Tissue culture and rapid propagation method of eucalyptus citrina

Abstract

The invention relates to the technical field of plant tissue culture, and particularly discloses a tissue culture and rapid propagation method of eucalyptus luminocarpa, which comprises the following steps: (1) selecting an explant, removing leaves, sterilizing the surface, and cutting small stem sections; (2) inoculating the small stem segments into an induction culture medium, and performing dark culture to obtain calluses; (3) transferring the calluses into a differentiation solid culture medium, and performing differentiation culture to obtain differentiated buds; (4) inoculating the differentiated buds into a proliferation culture medium, and carrying out suspension proliferation culture to obtain cluster buds; (5) selecting cluster bud single plants, inoculating the cluster bud single plants into a rooting culture medium, and inducing rooting culture to obtain tissue culture seedlings; and (6) selecting tissue culture seedlings, transferring the tissue culture seedlings to a greenhouse, opening a cover, hardening the seedlings, and transplanting the seedlings into a culture medium to obtain eucalyptus citrina plants. According to the tissue culture and rapid propagation method of the eucalyptus globulus, the explant pollution rate is low, the callus induction rate and the differentiation rate are high, the browning phenomenon in the tissue culture process can be inhibited, the tissue activity is protected, the robust development of a root system is promoted, and the rooting effect is improved.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, and more specifically, it relates to a method for rapid tissue culture propagation of Eucalyptus globulus. Background Technology

[0002] Eucalyptus nitens, belonging to the genus Eucalyptus in the family Myrtaceae, is a fast-growing tree species with both ecological and economic value. Its wood has a dense structure and moderate hardness, making it widely used in furniture manufacturing, papermaking, and architectural decoration. The essential oil extracted from its branches and leaves is rich in active ingredients such as eucalyptol, exhibiting significant antibacterial, anti-inflammatory, and insect-repellent effects, and is in high demand in the pharmaceutical, daily chemical, and agricultural and forestry protection industries. Furthermore, Eucalyptus nitens has a well-developed root system and strong adaptability, making it a preferred species for ecological afforestation, soil and water conservation, and degraded land restoration in southern mountainous areas, playing an important role in ecological protection and the development of the forestry industry.

[0003] However, the natural propagation of Eucalyptus globulus is mainly based on seed propagation. Its seeds have problems such as long dormancy period, hard seed coat and poor permeability, resulting in low natural germination rate. In addition, seedlings grown from seed exhibit trait segregation and cannot stably inherit the superior characteristics of the parent plant, resulting in inconsistent seedling quality. On the other hand, propagation by cuttings is strictly limited by the age of the mother tree, season and environmental conditions. Furthermore, cuttings are prone to carrying soil-borne pathogens (such as Phytophthora and Fusarium), resulting in low survival rate after transplanting and making it difficult to meet the needs of large-scale seedling production.

[0004] While tissue culture technology can achieve exponential proliferation under sterile conditions through in vitro explant culture and hormone regulation, overcoming seasonal limitations, the current tissue culture system for *Eucalyptus globulus* still faces significant technical bottlenecks: low bud induction rate, substandard proliferation coefficient, insufficient root differentiation efficiency, and high contamination rate. Existing *Eucalyptus globulus* tissue culture research largely employs a "bud-to-bud" proliferation model, obtaining complete plants through the induction of clustered buds combined with adventitious bud rooting. However, research on regeneration technologies based on callus organogenesis pathways is almost nonexistent, due to technical obstacles such as low callus induction rate, weak differentiation capacity, and high risk of genetic variation. Therefore, there is an urgent need to develop a rapid tissue culture propagation method with low contamination rate, high proliferation coefficient, rapid rooting, and high seedling survival rate to provide technical support for the industrialization of *Eucalyptus globulus*. Summary of the Invention

[0005] To address the technical problems mentioned in the background section, this application provides a rapid tissue culture propagation method for Eucalyptus globulus.

[0006] A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, sterilize the surface, rinse with sterile water, dry the surface moisture, and cut small stem segments with 1-2 axillary buds. (2) Inoculate the stem segments into the induction medium and culture in the dark for 20-25 days to obtain callus tissue; (3) Transfer the callus tissue to a differentiation solid medium and culture it for 25-30 days to differentiate into shoots; (4) Inoculate the differentiated buds into the proliferation medium and carry out suspension proliferation culture for 10-15 days to obtain clustered buds; (5) Select single plants with clustered buds, inoculate them into rooting medium, and induce rooting culture for 8-12 days to obtain tissue culture seedlings; (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3-5 days. Then transplant them into the culture medium and cultivate them for 20-30 days to obtain bright fruit eucalyptus plants.

