Method for sterilization and tissue culture of tectona grandis explants

Abstract

The present application relates to the technical field of seedling propagation, and particularly relates to a sterilization and tissue culture method of exogenous explants of Dalbergia hupeana Hance. The sterilization and tissue culture method of the exogenous explants of Dalbergia hupeana Hance provided by the present application realizes efficient control of browning by using activated carbon browning prevention composite material. The pores and surface of the activated carbon browning prevention composite material contain cysteine, which helps to combine the phenolic substances in the system faster, prevents the rapid occurrence of browning, and further combines the induction culture step, improves the survival rate of the exogenous explants of Dalbergia hupeana Hance, and obviously reduces the seedling propagation cost of Dalbergia hupeana Hance. This is of great significance for the large-scale planting and timber supply of Dalbergia hupeana Hance, and has good economic benefits.

Description

Technical Field

[0001] This invention relates to the field of seedling propagation technology, and in particular to a method for sterilizing and tissue culture explants of *Hylocereus undatus*. Background Technology

[0002] Red-flowered *Gynostemma pentaphyllum* is a plant belonging to the genus *Gynostemma* in the family Salicaceae. It prefers sunlight and thrives in areas with an average annual temperature of 22-24℃, a minimum coldest month temperature above 15℃, annual rainfall of 1500-2400 mm, and relative humidity of 75-85%. It has a well-developed root system, providing wind resistance. It prefers fertile, loose, and well-drained soil, growing well on gentle slopes with deep, humus-rich soils. It does not grow well in dry, infertile soils.

[0003] Red-flowered ebony is reddish-brown in color, with a hard yet resilient structure. It is of excellent quality, with a fine and dense texture, clear grain, and smooth cut surface. It does not warp or crack when drying, and is resistant to insects and decay. It is an important raw material for industries such as shipbuilding, construction, bridge building, and furniture making.

[0004] Therefore, developing a new method for sterilization and tissue culture of *Hylocereus undatus* explants is of great significance for the stable supply of *Hylocereus undatus* and the development of downstream industries. Summary of the Invention

[0005] In view of this, the present invention provides a method for sterilizing and tissue culture of *Hylocereus undatus* explants. The method for sterilizing and tissue culture of *Hylocereus undatus* explants provided by the present invention can significantly reduce browning and improve the survival rate of *Hylocereus undatus* explants.

[0006] This invention provides a method for sterilizing and tissue culture explants of *Hylocereus undatus*, comprising the following steps: (1) Select healthy, disease-free mother plants and cut semi-lignified young branches as explants of red-flowered jujube wood; (2) The explants of the red-flowered crape myrtle were cleaned, surface sterilized, aseptically washed, the leaves were removed, and a 2 cm stem segment with one axillary bud was cut for induction culture to obtain a single bud; (3) The single bud is cultured to produce clustered buds; (4) Inducing rooting culture in the single plant of the clustered buds to obtain tissue culture rooted seedlings; (5) The tissue culture rooted seedlings are transferred to a greenhouse for hardening. When the seedlings reach a height of 3-3.5cm, they are transplanted into a culture medium for further cultivation to obtain red flower bud seedlings.

[0007] Preferably, the surface sterilization includes sequential immersion in an ethanol solution and immersion in hydrogen peroxide; the volume concentration of the ethanol solution used for the ethanol solution immersion is 70-75%, and the immersion time is 30-40 seconds; the mass concentration of the hydrogen peroxide used for the hydrogen peroxide immersion is 3%, and the immersion time is 10-15 minutes.

[0008] Preferably, the induction culture is carried out in an induction medium; the components of the induction medium include 1600-1800 mg / L potassium nitrate, 650-750 mg / L ammonium nitrate, 250-270 mg / L potassium dihydrogen phosphate, 350-380 mg / L magnesium sulfate heptahydrate, 500-600 mg / L calcium nitrate, 0.5-0.8 mg / L 6-benzylaminopurine, 0.1-0.2 mg / L naphthaleneacetic acid, 300-500 mg / L activated carbon anti-browning composite material, 30 g / L sucrose, and 4.5-5 g / L agar; the activated carbon anti-browning composite material includes cysteine ​​and activated carbon, wherein the cysteine ​​is adsorbed on the surface of the activated carbon; the mass ratio of cysteine ​​to activated carbon is 2-3:10.

