CN112042541B

CN112042541B - Method for propagating taxillus through somatic embryogenesis

Info

Publication number: CN112042541B
Application number: CN202010969083.0A
Authority: CN
Inventors: 孔立生; 张金凤; 赵健; 高英; 崔莹; 李珊珊; 范英明
Original assignee: Shenzhou Lvpeng Agricultural Science & Technology Co ltd; Beijing Forestry University
Current assignee: Shenzhou Lvpeng Agricultural Science & Technology Co ltd; Beijing Forestry University
Priority date: 2020-09-15
Filing date: 2020-09-15
Publication date: 2023-03-07
Anticipated expiration: 2040-09-15
Also published as: CN112042541A

Abstract

The invention discloses a propagation method of taxus plants through somatic embryogenesis, which comprises the following steps of performing embryonic tissue induction culture, propagation culture, embryonic tissue adjustment culture, somatic embryo (somatic embryo) maturation culture, germination culture of mature embryos and seedling transformation culture on sterile explants of the taxus plants. By the method, a stable east China fir embryonic cell line and a large number of mature somatic embryos are obtained by a somatic embryogenesis induction way for the first time, and then a complete somatic embryo plant is obtained. With the method of the present invention, a large number of somatic embryos of yellow cedar in east China can be produced in a relatively short period of time. The technology is suitable for large-scale and industrial production of the cloned seedlings of the taxus chinensis.

Description

Method for propagating taxillus through somatic embryogenesis

Technical Field

The invention relates to a plant tissue culture method, in particular to a method for regenerating a plant of yellow cedar in east China through somatic embryo (somatic embryo) culture, belonging to the technical field of cell engineering seedling breeding in forestry.

Background

Flammulina huadonii (Pseudootsuga gaussenii Fleus) is an evergreen tree of the genus Flammulina of the family Pinaceae. The Taxus genus plants have 7 species, 5 species are Chinese specific tree species, and the other 2 species are distributed in North America. The native north american douglas fir (mirb.) Franco is a well-known superior tree species of forest trees in the world. The height of the east China fir can reach 40 meters, the bark is dark gray, the top of the leaf pillow is brown, the main branch has no hair or has loose hair, the lateral branch has brown dense hair, and the winter bud is oval or oval-shaped, conical and brown. The blades are strip-shaped and are arranged in two rows or extend on the main branch in a near-radiation manner, the upper part is dark green, and the lower part is provided with two white porous belts; cone-shaped spherical or oval, wide base, narrow upper part and slightly white powder; the middle part of the seed is shaped like a kidney with scales or a kidney with a transverse ellipse, the upper part of the bract scales is backwards extended, the median fissure is longer, the shape of a narrow triangle, the shape of a triangle with side fissure is narrow, the tip of the seed is sharp or blunt, the seed is in a triangular oval shape, the seed is slightly flat, and the seed wing is approximately equal to the seed in length. Blooming in 4-5 months, and ripening in 10 months. The distribution area of the yellow cedar in east China is located in the mountainous areas with elevation of 600-1500 m from the low mountain to the middle mountain of the subtropical zone in the east China. The weather in the distribution area is cool and moist, the annual average temperature is 9-15 ℃, the lowest temperature is-9 to-16 ℃, and the highest temperature is 32-35 ℃. Annual precipitation is 400-2000 mm, and the relative humidity is generally 90%. The soil is red soil and mountain yellow soil, and the pH value is 5.5-6.5. The taxus chinensis has the advantages of light brown sapwood, brown heartwood, straight texture, rough material, moderate hardness and specific gravity of 0.6, and can be used for buildings, furniture and the like. Can also be used as garden ornamental tree species.

The yellow fir (Pseudotsuga sinensis Dode) is similar to yellow fir of east China, and is mainly characterized in that the spherical fruit of the yellow fir is oval or elliptical, the middle part is wide, and the two ends are slightly narrow. The cones of the taxus chinensis in east China are usually in a conical oval shape, the base part is wide, the upper part is narrow, the middle part is in a kidney shape of scales or a kidney shape of a transverse ellipse, and the exposed parts of the scales are unhaired; the seed wings are approximately equal in length to the seeds.

The yellow fir and the yellow fir in east China generally blossoms and fruits in 15 years and has mature cones. The number of seeds that can be pregnant is very small, some are all infertile, and some embryos shrink or are damaged by insects. Can be used as afforestation tree species in mountainous regions with 500-1500 m altitude in Zhejiang, south Anhui, northern Fujian, west and east China. The taxus genus plants are distributed in east Asia and North America discontinuously, so that the taxus genus plants are well protected, and the taxus genus plants have academic significance for researching plant regions. In the environmental protection committee of the Chinese State Council, published in 7 months 1984 (rare endangered protection plant roster), the yellow fir in east China and the yellow fir belong to II-level protection and are gradually planted in critical conditions. Due to long-term felling and utilization, the seed fertility rate is very low, the tree species renewal capacity is very weak, and the forest trees are gradually reduced. In the producing area of the taxus plants, measures are urgently needed to protect the mother trees and promote natural updating and expand artificial planting (refer to China forestry science data center, plant annals).

The breeding of the taxus plants mainly adopts a seeding and seedling raising method, and seeds are mainly collected in the field or provided by a seed garden. China has a taxus plant seed garden, and the seed garden building is long in time and needs to invest a large amount of manpower and financial resources in the early stage. In addition, the problems of late seed setting, low seed yield, unstable quality, serious pest and disease damage and the like are the problems commonly existing in the current seed gardens. 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. Similar to other needle-leaved tree plants, the cutting and grafting technology of the taxus genus plant has many difficulties: such as strong age effect, low rooting rate of cuttage, low survival rate of grafting, and the like. The breeding difficulty of the taxus genus plant greatly limits the protection of the taxus genus plant and the popularization and application of improved varieties.

Tissue culture is a widely applied technology in plant breeding at present, and is a basis for realizing forest genetic transformation and transgenic technology. Plant somatic embryos resemble seed embryos with shoot tips and root tip meristems. Somatic embryogenesis is considered to be the most potent method and means of asexual propagation of conifers. The research of successfully establishing a somatic embryo system of the taxus chinensis for large-scale propagation is not reported yet.

The invention mainly aims to develop an efficient vegetative propagation technology for plant somatic embryos aiming at the current situations that the natural seed setting rate of the taxus chinensis and the cuttage is difficult, and the seedling propagation technology cannot meet the requirements of large-area afforestation, and provides a method with short period, high propagation rate and low cost for the protection, large-scale propagation and seedling raising of the taxus chinensis and the taxus chinensis.

Disclosure of Invention

The invention aims to provide a novel method for propagating taxus plants, aiming at the technical problems in the propagation process of the existing taxus plants.

In order to achieve the object of the present invention, in one aspect, the present invention provides a method for propagating taxus genus plant, comprising the following steps in sequence: performing embryogenic tissue induction culture, proliferation culture, embryonic tissue adjustment culture, somatic embryo maturation culture, mature somatic embryo germination culture, and seedling transformation culture on sterile explants of Taxus plants.

Wherein the plant of the genus Flammulina is selected from Flammulina velutipes, flammulina yezoensis, flammulina brevifolia, flammulina velutipes, or Flammulina velutipes, preferably Flammulina velutipes and Flammulina yezoensis.

