Efficient regeneration method with larix gmelinae cotyledons as explants

Abstract

The invention relates to a high-efficiency regeneration method taking larch cotyledons as explants. The method comprises the following steps: sowing, disinfecting, carrying out induction culture, carrying out elongation culture, carrying out rooting culture, hardening seedlings and transplanting. According to the technical scheme provided by the invention, the culture medium with a specific formula is adopted, so that the larch in North China shows the remarkable advantages of high differentiation efficiency, large multiplication coefficient and good rooting rate in the propagation process. In addition, the method has the advantages of being short in plant growth cycle, high in adaptability after transplanting and the like. And the cultivated seedlings have the characteristics of being robust, tall and straight, good in growth vigor, tidy and consistent. According to the technical scheme, the seedling culture period is remarkably shortened, and the production cost is effectively reduced. By establishing an adventitious bud induction system with larch cotyledons as a starting material and successfully obtaining regenerated plants, a new technical approach is provided for rapid propagation of larch. Meanwhile, the invention also lays a foundation for establishing an efficient Larix hupehensis transgenic system in the future.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to a high-efficiency regeneration method using cotyledons of Larix principis-rupprechtii as explants. Background Art

[0002] Larix gmelinii var. principis-rupprechtii (Mayr) Pilger belongs to the Pinaceae family and Larix genus. It is a unique larch variety in North China. It is mainly distributed in the high mountainous areas of North China, such as Taihang Mountain, Luliang Mountain and Wutai Mountain, with an altitude range of 1,400 to 2,800 meters. It is the main forest tree species in the alpine coniferous forest belt in North China and plays an important role in forestry and ecosystem. Larix gmelinii has many unique characteristics: strong adaptability, cold resistance, drought resistance, and barrenness resistance, and can adapt to the harsh environment of the high mountainous areas of North China; rapid growth, with an annual growth of more than 1 meter under suitable conditions; beautiful tree shape, conical, flat branches, soft needles, yellowing in autumn, with high ornamental value; excellent material, dense wood, and corrosion resistance, it is a high-quality building and furniture material. Larix principis-rupprechtii is not only an important forestry resource with high ecological benefits and economic value, but also can be used in many fields: it can be used as excellent materials for bridges, vehicles, buildings, etc.; it can be used for forest renewal and afforestation of barren hills in the distribution area and the alpine areas of the Yellow River Basin and the upper reaches of the Liaohe River; it is an excellent garden greening tree species, especially suitable for landscape design in northern regions; it can also be used to make larch arabinogalactan, which has the effect of enhancing immunity and anti-tumor. The research on genetic improvement of larch started relatively early, and many excellent larch varieties have been screened out through conventional breeding methods. Larch has problems such as long growth cycle and difficult genetic manipulation, and it is more affected by natural conditions. Compared with conventional breeding, genetic engineering breeding is more efficient and more accurate, and is the main trend of genetic improvement research. At present, there are few reports on the gene function of larch in China, and the research on genetic transformation and gene editing has been slow. The main reason is the lack of a good larch regeneration system. Therefore, the establishment of efficient and stable larch rapid propagation technology is the basic premise for genetic engineering improvement of larch.

[0003] The in vitro culture and genetic transformation system of conifers is an important research direction in the field of forestry biotechnology, which is of great significance to the genetic improvement and rapid propagation of Larix principis-rupprechtii. As a kind of conifer, Larix principis-rupprechtii faces unique challenges and opportunities in in vitro culture due to its special biological characteristics. The research on in vitro culture technology of Pinaceae species began in the 1970s. Since then, this field has received extensive attention and made continuous breakthroughs. This shows that the in vitro culture technology of conifers, including Larix principis-rupprechtii, has a long research history and has been continuously improved and developed in this process. As an important economic and ecological tree species, the research on in vitro culture and plant regeneration technology of Larix principis-rupprechtii has always been one of the difficult problems to solve in plant tissue culture research. With the increasing attention paid to asexual forestry, countries around the world have paid special attention to the tissue culture of conifers, including Larix principis-rupprechtii. In the past 20 years of development, the in vitro culture of conifers such as Larix principis-rupprechtii has expanded from tissue and organ culture to cell and protoplast culture, and has gradually realized the transformation from theoretical research to application. These advances provide important technical support for the genetic improvement and rapid propagation of Larix principis-rupprechtii, and help improve its application value in forestry production and ecological construction. However, due to the complexity of its growth characteristics and genetic background, its in vitro culture technology still faces many challenges and requires further research and breakthroughs.

