Four-stage culture and transplanting method for aseptic rapid propagation of eucalyptus regnans

Abstract

This invention relates to the field of seedling propagation technology, and more particularly to a four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus. The four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus provided by this invention includes four core stages: callus induction, bud differentiation, proliferation, and rooting. Through specific culture medium formulations and environmental conditions, it achieves highly efficient propagation, significantly improving production efficiency, reducing production costs, better meeting market demands, and is highly compatible with existing processes. It requires no development of new equipment or production lines, involves small investment, is suitable for industrial production applications, has broad application prospects, and yields significant economic and social benefits.

Description

Technical Field

[0001] This invention relates to the field of seedling propagation technology, and in particular to a four-stage cultivation and transplanting method for aseptic rapid propagation of Eucalyptus globulus. Background Technology

[0002] Eucalyptus globulus is a species of eucalyptus in the Myrtaceae family. It is a large tree, typically 40-70 meters tall and 1-2 meters in diameter. It generally has uniformly shaped, vertical stems that can cover more than half of its height, and smooth, grayish-white bark. Native to Australia, it grows at altitudes of 600-1600 meters and is a light-loving species. It prefers cool to warm, humid to semi-humid climates, and is intolerant of hot, dry winds and drought. It is one of the most frost-resistant, fast-growing timber species for industrial use, and can withstand temperatures as low as -14°C. Its suitable growing area has an average monthly temperature of 21-26°C for the hottest month and -5-2°C for the coldest month, with an average annual rainfall of 750-1750 mm. Eucalyptus globulus grows best in moist loam, but moderately fertile soils can also meet its growth requirements, especially clay soils as the best.

[0003] Eucalyptus globulus, a superior fast-growing industrial timber species, has a basic wood density of 0.45-0.61 kg / m³, making it suitable for general construction, flooring and interior decoration, engineered wood products, and pulp production. Rapid propagation of Eucalyptus globulus is primarily achieved through tissue culture, a technique that efficiently produces high-quality seedlings while maintaining varietal characteristics. As market demand for Eucalyptus globulus continues to rise, the need for supplying high-quality seedlings through rapid propagation is also increasing.

[0004] Therefore, providing a more efficient method for rapid propagation of Eucalyptus globulus via tissue culture is of great significance to the development of the industry, and this is precisely the problem faced in this field. Summary of the Invention

[0005] In view of this, the present invention provides a four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus. The four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus provided by the present invention can further improve the seedling propagation efficiency.

[0006] This invention provides a four-stage cultivation and transplanting method for aseptic rapid propagation of Eucalyptus globulus, comprising the following steps: (1) Using young stem segments with axillary buds of Eucalyptus globulus as explants, surface sterilization, aseptic washing, cutting 2 cm young stem segments with 1 axillary bud and induction culture were carried out in sequence to obtain callus tissue; (2) The callus tissue is subjected to differentiation culture to obtain differentiated buds; (3) The differentiated buds are cultured for proliferation to obtain clustered buds; (4) Inducing rooting culture in individual plants of the clustered buds to obtain tissue culture seedlings; (5) The tissue culture seedlings were moved to the greenhouse, washed, hardened off, and transplanted into the culture medium for further cultivation to obtain Eucalyptus globulus plants.

[0007] Preferably, the induction culture is carried out in an induction medium; the components of the induction medium include modified MS medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.3-0.5 mg / L naphthaleneacetic acid, 0.2-0.4 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

[0008] Preferably, the induction culture temperature is 24~26℃, the relative humidity is 40~50%, the pH value is 5.7~5.8, and the time is 24~26 days; the first 1 to 5 days of the induction culture are dark culture, and the 6th day is light culture, with a light intensity of 1000~1200 lx and a light duration of 14~15 h / d.

[0009] Preferably, the differentiation culture is carried out in a differentiation medium; the components of the differentiation medium include modified MS medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.05-0.2 mg / L naphthaleneacetic acid, 0.4-0.6 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

[0010] Preferably, the differentiation culture is carried out at a temperature of 24-26°C, a relative humidity of 40-50%, a pH of 5.7-5.8, and a duration of 24-26 days; the first to fifth days of the differentiation culture are dark culture, and the sixth day is light culture, with a light intensity of 1000-1600 lx and a light duration of 14-15 h / d.

[0011] Preferably, the proliferation culture is a suspension proliferation culture; the equipment for the suspension proliferation culture includes a pneumatic bioreactor; the pneumatic bioreactor has a power of 12-13W, a frequency of 50Hz, an aeration rate of 7-8L / min, an operating pressure of 0.03-0.04MPa, and a pH value of 5.7-5.8; the proliferation culture is carried out in a proliferation medium; the components of the proliferation medium include modified MS medium, 500-80... 0 mg / L calcium nitrate, 170~270 mg / L potassium dihydrogen phosphate, 1600~1750 mg / L potassium nitrate, 300~370 mg / L magnesium sulfate, 550~850 mg / L ammonium nitrate, 0.05~0.08 mg / L naphthaleneacetic acid, 0.25~0.35 mg / L 6-benzylaminopurine, 5~8 mg / L citric acid, 6~10 mg / L cysteine, 25~35 g / L sucrose, and 4.5~6 g / L agar.

