CN110896854B - Culture method for promoting development and maturation of somatic embryos of Chinese pine - Google Patents
Culture method for promoting development and maturation of somatic embryos of Chinese pine Download PDFInfo
- Publication number
- CN110896854B CN110896854B CN201910776447.0A CN201910776447A CN110896854B CN 110896854 B CN110896854 B CN 110896854B CN 201910776447 A CN201910776447 A CN 201910776447A CN 110896854 B CN110896854 B CN 110896854B
- Authority
- CN
- China
- Prior art keywords
- culture
- embryonic tissue
- embryo
- medium
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for promoting development and maturation of a somatic embryo (somatic embryo) of Chinese pine, which comprises the step of performing liquid pretreatment culture on a Chinese pine embryonic tissue, wherein a pretreatment culture medium of the embryonic tissue contains a polyamine synthesis inhibitor regulator. The method adds polyamine synthesis inhibitor regulators into a liquid pretreatment culture medium and an embryo maturation culture medium so as to achieve the purposes of inhibiting excessive proliferation of embryonic tissues in the embryo maturation process, promoting the development and maturation of somatic embryos and improving the quality and quantity of mature somatic embryos. The method can obtain more mature embryos and embryo seedlings from the genotype materials with weak embryo performance and excessive proliferation of the Chinese pine, is favorable for diversification of the genotype of the Chinese pine embryo seedlings, can overcome the limitation of clone forestry genotype, and can produce multi-genotype Chinese pine embryo seedlings in a large scale and in an industrialized manner.
Description
Technical Field
The invention relates to a plant tissue culture method, in particular to a culture method for promoting the development and maturity of pine somatic embryos, and belongs to the technical field of cell engineering seedling breeding in the forestry industry.
Background
Pinus tabulaeformis (Pinusta tabulaeformis C.), also called Pinus tabulaeformis, Pinus brevifolia and the like, belongs to Pinus of Pinaceae, the tree height can reach 30m, the diameter at breast can reach 1m, the bark is mostly grey brown, the surface is often chapped to be scaly, the needle leaves are usually a bundle of 2 needles, the edge is provided with fine saw teeth, the length is 9-14cm, the young period is light green, the back is dark green, the texture is hard, the small branches are thick, the small branches are yellow brown, the resin is trace, the branches are usually downwards inclined or flat, the crown is flat in the adult period, the leaf sheath is firstly light brown and gradually becomes brown black, the cones are oval or oval, the length is 3-8cm, the stems are short, the male peduncle flowers are cylindrical, the clusters are usually gathered at the lower parts of the new branches, the length is 1.3-1.8cm, the flowering period is 4-5 months, and the cones are mature in 10 months in the next year.
The nutrition propagation modes commonly applied in the propagation of the conifer mainly comprise cuttage, grafting and the like. Cuttage is a conventional and efficient method in plant cultivation. The propagation of the Chinese pine is mainly realized by adopting a seeding and seedling raising method, and seeds are mainly provided by a seed garden. Although China already has some Chinese pine seed gardens, the problems of long time consumption, large investment of manpower and financial resources in the early stage, late seed setting, unstable seed yield and quality, serious pest and disease damage and the like are the problems commonly existing in the current seed gardens. On the other hand, the cutting and grafting technology of the Chinese pine has strong age effect. The cuttage rooting rate is low, the grafting survival efficiency is low, great problems are brought to the fine variety breeding of the Chinese pine, the production requirements cannot be met, the popularization and the application of the fine variety of the Chinese pine are greatly limited, and the afforestation quality is influenced.
The relative lag of the fine variety breeding technology of the Chinese pine affects the production and the scientific research progress of the Chinese pine. Tissue culture is a mode widely applied to plant propagation at present, is the basis for realizing genetic transformation and transgenic technology of forest trees, is considered to be a method and means for the best potential fine variety propagation of conifers, and a somatic embryogenesis technology is the most advanced plant asexual propagation method at present.
The related research of the Chinese pine somatic embryo system is less, the related research reports basically focus on the research of the in vitro culture of the organogenesis, and the research aiming at the Chinese pine somatic embryogenesis is less. The invention patent 'a method for generating and regenerating a plant by a pine somatic embryo' (patent number ZL201410573714.1) discloses a method for generating and regenerating a pine somatic embryo, which comprises 1) collecting pine cones and sterilizing the surfaces of the pine cones to obtain sterile zygotic embryos; 2) inoculating the sterile zygotic embryo to an embryonic tissue induction culture medium for embryonic tissue induction culture; 3) inoculating the obtained embryonic tissue on a somatic embryo maturation culture medium to perform developmental maturation culture of a somatic embryo; 4) inoculating the mature somatic embryos into a germination culture medium for germination culture to obtain somatic embryo seedlings; 5) hardening and transplanting the somatic embryo seedlings. The method obtains a large amount of stably-growing Chinese pine somatic embryos by inducing embryonic tissues and obtains complete plantlets.
The existing research on the pine embryos mainly has the following technical defects: (1) the number of pinus tabulaeformis embryonic cell lines is limited, and the genotypes which can produce a large number of somatic embryos are fewer; (2) the mature effect of the somatic embryo is unstable, and the mature efficiency of the somatic embryo is influenced. These problems have limited the development and application of the pine somatic embryo technology to large-scale propagation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, namely the difficulty in obtaining mature somatic embryos from a genotype material (Chinese pine) with weak embryo performance, and provides a novel culture method for promoting the development and maturation of the Chinese pine somatic embryos (somatic embryos). The method of the invention promotes the maturation of the somatic embryos, increases the number of the somatic embryos, improves the quality and the germination rate of the somatic embryos, and can be used for large-scale and industrial production.
In order to achieve the object of the present invention, in one aspect, the present invention provides a culture method for promoting the development and maturation of a somatic embryo (somatic embryo) of pinus tabulaeformis, comprising performing pretreatment culture on embryonic tissue of pinus tabulaeformis, wherein a medium for the pretreatment culture of the embryonic tissue contains a regulator such as polyamine synthesis inhibitor.
Wherein the embryonic tissue of the Chinese pine is obtained by performing induction culture on an explant of the Chinese pine, and the culture medium of the induction culture is mLV culture medium +2, 4-D2-4 mg/L +6-BA1-2mg/L + casein hydrolysate 500mg/L + glutamine 500mg/L + sucrose 10-30g/L + plant gel 2.5-3.0g/L, preferably mLV culture medium +2, 4-D4 mg/L +6-BA 2mg/L + casein hydrolysate 500mg/L + glutamine 500mg/L + sucrose 20g/L + plant gel 2.5-3.0 g/L.
In particular, the culture conditions of the induction culture are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 50-60 days.
In particular, the Chinese pine explant selects a Chinese pine female gametophyte and a zygotic embryo.
Wherein the polyamine synthesis inhibitor is MGBG (methylglyoxal bis-guanylhydrazone; mitoguazone).