[0004] The research progress of somatic embryogenesis technology of conifers has important reference value for the in vitro culture and genetic improvement of Larix principis-rupprechtii. The breakthrough in this field began in 1976, when Durzan and Chalupa first reported the embryo-like structure of Pinus banksiana in vitro culture. In 1985, researchers successfully induced somatic embryos from immature embryos of Norway spruce (Picea abies) and obtained regenerated plants. This achievement opened up a new direction for the study of in vitro embryogenesis of conifers including Larix principis-rupprechtii. Since then, research in this field has rapidly emerged and made significant progress. At present, more than 40 species of conifers have been cultured to produce somatic embryos in this way, mainly concentrated in the genus Picea and Pinus. Although Larix principis-rupprechtii belongs to the genus Larix, these research results provide valuable experience and methods for the development of its somatic embryogenesis technology. By learning from and improving these technologies, researchers are expected to make breakthroughs in the somatic embryogenesis of Larix principis-rupprechtii, thereby providing a new way for its rapid reproduction and genetic improvement. This will not only improve the reproduction efficiency of Larix principis-rupprechtii, but also provide important experimental materials and technical support for research on its stress resistance improvement and wood quality enhancement, further enhancing its application value in forestry production and ecological construction.

[0005] In terms of in vitro regeneration of Larix principis-rupprechtii, the Center for Excellence in Molecular Plant Sciences of the Chinese Academy of Sciences has disclosed an efficient in vitro regeneration method for conifers. This method uses the thick conifers of Larix principis-rupprechtii as the starting material and can quickly produce a large number of adventitious buds and regenerated plants. The needles of Larix principis-rupprechtii are usually linear, 2 to 3 cm long, clustered on short branches, with 20 to 40 needles per cluster. This special needle structure provides a good basis for in vitro regeneration. This technology not only maintains the genetic phenotype of the parent, but can also be used as a receptor system for transgenics to prepare transgenic plants. It is worth noting that Larix principis-rupprechtii has strong cold and drought resistance. These excellent characteristics are retained during the in vitro regeneration process, providing favorable conditions for subsequent genetic improvement. This discovery is of great significance for the research on genetic improvement of conifers, cultivation of new varieties and propagation of high-quality seedlings, and has a far-reaching impact on the sustainable development of the forestry economy and narrowing the gap with developed countries in the field of basic forestry research. In addition, as Larix principis-rupprechtii is an important timber species and ecological protection tree species in my country, the breakthrough in its efficient in vitro regeneration technology has provided new technical support for the large-scale breeding and ecological restoration projects of this species.

[0006] The results of the present invention not only demonstrate the powerful regeneration ability of Larix principis-rupprechtii, but also make full use of its excellent genetic characteristics, such as strong cold and drought resistance. More importantly, the adventitious buds formed by this cotyledon regeneration system show great application potential: they are not only suitable for gene gun-mediated genetic transformation, but also for Agrobacterium-mediated genetic transformation system. This versatility has opened up broad prospects for the genetic improvement and new variety breeding of Larix principis-rupprechtii. Considering the important position of Larix principis-rupprechtii in my country's forestry economy and ecological construction, the breakthrough of this technology will undoubtedly promote the rapid development of my country in the fields of conifer genetic improvement, high-quality seedling propagation and ecological restoration projects, help narrow the gap with developed countries in basic forestry research, and provide strong support for the sustainable development of the forestry economy. Summary of the invention

[0007] The present invention aims to develop an efficient regeneration method using cotyledons of Larix principis-rupprechtii as explants. The method has the following advantages: convenient material acquisition, high cost-effectiveness, simplified operation process, fast propagation rate, and high propagation coefficient. The establishment of the method can provide an efficient regeneration system support for the genetic transformation research of Larix principis-rupprechtii.

