A tissue culture rapid propagation method for astringent crabapple and a special combined culture medium thereof

Abstract

The application discloses a tissue culture and rapid propagation method for Yangmei of Alocasia macrorrhiza and a special combined culture medium thereof, and the method comprises the steps of sterilizing an explant, carrying out primary culture and subculture and proliferation culture in stages, wherein the primary culture is performed by first inoculating a stem segment into an induction culture medium containing 6-BA 0.8-1.2 mg / L, IBA 0.05-0.15 mg / L and PVP-40 to induce germination, and then transferring the stem segment into an elongation culture medium containing 6-BA 0.1-0.5 mg / L, NAA 0.01-0.05 mg / L and PVP-40 to promote elongation; the new shoots are then transferred into a subculture medium containing 6-BA 1.0-1.8 mg / L, NAA 0.1-0.2 mg / L, GA3 0.5-1.5 mg / L and PVP-40 to proliferate. The application effectively solves the technical problems that Yangmei is prone to brown and death, and it is difficult to simultaneously achieve budding and high growth by means of two-stage hormone stepwise regulation and cooperation with a brown prevention agent, and realizes efficient and large-scale breeding of high-quality seedlings.

Description

Technical Field

[0001] This invention relates to the field of bayberry propagation technology, and more specifically, to a rapid propagation method for *Myrica rubra* var. *rubra* via tissue culture and its dedicated combined culture medium. Background Technology

[0002] Waxberry (Morella rubra) is a specialty fruit of southern my country. It is a perennial evergreen tree belonging to the Myricaceae family and the Myrica genus. Originally from southeastern provinces and the Yunnan-Guizhou Plateau, it has been cultivated in China for over two thousand years. The tree is robust, easy to cultivate, and has a long economic lifespan. Its fruit is highly nutritious, delicious, and possesses antioxidant and anti-aging properties, making it valuable for both food and medicine. There are more than ten varieties of waxberry, among which the four main cultivated varieties are the Water Chestnut variety, Dongkui waxberry, Ding'ao waxberry, and Late Rice waxberry. The Water Chestnut variety, named for its purplish-black fruit resembling a water chestnut when ripe, is widely adaptable, early-fruiting, and high-yielding, making it a major variety promoted in China.

[0003] However, traditional propagation methods for the Chinese bayberry variety, mainly including seed propagation, cutting propagation, and grafting, all have significant limitations. Seed (sexual) propagation produces offspring with severe phenotypic segregation, making it difficult to inherit the superior traits of the parent plant. Cutting propagation is greatly affected by the season, has difficulty rooting, and a low propagation coefficient. Grafting propagation faces compatibility issues, limited rootstock resources, and problems such as immature virus-free technology and high risk of pests and diseases. These problems severely restrict the large-scale and high-quality development of the Chinese bayberry industry. Plant tissue culture, as a novel asexual propagation technology, has significant advantages such as rapid propagation, exemption from seasonal restrictions, and the ability to be mass-produced, effectively overcoming the limitations of traditional asexual propagation. However, as a perennial woody plant, the Chinese bayberry has a high degree of lignification, a complex surface environment on its branches, numerous endophytic bacteria, and contains large amounts of tannins and phenolic substances. During tissue culture, explants are highly susceptible to severe browning (i.e., phenolic substances are oxidized to form browning substances, leading to explant toxicity and death), making sterilization extremely difficult. In addition, the explants of Myrica rubra are extremely sensitive to plant growth regulators, and it is often difficult to achieve both bud induction rate and bud elongation rate in the initial culture.

[0004] While some attempts have been made in existing techniques for tissue culture of bayberry, many shortcomings remain: Chinese patent application CN103651144A discloses a method for rapid propagation of *Myrica rubra* (Chinese bayberry). This method uses stem segments with buds as explants, disinfects the surface with a 0.1% mercuric chloride solution, and then induces callus formation, from which adventitious buds differentiate. However, this method has significant drawbacks: First, the callus development pathway is prone to somatic asexual variation during frequent division and proliferation, leading to phenotypic segregation in offspring and making it difficult to stably maintain the superior genetic traits of the *Myrica rubra* parent plant. Second, the method uses highly toxic mercuric chloride solution as a disinfectant, posing a significant health hazard to operators and easily causing environmental pollution, which does not meet the production requirements of modern green agriculture.

[0005] Chinese patent application CN118415071A discloses a method for tissue culture of bayberry. This method propagates the fruit through the axillary bud route and uses a sodium hypochlorite solution with a concentration as high as 15% during the sterilization stage. At the same time, the culture medium is added with extremely complex anti-browning and anti-contamination chemical components, including ProClin preservative, ascorbic acid, activated carbon, and polyvinylpyrrolidone. In addition, the initial culture uses a single culture medium for single-stage induction culture. This method also has obvious shortcomings: on the one hand, sodium hypochlorite at a concentration of up to 15% is a very high concentration of strong oxidant. Although it can reduce pollution, it will cause irreversible chemical damage to the tender stem cells of bayberry, thus accelerating tissue necrosis and browning. On the other hand, in order to compensate for the damage caused by disinfection, it is necessary to add a variety of chemical preservatives and antioxidants, which greatly increases the cost of reagents and the complexity of operation. More importantly, it adopts a single-stage primary culture mode, which cannot specifically solve the physiological contradiction between cytokinin and auxin in inducing multiple buds and promoting bud elongation. This often results in induced buds that are mostly clustered and short, which cannot meet the standards for efficient subsequent subculture proliferation.

[0006] In summary, the industry urgently needs a new, efficient, safe, environmentally friendly, and quality-controllable tissue culture rapid propagation system for *Myrica rubra* to overcome the technical bottlenecks of low propagation efficiency, easy browning and death of explants, and difficulty in balancing bud induction and elongation, thereby achieving large-scale, year-round production of high-quality seedlings. Summary of the Invention

[0007] One of the technical problems to be solved by the present invention is to provide a rapid propagation method for *Myrica rubra* tissue culture, in order to solve the problems in the prior art where *Myrica rubra* explants are prone to browning and death, and the first generation culture is limited by a single stage and a single formula, which leads to mutual constraints and difficulty in achieving both bud induction multiple and bud elongation rate, resulting in low overall propagation efficiency.

