Method for improving somatic embryo occurrence rate in later stage of picea crassifolia embryogenic tissue proliferation and obtaining regenerated plant

Abstract

The invention relates to a method for efficiently increasing the somatic embryo occurrence rate in the later stage of picea crassifolia embryonic tissue proliferation and continuously obtaining regenerated plants. The method mainly comprises the following steps of pretreatment before differentiation of embryonic tissues subjected to long-term subculture preservation, somatic embryo differentiation and somatic embryo germination and somatic embryo seedling transplanting. In order to solve the technical problem that excellent cell lines are difficult to continuously use due to the fact that the proliferation rate and the somatic embryo occurrence rate are obviously reduced after the picea crassifolia embryogenic tissue is subcultured for a long time, the invention constructs a set of efficient somatic embryo generation and plant regeneration technology suitable for the later proliferation period of the picea crassifolia embryogenic tissue. According to the method, the proliferation rate and somatic embryo occurrence rate of the embryogenic tissue are greatly improved by pretreating the embryogenic tissue in the later proliferation stage, and the problem that the proliferation rate and somatic embryo differentiation rate of the picea crassifolia embryogenic tissue are rapidly reduced along with the prolongation of subculture time is effectively solved by combining with an optimized culture medium formula in the somatic embryo differentiation stage.

Description

Technical Field

[0001] This invention relates to the field of cell-engineered seedling propagation in forestry, specifically to a method for efficiently increasing the somatic embryogenesis rate in the later stages of embryogenic tissue proliferation in Qinghai spruce and continuously obtaining regenerated plants. Background Technology

[0002] Qinghai Spruce ( Picea crassifolia The Qinghai spruce (Picea spp.) is an evergreen tree belonging to the genus Picea in the family Pinaceae. It is characterized by its cold resistance, drought tolerance, ability to thrive in poor soil, and strong adaptability, and is a tree species endemic to my country. The Qinghai spruce is known for its beautiful shape and year-round evergreen foliage. It holds significant ecological and economic value in soil and water conservation, climate regulation, water resource conservation, and maintaining ecological balance, making it an excellent species for landscaping and afforestation. Currently, Qinghai spruce is primarily propagated through traditional methods such as sowing and cuttings. These methods suffer from long growth cycles, high genetic variation rates, and poor seedling uniformity, severely impacting its large-scale production efficiency and the progress of variety improvement.

[0003] Somatic embryogenesis (SEP) refers to the process by which plant somatic cells, under specific conditions, develop directly into bipolar structures without the need for sex cell fusion; the product is called a somatic embryo. In spruce species, SEP often occurs indirectly, and its complete process includes four main stages: embryogenic tissue induction, embryogenic tissue subculture and proliferation, somatic embryo differentiation, and somatic embryo germination and plant regeneration. Embryogenic tissue induction and proliferation are the first steps in SEP, and the embryogenic cell lines formed through subculture are the key starting material for the SEP technology system. However, embryogenic cell lines that have been subcultured for a long time generally exhibit a significant decline in proliferation and SEP capacity: typically, the proliferation rate and somatic embryo differentiation rate begin to decline after six months of subculture, and most cell lines completely lose their SEP capacity after 1-3 years. This phenomenon prevents the long-term utilization of superior cell lines, severely restricting the continued large-scale industrial application of SEP technology.

[0004] Research on somatic embryogenesis technology in Qinghai spruce has been relatively slow, particularly due to problems such as low embryogenic tissue induction rate, significant decline in the proliferation and somatic embryogenesis capacity of embryogenic tissue after six months of subculturing, poor somatic embryo quality, and low germination rate. Therefore, the somatic embryogenesis technology system of Qinghai spruce urgently needs further optimization, especially the establishment of a highly efficient somatic embryogenesis and plant regeneration system in the later stages of embryogenic tissue proliferation. Brassinolides (BRs) are a class of bioactive plant hormones with chemical structures similar to animal steroid hormones. 2,4-epibrassinolide (EBR) is a synthetically produced highly active isomer of brassinolide. To date, the application of EBR in somatic embryogenesis of Qinghai spruce has not been reported, especially its application in long-term subcultured spruce embryogenic tissue.

