A tissue culture method of malus hallings

Abstract

The application discloses a tissue culture method of Loropetalum chinense, and belongs to the technical field of tissue culture of Loropetalum chinense. The method comprises the following steps: collecting and processing explants, disinfecting the explants, inducing culture, integrated proliferation and rooting culture, seedling raising and transplanting and the like. The medium is improved, integrated proliferation and rooting culture of Loropetalum chinense is realized, the tissue culture time is shortened, and the operation cost is reduced. The dry matter accumulation of the tissue culture seedlings of Loropetalum chinense is promoted, the hormone level, illumination time and intensity and temperature are controlled, the vitrification phenomenon is effectively inhibited, the problem that the propagation coefficient is reduced due to vitrification of the tissue culture seedlings in the tissue culture process of Loropetalum chinense is solved, efficient tissue culture of Loropetalum chinense is realized, and the development of the Loropetalum chinense industry is promoted.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology of *Senecio scandens*, and more particularly to a method for tissue culture of *Senecio scandens*. Background Technology

[0002] *Semiliquidambar cathayensis*, belonging to the genus *Semiliquidambar* in the family Hamamelidaceae, is a species endemic to my country, newly discovered in 1962. It is an excellent ornamental tree for landscaping, and its high-quality wood, with good turning properties, is a superior material for crafting turned products. Furthermore, its roots, branches, leaves, bark, and nectar all possess medicinal value, used to treat symptoms such as rheumatoid arthritis, lumbar muscle strain, and bruises, making it an effective local folk remedy. However, due to its low natural reproduction rate and frequent logging, wild resources are dwindling, and the species is facing endangerment. Therefore, protecting and expanding *Semiliquidambar cathayensis* resources has become a critical issue that urgently needs to be addressed.

[0003] Currently, the main propagation methods for *Senecio scandens* are seed propagation and cutting propagation. However, traditional seed propagation cannot produce a large number of high-quality seedlings in a short period of time, and the offspring cannot consistently maintain the superior traits of the parent plants. Furthermore, *Senecio scandens* is a species that is difficult to root, resulting in a low survival rate for cuttings. Therefore, neither seed propagation nor cutting propagation can meet the large-scale industrialization needs of superior *Senecio scandens* varieties, thus limiting the development of the *Senecio scandens* industry. Tissue culture, compared to seed propagation and cutting propagation, has advantages such as a shorter cycle, faster speed, and stable maintenance of superior parent traits. Therefore, establishing tissue culture technology for *Senecio scandens* is an effective way to solve the shortage of *Senecio scandens* resources and maintain its superior varieties. However, traditional tissue culture methods require multiple stages, including induction culture, proliferation culture, rooting culture, and hardening-off transplanting. The process is cumbersome, time-consuming, and costly. In addition, vitrification is prone to occur during the proliferation culture process of *Senecio scandens*, leading to a low propagation coefficient and poor quality of tissue culture seedlings.

[0004] Therefore, there is an urgent need to find a semi-maple lily tissue culture method to solve the problems of long culture time, complicated procedures, and low propagation coefficient and poor seedling quality caused by vitrification under conventional tissue culture methods. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a tissue culture method for *Senecio scandens*, which solves the problems of long culture time, complicated procedures, and low propagation coefficient and poor seedling quality caused by vitrification under conventional tissue culture methods.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] A method for tissue culture of *Senecio scandens*, the method being as follows:

[0008] (1) Explant collection and treatment: In spring, healthy new shoots are collected from superior mother trees. Semi-lignified stem segments are cut and leaves are removed. After washing off surface dirt in water, the explants are soaked in soapy water for 10-20 minutes and then rinsed with running water for 30 minutes to obtain the explants.

[0009] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1-2 axillary buds and 2-3 cm in length. Wash them once in sterile water and then soak them in 75% ethanol solution for 15-30 seconds. Then wash them 3-5 times with sterile water and then disinfect them in 0.1% mercuric chloride solution for 5-8 minutes. After disinfection, wash them 5-7 times with sterile water to obtain the disinfected explants.