[0008] The technical solution adopted by the present invention is as follows: An efficient regeneration method using Larix principis-rupprechtii cotyledons as explants comprises the following steps: S1: Sowing: Select mature and full seeds of Larix principis-rupprechtii, soak them in warm water for 24 hours and then sow them in the substrate. Take the seedlings that have germinated for 17 days and are in good growth condition as the materials for the next experiment; S2: Disinfection: After removing the seed shell and roots of the seedlings obtained in step S1, disinfection is performed to obtain disinfected seedlings; S3: Induction culture: cut the cotyledons of the seedlings disinfected in step S2 with a sterile blade in a clean bench, inoculate them into an adventitious bud induction medium for pre-culture, and determine whether there is bacterial or fungal contamination after 3 to 5 days. If no colonies appear on the culture medium, it indicates that the sample is not contaminated; take the uncontaminated cotyledons as the starting explants for subsequent experiments, and culture them in a new adventitious bud induction medium for 35 to 50 days to obtain adventitious buds; S4: elongation culture: the adventitious buds from step S3 are inoculated into an adventitious bud elongation medium and cultured for 30-50 days to obtain elongated adventitious buds; S5: Rooting culture: The adventitious buds elongated in step S4 are inoculated into a rooting medium and cultured for 15 to 30 days to obtain rooted seedlings; Among them, the formula of adventitious bud induction medium is: DCR+1.5mg / L 6-BA+0.01mg / LTDZ; The formula of adventitious bud elongation medium is: DCR+1.5mg / L 6BA+0.01mg / L TDZ+0.2g / L activated carbon; The formula of rooting medium is: DCR+0.05mg / LNAA+0.2g / L activated carbon.

[0009] Furthermore, in the above step S1, the matrix is ​​prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1.

[0010] Furthermore, in the above step S2, the disinfection method is: rinse with running water for 2 to 3 hours, then rinse with sterile water twice, then disinfect with 75% alcohol for 1 minute, then disinfect with 8% sodium hypochlorite for 6 minutes, and then rinse with sterile water for 3 to 5 times.

[0011] Furthermore, in the above step S3, the pre-culture conditions are: temperature 25°C, light rate 95 μmol·m -2 ·s -1 , light duration 16h / d, culture conditions: temperature 25℃, light rate 95μmol·m -2 ·s -1 , lighting time: 16h / d.

[0012] Furthermore, in the above step S4, the culture conditions are: temperature 25°C, light rate 95 μmol·m -2 ·s -1 , lighting time: 16h / d.

[0013] Furthermore, in the above step S5, the culture conditions are: temperature 25°C, light rate 95 μmol·m -2·s -1 , lighting time: 16h / d.

[0014] Furthermore, after the above step S5 is completed, the process further includes step S6: seedling hardening and transplanting.

[0015] The significant advantages of the present invention are: (1) Innovation in explant selection: The present invention selects cotyledons of Larix principis-rupprechtii seedlings germinated in a greenhouse environment as the explant source. This selection not only simplifies the material collection process, but also overcomes the limitations of time and quantity. In addition, the method also has significant advantages of saving time and labor, and lays a foundation for large-scale propagation.

[0016] (2) Specificity of culture medium formulation: The present invention provides the required specific hormone combination for the explants at different growth stages through a carefully designed culture medium formula. This customized culture system creates ideal conditions for rooting and seedling growth, effectively promotes the healthy growth of seedlings, and finally successfully obtains regenerated plants of Larix principis-rupprechtii.

