A high-efficiency regeneration method using larch (larix principis-rupprechtii) cotyledon as explant

Abstract

The present application relates to a kind of efficient regeneration method with the larch of north China as explant, the method comprises the following steps: sowing, disinfection, induction culture, elongation culture, rooting culture, seedling and transplanting.According to the technical scheme provided by the present application, the medium with specific formula is used, so that the larch of north China shows significant advantages in the process of propagation: high differentiation efficiency, large proliferation coefficient, good rooting rate.In addition, the method also has the characteristics of short plant growth cycle, strong adaptability after transplanting and the like.The seedling after cultivation presents the characteristics of robust and straight, good growth and uniformity.The technical scheme of the present application significantly reduces the seedling culture period, effectively reduces the production cost.By establishing the adventitious bud induction system with the larch of north China as starting material, and successfully obtaining regenerated plant, a new technical approach is provided for the rapid propagation of the larch of north China.Meanwhile, the present application also lays the foundation for the future establishment of efficient larch of north China transgenic system.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to an efficient regeneration method using larch leaves from North China as explants. Background Technology

[0002] *Larix gmelinii* var. *principis-rupprechtii* (Mayr) Pilger, belonging to the family Pinaceae and the genus *Larix*, is a larch variety endemic to North China. It is mainly distributed in the high-altitude regions of North China, including the Taihang Mountains, Lüliang Mountains, and Wutai Mountains, at altitudes ranging from 1400 to 2800 meters. It is a major forest tree species in the high-altitude coniferous forest belt of North China and plays an important role in forestry and the ecosystem. *Larix gmelinii* has several unique characteristics: strong adaptability, capable of withstanding cold, drought, and poor soil conditions, adapting to the harsh environment of the North China high-altitude regions; rapid growth, with an annual growth of over 1 meter under suitable conditions; beautiful tree shape, conical in shape, with spreading branches, soft needles that turn yellow in autumn, giving it high ornamental value; and excellent wood quality, with dense, rot-resistant timber, making it a high-quality building and furniture material. Larch, native to North China, is not only an important forestry resource with high ecological and economic value, but it can also be used in multiple fields: it serves as excellent timber for bridges, vehicles, and buildings; it can be used for forest regeneration and afforestation of barren hills in its distribution area, as well as in the high-altitude areas of the Yellow River Basin and the upper reaches of the Liao River; it is an excellent landscaping tree species, particularly suitable for landscape design in northern regions; and it can be used to produce larch arabinogalactan, which has immune-enhancing and anti-tumor effects. Research on larch genetic improvement began relatively early, and many superior larch varieties have been screened through conventional breeding methods. However, larch suffers from problems such as a long growth cycle, difficulties in genetic manipulation, and significant susceptibility to natural conditions. Compared with conventional breeding, genetic engineering breeding is more efficient and precise, and is the main trend in genetic improvement research. Currently, there are few reports on larch gene function research in China, and progress in genetic transformation and gene editing is slow. The main reason for this is the lack of a good larch regeneration system. Therefore, establishing a high-efficiency and stable rapid larch propagation technology is a fundamental prerequisite for larch genetic engineering improvement.

[0003] In vitro culture and genetic transformation systems for conifers are an important research direction in forestry biotechnology, and are of great significance for the genetic improvement and rapid propagation of *Larix chinensis*. As a conifer species, *Larix chinensis* faces unique challenges and opportunities in in vitro culture due to its special biological characteristics. Research on in vitro culture technology for pine species began in the 1970s, and since then, this field has received widespread attention and continuous breakthroughs. This indicates that in vitro culture technology for conifers, including *Larix chinensis*, has a long research history and has continuously progressed and developed. As an important economic and ecological tree species, the research on in vitro culture and plant regeneration technology of *Larix chinensis* has always been one of the most difficult problems to solve in plant tissue culture research. With the increasing emphasis on clonal forestry, countries around the world have paid special attention to the tissue culture of conifers, including *Larix chinensis*. In the past 20 years, the in vitro culture of conifers such as *Larix chinensis* has expanded from tissue and organ culture to cell and protoplast culture, and has gradually realized the transformation from theoretical research to application. These advancements have provided crucial technical support for the genetic improvement and rapid propagation of *Larix chinensis* in North China, contributing to its enhanced application value in forestry production and ecological construction. However, due to the complexity of the growth characteristics and genetic background of *Larix chinensis*, its in vitro culture technology still faces numerous challenges, requiring further research and breakthroughs.