[0010] (3) Induction culture: After sterilization, the explants were inoculated into the induction medium for induction culture to obtain adventitious shoots;

[0011] (4) Proliferation-rooting integrated culture: Single shoots are cut from the adventitious shoots obtained by induction culture and then inoculated into the proliferation-rooting integrated culture medium to obtain rooted seedlings;

[0012] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1cm and root number ≥3 obtained from the integrated propagation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 800-1000 times carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

[0013] Traditional tissue culture methods require steps such as induction culture, proliferation culture, rooting culture, and seedling hardening and transplanting, which are complex, costly, and time-consuming. Therefore, this invention prepares a proliferation-rooting integrated culture medium for integrated proliferation-rooting culture, which can effectively shorten the tissue culture time of *Senecio scandens* and reduce the cost of transfer operations, thereby improving the tissue culture propagation efficiency of *Senecio scandens*.

[0014] Furthermore, the induction culture medium consists of: MS + 1.0–1.2 mg / L 6-BA + 0.2–0.5 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8.

[0015] Furthermore, in step (4), the proliferation-rooting integrated culture medium is a solid-liquid bilayer culture medium, wherein the upper layer is a liquid proliferation culture medium and the lower layer is a solid rooting culture medium, and the proliferation-rooting integrated culture medium includes the following raw materials:

[0016] Upper liquid proliferation medium: MS + 0.2-0.3 mg / L 6-BA + 0.05-0.15 mg / L NAA + 30 g / L sucrose + 2-3 g / L cyclohexylamine + 2-3 g / L isopropyl acetate + 3-4 g / L polyethylene glycol + 8-15 g / L stabilizer, pH = 5.8;

[0017] The lower solid rooting medium consisted of MS medium containing 0.6–0.8 mg / L IBA, 0.4 mg / L NAA, 0.6 mg / L LABT, 30 g / L sucrose, and 6.8 g / L agar, with a pH of 5.8.

[0018] Furthermore, the preparation method of the integrated proliferation-rooting culture medium is as follows:

[0019] According to the composition ratio of the lower solid rooting medium, IBA, NAA, and ABT were added to MS medium and mixed thoroughly. Agar was then added, and the mixture was heated until the agar dissolved. Sucrose was then added and stirred until dissolved and mixed thoroughly. The pH was adjusted to 5.8, and the mixture was poured into culture flasks and sterilized at 121°C for 20 minutes. According to the composition ratio of the upper liquid proliferation medium, 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate, and polyethylene glycol were added to MS medium and mixed thoroughly. Stabilizer was then added and mixed thoroughly. The mixture was sterilized at 121°C for 20 minutes. After the lower solid rooting medium in the culture flask solidified, it was added to obtain the integrated proliferation-rooting medium.

[0020] Furthermore, the stabilizer comprises the following raw materials:

[0021] Sodium alginate, 6-BA, NAA, 2wt% calcium chloride solution, rice bran wax, triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin.

[0022] Furthermore, the method for preparing the stabilizer is as follows:

[0023] A: Dissolve sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 6-BA and NAA and mix well. Then slowly drop it into a 2wt% calcium chloride solution to gel and obtain sodium alginate-coated balls. Wash with sterile water 3 times and dry at 45℃ for 20-30 minutes to obtain semi-dry sodium alginate-coated balls for later use.

[0024] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put triethylamine, thiourea, and 2,4,6-tris(dimethylaminomethyl)phenol into a reaction vessel and mix evenly, heat to 110-130℃ and react for 0.5-1h, then cool to 55℃ and add epoxy resin and mix thoroughly, then add rice bran wax microparticles and mix evenly to obtain a coating solution;

[0025] C: Spray the coating liquid onto the dry sodium alginate coated ball surface, let it stand in a cool and ventilated place for 12-24 hours, then place it at 60-70℃ for 2-3 hours to allow the rice bran wax particles on the coated ball surface to melt and flow out, and then wash it with deionized water 3-5 times to obtain the stabilizer.

[0026] Furthermore, in step A, the mass ratio of sodium alginate solution, 6-BA, and NAA is (160-300):(0.03-0.05):(0.01-0.02).

[0027] Furthermore, in step B, the mass ratio of triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin, and rice bran wax microparticles is (8-16):(10-20):(10-20):(50-70):(10-20).

[0028] Furthermore, the induction culture temperature is 25–27℃, the light duration is 8–12 h / d, and the light intensity is 2000–3000 lx; ​​the integrated proliferation-rooting culture temperature is 25–27℃, the light duration is 8–12 h / d, and the light intensity is 4500–5000 lx.