Particularly, the concentration of polyamine synthesis inhibitor MGBG in the embryonic tissue pretreatment medium is 30-300. mu.M, preferably 50-200. mu.M.
Wherein the embryonic tissue pretreatment culture medium is mLV culture medium + MGBG30-300 μ M + ABA0-100 μ M + hydrolyzed casein 200 + 500mg/L + maltose 10-40g/L + sucrose 10-40g/L + glutamine 500mg/L, preferably mLV culture medium + MGBG50-200 μ M + ABA20-40 μ M + hydrolyzed casein 200 + maltose 20-30g/L + sucrose 10-20g/L + glutamine 500mg/L, more preferably mLV culture medium + MGBG50-200 μ M + ABA30 μ M + hydrolyzed casein 200 + maltose 500mg/L + maltose 20-30g/L + sucrose 10-20g/L + glutamine 500mg/L.
In particular, the culture conditions for the pre-treatment of the embryonic tissue are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 1-2 weeks.
In particular, the method comprises inoculating a pretreated embryonic tissue to an embryo maturation medium containing a polyamine synthesis inhibitor-based modulator, and culturing the embryo in somatic embryo maturation.
Wherein the polyamine synthesis inhibitor contained in the embryo maturation medium is MGBG.
In particular, the concentration of polyamine synthesis inhibitor MGBG in the embryo maturation medium is 30-150. mu.M, preferably 50-100. mu.M.
Wherein the embryo maturation culture medium is mLV culture medium + MGBG 30-150 μ M + ABA30-80 μ M + PEG400050-90g/L + hydrolyzed casein 200 + 500mg/L + maltose 30-40g/L + sucrose 10-30g/L + glutamine 200 + plant gel 4-7g/L, preferably mLV culture medium + MGBG50-100 μ M + ABA 50-70 μ M + PEG400050-90g/L + hydrolyzed casein 200 + maltose 500mg/L + maltose 30-40g/L + sucrose 10-30g/L + glutamine 200 + plant gel 500mg/L + plant gel 4-7g/L, and more preferably mLV culture medium + MGBG50-100 μ M + ABA60 μ M + PEG 400075 g/L + hydrolyzed casein 500mg/L + maltose 30-40g/L + sucrose 10-10 g/L 30g/L + glutamine 200mg/L + plant gel 5-6 g/L.
In particular, the culture conditions for embryo maturation culture are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 6-12 weeks.
The method uses liquid pretreatment and adds MGBG in a pretreatment culture medium and an embryo maturation culture medium to achieve the purposes of inhibiting excessive proliferation of embryonic tissues in the embryo maturation process and promoting the development and maturation of somatic embryos. The method of the invention can obtain relatively more mature somatic embryos and somatic embryo seedlings from pinus tabulaeformis genotype materials with weak embryo performance.
The invention also provides a culture method for promoting the development and maturation of the somatic embryos of the Chinese pine, which comprises the following steps in sequence:
1) inoculating the Chinese pine aseptic zygotic embryo (in a female gamete body) into an induction culture medium, and performing embryonic tissue induction culture to obtain an embryonic tissue;
2) inoculating the embryonic tissue into an embryonic tissue pretreatment culture medium, and performing embryonic tissue pretreatment culture to obtain a pretreated cultured embryonic tissue, wherein the embryonic tissue pretreatment culture medium contains MGBG;
3) inoculating the embryonic tissue after the pretreatment culture on an embryo maturation culture medium, and performing somatic embryo maturation culture to obtain a mature somatic embryo.
The induction rate of the embryonic tissue is obviously influenced by the explant embryo age of the Chinese pine, and the perfect embryonic tissue induction result cannot be obtained by the excessively tender or excessively mature zygotic embryo. According to the invention, immature seeds of the Chinese pine are collected according to a patent 'Chinese pine somatic embryogenesis and plant regeneration method' (patent number ZL201410573714.1), and the induction rate of the immature seeds on an induction culture medium is high.
The sterile zygotic embryo (female gametophyte) in the step 1) is prepared according to the invention patent method of the patent number ZL 201410573714.1.
Wherein the embryonic tissue induction culture medium in the step 1) is mLV culture medium +2, 4-D2-4 mg/L +6-BA1-2mg/L + casein hydrolysate 500mg/L + glutamine 500mg/L + sucrose 10-30g/L + plant gel 2.5-3.0 g/L.
In particular, the culture conditions for the embryogenic tissue induction culture in step 1) are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 10-20 days.
In particular, the subculture is carried out once every 20 to 25 days in the process of embryonic tissue induction culture;
in particular, it also includes subculturing the embryonic tissue every 2-4 weeks.
In particular, the embryonic tissue subculture medium is: mLV culture medium +2, 4-D0.2-2 mg/L +6-BA 0.1-1mg/L + hydrolyzed casein 500mg/L + glutamine 500mg/L + sucrose 10-30g/L + plant gel 2.5-3.0 g/L.
In particular, the culture conditions for the subculture were: under dark conditions, the culture temperature (25 +/-2) DEG C; the culture time is 2-4 weeks.
Particularly, the method further comprises the step 1A) of inoculating the embryonic tissue obtained by the culture in the step 1) to a multiplication culture medium, performing multiplication culture on the embryonic tissue to obtain a multiplication embryonic callus, then inoculating the multiplication embryonic callus to an embryonic tissue pretreatment culture medium, and performing the embryonic tissue pretreatment culture.
Wherein the embryonic tissue proliferation culture in the step 1A) comprises the following steps:
1A-1) inoculating the embryonic tissue to an embryonic tissue solid multiplication culture medium to carry out first-stage solid multiplication culture on the embryonic tissue of the Chinese pine;
1A-2) inoculating the embryonic tissue subjected to the first-stage solid multiplication culture into an embryonic tissue liquid multiplication culture medium, and performing second-stage liquid multiplication culture on the embryonic tissue of the Chinese pine to obtain the embryonic tissue multiplied by liquid culture.
Specifically, the solid medium for embryogenic tissue proliferation in step 1A-1) is mLV medium +2, 4-D0.2-2 mg/L +6-BA 0.1-1mg/L + hydrolyzed casein 500mg/L + sucrose 10-30g/L + phytogel 2.5-3.0g/L + glutamine 500mg/L, preferably mLV medium +2, 4-D0.5-2 mg/L +6-BA 0.25-1mg/L + hydrolyzed casein 500mg/L + sucrose 10-20g/L + phytogel 2.5-3.0g/L + glutamine 500mg/L.
Specifically, the liquid multiplication medium for embryonic tissue in step 1A-2) is mLV medium +2, 4-D0.2-2 mg/L +6-BA 0.1-1mg/L + casein hydrolysate 500mg/L + sucrose 10-30g/L + glutamine 500mg/L, preferably mLV medium +2, 4-D0.5-2 mg/L +6-BA 0.25-1mg/L + casein hydrolysate 500mg/L + sucrose 10-20g/L + glutamine 500mg/L.
