CN112042541A - Propagation of Douglas genus by somatic embryogenesis - Google Patents

Abstract

The invention discloses a method for propagating a Douglas genus plant through somatic embryogenesis, comprising the steps of inducing and culturing embryonic tissue, proliferating and culturing the sterile explants of the genus Douglas, adjusting and culturing embryonic tissue, and performing somatic cell culture. Embryos (somatic embryos) mature culture, germination culture of mature somatic embryos, transfer seedling culture. Through the method of the invention, stable embryogenic cell lines and a large number of mature somatic embryos are obtained by inducing somatic embryogenesis for the first time, and then complete somatic embryo plants are obtained. By using the method of the present invention, a large number of seedlings of Chinese orientalis can be produced in a relatively short period of time. The technology is suitable for the large-scale and factory production of P. chinensis clone seedlings.

Description

Propagation of Douglas genus by somatic embryogenesis

technical field

The invention relates to a method for plant tissue culture, in particular to a method for plant regeneration of Douglas orientalis through somatic embryo (somatic embryo) culture, and belongs to the technical field of cell engineering seedling breeding in forestry.

Background technique

Pseudotsuga gaussenii Flous is an evergreen tree of the Pinaceae family. There are 7 species of Douglas fir, 5 of which are endemic to China, and the other 2 are originally distributed in North America. Douglas fir (Pseudotsuga menziesii (Mirb.) Franco), native to North America, is a world-famous fine tree species. Douglas fir is up to 40 meters high, the bark is dark gray, the top of the leaf pillow is brown, the main branch is glabrous or sparsely hairy, the side branches have brown dense hairs, and the winter buds are oval or ovate-conical, brown. Leaf blade strip-shaped, arranged in two rows or nearly radiating on the main branch, dark green above and two white stomata below; cone-shaped oval or oval, broad at base, narrow at upper part, slightly White powder; middle seed scales reniform or horizontally elliptic kidney-shaped, upper part of bract scales retrograde backwards, mid-lobe longer, narrow triangular, lateral fissure triangular-shaped, apex pointed or obtuse, seeds triangular-oval, slightly flattened, The wings are nearly as long as the seeds. It blooms in April-May, and the fruit matures in October. The distribution area of Huangshan fir in East China is located in the eastern mid-subtropical low mountains to Zhongshan, with an altitude of 600-1500 meters. The climate in the distribution area is warm and humid, with an annual average temperature of 9-15 °C, a minimum temperature of -9 to -16 °C, and a maximum temperature of 32-35 °C. The annual precipitation is 400-2000 mm, and the relative humidity is generally 90%. The soil is red soil and mountain yellow soil, pH 5.5-6.5. The sapwood of Huangshan fir is light brown, the heartwood is brown, the texture is straight, the material is slightly thick, the hardness is moderate, and the specific gravity is 0.6, which can be used for construction and furniture. It can also be used as a garden ornamental tree.

Pseudotsuga sinensis Dode is similar to Pseudomonas sinensis, the main difference is that the cones of Pseudotusuga sinensis Dode are oval or elliptical, wide near the middle and slightly narrow at both ends. The cones of P. chinensis are often conical-oval, wide at the base and narrow at the top, and the middle seed scales are kidney-shaped or horizontally elliptical kidney-shaped, and the exposed part of the back of the scales is glabrous; the seed wings are nearly as long as the seeds.

Douglas fir and Douglas fir are generally 15-year-old with flowering and fruiting, and mature cones. There are very few fertile seeds, some are all infertile, and some have shrunk embryos or are infested by insects. In China's Zhejiang, southern Anhui, northern and western Fujian, and eastern Jiangxi mountains with an altitude of 500-1500 meters can be used as afforestation tree species. Douglas fir is distributed intermittently in East Asia and North America. Protecting this species is of academic significance to the study of flora. The Environmental Protection Committee of the State Council of China, in July 1984, announced the "List of Rare and Endangered Plants", and the genus Douglas fir and Douglas fir are Class II protected and endangered species. Due to long-term harvesting and utilization, coupled with the extremely low seed fertility rate, the tree species renewal ability is very weak, and the forest trees are decreasing day by day. In the production areas of Douglas fir, it is urgent to take measures to protect the mother tree, promote natural regeneration and expand artificial planting (Reference: China Forestry Science Data Center, Flora).

The breeding of Douglas fir mainly adopts the method of sowing seedlings, and the seeds are mainly collected from the field or provided by the seed garden. There are few Douglas fir seed gardens in my country, and the construction of seed gardens takes a long time and requires a lot of human and financial resources in the early stage. In addition, late fruiting, low seed yield and unstable quality, and serious pests and diseases are common problems in current seed orchards. The vegetative propagation methods often used in the breeding of conifers mainly include cuttings and grafting. Cuttings are a routine and effective method in plant cultivation. Similar to other conifers, the cutting and grafting techniques of Douglas fir have many difficulties: such as strong age effect, low rooting rate of cuttings, low survival efficiency of grafting, etc. The breeding difficulties of Douglas fir have greatly restricted the protection of this genus, as well as the promotion and application of improved varieties.

Tissue culture is a widely used technology in plant breeding at present, and it is the basis for realizing forest tree genetic transformation and transgenic technology. Plant somatic embryos are similar to seed embryos and have both shoot and root apex meristems. Somatic embryogenesis is considered to be the most potential asexual reproduction method and means of conifers. The successful establishment of the somatic embryo system of P. chinensis for large-scale reproduction has not been reported yet.

The main purpose of the present invention is to develop a high-efficiency plant embryo clonal propagation technology in view of the low natural seed setting rate, difficult cutting, and seedling propagation technology that cannot meet the needs of large-scale afforestation. Conservation and large-scale propagation of seedlings provide a method with a short cycle, high reproduction rate and low cost.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a new method for the reproduction of Douglas fir, aiming at the technical problems existing in the reproduction process of the existing Douglas genus, and the method of the present invention can form a large number of excellent somatic embryo test tubes in a relatively short period of time. The seedlings can be produced on a large scale and in a factory. The invention provides a method with short cycle, high reproduction rate and low cost for large-scale vegetative propagation and seedling raising of Douglas fir.

In order to achieve the purpose of the present invention, one aspect of the present invention provides a method for propagating Douglas fir plants, comprising the following steps in sequence: performing embryogenic tissue induction culture, proliferation culture, The adjustment culture of embryogenic tissue, the mature culture of somatic embryos, the germination and culture of mature somatic embryos, and the culture of transfer seedlings are obtained.

Wherein, the Douglas fir plants are selected from Douglas fir, Douglas fir, Douglas fir, Lancang fir, Douglas fir, Douglas fir or Taiwan Douglas fir, preferably Douglas fir and Douglas fir.

