Method for stimulating differentiation of pinus sylvestris through symbiosis of explant and callus

Abstract

The invention belongs to the field of plant regeneration, and particularly relates to a method for stimulating pinus sylvestris differentiation through explant and callus symbiosis. The method comprises the following steps: culturing mature pinus sylvestris seeds into complete seedlings, and taking stems with leaves as explants; inoculating the explants on a primary culture medium for culturing, so that calluses appear on the explants; and performing subculture on the explant with the callus, and performing alternate culture by using a differential culture medium and an elongation culture medium in each round of subculture to form a bud primordium. The invention further provides a culture medium for explant and callus symbiosis to stimulate pinus sylvestris differentiation. The culture medium comprises a primary culture medium, a differential culture medium and an elongation culture medium. According to the method, symbiotic induced differentiation of the explants and the calluses is adopted, differentiation of the calluses induced by pinus sylvestris is achieved, and the calluses are stimulated to differentiate a large number of bud primordia through interaction of the original explants and the newly generated calluses.

Description

Technical Field

[0001] The invention belongs to the field of plant regeneration, and particularly relates to a method for stimulating differentiation of Pinus sylvestris var. mongolica through symbiosis of explants and callus tissues. Background Art

[0002] Scotch pine (Pinus sylvestris var. mongolica), belonging to the genus Pinus of the Pinaceae family, is a large tree that can grow up to 30m high and have a breast diameter of up to 1m. It is mainly distributed in the Greater Khingan Range area of ​​Heilongjiang Province, China, in the mountains with an altitude range of 400 to 900m, and in the dune areas of the west and south of Hailar. Scotch pine is known for its excellent drought resistance, cold resistance and ability to adapt to poor soil. It is an important shelterbelt tree species and "four sides" (i.e. roadsides, waterside, village side, and house side) greening tree species in Northeast China. It is not only suitable as a garden ornamental plant, but also an ideal tree species for urban landscaping. The wood of this tree is of high quality and has strong adaptability. It can adapt to different environmental conditions in the north. It has the characteristics of a well-developed root system, cold resistance, drought resistance, light-loving, adaptability to sandy soil, excellent material, strong soil adaptability, and fast growth.

[0003] However, the fruiting interval of Pinus sylvestris is long, the empty grain rate is high, sowing and seedling raising are subject to certain restrictions, and cutting and other technical difficulties are relatively large, making reproduction difficult. Tissue culture technology provides an effective way for the rapid propagation of improved varieties of Pinus sylvestris. Most of the tissue culture methods reported in the past were direct organogenesis, that is, the explants directly generate multiple buds without going through the callus stage; this method is rarely reported on Pinus sylvestris, and most of the research in this field uses pure callus culture for subsequent proliferation and differentiation processes, and there are no reports on differentiation using the symbiotic method of explants and callus.

[0004] In the indirect organogenesis of plant tissue culture, the explant first forms callus tissue, which is regenerated in the form of regenerated stems through the proliferation, differentiation and rooting of callus tissue (i.e., explant-callus-regenerated stem occurrence). This is the way most plants achieve in vitro regeneration in genetic transformation. In this process, callus cells are omnipotent and can differentiate into regenerated buds after induction and proliferation. In the complete regeneration system, callus differentiation is the most difficult step. In order to break through the differentiation technology of Pinus sylvestris, predecessors have made a lot of efforts, but there has been no obvious effect in the differentiation of Pinus sylvestris.

[0005] For example, immature endosperm of Pinus sylvestris was used as explant, and callus was induced by heat shock, light culture, dark culture and endosperm wounding, but the callus failed to differentiate and sprout (Qi Liwang et al., 1996); embryonic callus was induced by mature embryos, and mature cotyledon embryos were differentiated, but the differentiation rate was low (Liang Yan et al., 2014); young needles of Pinus sylvestris were used as explants, and only dark green callus was proliferated without differentiation (Lu Peng, 2020); mature embryos of Pinus sylvestris were used as explants, and only callus formation was induced (Chirva et al., 2023). The reasons include different degrees of explant damage, insufficient nutrients, phytohormone imbalance, blocked callus differentiation, and lack of appropriate external stimuli (light, temperature and humidity), etc. These reasons may interact with each other and affect the differentiation of Pinus sylvestris callus. CN104186324B discloses a method for inducing somatic embryos of mature zygotic embryos of Pinus sylvestris. Callus proliferation culture successfully induced somatic embryos of Pinus sylvestris in a culture medium supplemented with abscisic acid, but no regenerated stems were differentiated.