In another aspect, the present invention provides a method for propagating Taxus genus plant, comprising the steps of:

1) Inoculating the sterilized explant of the taxus genus plant on an embryonic tissue induction culture medium for induction culture to obtain an embryonic tissue;

2) Inoculating the embryonic tissue on a proliferation culture medium to perform proliferation culture of the embryonic tissue;

3) Inoculating the embryonic tissue subjected to propagation culture into an embryonic tissue pretreatment culture medium, and performing embryonic tissue adjustment culture to obtain a pretreated embryonic tissue;

4) Inoculating the pretreated embryonic tissue on a somatic embryo maturation culture medium, and performing somatic embryo maturation culture to obtain a mature somatic embryo;

5) Inoculating the mature somatic embryos on a somatic embryo germination culture medium, and performing germination culture to obtain somatic embryo seedlings;

6) Hardening and transplanting the somatic embryo seedlings to obtain the seedling-hardening seedling-transplanting seedling-hardening seedling-transplanting seedling.

Wherein the sterilized explant in step 1) is obtained by the following method: and (3) carrying out surface sterilization treatment on the collected immature female cones of the taxus genus plant, then stripping, and taking out zygotic embryos, namely the sterilized explants.

In particular, the surface sterilization treatment comprises the following steps in the following order:

1-1) cleaning collected female strobilus of the genus Cunninghamia with a detergent, and then washing with tap water;

1-2) sucking water on the surface of the female cones, and taking out seeds from the female cones;

1-3) soaking the seeds in 75 vol% ethanol for 30-60s, taking out, washing with sterile water for 3-5 times, and adding 0.1% HgCl ₂ Soaking the seeds in the solution for 5-10min, taking out, and washing with sterile water for 5-6 times to obtain sterile seeds;

1-4) after moisture on the surface of the seeds is absorbed, peeling off the seed coat of the sterile seeds under a sterile state until a sterile embryo (namely a sterile zygotic embryo) is obtained.

In particular, the induction rate of embryogenic tissue is significantly affected by the explant embryonic age of the taxus genus plant. The invention selects a seed embryo with a growth and development stage between a multi-embryo stage and a cotyledon stage as an explant for inducing embryonic tissues. Collecting female cones at about 6-10 weeks after blooming and pollen scattering, continuously checking the development condition of the embryo of the seed from the sixth week of pollination, and selecting the embryo with a proper development stage as an explant.

In particular, the strobilus of the fir, six weeks after flowering loose powder, is collected sequentially to determine the appropriate explant, which is preferably a seed embryo with a developmental stage between the multi-embryonic stage and the cotyledon stage.

Wherein, the embryonic tissue induction culture medium in the step 1) is: mLV medium +2,4-D (2-6 mg/L) +6-BA (1-3 mg/L) + hydrolyzed casein (400-1000 mg/L) + glutamine (200-600 mg/L) + sucrose (10-30 g/L) + plant gel (2.5-3.0 g/L); preferably mLV medium +2,4-D (2-4 mg/L) +6-BA (1-2 mg/L) + hydrolysed casein (400-800 mg/L) + glutamine (200-500 mg/L) + sucrose (10-30 g/L) + plant gel (2.5-3.0 g/L), preferably mLV medium +2,4-D (4 mg/L) + BA (2 mg/L) + hydrolysed casein (500 mg/L) + glutamine (500 mg/L) + sucrose (10 g/L) + plant gel (2.75 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 4-6 weeks.

In particular, the embryogenic tissue is subcultured once every 2-3 weeks in the induction culture process; the number of subcultures was 1-3.

Wherein, the proliferation culture in the step 2) comprises the following steps in sequence:

2A) Inoculating the embryonic tissue to an embryonic tissue solid multiplication culture medium to carry out the first-stage solid multiplication culture of the embryonic tissue;

2B) 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 to obtain the multiplication-cultured embryonic tissue.

In particular, the liquid multiplication culture of the second stage is to inoculate the embryonic tissue after the solid multiplication culture of the first stage into a conical flask or a bioreactor filled with the liquid multiplication culture medium of the embryonic tissue for liquid multiplication.

In particular, the liquid proliferation may be performed by inoculating the embryogenic tissue after passing through the Erlenmeyer flask liquid proliferation medium into a pneumatic bioreactor containing the liquid proliferation medium for the embryogenic tissue.

Particularly, in the liquid proliferation culture process of the second stage using the pneumatic bioreactor in the step 2B), the aeration rate is controlled to be 50-400mL per minute per 1000mL volume of the reactor, and 100-500mL (preferably 200-400 mL) of the liquid proliferation culture medium is contained per 1000mL volume of the reactor; preferably, 100 to 200mL of air per minute, and more preferably 100 to 150mL of air per minute is introduced into the reactor having a volume of 1000 mL.

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.

In particular, the solid multiplication culture process of the embryonic tissue is subcultured once every 2 to 3 weeks; subculturing the embryonic tissue liquid in a proliferation culture process once a week.

Particularly, the rotating speed of the shaking table is controlled to be 100-100 r/min in the process of liquid proliferation culture by adopting the conical flask.

Wherein, the solid multiplication culture medium of the embryonic tissue in the step 2A) is mLV culture medium +2,4-D (1-3 mg/L) +6-BA (0.5-2 mg/L) + hydrolyzed casein (200-1000 mg/L) + glutamine (200-1000 mg/L) + sucrose (10-30 g/L) + plant gel (2.5-3.0 g/L); preferably 2,4-D (1-2 mg/L) +6-BA (0.5-1 mg/L) + casein hydrolysate (400-800 mg/L) + glutamine (400-600 mg/L) + sucrose (10-20 g/L) + plant gel (2.5-3.0 g/L) in mLV medium; more preferably, the medium is mLV medium +2,4-D (2 mg/L) +6-BA (1 mg/L) + casein hydrolysate (500 mg/L) + glutamine (500 mg/L) + sucrose (10 g/L) + vegetable gel (2.8 g/L).

Wherein, the liquid multiplication culture medium of the embryonic tissue in the step 2B) is: 2,4-D (1-3 mg/L) +6-BA (0.5-2 mg/L) + hydrolyzed casein (200-1000 mg/L) + glutamine (200-1000 mg/L) + sucrose (10-30 g/L) in mLV medium; preferably +2,4-D (1-2 mg/L) +6-BA (0.5-1 mg/L) + hydrolyzed casein 400-800 mg/L) + glutamine 400-600 mg/L) + sucrose (10-20 g/L) in mLV medium; more preferably, mLV medium +2,4-D (2 mg/L) +6-BA (1 mg/L) + hydrolyzed casein (500 mg/L) + glutamine (500 mg/L) + sucrose (10 g/L).

Wherein, the embryonic tissue pretreatment culture medium I in the step 3) is: mLV medium + Aza-dC (0-20. Mu.M) + ABA (20-60. Mu.M) + polyethylene glycol 4000 (30-80 g/L) + hydrolyzed casein (200-800 mg/L) + glutamine (100-500 mg/L) + sucrose (0-20 g/L) + maltose (10-30 g/L) + preferably mLV medium + Aza-dC (5-15. Mu.M) + ABA (20-60. Mu.M) + polyethylene glycol 4000 (30-80 g/L) + hydrolyzed casein (200-800 mg/L) + glutamine (100-500 mg/L) + sucrose (0-20 g/L) + maltose (10-30 g/L) + polyethylene glycol 4000 (30-30 g/L) + Glu preferably mLV medium + Aza-dC (5-15. Mu.M) + ABA (20-60. Mu.M) + polyethylene glycol 4000 (30-80 g/L) + hydrolyzed casein (200-30 mg/L) + sucrose (200-20 mg/L) + maltose (200-30 g/L) + hydrolyzed casein (200-30 mg/L) + 100-30 mg/L) + maltose (10 mg/L) + maltose) + glutamine (10 mg/L) + maltose (10-30 g/L) + preferably mLV medium + 20-30 mg/L) + maltose (10 mg/L) + Glu mLV medium + 20-30 g/L) + maltose (10 g/L) + ABA + 20-30 mg/L) + L; still more preferably, mLV medium + AZa-dC (5-10. Mu.M) + ABA (30. Mu.M) + polyethylene glycol 4000 60g/L) + hydrolyzed casein (400 mg/L) + glutamine (200 mg/L) + sucrose (10 g/L) + maltose (30 g/L).