[0029] Furthermore, the stabilizer has a particle size of 0.5-1 mm.

[0030] During the tissue culture of *Senecio scandens*, vitrification easily occurs during the proliferation stage. Vitrified seedlings exhibit high water content, low dry matter and cellulose accumulation, poor growth, difficulty in further rooting, and low survival rate after transplanting, significantly reducing the propagation coefficient. Therefore, this invention adds isopropyl acetate, cyclohexylamine, and polyethylene glycol to the upper liquid proliferation medium during the preparation of the integrated proliferation-rooting medium. Cyclohexylamine enhances the ion exchange between nutrients in the medium and the roots of the tissue culture seedlings by adjusting the charge properties of the medium. Furthermore, isopropyl acetate acts on the cell membrane, altering the cell membrane structure to increase cell membrane permeability. As a result, nutrients in the medium enter the tissue culture seedlings through the enhanced permeability of the cell membrane under stronger ion exchange, accelerating the accumulation of dry matter, promoting lignification, and improving seedling quality. Simultaneously, the added polyethylene glycol adjusts the osmotic potential of the medium to regulate the water content within the tissue culture seedlings. Thus, the combined effect of all components in the medium reduces vitrification of *Senecio scandens* tissue culture seedlings, promoting better rooting and yielding high-quality rooted seedlings.

[0031] To shorten tissue culture time and improve propagation efficiency, this invention prepares a double-layer culture medium for integrated proliferation-rooting culture. However, after a long period of time, the components in the upper liquid proliferation medium tend to deposit downwards and penetrate into the lower solid rooting medium. Among them, 6-BA and NAA are small amounts of added hormones. After penetrating into the lower rooting medium, they will lead to insufficient hormones required for proliferation. Adding more hormones to the upper liquid proliferation medium will further lead to vitrification.

[0032] To address the aforementioned issues, this invention employs sodium alginate gelation to embed a specific ratio of 6-BA and NAA to prepare a stabilizer, which is then added to the upper liquid proliferation medium. The 6-BA and NAA embedded in the sodium alginate gel are slowly released into the upper liquid proliferation medium for use by tissue culture seedlings, preventing them from accumulating in the lower rooting and proliferation medium over a long period, thus ensuring an appropriate supply of hormones in the medium. However, sodium alginate-coated pellets tend to rapidly absorb water and swell after entering the medium, causing a burst release of 6-BA and NAA. Therefore, this invention semi-dries the sodium alginate-coated pellets and then sprays them with an epoxy resin coating solution containing rice bran wax. After the epoxy resin cures, a hard shell layer is formed on the surface of the sodium alginate-coated pellets, thereby inhibiting swelling and effectively solving the problem of burst release due to swelling. The porous structure formed by the melting of rice bran wax allows the hormone components released internally to smoothly enter the medium for use by tissue culture seedlings.

[0033] Beneficial effects:

[0034] 1. This invention effectively controls the composition of the culture medium, the culture temperature, the time and intensity of light during the tissue culture process of *Senecio scandens*, inhibits the formation of vitrified seedlings, increases the tissue culture propagation coefficient, and improves the quality of tissue culture seedlings, which is of great significance to promoting the development of the *Senecio scandens* industry.

[0035] 2. The present invention prepares a proliferation-rooting integrated culture medium for integrated proliferation and rooting culture, which can effectively shorten the tissue culture time and reduce the cost of transfer operation; and further improves the upper liquid proliferation medium in the proliferation-rooting integrated culture medium to maintain an appropriate concentration of hormones in the culture medium, promote the accumulation of dry matter in tissue culture seedlings, inhibit the excessive increase of water content in seedlings, further inhibit vitrification of tissue culture seedlings, and improve the quality of tissue culture seedlings, thereby effectively improving the rooting rate and transplant survival rate, and increasing the propagation coefficient. Attached Figure Description

[0036] Figure 1 : This is a picture of the tissue culture seedling condition in Example 4 of the present invention. Detailed Implementation

[0037] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0038] Example 1: Stabilizer Preparation

[0039] A: Dissolve 230g of sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 0.04g of 6-BA and 0.015g of NAA and mix well. Then slowly drop the mixture into a 2wt% calcium chloride solution to gel and prepare sodium alginate-coated spheres with a particle size of about 1mm. After washing with sterile water three times, dry the spheres at 45℃ for 25min to obtain semi-dry sodium alginate-coated spheres for later use.