[0007] Preferably, in step (1), the explants are collected in spring, from March to April, and the collection site is a healthy branch in the upper part of a 3-5 year old Eucalyptus globulus mother tree that is free from pests and diseases.

[0008] Preferably, the surface sterilization method in step (1) is as follows: after soaking in alcohol with a volume fraction of 70-75% for 30-60 seconds, soak in sodium hypochlorite solution with a mass fraction of 0.1-0.2% for 8-12 minutes.

[0009] Preferably, in step (2), the induction medium is a modified MS medium with 0.5-1.0 mg / L 2,4-D, 0.08-0.12 mg / L TDZ, 1-3 mg / L VB2, 25-35 g / L sucrose, 5-9 g / L agar, and pH 5.8-6.0.

[0010] Preferably, the dark culture temperature in step (2) is 24-26℃ and the relative humidity is 50-70%.

[0011] Preferably, in step (3), the differentiation solid medium is modified MS as the basic medium, with TDZ 0.05-0.1 mg / L, IBA 0.1-0.2 mg / L, 6-BA 0.5-0.7 mg / L, PVP 0.3-0.5 g / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.0 added.

[0012] Preferably, in step (3), the differentiation culture temperature is 24-26℃, the light intensity is 1500-2500lx, and the light duration is 15-18h / d.

[0013] Preferably, in step (4), the proliferation medium is modified MS as the basic medium, with NAA 0.1-0.3 mg / L, 6-BA 0.3-0.5 mg / L, PVP 0.3-0.5 g / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.0 added.

[0014] Preferably, in step (4), the power of the pneumatic bioreactor is 12-15W, the frequency is 50-60Hz, and the air flow rate is 8-10L / min.

[0015] Preferably, the suspension proliferation culture conditions in step (4) are: temperature 23-25℃, light intensity 2500-3500lx, light duration 15-18h / d, and relative humidity 50-60%.

[0016] Preferably, in step (5), the rooting medium is 1 / 2 modified MS as the basic medium, with added ABT1 0.4-0.6 mg / L, IBA 0.5-1.0 mg / L, PVP 0.3-0.4 g / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.0.

[0017] Preferably, the rooting culture conditions in step (5) are: temperature 24-26℃, light intensity 2000-2500lx, light duration 14-16h / d, and relative humidity 50-60%.

[0018] Preferably, during the hardening-off period of the seedlings in step (6), ventilation is carried out 2-3 times a day for 30-60 minutes each time; spraying is carried out 2-4 times a day for 8-12 days before transplanting to maintain the moisture content of the culture medium at 60-70%.

[0019] Preferably, the humidity of the transplanting culture environment in step (6) is 70-80% and the temperature is 20-28℃.

[0020] Preferably, the culture medium in step (6) is composed of peat, perlite and vermiculite in a mass ratio of 3-5:1-2:1-3, and the culture medium is sterilized by high pressure steam at 121℃ for 30-40 minutes before use. The transplanted culture is sprayed with carbendazim solution once a week for 2-3 consecutive weeks.

[0021] In summary, this application has the following beneficial effects: This application systematically optimizes the components of four key culture media—induction, differentiation, proliferation, and rooting—by precisely targeting the core needs of each culture stage, forming a scientific formula system adapted to the tissue culture characteristics of Eucalyptus globulus. The specific advantages and mechanisms of action of each culture medium are as follows: In the induction medium, a synergistic system of "auxin-cytokinin-nutrient enhancement" was constructed using a nutritionally complete modified MS as the base carrier. Among them, 2,4-D, as a classic auxin hormone, can effectively activate the dedifferentiation program of explant somatic cells, promote the restoration of totipotency of stem segment cells and the formation of callus tissue; although TDZ belongs to the cytokinin class, it can specifically enhance the signal transduction efficiency of 2,4-D at low concentrations, solving the problem of "slow start-up and low efficiency" in single auxin induction, resulting in more uniform and full callus tissue formation; VB2, as a key coenzyme, directly participates in the carbohydrate metabolism and energy conversion of explants, providing sufficient energy supply for the dedifferentiation process and preventing yellowing and necrosis of explants due to nutrient depletion.

