CN110604055A - In-vitro preservation method for rubber grass germplasm resources - Google Patents
In-vitro preservation method for rubber grass germplasm resources Download PDFInfo
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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
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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
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Abstract
The invention belongs to the technical field of plant germplasm resource preservation and tissue culture, and particularly relates to an in vitro preservation method of a kokstroemia indica germplasm resource. The method specifically comprises the following steps: (1) preparing a culture medium; (2) preparing and inoculating a material; (3) inducing and differentiating the callus; (4) proliferating adventitious buds; (5) rooting culture; (6) preserving and culturing germplasm; (7) and (5) recovering culture. The invention adopts a germplasm preservation culture medium consisting of a special formula: the MS concentration of the basic culture medium is reduced to 1/2MS, mannitol and abscisic acid are added, compared with a conventional tissue culture method, the subculture period of the rubberella tabacum test-tube plantlet is prolonged to 10-12 months from 2 months, the limited growth storage of the test-tube plantlet is realized, the survival rate of the test-tube plantlet is still 95% after the test-tube plantlet is stored for 10 months, the plant grows normally, and the test-tube plantlet on a normal culture medium can only survive for 2 months.
Description
Technical Field
The invention belongs to the technical field of plant germplasm resource preservation and tissue culture, and particularly relates to an in vitro preservation method of a kokstroemia indica germplasm resource.
Background
The Taraxacumkok-saghyzRodin is perennial root herbaceous plant of taraxacum of Compositae, the natural rubber content in the root is averagely 22.4%, the quality of the Taraxacumkok-saghyzRodin is similar to that of the rubber produced by Brazilian rubber tree, and the taraxacum rubber also contains 25-40% of inulin, is recognized as one of excellent natural rubber plants with development potential, and is an ideal model plant for researching and regulating the molecular synthesis and storage processes of rubber.
The current germplasm resource preservation modes of the rubber grasses mainly comprise seed preservation and field planting preservation. In the process of propagation, the rubber grass is self-sterile, so that the seeds are the result of hybridization among individuals and different lines, and the progeny has heterozygosity, so that germplasm resources are easy to mix, and the excellent traits of the germplasm are difficult to maintain. And the workload of field planting and preservation is large, and the field planting and preservation is easily influenced by extreme natural disasters and plant diseases and insect pests. In order to overcome the defects of traditional seed preservation and field planting preservation of the kochia scoparia, the development of in vitro preservation research of kochia scoparia germplasm resources is very necessary.
The growth-limited in vitro preservation is a newly developed and effective method for preserving the germplasm in vitro in recent years, and can realize the purpose of preserving the plant germplasm resources in the middle period. The growth-limited in-vitro preservation is based on a tissue culture technology and conventional subculture, the growth of plants is limited by changing the growth environment of test-tube plantlets in bottles, and the subculture interval time is prolonged, so that the subculture times are reduced, the pollution or the initiation of germplasm variation caused by frequent subculture is avoided, and the genetic stability of germplasm can be effectively ensured. With the comprehensive development of the in vitro culture technology in recent decades, the in vitro subculture technology is increasingly becoming an important means for germplasm resource preservation. At present, reports on the aspect of in vitro preservation of the germplasm resources of the kochia scoparia are not found in China.
Disclosure of Invention
The invention aims to provide the method for preserving the rubber grass germplasm resources in vitro, which aims at overcoming the defects of the traditional seed preservation and field planting preservation of the rubber grass germplasm resources at present and the defects of large workload and the like caused by frequent subculture required by normal subculture preservation of tissue culture, realizes the in vitro preservation of the rubber grass germplasm resources at normal temperature, does not require frequent subculture, can preserve for 10-12 months, and can quickly recover the growth of the materials after in vitro preservation and propagate in large quantities.