[0006] In addition, the explants used in the above method for promoting differentiation of Pinus sylvestris var. mongolica are immature zygotic embryos, mature zygotic embryos, immature endosperms and young and tender tissues of the plant. Among them, the immature zygotic embryos, immature endosperms and young and tender tissues of the plant are restricted by seasons and need to be collected at a specific time, and the collection is difficult. All of the above explants can induce callus tissue, but it is difficult to differentiate the callus tissue into adventitious buds, which is still a key problem in the tissue culture of Pinus sylvestris var. mongolica. Moreover, the callus tissue will eventually turn brown, and the browned callus tissue cannot continue to differentiate. Summary of the invention

[0007] In order to solve the above technical problems, the first aspect of the present invention provides a method for symbiotically stimulating differentiation of Pinus sylvestris var. mongolica by explant and callus tissue, comprising the following steps:

[0008] (1) taking mature seeds of Pinus sylvestris var. mongolica, sterilizing the seeds and culturing complete seedlings, and taking stem segments with cotyledons as explants;

[0009] (2) sterilizing the explants of step (1) and inoculating them on a primary culture medium under sterile conditions to allow callus to form on the explants, wherein the primary culture medium comprises: a basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1-3 mg / L 2,4-D, 1-3 mg / L 6-BA, 1-3 mg / L IBA, 1-3 mg / L NAA, and 1-3 mg / L KT;

[0010] (3) subculturing the explants from which callus tissue has emerged in step (2), wherein each round of subculture is cultured alternately using a differentiation medium and an elongation medium;

[0011] The differentiation medium comprises: a basal medium MS medium, 2.0 mg / L 6-BA, 1.0 mg / L NAA, 6 g / L agar, and 20 g / L sucrose; the elongation medium comprises: a basal medium of 1.18 g / L MS+2.14 g / L WPM, 1.0 mg / L IBA, 6 g / L agar+20 g / L sucrose;

[0012] The explants that have callus tissue and are growing robustly in step (2) are treated as follows: the bottom callus is removed in each round of subculture, and the stem is continuously shortened until the cotyledon part contacts the culture medium, callus tissue is formed at the stem tip, and multiple bud primordia are formed; or the explants that have callus tissue and are growing poorly in step (2) are treated as follows: the bottom callus is continuously expanded and browned, and finally bud primordia are formed on the browned callus tissue.

[0013] Preferably, the primary culture medium comprises: basal culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1 mg / L 2,4-D, 2 mg / L 6-BA, 1 mg / L IBA, 1 mg / L NAA, and 3 mg / L KT.

[0014] In some embodiments, in step (1), the seeds are sterilized using a 3% CuSO4 solution for 1 to 3 hours.

[0015] In some embodiments, in step (2), the step of sterilizing the explant is as follows: sterilize the explant with 75% alcohol for 10s to 60s, rinse with sterile water for multiple times, sterilize with 2% NaClO for 5 to 20min, and then rinse with sterile water for multiple times.

[0016] In some embodiments, the culture conditions for the primary culture and the secondary culture are: in a constant temperature incubator, light culture is carried out, light 16h / darkness 8h, light intensity 2000-3000 lux, temperature 25±2°C, relative humidity 70%-80%.

[0017] In some embodiments, the primary culture time is 20-30 days.

[0018] In some embodiments, each round of subculture is 30-50 days in differentiation medium and 30-50 days in elongation medium.

[0019] In some embodiments, the subculture is cultured for 6-8 rounds.

[0020] The second aspect of the present invention also provides a culture medium for symbiotically stimulating differentiation of Pinus sylvestris var. mongolica by explants and callus tissues, comprising a primary culture medium, a differentiation culture medium and an elongation culture medium, wherein the primary culture medium is used to culture callus tissues from explants, and the differentiation culture medium and the elongation culture medium are cultured alternately to culture bud primordia from explants with callus tissues;

[0021] The primary culture medium comprises: a basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1-3 mg / L 2,4-D, 1-3 mg / L 6-BA, 1-3 mg / L IBA, 1-3 mg / L NAA, and 1-3 mg / L KT;

[0022] The differentiation medium comprises: a basic medium MS medium, 2.0 mg / L 6-BA, 1.0 mg / L NAA, 6 g / L agar, and 20 g / L sucrose;

[0023] The elongation medium comprises: a basic medium of 1.18 g / LMS+2.14 g / LWPM, 1.0 mg / L IBA, and 6 g / L agar+20 g / L sucrose.