[0009] Preferably, the induction culture is carried out at a temperature of 25-27°C, a relative humidity of 45-55%, a pH of 5.8, and a duration of 20-30 days; the induction culture includes sequential light-free induction culture and light-induced culture; the light-free induction culture is carried out under light-free conditions for 10 days; the light-induced culture is carried out under light conditions, with a light intensity of 2200-2400 lx and a daily light exposure time of 8-10 hours; the light-induced culture lasts for 10-20 days.

[0010] Preferably, the proliferation culture is a suspension proliferation culture; the proliferation culture is carried out in a proliferation medium; the components of the proliferation medium include 1600~1800 mg / L potassium nitrate, 650~750 mg / L ammonium nitrate, 250~270 mg / L potassium dihydrogen phosphate, 350~380 mg / L magnesium sulfate heptahydrate, 500~600 mg / L calcium nitrate, 200~300 mg / L hydrolyzed casein, 0.4~0.6 mg / L 6-benzylaminopurine, 0.05~0.1 mg / L naphthaleneacetic acid, 10~20 mg / L cysteine, 100~120 mg / L polyphenylenepyrrolidone, 30 g / L sucrose and 4.5~5 g / L agar.

[0011] Preferably, the temperature for the proliferation culture is 26~28℃, the relative humidity is 55~65%, the light intensity is 2200~2400lx, and the light duration is 13~15h / d; the proliferation culture time is 25~30 days.

[0012] Preferably, the rooting induction culture is carried out in a rooting induction medium; the components of the rooting induction medium include 500-650 mg / L potassium nitrate, 200-300 mg / L ammonium nitrate, 150-170 mg / L potassium dihydrogen phosphate, 270-380 mg / L magnesium sulfate heptahydrate, 300-350 mg / L calcium nitrate, 0.4-0.5 mg / L indolebutyric acid, 0.5-0.6 mg / L naphthaleneacetic acid, 10-20 mg / L cysteine, 30-50 mg / L activated carbon, 20 g / L sucrose, and 6-7 g / L carrageenan.

[0013] Preferably, the temperature for the induced rooting culture is 25-27℃, the relative humidity is 50-55%, the light intensity is 1700-2000 lx, the light duration is 14-16 h / d, and the pH value is 5.8; the induced rooting culture time is 5-10 days.

[0014] Preferably, the culture medium comprises peat moss, river sand and vermiculite in a mass ratio of 1:2:1; the water content of the culture medium is 75-85 wt%.

[0015] Preferably, the culture temperature in step (5) is 26~28℃ and the time is 10~15 days; the culture includes a first stage (2~3 days), a second stage (3~4 days) and a third stage (5~8 days) performed sequentially; the relative humidity of the environment in the first stage is 90~95% and the light intensity is 2500~3500lx; the relative humidity of the environment in the second stage is 75~85% and the light intensity is 3500~5000lx; the relative humidity of the environment in the third stage is 50~60% and the light intensity is not less than 5000lx.

[0016] Compared with existing technologies, the sterilization and tissue culture method for *Symplocos rubrum* explants provided by this invention achieves the following beneficial effects: The sterilization and tissue culture method for *Hylocereus undatus* explants provided by this invention achieves efficient control of browning by using activated carbon anti-browning composite material. The pores and surface of the activated carbon anti-browning composite material contain cysteine, which helps to bind phenolic substances in the system more quickly, preventing rapid browning. Furthermore, combined with induction culture and other steps, it improves the survival rate of *Hylocereus undatus* explants, significantly reducing the cost of *Hylocereus undatus* seedling propagation. This is of great significance for the large-scale planting and timber supply of *Hylocereus undatus*, and has good economic 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 This is a comparison chart of browning rate and survival rate for test example 1. Detailed Implementation

[0019] This invention provides a method for sterilizing and tissue culture explants of *Hylocereus undatus*, comprising the following steps: (1) Select healthy, disease-free mother plants and cut semi-lignified young branches as explants of red-flowered jujube wood; (2) The explants of the red-flowered crape myrtle were cleaned, surface sterilized, aseptically washed, the leaves were removed, and a 2 cm stem segment with one axillary bud was cut for induction culture to obtain a single bud; (3) The single bud is cultured to produce clustered buds; (4) Inducing rooting culture in the single plant of the clustered buds to obtain tissue culture rooted seedlings; (5) The tissue culture rooted seedlings are transferred to a greenhouse for hardening. When the seedlings reach a height of 3-3.5cm, they are transplanted into a culture medium for further cultivation to obtain red flower bud seedlings.