Particularly, the culture conditions of the solid multiplication culture and the liquid multiplication culture of the embryonic tissue are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 6-8 weeks; the liquid proliferation culture rotating speed is 100-120 r/min.
In particular, the solid multiplication culture process of the embryonic tissue is subcultured once every 3 to 4 weeks; the number of subcultures is 2-3; subculturing the embryonic tissue once a week in the liquid multiplication culture process; the number of subcultures was 1-2.
Wherein the concentration of MGBG in the embryonic tissue pretreatment culture medium in the step 2) is 30-300. mu.M, preferably 50-200. mu.M.
Particularly, the embryonic tissue pretreatment culture medium in the step 2) is mLV culture medium + MGBG30-300 μ M + ABA0-100 μ M + hydrolyzed casein 200- + 500mg/L + maltose 10-40g/L + sucrose 10-40g/L + glutamine 500mg/L, preferably mLV culture medium + MGBG50-200 μ M + ABA20-40 μ M + hydrolyzed casein 200- + 500mg/L + maltose 20-30g/L + sucrose 10-20g/L + glutamine 500mg/L, more preferably mLV medium + MGBG 50-200. mu.M + ABA 30. mu.M + hydrolyzed casein 500mg/L + maltose 20-30g/L + sucrose 10-20g/L + glutamine 500mg/L.
Particularly, the culture conditions for the embryonic tissue pretreatment culture in the step 2) are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 1-2 weeks.
Particularly, the inoculation amount in the pretreatment culture process of the embryonic tissue is 2-6g of the embryonic tissue inoculated in each 100ml of pretreatment culture medium of the embryonic tissue.
Wherein the embryo maturation medium in the step 3) contains a regulator MGBG.
In particular, the concentration of the regulator MGBG in the embryo maturation medium is 30-150. mu.M, preferably 50-100. mu.M.
Wherein the embryo maturation culture medium in the step 3) is mLV culture medium + MGBG 30-150 μ M + ABA30-80 μ M + PEG400050-90g/L + hydrolyzed casein 200 + 500mg/L + maltose 30-40g/L + sucrose 10-30g/L + glutamine 500mg/L + plant gel 4-7g/L, preferably mLV culture medium + MGBG50-100 μ M + ABA 50-70 μ M + PEG400050-90g/L + hydrolyzed casein 200 + glutamine 500mg/L + maltose 30-40g/L + sucrose 10-30g/L + glutamine 500mg/L + plant gel 4-7g/L, more preferably mLV culture medium + MGBG50-100 μ M + ABA60 μ M + PEG 3535 400075 g/L + hydrolyzed casein 500mg/L + maltose 30-40g/L + sucrose 10-30g/L g/L + glutamine 500mg/L + plant gel 5-6 g/L.
In particular, the culture conditions for the embryo maturation culture in step 3) are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 8-12 weeks.
The invention also provides a propagation method of the Chinese pine, which comprises the step of performing embryonic tissue pretreatment culture on the embryonic tissue of the Chinese pine, wherein the embryonic tissue pretreatment culture medium contains a regulator of polyamine synthesis inhibitors.
Wherein the regulator containing polyamine synthesis inhibitor in the embryonic tissue pretreatment culture medium is MGBG.
Particularly, the method also comprises the step of performing somatic embryo maturation culture on the pretreated embryonic tissue, wherein the embryo maturation culture medium contains a polyamine synthesis inhibitor regulator MGBG.
In another aspect, the present invention provides a method for propagating Chinese pine, comprising the following steps:
A) inoculating the Chinese pine aseptic zygotic embryo (in a female gamete body) to an induction culture medium, and performing embryonic tissue induction culture to obtain an embryonic tissue;
B) inoculating the embryonic tissue obtained by the induction culture to a proliferation culture medium for proliferation culture of the embryonic tissue;
C) inoculating the embryonic tissue after propagation culture into an embryonic tissue pretreatment culture medium, and performing embryonic tissue pretreatment culture, wherein the embryonic tissue pretreatment culture medium contains a regulator MGBG;
D) inoculating the embryonic tissue which is cultured by pretreatment on an embryo maturation culture medium, and performing somatic embryo maturation culture to obtain a somatic embryo;
E) inoculating the mature somatic embryos into a germination culture medium for germination culture to obtain somatic embryo seedlings (container seedlings);
F) hardening and transplanting the somatic embryo seedlings to obtain the seedling-hardening seedling-transplanting seedling-hardening seedling-transplanting seedling.
Wherein the germination culture medium in the step E) is mLV minimal medium, 1g/L of activated carbon, 10-20g/L of sucrose and 7-8g/L of agar.
In particular, said germination culture in step E) is carried out under the following conditions: under the illumination condition, the culture temperature is 25 +/-2 ℃.
Particularly, the culture time of germination and seedling transferring culture in the step E) is 40-60 days; the illumination intensity is 1500-.
And (5) hardening and transplanting the container body embryo seedlings to obtain Chinese pine seedlings. The hardening off and transplanting method is the same as the method disclosed in the patent No. ZL 201410573714.1.
In the process of plant somatic embryogenesis, the proliferation of embryonic tissue and the developmental maturation of somatic embryos are in succession in two distinct stages. The former promotes the increase of embryonic tissues, and the latter is to obtain more mature embryos. In the method, the 2,4-D and 6-BA in the embryogenic tissue multiplication culture medium can promote the multiplication of the embryogenic tissue, and the continuous multiplication of the embryogenic tissue can hinder the development and the maturation of the somatic embryo in the maturation culture process of the somatic embryo. Direct transfer of embryogenic tissue in multiplication culture to maturation medium results in continued multiplication of embryogenic tissue, which hinders the development of somatic embryos. The continuous proliferation of the embryogenic tissue in mature culture is mainly caused by the residual 2,4-D and 6-BA in the embryogenic tissue. The liquid pretreatment culture medium of the embryonic tissue does not contain 2,4-D and 6-BA, and the residual 2,4-D and 6-BA in the tissue can be effectively removed through the pretreatment suspension culture of the embryonic tissue; meanwhile, MGBG is added into the pretreatment culture medium, so that excessive proliferation of embryonic tissues caused by other reasons can be further inhibited, and better conditions are created for the next step of mature culture of somatic embryos; and MGBG is added in liquid pretreatment and embryo maturation culture medium, so that the development and maturation of somatic embryos can be further effectively promoted, more and better mature somatic embryos can be obtained, and good conditions are provided for subsequent somatic embryo germination and somatic embryo seedling growth.