[0024] Preferably, the primary culture medium comprises: basal culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1 mg / L 2,4-D, 2 mg / L 6-BA, 1 mg / L IBA, 1 mg / L NAA, and 3 mg / L KT.

[0025] Compared with the prior art, the beneficial effects of the present invention include at least:

[0026] 1. The present invention adopts the symbiotic induction differentiation of explants and callus tissues to achieve the differentiation of callus tissues induced by Pinus sylvestris var. mongolica. The original explants and the newly generated callus tissues are interacted to stimulate the callus tissues to differentiate into a large number of bud primordia.

[0027] 2. The present invention provides a new method for inducing callus tissue. Different from the traditional method of directly inducing callus tissue using various explants, the present invention uses stem segments with cotyledons to induce callus tissue at the root of the stem segments.

[0028] 3. Different from the existing teachings of avoiding or reducing the browning of callus tissue as much as possible, the present invention successfully induced the differentiation of shoot primordia from the browned callus tissue.

[0029] 4. The primary culture medium selected by the present invention can induce callus tissue at the root of the stem segment and proliferate it in large quantities. The provided differentiation culture medium and elongation culture medium can cause the callus tissue to differentiate into bud primordia by alternating cycles. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the process of cultivating explants of Pinus sylvestris var. mongolica stem segments. Figure 1 In the middle, a is the mature seed state. Figure 1 Middle b is the seedling state.

[0031] Figure 2 This is the state of the cotyledonary stem segment of Larix gmelinii after being disinfected and inoculated.

[0032] Figure 3 This is the condition of the plants after primary culture.

[0033] Figure 4 It is the formation of bud primordium after succession of healthy plants.

[0034] Figure 5 The callus tissue at the roots of weak seedlings continues to proliferate and turn brown.

[0035] Figure 6 The brown callus differentiates into bud primordia. Figure 6 Middle b shows a large number of buds capable of forming bud primordia in the brown callus tissue observed under a microscope.

[0036] Figure 7 The results of SOD determination in callus induced by Pinus sylvestris var. mongolica are shown in Figure 2. Figure 7 A in the middle is embryo-induced callus; Figure 7 B in the middle is the callus tissue induced from a healthy plant. Figure 7 Center C is the differentiated bud primordium.

[0037] Figure 8 The results of SS determination of callus induced by Pinus sylvestris var. mongolica are shown in Figure 1. Figure 8 A in the middle is embryo-induced callus tissue, Figure 8 B in the middle is the bud primordium, Figure 8 Center C is the callus tissue induced from a healthy plant.

[0038] Fig. 9 The process of separating the callus and the original plant from the differentiated bud primordium. Fig. 9 Figure b in the middle is the callus tissue with bud primordium after separation. Fig. 9 Middle c is a local enlargement. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0040] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0041] The following abbreviations are used in the culture medium:

[0042] Polyvinyl pyrrolidone (PVP), 2,4-dichlorophenoxyacetic acid (2,4-D), 6-Benzylaminopurine (6-BA), 3-Indolebutyric acid (IBA), 1-naphthlcetic acid (NAA), Indole acetic acid (IAA), Kinetin (KT).

[0043] Example 1: Cultivation and sterilization of explants

[0044] The basic material is selected from mature seeds of Pinus sylvestris var. mongolica in Gannan County, Heilongjiang Province, which are full of grains and uniform in size. They are sterilized with 3% CuSO4 solution for 1 hour, rinsed with clean water, and soaked in distilled water for 24 hours. After the seeds are sterilized, they are stored in a refrigerator at 4°C for later use. The sterilized seeds are placed in a warm and humid environment to cultivate complete seedlings (such as Figure 1 As shown), the stem segments with cotyledons were taken as explants.

[0045] Disinfect the workbench with ultraviolet light for 20 minutes, disinfect the stem segments with cotyledons with 75% alcohol for 30 seconds, and rinse with sterile water 3 to 4 times. Disinfect with 2% NaClO for 10 minutes, rinse with sterile water 3 to 4 times, take out for use, and finally inoculate the explants on the prepared culture medium (such as Figure 2 shown).