[0020] This invention selects healthy, disease-free mother plants and cuts semi-lignified young branches as explants for *Hylocereus undatus*. In this invention, the preferred season for cutting is spring.

[0021] After obtaining the *Pterocarya stenoptera* explants, this invention sequentially cleans, sterilizes, and sterilizes the explants with water, removes leaves, and cuts 2 cm stem segments with one axillary bud for induction culture to obtain single buds. In this invention, the cleaning preferably includes the following steps: rinsing the *Pterocarya stenoptera* explants with clean water for 25-35 minutes, more preferably 30 minutes.

[0022] In this invention, the surface sterilization preferably includes sequential immersion in an ethanol solution and immersion in hydrogen peroxide; the volume concentration of the ethanol solution used for the ethanol solution immersion is preferably 70-75%, more preferably 75%, and the immersion time is preferably 30-40 seconds, more preferably 30-35 seconds; the mass concentration of the hydrogen peroxide used for the hydrogen peroxide immersion is preferably 3%, and the immersion time is preferably 10-15 minutes, more preferably 12 minutes.

[0023] In this invention, the sterile water washing method is preferably rinsing; the rinsing time is preferably 3 to 5 minutes per rinse and the number of rinses is preferably 5 to 10.

[0024] In this invention, the aseptic washing process preferably includes absorbing the surface moisture of the aseptically washed safflower wood explant.

[0025] In this invention, the induction culture is preferably carried out in an induction medium; the components of the induction medium preferably include 1600-1800 mg / L potassium nitrate, 650-750 mg / L ammonium nitrate, 250-270 mg / L potassium dihydrogen phosphate, 350-380 mg / L magnesium sulfate heptahydrate, 500-600 mg / L calcium nitrate, 0.5-0.8 mg / L 6-benzylaminopurine, 0.1-0.2 mg / L naphthaleneacetic acid, 300-500 mg / L activated carbon anti-browning composite material, 30 g / L sucrose, and 4.5-5 g / L agar; the activated carbon anti-browning composite material preferably includes cysteine ​​and activated carbon, wherein the cysteine ​​is adsorbed on the surface of the activated carbon; the mass ratio of cysteine ​​to activated carbon is preferably 2-3:10, more preferably 2.5:10.

[0026] In this invention, the preferred temperature for the induction culture is 25-27°C, more preferably 26°C; the preferred relative humidity is 45-55%, more preferably 50%; the preferred pH value is 5.8; and the preferred time is 20-30 days, more preferably 24-27 days. The induction culture includes sequential light-free induction culture and light-induced culture. The light-free induction culture is preferably carried out under light-free conditions for 10 days. The light-induced culture is carried out under light conditions, with a light intensity of 2200-2400 lx and a daily light exposure time of 8-10 hours. The light-induced culture lasts for 10-20 days.

[0027] After obtaining a single bud, the present invention performs proliferation culture on the single bud 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 10~15W, the frequency is preferably 50~60Hz, the aeration rate is preferably 5~6L / min, and the pH value is preferably 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 1600~1800 mg / L potassium nitrate, 650~750 mg / L ammonium nitrate, 250~270 mg / L potassium dihydrogen phosphate, 350~380 mg / L magnesium sulfate heptahydrate, 500~600 mg / L calcium nitrate, 200~300 mg / L hydrolyzed casein, 0.4~0.6 mg / L 6-benzylaminopurine, 0.05~0.1 mg / L naphthaleneacetic acid, 10~20 mg / L cysteine, 100~120 mg / L polyphenylenepyrrolidone, 30 g / L sucrose and 4.5~5 g / L agar.

[0029] In this invention, the preferred temperature for the proliferation culture is 26-28°C, the preferred relative humidity is 55-65%, more preferably 60%, the preferred light intensity is 2200-2400 lx, and the preferred light duration is 13-15 h / d, more preferably 14 h / d; the preferred proliferation culture time is 25-30 days, more preferably 27-28 days.

[0030] After obtaining the clustered buds, the present invention induces rooting culture in individual plants of the clustered buds to obtain tissue-cultured rooted seedlings. In the present invention, the height of the individual plants of the clustered buds is preferably 2~2.2cm, more preferably 2.1cm.