The purpose of the invention is realized as follows:
an in vitro preservation method of a kokstroemia indica germplasm resource comprises the following steps:
(1) preparing a culture medium:
(1.1) preparing an induction culture medium, wherein the specific components are as follows: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
(1.2) preparing a differentiation medium, wherein the specific components are as follows: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
(1.3) preparing a proliferation culture medium, which comprises the following specific components: MS +6-BA1.0mg/L + NAA0.1mg/L;
(1.4) preparing a rooting culture medium, which comprises the following specific components: 1/2MS + NAA0.3mg/L;
(1.5) preparing a germplasm preservation culture medium, wherein the germplasm preservation culture medium comprises the following specific components: 1/2MS + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 2.0 mg/L;
wherein 30g/L of sucrose, 5.5g/L of agar powder and the pH value of the culture medium at each stage of (1.1) - (1.5) are added, and the pH value is 5.8-6.0;
(2) preparation of the material and inoculation: the material is tender leaves of the rubber grass, after being cleaned by the liquid detergent,flushing with flowing water for 2-4 h, disinfecting with 75% alcohol for 10s on a superclean bench, and then disinfecting with 0.1% HgCL2Sterilizing the solution for 5min, finally washing with sterile water for 5-6 times, and inoculating to an induction culture medium for culture;
(3) inducing and differentiating the callus: inoculating the callus formed by induction in the step (2) on a differentiation culture medium, and performing differentiation culture of adventitious buds;
(4) and (3) proliferation of adventitious buds: performing enrichment culture on the adventitious bud strains in the step (3) on an enrichment culture medium;
(5) rooting culture: dividing the cluster buds in the step (4) into single plants, and transferring the single plants to a rooting culture medium for rooting culture;
(6) and (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (5) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
(7) and (3) recovery culture: and (5) transferring the plants preserved and cultured in the step (6) for more than 10 months to the culture conditions in the same step (5) for recovering and culturing for one month.
The culture medium of each stage of (1.1) - (1.5) is added with 30g/L of sucrose, 5.5g/L of agar powder and the pH value is 5.8-6.0.
The germplasm preservation culture medium adopts a special culture medium formula as follows: the MS minimal medium is reduced to 1/2MS, mannitol with the content of 30mg/L is added, and abscisic acid with the content of 2.0mg/L is added.
And (3) placing the callus in the step (2) on a differentiation culture medium for culturing, and differentiating the callus to form adventitious buds after 4 weeks.
The culture conditions of the steps (1), (2), (3), (4), (5), (6) and (7) are that the culture temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
The invention has the beneficial effects that:
the invention establishes a method for in vitro preservation of a kokstroemia indica germplasm resource. The germplasm preservation culture medium composed of a special formula is adopted: the MS concentration of the basic culture medium is reduced to 1/2MS, mannitol and abscisic acid are added, compared with a conventional tissue culture method, the subculture period of the rubberella tabacum test-tube plantlet is prolonged to 10-12 months from 2 months, the limited growth storage of the test-tube plantlet is realized, the survival rate of the test-tube plantlet is still 95% after the test-tube plantlet is stored for 10 months, the plant grows normally, and the test-tube plantlet on a normal culture medium can only survive for 2 months. The subculture time is prolonged to 10-12 months from about 2 months of ordinary MS culture medium preservation, and the survival rate reaches 95% after growth recovery. Realizes the long-time in vitro preservation of the rubber grass germplasm resources under the normal temperature condition. The in vitro preserved material can quickly recover growth and can be propagated in large quantities, and compared with the test-tube plantlet which is not preserved, the test-tube plantlet obtained by mass propagation has no obvious difference in morphological characteristics and growth vigor and stable properties.
The invention firstly utilizes the method of adding mannitol and abscisic acid into the culture medium to preserve the rubber grass test-tube plantlet, compared with the traditional preservation of rubber grass seeds and field planting preservation, the invention can save the preservation space, has small workload, can prevent the seed degeneration and virus infection after multi-generation propagation, and ensures the excellent property and purity of the germplasm. Breaks through the limitation of the growing season of the plants, can provide materials at any time, and has the advantages of saving storage space, being convenient for transportation and communication, and the like. Can prevent loss of germplasm resources. The heavy subculture work in the production is reduced, the unnecessary waste of manpower and material resources in the production operation process is reduced, and a necessary way is provided for reducing the production cost and improving the economic benefit in the actual production. Greatly saves manpower and material resources for the transfer culture, solves the problem that the rubber grass individuals and seeds are easy to be hybridized and are difficult to store excellent individuals, and realizes the in vitro storage of the rubber grass germplasm resources.
Drawings
FIG. 1 is a flow chart of the method of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The method for in vitro preservation of the rubber grass germplasm resources comprises the steps of obtaining test-tube plantlets and in vitro preservation, wherein the in vitro preservation step is to transfer the obtained test-tube plantlets into a germplasm preservation culture medium for preservation after subculture for 30 days, wherein:
the germplasm preservation culture medium comprises the following formula: (1/3-1) MS + NAA 0.1-0.3 mg/L + mannitol 10-50 mg/L + abscisic acid 0.5-3.0 mg/L. The best choice is: 1/2MS + + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 2.0 mg/L. 30g/L of sucrose and 5.5g/L of agar powder are added into the culture medium at each stage, and the pH value is 5.8-6.0. The 1/2MS minimal medium is MS medium with half of macroelements.