[0040] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 12g triethylamine, 15g thiourea, and 15g 2,4,6-tris(dimethylaminomethyl)phenol into a reaction vessel and mix evenly. Heat to 120℃ and react for 0.7h. Then cool to 55℃ and add 60g epoxy resin and mix thoroughly. Then add 15g rice bran wax microparticles and mix evenly to obtain a coating solution.

[0041] C: Spray the coating liquid onto the surface of the dried sodium alginate coated balls with a coating thickness of about 0.02 mm. Let it stand in a cool and ventilated place for 18 hours, and then place it at 65°C for 2.5 hours to allow the rice bran wax particles on the surface of the coated balls to melt and flow out. Then wash it with deionized water 4 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0042] Example 2: Stabilizer Preparation

[0043] A: Dissolve 160g of sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 0.03g of 6-BA and 0.01g of NAA and mix well. Then slowly drop the mixture into a 2wt% calcium chloride solution to gel and prepare sodium alginate-coated spheres with a particle size of about 1mm. After washing with sterile water three times, dry the spheres at 45℃ for 20min to obtain semi-dry sodium alginate-coated spheres for later use.

[0044] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 8g triethylamine, 10g thiourea, and 10g 2,4,6-tris(dimethylaminomethyl)phenol into a reaction vessel and mix evenly. Heat to 110℃ and react for 0.5h. Then cool to 55℃ and add 50g epoxy resin and mix thoroughly. Then add 10g rice bran wax microparticles and mix evenly to obtain a coating solution.

[0045] C: Spray the coating liquid onto the surface of the dry sodium alginate coated balls with a coating thickness of about 0.02 mm. Let it stand in a cool and ventilated place for 12 hours, and then place it at 60°C for 3 hours to allow the rice bran wax particles on the surface of the coated balls to melt and flow out. Then wash it with deionized water 4 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0046] Example 3: Stabilizer Preparation

[0047] A: Dissolve 300g of sodium alginate in water to prepare a 5wt% sodium alginate solution, then add 0.05g of 6-BA and 0.02g of NAA and mix well. Then slowly drop the mixture into a 2wt% calcium chloride solution to gel and prepare sodium alginate-coated spheres with a particle size of about 1mm. Wash the spheres three times with sterile water and dry them at 45℃ for 30min to obtain semi-dry sodium alginate-coated spheres for later use.

[0048] B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put 16g triethylamine, 20g thiourea, and 20g 2,4,6-tris(dimethylaminomethyl)phenol into a reaction vessel and mix evenly. Heat to 130℃ and react for 1 hour. Then cool to 55℃ and add 70g epoxy resin and mix thoroughly. Then add 20g rice bran wax microparticles and mix evenly to obtain a coating solution.

[0049] C: Spray the coating liquid onto the surface of the dry sodium alginate coated balls with a coating thickness of about 0.02 mm. Let it stand in a cool and ventilated place for 24 hours, and then place it at 70°C for 2 hours to allow the rice bran wax particles on the surface of the coated balls to melt and flow out. Then wash it with deionized water 5 times to obtain a stabilizer with a particle size of about 0.8 cm.

[0050] After the stabilizer was prepared, the tissue culture of *Senecio scandens* was further carried out, as shown below.

[0051] Example 4: Method 1 for Tissue Culture of *Liriope muscari*

[0052] Before conducting the semi-maple lily tissue culture, an induction medium and a combined proliferation and rooting medium were prepared, as follows:

[0053] Induction medium: MS + 1.1 mg / L 6-BA + 0.3 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0054] Proliferation-rooting integrated culture medium:

[0055] Add IBA (0.7 mg / L), NAA (0.4 mg / L), and ABT (0.6 mg / L) to MS medium, mix well, then add agar (6.8 g / L), heat until the agar dissolves, then add sucrose (30 g / L), stir until dissolved and mix well. Adjust the pH to 5.8, then pour into a culture flask and sterilize at 121°C for 20 min to obtain the lower solid rooting medium. Add 6-BA (0.25 mg / L), NAA (0.1 mg / L), sucrose (30 g / L), cyclohexylamine (2.5 g / L), isopropyl acetate (2.5 g / L), and polyethylene glycol (3.5 g / L) to MS medium, stir well, then add the stabilizer prepared in Example 1 (11 g / L), mix well, sterilize at 121°C for 20 min, and add after the lower solid rooting medium in the culture flask has solidified to obtain the proliferation-rooting integrated medium.