[0008] To overcome the shortcomings of the existing technology, this invention provides a method for rapid propagation of *Myrica rubra* through tissue culture, comprising the following steps: S1: Explant disinfection: Select semi-lignified new shoots in spring, disinfect and sterilize them to obtain sterilized stem segments; S2: Initial generation training with functional phases: S21: Bud induction stage: Sterilized stem segments are inoculated into induction medium, and axillary buds are induced to sprout using a combination of cytokinin and auxin IBA. The induction medium is: WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; S22: Bud elongation stage: After the buds sprout, the explants are transferred to the elongation medium, the cytokinin concentration is reduced and the auxin is switched to NAA to promote bud elongation. The elongation medium is: WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; S3: Subculture and proliferation: The elongated new shoots are transferred to a subculture medium for proliferation. The subculture medium is: WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L.

[0009] Compared with existing technologies, the rapid propagation method of *Myrica rubra* tissue culture of this invention has the following advantages: In the S21 bud induction stage, the method of this invention utilizes a relatively high concentration of cytokinin (6-BA 0.8-1.2 mg / L) in combination with auxin IBA. The high concentration of cytokinin can effectively break apical dominance, stimulate a large number of axillary buds of the explant, and increase the number of buds and the induction multiple. In the S22 bud elongation stage, this invention timely transfers the explants, significantly reduces the concentration of cytokinin (6-BA to 0.1-0.5 mg / L), and switches the helper auxin from IBA to NAA. This eliminates the growth inhibition phenomena such as stunted buds and clustering caused by the explants being exposed to a high concentration of 6-BA for a long time. The method uses NAA in conjunction with a low concentration of 6-BA to specifically promote the longitudinal cell elongation of the buds, overcoming the contradiction between the induction rate and the elongation rate. In the S3 subculture, this invention further incorporated a specific concentration of GA3 (0.5-1.5 mg / L) into the 6-BA and NAA cultures. GA3, in synergy with cytokinin, further promoted the elongation and thickening of new shoots and stems, as well as leaf expansion, resulting in optimal new shoot development. The S2 and S3 stage culture media of this invention both contained the anti-browning agent PVP-40 (0.3-1.2 mg / L). (g / L) Because the *Myrica rubra* variety has a high degree of lignification and is rich in phenolic substances, explants are prone to producing polyphenol oxidase during cutting wounds and tissue culture, leading to browning. This invention combines the physical adsorption and anti-browning effect of PVP-40 with mild hormone regulation at each stage, effectively adsorbing harmful phenolic substances at each stage. It effectively solves the technical problems of easy browning and death of *Myrica rubra* explants and the difficulty in balancing budding and elongation in the background technology, obtaining a regeneration system with low pollution and browning rates, high proliferation efficiency, and good seedling quality, providing reliable technical support for the large-scale breeding of superior *Myrica rubra* varieties.

[0010] In one possible implementation, in step S1, the new shoots are first treated by removing the leaves and immersing them in sterile water at 4°C for 12-24 hours before disinfection and sterilization. Then, they are pruned into sections with a length of 3 cm and 2-3 buds.

[0011] Compared with existing technologies, the above-mentioned technical solution involves removing leaves from new shoot segments before disinfection and sterilization, immersing them in sterile water, and then maintaining them at 4°C. This low-temperature environment significantly inhibits the initial activity of polyphenol oxidase in the plant. Simultaneously, soaking promotes the elimination of phenolic and other metabolic substances from the explant and increases the moisture content of the branches. Subsequently, the segments are pruned to a length of about 3 cm with 2-3 buds. This preserves an appropriate number of axillary buds to ensure a sufficient number of germination sites, while avoiding excessively large individual wounds that would accelerate the release and accumulation of polyphenol oxidase. This reduces the initial browning of the explants, eliminates surface impurities from the wild, and enhances the physiological functions of the branches themselves, laying a good foundation for the success rate of subsequent surface sterilization and the efficient germination of the first generation of culture.

[0012] In one possible implementation, step S1, the disinfection and sterilization includes: treating the new shoot stem segment with 70% alcohol for 0.5-1.5 min, and then treating it with a sodium hypochlorite solution with a mass fraction of 4%-6% for 8-10 min for surface sterilization.

[0013] Compared with existing technologies, the above-mentioned technical solution addresses the issue that, as a perennial woody plant, the surface environment of the branches of *Myrica rubra* is complex, disinfection is difficult, and its high degree of lignification makes it prone to browning. If the concentration of sodium hypochlorite solution is too low, the contamination situation cannot be significantly improved; if the concentration is increased and the treatment time is not adjusted accordingly, the browning rate of the explants will increase significantly while the contamination rate decreases, thus negatively impacting the budding effect. This invention first treats the surface structure with 70% alcohol for 0.5-1.5 min to destroy it, and then precisely combines this with a 4%-6% sodium hypochlorite solution for 8-10 min. This replaces the traditional highly toxic combination, making the operation safer and reducing the chemical damage of strong oxidants to the explant material. While ensuring the best budding effect, it successfully reduces the contamination rate and browning rate of the explants.

[0014] In one possible implementation, in step S21, the induction medium is formulated as WPM + 6-BA 1.0 mg / L + IBA 0.1 mg / L + PVP-40 1.0 g / L; in step S22, the elongation medium is formulated as WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L + PVP-40 1.0 g / L.

[0015] Compared with existing technologies, the formulation using the above-mentioned technical solution further improves the propagation efficiency. By precisely locking the formulation of WPM + 6-BA 1.0 mg / L + IBA 0.1 mg / L in S21, the optimal synergistic ratio of cytokinin and auxin at this concentration is utilized to effectively break apical dominance and stimulate axillary bud germination while avoiding excessive hormone toxicity, achieving an excellent induction multiple of up to 3.13. Subsequently, in S22, the formulation is adjusted to WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L, reducing the 6-BA concentration and specifically introducing trace amounts of NAA, which relieves the physiological inhibition of the longitudinal growth of young buds by the high concentration of cytokinin in the early stage, and specifically promotes the elongation of internode cells, allowing the bud elongation rate to exceed 95%. In addition, 1.0 g / L of PVP-40 is constantly added in both stages, which precisely provides the optimal physical adsorption concentration while ensuring the high-frequency differentiation activity of explants, and removes phenolic toxic substances released from the cutting wound and under strong hormone stimulation.