The Chinese pine reproduction and regeneration method has the following advantages:
1. the method utilizes young zygotic embryos (female gametophytes) of the Chinese pine as explants to regenerate and propagate plants, after the multiplication culture of embryonic tissues is carried out, MGBG is added into a pretreatment culture medium and a maturation culture medium in the pretreatment culture medium and the maturation culture process of the embryonic tissues and the subsequent somatic embryo to inhibit the excessive proliferation of the Chinese pine embryonic tissues; MGBG is added into a maturation culture medium to promote the maturation of somatic embryos, improve the quality of the germinated somatic embryos, obtain more high-quality mature somatic embryos, further improve the germination rate of somatic embryo seedlings and provide good conditions for the growth of the somatic embryo seedlings.
MGBG (Methylglyoxal bis-guanylhydrazone, Chinese name: mitoguazone (methylglyoxaldiamidinhydrazone)) is an inhibitor of polyamine synthesis in plant tissues. Polyamines promote the proliferation of embryonic tissue and facilitate the production of somatic embryos, such as spermine. MGBG was not used in the current conifer somatic embryo culture process because it is generally accepted by academia that inhibition of polyamine synthesis also hinders somatic embryo development. The invention proves that the excessive proliferation of embryonic tissues can be reduced and the development and the maturation of somatic embryos can be promoted by adding a proper amount of MGBG in the liquid pretreatment and embryo maturation culture medium. By using the novel method for adding the MGBG, more high-quality mature somatic embryos can be obtained, so that good conditions are provided for subsequent somatic embryo germination and somatic embryo seedling growth. The invention is particularly suitable for obtaining relatively more mature somatic embryos and somatic embryo seedlings from pinus tabulaeformis genotype materials with weak embryo performance and excessive plant tissue proliferation.
2. MGBG added in the Chinese pine propagation method is a polyamine synthesis inhibitor, can reduce the synthesis amount of polyamine, and obviously inhibits the excessive proliferation of embryonic tissues in mature culture. The invention can obviously inhibit the proliferation of the embryonic tissue by the suspension pretreatment of the embryonic tissue and the addition of MGBG with proper concentration in a maturation culture medium, and the somatic embryo can well grow and develop into a morphological and normal-function mature somatic embryo in the MGBG culture medium with proper concentration. Unlike the prior art, the present invention uses a proper amount of polyamine synthesis inhibitor to promote the development and maturation of somatic embryos, and the general research suggests that polyamines are beneficial for the production of somatic embryos of plants, so MGBG is not used in the method for culturing conifer somatic embryos to avoid the reduction of endogenous polyamine synthesis.
Drawings
FIG. 1 is a view of the embryonic tissue of Pinus tabulaeformis.
FIG. 2 is the embryonic tissue and embryonic tissue structure of the liquid multiplication culture stage of the embryonic tissue of Chinese pine, wherein A is the embryonic tissue observed under the microscope of the body type; B. embryonic tissue structure observed microscopically after staining, wherein S means embryonal suspensor and em means embryoid body.
FIG. 3 is a comparison of the growth state of embryonic tissue after 1 week of culture before the embryonic tissue of pinus tabulaeformis.
FIG. 4 is the embryogenic status of the embryonic tissue of Pinus tabulaeformis on maturation medium, where A and B are the growth and development status of the somatic embryo on maturation medium containing MGBG: a shows that several proembryo-like stages of somatic embryos developed in the embryonic tissue of Pinus tabulaeformis; b is an immature embryo before cotyledon emergence; c is mature embryo of Chinese pine observed under a microscope.
FIG. 5 effect of MGBG in different concentrations in the medium on the number of normal mature somatic embryos of Pinus tabulaeformis. Wherein the abscissa represents the concentration of MGBG (μ M) in the maturation medium; in the figure, 0, 50, 100 and 200 shown in the right small squares indicate the concentration (. mu.M) of MGBG in the pretreatment medium, respectively.
FIG. 6 comparison of growth of four differently treated embryogenic tissues on MGBG-containing maturation medium (1 week).
FIG. 7 comparison of growth of four differently treated embryogenic tissues on MGBG-containing maturation medium (6 weeks).
FIG. 8 shows somatic embryos of germination cultures of pine cone embryos.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Example 1
First, test materials
1. Immature cones of Chinese pine
In 2017, the method selects Chinese pine which is healthy in growth, free of diseases and insect pests, high in fruit setting rate and free in pollination from the Chinese pine fine variety base of the Shanxi Lvliang national forestry administration as a material taking place, collects 100 fruit balls after 51 days of pollen scattering, and stores the collected fruit balls in an environment at 4 ℃ for refrigeration for later use.
2. Plant growth regulator
The plant growth regulating substance used in the present invention is selected from domestic 6-benzylaminopurine (6-BA), 2, 4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), naphthylacetic acid (NAA), abscisic acid (ABA), MGBG (methylglyoxal bis-guanylhydrazone; methylglyoxal diamidinylhydrazone).
3. Culture medium
(1) mLV culture medium
TABLE 1 mLV culture Medium formulation
mLV minimal medium (Kong L, von Aderkas P.2007. genomic effects on ABA complementation and acidic organization in the interior space (Piceaglauca xenlmanni). Journal of Experimental Botany 58: 1525. 1531) is optimized from LV minimal medium formulation (Litvay JD, Verma DC, Johnson MA.1985. influx of cloning pine (Pinus taeda L.) culture medium and ingredients on growth and acidic organization of the world Carrot (Daucus carota L.). Plant Cell Reports 4: 325. 328).
(2) Embryonic tissue induction medium: mLV the culture medium is added with 2, 4-D2-4 mg/L, 6-BA1-2mg/L, hydrolyzed casein 500mg/L, sucrose 10-30g/L, plant gel 2.5-3.0g/L, and glutamine 500mg/L, the pH value is adjusted to 5.8, and the mixture is sterilized at the constant temperature of 121 ℃ for 20 minutes. Cooling the culture medium to about 50 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.
(3) Embryonic tissue solid proliferation medium: mLV the culture medium is added with 2, 4-D0.2-2 mg/L, 6-BA 0.1-1mg/L, hydrolyzed casein 500mg/L, sucrose 10-30g/L, plant gel 2.5-3.0g/L, and glutamine 500mg/L, the pH value is adjusted to 5.8, and the mixture is sterilized at the constant temperature of 121 ℃ for 20 minutes. Filtering and sterilizing glutamine in a clean bench, adding the culture medium into the culture medium after the culture medium is cooled to about 50 ℃, and mixing and shaking up.
(4) Embryonic tissue liquid proliferation medium: mLV the culture medium is added with 2, 4-D0.2-2 mg/L, 6-BA 0.1-1mg/L, hydrolyzed casein 500mg/L, sucrose 10-30g/L, and glutamine 500mg/L, the pH value is adjusted to 5.8, and the mixture is sterilized at the constant temperature of 121 ℃ for 20 minutes. Filtering and sterilizing glutamine in a clean bench, adding the culture medium into the culture medium after the culture medium is cooled to about 50 ℃, and mixing and shaking up.