In the in vitro preservation method, the steps for obtaining the test-tube plantlet are the same as those of the prior conventional technology.
The induction culture medium is as follows: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
the differentiation culture medium is as follows: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
The proliferation culture medium is: MS +6-BA1.0mg/L + NAA0.1mg/L;
the rooting culture medium comprises: 1/2MS + NAA0.3mg/L;
germplasm preservation culture medium: 1/2MS + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 2.0 mg/L;
explant disinfection, inoculation and induction culture: selecting tender leaves of the Hevea brasiliensis as explant materials, cleaning with a detergent solution, flushing with running water for 2-4 hours, disinfecting with 75% (volume percentage) alcohol for 10s on a super-clean workbench, and disinfecting with 0.1% (mass percentage) HgCL2Sterilizing the solution for 5min, finally washing with sterile water for 5-6 times, and inoculating to an induction culture medium for culture;
differentiation culture: placing the callus in the step (1) on a differentiation culture medium for culturing, and differentiating the callus after 4 weeks to form adventitious buds;
and (3) proliferation culture: culturing the adventitious buds in the step (2) on an enrichment medium, wherein the subculture period is one month, and cutting the massive cluster buds into small blocks for propagation;
rooting culture: selecting the robust plantlets in the step (3), transferring the robust plantlets into rooting culture, and growing into 3-4 plantlets with white roots after 4 weeks;
and (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (4) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
and (3) recovery culture: and (4) transferring the plants preserved and cultured in the step (5) for more than 10 months to the same culture conditions in the step (4) for recovery and culture for one month.
The culture conditions of the steps (1), (2), (3), (4), (5) and (6) are that the culture temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
The object of the present invention is achieved by the following technical means. The method for in vitro preservation of the rubber grass germplasm resources comprises the following steps:
(1) preparation of a culture medium:
A. induction medium: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
B. differentiation medium: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
C. Proliferation culture medium: MS +6-BA1.0mg/L + NAA0.1mg/L;
D. rooting culture medium: 1/2MS + NAA0.3mg/L;
E. germplasm preservation culture medium: (1/3-1) MS + NAA 0.1-0.3 mg/L + mannitol 10-50 mg/L + abscisic acid 0.5-3.0 mg/L;
wherein 30g/L of sucrose and 5.5g/L of agar powder are added into the culture medium at each stage, and the pH value is 5.8-6.0;
(2) material and inoculation: the material is tender leaves of the rubber grass, and the disinfected leaves are inoculated on an induction culture medium under the aseptic condition for culture;
(3) inducing and differentiating the callus: inoculating the callus formed by induction in the step (2) on a differentiation culture medium, and performing differentiation culture of adventitious buds;
(4) proliferation of adventitious buds: performing enrichment culture on the adventitious bud strains in the step (3) on an enrichment culture medium;
(5) rooting culture: dividing the cluster buds in the step (4) into single plants, and transferring the single plants to a rooting culture medium for rooting culture.
(6) And (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (5) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
(7) and (3) recovery culture: and (5) transferring the plants preserved and cultured in the step (6) for more than 10 months to the culture conditions in the same step (5) for recovering and culturing for one month.
In the step (1), the culture medium comprises a basic culture medium and culture media of each stage of tissue culture, and specifically comprises the following components:
A. induction medium: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
B. differentiation medium: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
C. Proliferation culture medium: MS +6-BA1.0mg/L + NAA0.1mg/L;
D. rooting culture medium: 1/2MS + NAA0.3mg/L;
E. germplasm preservation culture medium: (1/3-1) MS + NAA 0.1-0.3 mg/L + mannitol 10-50 mg/L + abscisic acid 0.5-3.0 mg/L;
wherein 30g/L of sucrose and 5.5g/L of agar powder are added into the culture medium at each stage, and the pH value is 5.8-6.0;
in the step (2), tender leaves of the rubber grass are selected as explant materials, washed by a detergent solution, flushed with running water for 2-4 hours, sterilized by 75% (volume percentage) of alcohol for 10 seconds, then sterilized by 0.1% (mass percentage) of HgCL2 solution for 5 minutes on a super-clean workbench, finally flushed with sterile water for 5-6 times, and inoculated on an induction culture medium for culture.