[0016] In one possible implementation, in step S21, after the sterilized stem segments are inoculated into the induction medium, they are first cultured in the dark, and then transferred to light culture after the explants have stabilized.

[0017] Compared with existing technologies, the above-mentioned technical solution can effectively suppress severe browning in the early stage of inoculation and significantly improve the survival rate of explants. The wound surface of the explant is largest when it has just been cut and inoculated. If it is directly exposed to light at this time, the light will strongly stimulate the production and activity of polyphenol oxidase. This implementation method avoids the stimulation of polyphenol oxidase by light by performing dark culture for a period of time after inoculation, slows down the rate at which phenolic substances are oxidized into browning substances, and provides a mild physiological buffer period for the explants that have just been subjected to the dual stimulation of mechanical damage and chemical disinfection. This effectively avoids the explants from accelerating browning or even being poisoned and killed by light stimulation. After their condition stabilizes, they are then transferred to light culture. From the perspective of operation process, this greatly ensures the success rate of the first generation of induction.

[0018] In one possible implementation, the conditions for the light culture are: light intensity 1500 lux, photoperiod 12h, culture temperature 24℃, and relative humidity 60%.

[0019] Compared with existing technologies, the above-mentioned technical solution can provide the optimal external physical growth environment for bayberry tissue culture seedlings, achieving a precise balance between promoting organ regeneration and preventing browning. Bayberry tissue is extremely sensitive to light and temperature conditions. If the light intensity is too high (more than 1500 lux as set in this implementation plan), it will significantly increase the activity of polyphenol oxidase, leading to aggravated browning of already sprouting buds or stem segments. The 12-hour photoperiod and constant temperature of 24°C combined with 60% relative humidity precisely meet the basic metabolic needs of bayberry new bud cell division, elongation, and leaf unfolding. This implementation method ensures that new buds carry out necessary photosynthesis and material accumulation during the elongation and proliferation stages, while strictly avoiding the environmental threshold that induces severe browning, thereby cultivating high-quality tissue culture seedlings with healthy growth and no physiological diseases.

[0020] In one possible implementation, in step S3, the subculture medium is formulated as follows: WPM + 6-BA 1.2 mg / L + NAA 0.1 mg / L + GA3 1.0 mg / L + PVP-40 0.5 g / L.

[0021] Compared with existing technologies, the above-mentioned technical solution can maximize the proliferation efficiency of bayberry tissue culture seedlings and fundamentally improve the morphological quality of new shoots. In subculture, excessively high concentrations of cytokinin 6-BA (above 1.8 mg / L) can lead to a significant reduction in the growth vigor of new shoots, resulting in stunted shoots and curled leaves, which is physiologically toxic. Conversely, excessively low concentrations cannot effectively stimulate the proliferation of clustered shoots. In this embodiment, a 6-BA concentration of 1.2 mg / L is used, and with the assistance of NAA, the proportion of gibberellin GA3 is specifically increased to 1.0 mg / L. The moderate increase of GA3 and the specific concentration of 6-BA produce a physiological synergy, promoting cell elongation and thickening of new shoot stem segments and helping leaves to unfold smoothly. This overcomes the common problem of clustered dwarfing in tissue culture subculture, improves the proliferation coefficient and average shoot length, and results in numerous new shoot leaves with good unfolding, providing an excellent seedling foundation for subsequent rooting and high-survival-rate domestication and transplanting.

[0022] In one possible implementation, the induction medium, elongation medium, and subculture medium are all based on WPM and contain 30 g / L sucrose and 6 g / L agar, with the pH adjusted to 5.8.

[0023] Another technical problem to be solved by the present invention is to provide a special combined culture medium for rapid propagation of water chestnut-type bayberry tissue culture, so as to solve the problems in the prior art where water chestnut-type bayberry is limited by a single culture medium formula during tissue culture, resulting in mutual restriction between bud induction multiple and bud elongation rate, and the easy occurrence of new bud clustering and dwarfing in subculture, as well as the extreme susceptibility to browning and necrosis caused by polyphenol toxicity throughout the process.

[0024] This invention provides a specialized combined culture medium for rapid propagation of *Myrica rubra* var. *rubra* via tissue culture, comprising a combination of a functional staged culture medium for primary culture and a subculture medium: Induction medium: used for early axillary bud induction, its formula is WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; Elongation medium: used for later bud growth, its formula is WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; Subculture medium: used for new shoot proliferation, its formula is WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L; Furthermore, the induction medium, elongation medium, and subculture medium were all based on WPM, and all contained 30 g / L sucrose and 6 g / L agar, with the pH value adjusted to 5.8.

[0025] This invention provides a specialized combined culture medium for the rapid propagation of *Myrica rubra* var. *rubra* through tissue culture. Compared with existing technologies, this invention has the following advantages: It replaces the existing method of relying on a single formula for primary induction and culture with a functional, multi-stage combined culture medium system comprising induction medium, elongation medium, and subculture medium. The induction medium uses a specific ratio of high concentrations of cytokinin (6-BA 0.8-1.2 mg / L) and auxin IBA (0.05-0.15 mg / L). This high concentration of cytokinin effectively breaks the apical dominance of the woody plant explant, stimulating a large number of cell divisions in the lateral axillary bud meristems, significantly increasing the germination rate of the primary culture, with an induction fold reaching 3.13. Subsequently, the culture is transferred to the elongation medium, where the 6-BA concentration is significantly reduced to 0.1-0.5 mg / L, and the auxin is specifically switched from IBA to NAA (0.01-0.05 mg / L). The high concentration of cytokinin (mg / L) promptly relieved the physiological inhibition of longitudinal cell growth in buds, and NAA was used to promote longitudinal cell elongation, resulting in rapid growth of the large number of induced young buds, with a bud elongation rate of 95%. Furthermore, the subculture medium was supplemented with GA3 (0.5-1.5 mg / L) on the basis of moderate concentrations of 6-BA and NAA. Utilizing the properties of gibberellin to promote cell wall relaxation and internode elongation, the clustering and dwarfing of new buds during multiple graftings were prevented, achieving a proliferation coefficient as high as 4.29 and an average bud length of 1.53 cm, with excellent leaf expansion. The above-mentioned phased plant growth regulators, along with the addition of PVP-40 (0.3-1.2 mg / L) to each culture medium, further enhanced the effect. This combined culture medium system (g / L) works in close synergy with the physiological characteristics of *Myrica rubra* var. *rubra*, which has a high degree of lignification, is rich in phenolic substances, and is highly susceptible to fatal browning during strong hormone stimulation and tissue differentiation. Through continuous physical adsorption of the anti-browning agent throughout the entire process, it maintains high-frequency differentiation and rapid growth of *Myrica rubra* tissue while consistently suppressing the browning rate to below 8% during tissue culture. This combined culture medium system effectively solves the problems in the background technology, such as the difficulty in simultaneously achieving bud break and growth in *Myrica rubra* tissue culture, poor quality of subcultured seedlings, and easy browning and death. It provides product support with extremely high conversion rate and stability for the industrialized, large-scale rapid propagation of *Myrica rubra* var. *rubra*. Attached Figure Description