[0012] Preferably, the temperature for the proliferation culture is 24~26℃, the relative humidity is 40~50%, and the time is 24~26 days; the first 1 to 5 days of the proliferation culture are dark culture, and the 6th day is light culture, with a light intensity of 2400~2500 lx and a light duration of 15~16 h / d.

[0013] Preferably, the rooting induction culture is carried out in a rooting induction medium; the components of the rooting induction medium include modified MS medium, 300-450 mg / L calcium nitrate, 150-200 mg / L potassium dihydrogen phosphate, 600-850 mg / L potassium sulfate, 200-250 mg / L magnesium sulfate, 350-450 mg / L ammonium nitrate, 0.4-0.6 mg / L indolebutyric acid, 0.2-0.3 mg / L naphthaleneacetic acid, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 20-25 g / L sucrose, and 6-7 g / L carrageenan.

[0014] Preferably, the temperature for the induced rooting culture is 23-25℃, the relative humidity is 50-55%, the light intensity is 1600-1800 lx, the light duration is 15-16 h / d, and the pH value is 5.7-5.8; the induced rooting culture time is 21-23 days.

[0015] Preferably, the transplanting culture is carried out at a temperature of 25-28°C for 14 days; the transplanting culture includes a first stage (days 1-3), a second stage (days 4-7), and a third stage (days 8-14) performed sequentially; the relative humidity of the environment in the first stage is 90-95%, and the light intensity is 2500-3000 lx; ​​the relative humidity of the environment in the second stage is 70-80%, and the light intensity is 3000-5000 lx; ​​the relative humidity of the environment in the third stage is 50-65%, and the light intensity is not less than 5000 lx.

[0016] Compared with existing technologies, the four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus provided by this invention has the following beneficial effects: The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus provided by this invention includes four core stages: callus induction, bud differentiation, proliferation and rooting. Through specific culture medium formulations and environmental conditions, it achieves efficient propagation, has broad application prospects, and significant economic and social benefits. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of this invention, the accompanying drawings used in the embodiments of this invention or in the prior art are briefly described below. For those skilled in the art, other drawings can be derived from the following drawings without creative effort, and all such drawings are within the protection scope of this invention.

[0018] Figure 1 The graph shows a comparison of the browning rate and transplant survival rate of tissue culture seedlings from Examples 1-3 and Comparative Examples 1-10. Detailed Implementation

[0019] This invention provides a four-stage cultivation and transplanting method for aseptic rapid propagation of Eucalyptus globulus, comprising the following steps: (1) Using young stem segments with axillary buds of Eucalyptus globulus as explants, surface sterilization, aseptic washing, cutting 2 cm young stem segments with 1 axillary bud and induction culture were carried out in sequence to obtain callus tissue; (2) The callus tissue is subjected to differentiation culture to obtain differentiated buds; (3) The differentiated buds are cultured for proliferation to obtain clustered buds; (4) Inducing rooting culture in individual plants of the clustered buds to obtain tissue culture seedlings; (5) The tissue culture seedlings were moved to the greenhouse, washed, hardened off, and transplanted into the culture medium for further cultivation to obtain Eucalyptus globulus plants.

[0020] This invention uses young stem segments with axillary buds from *Eucalyptus globulus* as explants, which undergo sequential surface sterilization, aseptic washing, cutting of 2 cm young stem segments with one axillary bud, and induction culture to obtain callus tissue. In this invention, the surface sterilization preferably includes sequential soaking in an ethanol solution and then in a sodium hypochlorite solution; the volume concentration of the ethanol solution used for the ethanol solution soaking is preferably 70%, and the soaking time is preferably 30-35 seconds, more preferably 32 seconds.

[0021] In this invention, the mass concentration of the sodium hypochlorite solution used for soaking is preferably 0.5-1%, more preferably 0.7%; the soaking time of the sodium hypochlorite solution is preferably 8-10 minutes, more preferably 9 minutes.

[0022] In this invention, the sterile water washing method is preferably rinsing; the single rinsing time is preferably 10-20 seconds; and the number of rinsings is preferably 5-8 times.

[0023] In this invention, the induction culture is preferably carried out in an induction medium; the components of the induction medium preferably include modified MS (1 / 2 MS) medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.3-0.5 mg / L naphthaleneacetic acid, 0.2-0.4 mg / L 6-benzylaminopurine (6-BA), 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

[0024] In this invention, the preferred temperature for the induction culture is 24-26℃, more preferably 25℃; the preferred relative humidity is 40-50%, more preferably 45%; the preferred pH value is 5.7-5.8; and the preferred time is 24-26 days, more preferably 25 days. The induction culture is preferably carried out in the dark for the first 1-5 days, and then under light culture begins on the 6th day. The preferred light intensity for the under light culture is 1000-1200 lx, and the preferred light duration is 14-15 h / d.