Particularly, the sum of the concentrations of maltose and sucrose in the embryogenic tissue pretreatment medium is 40g/L.

In particular, the concentration of the methylation inhibitor Aza-dC DNA in the pretreatment medium is 5 to 15. Mu.M, preferably 5 to 10. Mu.M, and more preferably 5. Mu.M.

Wherein, the culture medium for the embryonic tissue pretreatment in the step 3) is: mLV medium + ABA (20-60 μ M) + polyethylene glycol 4000 (30-80 g/L) + hydrolyzed casein (200-800 mg/L) + glutamine (100-500 mg/L) + sucrose (0-20 g/L) + maltose (10-30 g/L), preferably mLV medium + ABA (30-50 μ M) + polyethylene glycol 4000 (50-75 g/L) + hydrolyzed casein (200-400 mg/L) + glutamine 100-200 mg/L) + sucrose (0-10 g/L) + maltose (10-30 g/L); more preferably: mLV medium + ABA (40. Mu.M) + polyethylene glycol 4000 (62.5 g/L) + hydrolysed casein (300 mg/L) + glutamine (150 mg/L) + sucrose (10 g/L) + maltose (30 g/L).

Particularly, the sum of the concentrations of maltose and sucrose in the tissue pretreatment culture medium is 30-40g/L.

In particular, the culture conditions for the embryonic tissue pretreatment culture in the step 3) are as follows: under dark condition, the culture temperature is 25 +/-2 ℃; the culture time is 7-21 days (1-3 weeks).

In particular, the inoculation amount in the pre-treatment culture process of the embryonic tissue is 2 to 20g, preferably 2 to 5g, of the embryonic tissue to 100ml of the embryonic tissue adjusting culture medium.

In particular, the embryonic tissue after passing through the multiplication medium is inoculated into a conical flask or a bioreactor containing an embryonic tissue pretreatment medium or an embryonic tissue pretreatment medium I, and then the embryonic tissue pretreatment (conditioned culture) is carried out.

Particularly, in the process of performing embryonic tissue pretreatment culture by adopting a pneumatic bioreactor, 50-400mL of air is introduced into a reactor with the volume of 1000mL per minute, and 100-500mL (preferably 200-400 mL) of embryonic tissue pretreatment culture medium or embryonic tissue pretreatment culture medium I is contained in the reactor with the volume of 1000 mL; preferably, 100 to 200mL of air per minute, more preferably 100 to 150mL of air per minute is introduced per 1000mL of the reactor.

Wherein, the somatic embryo maturation medium in the step 4) is mLV medium + ABA (30-100 mu M) + PEG4000 (50-100 g/L) + hydrolyzed casein (200-800 mg/L) + glutamine (100-400 mg/L) + sucrose (0-30 g/L) + maltose (20-40 g/L) + plant gel (3-8 g /) L; preferably mLV culture medium + ABA (50-70 μ M) + PEG4000 (60-80 g/L) + hydrolyzed casein 400-800 mg/L) + glutamine (200-400 mg/L) + sucrose (0-10 g/L) + maltose (30-40 g/L) + plant gel (4-8 g/L); more preferably, the medium is mLV medium + ABA (60. Mu.M) + PEG4000 (60 g/L) + hydrolyzed casein (400 mg/L) + glutamine (200 mg/L) + maltose (30 g/L) + sucrose (10 g/L) + plant gel (6 g/L).

In particular, when Aza-dC is contained in the pretreatment medium during the pretreatment culture of the embryonic tissue, the somatic embryo maturation medium is more preferably mLV medium + ABA (60. Mu.M) + PEG4000 (75 g/L) + Casein hydrolysate (400 mg/L) + Glutamine (200 mg/L) + maltose (30 g/L) + sucrose (10 g/L) + plant gel (6 g/L).

In particular, the culture conditions for the embryo maturation culture in the step 4) are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 8-10 weeks.

Wherein the germination medium in step 5) is: mLV medium + NH ₄ NO ₃ (0.5-1.5 g/L) + hydrolyzed casein (200-400 mg/L) + glutamine (0-200 mg/L) + sucrose (10-20 g/L) + activated carbon (1-3 g/L) + plant gel (2-4.0 g/L) or agar (5-9 g/L); preferably: mLV medium + NH ₄ NO ₃ (1 g/L) + hydrolyzed casein (400 mg/L) + glutamine (100 mg/L) + sucrose (10 g/L) + activated charcoal (2 g/L) + vegetable gel (3.0 g/L) or agar (7 g/L).

In particular, the culture conditions of the germination culture are as follows: carrying out germination culture at 25 +/-2 ℃ under the conditions that the photoperiod is 16h of illumination/8 h of darkness and the illumination intensity is 1500-2000 Lux; the culture time is 6-8 weeks.

Particularly, the method also comprises the step 5A) of culturing the somatic embryos before germination, and culturing the mature somatic embryos obtained by the mature culture in the step 4) together with a maturation culture medium in the dark for more than 1 week at low temperature (4-10 ℃); or under dark condition, inoculating mature somatic embryo obtained by maturation culture on somatic embryo maturation culture medium, and culturing at low temperature in dark for at least 1 week; preferably, the culture is carried out at 4 ℃ in the dark for 2 to 4 weeks.

In particular, the culture conditions for the pre-germination culture of the somatic embryos are: culturing at low temperature (4-10 deg.C) in dark for 2-4 weeks.

Wherein, the hardening off and transplanting in the step 6) comprises the steps of sequentially carrying out: opening a culture bottle cap, hardening and culturing seedlings in a transplanting room for 1 day, taking out the plants, washing agar culture medium remained at the root of the test-tube seedlings by tap water, transplanting the agar culture medium into a taxus chinensis soilless culture medium (perlite, vermiculite, and the volume ratio of the perlite to the vermiculite is 1) to culture in a container in the transplanting room. Maintaining relative humidity at 70-90% for one week after transplanting, culturing at 25 + -5 deg.C for 2-4 weeks to obtain yellow fir seedling.

In the transplanting process of the test-tube plantlets, the roots are not damaged when the test-tube plantlets are transplanted, and the thin plastic film is sleeved above the container, so that sufficient humidity is kept, and careful management is realized.

Compared with the prior art, the invention has the following advantages and benefits:

1. the invention provides a method for efficiently propagating taxillus (especially the eastern taxillus and other Chinese special taxillus) through the process of somatic embryogenesis for the first time.

2. The method combines the time period after pollination of cones, and accurately selects a proper ectosome to ensure the induction rate of embryonic tissues by observing the development stage of the embryo of the seed under a microscope.

3. In order to ensure that the technology is suitable for more different genotypes, the optimal time range of pretreatment and the concentration range of adding Aza-dC in a pretreatment culture medium are determined.

4. The invention also utilizes the pneumatic bioreactor to carry out high-efficiency proliferation of embryonic tissues and carries out continuous culture with the subsequent pretreatment, thereby greatly improving the production efficiency.

5. The embryo is treated before germination, which comprises low-temperature (4 ℃) treatment and semi-dry (empty dish) treatment, so as to improve the physiological maturity of the embryo and facilitate germination.

6. The method of the invention adjusts the component proportion of the culture medium through repeated experiments to be beneficial to the maturation, germination and seedling transformation of the somatic embryo.