[0026] Figure 1 This is a comparison chart of the effects of different hormone ratios in the initial culture after 30 days in the embodiments of the present invention; Figure 1 In the figure, a represents the initial culture effect of Comparative Example 7 using a hormone ratio of 0.2 mg / L 6-BA and 0.03 mg / L NAA; b is a graph showing the initial culture results of Comparative Example 11 using a hormone ratio of 1.0 mg / L 6-BA and 0.1 mg / L IBA. c shows the initial culture results of Comparative Example 7 using a hormone ratio of 0.2 mg / L 6-BA and 0.03 mg / L NAA. d shows the initial culture results of Comparative Example 11 using a hormone ratio of 1.0 mg / L 6-BA and 0.1 mg / L IBA. e is an image showing the effect of bud induction culture in Example 1, where buds were first induced and cultured with a hormone ratio of 1.0 mg / L 6-BA and 0.1 mg / L IBA. After all buds had sprouted, the culture was then transferred to a hormone ratio of 0.2 mg / L 6-BA and 0.03 mg / L NAA for bud elongation culture.

[0027] Figure 2 This is a comparison chart showing the growth of *Myrica rubra* var. *rubra* after 45 days of subculture under different hormone ratios in this embodiment of the invention. Figure 2 In the figure, a represents the subculture effect using a hormone ratio of 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 0.005 mg / L TDZ; b shows the subculture effect using a hormone ratio of 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 0.5 mg / L GA3; c shows the subculture effect using a hormone ratio of 1.2 mg / L 6-BA, 0.1 mg / L NAA, and 0.5 mg / L GA3; Figure d shows the subculture effect using a hormone ratio of 1.2 mg / L 6-BA, 0.1 mg / L NAA, and 1.0 mg / L GA3. e shows the effect of subculture using a hormone ratio of 1.8 mg / L 6-BA, 0.2 mg / L NAA, and 1.5 mg / L GA3; f shows the effect of continuous culture for 90 days using a hormone ratio of 0.6 mg / L 6-BA, 0.1 mg / L NAA, and 0.005 mg / L TDZ.

[0028] Figure 3 This is a schematic diagram illustrating the browning of explant stem segments and new shoots in an embodiment of the present invention; Figure 3 In the middle, a is a diagram showing the browning of the stem segment before bud sprouting in the first generation of culture (corresponding to the browning phenomenon comparison example 15). b shows the browning of stem segments after bud sprouting in the initial culture and some new buds after transfer to fresh culture medium (corresponding to browning phenomenon comparison example 15). c shows the browning of new shoots during subculture (corresponding to Comparative Figure 15 of browning phenomena). Detailed Implementation

[0029] First, those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make adjustments as needed to adapt to specific application scenarios.

[0030] The following are embodiments and comparative examples incorporating specific data to further illustrate the above-described technical solutions of the present invention: This invention provides a method for rapid propagation of *Myrica rubra* var. *rubra* via tissue culture, comprising the following steps: S1: Explant disinfection: Select semi-lignified new shoots in spring, disinfect and sterilize them to obtain sterilized stem segments; S2: Initial generation training with functional phases: S21: Bud induction stage: Sterilized stem segments are inoculated into induction medium, and axillary buds are induced to sprout using a combination of cytokinin and auxin IBA. The induction medium is: WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; S22: Bud elongation stage: After the buds sprout, the explants are transferred to the elongation medium, the cytokinin concentration is reduced and the auxin is switched to NAA to promote bud elongation. The elongation medium is: WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; S3: Subculture and proliferation: The elongated new shoots are transferred to a subculture medium for proliferation. The subculture medium is: WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L.

[0031] As a preferred embodiment, in step S1, the new shoots are first treated by removing the leaves and then being immersed in sterile water at 4°C for 12-24 hours before being disinfected and sterilized. After that, they are pruned into sections with a length of 3 cm and 2-3 buds.

[0032] As a preferred embodiment, in step S1, the disinfection and sterilization includes: treating the new shoot stem segments with 70% alcohol for 0.5-1.5 min, and then treating them with a sodium hypochlorite solution with a mass fraction of 4%-6% for 8-10 min for surface sterilization.

[0033] As a preferred embodiment, in step S21, the formulation of the induction medium is WPM + 6-BA 1.0 mg / L + IBA 0.1 mg / L + PVP-40 1.0 g / L; in step S22, the formulation of the elongation medium is WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L + PVP-40 1.0 g / L.

[0034] As a preferred embodiment, in step S21, after the sterilized stem segments are inoculated into the induction medium, they are first cultured in the dark, and then transferred to light culture after the explants have stabilized.

[0035] As a preferred embodiment, the conditions for light cultivation are: light intensity of 1500 lux, photoperiod of 12 h, cultivation temperature of 24℃, and relative humidity of 60%.

[0036] As a preferred embodiment, in step S3, the subculture medium is formulated as follows: WPM + 6-BA 1.2 mg / L + NAA 0.1 mg / L + GA3 1.0 mg / L + PVP-40 0.5 g / L.

[0037] As a preferred embodiment, the induction medium, elongation medium, and subculture medium are all based on WPM and contain 30 g / L sucrose and 6 g / L agar, with the pH value adjusted to 5.8.