[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 500-650 mg / L potassium nitrate, 200-300 mg / L ammonium nitrate, 150-170 mg / L potassium dihydrogen phosphate, 270-380 mg / L magnesium sulfate heptahydrate, 300-350 mg / L calcium nitrate, 0.4-0.5 mg / L indolebutyric acid, 0.5-0.6 mg / L naphthaleneacetic acid, 10-20 mg / L cysteine, 30-50 mg / L activated carbon, 20 g / L sucrose, and 6-7 g / L carrageenan.

[0032] In this invention, the preferred temperature for the induced rooting culture is 25-27°C, the preferred relative humidity is 50-55%, the preferred light intensity is 1700-2000 lx, the preferred light duration is 14-16 h / d, and the preferred pH value is 5.8; the preferred duration for the induced rooting culture is 5-10 days, more preferably 7-8 days.

[0033] After obtaining the tissue-cultured rooted seedlings, this invention transfers them to a greenhouse for hardening-off. Once the seedlings reach a height of 3-3.5 cm, they are transplanted into a culture medium for further cultivation, yielding *Hylocereus undatus* seedlings. In this invention, the hardening-off process preferably includes washing the seedlings; this washing is preferably performed using sterile water. By washing the seedlings, this invention removes residual culture medium such as agar from the roots, reducing the growth of fungi and protecting the roots.

[0034] In this invention, the preferred temperature for hardening off seedlings is 26-28°C, the preferred light intensity is 2200-2500 lx, and the preferred hardening time is 1-2 days.

[0035] In this invention, the culture medium preferably comprises peat moss, river sand and vermiculite in a mass ratio of 1:2:1; the culture medium is preferably disinfected with a 3 wt% potassium permanganate solution before use; and the water content of the culture medium is preferably 75-85 wt%.

[0036] In this invention, the culture temperature is preferably 26~28℃, and the culture time is preferably 10~15 days.

[0037] In this invention, the cultivation preferably includes a first stage (2-3 days), a second stage (3-4 days), and a third stage (5-8 days) performed sequentially; the relative humidity of the environment in the first stage is preferably 90-95%, and the light intensity is preferably 2500-3500 lx; ​​the relative humidity of the environment in the second stage is preferably 75-85%, and the light intensity is preferably 3500-5000 lx; ​​the relative humidity of the environment in the third stage is preferably 50-60%, 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] In a specific embodiment of the present invention, the preparation steps of the activated carbon anti-browning composite material are as follows: cysteine ​​and water are mixed to obtain a premixed solution with a mass concentration of 20wt%, and then activated carbon is immersed in the prepared premixed solution at 60 degrees Celsius for adsorption for 2 hours to obtain the activated carbon anti-browning composite material.

[0040] Example 1: This embodiment provides a method for sterilizing and tissue culture explants of *Safflower Wood*, the specific steps of which are as follows: In spring, select healthy, disease-free mother plants and cut semi-lignified young branches to use as explants for *Lysimachia christinae*.

[0041] The obtained *Symplocos rubra* explants were rinsed with clean water for 30 minutes to remove surface impurities. They were then successively immersed in a 75% ethanol solution for 35 seconds and in a 3% hydrogen peroxide solution for 12 minutes to complete surface sterilization. After rinsing with sterile water for 5 minutes (8 times), the leaves were removed, surface moisture was absorbed, and 2 cm stem segments with one axillary bud were cut. These segments were then induced in an induction culture medium consisting of 1700 mg / L potassium nitrate, 700 mg / L ammonium nitrate, 260 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 550 mg / L calcium nitrate, 0.7 mg / L 6-benzylaminopurine, 0.15 mg / L naphthaleneacetic acid, and 400 mg / L activated charcoal for browning prevention. The treatment consisted of a composite material, 30 g / L sucrose, and 4.5 g / L agar; the activated carbon anti-browning composite material was cysteine ​​and activated carbon, with cysteine ​​adsorbed on the surface of the activated carbon at a mass ratio of 2:10. The induction culture was conducted at 25℃, 55% relative humidity, and pH 5.8. The induction culture was first conducted under dark conditions for 10 days, followed by 15 days under light conditions with a light intensity of 2300 lx and a daily light duration of 9 hours, resulting in single buds.