[0025] After obtaining callus tissue, the present invention performs differentiation culture on the callus tissue to obtain differentiated shoots. In the present invention, the differentiation culture is preferably carried out in a differentiation medium; the components of the differentiation medium preferably include modified MS (3 / 4 MS) medium, 500~800 mg / L calcium nitrate, 170~270 mg / L potassium dihydrogen phosphate, 1600~1750 mg / L potassium nitrate, 300~370 mg / L magnesium sulfate, 550~850 mg / L ammonium nitrate, 0.05~0.2 mg / L naphthaleneacetic acid, 0.4~0.6 mg / L 6-benzylaminopurine, 5~8 mg / L citric acid, 6~10 mg / L cysteine, 25~35 g / L sucrose and 4.5~6 g / L agar.

[0026] In this invention, the temperature for differentiation culture is preferably 24-26℃, more preferably 25℃, the relative humidity is preferably 40-50%, more preferably 45%, and the pH value is preferably 5.7-5.8; the differentiation culture time is preferably 24-26 days, more preferably 25 days; the first to fifth days of differentiation culture are preferably dark culture, and light culture begins on the sixth day. The light intensity for light culture is preferably 1000-1600 lx, more preferably 1400 lx, and the light exposure time is preferably 14-15 h / d.

[0027] After obtaining differentiated buds, the present invention performs proliferation culture on the differentiated buds to obtain clustered buds. In the present invention, the proliferation culture is preferably a suspension proliferation culture; the equipment for the suspension proliferation culture preferably includes a pneumatic bioreactor; the power of the pneumatic bioreactor is preferably 12~13W, the frequency is preferably 50Hz, the aeration rate is preferably 7~8L / min, the operating pressure is preferably 0.03~0.04MPa, and the pH value is preferably 5.7~5.8.

[0028] In this invention, the proliferation culture is preferably carried out in a proliferation medium; the components of the proliferation medium preferably include modified MS (1 / 2 MS) medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.05-0.08 mg / L naphthaleneacetic acid, 0.25-0.35 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

[0029] In this invention, the temperature for proliferation culture is preferably 24~26℃, more preferably 25℃, and the relative humidity is preferably 40~50%, more preferably 45%; the proliferation culture time is preferably 24~26 days, more preferably 25 days; the first 1 to 5 days of proliferation culture are preferably dark culture, and light culture begins on the 6th day. The light intensity for light culture is preferably 2400~2500 lx, and the light duration is preferably 15~16 h / d, more preferably 15 h / d.

[0030] After obtaining the clustered buds, the present invention induces rooting culture in individual plants of the clustered buds to obtain tissue culture seedlings. In the present invention, the height of the individual plants of the clustered buds is preferably 2~2.5cm, more preferably 2~2.3cm.

[0031] In this invention, the rooting induction culture is preferably carried out in a rooting induction medium; the components of the rooting induction medium preferably include modified MS (1 / 2 MS) medium, 300-450 mg / L calcium nitrate, 150-200 mg / L potassium dihydrogen phosphate, 600-850 mg / L potassium sulfate, 200-250 mg / L magnesium sulfate, 350-450 mg / L ammonium nitrate, 0.4-0.6 mg / L indolebutyric acid, 0.2-0.3 mg / L naphthaleneacetic acid, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 20-25 g / L sucrose, and 6-7 g / L carrageenan.

[0032] In this invention, the preferred temperature for the induced rooting culture is 23-25℃, the preferred relative humidity is 50-55%, the preferred light intensity is 1600-1800 lx, the preferred light duration is 15-16 h / d, and the preferred pH value is 5.7-5.8; the preferred duration for the induced rooting culture is 21-23 days, more preferably 22 days.

[0033] After obtaining the tissue culture seedlings, this invention transfers them to a greenhouse, washes them, hardens them by opening the cover, and then transplants them into a culture medium for further cultivation, resulting in *Eucalyptus globulus* plants. In this invention, sterile water is preferably used for washing the seedlings. This washing process removes residual culture medium from the seedling roots.

[0034] In this invention, the temperature for hardening off the seedlings with the lid open is preferably 23~28℃, the light intensity is preferably 2000~2400lx, ventilation is carried out twice a day for 20~30 minutes each time, and the total time for hardening off the seedlings with the lid open is preferably 35~40 hours, more preferably 38 hours.

[0035] In this invention, the culture medium preferably comprises peat moss, perlite, and vermiculite in a mass ratio of 3:1:1; the culture medium is preferably autoclaved at 121°C for 30 minutes before use; and the moisture content of the culture medium is preferably 45-50 wt%. During transplanting culture, the culture medium is sprayed with a 0.1 wt% carbendazim solution once a week for two consecutive weeks.

[0036] In this invention, the temperature for transplanting and cultivation is preferably 25~28℃, more preferably 26~27℃, and the time is preferably 14 days.