[0004] Advances in somatic embryogenesis technology for conifers have significant reference value for the in vitro culture and genetic improvement of *Larix banksiana*. Breakthroughs in this field began in 1976 when Durzan and Chalupa first reported embryo-like structures in in vitro culture of *Pinus banksiana*. In 1985, researchers successfully induced somatic embryos from immature embryos of *Picea abies* and obtained regenerated plants, opening a new direction for in vitro embryogenesis research in conifers, including *Larix banksiana*. Since then, research in this field has rapidly developed and made significant progress. Currently, somatic embryos have been cultured from more than 40 conifer species using this method, mainly concentrated in the genera *Picea* and *Pinus*. Although *Larix banksiana* 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 techniques, researchers hope to achieve breakthroughs in somatic embryogenesis of *Larix banksiana*, thus providing new avenues for its rapid propagation and genetic improvement. This can not only improve the propagation efficiency of larch in North China, but also provide important experimental materials and technical support for research on its stress resistance improvement and timber quality enhancement, further enhancing its application value in forestry production and ecological construction.

[0005] A highly efficient method for in vitro regeneration of Larch (Pterocarpus stenoptera) needles has been developed by the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences. This method uses the thick needles of Larch as starting material, rapidly generating a large number of adventitious buds and regenerated plants. Larch needles are typically linear, 2–3 cm long, and clustered on short branches, with 20–40 needles per cluster. This unique needle structure provides a favorable foundation for in vitro regeneration. This technique not only preserves the genetic phenotype of the parent plant but can also be used as a recipient system for transgenic plants. Notably, Larch possesses strong cold and drought resistance, and these excellent traits are preserved during in vitro regeneration, providing favorable conditions for subsequent genetic improvement. This discovery is of great significance for research on conifer genetic improvement, new variety breeding, and high-quality seedling propagation, and will have a profound impact on the sustainable development of forestry and narrowing the gap with developed countries in basic forestry research. Furthermore, as an important timber and ecological protection tree species in my country, the breakthrough in efficient in vitro regeneration technology for larch in North China has provided new technical support for the large-scale propagation and ecological restoration projects of this tree species.

[0006] This invention not only demonstrates the powerful regeneration capacity of the North China larch but also fully utilizes its excellent genetic characteristics, such as strong cold and drought resistance. More importantly, the adventitious buds formed by this cotyledon regeneration system exhibit great application potential: they are suitable not only for gene gun-mediated genetic transformation but also for Agrobacterium-mediated genetic transformation systems. This versatility opens up broad prospects for the genetic improvement and new variety breeding of North China larch. Considering the important role of North China larch in my country's forestry economy and ecological construction, this technological breakthrough will undoubtedly promote the rapid development of my country in areas such as coniferous genetic improvement, high-quality seedling propagation, and ecological restoration projects, helping to narrow the gap with developed countries in basic forestry research and providing strong support for the sustainable development of the forestry economy. Summary of the Invention

[0007] The purpose of this invention is to develop an efficient regeneration method using larch cotyledons as explants. This method has the following advantages: convenient material acquisition, high cost-effectiveness, simplified operation process, rapid propagation rate, and high propagation coefficient. The establishment of this method can provide efficient regeneration system support for genetic transformation research on larch.

[0008] The technical solution adopted in this invention is as follows:

[0009] A highly efficient regeneration method using larch leaves from North China as explants includes the following steps:

[0010] S1: Sowing: Select mature and plump seeds of North China larch, soak them in warm water for 24 hours and then sow them in the substrate. Seedlings that have germinated for 17 days and are growing well are used as materials for the next experiment.

[0011] S2: Disinfection: After removing the seed coat and roots from the seedlings obtained in step S1, disinfect them to obtain disinfected seedlings.