[0022] The differentiation medium focuses on the triple goals of "bud formation, stability, and anti-browning," with optimized ratios of TDZ, IBA, 6-BA, and PVP. TDZ and 6-BA form a synergistic combination of cytokinins, with TDZ focusing on initiating the differentiation of bud primordia within the callus tissue, while 6-BA focuses on promoting bud elongation and robustness. Together, they achieve bud differentiation "from nothing to something, from weak to strong." IBA, as a mild auxin, can precisely regulate the balance of "cytokinin-auxin" ratio in the medium, avoiding excessive bud growth or differentiation disorder caused by cytokinin overdose, and enhancing the stability of the differentiation process. PVP, through its polar groups in its molecular structure, binds to phenolic substances secreted by the callus tissue, preventing the oxidation of phenols to form browning substances, thus fundamentally solving the common browning and seedling death problems in tissue culture.

[0023] The proliferation medium is designed with "high-efficiency propagation and maintenance of activity" as its core principle, combining NAA, 6-BA, and PVP. 6-BA, as the main proliferation hormone, efficiently stimulates the germination of axillary buds in differentiated shoots, promoting the differentiation of single buds into clusters of shoots. NAA, added at a low concentration, enhances the cell division stimulation effect of 6-BA and promotes nutrient absorption at the base of the shoot, avoiding the problems of weak and easily detached shoots caused by using 6-BA alone, thus significantly improving the quantity and quality of clustered shoots. PVP continuously exerts its anti-browning effect, especially in the suspended proliferation environment of the pneumatic bioreactor, reducing the release of phenols caused by tissue damage and ensuring high tissue activity during proliferation.

[0024] The rooting medium is designed to meet the specific needs of root development, using a 1 / 2 modified MS base supplemented with ABT1, IBA, and PVP. The 1 / 2 modified MS creates a low-osmotic-pressure environment by reducing the concentration of inorganic salts such as nitrogen, phosphorus, and potassium, preventing high salt concentrations from burning young roots and adapting to the root's preference for diluted fertilizer and aversion to high osmotic pressure. ABT1, as a dedicated rooting promoter, rapidly induces the formation of root primordia at the base of clustered buds, while IBA further promotes root primordia elongation and stimulates lateral root germination. Together, these two components form a robust root structure with numerous roots, significantly improving rooting quality. PVP protects meristematic tissues during root differentiation, preventing browning that could negatively impact root germination.

[0025] In summary, the core advantage of this application lies in the construction of a culture medium system that is "stage-adapted, component-synergistic, and pain point-targeted." The induction stage focuses on dedifferentiation efficiency, the differentiation stage takes into account both bud quality and anti-browning, the proliferation stage balances quantity and activity, and the rooting stage is adapted to root growth characteristics. Each culture medium independently solves the core problems of its corresponding stage, and is also connected through common technologies such as PVP and auxin / cytokinin ratio. Ultimately, this achieves high efficiency and stability throughout the entire tissue culture process of Eucalyptus globulus, providing key technical support for its large-scale rapid propagation. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the embodiments.

[0027] The 2,4-D used in the embodiments and comparative examples of this invention was purchased from Shandong Keyuan Chemical Co., Ltd.; TDZ was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.; VB2 was purchased from Shaanxi Chenming Biotechnology Co., Ltd.; modified MS was purchased from Beijing Kulaibo Technology Co., Ltd.; IBA was purchased from Hubei Langbowan Biomedical Co., Ltd.; 6-BA was purchased from Hubei Xinghengye Technology Co., Ltd.; PVP was purchased from Huzhou Shenhua Polymer Materials Co., Ltd.; NAA was purchased from Solarbio, a professional biochemical reagent supplier; ABT1 was purchased from Wuhan Sanying Biotechnology Co., Ltd.; peat, perlite (particle size: 6μm) and vermiculite (particle size: 40-60 mesh) were all purchased from Lingshou Yongshun Mineral Products Processing Plant.

[0028] Examples 1-3 provide a rapid tissue culture propagation method for Eucalyptus globulus.