[0037] In this invention, the transplanting culture preferably includes a first stage (days 1-3), a second stage (days 4-7), and a third stage (days 8-14) performed sequentially; the relative humidity of the environment in the first stage is preferably 90-95%, and the light intensity is preferably 2500-3000 lx; ​​the relative humidity of the environment in the second stage is preferably 70-80%, and the light intensity is preferably 3000-5000 lx; ​​the relative humidity of the environment in the third stage is preferably 50-65%, and the light intensity is preferably not less than 5000 lx.

[0038] To further illustrate the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments.

[0039] Example 1: This embodiment presents a four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus. The specific steps are as follows: Young stem segments with axillary buds of Eucalyptus globulus were used as explants. They were soaked in 70% ethanol solution for 32 seconds, then in 0.7% sodium hypochlorite solution for 9 minutes, rinsed with sterile water for 15 seconds, and repeated 7 times. Two-cm segments with one axillary bud were then cut and incubated on an induction medium (components 1 / 2). Callus tissue was induced and cultured for 25 days at 25°C, 45% relative humidity, and pH 5.7 in MS medium (650 mg / L calcium nitrate, 220 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.4 mg / L naphthaleneacetic acid, 0.3 mg / L 6-benzylaminopurine, 6.5 mg / L citric acid, 8 mg / L cysteine, 30 g / L sucrose, and 5 g / L agar). Days 1-5 were in the dark, and from day 6 onwards, light culture was conducted at an illumination intensity of 1100 lx for a preferred duration of 15 h / d.

[0040] The obtained callus tissue was cultured in differentiation medium, which consisted of 3 / 4 MS medium, 700 mg / L calcium nitrate, 200 mg / L potassium dihydrogen phosphate, 1700 mg / L potassium nitrate, 320 mg / L magnesium sulfate, 750 mg / L ammonium nitrate, 0.1 mg / L naphthaleneacetic acid, 0.5 mg / L 6-benzylaminopurine, 7 mg / L citric acid, 8 mg / L cysteine, 32 g / L sucrose, and 5 g / L agar. The differentiation culture temperature was 25℃, the relative humidity was 45%, the pH was 5.8, and the differentiation culture lasted for 25 days. The first 5 days of differentiation culture were in the dark, and the 6th day onwards were under light culture. The light intensity for light culture was 1200 lx, and the light duration was 15 h / d, which yielded differentiated shoots.

[0041] The obtained differentiated shoots were cultured in suspension in a pneumatic bioreactor and a proliferation medium. The pneumatic bioreactor had a power of 12W, a frequency of 50Hz, an aeration rate of 7L / min, an operating pressure of 0.04MPa, and a pH of 5.8. The proliferation medium consisted of 1 / 2... MS medium containing 600 mg / L calcium nitrate, 240 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 350 mg / L magnesium sulfate, 650 mg / L ammonium nitrate, 0.06 mg / L naphthaleneacetic acid, 0.3 mg / L 6-benzylaminopurine, 7 mg / L citric acid, 9 mg / L cysteine, 28 g / L sucrose, and 6 g / L agar was used for suspension proliferation culture at 25℃ and 45% relative humidity for 25 days. The first 5 days of proliferation culture were in the dark, and from the 6th day onwards, artificial light culture was conducted at an intensity of 2500 lx for 15 h / d, resulting in clustered shoots.

[0042] Individual plantlets with clustered shoots reaching 2 cm in height were induced to root in a rooting medium. The rooting medium consisted of 1 / 2 MS medium, 350 mg / L calcium nitrate, 170 mg / L potassium dihydrogen phosphate, 750 mg / L potassium sulfate, 220 mg / L magnesium sulfate, 400 mg / L ammonium nitrate, 0.5 mg / L indolebutyric acid, 0.25 mg / L naphthaleneacetic acid, 7 mg / L citric acid, 7 mg / L cysteine, 24 g / L sucrose, and 7 g / L carrageenan. The rooting culture was conducted at 25℃, with a relative humidity of 50%, a light intensity of 1700 lx, a photoperiod of 16 h / d, and a pH of 5.8. After 22 days of rooting culture, tissue culture seedlings were obtained.

[0043] Tissue culture seedlings were transferred to a greenhouse and sterilely washed to remove any remaining culture medium residue from the roots. They were then hardened off for 38 hours at 26°C and 2200 lx light intensity, with ventilation twice for 25 minutes each time. Afterward, they were transplanted into a 3:1:1 mixture of peat moss, perlite, and vermiculite with a moisture content of 45 wt%. The culture medium was autoclaved at 121°C for 30 minutes before use. During transplanting, the culture medium was sprayed weekly with a 0.1 wt% carbendazim solution for two consecutive weeks. The seedlings were then cultured at 25°C for 14 days (stage 1 (days 1-3): relative humidity 90%, light intensity 2500 lx; ​​stage 2 (days 4-7): relative humidity 70%, light intensity 3000 lx; ​​stage 3 (days 8-14): relative humidity 50%, light intensity 5200 lx) to obtain *Eucalyptus globulus* plants.