[0056] (1) Explant collection and treatment: In spring, healthy new shoots were collected from superior mother trees. Semi-lignified stem segments were cut and leaves were removed. The explants were washed in water to remove surface dirt, then soaked in soapy water for 15 minutes and rinsed with running water for 30 minutes.

[0057] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1-2 axillary buds and about 2 cm long. Wash them once in sterile water and then soak them in 75% ethanol solution for 25 seconds. Then wash them 4 times with sterile water and then disinfect them in 0.1% mercuric chloride solution for 6 minutes. After disinfection, wash them 6 times with sterile water to obtain the disinfected explants.

[0058] (3) Induction culture: After disinfection, the explants were inoculated into the induction culture medium and induced to obtain adventitious shoots under the conditions of 26℃, 10h / d light time and 2500lx light intensity.

[0059] (4) Proliferation-rooting integrated culture: Single shoots were cut from the adventitious shoots obtained by induction culture and then inoculated into the proliferation-rooting integrated culture medium. Proliferation-rooting integrated culture was carried out under the conditions of temperature of 26℃, light time of 10h / d and light intensity of 4700lx to obtain rooted seedlings.

[0060] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1cm and root number ≥3 obtained from the integrated propagation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the culture medium from the roots, soak them in 800 times carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

[0061] Example 5: Method 2 for Tissue Culture of *Liriope muscari*

[0062] Before conducting the semi-maple lily tissue culture, an induction medium and a combined proliferation and rooting medium were prepared, as follows:

[0063] Induction medium: MS + 1.0 mg / L 6-BA + 0.2 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0064] Proliferation-rooting integrated culture medium:

[0065] Add IBA (0.6 mg / L), NAA (0.4 mg / L), and ABT (0.6 mg / L) to MS medium, mix well, then add agar (6.8 g / L), heat until the agar dissolves, then add sucrose (30 g / L), stir until dissolved and mix well. Adjust the pH to 5.8, then pour into a culture flask and sterilize at 121°C for 20 min to obtain the lower solid rooting medium. Add 6-BA (0.2 g / L), NAA (0.05 mg / L), sucrose (30 g / L), cyclohexylamine (2 g / L), isopropyl acetate (2 g / L), and polyethylene glycol (3 g / L) to MS medium, stir well, then add the stabilizer prepared in Example 2 (8 g / L), mix well, sterilize at 121°C for 20 min, and add after the lower solid rooting medium in the culture flask has solidified to obtain the proliferation-rooting integrated medium.

[0066] (1) Explant collection and treatment: In spring, healthy new shoots were collected from superior mother trees. Semi-lignified stem segments were cut and leaves were removed. The explants were washed in water to remove surface dirt, then soaked in soapy water for 10 minutes and rinsed with running water for 30 minutes.

[0067] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1-2 axillary buds and about 2 cm long. Wash them once in sterile water and then soak them in 75% ethanol solution for 15 seconds. Then wash them three times with sterile water and then disinfect them in 0.1% mercuric chloride solution for 5 minutes. After disinfection, wash them five times with sterile water to obtain the disinfected explants.

[0068] (3) Induction culture: After disinfection, the explants were inoculated into the induction culture medium and induced to obtain adventitious shoots under the conditions of 25℃, 8h / d light time and 2000lx light intensity.

[0069] (4) Proliferation-rooting integrated culture: Single shoots were cut from the adventitious shoots obtained by induction culture and then inoculated into the proliferation-rooting integrated culture medium. Proliferation-rooting integrated culture was carried out under the conditions of 25℃, 8h / d light time and 5000lx light intensity to obtain rooted seedlings.