[0046] Example 2: Optimization of primary culture medium and primary culture

[0047] Culture conditions: The experiment was carried out in a constant temperature incubator under light culture, with 16h light / 8h dark, light intensity of 2000-3000 lux, temperature of 25±2℃, and relative humidity of 70%-80%.

[0048] The basic culture medium was DCR + 6g / L agar + 20g / L sucrose + 1g / L inositol + 0.3g / LPVP, and different types and concentrations of hormones were added. A five-factor four-level orthogonal test was used (Table 1). Four cotyledon stem segments were inoculated in each bottle, and each scheme was repeated four times. 20 bottles were cultured each time and placed in a constant temperature of 23±2℃. The growth rate and growth state of the callus were observed, and the callus induction rate was calculated after 14 days.

[0049] Table 1: Hormone concentration gradient settings and results for primary culture

[0050]

[0051] Through variance analysis, it was found that the five hormones had significant effects on it. According to the callus induction rate, it can be seen that when the concentration of the five hormones is 1-3 mg / L, a good induction rate can be obtained. Through range analysis, it was found that the optimal level of each hormone was 1.0 mg / L2,4-D, 2.0 mg / L 6-BA, 1.0 mg / L IBA, 1.0 mg / L NAA, 3.0 mg / L KT. The optimal level was taken as the final primary culture medium. The explants were cultured using the primary culture medium at the optimal level. After one week, callus appeared in the part of the plant stem that was in contact with the culture medium, showing two conditions ( Figure 3 a), a strong plant ( Figure 3 b), a weak strain with poor growth ( Figure 3 c) and classify them.

[0052] Example 3: Subculture medium optimization

[0053] Culture conditions: The experiment was carried out in a constant temperature incubator, with light culture, 16 hours of light / 8 hours of darkness, light intensity of 2000-3000 lux, temperature of 23±2℃, and relative humidity of 70%-80%. Subculture includes differentiation culture and elongation culture.

[0054] 1) Optimization of differentiation culture and elongation culture medium

[0055] The mongolica pine stem segments induced with callus tissue in primary culture were placed in subculture medium for cultivation.

[0056] In the differentiation medium, MS was designed as the basal medium, and 1.0 mg / L, 2.0 mg / L, 3.0 mg / L 6-BA and 1.0 mg / L NAA were added, and 0.3 g / L PVP, 6 g / L agar, and 20 g / L sucrose were added (Table 2). The time of emergence of bud primordia, the number of bud primordia, and the volume of callus produced at the hypocotyl were counted.

[0057] The bud primordium explants that grew well after differentiation culture were subjected to elongation culture. In the elongation medium, 1.18g / LMS+2.14g / LWPM was used as the basic medium, and 1.0mg / L IBA or 1.0mg / L2,3,5-Triiodobenzoicacid (TIBA) (Table 2) was added. 0.3g / L PVP, 6g / L agar, and 20g / L sucrose were also added.

[0058] Table 2: Hormone concentration gradient settings for subculture

[0059]

[0060] 2) Analysis of the results of bud primordium induction and elongation As shown in Table 3, in the primary culture stage, when no hormones were added to the culture medium, there was no obvious change in the explants, no bud primordium was produced, only the cotyledon part was elongated, and the color was mostly yellow-green. When cytokinin (6-BA) and auxin (NAA) were added to the culture medium, bud primordium appeared. As the ratio of the two increased, the average time for the appearance of bud primordium gradually shortened, and the results after one-way ANOVA showed significance (F=5.116, p=0.037*). The number of bud primordium gradually increased, and the results after one-way ANOVA showed significance (F=6.808, p=0.008**), so the bud primordium was affected by hormone changes. Although there were differences in the volume of callus produced at the hypocotyl, the results after one-way ANOVA did not show significance (F=2.041, p=0.167). Because a higher ratio (3:1) stunted the growth and development of explants and accelerated needle wilting, a ratio of 2:1 was determined to be the ideal hormone ratio for the differentiation culture stage.

[0061] Table 3: Effects of 6-BA and NAA on the growth status of explants

[0062]

[0063] As shown in Table 4, in the elongation culture, the bud primordium formed in the induction stage was used as an explant, which could elongate and grow needles in the culture medium supplemented with 1 mg / LIBA. The average number of needles (F=1.445, p=0.265) and the average needle length (F=3.728, p=0.047*) were negatively correlated with the 6-BA / NAA ratio in the induction period. When the primary culture medium was determined to be the same, the growth status of IBA and TIBA explants was compared. In the case of explants using TIBA without adding exogenous hormones and inhibiting endogenous hormones, the bud primordium could not grow into needles, while the explants using IBA could grow into needles (average length of 6.43 cm) (Table 4). In addition, explants without the addition of 6-BA and NAA hormones could not grow into needles because the bud primordium was not induced and only the elongation of the original cotyledons was maintained.