[0044] Example 2: This embodiment presents a four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus. The specific steps are as follows: Stem segments with axillary buds from *Eucalyptus globulus* were used as explants. They were soaked in 70% ethanol solution for 32 seconds, then in 0.7% sodium hypochlorite solution for 9 minutes, rinsed with sterile water for 15 seconds, and repeated 7 times. Stem segments with one axillary bud were then cut and inducing culture medium (components 1 / 2) The callus tissue was induced and cultured for 24 days in MS medium (600 mg / L calcium nitrate, 170 mg / L potassium dihydrogen phosphate, 1750 mg / L potassium nitrate, 300 mg / L magnesium sulfate, 550 mg / L ammonium nitrate, 0.5 mg / L naphthaleneacetic acid, 0.4 mg / L 6-benzylaminopurine, 8 mg / L citric acid, 6 mg / L cysteine, 35 g / L sucrose, and 5 g / L agar) at 26°C, 48% relative humidity, and pH 5.7. The first 5 days of induction culture were in the dark, and the 6th day onwards were under light culture with an illumination intensity of 1200 lx and a light exposure time of 14 h / d to obtain callus tissue.

[0045] The obtained callus tissue was cultured in differentiation medium, which consisted of 3 / 4 MS medium, 500 mg / L calcium nitrate, 270 mg / L potassium dihydrogen phosphate, 1600 mg / L potassium nitrate, 370 mg / L magnesium sulfate, 850 mg / L ammonium nitrate, 0.2 mg / L naphthaleneacetic acid, 0.4 mg / L 6-benzylaminopurine, 8 mg / L citric acid, 10 mg / L cysteine, 35 g / L sucrose, and 6 g / L agar. The differentiation culture was conducted at 26℃, with a relative humidity of 45% and a pH of 5.8 for 25 days. The first 5 days of differentiation culture were conducted in the dark, and the next 6 days were conducted under light. The light intensity for the light culture was 1400 lx, and the light duration was 15 h / d, which resulted in the formation of differentiated shoots.

[0046] The obtained differentiated shoots were cultured in suspension in a pneumatic bioreactor and a proliferation medium. The pneumatic bioreactor had a power of 13W, a frequency of 50Hz, an aeration rate of 8L / min, an operating pressure of 0.04MPa, and a pH of 5.8. The proliferation medium consisted of 1 / 2... MS medium containing 650 mg / L calcium nitrate, 230 mg / L potassium dihydrogen phosphate, 1710 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 730 mg / L ammonium nitrate, 0.08 mg / L naphthaleneacetic acid, 0.28 mg / L 6-benzylaminopurine, 7 mg / L citric acid, 9 mg / L cysteine, 33 g / L sucrose, and 6 g / L agar was used for suspension proliferation culture at 24℃ and 45% relative humidity for 26 days. The first 5 days of proliferation culture were in the dark, and from the 6th day onwards, artificial light culture was conducted at a light intensity of 2450 lx for 15 h / d, resulting in clustered shoots.

[0047] Individual plantlets with clustered shoots reaching 2 cm in height were induced to root in a rooting medium. The rooting medium consisted of 1 / 2 MS medium, 350 mg / L calcium nitrate, 160 mg / L potassium dihydrogen phosphate, 750 mg / L potassium sulfate, 240 mg / L magnesium sulfate, 380 mg / L ammonium nitrate, 0.55 mg / L indolebutyric acid, 0.22 mg / L naphthaleneacetic acid, 5.5 mg / L citric acid, 7.5 mg / L cysteine, 23 g / L sucrose, and 6.5 g / L carrageenan. The rooting culture was conducted at 25℃, with a relative humidity of 55%, a light intensity of 1700 lx, a photoperiod of 15 h / d, and a pH of 5.8. After 21 days of rooting culture, tissue culture seedlings were obtained.

[0048] Tissue culture seedlings were transferred to a greenhouse and sterilely washed to remove any remaining culture medium residue from the roots. They were then hardened off for 38 hours at 27°C and 2200 lx light intensity, with ventilation twice for 25 minutes each time. Afterward, they were transplanted into a 3:1:1 mixture of peat moss, perlite, and vermiculite with a moisture content of 47 wt%. The culture medium was autoclaved at 121°C for 30 minutes before use. During transplanting, the culture medium was sprayed weekly with a 0.1 wt% carbendazim solution for two consecutive weeks. The seedlings were then cultured at 26°C for 14 days (stage 1 (days 1-3): relative humidity 95%, light intensity 2700 lx; ​​stage 2 (days 4-7): relative humidity 80%, light intensity 4000 lx; ​​stage 3 (days 8-14): relative humidity 60%, light intensity 5100 lx) to obtain *Eucalyptus globulus* plants.