[0042] The obtained single buds were cultured in suspension in a pneumatic bioreactor with a power of 12W, a frequency of 60Hz, an aeration rate of 6L / min, and a pH of 5.8. The growth medium consisted of 1650 mg / L potassium nitrate, 680 mg / L ammonium nitrate, 270 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 600 mg / L calcium nitrate, 240 mg / L hydrolyzed casein, 0.5 mg / L 6-benzylaminopurine, 0.08 mg / L naphthaleneacetic acid, 15 mg / L cysteine, 110 mg / L polyphenylenepyrrolidone, 30 g / L sucrose, and 4.5 g / L agar. The culture temperature was 26℃, the relative humidity was 65%, the light intensity was 2200 lx, the photoperiod was 15 h / d, and the culture lasted for 25 days to obtain clustered buds.

[0043] The obtained 2cm tall clustered shoots were cultured in a rooting induction medium. The components of the rooting induction medium were 650mg / L potassium nitrate, 300mg / L ammonium nitrate, 170mg / L potassium dihydrogen phosphate, 270mg / L magnesium sulfate heptahydrate, 350mg / L calcium nitrate, 0.4mg / L indolebutyric acid, 0.5mg / L naphthaleneacetic acid, 10mg / L cysteine, 50mg / L activated carbon, 20g / L sucrose, and 7g / L carrageenan. The rooting induction culture was conducted at a temperature of 27℃, a relative humidity of 55%, a light intensity of 2000lx, a light duration of 14h / d, a pH of 5.8, and for 9 days, yielding tissue-cultured rooted seedlings.

[0044] The obtained tissue-cultured rooted seedlings were transferred to a greenhouse, where they were washed with sterile water and hardened off at a temperature of 27°C and a light intensity of 2500 lx for 2 days. Once the seedlings reached a height of 3-3.5 cm, they were transplanted into a culture medium consisting of peat moss, river sand, and vermiculite in a 1:2:1 mass ratio. The culture medium was sterilized with a 3 wt% potassium permanganate solution before use, and the moisture content of the culture medium was 80 wt%. The transplanting culture was conducted at a temperature of 28°C for 15 days, followed by three stages: a first stage (3 days), a second stage (4 days), and a third stage (8 days). The relative humidity in the first stage was 95%, and the light intensity was 3500 lx; ​​the relative humidity in the second stage was 75%, and the light intensity was 4000 lx; ​​and the relative humidity in the third stage was 60%, and the light intensity was 5000 lx. This yielded *Hylocereus undatus* seedlings.

[0045] Example 2: This embodiment provides a method for sterilizing and tissue culture explants of *Safflower Wood*, the specific steps of which are as follows: In spring, select healthy, disease-free mother plants and cut semi-lignified young branches to use as explants for *Lysimachia christinae*.

[0046] The obtained *Symplocos rubra* explants were rinsed with clean water for 30 minutes to remove surface impurities. They were then sequentially immersed in a 75% ethanol solution for 35 seconds and in a 3% hydrogen peroxide solution for 12 minutes to complete surface sterilization. After rinsing with sterile water for 5 minutes (8 times), the leaves were removed, surface moisture was absorbed, and 2 cm stem segments with one axillary bud were cut. These segments were then induced in an induction medium consisting of 1700 mg / L potassium nitrate, 700 mg / L ammonium nitrate, 260 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 550 mg / L calcium nitrate, 0.7 mg / L 6-benzylaminopurine, 0.15 mg / L naphthaleneacetic acid, and 400 mg / L activated charcoal. Browning-resistant composite material, 30 g / L sucrose and 5 g / L agar; activated carbon anti-browning composite material is cysteine ​​and activated carbon, with cysteine ​​adsorbed on the surface of activated carbon, and the mass ratio of cysteine ​​to activated carbon is 3:10. The induction culture temperature is 26℃, the relative humidity is 50%, and the pH value is 5.8. It is first induced cultured for 10 days under dark conditions, and then induced cultured for 17 days under light conditions with a light intensity of 2200 lx and a daily light duration of 10 hours to obtain single buds.

[0047] The obtained single buds were cultured in suspension in a pneumatic bioreactor with a power of 12W, a frequency of 60Hz, an aeration rate of 6L / min, and a pH of 5.8. The growth medium consisted of 1650 mg / L potassium nitrate, 700 mg / L ammonium nitrate, 270 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 600 mg / L calcium nitrate, 240 mg / L hydrolyzed casein, 0.5 mg / L 6-benzylaminopurine, 0.08 mg / L naphthaleneacetic acid, 15 mg / L cysteine, 110 mg / L polyphenylenepyrrolidone, 30 g / L sucrose, and 4.5 g / L agar. The culture temperature was 28℃, the relative humidity was 65%, the light intensity was 2400 lx, the photoperiod was 13 h / d, and the culture lasted for 28 days, resulting in clustered buds.