[0064] Based on the above results, the differentiation medium was determined to be: MS+2.0mg / L 6-BA+1.0mg / L NAA+6g / L agar+20g / L sucrose; the elongation medium was determined to be: 1 / 4MS+WPM+1.0mg / LIBA+6g / L agar+20g / L sucrose.

[0065] Table 4: Effect of IBA on explant growth status

[0066]

[0067] Example 4: Subculture of the plant from Example 2

[0068] Culture conditions: The experiment was carried out in a constant temperature incubator under light culture, with 16h light / 8h dark, light intensity of 2000-3000 lux, temperature of 25±2℃, and relative humidity of 70%-80%.

[0069] The robust plants of Example 2 were subcultured, and the differentiation medium was: MS + 2.0 mg / L 6-BA + 1.0 mg / L NAA + 6 g / L agar + 20 g / L sucrose; the elongation medium was: 1 / 4 MS + WPM + 1.0 mg / LI BA + 6 g / L agar + 20 g / L sucrose. The bottom callus was removed at each subculture, and the stem was continuously shortened until the cotyledon part was in contact with the culture medium ( Figure 4 In a), callus tissue forms at the stem tip, and multiple bud primordia are formed during the continuous conversion of differentiation medium and elongation medium ( Figure 4 (b)

[0070] The weak strain of Example 2 was subcultured to cultivate callus tissue, and was also cultured in differentiation medium and elongation medium. The weak strain was subcultured together with the callus tissue formed at its root, and the bottom callus continued to proliferate and browned ( Figure 5 ), and finally formed bud primordia on the brown callus ( Figure 6 ), during which the mother plant gradually dies, but provides the necessary nutrients for the bud primordium.

[0071] Example 5: Intrinsic mechanism of callus differentiation

[0072] In order to improve differentiation, the internal mechanism of explants was explored. The superoxide dismutase (SOD) and soluble sugar (SS) were measured in embryo-induced callus, callus induced by healthy plants, and differentiated bud primordia to explore the internal mechanism of callus differentiation.

[0073] Antioxidant system activity assay:

[0074] The results of variance analysis in this study show that ( Figure 7), there were significant differences in SOD activity (F=8.038, p=0.020**) in the three stages of explants A, B, and C. The specific activity comparison was: SOD activity, C (102.9U / g)>B (56.86U / g)>A (41.65U / g), that is, the differentiated bud primordium>the callus induced by healthy plants>the callus induced by embryos. Since the increase of SOD activity is conducive to the differentiation of callus, the SOD activity of the callus induced by healthy plants is higher than that of the callus induced by embryos, which is closer to the activity of the bud primordium, so the symbiosis between plants and callus is easier to stimulate differentiation.

[0075] Determination of soluble sugar (SS):

[0076] According to existing research, embryo development requires a high level of soluble sugar. In the study of soluble sugar in the process of callus differentiation of Pinus sylvestris var. mongolica, the soluble sugar content of the three treatments A, B, and C showed significant differences (F=27.352, p=0.000**). The specific comparison is ( Figure 8 ): Soluble sugar content, B (77.12 mg / g)>C (21.32 mg / g)>A (12.39 mg / g), that is, callus induced by healthy plants>bud primordium>embryo-induced callus. The soluble sugar content produced by the interaction between healthy plants and callus is six times that of the soluble sugar content in embryo-induced callus. Since the differentiation of bud primordium requires the accumulation of soluble sugar, healthy plants provide a large amount of soluble sugar to the callus symbiotic with it, thereby stimulating the differentiation of callus into bud primordium, while the soluble sugar content in embryo-induced callus is low and does not reach the soluble sugar content required for differentiation, so it cannot differentiate. After the differentiation of callus induced by healthy plants, the soluble sugar content in the bud primordium stage also decreases accordingly due to the consumption of nutrients in the callus, but it is still higher than the soluble sugar content in the embryo-induced callus.