[0049] Example 3: This embodiment presents a four-stage culture and transplanting method for the aseptic rapid propagation of Eucalyptus globulus. The specific steps are as follows: Stem segments with axillary buds from *Eucalyptus globulus* were used as explants. They were soaked in 70% ethanol solution for 32 seconds, then in 0.7% sodium hypochlorite solution for 9 minutes, rinsed with sterile water for 15 seconds, and repeated 7 times. Stem segments with one axillary bud were then cut and inducing culture medium (components 1 / 2) Callus tissue was induced and cultured for 25 days in MS medium (800 mg / L calcium nitrate, 270 mg / L potassium dihydrogen phosphate, 1600 mg / L potassium nitrate, 370 mg / L magnesium sulfate, 550 mg / L ammonium nitrate, 0.3 mg / L naphthaleneacetic acid, 0.4 mg / L 6-benzylaminopurine, 8 mg / L citric acid, 6 mg / L cysteine, 25 g / L sucrose, and 4.5 g / L agar) at 24°C, 42% relative humidity, and pH 5.8. The first 5 days of induction culture were in the dark, and from day 6 onwards, light culture was conducted at an intensity of 1000 lx for 15 h / d to obtain callus tissue.

[0050] The obtained callus tissue was cultured in differentiation medium, which consisted of 3 / 4 MS medium, 500 mg / L calcium nitrate, 260 mg / L potassium dihydrogen phosphate, 1680 mg / L potassium nitrate, 310 mg / L magnesium sulfate, 620 mg / L ammonium nitrate, 0.15 mg / L naphthaleneacetic acid, 0.45 mg / L 6-benzylaminopurine, 6.5 mg / L citric acid, 7.2 mg / L cysteine, 26 g / L sucrose, and 5.3 g / L agar. The differentiation culture was conducted at 25℃, with a relative humidity of 47% and a pH of 5.8 for 25 days. The first 5 days of differentiation culture were in the dark, and from day 6 onwards, artificial light culture was conducted at a light intensity of 1300 lx for 15 h / d to obtain differentiated shoots.

[0051] The obtained differentiated shoots were cultured in suspension in a pneumatic bioreactor and a proliferation medium. The pneumatic bioreactor had a power of 13W, a frequency of 50Hz, an aeration rate of 7L / min, an operating pressure of 0.03MPa, and a pH of 5.8. The proliferation medium consisted of 1 / 2... MS medium containing 600 mg / L calcium nitrate, 210 mg / L potassium dihydrogen phosphate, 1660 mg / L potassium nitrate, 360 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.06 mg / L naphthaleneacetic acid, 0.3 mg / L 6-benzylaminopurine, 8 mg / L citric acid, 6 mg / L cysteine, 31 g / L sucrose, and 5.5 g / L agar was used for suspension proliferation culture at 25℃ and 45% relative humidity for 24 days. The first 5 days of proliferation culture were in the dark, and from the 6th day onwards, artificial light culture was conducted at a light intensity of 2450 lx for 16 h / d, resulting in clustered shoots.

[0052] Individual shoots with a height of 2 cm were induced to root in a rooting medium containing 1 / 2 MS medium, 420 mg / L calcium nitrate, 180 mg / L potassium dihydrogen phosphate, 690 mg / L potassium sulfate, 230 mg / L magnesium sulfate, 390 mg / L ammonium nitrate, 0.5 mg / L indolebutyric acid, 0.25 mg / L naphthaleneacetic acid, 8 mg / L citric acid, 10 mg / L cysteine, 20 g / L sucrose, and 7 g / L carrageenan. The rooting culture was conducted at 25℃, with a relative humidity of 55%, a light intensity of 1800 lx, a photoperiod of 15 h / d, and a pH of 5.8 for 23 days to obtain tissue culture seedlings.

[0053] Tissue culture seedlings were transferred to a greenhouse and sterilely washed to remove any remaining culture medium residue from the roots. They were then hardened off for 38 hours at 26°C and 2200 lx light intensity, with ventilation twice for 25 minutes each time. Afterward, they were transplanted into a 3:1:1 mixture of peat moss, perlite, and vermiculite with a moisture content of 48 wt%. The culture medium was autoclaved at 121°C for 30 minutes before use. During transplanting, the culture medium was sprayed weekly with a 0.1 wt% carbendazim solution for two consecutive weeks. The seedlings were then cultured at 28°C for 14 days (stage 1 (days 1-3): relative humidity 92%, light intensity 2700 lx; ​​stage 2 (days 4-7): relative humidity 75%, light intensity 4000 lx; ​​stage 3 (days 8-14): relative humidity 55%, light intensity 5000 lx) to obtain *Eucalyptus globulus* plants.

[0054] Comparative Example 1: The preparation method of this comparative example is the same as that of Example 1, except that the components of the induction medium are: 1 / 2 MS medium, 650 mg / L calcium nitrate, 220 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.6 mg / L naphthaleneacetic acid, 0.6 mg / L 6-benzylaminopurine, 6.5 mg / L citric acid, 8 mg / L cysteine, 30 g / L sucrose and 5 g / L agar.

[0055] Comparative Example 2: The preparation method of this comparative example is the same as that of Example 1, except that the components of the induction medium are: 1 / 2 MS medium, 650 mg / L calcium nitrate, 220 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.3 mg / L 6-benzylaminopurine, 6.5 mg / L citric acid, 8 mg / L cysteine, 30 g / L sucrose and 5 g / L agar.