[0038] This invention provides a specialized combined culture medium for rapid propagation of *Myrica rubra* var. *rubra* via tissue culture, comprising a combination of a functional staged culture medium for primary culture and a subculture medium: Induction medium: used for early axillary bud induction, its formula is WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; Elongation medium: used for later bud growth, its formula is WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; Subculture medium: used for new shoot proliferation, its formula is WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L; Furthermore, the induction medium, elongation medium, and subculture medium were all based on WPM, and all contained 30 g / L sucrose and 6 g / L agar, with the pH value adjusted to 5.8.

[0039] The following are embodiments incorporating specific data to further elaborate on the above-described technical solutions of the present invention: Example 1 This invention provides a special combined culture medium for rapid propagation of water chestnut-type bayberry through tissue culture and a method for rapid propagation of water chestnut-type bayberry through tissue culture.

[0040] The specialized combined culture medium includes an induction medium, an elongation medium, and a subculture medium: Induction medium: WPM as the basal medium, with 30 g / L sucrose and 6 g / L agar added, pH adjusted to 5.8, with the following formula: WPM + 6-BA 1.0 mg / L + IBA 0.1 mg / L + PVP-40 1.0 g / L.

[0041] Elongation medium: WPM as the basal medium, with 30 g / L sucrose and 6 g / L agar added, pH adjusted to 5.8, with the following formula: WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L + PVP-40 1.0 g / L.

[0042] Subculture medium: WPM as the basal medium, with 30 g / L sucrose and 6 g / L agar added, pH adjusted to 5.8, with the following formula: WPM + 6-BA 1.2 mg / L + NAA 0.1 mg / L + GA3 1.0 mg / L + PVP-40 0.5 g / L.

[0043] The method for rapid propagation of water chestnut-based waxberry via tissue culture includes the following steps: S1: Explant disinfection Select semi-lignified new shoots (approximately 3-5 mm in diameter) from spring (April-May). After removing the leaves, insert them into sterile water and pre-treat at 4°C for 12-24 hours. Then, use sterilized scissors to cut off the necrotic ends, trimming them into small sections approximately 3 cm long with 2-3 buds. Treat the stem sections with 70% alcohol for 1 min, followed by 5% sodium hypochlorite solution for 9 min for surface sterilization, obtaining sterilized stem sections.

[0044] S2: Initial generation training in functional phases S21: Bud induction stage: The morphological lower end of the sterilized stem segment is inoculated into the induction medium and cultured in the dark for a period of time. The axillary buds are induced to sprout by using a combination of high concentrations of cytokinin and auxin IBA.

[0045] S22: Bud elongation stage: After all buds have germinated and the explants are stable, the explants are transferred to the elongation medium, the cytokinin concentration is reduced and the auxin is switched to NAA, and then transferred to light culture to promote bud elongation. The light culture conditions are: light intensity 1500 lux, photoperiod 12 h, culture temperature 24℃, and relative humidity 60%.

[0046] S3: Subculture The elongated new shoots were transferred to the subculture medium for proliferation culture, and the culture conditions were the same as the light culture conditions of the S22 stage.

[0047] The following describes multiple sets of comparative experiments, in conjunction with embodiments of the present invention and comparative examples, to further elaborate on the present invention: Group 1 Variable Experiment: Verification of Explant Disinfection Parameters This experiment mainly verifies the effect of different concentrations and times of sodium hypochlorite in step S1 on the disinfection effect.

[0048] Example 2-3 Examples 2 and 3 are similar to Example 1, except that the disinfection method in step S1 is different: the treatment time of the 5% sodium hypochlorite solution is adjusted to 8 min and 10 min respectively.

[0049] Comparative Examples 1-3 Comparative Examples 1 to 3 are similar to Example 1, except that the disinfection method in step S1 is different: 3% sodium hypochlorite solution was used, and the treatment time was set to 10 min, 15 min, and 20 min respectively.

[0050] Comparative Examples 4-6 Comparative Examples 4 to 6 are similar to Example 1, except that the disinfection method in step S1 is different: 7% sodium hypochlorite solution was used, and the treatment time was set to 6 min, 7 min, and 8 min respectively.

[0051] The disinfection effects of the above Examples 1-3 and Comparative Examples 1-6 were tested, and the results are shown in Table 1 below: Table 1: Disinfection effect of *Myrica rubra* explants from Examples 1-3 and Comparative Examples 1-4 (70% alcohol for 1 min): Note: Contamination rate = Number of contaminated plants / Number of inoculated plants; Germination rate = Number of germination plants / Number of inoculated plants; Browning rate = Number of browned plants / Number of inoculated plants Based on the experimental results in Table 1 above, it can be seen that the disinfection method for explants in the primary culture is crucial for *Myrica rubra* tissue culture. This invention, by investigating the effects of different concentrations and durations of sodium hypochlorite on the disinfection effect, confirmed significant characterization differences between the examples and the comparative examples: As shown in Example 1, the best results were achieved with 5% sodium hypochlorite treatment for 9 minutes during disinfection. At this time, the contamination rate was effectively controlled at 12%, the browning rate was as low as 8%, and the bud budding effect reached its optimal state. Meanwhile, comparison with other groups revealed that when the concentration of the sodium hypochlorite solution increased (Comparative Examples 5-7), if the treatment time was not adjusted accordingly, the browning rate of the explants would significantly increase while the contamination rate decreased, thus causing irreversible negative effects on the budding effect. Conversely, if the concentration of the sodium hypochlorite solution was too low (Comparative Examples 1-4), even with a corresponding extension of the treatment time, the contamination situation could not be significantly improved, and the bud budding effect of the explants was poor. As a perennial woody plant, *Myrica rubra* has a high degree of lignification, and the surface environment of its branches is complex, making disinfection extremely difficult. The embodiments of this invention flexibly and precisely coordinated the concentration of the disinfectant and the treatment time, achieving an ideal disinfection effect.