In the step (3), callus is formed after the leaves are cultured on the induction culture medium for 2-3 weeks, the callus with good growth state is selected and placed on a differentiation culture medium for culturing, and the callus is differentiated to form adventitious buds after 4 weeks.
In the step (4), the adventitious bud is inoculated on a multiplication medium for subculture for multiplication, and a cluster bud is formed after 4 weeks.
In the step (5), the cluster buds are divided into single plants and transferred to a rooting culture medium for rooting culture, and strong seedlings with root systems are formed after 2-3 weeks of culture.
In the step (6), the strong seedlings are transferred to a germ plasm preservation culture medium, and the preservation period is more than 10 months.
In the step (7), the ex vivo preservation test-tube plantlet is transferred to a rooting culture medium for recovery culture, and the survival rate reaches 95% after the growth is recovered.
In the steps (2), (3), (4), (5), (6) and (7), the culture conditions are that the culture temperature is 25 ℃ and 2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
Example 1
The invention provides a method for in vitro preservation of a kokstroemia indica germplasm resource, which comprises the following steps:
(1) preparation of culture Medium
A. Induction medium: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
B. differentiation medium: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
C. Proliferation culture medium: MS +6-BA1.0mg/L + NAA0.1mg/L;
D. rooting culture medium: 1/2MS + NAA0.3mg/L;
E. germplasm preservation culture medium: 1/2MS + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 1.0 mg/L;
wherein 30g/L of sucrose and 5.5g/L of agar powder are added into the culture medium at each stage, and the pH value is 5.8-6.0;
(2) materials and inoculation
Cleaning tender leaves made of rubber grass with a detergent solution, flushing with flowing water for 2-4 h, disinfecting with 75% (volume percentage) alcohol for 10s on a superclean bench, and then disinfecting with 0.1% (mass percentage) HgCL2Sterilizing the solution for 5min, finally washing with sterile water for 5-6 times, and inoculating the sterilized leaves on an induction culture medium under the sterile condition for culture; the culture temperature is 25 ℃ and 2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
(3) Induction of callus: inoculating the callus formed by induction in the step (2) on an induction culture medium, and inducing the callus; callus is formed after 2-3 weeks of culture on the induction culture medium,
(4) differentiation of adventitious buds: selecting the callus with good growth state in the step (3) to be cultured on a differentiation culture medium, and differentiating the callus into adventitious buds after 4 weeks;
(5) proliferation of adventitious buds: performing enrichment culture on the adventitious bud inoculum obtained in the step (4) on an enrichment medium, and forming cluster buds after about 3 weeks;
(6) rooting culture: dividing the cluster buds obtained in the step (5) into single plants, transferring the single plants to a rooting culture medium for rooting culture, and obtaining strong seedlings with root systems after 2-3 weeks;
(7) and (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (6) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
(8) and (3) recovery culture: transferring the plant preserved and cultured in the step (7) for more than 10 months to the culture condition in the same step (6) for restoring and culturing for one month;
example 2
The invention provides a method for in vitro preservation of a kokstroemia indica germplasm resource, which comprises the following steps:
(1) preparation of culture Medium
A. Induction medium: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
B. differentiation medium: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
C. Proliferation culture medium: MS +6-BA1.0mg/L + NAA0.1mg/L;
D. rooting culture medium: 1/2MS + NAA0.3mg/L;
E. germplasm preservation culture medium: 1/2MS + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 2.0 mg/L;
wherein 30g/L of sucrose and 5.5g/L of agar powder are added into the culture medium at each stage, and the pH value is 5.8-6.0;
(2) materials and inoculation
The material is tender leaves of the rubber grass. Cleaning tender leaves made of rubber grass with a detergent solution, flushing with flowing water for 2-4 h, disinfecting with 75% (volume percentage) alcohol for 10s on a superclean bench, and then disinfecting with 0.1% (mass percentage) HgCL2Sterilizing the solution for 5min, finally washing with sterile water for 5-6 times, and inoculating the sterilized leaves on an induction culture medium under the sterile condition for culture; the culture temperature is 25 ℃ and 2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
(3) Induction of callus: inoculating the callus formed by induction in the step (2) on an induction culture medium, and inducing the callus; callus is formed after 2-3 weeks of culture on the induction culture medium,
(4) differentiation of adventitious buds: selecting the callus with good growth state in the step (3) to be cultured on a differentiation culture medium, and differentiating the callus into adventitious buds after 4 weeks;
(5) proliferation of adventitious buds: performing enrichment culture on the adventitious bud inoculum obtained in the step (4) on an enrichment medium, and forming cluster buds after about 3 weeks;
(6) rooting culture: dividing the cluster buds obtained in the step (5) into single plants, transferring the single plants to a rooting culture medium for rooting culture, and obtaining strong seedlings with root systems after 2-3 weeks;
(7) and (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (6) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
(8) and (3) recovery culture: transferring the plant preserved and cultured in the step (7) for more than 10 months to the culture condition in the same step (6) for restoring and culturing for one month;
the method realizes the in vitro preservation of the rubber grass germplasm resources under the normal temperature condition, can effectively reduce the conventional subculture times of the rubber grass, has the preservation time of 10-12 months, and can quickly recover the growth of the material after in vitro preservation and propagate in large quantities. Greatly reduces the production cost of industrial seedling.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.