Another aspect of the present invention provides a method for reproduction of Douglas fir, comprising the steps of:

1) inoculating the sterilized explants of the Douglas fir plant on an embryogenic tissue induction medium, and performing induction culture to obtain embryogenic tissue;

2) inoculating the embryogenic tissue on the proliferation medium to carry out the proliferation culture of the embryogenic tissue;

3) inoculating the embryogenic tissue of the proliferation culture into the embryogenic tissue pretreatment medium, carrying out the adjustment culture of the embryogenic tissue, and obtaining the pretreated embryogenic tissue;

4) inoculating the pretreated embryonic tissue on the somatic embryo maturation medium, carrying out the somatic embryo maturation culture to obtain mature somatic embryos;

5) inoculating the mature somatic embryos on the somatic embryo germination medium, germinating and culturing to obtain somatic embryo seedlings;

6) The somatic embryo seedlings are hardened and transplanted to obtain the final product.

Wherein, the sterilized explants described in step 1) are obtained according to the following method: the collected immature female cones of Douglas fir are subjected to surface sterilization treatment, then peeled off, and the zygotic embryos taken out are the sterilized embryos Explant.

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

1-1) The collected female cones of Douglas fir are first washed with detergent, and then rinsed with tap water;

1-2) after the surface moisture of the female cone is sucked dry, the seeds are taken out therefrom;

1-3) The seeds are first soaked in 75% alcohol by volume for 30-60s and then taken out, then rinsed with sterile water for 3-5 times, and then soaked in 0.1% _HgCl solution for 5-10min and taken out , and then rinsed with sterile water for 5-6 times to obtain sterile seeds;

1-4) After absorbing the moisture on the surface of the seeds, in a sterile state, peel off the seed coats of the sterile seeds until sterile embryos (ie, sterile zygotic embryos) are obtained.

In particular, the embryonic age of the explants of the genus Douglas has a significant effect on the induction rate of embryogenic tissue. In the present invention, seed embryos whose growth and development stages are located between the polyembryonic stage and the cotyledon stage are selected as explants for inducing embryogenic tissue. Collect female cones around 6-10 weeks after pollination and pollination, and continuously check the development of seed embryos from the sixth week of pollination, and select embryos with appropriate developmental stages as explants.

In particular, Douglas fir cones after six weeks of flowering and powdering are continuously collected to determine suitable explants, which are preferably seed embryos whose developmental stage lies between the polyembryonic and cotyledonary stages.

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

In particular, the culture conditions for embryogenic tissue induction culture in step 1) are as follows: under dark conditions, the culture temperature is 25±2° C.; the culture time is 4-6 weeks.

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

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

2A) inoculating the embryogenic tissue on the embryogenic tissue solid proliferation medium to carry out the first stage solid proliferation culture of the embryogenic tissue;

2B) Inoculate the embryogenic tissue after the first-stage solid proliferation culture into the embryogenic tissue liquid proliferation medium, and perform the second-stage liquid proliferation culture of the embryogenic tissue to obtain the proliferation-cultured embryogenic tissue.

In particular, the second stage of liquid proliferation culture is to inoculate the embryogenic tissue after the first stage of solid proliferation culture into an Erlenmeyer flask or a bioreactor containing an embryogenic tissue liquid proliferation medium to perform liquid proliferation.

In particular, the embryonic tissue that has passed through the liquid proliferation medium in the Erlenmeyer flask can be re-inoculated into a pneumatic bioreactor containing the liquid proliferation medium for embryogenic tissue to perform liquid proliferation.

In particular, in the step 2B) using the pneumatic bioreactor to carry out the liquid proliferation culture process of the second stage, the controlled ventilation rate is 50-400 mL of air per minute per 1000 mL of the reactor, and every 1000 mL of air is introduced into the reactor. The volumetric reactor contains 100-500mL (preferably 200-400mL) liquid proliferation medium; preferably 100-200mL of air per minute per 1000mL capacity reactor, more preferably 100-150ml of air per minute.

In particular, the culture conditions of the embryonic tissue solid proliferation culture and liquid proliferation culture are as follows: under dark conditions, the culture temperature is 25±2°C; the culture time is 6-8 weeks.

In particular, the subculture is performed once every 2-3 weeks during the solid proliferation culture of the embryogenic tissue; the subculture is performed once a week during the liquid proliferation culture of the embryogenic tissue.

In particular, in the process of liquid proliferation culture in a conical flask, the rotational speed of the shaker is controlled to be 100-100 rpm.

Wherein, the embryogenic tissue solid proliferation medium described in step 2A) is in mLV medium+2,4-D (1-3mg/L)+6-BA (0.5-2mg/L)+hydrolyzed casein (200 -1000mg/L) + glutamine (200-1000mg/L) + sucrose (10-30g/L) + phytogel (2.5-3.0g/L); preferably +2,4-D in mLV medium (1-2mg/L)+6-BA (0.5-1mg/L)+hydrolyzed casein (400-800mg/L)+glutamine (400-600mg/L)+sucrose (10-20g/L)+ Plant gel 2.5-3.0g/L; more preferably mLV medium+2,4-D (2mg/L)+6-BA (1mg/L)+hydrolyzed casein (500mg/L)+glutamine ( 500mg/L)+sucrose (10g/L)+vegetative gel (2.8g/L).

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

Wherein, the embryogenic tissue pretreatment medium I in step 3) is: mLV medium + Aza-dC (0-20 μM) + ABA (20-60 μM) + polyethylene glycol 4000 (30-80 g/L) +Hydrolyzed casein (200-800mg/L)+glutamine (100-500mg/L)+sucrose (0-20g/L)+maltose (10-30g/L; preferably: mLV medium+Aza-dC (5-15μM) + ABA (20-60μM) + polyethylene glycol 4000 (30-80g/L) + hydrolyzed casein (200-800mg/L) + glutamine (100-500mg/L) + sucrose ( 0-20g/L)+maltose (10-30g/L); more preferably: mLV medium+Aza-dC (5-15μM)+ABA (20-60μM)+polyethylene glycol 4000 (30-80g/ L)+hydrolyzed casein (200-800mg/L)+glutamine (100-500mg/L)+sucrose (0-20g/L)+maltose 10-30g/L; more preferably: mLV medium+ Aza-dC(5-15μM)+ABA(30-50μM)+polyethylene glycol 4000(40-60g/L)+hydrolyzed casein(200-500mg/L)+glutamine 100-200mg/L)+ Sucrose 0-20g/L)+maltose 10-30g/L; further preferably mLV medium+AZa-dC(5-10μM)+ABA(30μM)+polyethylene glycol 4000 60g/L)+hydrolyzed casein (400mg/L)+glutamine (200mg/L)+sucrose (10g/L)+maltose (30g/L).

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

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

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

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

In particular, the culture conditions of the embryogenic tissue pretreatment culture in step 3) are as follows: under dark conditions, the culture temperature is 25±2° C.; the culture time is 7-21 days (1-3 weeks).

In particular, the inoculation amount during the pretreatment and culture of embryogenic tissue is 2-20 g, preferably 2-5 g, of embryogenic tissue per 100 ml of embryogenic tissue-adjusted medium.