[0029] Example 1 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, PVP 0.3g / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0030] Example 2 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 72% alcohol for 45 seconds, then soak them in 0.15% sodium hypochlorite solution for 10 minutes. After surface sterilization, rinse them 6 times with sterile water, dry the surface moisture, and cut off small stem segments with 2 axillary buds. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 22 days at a temperature of 25℃ and a relative humidity of 60% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.8 mg / L of 2,4-D, 0.10 mg / L of TDZ, 2 mg / L of VB2, 30 g / L of sucrose, 7 g / L of agar, and pH 5.9. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 28 days at a temperature of 25℃, a light intensity of 2000 lx, and a light duration of 17 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.08 mg / L, IBA 0.15 mg / L, 6-BA 0.6 mg / L, PVP 0.4 g / L, sucrose 30 g / L, agar 7 g / L, and pH 5.9. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 14W, a frequency of 55Hz and an aeration rate of 9L / min. The suspension proliferation culture was carried out for 12 days under the conditions of temperature of 25℃, light intensity of 3000lx, light duration of 17h / d and relative humidity of 55% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.2mg / L, 6-BA 0.4mg / L, PVP 0.4g / L, sucrose 30g / L, and pH 5.9. (5) Select single buds with a height of 3cm and inoculate them into rooting medium. Under the conditions of temperature 25℃, light intensity 2300lx, light duration 15h / d and relative humidity 55%, induce rooting culture for 10 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.5mg / L, IBA 0.8mg / L, PVP 0.35g / L, sucrose 30g / L, agar 7g / L, and pH 5.9. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 4 days. During the hardening period, ventilate 3 times a day for 45 minutes each time. Then transplant them into the culture medium. Spray 3 times a day for 10 days before transplanting to maintain the moisture content of the culture medium at 65%. Control the ambient humidity at 75% and the temperature at 25℃. 25 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 4:1.5:2. The culture medium is sterilized by high pressure steam at 121℃ for 35 minutes before use. Spray 0.15% carbendazim solution once a week for 3 consecutive weeks.

[0031] Example 3 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 75% alcohol for 60 seconds, then soak them in 0.2% sodium hypochlorite solution for 12 minutes. After surface sterilization, rinse them 7 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 25 days at a temperature of 26℃ and a relative humidity of 70% to obtain callus tissue. The induction medium was based on modified MS medium, with 1.0 mg / L of 2,4-D, 0.12 mg / L of TDZ, 3 mg / L of VB2, 35 g / L of sucrose, 9 g / L of agar, and pH 6.0. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 30 days at a temperature of 26℃, a light intensity of 2500 lx, and a light duration of 18 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added ingredients: TDZ 0.1 mg / L, IBA 0.2 mg / L, 6-BA 0.7 mg / L, PVP 0.5 g / L, sucrose 35 g / L, agar 9 g / L, and pH 6.0. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 15W, a frequency of 60Hz and an aeration rate of 10L / min. The suspension proliferation culture was carried out for 15 days under the conditions of temperature of 25℃, light intensity of 3500lx, light duration of 18h / d and relative humidity of 60% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.3mg / L, 6-BA 0.5mg / L, PVP 0.5g / L, sucrose 35g / L, and pH 6.0. (5) Select single buds with a height of 3cm and inoculate them into rooting medium. Under the conditions of temperature 26℃, light intensity 2500lx, light duration 16h / d and relative humidity 60%, induce rooting culture for 12 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.6mg / L, IBA 1.0mg / L, PVP 0.4g / L, sucrose 35g / L, agar 9g / L, and pH 6.0. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 5 days. During the hardening period, ventilate 3 times a day for 60 minutes each time. Then transplant them into the culture medium. Spray 4 times a day for 12 days before transplanting to maintain the moisture content of the culture medium at 70%. Control the ambient humidity at 80% and the temperature at 28℃. 30 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 5:2:3. The culture medium is sterilized by high pressure steam at 121℃ for 40 minutes before use. Spray 0.2% carbendazim solution once a week for 3 consecutive weeks.