[0005] Developing a method to improve the somatic embryogenesis rate in the late stage of embryogenic tissue proliferation of Qinghai spruce and to continuously obtain regenerated plants is of great significance for the stable and efficient production of high-quality somatic embryos and the continuous acquisition of regenerated plants using superior cell lines, and provides technical support for the continuous, stable and large-scale propagation of superior germplasm of Qinghai spruce. Summary of the Invention

[0006] To address the challenges of rapid decline in the proliferation rate and somatic embryogenesis rate of embryogenic tissues in the later stages of proliferation (after more than 12 generations) in existing Qinghai spruce somatic embryogenesis techniques, this invention provides a method to efficiently improve the somatic embryogenesis rate of embryogenic tissues in the later stages of proliferation and continuously obtain regenerated plants. For the first time, by using EBR in the pretreatment medium before differentiation and optimizing the differentiation medium, the proliferation rate and somatic embryogenesis rate of embryogenic tissues that have declined due to long-term subculturing are effectively improved, providing technical support for the stable and large-scale propagation of superior Qinghai spruce germplasm.

[0007] This invention provides a method for improving the somatic embryogenesis rate in the late stage of embryogenic tissue proliferation in Qinghai spruce and obtaining regenerated plants, comprising: embryogenic tissue proliferation culture to form a cell line and long-term subculture preservation; firstly, treating the long-term subcultured embryogenic tissue with EBR; and then using a preferred differentiation medium to generate high-quality somatic embryos and obtain regenerated plants. The firstly, treating the long-term subcultured embryogenic tissue with EBR and then using a preferred differentiation medium to generate high-quality somatic embryos and obtain regenerated plants includes three steps: a pretreatment step for long-term subcultured embryogenic tissue before differentiation, a somatic embryo differentiation step, and a somatic embryo germination and somatic embryo seedling transplantation step.

[0008] In the process of establishing cell lines through embryonic tissue proliferation culture and long-term subculture, the proliferation medium used was mLV basal medium + 1.1 mg·L⁻¹. –1 2,4-D +0.55 mg·L –1 6-BA+1g·L–1 Enzymatic hydrolysis of casein + 0.5 g·L –1 Glutamine; culture conditions are 23±1℃ in the dark. At regular intervals, select well-morphologically good embryogenic tissues and transfer them to fresh culture medium for subculture, with at least 12 subcultures. Subculture after 12 subcultures (approximately 0.5 years) is defined as long-term subculture preservation.

[0009] In the pre-differentiation pretreatment step for long-term subcultured embryonic tissues, the pre-differentiation pretreatment culture used mLV basal medium + 1.1 mg·L⁻¹. –1 2,4-D + 0.55 mg·L –1 6-BA+1g·L –1 Enzymatic hydrolysis of casein + 0.5 g·L –1 Glutamine + EBR; the EBR concentration is 0.25~1.5mM, preferably 0.5~1.0mM; the culture conditions are dark culture at 23±1℃, and after a period of time, the embryogenic tissue with good morphology is transferred to fresh culture medium for subculture.

[0010] During the pretreatment culture before subculture proliferation and differentiation, the filamentous culture on the surface of the tissue is gently picked up with tweezers and inoculated onto fresh culture medium. The inoculation is carried out every 10 to 18 days, preferably 12 to 16 days, more preferably 13 to 15 days, and most preferably 14 days. The initial inoculation amount for each tissue is 0.1 to 0.5 g, preferably 0.2 to 0.4 g, and more preferably 0.25 to 0.35 g.