[0012] S3: Induction Culture: In a clean bench, use a sterile blade to cut off the cotyledons of the seedlings disinfected in step S2, and inoculate them into the adventitious bud induction medium for pre-culture. After 3-5 days, determine whether there is bacterial or fungal contamination. If no colonies appear on the medium, it indicates that the sample is not contaminated. Take the uncontaminated cotyledons as the starting explants for subsequent experiments. Culture them in a new adventitious bud induction medium for 35-50 days to obtain adventitious buds.

[0013] S4: Elongation culture: Inoculate the adventitious buds from step S3 into the adventitious bud elongation culture medium and culture for 30-50 days to obtain elongated adventitious buds;

[0014] S5: Rooting culture: Inoculate the elongated adventitious buds from step S4 into the rooting medium and culture for 15-30 days to obtain rooted seedlings;

[0015] The formulation of the adventitious bud induction medium is: DCR + 1.5 mg / L 6-BA + 0.01 mg / L LTDZ;

[0016] The formulation of the adventitious shoot elongation medium is: DCR + 1.5 mg / L 6BA + 0.01 mg / L TDZ + 0.2 g / L activated carbon;

[0017] The rooting medium formula is: DCR + 0.05 mg / L NAA + 0.2 g / L activated carbon.

[0018] Furthermore, in step S1 above, the matrix is ​​prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1.

[0019] Furthermore, in step S2 above, the disinfection method is as follows: rinse with running water for 2 to 3 hours, then rinse twice with sterile water, 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.

[0020] Furthermore, in step S3 above, the pre-culture conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 The photoperiod was 16 h / d, and the culture conditions were: temperature 25℃, photoperiod 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day.

[0021] Furthermore, in step S4 above, the cultivation conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day.

[0022] Furthermore, in step S5 above, the cultivation conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day.

[0023] Furthermore, after step S5 is completed, step S6 is also included: hardening off the seedlings and transplanting.

[0024] The significant advantages of this invention are:

[0025] (1) Innovation in explant selection:

[0026] This invention uses cotyledons from larch seedlings germinated in a greenhouse environment as the explant source. This choice not only simplifies the material collection process but also overcomes time and quantity limitations. Furthermore, this method offers significant advantages in terms of time and labor savings, laying the foundation for large-scale propagation.

[0027] (2) Specificity of culture medium formulation:

[0028] This invention utilizes a carefully designed culture medium formula to provide explants with the specific hormone combinations required at different growth stages. This customized culture system creates ideal conditions for rooting and robust seedling growth, effectively promoting healthy seedling growth and ultimately successfully obtaining regenerated larch plants from North China.

[0029] (3) The practicality and efficiency of the method:

[0030] The cultivation method described in this invention has many advantages: it is convenient to obtain materials, which reduces experimental costs; the operation process is simplified, making it easy to promote and apply; the propagation speed is fast, which significantly improves the reproduction efficiency; and the reproduction coefficient is high, which greatly increases the yield.

[0031] (4) Broad application prospects:

[0032] The regeneration system established in this invention is not only suitable for conventional propagation, but also provides an ideal platform for genetic transformation research on North China larch. The development of this technology lays a solid foundation for future gene improvement and new variety breeding, and is expected to promote the further development of forestry biotechnology. Attached Figure Description

[0033] Figure 1The process of adventitious bud formation in cotyledons of North China larch. A: Newly inoculated cotyledons; 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 diagram is 1 cm.

[0034] Figure 2 Paraffin sections illustrating the process of adventitious bud formation in larch leaves from North China. A: Newly inoculated cotyledons; B: Meristematic formation; CE: Appearance of bud primordia; F: Adventitious bud formation. Scale bar in the figure is 100 μm.

[0035] Figure 3 Scanning electron microscope (SEM) images of adventitious bud formation in cotyledons of North China larch. A: Newly inoculated cotyledons; B, C: Meristematic formation; D, E: Appearance of bud primordia; F: Adventitious bud formation. Scale bar in the image is 1 mm.

[0036] Figure 4 The effect of NAA on adventitious bud formation in larch leaves in North China. Detailed Implementation

[0037] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0038] Example 1:

[0039] This embodiment provides a highly efficient regeneration method using larch leaves from North China as explants, which is carried out according to the following steps:

[0040] S1: Sowing: Select mature and plump seeds of North China larch, soak them in warm water at 25℃ for 24 hours, and then sow them in the substrate. Seedlings that have germinated for 17 days and are growing well are used as materials for the next experiment. The substrate is prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1.