[0052] In terms of material selection and pretreatment, this invention preferably uses new shoots of bayberry in spring (April-May). Since the plants grow in the wild environment for a long time, there are many pollutants attached to their surface. In the pretreatment stage, this invention first washes away surface impurities and dust, and then uses sterile water for 12-24 hours of maintenance. This effectively promotes the elimination of various metabolic substances in the tissue, increases the moisture of the branches and improves the vitality of the branches. Then, the branches are transferred to a clean bench for surface sterilization, which greatly reduces the initial contamination rate after sterilization from the source.

[0053] Regarding sterilization mechanisms and browning prevention, compared to the commonly used "ethanol + mercuric chloride" disinfectant combination in existing technologies, the "ethanol + sodium hypochlorite" disinfectant combination selected in this invention is not only safer for operators but also causes less chemical damage to explant materials. Studies have shown that browning of waxberry explants occurs more frequently in stem segments with higher lignification and terminal bud stem segments. This is because stem segments with higher lignification contain more and more active phenolic substances, while terminal bud stem segments have strong cell activity and are more sensitive to mechanical damage such as leaf removal. In addition, improper use of multiple disinfectants can easily lead to a decrease in branch vigor. Therefore, this invention effectively avoids excessive stimulation to explants caused by the superposition of reagent residues by carefully selecting stem segments and strictly limiting the transition window between 70% alcohol (0.5-1.5 min) and sodium hypochlorite (4%-6%, 8-10 min). This method ensures thorough sterilization while minimizing mechanical and chemical damage, effectively saving sterilization operation time, reducing reagent consumption, and significantly improving overall propagation efficiency.

[0054] Group 2 variable experiments: Formula validation for a single stage of primary culture This experiment mainly verifies the effects of different concentrations and times of culture medium formulations on bayberry in the initial culture stage of step S2.

[0055] Comparative Example 7 Comparative Example 7 is similar to Example 1, except that the functional phased culture mode of S21 to S22 of the present invention was not used in the initial culture stage. Instead, a single culture medium was used for continuous culture. The culture medium formula is: WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L.

[0056] Comparative Examples 8-9 Comparative Examples 8 and 9 are similar to Example 1, except that the initial culture stage did not use the functional phased culture mode of S21 to S22 of the present invention, but instead used a single culture medium for continuous culture. The culture medium formulations were: WPM + 6-BA 0.6 mg / L + NAA 0.03 mg / L and WPM + 6-BA 1.0 mg / L + NAA 0.03 mg / L, respectively.

[0057] Comparative Examples 10-12 Comparative Examples 10 to 12 are similar to Example 1, except that the initial culture stage did not use the functional phased culture mode of S21 to S22 of the present invention, but instead used a single culture medium for continuous culture. The culture medium formulas were: WPM + 6-BA 1.0 mg / L combined with IBA at concentrations of 0.05 mg / L, 0.1 mg / L, and 0.15 mg / L, respectively.

[0058] The statistical results are shown in Table 2 below: Table 2: Statistics on the initial cultivation effect of water chestnut-type waxberry (statistics after 30 days of cultivation, elongation standard ≥1cm) Note: Induction ratio = number of buds / number of bud points; bud elongation rate = bud elongation / number of buds; average bud length = total bud length / number of buds. Combined with Table 2 and Figure 1 The results showed that, in order to determine the optimal hormone ratio for the primary culture of *Myrica rubra* var. *rubra*, this invention set up multiple single-stage culture comparative examples (comparative examples 7 to 12) with different single hormone ratios to investigate their effects on the growth of *Myrica rubra* tissue culture seedlings. Combined with the data in Table 2, it can be found that, in the single-stage culture, comparative example 11 (using a formula of 1.0 mg / L 6-BA + 0.1 mg / L IBA) had the best effect on inducing buds; while comparative example 7 (using a formula of 0.2 mg / L 6-BA + 0.03 mg / L NAA) had the best effect on promoting bud elongation.

[0059] And through Figure 1 The comparison results shown further demonstrate the limitations of the above single-stage formulation: Figure 1 a and c (corresponding to a ratio of 7) and Figure 1 Comparative proportions b and d (corresponding to proportion 11) showed significant differences in characterization. Although the formulation of proportion 11 had an excellent effect on inducing shoots, the resulting physiological state was extremely unfavorable for subsequent shoot elongation culture. Although the formulation of proportion 7 had an excellent effect on promoting shoot elongation, the number of initial shoots induced was too small to meet the proliferation requirements of large-scale rapid propagation. The two formulations exhibited irreconcilable physiological contradictions in a single culture medium.

[0060] To address the technical problems in existing technologies where single-stage cultivation results in low bud height or high bud height but few shoots, this invention innovatively employs a functionally phased initial cultivation mode (i.e., the mode used in this invention and Example 1). For example... Figure 1 As shown in Figure e, in Example 1 of the present invention, the shoot induction culture was first carried out in stage S21 under the hormone ratio of 1.0 mg / L 6-BA + 0.1 mg / L IBA. After all the shoots had sprouted, they were promptly transferred to the hormone ratio of 0.2 mg / L 6-BA + 0.03 mg / L NAA for stage S22 shoot elongation culture. The results also showed that Example 1 of the present invention perfectly balanced high induction ratio and high elongation rate. The new shoots of the explant stem segments developed well and the growth was very impressive. The above comparison further revealed the underlying physiological response mechanism of the primary culture. The initial culture of this invention was based on WPM medium, using 6-BA combined with NAA or IBA as the basic plant growth regulator combination. The results of comparative examples 7 to 9 show that when the concentration of auxin (NAA) is relatively low and fixed, even a significant increase in the concentration of cytokinin (6-BA) does not significantly improve the induction effect. This indicates that NAA alone cannot play a synergistic dominant role in the induction of new shoots. However, the results of comparative examples 10 to 12 show that under the condition that high concentration of cytokinin (6-BA 1.0 mg / L) dominates, the combination of IBA in a specific concentration range (0.05-0.15 mg / L) as an auxiliary auxin can significantly improve the induction effect.

[0061] In summary, single formulations have irreconcilable limitations in terms of culture effects. However, the technical solution of this invention (including the examples) employs a phased combination and switching of specific formulations that promote bud induction and those that promote bud elongation. This not only reveals the sensitive response of *Myrica rubra* explants to specific hormone types at different developmental stages but also achieves unexpected and significant synergistic effects. It breaks through the core technical bottleneck in traditional *Myrica rubra* tissue culture propagation systems, providing highly valuable patented technical support for the efficient tissue culture of *Myrica rubra* and similar woody plants.