[0048] The obtained 2cm tall clustered shoots were cultured in a rooting induction medium. The components of the rooting induction medium were 600mg / L potassium nitrate, 250mg / L ammonium nitrate, 160mg / L potassium dihydrogen phosphate, 300mg / L magnesium sulfate heptahydrate, 330mg / L calcium nitrate, 0.5mg / L indolebutyric acid, 0.5mg / L naphthaleneacetic acid, 10mg / L cysteine, 40mg / L activated carbon, 20g / L sucrose, and 6g / L carrageenan. The rooting induction culture was conducted at a temperature of 27℃, a relative humidity of 55%, a light intensity of 2000lx, a light duration of 16h / d, a pH of 5.8, and for 7 days, resulting in tissue-cultured rooted seedlings.

[0049] The obtained tissue-cultured rooted seedlings were transferred to a greenhouse, where they were washed with sterile water and hardened off at a temperature of 28℃ and a light intensity of 2500 lx for 2 days. Once the seedlings reached a height of 3-3.5 cm, they were transplanted into a culture medium consisting of peat moss, river sand, and vermiculite in a mass ratio of 1:2:1. The culture medium was sterilized with a 3 wt% potassium permanganate solution before use, and the moisture content of the culture medium was 80 wt%. The transplanting culture was carried out at a temperature of 28℃ for 13 days. The transplanting culture was carried out in three stages: the first stage (3 days), the second stage (4 days), and the third stage (6 days). The relative humidity of the environment in the first stage was 90%, and the light intensity was 2500 lx. The relative humidity of the environment in the second stage was 85%, and the light intensity was 3500 lx. The relative humidity of the environment in the third stage was 60%, and the light intensity was 5000 lx. This yielded *Hylocereus undatus* seedlings.

[0050] Example 3: This embodiment provides a method for sterilizing and tissue culture explants of *Safflower Wood*, the specific steps of which are as follows: In spring, select healthy, disease-free mother plants and cut semi-lignified young branches to use as explants for *Lysimachia christinae*.

[0051] The obtained *Symplocos rubra* explants were rinsed with clean water for 30 minutes to remove surface impurities. They were then successively immersed in a 75% ethanol solution for 35 seconds and in a 3% hydrogen peroxide solution for 12 minutes to complete surface sterilization. After rinsing with sterile water for 5 minutes (8 times), the leaves were removed, surface moisture was absorbed, and 2 cm stem segments with one axillary bud were cut. These segments were then induced in an induction medium consisting of 1750 mg / L potassium nitrate, 720 mg / L ammonium nitrate, 260 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 550 mg / L calcium nitrate, 0.7 mg / L 6-benzylaminopurine, 0.15 mg / L naphthaleneacetic acid, and 400 mg / L activated charcoal for browning prevention. Composite material, 30 g / L sucrose and 4.5 g / L agar; activated carbon anti-browning composite material is cysteine ​​and activated carbon, cysteine ​​is adsorbed on the surface of activated carbon, the mass ratio of cysteine ​​to activated carbon is 2.5:10, the induction culture temperature is 26℃, the relative humidity is 55%, the pH value is 5.8, first induction culture for 10 days under dark conditions, and then induction culture for 18 days under light conditions, the light intensity is 2400 lx, the daily light time is 8 hours, and single buds are obtained.

[0052] The obtained single buds were cultured in suspension in a pneumatic bioreactor with a power of 15W, a frequency of 60Hz, an aeration rate of 6L / min, and a pH of 5.8. The growth medium consisted of 1650 mg / L potassium nitrate, 680 mg / L ammonium nitrate, 270 mg / L potassium dihydrogen phosphate, 360 mg / L magnesium sulfate heptahydrate, 600 mg / L calcium nitrate, 240 mg / L hydrolyzed casein, 0.5 mg / L 6-benzylaminopurine, 0.08 mg / L naphthaleneacetic acid, 15 mg / L cysteine, 110 mg / L polyphenylene pyrrolidone, 30 g / L sucrose, and 4.5 g / L agar. The culture temperature was 27℃, the relative humidity was 65%, the light intensity was 2400 lx, the photoperiod was 14 h / d, and the culture lasted for 26 days to obtain clustered buds.