In particular, the embryogenic tissue after the proliferation medium is inoculated into the Erlenmeyer flask or bioreactor containing the embryogenic tissue pretreatment medium or the embryogenic tissue pretreatment medium I, and the embryogenic tissue pretreatment ( adjusted culture).

In particular, during the pretreatment and culture of embryogenic tissue using a pneumatic bioreactor, the control ventilation rate is 50-400 mL of air per minute per 1000 mL of the reactor, and each 1000 mL of the reactor contains 100-500 mL of air. (preferably 200-400mL) embryogenic tissue pretreatment medium or embryogenic tissue pretreatment medium I; preferably 100-200mL of air per minute per 1000mL capacity reactor, more preferably 100mL per minute -150ml air.

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

In particular, if Aza-dC is contained in the pretreatment medium during the pretreatment culture of embryogenic tissue, the somatic embryo maturation medium is more preferably mLV medium + ABA (60 μM) + PEG4000 (75 g/L) + hydrolyzed cheese Protein (400mg/L)+glutamine 200mg/L)+maltose (30g/L)+sucrose (10g/L)+vegetative gel (6g/L).

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

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

In particular, the culture conditions for the germination culture are: 25±2°C, germination culture under the conditions of a photoperiod of 16h light/8h darkness, and a light intensity of 1500-2000Lux; the culture time is 6-8 weeks.

In particular, it also includes step 5A) pre-germination culturing of somatic embryos, under dark conditions, the mature somatic embryos obtained in step 4) are cultured in the dark together with the mature medium for more than 1 week at low temperature (4-10° C.); or Under dark conditions, mature somatic embryos obtained from mature culture are inoculated on somatic embryo maturation medium, and cultured in dark at low temperature for at least 1 week; preferably, at 4° C., cultured in dark for 2-4 weeks.

In particular, the culture conditions of the somatic embryos before germination are: low temperature (4-10° C.) and dark culture for 2-4 weeks.

Wherein, the hardening and transplanting described in step 6) include the steps performed in sequence: opening the cap of the culture bottle, refining the seedlings for 1 day in the transplanting room, taking out the plants, and washing the residual agar medium at the roots of the test-tube seedlings with tap water, Transplanted into Douglas fir soilless culture medium (perlite, vermiculite, the volume ratio of perlite to vermiculite is 1:1) for container culture in a transplanting room. Keep the relative humidity at 70-90% and the culture temperature at 25±5° C. one week after transplanting the seedlings, and obtain Douglas fir seedlings after 2-4 weeks.

In the process of transplanting test-tube seedlings, do not damage the root system when transplanting, cover the container with a thin plastic film, pay attention to maintaining sufficient humidity, and manage it carefully.

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

1. The present invention provides, for the first time, a method for efficiently multiplying Douglas fir plants (especially Douglas fir and other Chinese endemic Douglas fir plants) through the process of somatic embryogenesis.

2. The method of the present invention combines the time period after pollination of the cones, observes the stage of seed embryo development under a microscope, and accurately selects a suitable explant to ensure the induction rate of embryogenic tissue.

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

4. The present invention also utilizes a pneumatic bioreactor to efficiently proliferate the embryonic tissue, and performs continuous culture with subsequent pretreatment, thereby greatly improving the production efficiency.

5. The somatic embryos are treated before germination, including low temperature (4°C) treatment and semi-drying (empty dish) treatment to improve the physiological maturity of the somatic embryos and facilitate germination.

6. The method of the present invention adjusts the composition ratio of the medium through repeated experiments to facilitate the maturation, germination and seedling transfer of somatic embryos.

Description of drawings

Figure 1 shows the embryogenic tissue and proliferation culture of Dougus chinensis, in which: A is the solid proliferation culture culture dish of Douglas chinensis embryonic tissue; B is the liquid culture in the conical flask; C is the liquid culture in the pneumatic bioreactor; D is the East China Douglas fir embryogenic tissue; E is the embryogenic tissue of liquid proliferation culture;

Fig. 1a is the somatic embryo diagram of Douglas orientalis embryonic tissue after pretreatment;

Fig. 2 is the mature somatic embryo and the transferred seedling of the somatic embryo of P. chinensis, wherein A is the developing somatic embryo; B is the mature somatic embryo; C is the germination of the somatic embryo; D is the growing somatic embryo seedling;

Figure 3 is a graph showing the effect of adding Aza-dC to the embryogenic tissue pretreatment medium of Douglas orientalis on somatic embryo maturation;

Figure 4 shows the somatic embryo transfer seedlings of P. orientalis, wherein A is the somatic embryo germination; B is the sterile somatic embryo seedling; C is the somatic embryo seedling after hardening treatment (seeding, transplanting).

Detailed ways

The present invention will be further described below with reference to specific embodiments, and the advantages and characteristics of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Test material and culture medium

1. Test materials

1. Cone collection

The immature female cones of P. chinensis were harvested in Sanqingshan, Jiangxi Province in mid-to-late July 2019. Before the formal sampling, the embryonic development observation was carried out in mid-June. After 40 days of flowering and pollination, a small number of cones were collected every week. When it is between the polyembryonic stage and the cotyledon stage, it can be used as an explant. This is to ensure that the embryos within the seeds have clearly developed but no cotyledons have formed at the time of sampling. The mother cones are selected from 3-5 large trees with healthy growth and free pollination. The collected cones were stored at 4°C and refrigerated for later use.

The explant embryo age of Douglas orientalis has a significant effect on the induction rate of embryogenic tissue. In the present invention, seed embryos whose growth and development stages are located between the polyembryonic stage and the cotyledon stage are selected as explants for inducing embryogenic tissue. Collect female cones around 6-10 weeks after pollination and pollination, and continuously check the development of seed embryos from the sixth week of pollination, and select embryos with appropriate developmental stages as explants.

In the specific embodiment of the present invention, P. orientalis is taken as an example, and other P. chinensis are applicable to the present invention.

2. Plant Growth Regulators

The plant growth regulating substance used in the present invention adopts 6-benzylaminoadenine (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 medium adopts mLV medium

The formula of mLV medium is shown in Table 1.

Table 1 mLV medium formula

mLV minimal medium (Kong L, von Aderkas P. 2007. Genotype effects on ABAconsumption and somatic embryo maturation in interior spruce (Piceaglauca xengelmanni). Journal of Experimental Botany 58:1525–1531) is based on the LV minimal medium recipe (Litvay JD , Verma DC, Johnson MA. 1985. Influence of loblolly pine (Pinus taeda L.) culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucuscarota L.). Plant Cell Reports 4:325–328) optimized.