Drawings

FIG. 1 shows the embryonic tissue and proliferation culture of yellow cedar in east China, wherein: a is a culture dish for solid proliferation of embryonic tissue of the yellow cedar in east China; b is Erlenmeyer flask liquid culture; c is liquid culture of a pneumatic bioreactor; d is the embryonic tissue of the yellow cedar in east China; e is embryonic tissue cultured by liquid proliferation;

FIG. 1a is a diagram of somatic embryos of yellow cedar in east China after pretreatment of embryonic tissues;

FIG. 2 shows mature embryos and transferred seedlings of embryos of Yew fir, wherein A is a developing embryo; b is a mature somatic embryo; c, somatic embryo germination; d is a growing somatic embryo plantlet;

FIG. 3 is a graph showing the effect of Aza-dC addition to embryogenic tissue pretreatment medium of yellow cedar in east China on somatic embryo maturation;

FIG. 4 shows somatic embryo transferred seedlings of yellow cedar in east China, wherein A is somatic embryo germination; b is aseptic embryo seedling; c is the hardened (hardening, transplanting) somatic embryo seedling.

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 test materials and media

1. Test materials

1. Cone collection

Immature female strobilus of the east China fir is collected in san Qing mountain of Jiangxi province in the middle and late 7 months in 2019. Before formal sampling, embryo development observation is carried out in the middle 6 th of the month, a small amount of cones are collected every week after flowering and pollination for 40 days, the stage of seed embryo development is observed under a stereoscopic microscope, embryos are stripped and observed, and when the embryos develop to the period between the multi-embryo stage and the cotyledon stage, the embryos can be used as explants. To ensure that embryos within the seeds have developed significantly but no cotyledons have formed at the time of sampling. Selecting 3-5 big trees which grow healthily and pollinate freely from the cone stock plant. And storing the collected cones in an environment of 4 ℃ for refrigeration for later use.

The explant embryonic age of the east China fir has obvious influence on the induction rate of embryonic tissues. The invention selects a seed embryo with a growth and development stage between a multi-embryo stage and a cotyledon stage as an explant for inducing embryonic tissues. Collecting female cones at about 6-10 weeks after blooming and pollen scattering, continuously checking the development condition of the embryo of the seed from the sixth week of pollination, and selecting the embryo with a proper development stage as an explant.

In the specific embodiment of the present invention, the east China fir is taken as an example, and other firs are all applicable to the present invention.

2. Plant growth regulator

The plant growth regulating substance used in the invention adopts 6-benzylamino adenine (6-BA); 2, 4-dichlorophenoxyacetic acid (2, 4-D); abscisic acid (ABA); 2' -deoxycytidine (5-Aza-2-deoxycytidine, aza-dC, DNA methylation inhibitor).

2. Culture medium

1. Culture medium

(1) The basic culture medium adopts mLV culture medium

The formulation of mLV medium is shown in table 1.

TABLE 1 mLV medium recipe

mLV minimal medium (Kong L, von Aderkas P.2007.Genotype effects on ABA supplementation and nutritional supplementation in the interior space (Piceaglauca x engelmanni). Journal of Experimental Botany 58.

After the mLV culture medium mother liquor is prepared, the mLV culture medium mother liquor is respectively marked and then respectively stored in a refrigerator with the temperature of 4 ℃ for standby. According to the volume of the prepared culture medium, sequentially adding a needed macroelement mother liquor, a needed microelement mother liquor, a needed organic component mother liquor, and a weighed plant gel, cane sugar or/and maltose and hydrolyzed casein into deionized water, fully stirring each of the mother liquor, the plant gel, the cane sugar or/and the maltose and the hydrolyzed casein, finally adding water to fix the volume to the final volume of the culture medium, testing the alkalinity of cultured amino acid by using a pH meter, and adjusting the pH to 5.8 by using NaOH with the concentration of 1M or HCl with the concentration of 1M. The mixture was sterilized at a constant temperature of 121 ℃ for 15 minutes. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.

(2) Embryonic tissue induction medium: adding 2,4-D (2-6 mg/L), 6-BA (1-3 mg/L), hydrolyzed casein (400-1000 mg/L), glutamine (200-600 mg/L), sucrose (10-30 g/L) and plant gel (2.5-3.0 g/L) into mLV culture medium, adjusting pH to 5.8, and sterilizing at 121 deg.C for 20 min. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.

(3) Embryonic tissue solid proliferation medium: adding 2,4-D (1-3 mg/L), 6-BA (0.5-2 mg/L), hydrolyzed casein (200-1000 mg/L), glutamine (200-1000 mg/L), sucrose (10-30 g/L) and plant gel (2.5-3.0 g/L) into mLV culture medium, adjusting pH to 5.8, and sterilizing at 121 deg.C for 20 min. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.

(4) Embryonic tissue liquid proliferation medium: adding 2,4-D (1-3 mg/L), 6-BA (0.5-2 mg/L), hydrolyzed casein (200-1000 mg/L), glutamine (200-1000 mg/L) and sucrose (10-30 g/L) into mLV culture medium, adjusting pH to 5.8, and sterilizing at 121 deg.C for 20 min. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.

(5) Embryonic tissue pretreatment medium (pre-embryo maturation medium): ABA (20-60 mu M), polyethylene glycol (PEG 4000, 30-80 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-500 mg/L), sucrose (0-20 g/L) and maltose (10-30 g/L) are added into an mLV culture medium, the pH value is adjusted to 5.8, and the medium is sterilized at the constant temperature of 121 ℃ for 20 minutes. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine and Aza-dC in a super clean bench, adding into the culture medium, mixing, and shaking.

(5A) Embryonic tissue pretreatment medium i (pre-embryo maturation medium i): AZa-dC (0-20. Mu.M), ABA (20-60. Mu.M), polyethylene glycol (PEG 4000, 30-80 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-500 mg/L), sucrose (0-20 g/L) and maltose (10-30 g/L) were added to the mLV medium, the pH was adjusted to 5.8, and the medium was sterilized at a constant temperature of 121 ℃ for 20 minutes. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine and AZa-dC in a clean bench, adding into the culture medium, and mixing.

(6) Somatic embryo maturation medium: ABA (30-100 mu M), PEG4000 (50-100 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-400 mg/L), sucrose (0-30 g/L), maltose (20-40 g/L) and plant gel (3.0-8.0 g/L) are added into a mLV culture medium, the pH value is adjusted to 5.8, and the medium is sterilized at the constant temperature of 121 ℃ for 20 minutes. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking. Wherein the total mass concentration of the sucrose and the maltose is 30-60g/L.

(7) The culture medium for the somatic embryo pre-germination treatment is the same as the somatic embryo maturation culture medium.

(8) Somatic embryo germination culture medium: addition of NH to mLV medium ₄ NO ₃ (0.5-1.5 g/L), hydrolyzed casein (200-400 mg/L), glutamine (0-200 mg/L), sucrose (10-20 g/L), activated carbon (1-3 g/L), plant gel (2-4.0 g/L) or agar (5-9 g/L), adjusting the pH value to 5.8, and sterilizing at 121 ℃ for 20 minutes. Cooling the culture medium to about 60 deg.C, filtering and sterilizing glutamine in a clean bench, adding into the culture medium, mixing, and shaking.

2. Culture conditions

(1) Embryonic tissue induction culture: dark conditions; the culture temperature is 25 +/-2 ℃; subculturing for 1 time every 2 weeks; the culture time is 4-6 weeks.

(2) Solid multiplication culture of embryonic tissue: dark conditions; the culture temperature (25 + -2) deg.C, and subculture for 1 time in 2-3 weeks.

(3) Liquid multiplication culture of embryonic tissues: dark conditions; the culture temperature is 25 +/-2 ℃; the rotating speed is 100-110rpm; subcultured 1 time per week.

(4) Liquid proliferation culture of the bioreactor: dark conditions; the culture temperature (25 +/-2) DEG C; the ventilation amount is 50-400ml/min in each reactor with the volume of 1000 ml; subcultured 1 time per week.