[0070] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1cm and root number ≥3 obtained from the integrated propagation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 1000 times carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

[0071] Example 6: Method 3 for Tissue Culture of *Liriope muscari*

[0072] Before conducting the semi-maple lily tissue culture, an induction medium and a combined proliferation and rooting medium were prepared, as follows:

[0073] Induction medium: MS + 1.2 mg / L 6-BA + 0.5 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH = 5.8;

[0074] Proliferation-rooting integrated culture medium:

[0075] Add IBA (0.8 mg / L), NAA (0.4 mg / L), and ABT (0.6 mg / L) to MS medium, mix well, then add agar (6.8 g / L), heat until the agar dissolves, then add sucrose (30 g / L), stir until dissolved and mix well. Adjust the pH to 5.8, then pour into a culture flask and sterilize at 121°C for 20 min to obtain the lower solid rooting medium. Add 6-BA (0.3 mg / L), NAA (0.15 mg / L), sucrose (30 g / L), cyclohexylamine (3 g / L), isopropyl acetate (3 g / L), and polyethylene glycol (4 g / L) to MS medium, stir well, then add the stabilizer prepared in Example 1 (15 g / L), mix well, sterilize at 121°C for 20 min, and add after the lower solid rooting medium in the culture flask has solidified to obtain the proliferation-rooting integrated medium.

[0076] (1) Explant collection and treatment: In spring, healthy new shoots were collected from superior mother trees. Semi-lignified stem segments were cut and leaves were removed. The explants were washed in water to remove surface dirt, then soaked in soapy water for 20 minutes and rinsed with running water for 30 minutes.

[0077] (2) Explant disinfection: On a clean bench, cut the explant into stem segments with 1-2 axillary buds and about 3cm in length. Wash them once in sterile water and then soak them in a 75% ethanol solution for 30 seconds. Then wash them 5 times with sterile water and then disinfect them in a 0.1% mercuric chloride solution for 5 minutes. After disinfection, wash them 5 times with sterile water to obtain the disinfected explants.

[0078] (3) Induction culture: After disinfection, the explants were inoculated into the induction culture medium and induced to obtain adventitious shoots under the conditions of 27℃, 12h / d light time and 3000lx light intensity.

[0079] (4) Proliferation-rooting integrated culture: Single shoots were cut from the adventitious shoots obtained by induction culture and then inoculated into the proliferation-rooting integrated culture medium. Proliferation-rooting integrated culture was carried out under the conditions of 27℃, 12h / d light time and 4500lx light intensity to obtain rooted seedlings.

[0080] (5) Hardening and transplanting: Harden the rooted seedlings with root length ≥1cm and root number ≥3 obtained from the integrated propagation-rooting culture. After hardening, take the rooted seedlings out of the tissue culture bottle, wash off the root culture medium, soak them in 1000 times carbendazim solution for 20 minutes, and then transplant them into the substrate tray.

[0081] Comparative Example 1: Tissue Culture Methods of *Senecio scandens*

[0082] Compared with Example 4, the only difference is that cyclohexylamine was not added to the proliferation-rooting medium when performing tissue culture of *Symplocos rubrum* in Comparative Example 1. All other steps were the same as in Example 4.

[0083] Comparative Example 2: Tissue Culture Methods of *Senecio scandens*

[0084] Compared with Example 4, the only difference is that isopropyl acetate was not added to the proliferation-rooting medium when performing tissue culture of *Symplocos rubrum* in Comparative Example 2. All other steps were the same as in Example 4.

[0085] Comparative Example 3: Tissue Culture Methods of *Senecio scandens*

[0086] Compared with Example 4, the only difference is that no stabilizer was added to the proliferation-rooting medium when performing tissue culture of *Symplocos rubrum* in Comparative Example 3. All other steps were the same as in Example 4.

[0087] Comparative Example 4: Tissue Culture Methods of *Senecio scandens*

[0088] Compared with Example 4, the only difference is that polyethylene glycol was not added to the proliferation-rooting medium during the tissue culture of *Symplocos rubrum* in Comparative Example 4; all other steps were the same as in Example 4.

[0089] Comparative Example 5: Tissue Culture Methods of *Senecio scandens*

[0090] Compared with Example 4, the only difference is that the amount of isopropyl acetate added to the proliferation-rooting medium in Comparative Example 5 during the tissue culture of *Symplocos rubrum* is 5 g / L. All other steps are the same as in Example 4.