[0017] (3) Practicality and efficiency of the method: The cultivation method described in the present invention has many advantages: convenient material acquisition, reduced experimental costs; simplified operation process, easy to promote and apply; fast propagation speed, significantly improved breeding efficiency; high breeding coefficient, achieved a substantial increase in yield.

[0018] (4) Broad application prospects: The regeneration system established by the present invention is not only suitable for conventional reproduction, but also provides an ideal platform for genetic transformation research of Larix principis-rupprechtii. The development of this technology has laid a solid foundation for future genetic improvement and new variety breeding, and is expected to promote the further development of forestry biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : The process of adventitious bud formation in Larix principis-rupprechtii cotyledons. A: Cotyledons just inoculated; B: Cotyledons 10-15 days after inoculation; C: Cotyledons 20-23 days after inoculation; D, E: Cotyledons 28-42 days after inoculation; F: Cotyledons 50 days after inoculation. The scale bar in the figure is 1 cm.

[0020] Figure 2 :Paraffin sections of adventitious bud formation in Larix principis-rupprechtii cotyledons. A: Freshly inoculated cotyledons; B: meristem formation; CE: bud primordium emergence; F: adventitious bud formation. The scale bar in the figure is 100 μm.

[0021] Figure 3: Scanning electron micrographs of the process of adventitious bud formation in Larix principis-rupprechtii cotyledons. A: freshly inoculated cotyledons; B, C: meristem formation; D, E: emergence of bud primordia; F: adventitious bud formation. The scale bar in the figure is 1 mm.

[0022] Figure 4 :Effects of NAA on adventitious bud formation on cotyledons of Larix principis-rupprechtii. DETAILED DESCRIPTION

[0023] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0024] Embodiment 1: This embodiment provides a high-efficiency regeneration method using Larix principis-rupprechtii cotyledons as explants, which is carried out in the following steps: S1: Sowing: Select mature and full seeds of Larix principis-rupprechtii, soak them in warm water at 25℃ for 24 hours, and sow them in the substrate. Take the seedlings that have germinated for 17 days and are in good growth condition as the material for the next experiment; the substrate is made by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1.

[0025] S2: Disinfection: After removing the seed coat and roots of the seedlings obtained in step S1, rinse with running water for 2 to 3 hours, then rinse with sterile water twice, each time for 1 minute, then disinfect with 75% alcohol for 1 minute, then disinfect with 8% sodium hypochlorite for 6 minutes, then rinse with sterile water 3 to 5 times, each time for 1 minute, and set aside.

[0026] S3: Induction culture: In a clean bench, the cotyledons of the seedlings disinfected in step S2 were cut with a sterile blade and inoculated into adventitious bud induction medium. The culture medium was kept at 25°C and a light rate of 95 μmol·m -2 ·s -1 , pre-culture under the condition of 16h / d light time, and judge whether there is bacterial contamination after 5 days. If no colonies appear on the culture medium, it indicates that the sample is not contaminated; take uncontaminated cotyledons as starting explants for subsequent experiments, and culture them in new adventitious bud induction medium for 50 days to obtain adventitious buds; among them, the formula of adventitious bud induction medium is: DCR+1.5mg / L 6-BA+0.01mg / L TDZ.

[0027] S4: elongation culture: The adventitious buds induced in step S3 were inoculated into the adventitious bud elongation medium at a temperature of 25°C and a light rate of 95 μmol·m -2 ·s -1, culture for 30 days under the condition of 16h / d of light time until the adventitious buds elongate to more than 0.5cm, and obtain elongated adventitious buds; wherein, the formula of the adventitious bud elongation culture medium is: DCR+1.5mg / L 6BA+0.01mg / LTDZ+0.2g / L activated carbon.

[0028] S5: Rooting culture: The adventitious buds elongated in step S4 were inoculated into the rooting medium at a temperature of 25°C and a light rate of 95 μmol·m -2 ·s -1 , and cultured for 30 days under the condition of 16h / d of light, and rooted seedlings were obtained; wherein, the formula of the rooting medium is: DCR+0.05mg / LNAA+0.2g / L activated carbon. According to statistics, the rooting rate is 50%.