[0056] Comparative Example 3: The preparation method of this comparative example is the same as that of Example 1, except that the components of the induction medium are 1 / 2 MS medium, 650 mg / L calcium nitrate, 220 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.4 mg / L naphthaleneacetic acid, 6.5 mg / L citric acid, 8 mg / L cysteine, 30 g / L sucrose and 5 g / L agar.

[0057] Comparative Example 4: The preparation method of this comparative example is the same as that of Example 1, except that the components of the induction medium are: 1 / 2 MS medium, 650 mg / L calcium nitrate, 220 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 340 mg / L magnesium sulfate, 700 mg / L ammonium nitrate, 0.4 mg / L naphthaleneacetic acid, 0.3 mg / L 6-benzylaminopurine, 9 mg / L citric acid, 8 mg / L cysteine, 30 g / L sucrose and 5 g / L agar.

[0058] Comparative Example 5: The preparation method of this comparative example is the same as that of Example 1, except that the components of the differentiation medium are 3 / 4 MS medium, 700 mg / L calcium nitrate, 200 mg / L potassium dihydrogen phosphate, 1700 mg / L potassium nitrate, 320 mg / L magnesium sulfate, 750 mg / L ammonium nitrate, 0.5 mg / L 6-benzylaminopurine, 7 mg / L citric acid, 8 mg / L cysteine, 32 g / L sucrose and 5 g / L agar.

[0059] Comparative Example 6: The preparation method of this comparative example is the same as that of Example 1, except that the components of the differentiation medium are 3 / 4 MS medium, 700 mg / L calcium nitrate, 200 mg / L potassium dihydrogen phosphate, 1700 mg / L potassium nitrate, 320 mg / L magnesium sulfate, 750 mg / L ammonium nitrate, 0.1 mg / L naphthaleneacetic acid, 7 mg / L citric acid, 8 mg / L cysteine, 32 g / L sucrose and 5 g / L agar.

[0060] Comparative Example 7: The preparation method of this comparative example is the same as that of Example 1, except that the components of the proliferation medium are 1 / 2 MS medium, 600 mg / L calcium nitrate, 240 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 350 mg / L magnesium sulfate, 650 mg / L ammonium nitrate, 0.3 mg / L 6-benzylaminopurine, 7 mg / L citric acid, 9 mg / L cysteine, 28 g / L sucrose and 6 g / L agar.

[0061] Comparative Example 8: The preparation method of this comparative example is the same as that of Example 1, except that the components of the proliferation medium are 1 / 2 MS medium, 600 mg / L calcium nitrate, 240 mg / L potassium dihydrogen phosphate, 1650 mg / L potassium nitrate, 350 mg / L magnesium sulfate, 650 mg / L ammonium nitrate, 0.06 mg / L naphthaleneacetic acid, 7 mg / L citric acid, 9 mg / L cysteine, 28 g / L sucrose and 6 g / L agar.

[0062] Comparative Example 9: The preparation method of this comparative example is the same as that of Example 1, except that the components of the rooting induction medium are 1 / 2 MS medium, 350 mg / L calcium nitrate, 170 mg / L potassium dihydrogen phosphate, 750 mg / L potassium sulfate, 220 mg / L magnesium sulfate, 400 mg / L ammonium nitrate, 0.25 mg / L naphthaleneacetic acid, 7 mg / L citric acid, 7 mg / L cysteine, 24 g / L sucrose and 7 g / L carrageenan.

[0063] Comparative Example 10: The preparation method of this comparative example is the same as that of Example 1, except that the components of the rooting induction medium are 1 / 2 MS medium, 350 mg / L calcium nitrate, 170 mg / L potassium dihydrogen phosphate, 750 mg / L potassium sulfate, 220 mg / L magnesium sulfate, 400 mg / L ammonium nitrate, 0.5 mg / L indolebutyric acid, 7 mg / L citric acid, 7 mg / L cysteine, 24 g / L sucrose and 7 g / L carrageenan.

[0064] Test Example 1: The effects of Examples 1-3 and Comparative Examples 1-10 were tested. Each group was inoculated with 50 explants, and the same testing conditions and instruments were used, as detailed below: 1) Explant contamination rate: The number of contaminated explants (surface bacterial growth or internal rot) was counted 7 days after inoculation; contamination rate = (number of contaminated explants / total number of inoculated explants) × 100%; 2) 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%; 3) 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%; 4) Proliferation coefficient: After the proliferation culture is completed, the total number of clustered shoots is counted; Proliferation coefficient = (Total number of clustered shoots / Number of inoculated differentiated shoots) × 100%; 5) Rooting rate: After the rooting culture is completed, count the number of rooted shoot clusters (root length ≥ 0.3 cm); Rooting rate = (number of roots / number of inoculated shoot clusters) × 100%; 6) Browning rate of tissue culture seedlings: Count the number of browned explants / callus / bud clusters; Browning rate = (number of browned explants / total number of treatments) × 100%; 7) Survival rate of transplanting: 30 days after transplanting, count the number of surviving plants; survival rate = (number of surviving plants / number of transplanted plants) × 100%.

[0065] The test results are shown in Table 1 and Figure 1 As shown.