[0091] Comparative Example 6: Tissue Culture Methods of *Senecio scandens*

[0092] Compared with Example 4, the only difference is that in Comparative Example 6, the stabilizer added to the proliferation-rooting integrated culture medium for *Symplocos rubrum* tissue culture was not prepared by spraying with a coating solution. All other steps were the same as in Example 4. The specific preparation method of the stabilizer used is as follows:

[0093] 230g of sodium alginate was dissolved in water to prepare a 5wt% sodium alginate solution. Then, 0.04g of 6-BA and 0.015g of NAA were added and mixed evenly. The mixture was then slowly added dropwise to a 2wt% calcium chloride solution to gel and prepare sodium alginate-coated spheres with a particle size of about 1mm. After washing three times with sterile water, the spheres were dried at 45℃ for 25min to obtain semi-dry sodium alginate-coated spheres, which served as the stabilizer.

[0094] Experiment: Half-Maple Leaf Tissue Culture Experiment

[0095] The vitrification rate (when transferred for proliferation and rooting culture), rooting rate (after 38 days of integrated proliferation-rooting culture), and transplant survival rate (after 30 days of transplanting) of tissue culture seedlings from Examples 4-6 and Comparative Examples 1-6 were recorded. To ensure the accuracy of the experimental structure, the experiments were repeated twice more according to the methods of Examples 4-6 and Comparative Examples 1-6, for a total of 3 experiments. The final average data are shown in Table 1.

[0096] Table 1

[0097]

[0098] Based on the data analysis in Table 1, we can conclude that:

[0099] (1) The tissue culture of *Symplocos lumbricoides* according to the method of the present invention has a low vitrification rate. After 38 days of integrated proliferation-rooting culture, the rooting rate reached more than 85%, and the transplant survival rate was high. This indicates that the method of the present invention can effectively reduce the vitrification rate of *Symplocos lumbricoides*. Through integrated proliferation-rooting culture, most of the roots can be formed in a short time, and the rooted seedlings have a high survival rate after transplanting. This can better realize the efficient tissue culture of *Symplocos lumbricoides* and promote the development of the *Symplocos lumbricoides* industry.

[0100] (2) No cyclohexylamine was added to the proliferation-rooting integrated culture medium during the tissue culture of Comparative Example 1, and no isopropyl acetate was added to the proliferation-rooting integrated culture medium during the tissue culture of Comparative Example 2. The tissue culture seedlings in Comparative Examples 1 and 2 had weak absorption capacity of substances in the culture medium, low dry matter accumulation, and low lignification degree, so the proportion of vitrified seedlings obtained during the transfer was high.

[0101] (3) In Comparative Example 3, no stabilizer was added during the tissue culture of *Senecio scandens*, resulting in a low concentration of cytokinin in the culture medium and a reduction in vitrification. However, this limited the growth of the tissue culture seedlings, leading to a low rooting rate after transfer. In Comparative Example 4, no polyethylene glycol was added during the tissue culture of *Senecio scandens*, resulting in increased water absorption by the tissue culture seedlings and promoting the formation of vitrified seedlings. In Comparative Example 5, excessive addition of isopropyl acetate during the tissue culture of *Senecio scandens* caused excessive damage to the cell membranes of the tissue culture seedlings, which in turn affected their growth and seedling quality. In Comparative Example 6, no epoxy resin coating solution was used for spraying during the tissue culture of *Senecio scandens*. After being added to the culture medium, the seedlings rapidly absorbed water, swelled, and released most of the 6-BA. The excessive absorption of 6-BA by the tissue culture seedlings in a short period of time exacerbated the vitrification phenomenon.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for tissue culture of *Senecio scandens*, characterized in that, The method is as follows: (1) Explant collection and treatment: branches were collected from the mother tree, lignified stem segments were cut and leaves were removed, and the explants were obtained after washing. (2) Explant disinfection: Cut the explant into stem segments with 1-2 axillary buds and 2-3 cm long, wash with sterile water, and then disinfect them in ethanol solution and mercuric chloride solution to obtain disinfected explants; (3) Induction culture: After sterilization, the explants were inoculated into the induction medium for induction culture to obtain adventitious shoots; (4) Proliferation-rooting integrated culture: Single shoots are cut from the adventitious shoots obtained by induction culture and then inoculated into the proliferation-rooting integrated culture medium to obtain rooted seedlings; (5) Hardening and transplanting: Harden the rooted seedlings. After hardening, remove the rooted seedlings from the tissue culture bottle, wash off the culture medium from the roots, soak them in carbendazim solution for 20 minutes, and then transplant them into the substrate tray. The induction culture medium in step (3) consists of: MS + 1.0-1.2 mg / L 6-BA + 0.2-0.5 mg / L NAA + 30 g / L sucrose + 6.8 g / L agar, pH=5.8; In step (4), the proliferation-rooting integrated culture medium is a solid-liquid bilayer culture medium, wherein the upper layer is a liquid proliferation culture medium and the lower layer is a solid rooting culture medium. The proliferation-rooting integrated culture medium includes the following raw materials: Upper liquid proliferation medium: MS + 0.2-0.3 mg / L 6-BA + 0.05-0.15 mg / L NAA + 30 g / L sucrose + 2-3 g / L cyclohexylamine + 2-3 g / L isopropyl acetate + 3-4 g / L polyethylene glycol + 8-15 g / L stabilizer, pH=5.8; The lower solid rooting medium consisted of MS medium containing 0.6–0.8 mg / L IBA, 0.4 mg / L NAA, 0.6 mg / L ABT, 30 g / L sucrose, and 6.8 g / L agar, with a pH of 5.