[0032] Comparative Example 1 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium with 0.5 mg / L 2,4-D, 1 mg / L VB2, 25 g / L sucrose, 5 g / L agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, PVP 0.3g / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0033] Comparative Example 2 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the addition of TDZ 0.05 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, PVP 0.3g / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0034] Comparative Example 3 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was based on modified MS medium with 0.3mg / L 6-BA, 0.3g / L PVP, 25g / L sucrose, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0035] Comparative Example 4 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut small stem segments with 1-2 axillary buds. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0036] Comparative Example 5 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, PVP 0.3g / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is based on 1 / 2 modified MS medium, with the addition of IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0037] Comparative Example 6 A rapid tissue culture propagation method for Eucalyptus globulus includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, soak them in 70% alcohol for 30 seconds, then soak them in 0.1% sodium hypochlorite solution for 8 minutes. After surface sterilization, rinse them 5 times with sterile water, dry the surface moisture, and cut off small stem segments with one axillary bud. (2) The stem segments were inoculated into the induction medium and cultured in the dark for 20 days at a temperature of 24℃ and a relative humidity of 50% to obtain callus tissue. The induction medium was based on modified MS medium, with 0.5 mg / L of 2,4-D, 0.08 mg / L of TDZ, 1 mg / L of VB2, 25 g / L of sucrose, 5 g / L of agar, and pH 5.8. (3) The callus tissue was transferred to the differentiation solid medium and cultured for 25 days at a temperature of 24℃, a light intensity of 1500 lx, and a light duration of 15 h / d to differentiate into shoots. The differentiation solid medium was based on modified MS medium, with the following added: TDZ 0.05 mg / L, IBA 0.1 mg / L, 6-BA 0.5 mg / L, PVP 0.3 g / L, sucrose 25 g / L, agar 5 g / L, and pH 5.8. (4) The differentiated shoots were inoculated into the proliferation medium and transferred to a pneumatic bioreactor with a power of 12W, a frequency of 50Hz, and an aeration rate of 8L / min. The suspension proliferation culture was carried out for 10 days under the conditions of temperature 23℃, light intensity 2500lx, light duration 15h / d, and relative humidity 50% to obtain clustered shoots. The proliferation medium was modified MS as the basic medium, with the addition of NAA 0.1mg / L, 6-BA 0.3mg / L, PVP 0.3g / L, sucrose 25g / L, and pH 5.8. (5) Select single buds with a height of 2cm and inoculate them into rooting medium. Under the conditions of temperature 24℃, light intensity 2000lx, light duration 14h / d and relative humidity 50%, induce rooting culture for 8 days to obtain tissue culture seedlings. The rooting medium is modified MS as the basic medium, with the addition of ABT1 0.4mg / L, IBA 0.5mg / L, PVP 0.3g / L, sucrose 25g / L, agar 5g / L, and pH 5.8. (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3 days. During the hardening period, ventilate twice a day for 30 minutes each time. Then transplant them into the culture medium. Spray twice a day for 8 days before transplanting to maintain the moisture content of the culture medium at 60%. Control the ambient humidity at 70% and the temperature at 20℃. 20 days after transplanting, bright fruit eucalyptus plants are obtained. The culture medium is composed of peat moss, perlite and vermiculite in a mass ratio of 3:1:1. The culture medium is sterilized by high pressure steam at 121℃ for 30 minutes before use. Spray the transplanted seedlings with a 0.1% carbendazim solution once a week for 2 consecutive weeks.

[0038] Performance testing The induction effect of the tissue culture rapid propagation methods of Eucalyptus globulus provided in Examples 1-3 and Comparative Examples 1-6 of this application was tested. Each example / comparative example had 3 parallel experiments, with 50 explants inoculated in each group. The same testing environment and instruments were used uniformly. The specific test indicators and methods are as follows: Explant contamination rate: The number of contaminated explants (surface bacteria growth or internal rot) was counted 7 days after inoculation. Contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%; Callus induction rate: After the induction culture is completed, the number of surviving explants that form callus is counted. Induction rate = (number of callus formed / number of surviving explants) × 100%; Callus differentiation rate: After differentiation culture, the number of callus tissues that differentiated into shoots (shoot height ≥ 0.5 cm) was counted. Differentiation rate = (number of callus tissues that differentiated into shoots / total number of callus tissues) × 100%; Proliferation coefficient: After the proliferation culture is completed, the total number of clustered buds is counted. Proliferation coefficient = (total number of clustered buds / number of inoculated differentiated buds). Rooting rate: After the rooting culture is completed, the number of rooted shoot clusters (root length ≥ 0.3 cm) is counted. Rooting rate = (number of roots / number of inoculated shoot clusters) × 100%; Browning rate of tissue culture seedlings: The number of browned explants / callus / bud clusters was counted, and the browning rate was calculated as (number of browned explants / total number of treatments) × 100%. Transplant survival rate: 30 days after transplanting, the number of surviving plants is counted, and the survival rate = (number of surviving plants / number of transplanted plants) × 100%; The specific statistical results are shown in Table 1.