(5) Pre-treatment culture for somatic embryo maturation (triangular flask): dark conditions; the culture temperature (25 +/-2) DEG C; the rotating speed is 100-110rpm; culturing for 1-2 weeks.

(5A) Pre-treatment culture (bioreactor) for embryo maturation: dark conditions; the culture temperature is 25 +/-2 ℃; the ventilation amount is 50-400ml/min in each reactor with the volume of 1000 ml; culturing for 1-2 weeks.

(6) Somatic embryo maturation culture (petri dish): dark conditions; the culture temperature (25 +/-2) DEG C; culturing for 6-8 weeks.

(7) Pre-treatment culture (culture dish) for somatic embryo germination: dark conditions; the culture temperature is 4-10 ℃; culturing for at least 1 week.

(8) And (3) germination culture of somatic embryos: the culture temperature is 25 + -2 deg.C, the illumination intensity is 1500-2000lx, and the illumination period is 10-16 hr illumination/8-14 hr darkness.

Example 2 (Induction of embryonic tissue)

1. Sterilization of explants

Washing the surface of the refrigerated fir cones in the environment of 4 ℃ with detergent, then washing the cones with tap water, splitting the cones, and taking out seeds; soaking the seeds of the yellow fir in 75 percent alcohol for 30-60s on a superclean workbench, fishing the seeds, washing the seeds with sterile water for 3-5 times, and then putting 0.1 percent HgCl ₂ Soaking in the solution for 5-10min, taking out seeds, washing with sterile water for 5-6 times, placing the washed seeds on sterilized filter paper, removing water, and opening seed coat with tweezers and scissors in a super clean bench to obtain female gametophyte. Further opening the female gametophyte to obtain sterile immature zygotic embryo of the yellow fir for use.

2. Embryonic tissue induction culture

1) Inoculating the whole sterile fir immature zygotic embryo as an explant into an embryonic tissue induction culture medium in a clean bench, carrying out the induction culture of the embryonic tissue under the dark condition, subculturing once every 2 weeks, namely taking out the explant after 2 weeks of culture, placing the explant into another fresh embryonic tissue induction culture medium, and continuing the induction culture of the embryonic tissue so as to keep the culture medium containing sufficient nutrients and water, wherein for convenient counting statistics, 10 female gametophytes are inoculated into each dish and more than 5 repeats are arranged.

In this example, the embryonic tissue induction medium contains 2,4-D (4 mg/L), 6-BA (2 mg/L), hydrolyzed casein (500 mg/L), glutamine (500 mg/L), sucrose (10 g/L), and vegetable gel (2.75 g/L) as an example, and other embryonic tissue induction media contain 2,4-D (2-6 mg/L), 6-BA (1-3 mg/L), hydrolyzed casein (400-1000 mg/L), glutamine (200-600 mg/L), sucrose (10-30 g/L), and vegetable gel (2.5-3.0 g/L), and are all suitable for the present invention; wherein the embryogenic tissue induction medium preferably contains 2,4-D (2-4 mg/L), 6-BA (1-2 mg/L), hydrolyzed casein (400-800 mg/L), glutamine (200-600 mg/L), sucrose (10 g/L), and plant gel (2.75 g/L). After 2-3 weeks of induction culture, the tissue outgrowth from the explants was loose and translucent (see FIG. 1D), and the surface filamentous protrusions were observed under a dissecting mirror.

Changing a new induction culture medium every 2 weeks in induction culture, carrying out induction culture for 6 weeks to obtain embryonic tissues, counting the number of explants of the obtained embryonic tissues, calculating the induction rate of the embryonic tissues, and analyzing the results shown in Table 2, wherein:

embryogenic tissue induction rate (%) = embryogenic tissue number/total number of explants (embryos) × 100%

TABLE 2 embryogenic tissue induction rate

Tree species	Time of acquisition	Explant (embryo) developmental stage	Inductivity (%)
				Ramulus et folium taxi Cuspidatae	7.20-7.30	After the multiple embryo stage, before the cotyledon stage	21.4

The developmental stage of the female gametophyte (time to cone harvest) has a key effect on the induction of somatic embryogenesis. The embryo is not formed before the collection time is too early, the zygotic embryo which is collected too late is developed and matured or nearly matured, the differentiation degree is too high, and the induction of the somatic embryo is not facilitated. The invention adopts a method of directly observing and monitoring the development stage of the embryo to accurately determine the acquisition time of cones. The results of embryogenic tissue induction demonstrated that the zygotic embryos before the cotyledon stage were suitable explants for embryogenic tissue induction after the polyembryony stage.

Example 3 (multiplication culture of embryonic tissue)

Performing induction culture on the collected cones of the taxus chinensis with different genotypes (YDF-A and YDF-B) according to the method of the embodiment 2 for 4-6 weeks to obtain corresponding embryonic tissues

1. Multiplication culture of embryonic tissue

1) Solid multiplication culture of embryonic tissue

The embryonic tissue obtained by induction is separated from the explant, and is divided into small blocks of 0.5cm multiplied by 0.5cm, and the small blocks are inoculated on an embryonic tissue solid multiplication culture medium (as shown in figure 1A), the first-stage solid multiplication culture of the embryonic tissue of the yellow fir is carried out under the dark condition, wherein the culture temperature is (25 +/-2) DEG C, the 2,4-D used in the embryonic tissue solid multiplication culture medium is (2 mg/L), the 6-BA used in the embryonic tissue solid multiplication culture medium is (1 mg/L), the casein hydrolysate is (500 mg/L), the glutamine is (500 mg/L), the sucrose is (10 g/L), the plant gel is (2.8 g/L), and a large amount of the solid multiplication embryonic tissue of the yellow fir is obtained after subculture for 2 weeks and a plurality of subcultures (2-6 times) in the multiplication culture process of the somatic embryo of the yellow fir.

Because plant materials with different genotypes react differently to the method for the propagation culture of the same liquid, the experiment adopts two different genotypes (YDF-A and YDF-B) of the yellow cedar in east ChinSub>A as experimental materials. The embryonic tissue is weighed before and after solid proliferation culture for one week, and the peripheral proliferation rate of the embryonic tissue is calculated according to the following formula (1). Each treatment was 3 replicates. The average in table 3 is based on at least three replicate experimental data.

Proliferation rate (%) = fresh weight gain (g FW)/initial inoculum size (g FW) × 100% (1)

TABLE 3 Effect of different culture methods on the peri-proliferation (%) of embryonic tissue of Huang-Dong shirt

In this example, the embryogenic tissue solid multiplication medium contains 2,4-D (2 mg/L), 6-BA (1 mg/L), hydrolyzed casein (500 mg/L), glutamine (500 mg/L), sucrose (10 g/L), and plant gel (2.8 g/L) as examples, and other solid multiplication media containing 2,4-D (1-3 mg/L), 6-BA (0.5-2 mg/L), hydrolyzed casein (200-1000 mg/L), glutamine (200-1000 mg/L), sucrose (10-30 g/L), and plant gel (2.5-3.0 g/L) are all suitable for the present invention, wherein the solid multiplication medium preferably contains 2,4-D (1-2 mg/L), 6-BA (0.5-1 mg/L), hydrolyzed casein (400-800 mg/L), glutamine (400-600 mg/L), sucrose (10-20 g/L), and plant gel (2.5-3.0 g/L).