[0041] S2: Disinfection: After removing the seed coat and roots from the seedlings obtained in step S1, rinse them with running water for 2-3 hours, then rinse them twice with sterile water for 1 minute each time, then disinfect them with 75% alcohol for 1 minute, then disinfect them with 8% sodium hypochlorite for 6 minutes, and then rinse them with sterile water 3-5 times for 1 minute each time, and set them aside for use.

[0042] S3: Induction Culture: In a clean bench, use a sterile blade to cut off the cotyledons of the seedlings disinfected in step S2, and inoculate them into the adventitious bud induction medium. Incubate at 25℃ and a light rate of 95 μmol·m⁻¹. -2 ·s -1Pre-culture was performed under a light exposure of 16 h / d. After 5 days, bacterial contamination was assessed. If no colonies appeared on the culture medium, the sample was considered uncontaminated. Uncontaminated cotyledons were used as starting explants for subsequent experiments. Adventitious buds were obtained by culturing them in a new adventitious bud induction medium for 50 days. The formulation of the adventitious bud induction medium was: DCR + 1.5 mg / L 6-BA + 0.01 mg / L TDZ.

[0043] S4: Elongation Culture: The adventitious shoots induced in step S3 were inoculated into an adventitious shoot elongation medium and cultured at a temperature of 25℃ and a light rate of 95 μmol·m⁻¹. -2 ·s -1 Under a light exposure of 16 h / d, the adventitious buds were cultured for 30 days until they elongated to more than 0.5 cm. The elongated adventitious buds were obtained. The formula of the adventitious bud elongation medium was: DCR + 1.5 mg / L 6BA + 0.01 mg / L LTDZ + 0.2 g / L activated carbon.

[0044] S5: Rooting Culture: The adventitious shoots elongated in step S4 were inoculated into the rooting medium and cultured at a temperature of 25℃ and a light rate of 95 μmol·m⁻¹. -2 ·s -1 Rooted seedlings were obtained after culturing under a light intensity of 16 hours / day for 30 days. The rooting medium was formulated as follows: DCR + 0.05 mg / L NAA + 0.2 g / L activated charcoal. The rooting rate was 50%.

[0045] S6: Hardening off the seedlings: Place the rooted seedlings from step S5 in tap water in a partially opened state to harden off the seedlings. The hardening-off room temperature is 25℃, the light duration is 16h / d, and the light rate is 95μmol·m. -2 ·s -1 The humidity is 65%.

[0046] S7: Transplanting: After 10 days of hardening off, when the roots turn from white to grayish-white, transplant the rooted seedlings. Before transplanting, rinse the rooted seedlings in clean water to remove the culture medium from the roots, then dry the surface moisture of the roots. Transplant the seedlings into containers filled with substrate and cultivate them in a greenhouse for 1-2 weeks. The greenhouse cultivation conditions are: 16 hours / day light, 2000 lux light intensity, and 25℃ temperature. Afterward, cultivate under shade with diffused light, spraying water daily to maintain humidity at 40%-60%. The substrate is prepared by mixing nutrient soil, vermiculite, and perlite in a volume ratio of 2:2:1. The transplant survival rate is 85%.

[0047] Figure 1 This embodiment illustrates the process of adventitious bud formation in larch leaves in North China. Figure 2 Paraffin sections are used to illustrate the process of adventitious bud formation in larch leaves in North China in this embodiment. Figure 3This is a scanning electron microscope image of the adventitious bud formation process of *Pinus thunbergii* leaves in this embodiment. Figure 1 It was observed that after culturing cotyledons in adventitious bud induction medium for 10–15 days, the cotyledons began to swell, with cells tightly 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 bud formation; after 28–42 days of culture, bud primordia gradually formed; and after 50 days of culture, adventitious buds were fully formed, reaching a stage where they could be transferred to elongation medium. To gain a deeper understanding of the histological changes, paraffin sectioning was used to observe cotyledons at six different growth stages. Figure 2 Early stage: Under the influence of plant hormones, cells on the surface and subsurface of the cotyledon rapidly divide, leading to cotyledon swelling; Middle stage: Entering a period of vigorous DNA division, meristem forms, protruding outwards due to cell density; Late stage: Cells on both sides of the meristem divide rapidly under hormone stimulation, forming bud primordia; Subsequently, adventitious buds visible to the naked eye are observed; Elongation stage: Adventitious buds of a certain height are transferred to an elongation medium, and root primordia are visible after a period of time. Throughout the regeneration process, the cotyledons do not undergo callus and embryoid stages, but directly differentiate from the explant to form new organs. The adventitious buds maintain vascular connections with the parent plant, indicating that the regeneration pathway of North China larch cotyledons belongs to direct organogenesis. Figure 2 (C, 2D, 2E). This invention, through systematic histological observation, reveals the entire process of adventitious bud formation in larch leaves in North China, providing an important theoretical basis for optimizing regeneration technology.