[0053] The obtained 2cm tall clustered shoots were cultured in a rooting induction medium. The components of the rooting induction medium were 550mg / L potassium nitrate, 300mg / L ammonium nitrate, 150mg / L potassium dihydrogen phosphate, 350mg / L magnesium sulfate heptahydrate, 320mg / L calcium nitrate, 0.4mg / L indolebutyric acid, 0.5mg / L naphthaleneacetic acid, 20mg / L cysteine, 50mg / L activated carbon, 20g / L sucrose, and 7g / L carrageenan. The rooting induction culture was conducted at a temperature of 27℃, a relative humidity of 55%, a light intensity of 2000lx, a light duration of 16h / d, a pH of 5.8, and for 6 days, yielding tissue-cultured rooted seedlings.

[0054] The obtained tissue-cultured rooted seedlings were transferred to a greenhouse, where they were washed with sterile water and hardened off at a temperature of 28℃ and a light intensity of 2500 lx for one day. Once the seedlings reached a height of 3-3.5 cm, they were transplanted into a culture medium consisting of peat moss, river sand, and vermiculite in a mass ratio of 1:2:1. The culture medium was sterilized with a 3 wt% potassium permanganate solution before use, and the moisture content of the culture medium was 80 wt%. The transplanting culture was conducted at a temperature of 28℃ for 14 days, followed by three stages: a first stage (2 days), a second stage (4 days), and a third stage (8 days). The relative humidity in the first stage was 95%, and the light intensity was 3500 lx; ​​the relative humidity in the second stage was 85%, and the light intensity was 5000 lx; ​​and the relative humidity in the third stage was 60%, and the light intensity was 5300 lx. This yielded *Hylocereus undatus* seedlings.

[0055] Comparative Example 1: The preparation method of this comparative example is the same as that of Example 1, except that cysteine ​​of equal mass is used to replace the activated carbon anti-browning composite material.

[0056] Comparative Example 2: The preparation method of this comparative example is the same as that of Example 1, except that an equal mass of activated carbon is used to replace the activated carbon anti-browning composite material.

[0057] Comparative Example 3: The preparation method of this comparative example is the same as that of Example 1, except that a mixture of cysteine ​​and activated carbon of equal mass and proportion is used to replace the activated carbon anti-browning composite material.

[0058] Test Example 1: The effects of Examples 1-3 and Comparative Examples 1-3 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) Single bud induction rate: After the induction culture is completed, the number of surviving explants that formed single buds is counted; Induction rate = (number of single buds formed / number of surviving explants) × 100%; 3) 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%; 4) 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%; 5) Browning rate of tissue-cultured rooted seedlings: Count the number of browned explants / single buds / clumps of buds; Browning rate = (number of browned explants / total number of treatments) × 100%; 6) 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%.

[0059] The results are shown in Table 1 and Figure 1 As shown.

[0060] Table 1. Test results for Test Example 1:

[0061] According to Table 1 and Figure 1 It can be seen that the sterilization and tissue culture method for *Rhizophora stylosa* explants provided by this invention achieves efficient control of browning by using activated carbon anti-browning composite material. Furthermore, by combining steps such as induction culture, the survival rate of *Rhizophora stylosa* explants is improved, resulting in a significant reduction in the seedling propagation cost of *Rhizophora stylosa*. This is of great significance for the large-scale planting of *Rhizophora stylosa* and the supply of timber, and has good economic benefits.

[0062] 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 method for sterilizing and tissue culture explants of *Hylocereus undatus*, characterized in that, Includes the following steps: (1) Select healthy, disease-free mother plants and cut semi-lignified young branches as explants of red-flowered jujube wood; (2) The explants of the red-flowered crape myrtle were cleaned, surface sterilized, aseptically washed, the leaves were removed, and a 2 cm stem segment with one axillary bud was cut for induction culture to obtain a single bud; (3) The single bud is cultured to produce clustered buds; (4) Inducing rooting culture in the single plant of the clustered buds to obtain tissue culture rooted seedlings; (5) The tissue culture rooted seedlings are transferred to a greenhouse for hardening. When the seedlings reach a height of 3-3.5cm, they are transplanted into a culture medium for further cultivation to obtain red flower bud seedlings.

2. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1, characterized in that, The surface sterilization includes sequential immersion in an ethanol solution and immersion in hydrogen peroxide; the volume concentration of the ethanol solution used for the ethanol solution immersion is 70-75%, and the immersion time is 30-40 seconds; the mass concentration of the hydrogen peroxide used for the hydrogen peroxide immersion is 3%, and the immersion time is 10-15 minutes.

3. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1, characterized in that, The induction culture is carried out in an induction medium; the components of the induction medium include 1600~1800 mg / L potassium nitrate, 650~750 mg / L ammonium nitrate, 250~270 mg / L potassium dihydrogen phosphate, 350~380 mg / L magnesium sulfate heptahydrate, 500~600 mg / L calcium nitrate, 0.5~0.8 mg / L 6-benzylaminopurine, 0.1~0.2 mg / L naphthaleneacetic acid, 300~500 mg / L activated carbon anti-browning composite material, 30 g / L sucrose, and 4.5~5 g / L agar; the activated carbon anti-browning composite material includes cysteine ​​and activated carbon, and the cysteine ​​is adsorbed on the surface of the activated carbon; the mass ratio of cysteine ​​to activated carbon is 2~3:

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4. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1 or 3, characterized in that, The induction culture is carried out at a temperature of 25-27°C, a relative humidity of 45-55%, a pH of 5.8, and a duration of 20-30 days. The induction culture includes sequential light-free induction culture and light-induced culture. The light-free induction culture is carried out under light-free conditions for 10 days. The light-induced culture is carried out under light conditions with a light intensity of 2200-2400 lx and a daily light exposure time of 8-10 hours. The light-induced culture lasts for 10-20 days.

5. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1, characterized in that, The proliferation culture is a suspension proliferation culture; the proliferation culture is carried out in a proliferation medium; the components of the proliferation medium include 1600~1800 mg / L potassium nitrate, 650~750 mg / L ammonium nitrate, 250~270 mg / L potassium dihydrogen phosphate, 350~380 mg / L magnesium sulfate heptahydrate, 500~600 mg / L calcium nitrate, 200~300 mg / L hydrolyzed casein, 0.4~0.6 mg / L 6-benzylaminopurine, 0.05~0.1 mg / L naphthaleneacetic acid, 10~20 mg / L cysteine, 100~120 mg / L polyphenylenepyrrolidone, 30 g / L sucrose and 4.5~5 g / L agar.

6. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1 or 5, characterized in that, The culture temperature for proliferation is 26-28℃, the relative humidity is 55-65%, the light intensity is 2200-2400 lx, and the light duration is 13-15 h / d; the culture time for proliferation is 25-30 days.

7. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1, characterized in that, The rooting induction culture is carried out in a rooting induction medium; the components of the rooting induction medium include 500-650 mg / L potassium nitrate, 200-300 mg / L ammonium nitrate, 150-170 mg / L potassium dihydrogen phosphate, 270-380 mg / L magnesium sulfate heptahydrate, 300-350 mg / L calcium nitrate, 0.4-0.5 mg / L indolebutyric acid, 0.5-0.6 mg / L naphthaleneacetic acid, 10-20 mg / L cysteine, 30-50 mg / L activated carbon, 20 g / L sucrose, and 6-7 g / L carrageenan.

8. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1 or 7, characterized in that, The temperature for the induced rooting culture is 25-27℃, the relative humidity is 50-55%, the light intensity is 1700-2000 lx, the light duration is 14-16 h / d, and the pH value is 5.8; the induced rooting culture time is 5-10 days.

9. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1, characterized in that, The culture medium comprises peat moss, river sand, and vermiculite in a mass ratio of 1:2:1; the water content of the culture medium is 75-85 wt%.

10. The method for sterilization and tissue culture of *Hylocereus undatus* explants according to claim 1 or 9, characterized in that, The culture temperature in step (5) is 26~28℃, and the time is 10~15 days. The culture includes a first stage, a second stage and a third stage in sequence. The relative humidity of the environment in the first stage is 90~95%, the light intensity is 2500~3500lx, and the time is 2~3 days. The relative humidity of the environment in the second stage is 75~85%, the light intensity is 3500~5000lx, and the time is 3~4 days. The relative humidity of the environment in the third stage is 50~60%, the light intensity is not less than 5000lx, and the time is 5~8 days.

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