2) Liquid multiplication culture of embryonic tissue

The liquid multiplication culture can be carried out by adopting the following 2 ways: conical flask (triangular flask) suspension culture and pneumatic bioreactor suspension culture

2-1) Erlenmeyer flask suspension culture

Inoculating 2g of the embryonic tissue obtained from the first stage multiplication culture into a conical flask in 100ml of an embryonic tissue liquid multiplication culture medium, placing the conical flask on a shaker (100-110 rpm), and carrying out the second stage liquid multiplication culture of the embryonic tissue of the yellow fir under the dark condition (figure 1B), wherein the culture temperature is (25 +/-2) DEG C, 2,4-D used in the embryonic tissue liquid multiplication culture medium is 2mg/L,6-BA used in the embryonic tissue liquid multiplication culture medium is 1mg/L, casein hydrolysate is 500mg/L, glutamine is 500mg/L, and sucrose is 10g/L, and a large amount of the embryonic tissue of the yellow fir is obtained after 3-5 times of subculture once per week in the yellow fir embryonic tissue multiplication culture process (as shown in figure 1E).

The embryonic tissue is weighed before liquid enrichment culture and after liquid enrichment culture for one week, and then the peri-proliferation rate of the embryonic tissue is calculated according to the formula (1). The measurement results are shown in Table 3. The average in table 3 is based on at least three replicate experimental data.

The long-term addition of high concentration of 2,4-D during embryogenic tissue proliferation culture is detrimental to late embryonic development and differentiation, and the concentration of 2,4-D in the tissue proliferation culture medium during the tissue proliferation culture of the Flaveria yezoensis is 1-3mg/L, preferably 2mg/L. The concentration of BA is 0.5-2mg/L, preferably 1mg/L.

2-2) pneumatic bioreactor suspension multiplication culture

Inoculating the embryonic tissue obtained from the first-stage multiplication culture into a pneumatic bioreactor reaction bottle (figure 1C) filled with a liquid multiplication culture medium, and carrying out second-stage liquid multiplication culture, wherein 300mL (generally 100-500mL, preferably 200-400 mL) of the liquid multiplication culture medium is added into a pneumatic bioreactor with the capacity of 1000 mL; the inoculation amount is 2g of embryonic tissue (usually 1-4g/100 ml) per 100ml of liquid multiplication medium; the aeration rate per 1000mL volume of pneumatic bioreactor is 100-150mL/min (usually 50-400mL/min, preferably 100-200 mL/min) to ensure that the plant material can run uniformly in the liquid culture medium. The pneumatic bioreactor is placed at 25 +/-2 ℃ and cultured in the dark. 2,4-D used in the liquid multiplication medium of the embryonic tissue is 2mg/L,6-BA is 1mg/L, hydrolyzed casein is 500mg/L, glutamine is 500mg/L, sucrose is 10g/L, subculture is carried out once a week in the multiplication culture process of the embryonic tissue of the yellow fir, and a large amount of the embryonic tissue of the yellow fir is obtained after 3-5 subcultures (as shown in figure 1E).

The embryonic tissue is weighed before liquid enrichment culture and after liquid enrichment culture for one week, and then the peri-proliferation rate of the embryonic tissue is calculated according to the formula (1). The measurement results are shown in Table 3.

The embryonic tissue after the conical flask suspension culture can be inoculated into a pneumatic bioreactor reaction bottle filled with a liquid multiplication culture medium to continue the liquid suspension culture, and the culture condition, the culture medium, the inoculation quantity and the subculture period are equal to those of the pneumatic bioreactor.

The pneumatic bioreactor used in the invention is suitable for other plant tissue culture pneumatic bioreactors except the bioreactor disclosed in the Chinese utility model patent with the publication number of CN203840895U and the name of "bioreactor system".

In this example, the liquid multiplication medium for embryonic tissue contains 2,4-D (2 mg/L), 6-BA (1 mg/L), hydrolyzed casein (500 mg/L), glutamine (500 mg/L) and sucrose (10 g/L), and the solid multiplication medium preferably contains 2,4-D (1-3 mg/L), 6-BA (0.5-2 mg/L), hydrolyzed casein (200-1000 mg/L), glutamine (200-1000 mg/L) and sucrose (10-30 g/L) in the amounts of 2,4-D (1-2 mg/L), 6-BA (0.5-1 mg/L), hydrolyzed casein (400-800 mg/L), glutamine (400-600 mg/L) and sucrose (10-20 g/L).

EXAMPLE 4 pretreatment of embryonic tissue (conditioned culture)

The pretreatment culture of the embryonic tissue is an adjustment culture before the somatic embryo maturation culture of the embryonic tissue.

2-5g of the embryogenic tissue obtained in example 3 after liquid multiplication culture was placed in a 100mL conical flask, 30-40mL of the embryogenic tissue pretreatment medium was added to the conical flask, and suspension culture was performed in the conical flask under dark conditions, i.e., a pretreatment (conditioned culture) of the embryogenic tissue of Flaveria yezoensis was performed, wherein the culture temperature was (25. + -.2) ° C, and the embryogenic tissue pretreatment medium contained ABA (40. Mu.M), polyethylene glycol (PEG 4000) (62.5 g/L), hydrolyzed casein (300 mg/L), glutamine (150 mg/L), sucrose (10 g/L), maltose (30 g/L), and the conical flask was placed at 100 r.min ^–1 Dark treatment on a constant temperature shaker at 25 +/-2 ℃ for 1-2 weeks.

During the adjustment culture process, the differentiation of embryo head and embryo stem can be clearly seen by observing the embryonic tissue structure under a stereomicroscope (see FIG. 1 a).

In the examples of the present invention, the pretreatment medium is exemplified by ABA (40. Mu.M), polyethylene glycol (PEG 4000) (62.5 g/L), hydrolyzed casein (300 mg/L), glutamine (150 mg/L), sucrose (10 g/L), maltose (30 g/L), and others are exemplified by ABA (20-60. Mu.M), polyethylene glycol (PEG 4000) (30-80 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-500 mg/L), sucrose (0-20 g/L), maltose (10-30 g/L) in the medium; the pretreatment medium is preferably ABA (30-50. Mu.M), polyethylene glycol (PEG 4000) (50-75 g/L), hydrolyzed casein (200-400 mg/L), glutamine (100-200 mg/L), sucrose (0-10 g/L) or maltose (10-30 g/L).

EXAMPLE 4A pretreatment of embryonic tissue (conditioned culture)

Placing 2-5g of the embryonic tissue obtained after the liquid multiplication culture in example 3 into a 100mL conical flask, adding 30-40mL of embryonic tissue pretreatment culture medium I into the conical flask, and performing suspension culture in the conical flask under dark conditions, namely performing yellow cedar embryonic tissue pretreatment (adjustment culture), wherein the embryonic tissue pretreatment culture medium I contains ABA30 [ mu ] M, polyethylene glycol (PEG 4000) 60g/L, casein hydrolysate 400mg/L, glutamine 200mg/L, sucrose 10g/L and maltose 30g/L, and contains Aza-dC, and the dosage of the Aza-dC is 0, 5, 10 and 15 [ mu ] M respectively; placing the conical flask for 100 r.min ^–1 Dark treatment on a constant temperature shaking bed at 25 +/-2 ℃ for 1-2 weeks.

During the adjustment and culture process, the embryonic tissue structure is observed under a body type microscope, and the differentiation of the embryonic head and the embryonic stalk can be clearly seen.

In the embodiment of the invention, the pretreatment culture medium I takes ABA 30. Mu.M, polyethylene glycol (PEG 4000) 60g/L, hydrolyzed casein 400mg/L, glutamine 200mg/L, sucrose 10g/L, maltose 30g/L and Aza-dC (0, 5, 10 and 15. Mu.M) as examples, and the other pretreatment culture medium I takes ABA (20-60. Mu.M), polyethylene glycol (PEG 4000) (30-80 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-500 mg/L), sucrose (0-20 g/L), maltose (10-30 g/L) and Aza-dC (0-20. Mu.M) as examples; the pretreatment medium I is preferably ABA (20-60. Mu.M), polyethylene glycol (PEG 4000) (30-80 g/L), hydrolyzed casein (200-800 mg/L), glutamine (100-500 mg/L), sucrose (0-20 g/L), maltose (10-30 g/L), aza-dC (5-15. Mu.M), and more preferably: ABA (30-50 mu M), polyethylene glycol (PEG 4000) (40-60 g/L), hydrolyzed casein (200-500 mg/L), glutamine (100-200 mg/L), sucrose (0-10 g/L), maltose (10-30 g/L), aza-dC (5-15 mu M); more preferably: ABA (30. Mu.M), polyethylene glycol (PEG 4000) (60 g/L), hydrolyzed casein (400 mg/L), glutamine (200 mg/L), sucrose (10 g/L), maltose (30 g/L), aza-dC (5-10. Mu.M).