After the above-mentioned mLV medium stock solution was prepared, it was marked respectively, and then stored in a refrigerator with a temperature of 4° C. for later use. According to the volume of the preparation medium, the required macro-element mother solution, trace element mother solution and organic component mother solution and the weighed phytogel, sucrose or/and maltose, hydrolyzed casein were added to the deionized water in sequence, and each added one All were stirred well, and finally water was added to the final volume of the medium, the pH of the medium was tested with a pH meter, and the pH was adjusted to 5.8 with 1M NaOH or 1M HCl. Sterilize at a temperature of 121°C for 15 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well.

(2) Embryogenic tissue induction medium: add 2,4-D (2-6mg/L), 6-BA (1-3mg/L), hydrolyzed casein (400-1000mg/L), Glutamine (200-600mg/L), sucrose (10-30g/L), phytogel (2.5-3.0g/L), adjust pH to 5.8, and sterilize at 121°C for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well.

(3) Embryogenic tissue solid proliferation medium: add 2,4-D (1-3mg/L), 6-BA (0.5-2mg/L), hydrolyzed casein (200-1000mg/L) to mLV medium , glutamine (200-1000mg/L), sucrose (10-30g/L), plant gel (2.5-3.0g/L), adjust the pH value to 5.8, and sterilize at a constant temperature of 121°C for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well.

(4) Embryogenic tissue liquid proliferation medium: add 2,4-D (1-3mg/L), 6-BA (0.5-2mg/L), hydrolyzed casein (200-1000mg/L) to mLV medium , glutamine (200-1000mg/L), sucrose (10-30g/L), adjust the pH value to 5.8, and sterilize at a constant temperature of 121°C for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well.

(5) Embryogenic tissue pretreatment medium (pre-embryo maturation medium): add ABA (20-60 μM), polyethylene glycol (PEG4000, 30-80 g/L), hydrolyzed casein to mLV medium (200-800mg/L), glutamine (100-500mg/L), sucrose (0-20g/L), maltose (10-30g/L), adjust the pH value to 5.8, and keep the temperature at 121°C to keep the temperature off. bacteria for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine and Aza-dC in an ultra-clean workbench, add them to the medium, mix and shake well.

(5A) Embryogenic tissue pretreatment medium I (pre-embryo maturation medium I): AZa-dC (0-20 μM), ABA (20-60 μM), polyethylene glycol (PEG4000) were added to the mLV medium. , 30-80g/L), hydrolyzed casein (200-800mg/L), glutamine (100-500mg/L), sucrose (0-20g/L), maltose (10-30g/L), adjust pH The value was 5.8, and it was sterilized at a constant temperature of 121°C for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine and AZa-dC in an ultra-clean workbench, add them to the medium, mix and shake well.

(6) Somatic embryo maturation medium: add ABA (30-100μM), PEG4000 (50-100g/L), hydrolyzed casein (200-800mg/L), glutamine (100-400mg/L) to mLV medium ), sucrose (0-30g/L), maltose (20-40g/L), plant gel (3.0-8.0g/L), adjust the pH value to 5.8, and sterilize at a constant temperature of 121°C for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well. Wherein, the total mass concentration of sucrose and maltose is 30-60g/L.

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

(8) Somatic embryo germination medium: add 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), phytogel (2-4.0g/L) or agar (5-9g/L), adjusted to pH 5.8, at a temperature of 121°C Sterilize at constant temperature for 20 minutes. After the medium is cooled to about 60°C, filter and sterilize glutamine in an ultra-clean workbench, add it to the medium, mix and shake well.

2. Cultivation conditions

(1) Embryogenic tissue induction culture: dark conditions; culture temperature (25±2)°C; subculture once every 2 weeks; culture time 4-6 weeks.

(2) Embryogenic tissue solid proliferation culture: dark conditions; culture temperature (25±2)°C, subculture once every 2-3 weeks.

(3) Embryogenic tissue liquid proliferation culture: dark conditions; culture temperature (25±2)° C.; rotation speed 100-110 rpm; subculture once a week.

(4) Bioreactor liquid proliferation culture: dark conditions; culture temperature (25±2)° C.; ventilation rate of 50-400ml/min per 1000ml capacity of the reactor; subculture once a week.

(5) Pre-treatment culture of somatic embryos (triangle flask): dark conditions; culture temperature (25±2)° C.; rotation speed 100-110 rpm; culture for 1-2 weeks.

(5A) Somatic embryo maturation pre-treatment culture (bioreactor): dark conditions; culture temperature (25±2)° C.; aeration volume of 50-400ml/min per 1000ml capacity reactor; culture for 1-2 weeks.

(6) Somatic embryo maturation culture (Petri dish): dark conditions; culture temperature (25±2)° C.; culture for 6-8 weeks.

(7) Pre-germination culture of somatic embryos (Petri dish): dark conditions; culture temperature (4-10)° C.; culture for at least 1 week.

(8) Somatic embryo germination culture: the culture temperature is (25±2)°C, the light intensity is 1500-2000lx, and the light cycle is 10-16 hours light/8-14 hours dark.

Example 2 (Induction of embryogenic tissue)

1. Sterilization of explants

First wash the surface of the Douglas fir cones refrigerated at 4°C, then wash them with tap water, cut open the cones, and take out the seeds; use alcohol with a concentration of 75% by volume on the ultra-clean workbench. Soak the seeds of Douglas fir for 30-60s, then wash the seeds with sterile water for 3-5 times, then put them in 0.1% HgCl ₂ solution for 5-10min, then wash the seeds with sterile water for 5-6 times , put the rinsed seeds on the sterilized filter paper to absorb the water, open the seed coat with tweezers and scissors in the ultra-clean workbench, and obtain the female gametophyte. The female gametophytes were further opened to obtain aseptic and immature zygotic embryos of Douglas fir, which were used for later use.

2. Embryogenic tissue induction culture

1) Inoculate the whole sterile Douglas fir immature zygotic embryos as explants in the embryogenic tissue induction medium in the ultra-clean workbench, and carry out the induction culture of embryogenic tissue under dark conditions, and subculture once every 2 weeks, The explants were taken out after 2 weeks of culture, and placed in another fresh embryogenic tissue induction medium to continue the induction culture of embryogenic tissue to keep the medium containing sufficient nutrients and water. For the convenience of counting and statistics, Each dish was inoculated with 10 female gametophytes and more than 5 replicates were set up.

In this example, the embryogenic tissue induction medium contains 2,4-D (4mg/L), 6-BA (2mg/L), hydrolyzed casein (500mg/L), glutamine (500mg/L) , sucrose (10g/L), phytogel (2.75g/L) as an example to illustrate, in other embryogenic tissue induction medium 2,4-D (2-6mg/L), 6-BA (1-3mg /L), hydrolyzed casein (400-1000mg/L), glutamine (200-600mg/L), sucrose (10-30g/L), vegetable gel (2.5-3.0g/L), all suitable for The present invention; wherein the embryogenic tissue induction medium preferably contains 2,4-D (2-4mg/L), 6-BA (1-2mg/L), hydrolyzed casein (400-800mg/L), glutamine Amide (200-600mg/L), sucrose (10g/L), phytogel (2.75g/L). After 2-3 weeks of induction and culture, the tissue structure grown from the explants was loose and translucent, (as shown in Figure 1D), and observed under a dissecting microscope, with multiple filamentous protrusions on the surface.