(5) Embryonic tissue pretreatment culture medium: mLV the culture medium is added with MGBG50-200 mu M, ABA20-40 mu M, hydrolyzed casein 200mg/L, glutamine 500mg/L, maltose 20-30g/L and cane sugar 10-20g/L, the pH value is adjusted to 5.8, and the mixture is sterilized at the constant temperature of 121 ℃ for 20 minutes. Filtering and sterilizing glutamine, ABA and MGBG in a super clean bench, adding the culture medium into the culture medium after the culture medium is cooled to about 50 ℃, and mixing and shaking up the culture medium.
(6) Embryo maturation medium: mLV the culture medium is added with MGBG50-100 mu M, ABA 50-70 mu M, PEG400050-90g/L, hydrolyzed casein 200-500mg/L, glutamine 500mg/L, maltose 30-40g/L, sucrose 10-30g/L and plant gel 4-6g/L, the pH value is adjusted to 5.8, and the mixture is sterilized at the constant temperature of 121 ℃ for 20 minutes. Filtering and sterilizing glutamine, ABA and MGBG in a super clean bench, adding the culture medium into the culture medium after the culture medium is cooled to about 50 ℃, and mixing and shaking up the culture medium.
(7) 1-2g/L of active carbon, 10-20g/L of cane sugar and 8g/L of agar are added into a basic culture medium mLV of the somatic cell embryo germination, the pH value is adjusted to 5.8, and the somatic cell embryo germination culture medium is sterilized for 15 minutes at the constant temperature of 121 ℃.
Example 2
1. Sterilization of explants
The explant sterilization method is the same as that disclosed in patent number ZL 201410573714.1.
Washing the 51 st day pine cone after the loose powder is stored at 4 ℃ with a detergent to remove oil stains on the surface of the cone, then washing the cone with tap water, splitting the cone, and taking out seeds; soaking Chinese pine cone seeds in 75 vol% alcohol for 30-60s on a clean bench, taking out seeds, washing with sterile water for 3-5 times, and adding HgCl 0.1 wt%2Soaking in the solution for 5-10min, taking out seeds, washing with sterile water for several times (5-6 times), placing the washed seeds on filter paper sterilized at high temperature and high pressure, removing water, and opening seed coat with forceps and scissors to obtain female gametophyte (containing immature zygotic embryo) for use.
2. Embryonic tissue induction culture
Embryonic tissue induction culture was the same as disclosed in patent No. ZL 201410573714.1.
Inoculating 51 days after blooming and powder scattering of surface sterilized pinus tabulaeformis immature zygotic embryos serving as explants in an embryonic tissue induction culture medium, carrying out induction culture on the embryonic tissue under dark conditions, subculturing once every 20 days, wherein the culture temperature is (25 +/-2) ° c, 2,4-D used in the embryonic tissue induction culture medium is 2mg/L, 6-BA used in the embryonic tissue induction culture medium is 1mg/L, the natural compound is hydrolyzed casein 500mg/L, glutamine is 500mg/L, sucrose is 30g/L, plant gel is 2.8g/L, and after about 15 days of induction culture, the embryonic tissue grows from the end of a bead hole; about 60 days of culture, embryonic cell mass with a diameter of 1cm can be formed, as shown in FIG. 1.
3. Multiplication culture of embryonic tissue
3-1) solid multiplication culture of embryonic tissue
The solid multiplication culture of the embryonic tissue is the same as that disclosed in patent No. ZL 201410573714.1.
Inoculating the embryonic tissue to an embryonic tissue solid proliferation culture medium, and carrying out first-stage solid proliferation culture on the embryonic tissue of the pinus tabulaeformis under the dark condition, wherein the culture temperature is (25 +/-2) ° C, the 2,4-D used in the embryonic tissue solid proliferation culture medium is 1.0mg/L, the 6-BA is 0.5mg/L, the casein hydrolysate is 500mg/L, the glutamine is 500mg/L, the sucrose is 20g/L, and the plant gel is 2.8g/L, and after subculture is carried out for 2 times every 3-4 weeks in the process of solid proliferation culture of the embryonic cells of the pinus tabulaeformis, a large amount of translucent pinus tabulaeformis embryonic tissue is obtained, and is shown in figure 1.
The concentration of 2,4-D in the solid multiplication medium is not only 2mg/L, but also 0.2-2mg/L of 2,4-D is suitable for the present invention; concentrations of 6-BA in addition to 1mg/L, concentrations of 6-BA of 0.1 to 1mg/L are suitable for use in the present invention.
The embryonic tissue obtained after solid multiplication culture is in a semitransparent, loose and granular structure. Many small embryos with filamentous protrusions, including the structure of the embryo heads and the embryo stalks of the small embryos, can be seen under a stereoscopic microscope.
3-2) liquid multiplication culture of embryonic tissue
Inoculating 5-10g of embryonic tissue obtained by the first stage solid multiplication culture into 100ml of an embryonic tissue liquid multiplication culture medium, and performing second stage liquid multiplication culture of the embryonic tissue of the Chinese pine under the dark condition, wherein the culture temperature is (25 +/-2) DEG C, 2,4-D used in the embryonic tissue liquid multiplication culture medium is 1.0mg/L, 6-BA is 0.5mg/L, casein hydrolysate is 500mg/L, glutamine is 500mg/L, and sucrose is 20g/L, the embryonic tissue is subcultured once per week in the liquid multiplication culture process of the embryonic cells of the Chinese pine embryonic, the subculture is carried out to keep the culture medium to contain sufficient nutrients and water, and after the subculture is carried out for 2 times, the growth condition, the multiplication rate and the morphology of the embryonic tissue of the liquid multiplication are counted, and the embryonic tissue of the liquid culture medium are obviously layered, obtaining a large amount of semi-transparent, loose and granular structure embryonic proliferative embryonic tissues of the Chinese pine. The embryonic tissue observed under the stereomicroscope is shown in FIG. 2A; the differentiation of the embryoid body and the embryonal suspensor can be clearly seen by observing the embryogenic tissue structure under a microscope after staining with Carbonisatus (DZ 0040; LEAGENE) (see FIG. 2B).
The concentration of 2,4-D in the liquid multiplication medium is not only 2mg/L, but also 0.2-2mg/L of 2,4-D is suitable for the present invention; concentrations of 6-BA in addition to 1mg/L, concentrations of 6-BA of 0.1 to 1mg/L are suitable for use in the present invention.
EXAMPLE 3 Pre-treatment culture of embryonic tissue
Inoculating 2g (usually 2-5g) of semi-transparent embryonic tissue (surface is granular or filamentous) after liquid multiplication culture into a 100mL conical flask, adding 30-40mL of embryonic tissue pretreatment culture medium into the conical flask, and performing suspension culture in the conical flask under dark condition, namely performing Pinus tabulaeformis embryonic tissue pretreatment culture, wherein the culture temperature is (25 + -2) ° C, the dosage of MGBG used in the embryonic tissue pretreatment culture medium is shown in Table 2, ABA 30. mu.M, casein hydrolysate 500mg/L, glutamine 500mg/L, sucrose 10g/L and maltose 20g/L, and placing the conical flask in 100 r.min–1Dark treatment on a constant temperature shaker at 25 ℃ for 1 week, repeating each experiment for 3 times, setting blank control, observing and recording the growth condition and the morphology of the embryonic tissue.