[0077] Comparative Example 1: Callus Isolation and Culture

[0078] The stem segments of Pinus sylvestris seedlings were used as explants and inserted vertically into the culture medium. The callus formed near the explants was taken out for differentiation culture. It was found that the plants grew well in the late culture period, and new callus began to grow around the contact between the explants and the culture medium. Through the continuous accumulation of callus, bud primordia reappeared under light conditions. The newly generated callus and differentiated bud primordia were taken out and cultured separately ( Fig. 9 ), after 20 days, the growth state of the bud primordium is not as good as before removal, and it cannot be well preserved, and the callus tissue no longer differentiates into new bud primordium. Therefore, callus tissue cannot differentiate into bud primordium alone, and the original explant needs to be symbiotic with it to stimulate callus differentiation.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for stimulating differentiation of Pinus sylvestris var. mongolica by symbiosis of explant and callus, characterized in that: The steps include: (1) taking mature seeds of Pinus sylvestris var. mongolica, sterilizing the seeds and culturing complete seedlings, and taking stem segments with cotyledons as explants; (2) sterilizing the explants of step (1) and inoculating them on a primary culture medium under sterile conditions to allow callus to form on the explants, wherein the primary culture medium comprises: a basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1-3 mg / L 2,4-D, 1-3 mg / L 6-BA, 1-3 mg / L IBA, 1-3 mg / L NAA, and 1-3 mg / L KT; (3) subculturing the explants from which callus tissue has emerged in step (2), wherein each round of subculture is cultured alternately using a differentiation medium and an elongation medium; The differentiation medium comprises: a basic medium MS medium, 2.0 mg / L 6-BA, 1.0 mg / L NAA, 6 g / L agar, and 20 g / L sucrose; the elongation medium comprises: a basic medium prepared with 1.18 g / L MS + 2.14 g / L WPM, 1.0 mg / L IBA, 6 g / L agar + 20 g / L sucrose; The explants that have callus tissue and are growing robustly in step (2) are treated as follows: the bottom callus is removed in each round of subculture, and the stem is continuously shortened until the cotyledon part contacts the culture medium, callus tissue is formed at the stem tip, and multiple bud primordia are formed; or the explants that have callus tissue and are growing poorly in step (2) are treated as follows: the bottom callus is continuously expanded and browned, and finally bud primordia are formed on the browned callus tissue.

2. The method according to claim 1, characterized in that The primary culture medium comprises: basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1 mg / L 2,4-D, 2 mg / L 6-BA, 1 mg / L IBA, 1 mg / L NAA, and 3 mg / L KT.

3. The method according to claim 1, characterized in that In step (1), the seeds are disinfected using a 3% CuSO4 solution for 1 to 3 hours.

4. The method according to claim 1, characterized in that In step (2), the step of explant disinfection is as follows: the explant is disinfected with 75% alcohol for 10s to 60s, rinsed with sterile water for multiple times, then disinfected with 2% NaClO for 5 to 20min, and then rinsed with sterile water for multiple times.

5. The method according to claim 1, characterized in that The culture conditions for the primary culture and the secondary culture are: in a constant temperature incubator, light culture is carried out with 16 hours of light / 8 hours of darkness, light intensity of 2000-3000 lux, temperature of 23±2° C., and relative humidity of 70%-80%.

6. A culture medium for symbiotically stimulating differentiation of Pinus sylvestris var. mongolica, characterized in that: It comprises a primary culture medium, a differentiation culture medium and an elongation culture medium, wherein the primary culture medium is used to culture callus tissue from explants, and the differentiation culture medium and the elongation culture medium are cultured alternately to culture bud primordia from explants with callus tissue; The primary culture medium comprises: a basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1-3 mg / L 2,4-D, 1-3 mg / L 6-BA, 1-3 mg / L IBA, 1-3 mg / L NAA, and 1-3 mg / L KT; The differentiation medium comprises: a basic medium MS medium, 2.0 mg / L 6-BA, 1.0 mg / L NAA, 6 g / L agar, and 20 g / L sucrose; The elongation medium comprises: a basic medium of 1.18 g / LMS+2.14 g / LWPM, 1.0 mg / L IBA, 6 g / L agar, and 20 g / L sucrose.

7. The culture medium according to claim 6, characterized in that The primary culture medium comprises: basic culture medium DCR medium, 6 g / L agar, 20 g / L sucrose, 1 g / L inositol, 0.3 g / L PVP, 1 mg / L 2,4-D, 2 mg / L 6-BA, 1 mg / L IBA, 1 mg / L NAA, and 3 mg / L KT.

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