The induction culture was replaced with a new induction medium every 2 weeks, and the induction culture was carried out for 6 weeks to obtain embryogenic tissue. The number of explants of the obtained embryogenic tissue was counted, and the induction rate of embryogenic tissue was calculated. The analysis results are shown in Table 2, among which:

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

Table 2 Embryogenic tissue induction rate

species Acquisition time Explant (embryo) developmental stage Induction rate (%) East China fir 7.20-7.30 After polyembryonic stage, before cotyledon stage 21.4

The developmental stage of the female gametophyte (cone collection time) has a critical influence on the induction of somatic embryogenesis. If the collection time is too early, the immature embryo has not yet formed, and if the late zygotic embryo is collected, it has matured or is close to maturity, and the degree of differentiation is too high, which is not conducive to the induction of somatic embryos. The present invention adopts the method of direct observation and monitoring of the developmental stage of the embryo to accurately determine the collection time of the cone. The results of embryogenic tissue induction proved that after the polyembryonic stage, the zygotic embryos before the cotyledon stage were suitable explants for embryogenic tissue induction.

Example 3 (Proliferation and Culture of Embryogenic Tissue)

The collected cones of the different genotypes (YDF-A, YDF-B) of Douglas orientalis were induced and cultured according to the method in Example 2, and the induced culture was carried out for 4-6 weeks to obtain the corresponding embryogenic tissues.

1. Embryogenic tissue proliferation culture

1) Embryogenic tissue solid proliferation culture

The embryogenic tissue obtained by induction was separated from the explant, and divided into small pieces of 0.5cm × 0.5cm, inoculated on the solid proliferation medium of embryogenic tissue (as shown in Figure 1A), and the Douglas fir embryos were carried out under dark conditions. The first stage of solid proliferation culture of embryogenic tissue, wherein the culture temperature is (25±2)°C, 2,4-D used in the solid proliferation medium of embryogenic tissue is (2mg/L), and 6-BA is (1mg/L) L), hydrolyzed casein (500mg/L), glutamine (500mg/L), sucrose (10g/L), phytogel (2.8g/L), during the proliferation and culture of Douglas fir embryogenic somatic embryos Subculture once every 2 weeks, and after subculture several times (2-6 times), a large amount of solid proliferating embryogenic tissue of Dougus fir was obtained.

Because plant materials of different genotypes respond differently to the same liquid proliferation culture method, two different genotypes (YDF-A, YDF-B) of Douglas chinensis were used as experimental materials in this experiment. The embryogenic tissue was weighed before and after the solid proliferation culture of the embryogenic tissue for one week, and then the weekly proliferation rate of the embryogenic tissue was calculated according to the following formula (1). 3 replicates per treatment. The averages in Table 3 are based on data from at least three replicates.

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

Table 3 Effects of different culture methods on the weekly proliferation rate (%) of embryogenic tissue in East China yellow shirt

In this example, the solid proliferation medium for embryogenic tissue in the medium contains 2,4-D (2mg/L), 6-BA (1mg/L), hydrolyzed casein (500mg/L), glutamine Amide (500mg/L), sucrose (10g/L), phytogel (2.8g/L) as examples, 2,4-D (1-3mg/L), 6-BA (0.5g/L) in other solid proliferation medium -2mg/L), hydrolyzed casein (200-1000mg/L), glutamine (200-1000mg/L), sucrose (10-30g/L), vegetable gel (2.5-3.0g/L) are all suitable In the present invention, the solid growth medium preferably contains 2,4-D (1-2mg/L), 6-BA (0.5-1mg/L), hydrolyzed casein (400-800mg/L), gluten (400-800mg/L). Amino amide (400-600mg/L), sucrose (10-20g/L), vegetable gel (2.5-3.0g/L).

2) Embryogenic tissue liquid proliferation culture

Liquid proliferation culture can be cultured in the following two ways: conical flask (erlenmeyer flask) suspension culture, pneumatic bioreactor suspension culture

2-1) Conical flask suspension culture

The embryogenic tissue (2g) obtained from the first stage of proliferation culture was inoculated into a Erlenmeyer flask in 100ml of embryogenic tissue liquid proliferation medium, placed on a shaker (100-110 revolutions per minute), and carried out under dark conditions The second stage of liquid proliferation culture of Douglas fir embryonic tissue (Fig. 1B), wherein the culture temperature is (25±2) °C, the 2,4-D used in the embryogenic tissue liquid proliferation medium is 2 mg/L, 6- BA is 1mg/L, hydrolyzed casein 500mg/L, glutamine 500mg/L, sucrose 10g/L, subculture once a week in the process of embryogenic tissue proliferation culture of Dougus fir, and obtain a large amount of cells after 3-5 subcultures. Douglas fir embryogenic tissue (Figure 1E).

The embryogenic tissue was weighed before the liquid proliferation culture of the embryogenic tissue and after the liquid proliferation culture for one week, and then the weekly proliferation rate of the embryogenic tissue was calculated according to formula (1). The measurement results are shown in Table 3. The averages in Table 3 are based on data from at least three replicates.

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

In the process of embryogenic tissue proliferation and culture, adding high concentrations of 2,4-D for a long time is not conducive to the development and differentiation of late embryos. /L, preferably 2 mg/L. The BA concentration is 0.5-2 mg/L, preferably 1 mg/L.

2-2) Pneumatic bioreactor suspension proliferation culture

The embryogenic tissue obtained in the first stage of proliferation culture was inoculated into a pneumatic bioreactor reaction flask containing liquid proliferation medium (Figure 1C), and the second stage of liquid proliferation culture was carried out, in which the pneumatic bioreactor with a volume of 1000ml was used. In the container, add 300mL (usually 100-500mL, preferably 200-400mL) liquid proliferation medium; the inoculation amount is 2g embryogenic tissue (usually 1-4g/100ml) per 100ml liquid proliferation medium; per 1000mL capacity The aeration rate of the pneumatic bioreactor is 100-150ml/min (usually 50-400ml/min, preferably 100-200ml/min), to ensure that the plant material can run uniformly in the liquid medium. Pneumatic bioreactors were placed at 25±2°C and cultured in the dark. Embryogenic tissue liquid proliferation medium used 2,4-D 2mg/L, 6-BA 1mg/L, hydrolyzed casein 500mg/L, glutamine 500mg/L, sucrose 10g/L, in Douglas fir embryos In the process of sexual tissue proliferation and culture, subculture was performed once a week, and a large number of Douglas fir embryonic tissues were obtained after 3-5 subcultures (as shown in Figure 1E).

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

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

The embryonic tissue after suspension culture in the conical flask can also be inoculated into a pneumatic bioreactor reaction flask equipped with a liquid proliferation medium, and the liquid suspension culture can be continued. The same is true for pneumatic bioreactors.