[0011] In the somatic embryo differentiation step, a certain amount of viable filamentous embryonic tissue is gently picked up with forceps and inoculated into the somatic embryo differentiation medium. The somatic embryo differentiation medium is mLV basal medium + abscisic acid + polyethylene glycol + sucrose + activated carbon + gel, and the concentration of abscisic acid is 0~32 mg·L. -1 The preferred concentration is 4~24 mg·L. -1 More preferably 8~16 mg·L -1 The concentration of polyethylene glycol is 0~100 g·L. -1 The preferred concentration is 25~75 g·L. -1 More preferably 40~60g·L -1 Sucrose concentration is 5-60 g / L. -1 Preferred concentration: 10~45 g·L -1 More preferably 25~30g·L -1 The activated carbon concentration is 0~2.5 g·L. -1 Preferred concentration: 0.5~2 g·L -1 More preferably 1 g·L -1 The gel concentration is 2~10 g·L. -1 Preferred concentration: 3~8 g·L -1 More preferably 4~6 g·L-1 The somatic embryo differentiation culture time is 28-63 days, preferably 30-56 days, more preferably 32-49 days, and most preferably 35-42 days.

[0012] The inoculation amount of the filamentous embryonic tissue is 0.1~1g, 0.2~0.8g, more preferably 0.3~0.7g, and most preferably 0.4~0.6g per culture dish.

[0013] In the somatic embryo germination step, the somatic embryo germination medium used was 1 / 2 mL basal medium + 1 g·L⁻¹. –1 Acid-hydrolyzed casein + 0.5 g / L –1 Glutamine + 8g·L -1 Sucrose + 2 g·L -1 Activated carbon + 4~12 g·L -1 Plant gel, pH 5.8±0.01, 23±1℃, under light.

[0014] The embryo germination culture time is 15-60 days, preferably 20-50 days, more preferably 25-40 days, and most preferably 28-35 days.

[0015] In the in vivo embryo transplanting step, seedlings with normally unfolded cotyledons and normally elongated radicles are selected from germinating in vivo embryos and transplanted into culture bottles containing sterilized cultivation substrate. They are first cultured under sterile, closed, and light conditions for 30 to 60 days. After the seedlings resume growth, the bottle caps are gradually opened to harden the seedlings, and then they are transferred to a plant growth culture room for continued cultivation.

[0016] In one specific embodiment of the present invention, (1) Collect immature cones of free-pollinating clones of Qinghai spruce in June-September. After collection, store them in the dark at 4℃ for 0-30 days to improve the embryogenic tissue induction rate.

[0017] (2) Using a scalpel, remove the seeds one by one from the immature cones of Qinghai spruce. Then, visually inspect and press with your fingertips to initially remove shriveled or damaged seeds, retaining plump and firm seeds for subsequent operations. In a clean bench, first disinfect by immersing in 70-75% ethanol for 30-45 seconds, then rinse the surface with sterile water 2-5 times to complete the initial disinfection; then disinfect by immersing in 4% sodium hypochlorite for 6-20 minutes, and then rinse the surface with sterile water 3-5 times to remove the sodium hypochlorite. This disinfection method can control the contamination rate to within 0.1%, and is low in cost and causes minimal environmental pollution, meeting the needs of practical operation.

[0018] (3) Embryogenic tissue induction stage: In a clean bench, immature zygotic embryos from the seeds sterilized in step (2) are extracted using a scalpel and inoculated into 90 mm glass culture dishes containing approximately 30-35 mL of induction medium. 10-15 immature zygotic embryos are evenly inoculated into each dish. The inoculated explants are then cultured in the dark at (23±1)℃ for approximately 30-60 days to induce the formation of white, translucent, filamentous embryogenic tissue and slightly brownish or slightly white granular non-embryonic callus. The induction medium is as follows: mLV basal medium + 1.1 mg·L⁻¹ –1 2,4-Dichlorophenoxyacetic acid (hereinafter referred to as 2,4-D) + 0.55 mg·L –1 6-Benzylaminopurine (hereinafter referred to as 6-BA), with an additional 1 g·L –1 Enzymatic hydrolysis of casein, 0.5 g·L –1 Glutamine.

[0019] (4) Embryonic tissue proliferation culture to form cell lines and long-term subculture preservation stage: The embryonic tissue described in step (3) is transferred to proliferation medium for subculture to form a stable cell line. Fresh medium is transferred every 10-18 days. During subculture, filamentous tissues with good growth status and quality are selected and placed in the dark at (23±1)℃ to form an embryonic cell line for long-term subculture proliferation. The subculture proliferation medium is as follows: mLV basal medium + 1.1 mg·L⁻¹ –1 2,4-D + 0.55 mg·L –1 6-BA, with an additional 1 g·L –1 Enzymatic hydrolysis of casein, 0.5 g·L –1 Glutamine.

[0020] (5) Pre-differentiation pretreatment stage of long-term subcultured embryogenic tissues: Long-term subcultured late-stage proliferative embryogenic tissues were transferred to pre-differentiation pretreatment medium and cultured. After 10-18 days, they were transferred to fresh medium. At the time of transfer, edge filamentous tissues with good growth status and quality were selected and cultured in the dark at (23±1)℃. The pre-differentiation pretreatment medium was as follows: mLV basal medium + 1.1 mg·L⁻¹ –1 2,4-D + 0.55 mg·L –1 6-BA, with an additional 1 g·L –1 Enzymatic hydrolysis of casein, 0.5 g·L –1 Glutamine and 0~2.0mM EBR.

[0021] (6) Somatic embryo differentiation stage: Somatic embryo differentiation was performed using the "filter paper differentiation" method. A filter paper was placed on the surface of each somatic embryo differentiation medium. Approximately 0.1–1 g of the embryonic tissue pretreated in step (5) was weighed using a balance and gently spread evenly on the filter paper with tweezers. The embryonic tissue was then cultured in the dark at (23±1)℃ for 28–63 days to obtain high-quality, pale yellow somatic embryos with intact cotyledons, no deformities, and robust embryos. The somatic embryo differentiation medium was as follows: mLV basal medium + 1 g·L⁻¹ –1 Acid-hydrolyzed casein + 0.5 g / L –1 Glutamine + 0~32 mg·L -1 abscisic acid + 0~100g·L -1 Polyethylene glycol + 5~60 g·L -1 Sucrose + 0~3g·L -1 Activated carbon + 2~10 g·L -1 Plant gel.

[0022] (7) Somatic embryo germination and transplanting stage: The high-quality somatic embryos described in step (6) were inoculated using the flat inoculation and oblique placement method and cultured in a germination medium under low light conditions, with a photoperiod of 16h light / 8h darkness. The somatic embryo germination medium was as follows: 1 / 2 mL V basal medium + 1 g·L⁻¹ –1 Acid-hydrolyzed casein + 0.5 g / L –1 Glutamine + 8g·L -1 Sucrose + 2 g·L -1 Activated carbon + 4~12 g·L -1 Plant gel. After 15-60 days of somatic embryo germination, remove the culture medium from the seedling surface and transplant them into a peat moss:vermiculite:perlite = 3:1:1 cultivation substrate. Initially, culture in sterile, sealed culture bottles under light for 30-60 days, with a photoperiod of 16 hours light / 8 hours dark. Once the seedlings have resumed growth, gradually open the bottle caps to harden them off, and then transfer them to a plant growth chamber for continued cultivation.

[0023] In the preparation of the culture media for each stage of the present invention, after adding and completely dissolving all components except glutamine, ABA, and EBR, the pH value is adjusted to 5.8±0.01, and then a certain mass of plant gel is added. Then, it is sterilized at 121℃ under high temperature and pressure for 20 minutes, and then naturally cooled to about 60℃. Under aseptic conditions, filtered sterilized glutamine, ABA, and EBR are added, mixed evenly, dispensed and solidified, and stored in a dark environment at (23±1)℃ for later use.

[0024] This invention provides a method for efficiently improving the somatic embryogenesis rate in the late stage of embryogenic tissue proliferation in *Picea qinghaiensis* and continuously obtaining regenerated plants. This technology significantly improves the proliferation rate and somatic embryo differentiation rate of embryogenic tissue in the late stage of proliferation in *Picea qinghaiensis*: the proliferation rate of materials subcultured for 0.5 years can reach more than 1.5 times that of the control group, and the somatic embryo differentiation rate can reach more than 2 times that of the control group; the somatic embryo differentiation rate of materials subcultured for 1.5 years can reach more than 2.7 times that of the control group. By further optimizing differentiation and germination conditions, a stable and efficient system for somatic embryogenesis and plant regeneration in the late stage of embryogenic tissue proliferation in *Picea qinghaiensis* was constructed.

[0025] This invention is the first to confirm that adding EBR to the pretreatment medium before differentiation can effectively improve the proliferation rate and somatic embryogenesis rate of embryogenic tissues in the later stages of proliferation, effectively solving the technical problem of significantly declining proliferation and somatic embryogenesis capabilities in long-term subcultured embryogenic cell lines. Simultaneously, this pretreatment method can significantly improve somatic embryo quality and reduce the incidence of deformed embryos. This invention clarifies the optimal conditions for stable proliferation, somatic embryogenesis, and germination of embryogenic tissues in long-term subculture, and constructs a method for efficient somatic embryogenesis in the later stages of embryogenic tissue proliferation and the continuous acquisition of regenerated plants, providing technical support for the efficient propagation of high-quality germplasm resources and variety improvement. This invention can be extended to somatic embryogenesis and plant regeneration systems for other coniferous plants, and has important reference value for the breeding of superior coniferous varieties. Attached Figure Description

[0026] Figure 1 These are embryogenic and non-embryonic tissues induced by the first generation of Qinghai spruce.

[0027] Figure 2 It is an embryonic tissue of the Qinghai spruce embryonic cell line.

[0028] Figure 3 It is a mature somatic embryo of Qinghai spruce.

[0029] Figure 4 Germinated embryos of Qinghai spruce. Specific implementation methods

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0031] The mLV basal medium used in this invention has the same components as publicly available basal media. The 1 / 2 mLV basal medium used is obtained by halving the inorganic nutrient components of the mLV basal medium, while keeping other components unchanged. The basal medium used in this invention is a semi-solid medium obtained by adding plant gels and other components required for the corresponding culture stages to the above-mentioned liquid medium.

[0032] The present invention is a synthesis of experimental data from 2023 to 2025, and the experimental results are stable and reliable. The experimental material was Qinghai spruce collected from the Shaba Experimental Base of the Xiaolongshan Forestry Research Institute in Gansu Province.

[0033] Example 1: Isolation and disinfection of explants Immature cones were collected from the Shaba Experimental Base of the Xiaolongshan Forestry Research Institute in Gansu Province in July and August. After collection, they were wrapped in newspaper and then placed in resealable bags, stored at 4℃ in the dark for 14 days. Seeds were removed one by one from the immature cones using a scalpel. Subsequently, shriveled or damaged seeds were initially removed by visual inspection and fingertip pressing, retaining plump and firm seeds for further processing. The seeds were first immersed in 75% ethanol for 30-45 seconds in a clean bench for preliminary disinfection, followed by rinsing three times with sterile water. Then, they were immersed in 4% sodium hypochlorite for 12 minutes for disinfection, followed by rinsing three times with sterile water to complete the final disinfection.

[0034] Example 2 Induction of embryonic tissue In a clean bench, immature zygotic embryos were extracted from the seeds sterilized as described in Example 1 using a scalpel. These embryos were then inoculated into 90 mm glass culture dishes containing approximately 30–35 mL of embryogenic tissue induction medium, with 10 immature zygotic embryos evenly inoculated per dish. After inoculation, the dishes were incubated in the dark at (23±1)℃ for approximately 30–60 days. The induced tissues were identified as embryogenic tissues based on morphology and cell structure, and the embryogenic tissue induction rate was recorded. The induction medium consisted of mLV basal medium + 1.1 mg·L⁻¹. –1 2,4-D + 0.55 mg·L –1 6-BA, with an additional 1 g·L –1 Enzymatic hydrolysis of casein, 10 g·L –1 Sucrose, 2 g·L –1 Gel, 0.5 g·L –1 Glutamine (filtered and sterilized), pH 5.8 ± 0.01.

[0035] Observation of the morphological characteristics of explant-induced products: embryogenic tissues from primary induction ( Figure 1 A) Appears as a white, semi-transparent, tightly adhered filamentous tissue; non-embryonic tissue ( Figure 1 B) It appears as a loose, irregularly shaped, granular substance, often in a slightly brownish or slightly white granular form. Microscopic observation shows that the embryonic tissue has a typical bipolar structure, consisting of two parts: the upper part of the embryo and the stalk. The upper part of the embryo consists of meristematic cells with dense cytoplasm and a high nucleocytoplasmic ratio, staining red; the stalk consists of highly vacuolated elongated cells, staining blue. Figure 1C). Non-embryonic tissues lack the embryonic stalk mass structure; their cells are large and disordered in arrangement, irregular in shape, and their intracellular vacuoles are abnormally enlarged, almost occupying the entire cell space. Figure 1 D).

[0036] Example 3: Formation of embryonic cell lines The embryonic tissues obtained in Example 2 were transferred to subculture medium for subculture. Fresh medium was added every 14 days. During subculture, filamentous tissues with good growth and quality were selected and cultured in the dark at (23±1)℃ to form a stable proliferating embryonic cell line. Figure 2 The subculture culture medium was the same as the induction culture medium in Example 2.

[0037] Pretreatment stage before differentiation of embryonic tissues in the late proliferation phase: After 12 generations of continuous subculture (approximately 0.5 years), four embryonic cell lines with strong proliferative capacity and typical morphological characteristics were selected and named cell lines A, B, C, and D, respectively. Embryonic tissues from embryonic cell line AD were inoculated into pretreatment medium containing different concentrations of EBR, with six replicates for each treatment. The cells were incubated in the dark at (23±1) ℃ for 14 days, and the proliferation rate of embryonic tissues in each group was calculated. The pretreatment medium was as follows: mLV basal medium + 1.1 mg·L⁻¹ –1 2,4-D + 0.55 mg·L –1 6-BA, with an additional 1 g·L –1 Enzymatic hydrolysis of casein, 10 g·L –1 Sucrose, 0.5 g·L –1 Glutamine and different concentrations of EBR (0.0, 0.25, 0.5, 1.0, 2.0 mM), 3.5 g·L –1 Plant gel, pH 5.8 ± 0.01.

[0038] Effects of EBR on the proliferation rate of embryogenic tissue in the late stage of Qinghai spruce proliferation: The proliferation rate of the EBR-treated groups was higher than that of the control group without EBR, and the proliferation rate of embryogenic tissue generally showed a trend of first increasing and then decreasing with increasing EBR concentration. This indicates that EBR has a generally positive promoting effect on the proliferation rate of embryogenic tissue in the late stage of proliferation.

[0039] Table 1. Effects of different concentrations of EBR on the proliferation rate of embryonic tissues in various cell lines.

[0040] Example 4: Somatic Embryo Differentiation The "filter paper differentiation" method was used to perform somatic embryo-embryo differentiation on the embryogenic tissues pretreated for two culture cycles in Example 3. Specifically, a sheet of filter paper was placed on the surface of each somatic embryo-embryo differentiation medium, and approximately 0.4–0.6 g of pretreated embryogenic tissue was weighed using a balance and gently spread evenly onto the filter paper using tweezers. The differentiation medium used was mLV basal medium + 1 g·L⁻¹. –1 Acid-hydrolyzed casein + 0.5 g·L –1 Glutamine + 16 mg·L -1 abscisic acid + 50g·L -1 PEG4000+ 30g·L -1 Sucrose + 0.5g·L -1 Activated carbon + 4g·L -1 Gel, pH 5.8±0.01. Seven replicates were set up for each treatment, one replicate per dish, and cultured in the dark at (23±1)℃ for 28~63 days. The number of mature embryos in each culture dish was counted.

[0041] Effects of EBR pretreatment on the differentiation rate of embryogenic tissues in the late proliferation stage of *Picea qinghaiensis*: After 12 subcultures (approximately 0.5 years), the somatic embryo differentiation rate of all cell lines decreased compared to the early proliferation stage (within 4 subcultures), with the highest decrease reaching 54.91%. The overall high-quality somatic embryo differentiation rate in the EBR pretreated group was higher than that in the untreated group (Table 2). This indicates that EBR pretreatment before differentiation has a significant effect on improving the high-quality somatic embryo differentiation rate of embryogenic tissues in the late proliferation stage. After 36 subcultures (approximately 1.5 years), the somatic embryo differentiation rate of embryogenic tissues further decreased significantly, and EBR pretreatment could still effectively improve its somatic embryo differentiation rate (Table 3).

[0042] Table 2. Differentiation rate of high-quality somatic embryos in different cell lines at different proliferation stages and with different concentrations of EBR pretreatment groups.

[0043] Table 3. Somatic embryonic differentiation rate of cell line A after 36 passages and pretreatment with different concentrations of EBR.

[0044] Example 5: Optimization of Somatic Embryo Differentiation Somatic embryogenic differentiation was performed on embryogenic tissues pretreated for two culture cycles in Example 3 using different differentiation media. A 5-factor, 4-level orthogonal experimental design was employed. 16 (4 5 Sixteen differentiation media (A1-A16) were designed (Table 4), which consisted of mLV basal medium supplemented with different concentrations of ABA (8, 16, 24 and 32 mg·L⁻¹). –1 PEG4000 (0, 25, 50 and 75 g·L) –1), sucrose (5, 10, 30 and 45 g·L) –1 AC (0, 0.5, 1 and 3 g·L) –1 ), gels (2, 4, 6 and 8 g·L) –1 ) and 1 g·L –1 Acid-hydrolyzed casein, 0.5 g·L –1 Glutamine, pH 5.8 ± 0.01. Optimal combinations of different components were screened, and the differentiation culture method and statistical methods were the same as in Example 4. Different concentrations of abscisic acid, polyethylene glycol, sucrose, activated charcoal, and gel all had extremely significant effects on somatic embryo maturation, and the pairwise interactions among the five factors also had extremely significant effects on somatic embryo maturation (Table 5). The degree of influence of these five factors was in the order of D (sucrose) > B (abscisic acid) > E (activated charcoal) > F (gel) > C (polyethylene glycol) (Table 6). In this example, the optimal differentiation culture medium combination (A3) was 8 mg·L⁻¹. –1 ABA, 50 g·L –1 PEG4000, 30 g·L –1 Sucrose, 1 g·L –1 AC and 6 g·L –1 The gel yielded robust, uniformly sized somatic embryos with distinct hypocotyls and an overall pale yellow color, consistent with the characteristics of high-quality mature somatic embryos (Table 4). Figure 3 ).

[0045] Table 4. Results of embryo maturation of Qinghai spruce under different treatment combinations

[0046] Table 5. Analysis of variance on the effects of different factors on somatic embryo maturation of Qinghai spruce.

[0047] Table 6 Range analysis of five factors affecting embryo maturation in Qinghai spruce.

[0048] Example 6: Somatic Embryo Germination and Somatic Seedling Transplantation The high-quality somatic embryos obtained in Example 5 were inoculated into the germination medium using the flat-inoculation oblique placement method. Figure 4 The somatic embryo germination medium is as follows: 1 / 2 mL basal medium + 1 g·L⁻¹ –1 Acid-hydrolyzed casein + 0.5 g·L –1 Glutamine + 8 g·L -1 Sucrose + 2 g·L -1 Activated carbon + 8 g·L -1Plant gel culture, 23±1℃, under light, with a photoperiod of 16h light / 8h dark. After 30 days of somatic embryo germination, the culture medium on the seedling surface was removed, and the seedlings were transplanted into a sterilized cultivation substrate of peat moss:vermiculite:perlite = 3:1:1. They were initially cultured under sterile light in sealed culture bottles for 30-60 days. Once the seedlings resumed growth, the bottle caps were gradually opened to harden them off, and then they were transferred to a plant growth chamber for further cultivation. Figure 4 ).

