Culture method for promoting development and maturation of somatic embryos of Chinese pine
阅读说明：本技术 一种促进油松体细胞胚胎发育成熟的培养方法 (Culture method for promoting development and maturation of somatic embryos of Chinese pine ) 是由 孔立生 张金凤 赵健 李亦轩 崔莹 范英明 于 2019-08-22 设计创作，主要内容包括：本发明公开了一种促进油松体细胞胚胎(体胚)发育成熟的方法,包括对油松胚性组织进行液体前处理培养,其中所述胚性组织前处理培养基中含有多胺合成抑制剂类调节剂。本发明方法在液体前处理培养基和胚胎成熟培养基里添加多胺合成抑制剂类调节剂,以达到抑制胚性组织在胚胎成熟过程中的过量增殖,促进体胚的发育和成熟,提高成熟体细胞胚胎的质量和数量的目的。使用本发明的方法能从油松胚性较弱,增殖过量的基因型材料中获得更多的成熟体胚和体胚苗,有利于油松体胚苗基因型的多元化,从而能克服无性系林业基因型的局限性,规模化、工厂化生产多基因型油松体胚苗。(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.)
1. A culture method for promoting the development and maturation of the embryo of the somatic cell of the Chinese pine is characterized by comprising the step of carrying out pretreatment culture on the embryonic tissue of the Chinese pine, wherein a culture medium for the pretreatment culture of the embryonic tissue contains a regulator of polyamine synthesis inhibitors.
2. The method according to claim 1, further comprising inoculating the pre-treated and cultured embryonic tissue to an embryo maturation medium containing a polyamine synthesis inhibitor-based regulator, and culturing the somatic embryos for maturation.
3. 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) 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);
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.
4. The method according to claim 3, wherein the concentration of MGBG in the embryonic tissue pretreatment medium in step 2) is 30-300. mu.M.
5. The method as set forth in claim 3, wherein the embryonic tissue pretreatment medium in step 2) is mLV medium + MGBG 30-300. mu.M + ABA 0-100. mu.M + hydrolyzed casein 200-500mg/L + maltose 10-40g/L + sucrose 10-40g/L + glutamine 500mg/L.
6. A method according to any one of claims 3 to 5, wherein the embryo maturation medium in step 3) contains the modulator MGBG.
7. The method according to any one of claims 3 to 5, wherein the embryo maturation medium in step 3) is mLV 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 + phytogel 4-7 g/L.
8. The method according to any one of claims 3 to 5, further comprising a step 1A) of inoculating the embryogenic tissue obtained by the culture in the step 1) to a multiplication medium, performing embryogenic tissue multiplication culture, obtaining a multiplied embryogenic callus, inoculating the multiplied embryogenic callus to an embryogenic tissue pretreatment medium, and performing the embryogenic tissue pretreatment culture.
9. The method according to claim 8, wherein said embryogenic tissue proliferation culture in step 1A) comprises the steps of:
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.
10. A propagation method of Chinese pine is characterized by comprising the pretreatment of liquid suspension culture of embryonic tissue of Chinese pine, wherein a modifier of polyamine synthesis inhibitor is contained in a culture medium for pretreatment of the embryonic tissue.
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
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
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
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
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.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种变叶榕组织培养与快速繁殖的方法