The pneumatic bioreactor used in the present invention is not only the bioreactor disclosed in the Chinese utility model patent with the authorization announcement number CN203840895U and the invention name is "bioreactor system", other plant tissue culture pneumatic bioreactors All devices are suitable for the present invention.

In this example, the medium for embryogenic tissue liquid proliferation medium contains 2,4-D (2mg/L), 6-BA (1mg/L), hydrolyzed casein (500mg/L), glutamine Amide (500mg/L), sucrose (10g/L) as an example, 2,4-D (1-3mg/L), 6-BA (0.5-2mg/L), hydrolyzed casein ( 200-1000mg/L), glutamine (200-1000mg/L), sucrose (10-30g/L) are all suitable for the present invention, wherein the solid proliferation medium preferably contains 2,4-D as (1-2mg /L), 6-BA (0.5-1mg/L), hydrolyzed casein (400-800mg/L), glutamine (400-600mg/L), sucrose (10-20g/L).

Example 4 Pretreatment of embryogenic tissue (adjusted culture)

The pretreatment culture of embryogenic tissue is a conditioning culture before the embryogenic tissue is cultured for somatic embryo maturation.

Put 2-5 g of embryogenic tissue obtained after liquid proliferation and culture in Example 3 into a 100-mL conical flask, add 30-40 mL of embryogenic tissue pretreatment medium to the conical flask, and under dark conditions, place it in a conical flask. Suspension culture in a bottle, that is, pre-treatment (adjusted culture) of Douglas fir embryonic tissue, where the culture temperature is (25±2) °C, and the embryogenic tissue pre-treatment medium contains ABA (40 μM), polyethylene glycol ( PEG4000) (62.5g/L), hydrolyzed casein (300mg/L), glutamine (150mg/L), sucrose (10g/L), maltose (30g/L), place the conical flask for 100r min ^{– 1.} Treat in the dark on an incubator shaker at 25±2°C for 1-2 weeks.

During the adjustment and culture process, the embryonic tissue structure was observed under a stereomicroscope, and the differentiation of the embryo head and the embryo stalk could be clearly seen (as shown in Figure 1a).

In the embodiment of the present invention, the pretreatment medium is ABA (40 μM), polyethylene glycol (PEG4000) (62.5g/L), hydrolyzed casein (300mg/L), glutamine (150mg/L) in the medium, For example, sucrose (10g/L) and maltose (30g/L), other mediums contain ABA (20-60μM), polyethylene glycol (PEG4000) (30-80g/L), hydrolyzed casein (200- 800mg/L), glutamine (100-500mg/L), sucrose (0-20g/L), maltose (10-30g/L) are all suitable for the present invention; pretreatment medium is preferably ABA (30-50μM ), polyethylene glycol (PEG4000) (50-75g/L), hydrolyzed casein (200-400mg/L), glutamine (100-200mg/L), sucrose (0-10g/L), maltose ( 10-30g/L).

Example 4A Pretreatment of embryogenic tissue (adjusted culture)

Put 2-5 g of embryogenic tissue obtained after liquid proliferation and culture in Example 3 into a 100-mL conical flask, add 30-40 mL of embryogenic tissue pretreatment medium I to the conical flask, and under dark conditions, in Suspension culture in Erlenmeyer flasks, that is, pretreatment (adjusted culture) of Douglas fir embryonic tissue, wherein, embryogenic tissue pretreatment medium I contains ABA 30μM, polyethylene glycol (PEG4000) 60g/L, hydrolyzed casein 400mg /L, glutamine 200mg/l, sucrose 10g/L, maltose 30g/L, and Aza-dC. The dosage of Aza-dC was 0, 5, 10, and 15 μM, respectively; place the conical flask for 100 r·min ^{– 1.} Treat in the dark on an incubator shaker at 25±2°C for 1-2 weeks.

During the adjustment and culture process, the embryonic tissue structure was observed under a stereomicroscope, and the differentiation of the embryo head and the embryo stalk could be clearly seen.

In the embodiment of the present invention, the pretreatment medium I contains ABA 30 μM, polyethylene glycol (PEG4000) 60 g/L, hydrolyzed casein 400 mg/L, glutamine 200 mg/L, sucrose 10 g/L, and maltose 30 g/L in the medium. , Aza-dC (0, 5, 10, 15μM) as an example, other medium contains ABA (20-60μM), polyethylene glycol (PEG4000) (30-80g/L), hydrolyzed casein (200- 800mg/L), glutamine (100-500mg/L), sucrose (0-20g/L), maltose (10-30g/L), Aza-dC (0-20μM) are all suitable for the present invention; pretreatment Medium I is preferably ABA (20-60μM), polyethylene glycol (PEG4000) (30-80g/L), hydrolyzed casein (200-800mg/L), glutamine (100-500mg/L), sucrose (0-20g/L), maltose (10-30g/L), Aza-dC (5-15μM), more preferably: ABA (30-50μM), polyethylene glycol (PEG4000) (40-60g/L) ), hydrolyzed casein (200-500mg/L), glutamine (100-200mg/L), sucrose (0-10g/L), maltose (10-30g/L), Aza-dC (5-15μM) ; More preferably: ABA (30 μM), polyethylene glycol (PEG4000) (60g/L), hydrolyzed casein (400mg/L), glutamine (200mg/L), sucrose (10g/L), maltose (30 g/L), Aza-dC (5-10 μM).

Example 4B Pretreatment of embryogenic tissue (adjusted culture)

Inoculate 2-5 g of the embryogenic tissue obtained after the liquid proliferation culture in Example 3 into the reaction flask of the pneumatic bioreactor equipped with the embryogenic tissue pretreatment medium, under dark conditions, in the bioreactor. Suspension culture in the reaction flask, that is, pretreatment (adjusted culture) of Douglas fir embryonic tissue, wherein the embryogenic tissue pretreatment medium contains ABA 30 μM, polyethylene glycol (PEG4000) 60 g/L, hydrolyzed casein 400 mg/L , Glutamine 200mg/L, Sucrose 10g/L, Maltose 30g/L;

In a pneumatic bioreactor with a capacity of 1000ml, add 300mL (usually 100-500mL, preferably 200-400mL) embryogenic tissue pretreatment medium; the aeration volume of the pneumatic bioreactor per 1000mL capacity is 100-150ml/ min (usually 50-400ml/min, preferably 100-200ml/min), to ensure that the proliferating and culturing embryogenic tissue can run evenly in the embryogenic tissue pretreatment medium. Pneumatic bioreactors were placed at 25±2°C and cultured in the dark for 1-2 weeks.

Embryogenic tissue pretreatment medium I can also be used as the medium in the pneumatic bioreactor.