[0062] Group 3 Variable Experiments: Validation of Subculture Formula This group of experiments mainly verifies the effects of different hormone combinations and concentration boundaries in the S3 step subculture medium on the proliferation effect of tissue culture seedlings.

[0063] Example 4 Example 4 is similar to Example 1, except that the subculture medium formulation in step S3 is different: the GA3 concentration is adjusted to 0.5 mg / L, that is, the formulation is WPM + 6-BA 1.2 mg / L + NAA 0.1 mg / L + GA3 0.5 mg / L.

[0064] Example 5 Example 5 is similar to Example 1, except that the subculture medium formulation in step S3 is different: the formulation is adjusted to WPM + 6-BA 1.8 mg / L + NAA 0.2 mg / L + GA3 1.5 mg / L.

[0065] Comparative Example 13 Comparative Example 13 is similar to Example 1, except that the subculture medium formulation in step S3 does not fall within the protection scope of this invention: the formulation is WPM + 6-BA 0.6 mg / L + NAA 0.1 mg / L + GA3 0.5 mg / L.

[0066] Comparative Example 14 Comparative Example 14 is similar to Example 1, except that the subculture medium formulation in step S3 does not fall within the protection range of this invention: TDZ is used instead of GA3, and the concentration of 6-BA is also below the lower limit, i.e., the formulation is WPM + 6-BA 0.6 mg / L + NAA 0.1 mg / L + TDZ 0.005 mg / L.

[0067] The statistical results are shown in Table 3 below: Table 3. Statistics on the effects of subculture of *Myrica rubra* from *Myrica rubra* (cultured for 45 days). Note: Proliferation coefficient = number of new shoots / number of inoculated shoots; average shoot length = total shoot length / number of new shoots Through Table 3 and Figure 2 The experimental results show that, in order to clarify the optimal culture medium formula for the subculture and propagation of *Myrica rubra* var. *rubra*, this invention further set up multiple different combinations of plant growth regulators and investigated their effects on the growth of tissue culture seedlings. The results showed that there were significant differences in the subculture effects under different hormone ratios: Compared with control example 13 (6-BA concentration of 0.6 mg / L), its growth was as follows: Figure 2 As shown in Figure b) and Example 4 (6-BA concentration of 1.2 mg / L), their growth is as follows: Figure 2 As shown in Figure c), under the same basic conditions, appropriately increasing the concentration of cytokinin (6-BA) can effectively relieve proliferation inhibition, thereby significantly promoting the growth and increase in the number of new shoots and stems. Based on the confirmed optimal 6-BA concentration, a comparison was made between Example 4 and Example 1 (GA3 concentration was 1.0 mg / L), showing the same growth rate. Figure 2 As shown in Figure d), by appropriately increasing the proportion of gibberellin (GA3), the elongation and thickening of new shoots and stems and the unfolding of leaves can be significantly promoted; and Example 1, as the optimal ratio of the present invention, achieved a proliferation coefficient as high as 4.29 and an optimal overall development length of 1.53 cm.

[0068] However, higher hormone concentrations are not always better. Comparing Example 1 and Example 5 (where the 6-BA concentration reached 1.8 mg / L), their growth was as follows... Figure 2 As shown in Figure e), it can be observed that when the concentration of 6-BA reaches or exceeds the upper limit of the protection range of this invention, the growth potential of new shoots will be significantly reduced, manifested as stunted shoots and curled leaves. This verifies the critical significance of the upper limit of 6-BA concentration in subculture of this invention: that is, excessively high cytokinin concentration will lead to clustering and dwarfing toxicity in tissue culture seedlings.

[0069] This invention also compared the effects of introducing the novel plant growth regulator TDZ, as shown in Comparative Example 14. Although TDZ may have some potential to promote bud growth during long-term culture (e.g., 90 days), within the efficient and rapid propagation cycle (45 days) pursued in this invention, the formulation using TDZ to replace GA3 and reducing the concentration of 6-BA showed the worst performance in terms of proliferation coefficient (only 2.57) and average bud length (only 0.44 cm). The new buds were stunted and the leaves did not unfold, further confirming that TDZ does not have short-term applicability in the tissue culture rapid propagation system of water chestnut-type bayberry. The basic synergistic formulation of 6-BA + NAA + GA3 at a specific concentration established in this invention is a necessary and optimal technical choice for achieving high-quality, short-cycle seedling propagation.

[0070] Group 4 Variable Experiment: Comprehensive Anti-Browning Measures Comparison and Verification This group of experiments mainly verifies the comprehensive synergistic effect of specific environmental conditions and physical operations combined with anti-browning agents in inhibiting browning in this invention.

[0071] Comparative Example 15 Comparative Example 15 is similar to Example 1, except that the comprehensive anti-browning measures of the present invention were not adopted throughout the entire process: that is, after removing the leaves in step S1, the plants were directly kept in water at room temperature without undergoing a 4°C low-temperature pretreatment; the pruning was not strictly done with flat cutting; in the initial stage of induction in step S21, the plants were not cultured in the dark, but directly entered light culture; and the anti-browning agent PVP-40 was not added to the culture medium in each stage of steps S2 and S3.

[0072] Combined with Comparative Example 15 and Figure 3 The characterization of browning revealed that the lack of systematic anti-browning measures for the severe browning phenomenon that easily occurs in *Myrica rubra* explants often leads to disastrous consequences. In Comparative Example 15, which did not employ comprehensive anti-browning control, observation of the browning status of primary and subculture cultures revealed that the browning process of most explant stem segments first started from the cut ends, gradually expanding the affected area from the upper end to the lower end. Due to the larger contact area between the lower end and the culture medium, the degree of browning caused by phenolic toxicity was more severe (e.g., ...). Figure 3 (As shown in a and b); however, after being transferred to a fresh culture medium, the browning of the new shoots first started from the leaf tips and gradually spread to the entire shoot, eventually leading to stunted growth or even death of the shoots (as shown in a and b). Figure 3 (as shown in c).