[0048] Example 2:

[0049] This embodiment investigated the effects of different concentrations of 6-BA and TDZ on the induction of adventitious buds from larch leaves in North China. The specific steps were the same as steps S1 to S3 in Example 1, except that the concentration of 6-BA in the adventitious bud induction medium was adjusted to 0.75, 1.5, and 3.0 mg / L, and the concentration of TDZ was adjusted to 0.001, 0.01, and 0.05 mg / L.

[0050] Experimental results showed that the induction efficiency of adventitious buds in cotyledons first increased and then decreased with increasing 6-BA concentration. When the 6-BA concentration reached 3 mg / L, the induction rate and subsequent differentiation number of adventitious buds were both low. Further analysis revealed that under the condition of 1.5 mg / L 6-BA concentration, the adventitious bud induction rate also showed a trend of first increasing and then decreasing with the change of TDZ concentration, while the average number of differentiated adventitious buds gradually decreased. When the TDZ concentration was 0.01 mg / L, the adventitious bud induction rate reached its 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 optimal formulation for inducing adventitious buds in cotyledons of Larix principis-Pinus koraiensis. The plant growth regulators 6-BA and TDZ played a positive regulatory role in the occurrence of adventitious buds, especially in promoting adventitious bud formation and increasing the number of adventitious buds. Using DCR as the basal medium, cotyledons were inoculated onto DCR medium containing different concentrations of 6-BA and TDZ. Observations showed that the location of adventitious buds emerging from the cotyledons of seedlings exhibited a clear polarity, with the side facing upwards more likely to produce adventitious buds, and in greater numbers. Statistical analysis indicated that TDZ concentration significantly affected the bud induction frequency. At a 6-BA concentration of 1.5 mg / L, the adventitious bud induction rate and average differentiation number showed a trend of first increasing and then decreasing with changes in TDZ concentration. In conclusion, the optimal medium formulation for inducing adventitious buds from larch cotyledons in North China was determined to be DCR + 1.5 mg / L 6-BA + 0.01 mg / L TDZ. Under this formulation, the adventitious bud induction rate reached 92%, with an average of 4.06 adventitious buds.

[0051]

[0052]

[0053] Example 3:

[0054] 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 analogue, NAA has high stability, while auxins in plants can stimulate cell division and elongation. To further improve the occurrence rate of cotyledon adventitious buds, this example, based on Example 2, investigated 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 formulation of the adventitious bud induction medium on the occurrence rate of cotyledon adventitious buds in *L. pülensis*. The experimental results showed that the induction rate of *L. pülensis* cotyledon adventitious buds reached its highest value under the condition of no NAA addition, and the number of adventitious buds produced on each cotyledon was the largest. With the increase of NAA concentration, the induction efficiency and number of adventitious buds both showed a decreasing trend. When the NAA concentration reached 0.5 mg / L, it began to inhibit the formation of adventitious buds, and even the formed adventitious buds were prone to browning and then death. This phenomenon indicates that adding NAA does not improve the induction efficiency of *L. pülensis* cotyledon adventitious buds. Low concentrations of NAA failed to promote the formation of adventitious buds in cotyledons, while high concentrations inhibited their formation. Considering the induction effect of NAA on adventitious buds in *L. chinensis* cotyledons, subsequent experiments in this study will not add NAA. In summary, this example determined the optimal culture medium formula for inducing adventitious buds in *L. chinensis* cotyledons to be DCR + 1.5 mg / L 6-BA + 0.01 mg / L TDZ. This formula effectively promotes the induction and growth of adventitious buds without the addition of NAA.