Example 5 Mature culture of somatic embryos

The embryogenic tissues that have been pre-treated for 1-2 weeks (usually 7-14 days) in Example 4 were taken out with a truncated pipette, and the pre-treatment medium was evenly sprinkled on sterile filter paper, and then suction filtered. Remove excess pretreatment medium;

Next, the filter paper and the pretreated embryogenic tissue (about 0.1 g fresh weight) on it were placed on the surface (Petri dish) of a solid (or semi-solid) somatic embryo maturation medium, and the somatic embryos of Douglas fir were carried out. Mature culture; wherein, the somatic embryo maturation medium contains ABA60μM, polyethylene glycol (PEG4000, 60g/L), hydrolyzed casein (400mg/L), glutamine (200mg/L), sucrose (10g/L) , maltose (30g/L), phytogel (6g/L); under dark conditions, the culture temperature is 25±2℃, cultured for 6-8 weeks, it can be observed that the somatic embryo develops from the pre-cotyledon embryo stage to the cotyledon At the embryo stage, mature somatic embryos (morphologically mature somatic embryos) were obtained (Fig. 2A-B).

After 8 weeks of mature culture, the morphology of mature somatic embryos observed under a stereomicroscope is shown in Figure 2B, and the morphological mature somatic embryos in each dish were counted according to the embryonic developmental morphology. 5 dishes were counted for each treatment to calculate the average number of mature somatic embryos per dish. The measurement results are shown in Table 4.

Table 4 Effects of different culture methods and maturation medium components on somatic embryo maturation of P. orientalis

*Spruce maturation medium (without polyethylene glycol, maltose, sucrose); ^★ Spruce improved maturation medium (without maltose, with polyethylene glycol, sucrose)

The experimental data show that the number of mature embryos obtained in each dish is different depending on the treatment. Pretreatment for 2 weeks and use of Douglas fir maturation medium both increased the number of mature somatic embryos. The experimental results also proved the importance of using PEG and maltose in the Douglas fir maturation medium. Without PEG in the medium, mature embryos could not be obtained; without maltose, although mature somatic embryos could be obtained, it would lead to mature embryos. The number of embryos was significantly reduced, and the quality of somatic embryos was poor.

The mature culture of embodiment 5A somatic embryo

The embryogenic tissue after adjusting and culturing for 1-2 weeks in Example 4A was taken out with a truncated pipette, evenly sprinkled on sterile filter paper together with the pre-treatment medium, and then the excess pre-treatment medium was filtered off with suction;

Next, the filter paper and the pretreated embryogenic tissue (about 0.1 g fresh weight) on it were placed on the surface (Petri dish) of the embryo maturation medium, and under dark conditions, the embryogenic tissue maturation culture of Douglas fir was carried out, Among them, ABA (60μM), polyethylene glycol (PEG4000, 75g/L), hydrolyzed casein (400mg/L), glutamine (200mg/L), sucrose (10g/L) used in the somatic embryo maturation medium , maltose (30g/L), vegetable gel (6g/L). The cells were cultured in the dark at (25±2)°C, and observed under a stereomicroscope after 8 weeks, and the number of morphologically mature somatic embryos was counted.

Aza-dC can significantly inhibit the excessive proliferation of non-embryonic tissues in mature culture, providing space for the growth of mature embryos; when the embryogenic tissue pretreatment suspension liquid medium in Example 4A was added with 5 μM and 10 μM Aza-dC, respectively dC can significantly inhibit the growth of non-embryonic tissues, increase the number of normal mature embryos (Figure 3), and mature embryos can grow and develop well.

The results in Figure 3 show that when the embryogenic tissue pretreatment medium was supplemented with 5 μM, or 10 μM Aza-dC, the highest number of normal mature somatic embryos (morphologically mature somatic embryos) could be obtained from each culture dish. When the concentration of Aza-dC was 0, it could not inhibit the non-embryogenic tissue, while its concentration of 15 μM was too high, which partially inhibited the growth of normal embryogenic tissue. In both cases (at Aza-dC concentrations of 0, 15 μM), the number of normal mature somatic embryos produced per dish was low (Figure 3).

The mature culture of embodiment 5B somatic embryo

In order to test whether a relatively simple and economical spruce maturation medium (without PEG4000, with sucrose but without maltose) could be used, the pretreated embryogenic tissues were also inoculated on the spruce maturation medium at the same time.

The embryogenic tissues that have been pre-treated for 1-2 weeks (usually 7-14 days) in Example 4 were taken out with a truncated pipette, and the pre-treatment medium was evenly sprinkled on sterile filter paper, and then suction filtered. Remove excess pretreatment medium;

In addition to placing the filter paper and the pretreated embryogenic tissue (about 0.1 g fresh weight) on the surface of the solid spruce somatic embryo maturation medium (Petri dish) to carry out the maturation culture of Douglas fir somatic embryos, The rest are the same as in Example 5, wherein the spruce maturation medium contains no polyethylene glycol (PEG4000), no maltose, and sucrose (30 g/L); other components are the same as the somatic embryo maturation medium of the present invention.

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

The mature culture of embodiment 5C somatic embryo

The embryogenic tissues that have been pre-treated for 1-2 weeks (usually 7-14 days) in Example 4 were taken out with a truncated pipette, and the pre-treatment medium was evenly sprinkled on sterile filter paper, and then suction filtered. Remove excess pretreatment medium;

Except that the filter paper and the pretreated embryogenic tissue (about 0.1 g fresh weight) thereon are placed on the surface (Petri dish) of the solid spruce somatic embryo modified maturation medium to carry out the maturation culture of the spruce somatic embryos , the rest are the same as in Example 5, wherein, the spruce improved maturation medium contains polyethylene glycol (PEG4000, 60g/L), without maltose, containing sucrose (30g/L); other components are the same as the somatic embryo maturation medium of the present invention. same.

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

Example 6 Germination of somatic embryos, transplantation of somatic embryo seedlings

1) Pre-germination treatment

The mature somatic embryos are treated before germination, that is, the mature somatic embryos are placed together with the mature medium at low temperature (4°C, usually 4-10°C) for dark culture; the mature somatic embryos can also be placed together with the filter paper below. Placed in an empty sterile petri dish, then placed at low temperature (4°C, usually 4-10°C) for dark incubation. Generally, the temperature of pre-germination treatment of mature somatic embryos is preferably 4°C, and the pre-germination treatment time is 2-4 weeks. Somatic embryos treated before germination become physiologically more mature and have stronger germination ability.

2) Germination culture

The mature somatic embryos that have undergone pre-germination treatment are transferred to the somatic embryo germination medium, and the germination culture is carried out under the conditions of 25 ± 2 °C, under the condition of a photoperiod of 16h light/8h dark, and a light intensity of 1500-2000Lux. : The germination medium is based on mLV medium, supplemented with NH ₄ NO ₃ (1g/L), hydrolyzed casein (400mg/L), glutamine (100mg/L), sucrose (10g/L), activated carbon (2g/L), agar 7g/L (or vegetable gel 3.0g/L).