Proliferation rate (%) -fresh weight gain (g)/initial inoculum (g). 100
The growth status and morphology observation results of the embryonic tissue 1 week after the pretreatment culture are shown in fig. 3, wherein a is that MGBG is not added in the left-side conical flask culture medium, and the amount of the embryonic tissue after proliferation is large; and B is that the concentration of MGBG in the right conical flask is 200 mu M, the amount of the embryonic tissues in the flask after proliferation is less than that of a control (without MGBG), the proliferation amount of the embryonic tissues in the flask is obviously different, the proliferation amount of the embryonic tissues in a culture medium added with MGBG is obviously reduced, and the result shows that the MGBG plays a certain role in inhibiting the proliferation of the embryonic tissues in suspension culture. In addition, after pretreatment culture, many small somatic embryos corresponding to proembryos were cultured in the medium containing MGBG, the somatic embryo structure was normal, and the somatic embryos and suspensor were clear.
In the pretreatment culture of the embryonic tissue of the present invention, the embryonic tissue after the propagation culture may be used, or the embryonic tissue obtained by the induction culture may be directly subjected to the pretreatment culture.
EXAMPLE 4 maturation culture of embryogenic tissue
The embryonic tissue after the pretreatment culture for 1 week (usually 1-2 weeks) is taken out, placed on a maturation medium, and the total mass of the medium and the embryonic tissue is measured. And (3) carrying out embryo maturation culture on the embryonic tissue of the Chinese pine under the dark condition, wherein the culture temperature is (25 +/-2) DEG C.
The dosage of MGBG used in the embryonic tissue maturation medium is shown in Table 2, ABA 60. mu.M, polyethylene glycol (PEG4000)75g/L, hydrolyzed casein 500mg/L, glutamine 500mg/L, maltose 30g/L, and sucrose 10 g/L. Dark cultures were repeated 3 times per experiment and the total mass of mature medium and embryonic tissue was weighed weekly.
The method for determining the proliferation amount of the embryonic tissue comprises the steps of weighing the weight of the mature culture medium with the embryonic tissue at the initial stage and recording the weight as W0After one week the entire maturation medium was weighed again and recorded as W1，W1And W0The difference of (a) is the proliferation weight of the embryonic tissue within the week, and so on (the water loss in the culture medium during the culture process is ignored), and the proliferation amount of the embryonic tissue per week is counted. After culturing for 6-8 weeks, somatic embryos can be observed to develop from early immature somatic embryos to the stage of cotyledon embryos, and mature somatic embryos are obtained.
After 6-8 weeks, the number of mature embryos per dish was counted, and the results are shown in FIG. 5, in which the abscissa represents the concentration of MGBG in the maturation medium; in the figure, 0, 50, 100 and 200 shown in the right small squares indicate the concentration (. mu.M) of MGBG in the pretreatment medium, respectively.
Growth of mature embryos in mature media containing MGBG observed under microscope as FIG. 4; wherein A and B are the growth conditions of somatic embryos in MGBG-containing maturation medium; c is mature embryo of Chinese pine observed under a microscope. The scale in the figure is 1 mm.
TABLE 2 Effect of MGBG concentration on embryonic tissue maturation
As can be seen from table 2, the proliferation amount of the embryonic tissue which is not treated by the addition of MGBG in the embryonic tissue pretreatment culture and the embryo maturation culture is much higher than that of the other treatment groups in the whole culture process, while the proliferation amount of the embryonic tissue is significantly reduced in the other treatment groups to which MGBG is added; the proliferation amount of the embryonic tissue is gradually reduced along with the increase of the MGBG concentration, and the proliferation amount of the embryonic tissue is slowly increased after the proliferation amount of the embryonic tissue is increased to a certain concentration. It is shown that the optimum concentration is found in the experiment because the MGBG concentration reaches a certain value and has an inhibitory effect on the proliferation of embryonic tissues.
As can be seen from table 2, MGBG showed a significant inhibitory effect on the amount of growth of embryonic tissue, and the number of mature embryos was significantly increased although the proliferation of embryonic tissue was inhibited, as shown in fig. 3.
As can be seen from fig. 5, in the three treatments, the number of mature embryos increased gradually with the increase of the concentration of MGBG solution in the embryonic tissue pretreatment medium, but when the concentration of MGBG in the pretreatment medium was 200 μ M, the number of mature somatic embryos decreased to be lower than the number of mature somatic embryos with MGBG concentration of 100 μ M; wherein the number of mature embryos was the greatest when the MGBG solution concentration in the maturation medium was 50. mu.M (group 2 treatment in FIG. 5).
The concentration of the MGBG solution in the current treatment culture medium is 100 mu M, the number of mature embryos reaches the highest when the concentration of the MGBG solution in the maturation culture medium is 50 mu M, which indicates that the MGBG solution in the current treatment culture medium is a proper liquid suspension pretreatment concentration when the concentration of the MGBG solution in the pretreatment culture medium is 100 mu M, and the MGBG solution is more suitable for the conversion of the embryogenic tissue to the growth and development of the maturation stage, and in combination with the table 2, the proliferation amount of the embryogenic tissue is obviously reduced under the treatment, so that the MGBG solution has an inhibition effect on the proliferation of the embryogenic tissue, and has a promotion effect on the embryogenic development of the embryogenic tissue and the growth and development of the embryogenic tissue to the maturation stage to a certain extent.
As can be seen from FIG. 5, the pinus tabulaeformis embryonic tissue treated completely without MGBG produced only a small number of normal mature somatic embryos, whereas the number of normal mature somatic embryos increased 10-fold after treatment with the appropriate concentration of MGBG (pretreatment culture 100/maturation culture 50) (FIG. 5). The total number of somatic embryos is also increased by more than 10 times.
In both the pretreatment culture and the maturation culture, when the MGBG concentration reached a certain concentration (200. mu.M), the number of mature embryos increased insignificantly, while the amount of embryogenic tissue proliferation decreased, the growth state was unhealthy, the tissue was watery, not loosely bonded, hardened and browned. Indicating that excess MGBG is not suitable for proliferation and mature development of embryonic tissue. According to the results of this experiment, the effect and quality of the embryos were considered comprehensively, and the MGBG concentration was 100. mu.M in the pretreatment culture and the effect was the best at 50. mu.M in the maturation culture.