8. The stabilizer comprises the following raw materials: Sodium alginate, 6-BA, NAA, 2wt% calcium chloride solution, rice bran wax, triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin; The stabilizer is prepared as follows: A: Dissolve sodium alginate in water to make a 5wt% sodium alginate solution, then add 6-BA and NAA and mix well. Then slowly drop it into a 2wt% calcium chloride solution to gel and obtain sodium alginate-coated balls. Wash with sterile water 3 times and dry at 45℃ for 20-30 minutes to obtain semi-dry sodium alginate-coated balls for later use. B: Grind rice bran wax through a 300-mesh sieve to obtain rice bran wax microparticles; put triethylamine, thiourea, and 2,4,6-tris(dimethylaminomethyl)phenol into a reaction vessel and mix evenly, heat to 110-130℃ and react for 0.5-1h, then cool to 55℃ and add epoxy resin and mix thoroughly, then add rice bran wax microparticles and mix evenly to obtain a coating solution; C: Spray the coating liquid onto the surface of the semi-dry sodium alginate coated ball, let it stand in a cool and ventilated place for 12-24 hours, and then place it at 60-70℃ for 2-3 hours to allow the rice bran wax particles on the surface of the coated ball to melt and flow out. Then wash it with deionized water 3-5 times to obtain the stabilizer. In step A, the mass ratio of sodium alginate solution, 6-BA, and NAA is (160-300):(0.03-0.05):(0.01-0.02). In step B, the mass ratio of triethylamine, thiourea, 2,4,6-tris(dimethylaminomethyl)phenol, epoxy resin, and rice bran wax microparticles is (8-16):(10-20):(10-20):(50-70):(10-20).

2. The method for tissue culture of *Symplocos pubescens* according to claim 1, characterized in that, The method for preparing the integrated proliferation-rooting culture medium is as follows: According to the composition ratio of the lower solid rooting medium, IBA, NAA, and ABT were added to MS medium and mixed thoroughly. Agar was then added, and the mixture was heated until the agar dissolved. Sucrose was then added and stirred until dissolved and mixed thoroughly. The pH was adjusted to 5.8, and the mixture was poured into culture flasks and sterilized at 121°C for 20 minutes. According to the composition ratio of the upper liquid proliferation medium, 6-BA, NAA, sucrose, cyclohexylamine, isopropyl acetate, and polyethylene glycol were added to MS medium and mixed thoroughly. Stabilizer was then added and mixed thoroughly. The mixture was sterilized at 121°C for 20 minutes. After the lower solid rooting medium in the culture flask solidified, it was added to obtain the integrated proliferation-rooting medium.

3. The method for tissue culture of *Symplocos pubescens* according to claim 2, characterized in that, The induction culture temperature is 25–27℃, the light duration is 8–12 h / d, and the light intensity is 2000–3000 lx; ​​the integrated proliferation-rooting culture temperature is 25–27℃, the light duration is 8–12 h / d, and the light intensity is 4500–5000 lx.

4. The method for tissue culture of *Symplocos pubescens* according to claim 3, characterized in that, The stabilizer has a particle size of 0.5-1 mm.

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