Somatic embryo germination 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), agar (5-9g/L) or vegetable gel (2-4g/L) are suitable. The cotyledons of the mature somatic embryos quickly turned green, and the hypocotyls elongated and grew. After 8 weeks of culture, the cotyledons at the top of the mature somatic embryos unfolded, and the other end differentiated into a distinct root growth end, forming a somatic embryo seedling (as shown in Figure 2C- D; Figure 4A-B), the obtained somatic embryo seedlings were counted, and the germination rate of somatic embryos was 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 fetuses was 85-98%, and the seedling transfer rate was 32-71%.

3) Transplantation of somatic embryo seedlings

When the height of the somatic embryo seedlings reaches more than 2 cm and the roots are thick and strong, open the cap of the culture bottle, and after cultivating the seedlings in the transplanting room for 1 day, take out the plants, wash the agar medium remaining on the roots of the test-tube seedlings with tap water, and transplant them. To Douglas fir soilless culture medium (perlite, vermiculite, the volume ratio of perlite to vermiculite is 1:1), container culture is carried out in a transplanting room. The relative humidity was kept at 80-90% within 2 weeks after seedling transplanting, and gradually decreased to 60-70%, and the culture temperature was 25±5° C. After 6-8 weeks, the seedlings of Douglas fir were obtained (Fig. 4C).

In the specific embodiment of the present invention, a seed embryo that has obviously developed but has not yet formed cotyledons is used as an explant for inducing embryogenic tissue. Other female gametophytes (including embryos) in Douglas fir cones 40-70 days after flowering and powdering can be used in the present invention. The method for propagating Douglas fir in the invention provides a method for artificial large-scale industrial asexual propagation and seedling raising of Douglas fir, which has a short cycle and high reproduction rate, breaks through the difficulty of vegetative propagation of Douglas fir, has a long growth cycle, large investment in seed orchards, and late fruiting. Restrictions such as limited area have become a new way to maintain good varieties of forest trees, multiply quickly and improve varieties.

Claims (11)

1. a kind of reproduction method of Dougus chinensis by somatic embryogenesis, it is characterized in that, comprise the steps that carry out successively as follows: the sterile explant of Dougus chinensis is carried out embryogenic tissue inductive culture, proliferation culture, embryo The adjustment culture of sexual tissue, the maturation culture of somatic embryos, the germination culture of mature somatic embryos, and the culture of transfer seedlings are obtained. 2. a kind of reproduction method of Douglas fir by somatic embryogenesis, it is characterized in that: comprise the steps: 1) inoculating the sterilized explants of the Douglas fir plant on an embryogenic tissue induction medium, and performing induction culture to obtain embryogenic tissue; 2) inoculating the embryogenic tissue on the proliferation medium to carry out the proliferation culture of the embryogenic tissue; 3) inoculating the proliferated embryonic tissue into the embryogenic tissue pretreatment medium or embryogenic tissue pretreatment medium I, and carrying out the adjustment culture of the embryogenic tissue to obtain the pretreated embryogenic tissue; 4) inoculating the pretreated embryonic tissue on the somatic embryo maturation medium, carrying out the somatic embryo maturation culture to obtain mature somatic embryos; 5) inoculating the mature somatic embryos on the somatic embryo germination medium, germinating and culturing to obtain somatic embryo seedlings; 6) The somatic embryo seedlings are hardened and transplanted to obtain the final product. 3. method as claimed in claim 2 is characterized in that, described in step 1) embryogenic tissue induction medium is mLV medium+2,4-D (2-6mg/L)+6-BA (1 -3mg/L)+hydrolyzed casein (400-1000mg/L)+glutamine (200-600mg/L)+sucrose (10-30g/L)+vegetative gel (2.5-3.0g/L). 4. The method according to claim 2 or 3, wherein the proliferation culture described in step 2) comprises the steps of performing in the following order: 2A) inoculating the embryogenic tissue on the embryogenic tissue solid proliferation medium to carry out the first stage solid proliferation culture of the embryogenic tissue; 2B) Inoculate the embryogenic tissue after the first-stage solid proliferation culture into the embryogenic tissue liquid proliferation medium, and perform the second-stage liquid proliferation culture of the embryogenic tissue to obtain the proliferation-cultured embryogenic tissue. 5. method as claimed in claim 4 is characterized in that, described in step 2A) in embryogenic tissue solid proliferation medium is+2,4-D (1-3mg/L)+6-BA in mLV medium (0.5-2mg/L)+hydrolyzed casein (200-1000mg/L)+glutamine (200-1000mg/L)+sucrose (10-30g/L)+vegetable gel (2.5-3.0g/L) ; Embryogenic tissue liquid proliferation medium described in step 2B) is in mLV medium+2,4-D (1-3mg/L)+6-BA (0.5-2mg/L)+hydrolyzed casein (200- 1000mg/L) + glutamine (200-1000mg/L) + sucrose (10-30g/L). 6. The method according to claim 2 or 3, wherein the embryogenic tissue pretreatment medium I in step 3) is: mLV medium+AZa-dC (0-20 μM)+ABA (20- 60μM) + polyethylene glycol 4000 (30-80g/L) + hydrolyzed casein (200-800mg/L) + glutamine (100-500mg/L) + sucrose (0-20g/L) + maltose (10 -30g/L). 7. The method according to claim 2 or 3, wherein the embryogenic tissue pretreatment medium described in step 3) is: mLV medium+ABA (20-60 μM)+polyethylene glycol 4000 (30 μM) -80g/L)+hydrolyzed casein (200-800mg/L)+glutamine (100-500mg/L)+sucrose (0-20g/L)+maltose (10-30g/L). 8. The method according to claim 2 or 3, wherein the somatic embryo maturation medium described in step 4) is mLV medium+ABA (30-100 μM)+PEG4000 (50-100g/L)+hydrolysis Casein (200-800mg/L)+glutamine (100-400mg/L)+sucrose (0-30g/L)+maltose (20-40g/L)+vegetative gel (3-8g/L). 9. The method according to claim 2 or 3, wherein the germination medium described in step 5) is mLV 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 (2.0-4.0g/L) or agar (5.0- 9.0g/L). 10. method as claimed in claim 1 or 2 is characterized in that, also comprises step 5A) culture before somatic embryo germination, under dark conditions, step 4) mature culture is obtained mature somatic embryo together with mature medium at low temperature. (4-10°C) in dark for more than 1 week; or in dark conditions, inoculate mature somatic embryos obtained from mature culture on somatic embryo maturation medium, and culture in dark for at least 1 week at low temperature; preferably at 4°C, Incubate in the dark for 2-4 weeks. 11. The method of claim 2 or 3, wherein the induction culture of embryogenic tissue described in step 1), the proliferation culture of embryogenic tissue described in step 2), the embryo described in step 3) The culture conditions of the somatic embryo maturation culture described in step 4) are as follows: under dark conditions, the culture temperature is 25±2°C.

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