-5% in the first week, 80 +/-5% in the second week, 70 +/-5% in the third week, 60 +/-5% in the fourth week and 40% -50% in the fifth week.
In the present example, the strong seedling rooting is performed according to the method described above, and simultaneously, the strong seedling rooting is performed according to the method described in document 1 (jiang-xiang, wang-jiaya, river village jiayi lang, etc.. Study on the tissue culture technology of eucommia ulmoides [ J ]. The science and technology of forestry in north and Hu, 2000 (supplement): 96-98), and the occurrence of bacterial contamination during the rooting process is recorded as shown in the following table.
TABLE 3 rooting condition of strong seedling for each experiment number
5. Transplanting
In the sixth week after the strong seedlings take root, the seedlings with root systems are transplanted, and the transplanting survival rate is shown in the table below.
TABLE 4 survival of seedlings of eucommia ulmoides of each test number
The above results show that the method of this example and the medium composition of test No. 4 provide the best seedling raising effect.
Example 2
An eucommia ulmoides seedling culture method is basically the same as the eucommia ulmoides seedling culture method in example 1, wherein germination medium and subculture medium are selected from medium components of test number 4. The difference lies in that: in the rooting process of the strong seedlings cultured in the embodiment, the humidity is kept between 80 and 90 percent in the whole process.
The results show that: the air humidity is too high, bacteria are easy to generate, the stomata are in a closed or opened state for a long time, the development is incomplete, once the stomata are transplanted to the natural environment, the humidity is suddenly reduced, and large-area new seedlings die due to water shortage.
Example 3
A seedling raising method of eucommia ulmoides is substantially the same as the seedling raising method of eucommia ulmoides in example 1, wherein the germination medium and the subculture medium are culture medium components of test No. 4. The difference lies in that: in the rooting process of the strong seedlings cultured in the embodiment, the humidity is controlled to be 90 +/-5% in the first week, 70 +/-5% in the second week and 40% -50% in the third and later weeks.
The results show that: because the humidity of air influences the transpiration of plant, air humidity has the anti-proportional relation with the transpiration, in case air humidity descends too fast, in this embodiment, the moisture that the transpiration evaporates is greater than the absorptive moisture of plant root, has aroused that plant leaf inside supplies water inadequately to die.
Example 4
A seedling raising method of eucommia ulmoides is substantially the same as the seedling raising method of eucommia ulmoides in example 1, wherein the germination medium and the subculture medium are culture medium components of test No. 4. The difference lies in that: the humidity is controlled to be 90 +/-5% in the first week, 85 +/-5% in the second week, 80 +/-5% in the third week, 75 +/-5% in the fourth week and 70% -60% in the fifth week;
the results show that: high humidity, too slow reduction, resulting in closed stomata and CO 2 Fails to enter mesophyll cells, photosynthesis slows down or even stops, and causes an increase in the occurrence of bacteria.
Comparative example 1
A seedling raising method of eucommia ulmoides is substantially the same as the seedling raising method of eucommia ulmoides in example 1, wherein the germination medium and the subculture medium are culture medium components of test No. 4. The difference lies in that: no CO was introduced during the culture in this comparative example 2 。
The results show that: the carbon source is essential in the growth of plants, in the traditional example, the carbon source of the plants is white sugar, and the invention directly uses CO 2 Instead of white sugar, CO 2 Can inhibit the growth of bacteria to a certain extent. Without introducing CO 2 And because the container has no forced gas exchange and the air humidity is too high, the development of stomata is influenced, and bacteria are more easily generated on a culture medium taking sugar as a carbon source, so that the final pollution rate is greatly improved.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (4)
1. A eucommia ulmoides seedling raising method is characterized by comprising the following steps:
pretreatment: taking new shoots of eucommia ulmoides containing axillary buds, sterilizing, and cutting into stem segments containing the axillary buds for later use;
inoculation: inoculating the axillary bud-containing stem segments to a germination culture medium, and inducing germination;
and (3) cluster bud multiplication: inoculating the germinated tissue to a subculture medium, and performing cluster bud proliferation to obtain a rootless bud section;
strengthening seedlings and rooting: transferring the rootless bud segments into a container filled with a mixed matrix of vermiculite and river sand, wherein the weight part ratio of the vermiculite to the river sand in the mixed matrix is 3 +/-1, adding a culture solution for culturing, wherein the volume ratio of the culture solution to the mixed matrix is 1.5 +/-0.5, and introducing CO in the culture process 2 Keeping illumination and controlling humidity to enable the rootless bud segments to grow root systems to obtain eucommia seedlings; humidity was controlled as follows: controlling the humidity to be 90 +/-5% in the first week, 80 +/-5% in the second week, 70 +/-5% in the third week, 60 +/-5% in the fourth week and 40% -50% in the fifth week; introducing CO 2 The time of (2) is 7 2 The concentration of (A) is kept between 900 and 1100ppm; the illumination intensity is 3500lx, and the illumination time is 8; the temperature is kept at 26 +/-4 ℃;
transplanting: transplanting the seedlings with the root systems in the sixth week after the strong seedlings take root;
the germination culture medium is an MS culture medium and comprises the following components:
the subculture medium is an MS culture medium and comprises the following components:
the culture solution comprises the following components:
2. a seedling raising method of eucommia ulmoides as claimed in claim 1, wherein in the pretreatment step, the disinfection process comprises: soaking young shoots containing axillary buds of eucommia ulmoides in 10 + -2% hydrogen peroxide solution for 5 + -2 min, washing with water for 30 + -5 min, soaking and sterilizing in mixed solution of 1 + -0.2% sodium hypochlorite and 10 + -2 mg/L Vc for 20 + -5 min in a super clean bench, and washing with sterile water.
3. A seedling raising method of eucommia ulmoides as claimed in claim 1, wherein in the pretreatment step, the length of the axillary bud-containing stem is 0.5-1cm.
4. A seedling raising method according to claim 1, wherein in the step of rooting and strengthening eucommia ulmoides, the mixed matrix is sterilized at 121 +/-5 ℃ for 30 +/-5 min before being used.
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