[0029] S6: Hardening the seedlings: Put the rooted seedlings from step S5 into tap water in a half-open bottle for hardening. The temperature in the hardening room is 25°C, the light duration is 16h / d, and the light rate is 95μmol·m -2 ·s -1 , humidity is 65%.

[0030] S7: Transplantation: After 10 days of hardening, when the root system changes from white to grayish white, the rooted seedlings are transplanted. When transplanting, the rooted seedlings are first rinsed in clean water to remove the culture medium of the roots, and then the surface moisture of the roots is dried, and then the rooted seedlings are transplanted into a container with a substrate, and cultured in the greenhouse for 1 to 2 weeks. The greenhouse culture conditions are: light time 16h / d, light intensity 2000lux, temperature 25℃, followed by shade and diffuse light culture, spraying water every day, and maintaining humidity at 40% to 60%; the substrate is made by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1. According to statistics, the transplant survival rate is 85%.

[0031] Figure 1 This is the process of adventitious bud formation on the cotyledons of Larix principis-rupprechtii in this example. Figure 2 This is a paraffin section of the adventitious bud formation process of Larix principis-rupprechtii cotyledons in this example. Figure 3 This is a scanning electron microscope image of the adventitious bud formation process of Larix principis-rupprechtii cotyledons in this embodiment. Figure 1 It can be seen that after 10-15 days of culture in the adventitious bud induction medium, the cotyledons began to swell and the cells were closely arranged around the cell wall; after 20-23 days of culture, small protrusions gradually appeared on the surface of the cotyledons, marking the beginning of adventitious buds; after 28-42 days of culture, bud primordia gradually formed; after 50 days of culture, the adventitious buds were fully formed and reached the level of transfer to the elongation medium. In order to gain a deeper understanding of the histological changes, paraffin section technology was used to observe the cotyledons at 6 different growth stages ( Figure 2): Initial stage: Under the action of plant hormones, cells on the surface and below the surface of cotyledons divide rapidly, causing the cotyledons to swell; mid-stage: Entering the period of vigorous DNA division, meristem is formed, and protrudes outward due to dense cells; late stage: cells on both sides of the meristem divide faster under hormone stimulation to form bud primordia; subsequently, adventitious buds visible to the naked eye are observed; elongation stage: adventitious buds of a certain height are transferred to the elongation medium, and root primordia can be seen forming after a period of time. During the entire regeneration process, the cotyledons did not go through the callus and embryoid stages, but directly differentiated from the explants to form new organs. The adventitious buds maintain vascular contact with the mother plant, which indicates that the regeneration pathway of cotyledons of Larix principis-rupprechtii belongs to direct organogenesis ( Figure 2 C, 2D, 2E). The present invention reveals the whole process of adventitious bud formation of Larix principis-rupprechtii cotyledons through systematic histological observation, and provides an important theoretical basis for optimizing regeneration technology.

[0032] Embodiment 2: This example explores the effects of different concentration combinations of 6-BA and TDZ on the induction of adventitious buds from cotyledons of Larix principis-rupprechtii. The specific steps are the same as steps S1 to S3 of Example 1, except that the concentration of 6-BA in the adventitious bud induction medium is adjusted to 0.75, 1.5, and 3.0 mg / L, and the concentration of TDZ is adjusted to 0.001, 0.01, and 0.05 mg / L.