[0073] To completely overcome the aforementioned problem of frequent browning-induced death, this invention (as shown in Example 1) not only precisely adds a specific concentration of PVP-40 to the culture medium at each stage to chemically adsorb harmful phenolic substances, but also systematically supplements multi-dimensional physical and environmental browning prevention measures: This invention creatively employs a 4°C low-temperature pretreatment during pretreatment, effectively inhibiting enzyme activity and reducing initial browning; and during inoculation and trimming, strictly adheres to the use of sterile scissors for flat cutting, ensuring clean and crisp cuts and avoiding large new wound surfaces, thereby greatly facilitating the regeneration and healing of explant materials and reducing the browning rate of the cut; and in the early stage of primary culture (S21 stage), a period of dark culture is first conducted, effectively preventing the explant materials from being directly stimulated by light in the early stages of cutting damage, thus avoiding excessive production of polyphenol oxidase. During dark culture, the contamination situation can be observed in a timely manner, and browned explants can be transferred. Light culture is only started after the physiological state of the explants has completely stabilized. This invention utilizes a comprehensive anti-browning synergistic mechanism, including PVP-40 chemical adsorption, 4℃ low-temperature inhibition, mechanical flat cutting for minimal damage, and phased dark culture buffering, to completely cut off the catalytic pathway of polyphenol oxidase from three levels: source trauma, process oxidation, and environmental stimulation. This effectively suppresses the browning rate throughout the tissue culture process to an extremely low level, thus completely eliminating the biggest obstacle to the efficient regeneration of water chestnut-based bayberries.

[0074] In summary, the rapid propagation method and dedicated culture medium for *Myrica rubra* var. *rubra* provided by this invention, through detailed comparative data from the above-mentioned multiple examples and comparative examples, fully demonstrate that this invention successfully breaks through the limitations of the single-stage, single-culture system in the prior art, and reveals the specific response mechanism of *Myrica rubra* var. *rubra* explants to plant growth regulators at different developmental stages. The two-stage hormone step-regulation system provided by this invention solves the problem of balancing bud induction fold and elongation rate in primary culture. By precisely defining the optimal synergistic concentration range of 6-BA, NAA, and GA3 in subculture, it completely overcomes the problems of easy clustering, dwarfing, and failure of leaves to unfold in tissue culture seedlings. This invention successfully interrupts the catalytic pathway of polyphenol oxidase from multiple dimensions, including physical, environmental, and chemical aspects, through a four-dimensional comprehensive anti-browning mechanism of PVP-40 chemical adsorption, 4℃ low-temperature pretreatment, aseptic flat cutting, and staged dark culture. The above-mentioned processes are closely linked and synergistic, ultimately overcoming the numerous technical problems of high pollution, easy browning and death, and low propagation efficiency in the tissue culture of Myrica rubra var. rubra., establishing a highly valuable technology for the large-scale, industrialized rapid propagation of high-quality seedlings.

[0075] In the description of this invention, the references to "one embodiment," "some embodiments," "in this embodiment," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0076] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for rapid propagation of *Myrica rubra* var. *rubra* via tissue culture, characterized in that, Includes the following steps: S1: Explant disinfection: Select semi-lignified new shoots in spring, disinfect and sterilize them to obtain sterilized stem segments; S2: Initial generation training with functional phases: S21: Bud induction stage: Sterilized stem segments are inoculated into induction medium, and axillary buds are induced to sprout using a combination of cytokinin and auxin IBA. The induction medium is: WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; S22: Bud elongation stage: After the buds sprout, the explants are transferred to the elongation medium, the cytokinin concentration is reduced and the auxin is switched to NAA to promote bud elongation. The elongation medium is: WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; S3: Subculture and proliferation: The elongated new shoots are transferred to a subculture medium for proliferation. The subculture medium is: WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L.

2. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, In step S1, before disinfection and sterilization, the new shoots need to have their leaves removed and be pre-treated in sterile water at 4°C for 12-24 hours. Then, they are pruned into sections with a length of 3 cm and 2-3 buds.

3. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, In step S1, the disinfection and sterilization includes: treating the new shoot stem segments with 70% alcohol for 0.5-1.5 min, and then treating them with a sodium hypochlorite solution with a mass fraction of 4%-6% for 8-10 min for surface sterilization.

4. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, In step S21, the induction medium is formulated as WPM + 6-BA 1.0 mg / L + IBA 0.1 mg / L + PVP-40 1.0 g / L; in step S22, the elongation medium is formulated as WPM + 6-BA 0.2 mg / L + NAA 0.03 mg / L + PVP-40 1.0 g / L.

5. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, In step S21, after the sterilized stem segments are inoculated into the induction medium, they are first cultured in the dark, and then transferred to light culture after the explants are in a stable state.

6. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 5, characterized in that, The conditions for the light culture were: light intensity of 1500 lux, photoperiod of 12 h, culture temperature of 24℃, and relative humidity of 60%.

7. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, In step S3, the subculture medium is formulated as follows: WPM + 6-BA 1.2 mg / L + NAA 0.1 mg / L + GA3 1.0 mg / L + PVP-40 0.5 g / L.

8. The method for rapid propagation of *Myrica rubra* by tissue culture according to claim 1, characterized in that, The induction medium, elongation medium, and subculture medium were all based on WPM and contained 30 g / L sucrose and 6 g / L agar, with the pH adjusted to 5.

8.

9. A special composite culture medium for rapid propagation of *Myrica rubra* var. *rubra* via tissue culture, characterized in that, This includes a combination of functional staged culture media for primary culture and subculture media: Induction medium: used for early axillary bud induction, its formula is WPM + 6-BA 0.8-1.2 mg / L + IBA 0.05-0.15 mg / L + PVP-40 0.8-1.2 g / L; Elongation medium: used for later bud elongation, its formula is WPM + 6-BA 0.1-0.5 mg / L + NAA 0.01-0.05 mg / L + PVP-40 0.8-1.2 g / L; Subculture medium: used for new shoot proliferation, its formula is WPM + 6-BA 1.0-1.8 mg / L + NAA 0.1-0.2 mg / L + GA3 0.5-1.5 mg / L + PVP-40 0.3-0.7 g / L; Furthermore, the induction medium, elongation medium, and subculture medium were all based on WPM, and all contained 30 g / L sucrose and 6 g / L agar, with the pH value adjusted to 5.8.

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