EXAMPLE 4B pretreatment of embryonic tissue (conditioned culture)

Inoculating 2-5g of the embryonic tissue obtained by liquid multiplication culture in example 3 into a reaction bottle of a pneumatic bioreactor filled with an embryonic tissue pretreatment culture medium, and performing suspension culture in the reaction bottle of the bioreactor under dark conditions to perform yellow cedar embryonic tissue pretreatment (adjustment culture), wherein the embryonic tissue pretreatment culture medium contains ABA30 [ mu ] M, polyethylene glycol (PEG 4000) 60g/L, hydrolyzed casein 400mg/L, glutamine 200mg/L, sucrose 10g/L and maltose 30g/L;

300mL (usually 100-500mL, preferably 200-400 mL) of embryonic tissue pretreatment medium is added into a pneumatic bioreactor with the capacity of 1000 mL; the ventilation amount per 1000mL pneumatic bioreactor is 100-150mL/min (usually 50-400mL/min, preferably 100-200 mL/min) to ensure that the embryonic tissue of the propagation culture can uniformly run in the embryonic tissue pretreatment culture medium. The pneumatic bioreactor is placed at 25 +/-2 ℃ and cultured for 1-2 weeks in the dark.

The culture medium in the pneumatic bioreactor can also adopt an embryonic tissue pretreatment culture medium I.

EXAMPLE 5 maturation culture of somatic embryos

Taking out embryonic tissues pretreated for 1-2 weeks (usually 7-14 days) in example 4 by using a truncated pipette, uniformly scattering the embryonic tissues and the pretreatment culture medium on sterile filter paper, and then filtering off the redundant pretreatment culture medium;

then, putting the filter paper and the embryonic tissue (about 0.1g fresh weight) pretreated on the filter paper on the surface of a solid (or semi-solid) somatic embryo maturation culture medium (a culture dish) together for maturation culture of the somatic embryo of the taxus chinensis; wherein the somatic embryo maturation medium contains ABA60 μ M, polyethylene glycol (PEG 4000, 60 g/L), hydrolyzed casein (400 mg/L), glutamine (200 mg/L), sucrose (10 g/L), maltose (30 g/L) and plant gel (6 g/L); under dark conditions, the somatic embryos were observed to develop from the pre-cotyledonary embryo stage to the cotyledonary embryo stage at a culture temperature of 25. + -. 2 ℃ for 6-8 weeks, and mature somatic embryos (morphologically mature somatic embryos) were obtained (FIGS. 2A-B).

After 8 weeks of maturation, the morphology of mature embryos observed under a somatic microscope is shown in fig. 2B, and the number of mature embryos per dish was counted according to the developmental morphology of the embryos. 5 dishes were counted for each treatment to calculate the average number of mature somatic embryos per dish, and the results are shown in Table 4.

TABLE 4 Effect of different cultivation methods and maturation Medium composition on the maturation of somatic embryos of Flammulina Veitchii

* Spruce maturation medium (no polyethylene glycol, maltose, sucrose); ^★ spruce improved mature culture medium (maltose-free, polyethylene glycol, sucrose)

The experimental data show that: the number of mature embryos obtained per dish varied from treatment to treatment. The number of mature somatic embryos can be increased by pretreatment for 2 weeks and by using a picea chinensis maturation medium. The experimental result also proves the importance of using PEG and maltose in the mature culture medium of the yellow fir, and the culture medium does not contain PEG, so that the mature embryo can not be obtained; and does not contain maltose, but can obtain mature somatic embryos, but results in the remarkable reduction of the number of the mature somatic embryos and poor quality of the somatic embryos.

EXAMPLE 5A mature culture of somatic embryos

Taking out the embryonic tissue which is subjected to the adjustment culture for 1 to 2 weeks in the example 4A by using a truncated pipette, uniformly scattering the embryonic tissue and the pretreatment culture medium on sterile filter paper, and then filtering out the redundant pretreatment culture medium;

then, filters and the embryonic tissues pretreated thereon (fresh weight of about 0.1 g) were placed on the surface of an embryo maturation medium (Petri dish) and subjected to maturation culture of the embryonic tissues of Yew yellow cedar under dark conditions, wherein ABA (60. Mu.M), polyethylene glycol (PEG 4000, 75 g/L), casein hydrolysate (400 mg/L), glutamine (200 mg/L), sucrose (10 g/L), maltose (30 g/L) and plant gel (6 g/L) were used in the embryo maturation medium. Culturing in dark at 25 + -2 deg.C for 8 weeks, observing under microscope, and counting the number of morphologically mature embryos.

Aza-dC can obviously inhibit the excessive proliferation of non-embryonic tissues in mature culture and provide space for the growth of mature embryos; when 5. Mu.M and 10. Mu.M Aza-dC were added to the suspension medium for embryogenic tissue pretreatment in example 4A, respectively, significant inhibition of the growth of non-embryogenic tissue and an increase in the number of normal mature embryos (FIG. 3) could be achieved, and the mature embryos could grow and develop well.

The results in FIG. 3 show that the highest number of normal mature somatic embryos (morphologically mature somatic embryos) were obtained from each dish when 5. Mu.M, or 10. Mu.M Aza-dC was added to the embryogenic tissue pretreatment medium. When the concentration of Aza-dC is 0, it cannot inhibit non-embryonic tissue, and when it is too high at 15. Mu.M, it partially inhibits the growth of normal embryonic tissue. In both cases (Aza-

dC concentration

0, 15. Mu.M), the number of normal mature somatic embryos produced per dish was low (FIG. 3).

EXAMPLE 5 mature culture of somatic embryos

To test whether a simpler, economical spruce maturation medium (without PEG4000, sucrose without maltose) could be used, the pretreated embryonic tissue was also inoculated onto spruce maturation medium.

the same as example 5 was repeated, except that the filter paper and the pretreated embryonic tissue (fresh weight: about 0.1 g) thereon were placed together on the surface of a solid spruce somatic embryo maturation medium (petri dish) to conduct maturation culture of the spruce somatic embryos, wherein the spruce maturation medium contained no polyethylene glycol (PEG 4000), no maltose and sucrose (30 g/L); the other components are the same as those of the somatic embryo maturation medium of the present invention.

After 8 weeks of maturation, the morphology of mature embryos observed under a stereomicroscope was counted for each dish according to the embryo development morphology. 5 dishes were counted for each treatment to calculate the average number of mature somatic embryos per dish, and the results are shown in Table 4.

EXAMPLE 5C mature culture of somatic embryos

Taking out the embryonic tissue pretreated for 1-2 weeks (usually 7-14 days) in example 4 by using a truncated pipette, uniformly scattering the embryonic tissue and the pretreatment culture medium on sterile filter paper, and then filtering out the redundant pretreatment culture medium;

the same as example 5 was repeated, except that the filter paper and the pretreated embryonic tissue (fresh weight: about 0.1 g) were placed together on the surface of the solid spruce somatic embryo modified maturation medium (Petri dish) to conduct maturation culture of the spruce somatic embryos, wherein the spruce somatic embryo modified maturation medium contained polyethylene glycol (PEG 4000, 60 g/L), no maltose, and sucrose (30 g/L); the other components are the same as those of the somatic embryo maturation medium of the present invention.