[0033] The experimental results showed that with the increase of 6-BA concentration, the induction efficiency of cotyledon adventitious buds showed a trend of first rising and then falling. When the 6-BA concentration reached 3 mg / L, the induction rate and subsequent differentiation number of adventitious buds were low. Further analysis found that under the condition of 6-BA concentration of 1.5 mg / L, the adventitious bud induction rate also showed a trend of first rising and then falling with the change of TDZ concentration, and the average number of differentiated adventitious buds gradually decreased. When the TDZ concentration was 0.01 mg / L, the adventitious bud induction rate reached a maximum value of 92%, and the average number of adventitious buds was 4.06. Therefore, the combination of 1.5 mg / L 6-BA and 0.01 mg / L TDZ was determined to be the best formula for inducing adventitious buds in the cotyledons of Larix principis-rupprechtii. Plant growth regulators 6-BA and TDZ played a positive regulatory role in the occurrence of adventitious buds, especially in promoting the formation of adventitious buds and increasing the number of adventitious buds. Using DCR as the basic culture medium, the cotyledons were inoculated on the DCR culture medium containing the above-mentioned different concentrations of 6-BA and TDZ. The observation results showed that the position of the adventitious buds growing from the cotyledons of the seedlings had obvious polarity, and the side of the cotyledons facing upward was more likely to generate adventitious buds, and the number was larger. Statistical analysis showed that the concentration of TDZ had a significant effect on the frequency of bud induction. When the concentration of 6-BA was 1.5 mg / L, the induction rate of adventitious buds and the average number of differentiation showed a trend of first increasing and then decreasing with the change of TDZ concentration. In summary, the optimal culture medium formula for inducing adventitious buds from the cotyledons of Larix principis-rupprechtii was determined to be DCR+1.5 mg / L 6-BA+0.01 mg / LTDZ. Under this formula, the induction rate of adventitious buds can reach 92%, and the average number of adventitious buds is 4.06.

[0034] Embodiment 3: In tissue culture, the combined use of TDZ and NAA is often used to promote the induction and growth of adventitious buds. As an auxin analog, NAA has high stability, and auxin in plants can stimulate cell division and elongation. In order to further improve the incidence of cotyledon adventitious buds, this example, based on Example 2, explores the effect of adding different final concentrations of NAA (0, 0.1, 0.3, 0.5, 0.7, 1.0 mg / L) to the optimal formula of adventitious bud induction medium on the incidence of adventitious buds in Larix principis-rupprechtii cotyledons. The experimental results show that under the condition of not adding NAA, the induction rate of adventitious buds in Larix principis-rupprechtii cotyledons reaches the highest value, and the number of adventitious buds produced on each cotyledon is the largest. With the increase of NAA concentration, the induction efficiency and quantity of adventitious buds show a downward trend. When the NAA concentration reaches 0.5 mg / L, the formation of adventitious buds begins to be inhibited, and even the formed adventitious buds are prone to browning and then death. This phenomenon shows that the addition of NAA cannot improve the induction efficiency of adventitious buds in Larix principis-rupprechtii cotyledons. Low concentrations of NAA failed to promote the occurrence of cotyledon adventitious buds, while high concentrations of NAA inhibited the formation of adventitious buds. Considering that the induction effect of NAA on adventitious buds of cotyledons of Larix principis-rupprechtii is not significant, NAA will not be added in subsequent experiments of this study. In summary, this embodiment determines that the optimal culture medium formula for inducing adventitious buds of cotyledons of Larix principis-rupprechtii is DCR+1.5mg / L 6-BA+0.01mg / L TDZ. This formula can effectively promote the induction and growth of adventitious buds without the addition of NAA.

[0035] Embodiment 3: Introducing an appropriate amount of adsorptive substances (such as activated carbon) into the culture medium can promote the extension of adventitious buds. Such substances can effectively control and absorb harmful components, reducing their adverse effects on plant organs or tissues. In addition, it helps organ differentiation and morphogenesis to a certain extent, and also has a positive effect on the induction of adventitious roots. In order to further optimize the elongation effect, this embodiment investigates the effect of activated carbon of different concentrations on the elongation of adventitious buds of Larix principis-rupprechtii cotyledons. The specific steps are the same as steps S1 to S4 of Example 1, except that the concentration of activated carbon in the adventitious bud elongation culture medium is adjusted to 0, 0.2, 0.5, and 1 g / L.