[0055] Example 3:

[0056] Introducing appropriate amounts of adsorbent substances (such as activated carbon) into the culture medium can promote the elongation of adventitious buds. These substances can effectively control and absorb harmful components, reducing their adverse effects on plant organs or tissues. Furthermore, they contribute to organ differentiation and morphogenesis to some extent, and also have a positive effect on the induction of adventitious roots. To further optimize the elongation effect, this example investigated the influence of different concentrations of activated carbon on the elongation of adventitious buds from larch leaves in North China. The specific steps were the same as steps S1 to S4 of Example 1, except that the concentration of activated carbon in the adventitious bud elongation culture medium was adjusted to 0, 0.2, 0.5, and 1 g / L.

[0057] Experimental data showed that without activated carbon, adventitious buds stopped growing after reaching a certain length, accompanied by continuous enlargement of callus tissue. With increasing activated carbon concentration, the elongation rate of adventitious buds initially increased and then decreased. When the activated carbon concentration was 0.2 g / L, the elongation rate reached its highest value, exhibiting vigorous and rapid growth. Further increasing the activated carbon content led to a decrease in the elongation rate. When the concentration reached 1.0 g / L, the growth activity of the adventitious buds weakened, possibly due to the excessively high concentration of activated carbon inhibiting their growth. Given that the optimal activated carbon concentration of 0.2 g / L achieved the best effect on the elongation rate of adventitious buds, this concentration was used in subsequent experiments. In conclusion, this study determined that the optimal activated carbon content for the elongation of adventitious buds in North China larch leaves is 0.2 g / L, under which the elongation rate can reach 188.67%.

[0058]

[0059] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A highly efficient regeneration method using larch leaves from North China as explants, characterized in that: Includes the following steps: S1: Sowing: Select mature and plump seeds of North China larch, soak them in warm water for 24 hours and then sow them in the substrate. Seedlings that have germinated for 17 days and are growing well are used as materials for the next experiment. S2: Disinfection: After removing the seed coat and roots from the seedlings obtained in step S1, disinfect them to obtain disinfected seedlings. S3: Induction Culture: In a clean bench, use a sterile blade to cut off the cotyledons of the seedlings disinfected in step S2, and inoculate them into the adventitious bud induction medium for pre-culture. After 3-5 days, determine whether there is bacterial or fungal contamination. If no colonies appear on the 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 35-50 days to obtain adventitious buds. S4: Elongation culture: The adventitious buds obtained in step S3 are inoculated into the adventitious bud elongation culture medium and cultured for 30-50 days to obtain elongated adventitious buds; S5: Rooting culture: Inoculate the elongated adventitious buds obtained in step S4 into the rooting culture medium and culture for 15-30 days to obtain rooted seedlings; The formulation of the adventitious bud induction medium is: DCR + 1.5 mg / L 6-BA + 0.01 mg / L TDZ; The formulation of the adventitious shoot elongation medium is: DCR + 1.5 mg / L 6-BA + 0.01 mg / L TDZ + 0.2 g / L activated carbon; The rooting medium formula is: DCR + 0.05 mg / L NAA + 0.2 g / L activated carbon; In step S1, the substrate is prepared by mixing nutrient soil, vermiculite and perlite in a volume ratio of 2:2:1; In step S2, the disinfection method is as follows: rinse with running water for 2-3 hours, then rinse twice with sterile water, then disinfect with 75% alcohol for 1 minute, then disinfect with 8% sodium hypochlorite for 6 minutes, and then rinse with sterile water 3-5 times. In step S3, the pre-culture conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 The photoperiod was 16 h / d, and the culture conditions were: temperature 25℃, photoperiod 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day; In step S4, the cultivation conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day; In step S5, the cultivation conditions are: temperature 25℃, light rate 95 μmol·m⁻¹. -2 ·s -1 Light exposure time: 16 hours / day.

2. The efficient regeneration method according to claim 1, characterized in that: After step S5 is completed, step S6 is also included: hardening off and transplanting.

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