After 8 weeks of maturation culture, the mature embryos observed under a somatic microscope were counted for each dish according to their developmental morphology. 5 dishes were counted for each treatment to calculate the average number of mature somatic embryos per dish, and the results are shown in Table 4.

Example 6 somatic embryo Germination and somatic embryo seedling transplantation

1) Pre-treatment for germination

Carrying out pre-germination treatment on mature somatic embryos, namely placing the morphologically mature somatic embryos and a maturation culture medium at a low temperature (4 ℃, usually 4-10 ℃) for dark culture; mature somatic embryos can also be placed in an empty sterile petri dish, along with underlying filter paper, and then placed at low temperature (4 ℃, typically 4-10 ℃) for dark culture. In general, the temperature of the pre-germination treatment of the mature somatic embryos is preferably 4 ℃ and the time of the pre-germination treatment is 2 to 4 weeks. The somatic embryo after pre-treatment of germination becomes more mature physiologically and has stronger germination capacity.

2) Germination culture

Pre-treated for germinationTransferring the mature somatic embryos to a somatic embryo germination culture medium, and performing germination culture under the conditions of a photoperiod of 16h light/8 h dark and a light intensity of 1500-2000Lux at 25 +/-2 ℃, wherein: the germination culture medium takes mLV culture medium as basic culture medium, and NH is added ₄ NO ₃ (1 g/L), hydrolyzed casein (400 mg/L), glutamine (100 mg/L), sucrose (10 g/L), activated carbon (2 g/L), agar 7g/L (or vegetable gel 3.0 g/L).

NH in somatic embryo germination medium ₄ NO ₃ (0.5-1.5 g/L), hydrolyzed casein (200-400 mg/L), glutamine (0-200 mg/L), sucrose (10-20 g/L), activated carbon (1-3 g/L), agar (5-9 g/L) or plant gel (2-4 g/L) are all suitable. The cotyledons of the mature somatic embryos quickly turn green, the hypocotyls grow in an elongated manner, the cotyledons at the top ends of the mature somatic embryos expand after 8 weeks of culture, obvious root growing ends are differentiated from the other ends of the mature somatic embryos to form somatic embryo seedlings (as shown in figures 2C-D and 4A-B), the obtained somatic embryo seedlings are counted, and the germination rate of the somatic embryos is calculated: somatic embryo germination rate (%) = number of germinated somatic embryos/number of inoculated somatic embryos × 100%. In different genotypes, the germination rate of normal mature embryos is 85-98%, and the seedling transformation rate is 32-71%.

3) Somatic embryo seedling transplantation

When the height of the somatic embryo seedlings reaches more than 2 cm and the roots are thick and strong, opening the bottle caps of culture bottles, hardening and culturing for 1 day in a transplanting chamber, 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 a soilless culture substrate (perlite, vermiculite, and the volume ratio of the perlite to the vermiculite is 1) of the picea chinensis, and performing container culture in the transplanting chamber. Maintaining relative humidity of 80-90% within 2 weeks after seedling transplantation, gradually reducing to 60-70%, culturing at 25 + -5 deg.C for 6-8 weeks to obtain fir seedling (see figure 4C).

In a specific embodiment of the present invention, a seed embryo that has developed significantly but has not formed a cotyledon is used as an exoplant for inducing embryogenic tissue. Other female gametophytes (including embryos) in the strobilus of the fir 40-70 days after blooming and pollen scattering can be used for the invention. The yellow fir breeding method provides a method with short period and high breeding rate for the yellow fir artificial large-scale factory asexual propagation seedling, breaks through the limitations of difficult yellow fir vegetative propagation, long growth period, large seed orchard investment, late fructification, limited area and the like, and becomes a new way for maintaining and quickly breeding improved varieties of forest trees.

Claims

1. A method for propagating taxina through somatic embryogenesis is characterized in that: the method comprises the following steps:

1) Inoculating a sterilized explant of a taxus genus plant on an embryonic tissue induction culture medium, and carrying out induction culture to obtain an embryonic tissue, wherein a seed embryo with a growth and development period between a multiple embryo period and a cotyledon period is selected as the explant of the induced embryonic tissue; the embryonic tissue induction culture medium is as follows: mLV culture medium +2, 4-D2-6 mg/L +6-BA 1-3mg/L + hydrolyzed casein 400-1000mg/L + glutamine 200-600mg/L + sucrose 10-30g/L + plant gel 2.5-3.0g/L;

2) Inoculating the embryonic tissue on a proliferation culture medium to carry out proliferation culture of the embryonic tissue, wherein the proliferation culture comprises the following steps in sequence:

2A) Inoculating the embryonic tissue to an embryonic tissue solid multiplication culture medium for carrying out first-stage solid multiplication culture on the embryonic tissue, wherein the embryonic tissue solid multiplication culture medium is mLV culture medium +2, 4-D1-3 mg/L +6-BA 0.5-2mg/L + casein hydrolysate 200-1000mg/L + glutamine 200-1000mg/L + sucrose 10-30g/L + plant gel 2.5-3.0g/L;

2B) 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 to obtain a multiplication-cultured embryonic tissue; the embryonic tissue liquid proliferation culture medium is mLV culture medium +2, 4-D1-3 mg/L +6-BA 0.5-2mg/L + hydrolyzed casein 200-1000mg/L + glutamine 200-1000mg/L + sucrose 10-30g/L;

3) Inoculating the embryogenic tissue subjected to propagation culture into an embryogenic tissue pretreatment culture medium I, and performing embryonic tissue adjustment culture to obtain a pretreated embryogenic tissue, wherein the embryogenic tissue pretreatment culture medium I is as follows: mLV culture medium + AZa-dC 5-10. Mu.M + ABA 30. Mu.M + polyethylene glycol 4000 60g/L + hydrolyzed casein 400mg/L + glutamine 200mg/L + sucrose 10g/L + maltose 30g/L;

4) Inoculating the pretreated embryogenic tissue on a somatic embryo maturation culture medium, and performing somatic embryo maturation culture to obtain a mature somatic embryo, wherein the somatic embryo maturation culture medium is mLV culture medium, ABA 30-100 mu M, PEG4000 50-100g/L, hydrolyzed casein 200-800mg/L, glutamine 100-400mg/L, sucrose 0-30g/L, maltose 20-40g/L and plant gel 3-8g/L;

5) Inoculating the mature somatic embryos on a somatic embryo germination culture medium for germination culture to obtain somatic embryo seedlings, wherein the germination culture medium is mLV culture medium + NH ₄ NO ₃ 0.5-1.5g/L + hydrolyzed casein 200-400mg/L + glutamine 0-200mg/L + sucrose 10-20g/L + activated carbon 1-3g/L + plant gel 2.0-4.0 g/L or agar 5.0-9.0g/L;

2. The method as claimed in claim 1, further comprising the steps of 5A) culturing the somatic embryos before germination, and culturing the mature embryos obtained in step 4) in the dark together with a maturation medium at 4-10 ℃ for more than 1 week in the dark under dark conditions; or inoculating mature somatic embryo obtained by mature culture on somatic embryo maturation medium under dark condition, and culturing at 4-10 deg.C in dark for at least 1 week.

3. The method according to claim 2, wherein the dark culture is carried out at 4 ℃ for 2 to 4 weeks.

4. The method according to claim 1 or 2, wherein the conditions for the induction culture of the embryonic tissue in step 1), the propagation culture of the embryonic tissue in step 2), the adjustment culture of the embryonic tissue in step 3), and the maturation culture of the somatic embryo in step 4) are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃.