Culture medium and method for promoting somatic embryo maturation of pinus massoniana

Abstract

This invention relates to the field of plant breeding technology, and discloses a culture medium and method for promoting somatic embryo maturation in *Pinus massoniana*. Somatic embryogenic callus tissue from *Pinus massoniana* is inoculated into a somatic embryo maturation culture medium containing betaine, polyvinylpyrrolidone, or dimethyl thiourea for somatic embryo induction culture, which can significantly promote the early maturation of *Pinus massoniana* somatic embryos. Experimental results show that morphological observation of the somatic embryogenic callus at different time points during somatic embryo induction revealed that by 60 days of culture, most cotyledonary embryos had matured, and a large number of mature embryos appeared. The number of mature somatic embryos was 250–3500 per gram of somatic callus, and the total number of embryos was 3333–9000 per gram of somatic callus. This indicates that the method of this invention can efficiently promote somatic embryo maturation in *Pinus massoniana*, with high somatic embryo induction efficiency and good practicality.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to a culture medium and method for promoting the maturation of pine embryos. Background Technology

[0002] Somatic embryogenesis, as a highly efficient biotechnology, plays a vital role in the large-scale propagation of high-value plantation seedlings, enhancing forest productivity, and promoting sustainable forestry development, and has become a cutting-edge direction in forestry scientific research. Masson pine, an important native tree species endemic to my country (…),… Pinus massoniana Masson pine occupies an important position in southern forest resources, accounting for a large portion of the country's forest stock and producing a significant amount of resin annually, providing a solid raw material for industries such as timber processing, papermaking, and forest chemical products. Research on somatic embryogenesis of Masson pine began in 1995, when Huang Jianqiu first obtained regenerated plants using mature zygote embryos as explants. Subsequently, a preliminary somatic embryogenesis system for immature embryos was established, methods for inducing embryogenic cell masses were optimized, and techniques for the proliferation and preservation of embryogenic callus were improved. Nevertheless, this technology still faces bottlenecks such as low induction and preservation rates of embryogenic callus, low efficiency in somatic embryo maturation, and poor synchronicity, hindering its industrial application.

[0003] Currently, research on the effects of reducing agents on somatic embryogenesis in pine or coniferous plants is still relatively scarce, and a systematic literature report has not yet been formed. However, some studies have indirectly shown that conifers such as Japanese larch (… Larix kaempferi Conifers are highly sensitive to environmental stress and oxidative stress during somatic embryogenesis, and their somatic embryogenesis induction efficiency is significantly affected by endogenous hormone balance and antioxidant capacity. Therefore, rationally regulating redox status may be an important strategy to improve the somatic embryogenesis efficiency of conifers.

[0004] In summary, how to provide a culture medium and method to promote the maturation of somatic embryos in Pinus massoniana and improve the efficiency of somatic embryo induction is an urgent problem to be solved. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a culture medium and method for promoting somatic embryo maturation of Pinus massoniana. The embryogenic callus of Pinus massoniana is inoculated into a somatic embryo maturation culture medium containing betaine, polyvinylpyrrolidone (PVP), or dimethyl thiourea (DMTU) for somatic embryo induction culture. This method can regulate the somatic embryo maturation rate of Pinus massoniana, efficiently promote somatic embryo maturation, and improve the somatic embryo induction efficiency.

[0006] In this invention, betaine, as an osmotic regulator and methyl donor, can alleviate oxidative stress, stabilize proteins and membrane structures, and promote somatic embryogenesis in pine trees. Dimethylthiourea (DMTU), as a hydroxyl radical scavenger, can alleviate oxidative damage. Polyvinylpyrrolidone (PVP) and cross-linked polyvinylpyrrolidone (PVPP), as adsorbents of phenolic substances, can reduce oxidative browning and improve callus formation and somatic embryogenesis induction environment in tissue culture.

[0007] The first objective of this invention is to provide a culture medium that promotes the maturation of pine embryos, and to study the effects of four substances—betaine, DMTU, PVP, and PVPP—on pine embryogenesis.

[0008] To achieve the first objective, the present invention adopts the following technical solution: A culture medium for promoting somatic embryo maturation of Pinus massoniana includes a somatic embryo maturation medium, wherein betaine, polyvinylpyrrolidone (PVP), or dimethylthiourea (DMTU) is added to the somatic embryo maturation medium; wherein the somatic embryo maturation medium does not include cross-linked polyvinylpyrrolidone (PVPP).

[0009] The culture medium includes at least one of the following conditions: (1) The concentration of betaine in the somatic embryo maturation medium is not higher than 2.5 g / L; (2) The concentration of the polyvinylpyrrolidone in the somatic embryo maturation medium shall not exceed 300 mg / L; (3) The concentration of the dimethylthiourea in the embryo maturation medium shall not exceed 0.3 mg / L.

[0010] Preferably, the culture medium includes at least one of the following conditions: (1) The concentration of betaine in the somatic embryo maturation medium is 0.5–2.0 g / L; (2) The concentration of the polyvinylpyrrolidone in the somatic embryo maturation medium is 100-200 mg / L; (3) The concentration of the dimethylthiourea in the somatic embryo maturation medium is 0.1-0.2 mg / L.

[0011] More preferably, the culture medium includes at least one of the following conditions: (1) The concentration of betaine in the somatic embryo maturation medium is 0.5 g / L; (2) The concentration of the polyvinylpyrrolidone in the somatic embryo maturation medium is 200 mg / L; (3) The concentration of the dimethylthiourea in the somatic embryo maturation medium is 0.1 mg / L.

[0012] Preferably, the formulation of the somatic embryo maturation culture medium includes: basic medium mLP: L-glutamine 400-500 mg / L, hydrolyzed casein 400-600 mg / L, inositol 400-600 mg / L, 2-(N-morpholine) ethanesulfonic acid monohydrate 200-300 mg / L, and the following components are added at the following concentrations: maltose 20-40 g / L, abscisic acid (ABA) 8-10 mg / L, polyethylene glycol (PEG 8000) 100-150 g / L, and gellan gum 3-5 g / L.

[0013] More preferably, the formulation of the somatic embryo maturation culture medium includes: basic medium mLP: L-glutamine 450 mg / L, hydrolyzed casein 500 mg / L, inositol 500 mg / L, 2-(N-morpholine) ethanesulfonic acid monohydrate 250 mg / L, and the following components are added at the following concentrations: maltose 30 g / L, abscisic acid (ABA) 9 mg / L, polyethylene glycol (PEG 8000) 120 g / L, and gellan gum 4 g / L.

[0014] Preferably, the molecular weight of the polyvinylpyrrolidone is 10,000 to 500,000, and more preferably, the molecular weight is 30,000 to 100,000.

[0015] Preferably, the crosslinked polyvinylpyrrolidone has a molecular weight greater than 1 million.

[0016] The second objective of this invention is to provide a method for promoting the maturation of pine embryos, and to study how to use the above-mentioned culture medium to promote the maturation of pine embryos.

[0017] To achieve the second objective, the present invention adopts the following technical solution: A method for promoting somatic embryo maturation of Pinus massoniana involves inoculating embryogenic callus tissue of Pinus massoniana into the aforementioned culture medium for somatic embryo induction culture.

[0018] Preferably, the culture conditions for the pine embryogenic callus include at least one of the following: A. The inoculation amount of the embryogenic callus from *Pinus massoniana* is 4–20 g / L; B. Incubate at 20-28℃; C. Cultivate in the dark; D. Replacing every 20-40 days.

[0019] More preferably, the culture conditions for the pine embryogenic callus include at least one of the following: A. The inoculation amount of the embryogenic callus from *Pinus massoniana* is 5 g / L; B. Incubate at 24℃; C. Cultivate in the dark; D. Replacing every 30 days.

[0020] Preferably, the number of embryos is counted at 60 days after induction of somatic embryos, with the number of mature somatic embryos being 250-3500 per gram of embryogenic callus and the total number of embryos being 3333-9000 per gram of embryogenic callus.

[0021] More preferably, the embryogenic callus tissue of Pinus massoniana was inoculated into a somatic embryo maturation medium containing 0.5 g / L betaine for somatic embryo induction culture. The number of embryos was counted 60 days after somatic embryo induction. The number of mature somatic embryos was 3500 per gram of embryogenic callus, and the total number of embryos was 9000 per gram of embryogenic callus.

[0022] More preferably, the embryogenic callus tissue of Pinus massoniana was inoculated into a somatic embryo maturation medium containing 200 mg / L PVP for somatic embryo induction culture. The number of embryos was counted 60 days after somatic embryo induction. The number of mature somatic embryos was 250 per gram of embryogenic callus, and the total number of embryos was 3333.33 per gram of embryogenic callus.

[0023] More preferably, the embryogenic callus of Pinus massoniana was inoculated into a somatic embryo maturation medium containing 0.1 mg / L DMTU for somatic embryo induction culture, and the number of embryos was counted 60 days after somatic embryo induction, with a total number of 4500 embryogenic callus per gram.

[0024] Preferably, the embryogenic callus of *Pinus massoniana* is induced using immature embryos of resistant *Pinus massoniana*. The specific method includes: collecting *Pinus massoniana* cones, selecting incompletely developed seeds, disinfecting and drying them, peeling off the seed coat, and inoculating the immature embryos into basic medium mLP (10 immature embryos are added to every 20 mL of basic medium mLP), and culturing in the dark at 24°C to induce embryogenic callus. The callus is obtained after two months of induction.

[0025] The present invention also provides the application of the culture medium for promoting the somatic embryo maturation of Masson pine in the cultivation of Masson pine.

[0026] The present invention also provides the application of the method for promoting the somatic embryo maturation of Masson pine in the cultivation of Masson pine.

[0027] Technical effects of the present invention: 1. Inoculating the embryogenic callus of *Pinus massoniana* into a somatic embryo maturation medium containing betaine, polyvinylpyrrolidone (PVP), or dimethylthiourea (DMTU) for somatic embryo induction culture can regulate the somatic embryo maturation rate of *Pinus massoniana*, efficiently promote somatic embryo maturation, and improve the somatic embryo induction efficiency.

[0028] The experimental results showed that morphological observation of embryogenic callus at different time points during somatic embryo induction revealed that most cotyledonary embryos matured by day 60, resulting in a large number of mature embryos. The number of mature somatic embryos was 250–3500 per gram of embryogenic callus, and the total number of embryos was 3333–9000 per gram of embryogenic callus. This indicates that the method of the present invention can efficiently promote somatic embryo maturation in Pinus massoniana, with high somatic embryo induction efficiency and good practicality.

[0029] 2. The optimal concentration of betaine for inducing somatic embryo maturation in Pinus massoniana in this invention is 0.5 g / L. After 60 days of dark culture at 24°C, the number of mature somatic embryos reaches 3500 per gram of embryogenic callus, and the total number of embryos reaches 9000 per gram of embryogenic callus. The embryogenic callus tissue is in good condition, the somatic embryos of Pinus massoniana mature earlier, and the development speed is significantly faster than other treatment groups. The somatic embryo induction efficiency is significantly higher than other treatment groups.

[0030] 3. The optimal concentration of PVP for inducing somatic embryo maturation in Pinus massoniana in this invention is 200 mg / L. After 60 days of dark culture at 24°C, it promotes the early maturation of somatic embryos in Pinus massoniana. The number of mature somatic embryos is as high as 250 per gram of embryogenic callus, and the total number of embryos is as high as 3333.33 per gram of embryogenic callus. The development speed is significantly faster than that of other treatment groups.

[0031] 4. The optimal concentration of DMTU for inducing immature embryos of Pinus massoniana in this invention is 0.1 mg / L. After 60 days of dark culture at 24°C, the total number of embryos reaches 4500 embryogenic callus per gram, and the development speed is significantly faster than that of other treatment groups.

[0032] 5. The optimal concentration of PVPP for inducing somatic embryo maturation in Pinus massoniana is 0 mg / L. After 60 days of dark culture at 24°C, the somatic embryos of Pinus massoniana mature earlier, with a total number of embryos as high as 3166.67 per gram of embryogenic callus, and the development speed is significantly faster than other treatment groups. Attached Figure Description

[0033] Figure 1 The preparation of the Y23-23-1 embryogenic callus cell line material of *Pinus massoniana* used in the embodiments of the present invention is shown in Figure 1 (A is the incompletely developed seeds taken from *Pinus massoniana* cones, and B is the induction of the Y23-23-1 embryogenic callus cell line material of *Pinus massoniana*). Figure 2The images (A-F) show the morphological observations and embryo counts (G-I) of *Pinus massoniana* embryogenic callus cultured for 60 days in somatic embryo maturation media supplemented with different concentrations (0 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L) of betaine. In Example 1 of this invention, G represents the number of mature somatic embryos, H represents the number of embryos of other morphologies, and I represents the total number of embryos. Different letters in the same image indicate significant differences between treatment groups. P <0.05, the presence of identical letters in the same graph indicates no significant difference between treatment groups. P >0.05); Figure 3 The images (A-D) and embryo count results (E-G) are shown for 60 days after culturing *Pinus massoniana* embryogenic callus in Example 2 of this invention in somatic embryo maturation media supplemented with different concentrations (0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) of PVP. E represents the number of mature somatic embryos, F represents the number of embryos with other morphologies, and G represents the total number of embryos. Different letters in the same image indicate significant differences between treatment groups. P <0.05, the presence of identical letters in the same graph indicates no significant difference between treatment groups. P >0.05); Figure 4 The images (A-D) show the morphological observations and embryo counts (E-G) of *Pinus massoniana* embryogenic callus cultured for 60 days in somatic embryo maturation media supplemented with different concentrations (0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L) of DMTU in Example 3 of this invention. (E represents the number of mature somatic embryos, F represents the number of embryos of other morphologies, and G represents the total number of embryos. Different letters in the same image indicate significant differences between treatment groups.) P <0.05, the presence of identical letters in the same graph indicates no significant difference between treatment groups. P >0.05); Figure 5 The images (A-D) and embryo count results (E-G) are shown for 60 days after culturing *Pinus massoniana* embryogenic callus in Example 4 of this invention in somatic embryo maturation media supplemented with different concentrations (0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) of PVPP. E represents the number of mature somatic embryos, F represents the number of embryos of other morphologies, and G represents the total number of embryos. Different letters in the same image indicate significant differences between treatment groups. P <0.05, the presence of identical letters in the same graph indicates no significant difference between treatment groups. P >0.05). Detailed Implementation

[0034] The above-mentioned solution will be further described below with reference to specific embodiments; it should be understood that these embodiments are used to illustrate the basic principles, main features and advantages of the present invention, and the present invention is not limited to the scope of the following embodiments; the implementation conditions used in the embodiments can be further adjusted according to specific requirements, and the implementation conditions not specified are usually the conditions in conventional experiments.

[0035] Unless otherwise specified in the following examples, all raw materials were prepared by conventional methods in the art.

[0036] The Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus used in the following examples was obtained by the applicant of this invention through induction using immature embryos of resistant *Pinus massoniana*. The specific method included: Collect pine cones and store them in a 4°C refrigerator for subsequent processing. Select incompletely developed seeds (see...). Figure 1 (A) Transfer to a clean bench for sterilization. First, disinfect with 75% alcohol for 30 seconds, then treat with 10% sodium hypochlorite for 10 minutes, rinse 5 times, and continuously stir to ensure complete contact. After drying with sterile filter paper, peel off the seed coat and inoculate the immature embryos into basal medium mLP (10 immature embryos per 20 mL basal medium mLP). Incubate in the dark at 24℃ to induce embryogenic callus. After two months of induction, the Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus is obtained. Its structure is compact and dense, with moderate viscosity, and is milky white and translucent (see...). Figure 1 (B in the middle).

[0037] The somatic embryo maturation medium used in the following examples has the following basic formulation: basic medium mLP: L-glutamine (Merck, Darmstadt, Germany) 450 mg / L, hydrolyzed casein (Merck, Darmstadt, Germany) 500 mg / L, inositol (Merck, Darmstadt, Germany) 500 mg / L, 2-(N-morpholine) ethanesulfonic acid monohydrate (Sangon Biotech, Shanghai, China) 250 mg / L, with the following added concentrations: maltose (Sangon Biotech, Shanghai, China) 30 g / L, ABA (Merck, Darmstadt, Germany) 9 mg / L, PEG 8000 (Merck, Darmstadt, Germany) 120 g / L, and gellan gum (Sangon Biotech, Shanghai, China) 4 g / L. Example 1

[0038] 1. Preparation of somatic embryo maturation medium containing betaine: Different concentrations (0 g / L, 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L) of betaine were added to the somatic embryo maturation medium to prepare a betaine-containing somatic embryo maturation medium.

[0039] 2. Methods to promote the maturation of pine embryos: The Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus was inoculated into somatic embryo maturation medium containing different concentrations of betaine for somatic embryo induction culture. At least 3 plates were inoculated for each concentration, and each plate contained 20 mL of medium and 0.1 g of embryogenic callus for somatic embryo induction. The cells were cultured in the dark at 24°C and subcultured every 30 days.

[0040] 3. Results Morphological observation of embryogenic callus at different time points during somatic embryo induction was conducted. In the Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus, after 25 days of culture on a somatic embryo maturation medium containing betaine, numerous spherical embryos began to appear on the callus surface; around 40 days, cotyledonary embryos appeared, and numerous spherical embryos continued to be produced; by 50 days of culture, some somatic embryos had matured, while the remaining embryoids continued to grow and develop; Figure 2 As shown in (A-F), most of the cotyledon embryos matured after 60 days of culture, and a large number of mature embryos appeared.

[0041] At 60 days after somatic embryonic maturation induction, somatic embryos at each developmental stage were counted as the total embryo number. The total embryo number refers to embryoids that are smooth, easily separable, and spherical, ellipsoidal, or cotyledon-shaped after the embryonic callus tissue was flattened and dispersed following somatic embryonic induction treatment. Somatic embryos that have developed into cotyledon-shaped embryos are classified as mature somatic embryos and counted as the number of mature somatic embryos. The remaining embryoids are immature embryos and counted as the number of embryos of other morphologies.

[0042] The results are as follows Figure 2 As shown in (G~I), betaine can promote the somatic embryo maturation of Pinus massoniana at a concentration not higher than 2.5 g / L. The optimal concentration for inducing somatic embryo maturation is 0.5~2.0 g / L. Especially at a concentration of 0.5 g / L, the number of mature somatic embryos is as high as 3500 per gram of embryogenic callus, and the total number of somatic embryos is as high as 9000 per gram of embryogenic callus. The embryogenic callus tissue is in good condition, and the somatic embryos of Pinus massoniana mature earlier and develop at a significantly faster rate than other treatment groups. The somatic embryo induction efficiency is significantly higher than other treatment groups.

[0043] In conclusion, adding an appropriate concentration of betaine can promote the formation of somatic embryos in Pinus massoniana, accelerate the development process of somatic embryos, and improve the efficiency of somatic embryo maturation. Example 2

[0044] 1. Preparation of PVP-containing somatic embryo maturation medium: Different concentrations (0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) of PVP were added to the somatic embryo maturation medium to prepare PVP-containing somatic embryo maturation medium.

[0045] 2. Methods to promote the maturation of pine embryos: The Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus was seeded into somatic embryo maturation medium containing different concentrations of PVP for somatic embryo induction culture. At least 3 plates were seeded for each concentration, and each plate contained 20 mL of medium and 0.1 g of embryogenic callus for somatic embryo induction. The cells were cultured in the dark at 24°C and subcultured every 30 days.

[0046] 3. Results The results are as follows Figure 3 As shown in (A-D), the Y23-23-1 cell line of pine embryogenic callus produced a large number of mature embryos after 60 days of culture in a medium containing PVP. Figure 3 As shown in (E-G), PVP can promote somatic embryo maturation of Pinus massoniana at a concentration not higher than 300 mg / L. The optimal concentration for inducing somatic embryo maturation is 100-200 mg / L. Especially at a concentration of 200 mg / L, the number of mature somatic embryos is as high as 250 per gram of embryogenic callus, and the total number of somatic embryos is as high as 3333.33 per gram of embryogenic callus. Most of the somatic embryos have matured, and the development speed is significantly faster than that of other treatment groups.

[0047] In conclusion, adding an appropriate concentration of PVP can promote the formation of somatic embryos in Pinus massoniana, accelerate the development process of somatic embryos, and improve the efficiency of somatic embryo maturation. Example 3

[0048] 1. Preparation of DMTU-containing somatic embryo maturation medium: Different concentrations (0 mg / L, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L) of DMTU were added to the somatic embryo maturation medium to prepare DMTU-containing somatic embryo maturation medium.

[0049] 2. Methods to promote the maturation of pine embryos: The Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus was seeded into somatic embryo maturation medium containing different concentrations of DMTU for somatic embryo induction culture. At least 3 dishes were seeded for each concentration, and each dish contained 20 mL of medium and 0.1 g of embryogenic callus for somatic embryo induction. The cells were cultured in the dark at 24°C and subcultured every 30 days.

[0050] 3. Results The results are as follows Figure 4As shown in (A-D), the Y23-23-1 cell line of pine embryogenic callus, when cultured in DMTU-containing medium for 60 days, produced a large number of spherical embryos. Figure 4 As shown in (E-G), DMTU can promote somatic embryo maturation in *Pinus massoniana* at concentrations not exceeding 0.3 mg / L. The optimal concentration for inducing somatic embryo maturation is 0.1–0.2 mg / L. Particularly at a concentration of 0.1 mg / L, the total number of somatic embryos induced over 60 days reached as high as 4500 per gram of embryogenic callus, and the somatic embryo development rate was significantly faster than in other treatment groups. In conclusion, adding an appropriate concentration of DMTU can promote somatic embryo formation in *Pinus massoniana*. Example 4

[0051] 1. Preparation of PVPP-containing somatic embryo maturation medium: Different concentrations (0 mg / L, 100 mg / L, 200 mg / L, 300 mg / L) of PVPP were added to the somatic embryo maturation medium to prepare PVPP-containing somatic embryo maturation medium.

[0052] 2. Methods to promote the maturation of pine embryos: The Y23-23-1 cell line material of *Pinus massoniana* embryogenic callus was seeded into somatic embryo maturation medium containing different concentrations of PVPP for somatic embryo induction culture. At least 3 plates were seeded for each concentration, and each plate contained 20 mL of medium and 0.1 g of embryogenic callus for somatic embryo induction. The cells were cultured in the dark at 24°C and subcultured every 30 days.

[0053] 3. Results Morphological observation of embryogenic callus at different time points during in vivo embryonic induction was performed. When the Y23-23-1 cell line of *Pinus massoniana* embryogenic callus was cultured on medium containing 0 mg / L PVPP for 45 days, spherical embryos began to appear on the surface of the callus; the results are as follows. Figure 5 As shown in (A-D), when cultured for 60 days, the embryos continue to develop and a large number of spherical embryos appear.

[0054] Somatic embryos at each developmental stage were statistically analyzed 60 days after somatic embryonic maturation induction, and the results are as follows: Figure 5 As shown in (E-G), the optimal concentration of PVPP was 0 mg / L, at which point the total number of somatic embryos was 3166.67 per gram of embryogenic callus. The embryogenic callus tissue was in good condition and its development rate was significantly faster than that of other treatment groups. In conclusion, the number of somatic embryos induced in *Pinus massoniana* on a PVPP-free medium was high.

[0055] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for promoting the somatic embryo maturation of Pinus massoniana, characterized in that: The culture medium includes a somatic embryo maturation medium, wherein betaine is added to the somatic embryo maturation medium; the somatic embryo maturation medium does not include cross-linked polyvinylpyrrolidone. The concentration of betaine in the somatic embryo maturation medium is 0.5–2.0 g / L; The formulation of the somatic embryo maturation culture medium is as follows: basic medium mLP, L-glutamine 400-500 mg / L, hydrolyzed casein 400-600 mg / L, inositol 400-600 mg / L, 2-(N-morpholine)ethanesulfonic acid monohydrate 200-300 mg / L, maltose 20-40 g / L, abscisic acid 8-10 mg / L, polyethylene glycol 100-150 g / L, and gellan gum 3-5 g / L.

2. The culture medium according to claim 1, characterized in that: The concentration of betaine in the embryo maturation medium is 0.5 g / L.

3. A method for promoting the maturation of pine embryos, characterized in that: Embryogenic callus from Pinus massoniana was inoculated into the culture medium described in any one of claims 1-2 for somatic embryo induction culture.

4. The method according to claim 3, characterized in that: The culture conditions for the pine embryogenic callus include at least one of the following: A. The inoculation amount of the embryogenic callus from *Pinus massoniana* is 4–20 g / L; B. Incubate at 20-28℃; C. Cultivate in the dark; D. Replacing every 20-40 days.

5. The method according to claim 3, characterized in that: The number of embryos was counted at 60 days of in vitro induction. The number of mature embryos was 250-3500 per gram of embryogenic callus, and the total number of embryos was 3333-9000 per gram of embryogenic callus.

6. The method according to claim 4, characterized in that: The culture conditions for the pine embryogenic callus include at least one of the following: A. The inoculation amount of the embryogenic callus from *Pinus massoniana* is 5 g / L; B. Incubate at 24℃; D. Replacing every 30 days.

7. The application of the culture medium of claim 1 or the method of claim 3 in the cultivation of Masson pine.

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