[0036] Experimental data showed that without the addition of activated carbon, the adventitious buds stopped growing after reaching a certain length, and the callus continued to grow. With the increase of activated carbon concentration, the elongation rate of adventitious buds showed a trend of increasing first and then decreasing. When the amount of activated carbon added was 0.2g / L, the elongation rate of adventitious buds reached the highest value, and the growth was vigorous and rapid. With the continued increase in the content of activated carbon, the elongation rate of adventitious buds began to decline. When the concentration rose to 1.0g / L, the adventitious buds showed a state of weakened growth activity, which may be due to the excessively high concentration of activated carbon inhibiting its growth. In view of the fact that the elongation rate of adventitious buds reached the best effect at an activated carbon concentration of 0.2g / L, this concentration was used in subsequent experiments. In summary, this study determined that the optimal activated carbon content suitable for the elongation of adventitious buds of Larix principis-rupprechtii cotyledons is 0.2g / L. Under this condition, the elongation rate of adventitious buds can reach 188.67%.

[0037] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An efficient regeneration method using Larix principis-rupprechtii cotyledons as explants, characterized in that: The steps include: S1: Sowing: Select mature and full seeds of Larix principis-rupprechtii, soak them in warm water for 24 hours and then sow them in the substrate. Take the seedlings that have germinated for 17 days and are in good growth condition as the materials for the next experiment; S2: Disinfection: After removing the seed shell and roots of the seedlings obtained in step S1, disinfection is performed to obtain disinfected seedlings; S3: Induction culture: cut the cotyledons of the seedlings disinfected in step S2 with a sterile blade in a clean bench, inoculate them into an adventitious bud induction medium for pre-culture, and determine whether there is bacterial or fungal contamination after 3 to 5 days. If no colonies appear on the culture medium, it indicates that the sample is not contaminated; take the uncontaminated cotyledons as the starting explants for subsequent experiments, and culture them in a new adventitious bud induction medium for 35 to 50 days to obtain adventitious buds; S4: elongation culture: the adventitious buds obtained in step S3 are inoculated into an adventitious bud elongation medium and cultured for 30 to 50 days to obtain elongated adventitious buds; S5: Rooting culture: inoculate the elongated adventitious buds obtained in step S4 into a rooting medium and culture for 15 to 30 days to obtain rooted seedlings; Among them, the formula of adventitious bud induction medium is: DCR+1.5mg / L 6-BA+0.01mg / L TDZ; The formula of adventitious bud elongation medium is: DCR+1.5mg / L 6BA+0.01mg / L TDZ+0.2g / L activated carbon; The formula of rooting medium is: DCR+0.05mg / L NAA+0.2g / L activated carbon.

2. The high-efficiency regeneration method according to claim 1, characterized in that: In step S1, the matrix is ​​prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:

1.

3. The high-efficiency regeneration method according to claim 1, characterized in that: In step S2, the disinfection method is: rinse with running water for 2 to 3 hours, then rinse with sterile water twice, then disinfect with 75% alcohol for 1 minute, then disinfect with 8% sodium hypochlorite for 6 minutes, and then rinse with sterile water 3 to 5 times.

4. The high-efficiency regeneration method according to claim 1, characterized in that: In step S3, the pre-culture conditions are: temperature 25°C, light rate 95 μmol·m -2 ·s -1 , light duration 16h / d, culture conditions: temperature 25℃, light rate 95μmol·m -2 ·s -1 , lighting time: 16h / d.

5. The high-efficiency regeneration method according to claim 1, characterized in that: In step S4, the culture conditions are: temperature 25°C, light rate 95 μmol·m -2 ·s -1 , lighting time: 16h / d.

6. The high-efficiency regeneration method according to claim 1, characterized in that: In step S5, the culture conditions are: temperature 25°C, light rate 95 μmol·m -2 ·s -1 , lighting time: 16h / d.

7. The high-efficiency regeneration method according to claim 1, characterized in that: After step S5 is completed, step S6 is also included: seedling hardening and transplanting.

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