[0066] Table 1. Test results for Test Example 1:

[0067] According to Table 1 and Figure 1 It can be seen that this invention achieves efficient propagation, low contamination rate, and significantly better induction effect than comparative examples 1-10 through specific culture medium formulation and environmental conditions. The browning rate is below 1.6%, the survival rate is above 96%, and the propagation cycle is shorter, which greatly improves production efficiency, reduces production costs, and can better meet market demand. Furthermore, it is highly compatible with existing processes, requires no development of new equipment and production lines, requires a small investment, is suitable for industrial production applications, has broad application prospects, and has significant economic and social benefits.

[0068] The embodiments of the present invention have been described above; however, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the above embodiments of the present invention without inventive effort are within the protection scope of the present invention.

Claims

1. A four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus, characterized in that, Includes the following steps: (1) Using young stem segments with axillary buds of Eucalyptus globulus as explants, surface sterilization, aseptic washing, cutting 2 cm young stem segments with 1 axillary bud and induction culture were carried out in sequence to obtain callus tissue; (2) The callus tissue is cultured to differentiate and obtain differentiated buds; (3) The differentiated buds are cultured for proliferation to obtain clustered buds; (4) Inducing rooting culture in individual plants of the clustered buds to obtain tissue culture seedlings; (5) The tissue culture seedlings were moved to the greenhouse, washed, hardened off, and transplanted into the culture medium for further cultivation to obtain Eucalyptus globulus plants.

2. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The induction culture was carried out in an induction medium; The induction medium comprises modified MS medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.3-0.5 mg / L naphthaleneacetic acid, 0.2-0.4 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

3. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The induction culture was conducted at a temperature of 24-26°C, a relative humidity of 40-50%, a pH of 5.7-5.8, and for 24-26 days. The induction culture was carried out in the dark for 1-5 days, and then under light culture began on the 6th day. The light intensity for the light culture was 1000-1200 lx, and the light duration was 14-15 h / d.

4. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The differentiation culture was carried out in a differentiation medium; The differentiation medium comprises modified MS medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.05-0.2 mg / L naphthaleneacetic acid, 0.4-0.6 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

5. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The differentiation culture was conducted at a temperature of 24-26℃, a relative humidity of 40-50%, a pH of 5.7-5.8, and for 24-26 days. The differentiation culture was carried out in the dark for 1-5 days, and then under light culture began on day 6. The light intensity for light culture was 1000-1600 lx and the light duration was 14-15 h / d.

6. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The proliferation culture is a suspension proliferation culture; The equipment for suspension proliferation culture includes a pneumatic bioreactor; The pneumatic bioreactor has a power of 12-13W, a frequency of 50Hz, an air flow rate of 7-8L / min, an operating pressure of 0.03-0.04MPa, and a pH value of 5.7-5.

8. The proliferation culture was carried out in a proliferation medium; The proliferation medium comprises modified MS medium, 500-800 mg / L calcium nitrate, 170-270 mg / L potassium dihydrogen phosphate, 1600-1750 mg / L potassium nitrate, 300-370 mg / L magnesium sulfate, 550-850 mg / L ammonium nitrate, 0.05-0.08 mg / L naphthaleneacetic acid, 0.25-0.35 mg / L 6-benzylaminopurine, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 25-35 g / L sucrose, and 4.5-6 g / L agar.

7. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The culture temperature was 24-26℃, the relative humidity was 40-50%, and the time was 24-26 days. The proliferation culture was carried out in the dark for 1-5 days, and then under light culture began on the 6th day. The light intensity for the light culture was 2400-2500 lx, and the light duration was 15-16 h / d.

8. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The rooting induction culture was carried out in a rooting induction medium; The rooting induction medium comprises modified MS medium, 300-450 mg / L calcium nitrate, 150-200 mg / L potassium dihydrogen phosphate, 600-850 mg / L potassium sulfate, 200-250 mg / L magnesium sulfate, 350-450 mg / L ammonium nitrate, 0.4-0.6 mg / L indolebutyric acid, 0.2-0.3 mg / L naphthaleneacetic acid, 5-8 mg / L citric acid, 6-10 mg / L cysteine, 20-25 g / L sucrose, and 6-7 g / L carrageenan.

9. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The temperature for the induced rooting culture was 23-25℃, the relative humidity was 50-55%, the light intensity was 1600-1800 lx, the light duration was 15-16 h / d, and the pH value was 5.7-5.

8. The induction rooting culture time is 21-23 days.

10. The four-stage culture and transplanting method for aseptic rapid propagation of Eucalyptus globulus according to claim 1, characterized in that, The transplanting culture was carried out at a temperature of 25-28℃ for 14 days. The transplanting culture includes a first stage, a second stage, and a third stage performed sequentially; The first stage involves an environment with a relative humidity of 90-95% and a light intensity of 2500-3000 lx for 3 days. The second stage involves an environment with a relative humidity of 70-80% and a light intensity of 3000-5000 lx, lasting for 4 days. The third stage involves an environment with a relative humidity of 50-65% and a light intensity of no less than 5000 lx for 7 days.

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