[0049] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for increasing the somatic embryogenesis rate in the late stage of embryogenic tissue proliferation in Qinghai spruce and obtaining regenerated plants, comprising: Embryogenic tissues are proliferated and cultured to form cell lines and preserved long-term. First, the long-term subcultured embryogenic tissues are treated with EBR, and then high-quality somatic embryos are generated and regenerated plants are obtained using differentiation medium. The process of first treating the long-term subcultured embryogenic tissues with EBR and then using differentiation medium to generate high-quality somatic embryos and obtain regenerated plants includes three steps: pretreatment step of long-term subcultured embryogenic tissues before differentiation step, somatic embryo differentiation step, and somatic embryo germination and somatic embryo seedling transplantation step.

2. The method according to claim 1, characterized in that, In the proliferation culture of embryonic tissues to form cell lines and for long-term subculture, the proliferation medium used was mLV basal medium + 1.1 mg·L⁻¹. –1 2,4-D +0.55 mg·L –1 6-BA+1g·L –1 Enzymatic hydrolysis of casein + 0.5 g·L –1 Glutamine; culture conditions are 23±1℃ in the dark, and at regular intervals, well-formed embryogenic tissues are selected and transferred to fresh culture medium for subculture, with more than 12 subcultures.

3. The method according to claim 1, characterized in that, In the pre-differentiation pretreatment step for long-term subcultured embryonic tissues, the pre-differentiation pretreatment culture was performed using mLV basal medium + 1.1 mg·L⁻¹. –1 2,4-D + 0.55 mg·L –1 6-BA+1g·L –1 Enzymatic hydrolysis of casein + 0.5 g·L –1 Glutamine + EBR; the concentration of EBR is 0.25~1.5mM, preferably 0.5~1.0mM; the culture conditions are dark culture at 23±1℃, and after a period of time, well-morphologically good embryogenic tissues are selected and transferred to fresh culture medium for subculture.

4. The method according to claim 1, characterized in that, During the pretreatment culture before subculture proliferation and differentiation, the filamentous culture on the surface of the tissue is gently picked up with tweezers and inoculated onto fresh culture medium. The inoculation is carried out every 10 to 18 days, preferably 12 to 16 days, more preferably 13 to 15 days, and most preferably 14 days. The initial inoculation amount for each tissue is 0.1 to 0.5 g, preferably 0.2 to 0.4 g, and more preferably 0.25 to 0.35 g.

5. The method according to claim 1, characterized in that, In the somatic embryo differentiation step, a certain amount of viable filamentous embryonic tissue is gently picked up with forceps and inoculated into the somatic embryo differentiation medium in dark medium. The somatic embryo differentiation medium is mLV basal medium + abscisic acid 4~24 mg·L. -1 +25~75 g·L of polyethylene glycol -1 + 10~45 g·L of sucrose -1 +0.5~2g·L of activated carbon -1 + Gel 3~8g·L -1 .

6. The method according to claim 5, characterized in that, The inoculation amount of the filamentous embryonic tissue is 0.1~1g, 0.2~0.8g, more preferably 0.3~0.7g, and most preferably 0.4~0.6g per culture dish.

7. The method according to any one of claims 1, characterized in that, In the somatic embryo germination step, the somatic embryo germination medium was 1 / 2 mL V basal medium + 1 g·L⁻¹. –1 Acid-hydrolyzed casein + 0.5 g / L –1 Glutamine + 8g·L -1 Sucrose + 2 g·L -1 Activated carbon + 4~12 g·L -1 Plant gel, pH 5.8±0.01, 23±1℃, under light.

8. The method according to claim 7, characterized in that, The somatic embryo germination culture time is 15-60 days, preferably 20-50 days, more preferably 25-40 days, and most preferably 28-35 days.

9. The method according to any one of claims 1-8, characterized in that, In the transplanting of somatic embryos, seedlings with normally unfolded cotyledons and normally elongated radicles are selected from germinating somatic embryos and transplanted into culture bottles containing sterilized culture substrate. They are first cultured under sterile, closed, and light conditions for 30 to 60 days. After the seedlings resume growth, the bottle caps are opened to harden them off, and then they are transferred to a plant growth culture room for further cultivation.

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