[0039] Table 1. Induction effects of the tissue culture rapid propagation method for Eucalyptus globulus in Examples 1-3 and Comparative Examples 1-6.

[0040] As shown in Table 1, the proposed rapid propagation method for Eucalyptus globulus via tissue culture exhibits low explant contamination rates, high callus induction and differentiation rates, inhibits browning during tissue culture, protects tissue activity, promotes robust root development, and enhances rooting efficiency. Furthermore, the optimized hardening-off and transplanting processes improve the adaptability of tissue-cultured seedlings to the external environment, significantly increasing transplant survival rates and ensuring plant growth stability. This method enables efficient and stable large-scale rapid propagation of Eucalyptus globulus, providing reliable technical support for its germplasm resource conservation and industrial cultivation.

[0041] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A rapid tissue culture propagation method for Eucalyptus globulus, characterized in that, Includes the following steps: (1) Select healthy stem segments of Eucalyptus globulus as explants. After removing the leaves, sterilize the surface, rinse with sterile water, dry the surface moisture, and cut small stem segments with 1-2 axillary buds. (2) Inoculate the stem segments into the induction medium and culture in the dark for 20-25 days to obtain callus tissue; (3) Transfer the callus tissue to a differentiation solid medium and culture it for 25-30 days to differentiate into shoots; (4) Inoculate the differentiated buds into the proliferation medium and carry out suspension proliferation culture for 10-15 days to obtain clustered buds; (5) Select single plants with clustered buds, inoculate them into rooting medium, and induce rooting culture for 8-12 days to obtain tissue culture seedlings; (6) Select tissue culture seedlings and move them to the greenhouse. Open the cover and harden the seedlings for 3-5 days. Then transplant them into the culture medium and cultivate them for 20-30 days to obtain bright fruit eucalyptus plants.

2. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, The surface sterilization method in step (1) is as follows: after soaking in alcohol with a volume fraction of 70-75% for 30-60 seconds, soak in sodium hypochlorite solution with a mass fraction of 0.1-0.2% for 8-12 minutes.

3. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, In step (2), the induction medium is modified MS as the basic medium, with the following added: 2,4-D 0.5-1.0 mg / L, TDZ 0.08-0.12 mg / L, VB2 1-3 mg / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.

0.

4. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, In step (3), the differentiation solid medium is modified MS as the basic medium, with TDZ 0.05-0.1 mg / L, IBA 0.1-0.2 mg / L, 6-BA 0.5-0.7 mg / L, PVP 0.3-0.5 g / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.0 added.

5. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, In step (4), the proliferation medium is modified MS as the basic medium, with NAA 0.1-0.3 mg / L, 6-BA 0.3-0.5 mg / L, PVP 0.3-0.5 g / L, sucrose 25-35 g / L added, and pH 5.8-6.

0.

6. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, The suspension proliferation culture conditions in step (4) are: temperature 23-25℃, light intensity 2500-3500lx, light duration 15-18h / d, and relative humidity 50-60%.

7. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, In step (5), the rooting medium is based on 1 / 2 modified MS, with added ABT1 0.4-0.6 mg / L, IBA 0.5-1.0 mg / L, PVP 0.3-0.4 g / L, sucrose 25-35 g / L, agar 5-9 g / L, and pH 5.8-6.

0.

8. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, The rooting culture conditions in step (5) are: temperature 24-26℃, light intensity 2000-2500lx, light duration 14-16h / d, and relative humidity 50-60%.

9. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, During the hardening-off period of the seedlings in step (6), ventilate 2-3 times a day for 30-60 minutes each time; spray 2-4 times a day for 8-12 days before transplanting to maintain the moisture content of the culture medium at 60-70%.

10. The method for rapid tissue culture propagation of Eucalyptus globulus according to claim 1, characterized in that, In step (6), the culture medium is composed of peat, perlite and vermiculite in a mass ratio of 3-5:1-2:1-3. The culture medium is sterilized by high pressure steam at 121℃ for 30-40 minutes before use. The transplanted culture is sprayed with carbendazim solution once a week for 2-3 consecutive weeks.

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