Wherein: the conditions of the embryonic tissues of the 4 treatments (A, B, C, D) in the maturation culture in this example after 1 week and 6 weeks of the maturation culture treatment are shown in FIGS. 6 and 7, respectively, wherein the growth conditions of the embryonic tissues after 1 week of the treatment are shown in FIG. 6, and the growth conditions of the embryonic tissues after 6 weeks of the treatment are shown in FIG. 7, wherein:
the treatment A is that the MGBG concentration in the embryonic tissue pretreatment culture process is 0 mu M, and the MGBG concentration in the somatic cell embryo maturation culture process is 0 mu M;
the treatment B is that the MGBG concentration in the embryonic tissue pretreatment culture process is 50 MuM, and the MGBG concentration in the somatic cell embryo maturation culture process is 0 MuM;
c treatment is that the MGBG concentration in the embryonic tissue pretreatment culture process is 100 mu M, and the MGBG concentration in the somatic cell embryo maturation culture process is 50 mu M;
d, the treatment is that the MGBG concentration in the embryonic tissue pretreatment culture process is 200 mu M, and the MGBG concentration in the somatic cell embryo maturation culture process is 100 mu M;
as can be seen from fig. 6 and 7, the proliferation amount of the embryonic tissue after 6 weeks of culture varied differently for each group of treatments, and the four treatments were supplemented with the same amount of embryonic tissue at the initial stage of culture, and the gradual decrease in proliferation amount of the embryonic tissue was clearly observed after 6 weeks, which is illustrated in fig. 7, that MGBG plays a partial inhibitory role in the proliferation of the embryonic tissue.
The results of the comparative tests show that: MGBG can obviously inhibit excessive proliferation of embryonic tissues in mature culture and provide space for the growth of mature embryos; when MGBG mother liquor is added into a liquid culture medium and a mature solid culture medium before suspension treatment to 100 mu M and 50 mu M respectively, the purpose of obviously inhibiting the proliferation of embryonic tissues can be achieved, and mature embryos can grow and develop well.
Example 5 somatic embryo Germination and somatic embryo transplantation
Separating the mature somatic embryos cultured in the treatment 8 of the example 5 from embryonic tissues, transferring the separated mature somatic embryos to a somatic embryo germination culture medium, culturing the mature somatic embryos under the conditions of 25 +/-2 ℃ and light cycle of 16h light/8 h dark and light intensity of 1500-2000Lux, wherein the relative humidity in the germination culture process is 60-75%, the active carbon content in the germination culture medium is 1g/L, the sucrose content in the germination culture medium is 10-20g/L, the agar content in the germination culture medium is 7g/L, the cotyledons of the mature somatic embryos quickly turn green, the hypocotyls grow in an elongated manner, and the cotyledons at the top ends of the mature somatic embryos expand after 8 weeks of culture. When the somatic embryo germinates, one end of the somatic embryo differentiates to form a root growing end, and the other end of the somatic embryo germinates to form a somatic embryo seedling (as shown in figure 8).
The somatic embryo germination rate (%) -. germinated somatic embryo number/inoculated somatic embryo number X100%
In the process of somatic embryo germination culture, 10-12 somatic embryos are inoculated in each treatment, and the treatment is repeated for 3 times. The germination rate of somatic embryo is 94%, and the seedling transferring rate is 72%.
When the somatic embryo seedlings grow to be 2 cm high and the roots are thick and strong, opening the bottle caps of culture bottles, hardening and culturing for 1 day in a transplanting room, taking out the plants, washing agar culture medium remained at the roots of the test-tube seedlings by tap water, transplanting the test-tube seedlings into an Chinese pine soilless culture substrate (perlite, vermiculite and the volume ratio of the perlite to the vermiculite is 1:1), and performing container culture in the transplanting room. Keeping the relative humidity at 70-90% for one week after seedling transplantation, and culturing at 25 + -5 deg.C for 7-10 days to obtain Chinese pine seedling.
In the present invention, if only liquid pretreatment culture is performed, MGBG is not added to the maturation medium, and the embryonic tissue of Pinus tabulaeformis can only produce a few normal mature somatic embryos, as shown in FIG. 5.
In the present invention, if only maturation culture is performed without performing pretreatment culture of liquid maturation culture, the tissues of the embryonic plant of Pinus tabulaeformis cannot produce normal mature somatic embryos.
In the method, in the process of transforming the pine oleostock into the somatic embryo after the propagation culture, the pretreatment adjustment culture of the oleostock is firstly carried out, then the maturation culture of the somatic embryo is carried out, and polyamine synthesis inhibitor MGBG is added into a pretreatment culture medium and a maturation culture medium, so that the excessive propagation of the oleostock is avoided; the proliferation of the embryonic tissue is inhibited, the excessive proliferation of the embryonic tissue is reduced, better conditions are created for the mature culture of somatic embryos, more high-quality mature somatic embryos are obtained, and the yield of somatic embryo seedlings is improved. The Chinese pine propagation method provides an effective method for multi-gene type large-scale asexual seedling culture of the Chinese pine.
Claims (6)
1. A culture method for promoting development and maturation of a somatic embryo of Chinese pine is characterized by comprising the following steps in sequence:
1) inoculating the pinus tabulaeformis sterile female gametophyte into an induction culture medium, and performing embryonic tissue induction culture to obtain an embryonic tissue;
2) performing proliferation culture on the embryonic tissue in a proliferation culture medium;
3) inoculating the embryonic tissue into an embryonic tissue pretreatment culture medium, and performing embryonic tissue pretreatment culture to obtain a pretreated and cultured embryonic tissue, wherein the embryonic tissue pretreatment culture medium contains a regulator MGBG (methylglyoxal bis-guanylhydrazone);
4) and inoculating the embryonic tissue after the pretreatment culture on an embryo maturation culture medium, and performing somatic embryo maturation culture to obtain a mature somatic embryo, wherein the embryo maturation culture medium contains a regulator MGBG.
2. The method according to claim 1, wherein the concentration of MGBG in the embryonic tissue pretreatment medium in step 3) is 30-200. mu.M.
3. The method as set forth in claim 1, wherein the embryonic tissue pretreatment medium in step 3) is mLV medium + MGBG 30-200. mu.M + ABA 0-100. mu.M + hydrolyzed casein 200-500mg/L + maltose 10-40g/L + sucrose 10-40g/L + glutamine 500mg/L.
4. The method as claimed in claim 1, wherein the embryo maturation medium in step 4) contains the regulator MGBG50-100 μ M.
5. The method as claimed in claim 1, wherein the embryo maturation medium in step 4) is mLV medium + MGBG50-100 μ M + ABA30-80 μ M + PEG400050-90g/L + hydrolyzed casein 200-500mg/L + maltose 30-40g/L + sucrose 10-30g/L + glutamine 500mg/L + phytogel 4-7 g/L.