Claims (5)
1. The method for in vitro preservation of the rubber grass germplasm resources is characterized by comprising the following steps:
(1) preparing a culture medium:
(1.1) preparing an induction culture medium, wherein the specific components are as follows: 1.5-2.0 mg/L of MS +6-BA and 0.1-0.2 mg/L of NAA;
(1.2) preparing a differentiation medium, wherein the specific components are as follows: MS +6-BA 0.5-1.0 mg/L + NAA 0.05-0.1 mg/L + GA30.1～0.2mg/L；
(1.3) preparing a proliferation culture medium, which comprises the following specific components: MS +6-BA1.0mg/L + NAA0.1mg/L;
(1.4) preparing a rooting culture medium, which comprises the following specific components: 1/2MS + NAA0.3mg/L;
(1.5) preparing a germplasm preservation culture medium, wherein the germplasm preservation culture medium comprises the following specific components: 1/2MS + NAA0.3mg/L + mannitol 30mg/L + abscisic acid 2.0 mg/L;
wherein 30g/L of sucrose, 5.5g/L of agar powder and the pH value of the culture medium at each stage of (1.1) - (1.5) are added, and the pH value is 5.8-6.0;
(2) preparation of the material and inoculation: cleaning tender leaves made of rubber grass by using a detergent solution, flushing for 2-4 h by using flowing water, disinfecting for 10s by using 75% alcohol by volume on a superclean bench, and then using HgCL with the mass percent of 0.1%2Sterilizing the solution for 5min, finally washing with sterile water for 5-6 times, and inoculating to an induction culture medium for culture;
(3) inducing and differentiating the callus: inoculating the callus formed by induction in the step (2) on a differentiation culture medium, and performing differentiation culture of adventitious buds;
(4) and (3) proliferation of adventitious buds: performing enrichment culture on the adventitious bud strains in the step (3) on an enrichment culture medium;
(5) rooting culture: dividing the cluster buds in the step (4) into single plants, and transferring the single plants to a rooting culture medium for rooting culture;
(6) and (3) germplasm preservation and culture: selecting the healthy and strong seedlings in the step (5) and transferring the healthy and strong seedlings into a germ plasm preservation culture medium, wherein the preservation period is more than 10 months;
(7) and (3) recovery culture: and (5) transferring the plants preserved and cultured in the step (6) for more than 10 months to the culture conditions in the same step (5) for recovering and culturing for one month.
2. The method for in vitro preservation of kokstroemia indica germplasm resources according to claim 1, wherein the method comprises the following steps: the culture medium of each stage of (1.1) - (1.5) is added with 30g/L of sucrose, 5.5g/L of agar powder and the pH value is 5.8-6.0.
3. The method for in vitro preservation of kokstroemia indica germplasm resources according to claim 1, wherein the method comprises the following steps: the germplasm preservation culture medium adopts a special culture medium formula as follows: the MS minimal medium is reduced to 1/2MS, mannitol with the content of 30mg/L is added, and abscisic acid with the content of 2.0mg/L is added.
4. The method for in vitro preservation of kokstroemia indica germplasm resources according to claim 1, wherein the method comprises the following steps: and (3) placing the callus in the step (2) on a differentiation culture medium for culturing, and differentiating the callus to form adventitious buds after 4 weeks.
5. The method for in vitro preservation of kokstroemia indica germplasm resources according to claim 1, wherein the method comprises the following steps: the culture conditions of the steps (1), (2), (3), (4), (5), (6) and (7) are that the culture temperature is 25 +/-2 ℃, the illumination intensity is 1500-2000 lx, and the illumination time is 16 h/d.
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