6. The method according to claim 1, wherein said embryogenic tissue proliferation culture in step 2) comprises the steps of:
a) inoculating the embryonic tissue to an embryonic tissue solid multiplication culture medium to carry out first-stage solid multiplication culture on the embryonic tissue of the Chinese pine;
b) inoculating the embryonic tissue subjected to the first-stage solid multiplication culture into an embryonic tissue liquid multiplication culture medium, and performing second-stage liquid multiplication culture on the embryonic tissue of the Chinese pine to obtain the embryonic tissue multiplied by liquid culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910776447.0A CN110896854B (en) | 2019-08-22 | 2019-08-22 | Culture method for promoting development and maturation of somatic embryos of Chinese pine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910776447.0A CN110896854B (en) | 2019-08-22 | 2019-08-22 | Culture method for promoting development and maturation of somatic embryos of Chinese pine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110896854A CN110896854A (en) | 2020-03-24 |
| CN110896854B true CN110896854B (en) | 2021-05-11 |
Family
ID=69814555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910776447.0A Active CN110896854B (en) | 2019-08-22 | 2019-08-22 | Culture method for promoting development and maturation of somatic embryos of Chinese pine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110896854B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112042541B (en) * | 2020-09-15 | 2023-03-07 | 北京林业大学 | Method for propagating taxillus through somatic embryogenesis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461119A (en) * | 2013-08-20 | 2013-12-25 | 中国林业科学研究院林业研究所 | Somatic embryogenesis and plant regeneration method for Picea asperata Mast |
| CN104381131A (en) * | 2014-10-24 | 2015-03-04 | 北京林业大学 | Somatic embryogenesis and plant regeneration method for pinus tabulaeformis |
| CN108094207A (en) * | 2017-12-26 | 2018-06-01 | 广东省林业科学研究院 | Wet-land pine tree somatic embryo occurs and the method for plant regeneration |
| CN109156185A (en) * | 2018-09-03 | 2019-01-08 | 广西壮族自治区林业科学研究院 | A kind of masson pine Water culture method for culturing seedlings |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3775506B2 (en) * | 2002-04-08 | 2006-05-17 | 東洋紡績株式会社 | Plant with improved organ formation and method for producing the same |
| KR101899140B1 (en) * | 2016-04-28 | 2018-09-14 | 충청북도 (관리부서:충청북도 농업기술원) | Method of plant culture for mass propagation of Aralia elata Seem |
| CN108208149B (en) * | 2018-01-31 | 2021-06-15 | 河南农业大学 | Application of polyamine decomposition inhibitor aminoguanidine in fruit preservation and use method thereof |
| CN109220809B (en) * | 2018-11-13 | 2022-01-04 | 北京林业大学 | Koelreuteria paniculata somatic embryogenesis and plant regeneration culture method |
-
2019
- 2019-08-22 CN CN201910776447.0A patent/CN110896854B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103461119A (en) * | 2013-08-20 | 2013-12-25 | 中国林业科学研究院林业研究所 | Somatic embryogenesis and plant regeneration method for Picea asperata Mast |
| CN104381131A (en) * | 2014-10-24 | 2015-03-04 | 北京林业大学 | Somatic embryogenesis and plant regeneration method for pinus tabulaeformis |
| CN108094207A (en) * | 2017-12-26 | 2018-06-01 | 广东省林业科学研究院 | Wet-land pine tree somatic embryo occurs and the method for plant regeneration |
| CN109156185A (en) * | 2018-09-03 | 2019-01-08 | 广西壮族自治区林业科学研究院 | A kind of masson pine Water culture method for culturing seedlings |
Non-Patent Citations (3)
| Title |
|---|
| "Polyamines influence morphogenesis and caffeine biosynthesis in in vitro cultures of Coffea canephora P. ex Fr.";Vinod Kumar等;《Acta Physiol Plant》;20080315(第30期);第217-223页 * |
| "多胺对宁夏枸杞愈伤组织器官发生和体细胞胚发生的影响";胡忠等;《实验生物学报》;20010815;第34卷(第3期);第191页摘要 * |
| "植物体中多胺代谢及其功能研究进展";马瑛等;《陕西理工学院学报（自然科学版）》;20100615;第26卷(第2期);第49页第2.1.2.4节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110896854A (en) | 2020-03-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100889342B1 (en) | Propagation method of liriodendron tulipifera using somatic embryogenesis technique | |
| Eccher et al. | Comparison between 2iP and zeatin in the micropropagation of highbush blueberry (Vaccinium corymbosum) | |
| Aliyu | Application of tissue culture to cashew (Anacardium occidentale L.) breeding: An appraisal | |
| Kim et al. | Somatic embryogenesis and plant regeneration from immature embryos of five families of Quercus acutissima | |
| CN104381131B (en) | A kind of Pinus tabuliformis somatic embryo occurs and plant regeneration method | |
| Onay | In Vitro organogenesis and embryogenesis of pistachio, Pistacia vera L. | |
| Devi et al. | In vitro propagation of Arundinaria callosa Munro-an edible bamboo from nodal explants of mature plants | |
| US7932086B2 (en) | Commercially viable process for in vitro mass culture of Jatropha curcas | |
| Hossain et al. | High efficiency plant regeneration from petiole explants of Carica papaya L. through organogenesis | |
| JPH07308137A (en) | Method for producing body of plant belonging to section leuce | |
| CN110896854B (en) | Culture method for promoting development and maturation of somatic embryos of Chinese pine | |
| Häggman et al. | Early-flowering Scots pines through tissue culture for accelerating tree breeding | |
| Hosseinpour et al. | High frequency in vitro propagation of M× M60, a cherry rootstock: the effects of culture media and growth regulators | |
| Agnihotri et al. | In vitro cloning of female and male Carica papaya through tips of shoots and inflorescences | |
| CN112042541A (en) | Method for propagating taxillus through somatic embryogenesis | |
| Onay | Micropropagation of pistachio | |
| EP1817956A2 (en) | Commercially viable process for in-vitro mass culture of jatropha curcas | |
| Abo El-Fadl et al. | In vitro propagation of avocado (Persea americana Mill.) | |
| Tao et al. | Comparison of growth and rooting characteristics of micropropagated adult plants and juvenile seedlings of persimmon (Diospyros kaki L.) | |
| Prabhuling et al. | In Vitro Regeneration in Pomegranate (Punica granatum L.) cv. Bhagwa using Double Nodal Segments | |
| Arya et al. | In vitro shoot proliferation and somatic embryogenesis: means of rapid bamboo multiplication | |
| Yong-Wook | Somatic embryogenesis in Quercus acutissima | |
| Schwarz | 6. Plant growth regulator effects in the in vitro propagation of three hardwood tree genera: Castanea, Juglans, and Quercus | |
| CN114467753B (en) | Tissue culture method of silvery deer maple | |
| Tangolar et al. | High efficiency somatic embryogenesis from immature zygotic embryos of grapevine: The effect of genotype, media, 2, 4-D, and incubation conditions |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |