Tissue culture method of limonium bicolor

Abstract

This invention relates to the field of plant tissue culture technology, specifically a method for culturing *Limonium bicolor* tissue, comprising five steps: explant preparation and sterilization, callus induction culture, adventitious shoot proliferation culture, rooting culture, and hardening-off transplanting. Modified polyvinylpyrrolidone (PVP) and modified chitosan form a composite material through borate ester bonds, which accumulates around the explant. MES maintains pH stability, allowing PPVP to continuously diffuse into the wound. Modified chitosan blocks oxygen from direct contact with the wound surface, protecting endogenous phenols; its catechol groups chelate the PPO cofactor Cu. 2+ Activated carbon adsorbs free phenolic substrates in the culture medium, reducing quinone formation at its source. Modified polyvinylpyrrolidone inhibits PPO catalytic activity; grafted ascorbic acid can reduce already formed o-quinones back to catechol, directly removing brown pigment precursors.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically a method for tissue culture of Limonium bicolor. Background Technology

[0002] *Limonium bicolor*, a perennial herb belonging to the genus *Limonium* in the family Plumbaginaceae, is widely distributed in saline-alkali areas of Northeast, North, and Northwest my country. It possesses high ornamental, ecological restoration, and medicinal value. *Limonium bicolor* shows significant application potential in landscaping, the dried flower industry, and the ecological restoration of saline-alkali land, with continuously growing market demand. However, *Limonium bicolor* suffers from low seed germination rates and a small natural propagation coefficient, making traditional sexual reproduction insufficient for large-scale production. Tissue culture technology is a crucial means of achieving rapid propagation, but in practice, *Limonium bicolor* explants are prone to severe browning, becoming a major bottleneck restricting the establishment of its tissue culture system.

[0003] During plant tissue culture, the compartmental structure of cells is disrupted after explant cutting, allowing phenolic compounds stored in vacuoles to come into contact with polyphenol oxidase in the cytoplasm. Under aerobic conditions, polyphenol oxidase (PPO), with copper ions as a cofactor, catalyzes the oxidation of catechol substrates (such as chlorogenic acid and catechins) into o-quinone compounds. The resulting quinones are chemically reactive and can rapidly undergo spontaneous polymerization or Michael addition reactions with proteins and amino acids, ultimately generating high-molecular-weight brown pigments (melanin-like substances) that deposit on the explant wound surface, forming browning lesions. Mild browning inhibits explant differentiation, while severe browning leads to complete explant necrosis, severely affecting callus induction rate and adventitious shoot differentiation efficiency. Summary of the Invention

[0004] (1) Technical problems to be solved The purpose of this invention is to provide a method for tissue culture of *Limonium bicolor*, aiming to solve the problem of browning and death of *Limonium bicolor* explants.

[0005] (2) Technical solution To achieve the above objectives, the present invention provides a method for tissue culture of *Limonium sibiricum*, comprising the following steps: S1. Preparation and disinfection of explants: Take leaves of Limonium bicolor, and wash and disinfect them in sequence to obtain sterile explants; S2. Callus induction culture: The sterile explants obtained in step S1 are inoculated onto the callus induction culture medium and cultured to induce callus tissue. S3. Adventitious bud proliferation culture: The callus tissue obtained in step S2 is inoculated onto adventitious bud proliferation medium and cultured to differentiate and proliferate adventitious buds; S4. Rooting culture: The adventitious buds obtained in step S3 are divided and inoculated onto rooting medium for culture to induce rooting and obtain tissue culture seedlings; S5. Hardening and Transplanting: After hardening the tissue culture seedlings obtained in step S4, transplant them into the substrate; The callus induction culture medium includes a basic culture medium and additives; The basic culture medium includes: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-benzylaminopurine and 0.2 mg / L naphthaleneacetic acid; The additives include: MES at a concentration of 5-15 mM, modified chitosan at a concentration of 0.5-1.2 g / L, and modified polyvinylpyrrolidone at a concentration of 1.0-2.0 g / L. The modified chitosan is a caffeic acid-grafted chitosan-coated activated carbon composite material. The modified polyvinylpyrrolidone is phenylboronic acid-modified ascorbic acid-grafted polyvinylpyrrolidone.

[0006] Furthermore, the preparation method of the modified chitosan includes the following steps: S11. Dissolve chitosan in an aqueous acetic acid solution and stir until completely dissolved. Add caffeic acid, EDC·HCl and NHS in sequence and stir to dissolve. Adjust the pH with NaOH and stir the reaction under nitrogen protection. Purify the reaction solution by dialyzing and freeze-dry to obtain caffeic acid-chitosan. S12. Add granular activated carbon to dilute hydrochloric acid for soaking, filter, wash with deionized water, and dry at 80°C to obtain pretreated activated carbon. S13. Dissolve caffeic acid-chitosan in an aqueous acetic acid solution and stir until completely dissolved. Add pretreated activated carbon, disperse ultrasonically, and impregnate with magnetic stirring. Slowly add NaOH solution while stirring to adjust the pH. Continue stirring, filter, wash with deionized water, and vacuum dry to obtain modified chitosan.

[0007] Furthermore, the preparation method of the modified polyvinylpyrrolidone includes the following steps: S21. Dissolve polyvinylpyrrolidone in deionized water and stir until completely dissolved. Purge with nitrogen to remove oxygen. Add L-ascorbic acid and potassium persulfate and stir to dissolve. Under nitrogen protection, heat in a water bath and stir magnetically. After the reaction is complete, cool to room temperature, dialysis to purify the reaction solution, and freeze-dry to obtain ascorbic acid-polyvinylpyrrolidone. S22. Dissolve ascorbic acid-polyvinylpyrrolidone in PBS buffer, stir until completely dissolved, purge with nitrogen to remove oxygen, add 3-acrylamidophenylboronic acid and potassium persulfate, stir to dissolve, heat in a water bath under nitrogen protection, and stir magnetically to react. After the reaction is complete, cool to room temperature, dialysis to purify the reaction solution, and freeze-dry to obtain modified polyvinylpyrrolidone.

[0008] Furthermore, the method for preparing the callus induction culture medium includes the following steps: S31. Modified chitosan and modified polyvinylpyrrolidone were dissolved in PBS buffer, magnetically stirred, and filtered through a 0.22 μm filter membrane for sterilization to obtain the composite material; S32. Take MS basal medium, sucrose, and agar, add 6-benzylaminopurine and naphthaleneacetic acid, then add MES, adjust the pH, autoclave, cool, add the composite material, mix thoroughly, dispense into culture flasks, and let solidify for later use.

[0009] Furthermore, the adventitious bud proliferation medium in S3 comprises: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-benzylaminopurine, and 0.1 mg / L naphthaleneacetic acid.

[0010] Furthermore, the rooting medium in S4 comprises 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, and 0.2 mg / L indolebutyric acid.

[0011] Furthermore, the hardening-off treatment in S5 includes low-light hardening-off, weak-light hardening-off, normal-light hardening-off, and strong-light hardening-off, performed sequentially; the low-light hardening-off involves placing the tissue culture seedlings under a light intensity of 3000 Lux for 12 hours / day in closed bottles for 2 days; the weak-light hardening-off involves placing the tissue culture seedlings under a light intensity of 5000 Lux for 12 hours / day in closed bottles for 2 days; the normal-light hardening-off involves placing the tissue culture seedlings under a light intensity of 15000 Lux for 12 hours / day in open bottles for 3 days; and the strong-light hardening-off involves placing the tissue culture seedlings under a light intensity of 20000 Lux for 12 hours / day in open bottles for 4 days.

[0012] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. Under near-neutral conditions, phenylboronic acid grafted with modified polyvinylpyrrolidone (MES) forms a reversible borate ester bond with catechol of caffeic acid, anchoring the modified MES to the surface of modified chitosan. The modified chitosan accumulates around the explant through gravity sedimentation and the binding of the positive charge of chitosan with the negative charge of pectin in the cell wall. After the explant is cut, the release of organic acids causes a local pH drop. MES maintains pH stability through its buffering capacity, which is conducive to the slow hydrolysis of the borate ester bond, thereby allowing the modified MES to continuously diffuse and release into the cut.

[0013] 2. The modified chitosan coating forms a biocompatible film on the surface of the explant incision, preventing oxygen from directly contacting the wound; its grafted caffeic acid contains a catechol structure, which can efficiently chelate the PPO cofactor Cu. 2+ This inhibits the catalytic activity of PPO, reducing the oxidation of endogenous phenols. Simultaneously, the catechol groups of caffeic acid can act as competitive substrates when PPO activity is not completely inhibited, preferentially contacting PPO and delaying the oxidation process of endogenous phenols. Activated carbon can adsorb free phenolic substrates in the culture medium, reducing the concentration of substrates accessible to PPO and decreasing quinone formation at the source.

[0014] 3. The modified polyvinylpyrrolidone skeleton binds to PPO protein through hydrogen bonds and hydrophobic interactions, covering its active site and inhibiting PPO catalytic activity; the grafted ascorbic acid can reduce the generated o-quinone back to catechol, directly removing the brown pigment precursor. Attached Figure Description

[0015] Figure 1 This is a comparison diagram of the tissue growth status of the two-colored blood-tonifying herb of the present invention. Detailed Implementation

[0016] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0017] Example 1: This example discloses a method for tissue culture of *Hypericum divaricata*, including the following steps: S1. Preparation and disinfection of explants: Young leaves of *Limonium bicolor* were collected as explants and rinsed 3-5 times with tap water to remove surface soil. In a clean bench, the washed leaves were soaked in 75% (volume) alcohol for 30 seconds, the alcohol was discarded, and then soaked in 0.1% mercuric chloride solution for 8 minutes, gently shaking during the process. The mercuric chloride solution was discarded, and the leaves were rinsed 4 times with sterile water. The disinfected leaves were placed on sterile filter paper to absorb the moisture and cut into 0.5cm×1cm rectangular pieces with sterile scissors for later use.

[0018] S2. Callus induction culture: The leaf explants prepared in step S1 are inoculated onto callus induction culture medium. 3-4 explants are inoculated into each culture bottle.

[0019] The method for preparing the callus induction culture medium is as follows: S31. Dissolve 0.8 g / L modified chitosan and 1.5 g / L modified polyvinylpyrrolidone in 0.1 M PBS buffer (pH 7.5), and stir magnetically at room temperature for 2 h to allow phenylboronic acid and caffeic acid catechol to fully form reversible borate ester bonds under near-neutral conditions. Filter through a 0.22 μm filter membrane to remove bacteria and obtain the composite material. S32. Take 4.74 g / L MS basal medium, 30 g / L sucrose, and 7 g / L agar, add 1.0 mg / L 6-benzylaminopurine and 0.2 mg / L naphthaleneacetic acid, then add 10 mM MES to adjust the pH to 6.0. Autoclave at 121℃ for 20 min, cool to below 50℃, add the composite material, mix thoroughly, and dispense into culture flasks (approximately 40 mL per flask). Allow to solidify for later use.

[0020] The cultivation conditions were: temperature 26℃, light intensity 2000 Lux, light duration 12 hours / day, and cultivation for 20 days.

[0021] S3. Adventitious bud proliferation culture: The well-grown callus tissue induced in step S2 was cut into 0.5cm sections. 3 Small pieces were inoculated onto adventitious bud proliferation medium.

[0022] The adventitious bud proliferation medium formula is: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, with the addition of 1.0 mg / L 6-benzylaminopurine and 0.1 mg / L naphthaleneacetic acid, adjusted to pH 6.0, and autoclaved at 121℃ for 20 min.

[0023] The cultivation conditions were: temperature 26℃, light intensity 2000 Lux, light duration 12 hours / day, and cultivation for 30 days. S4. Rooting culture: Cut off individual adventitious buds that are 2-3 cm in height and growing well from step S3 and inoculate them onto rooting culture medium.

[0024] The rooting medium formula is: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar and 0.2 mg / L indolebutyric acid, pH 6.0.

[0025] The cultivation conditions were: temperature 26℃, light intensity 2000 Lux, light duration 12 hours / day, and cultivation for 35 days.

[0026] S5. Hardening off and transplanting: The rooted tissue culture seedlings obtained in step S4 are first placed in a closed bottle under low light conditions of 3000 Lux for 12 hours / day for 2 days, then transferred to a closed bottle under weak light conditions of 5000 Lux for 12 hours / day for 2 days, then transferred to a normal light condition of 15000 Lux for 12 hours / day for 3 days, and finally hardened off in a closed bottle under strong light conditions of 20000 Lux for 12 hours / day for 4 days.

[0027] The preparation method of the modified chitosan includes the following steps: S11. Dissolve 2g of chitosan in 200mL of 1% acetic acid aqueous solution and stir until completely dissolved. Then add 1g of caffeic acid, 0.8g of EDC·HCl and 0.4g of NHS in sequence and stir to dissolve. Adjust the pH to 5.5 with 0.1M NaOH. Under nitrogen protection, stir and react at 25℃ for 16 hours. Dilute the reaction solution by dialysis and freeze dry to obtain caffeic acid-chitosan. S12. Soak 10g of granular activated carbon in 5% dilute hydrochloric acid for 2 hours, filter, wash repeatedly with deionized water until the pH of the filtrate is neutral, and dry at 80℃ to obtain pretreated activated carbon. S13. Dissolve 2g of caffeic acid-chitosan in 200mL of 1% acetic acid aqueous solution, stir until completely dissolved, add pretreated activated carbon, ultrasonically disperse for 10min, magnetically stir and impregnate at room temperature for 12h, slowly add 10% NaOH solution dropwise while stirring, adjust pH to 8.5-9.0, continue stirring for 1h, filter, wash repeatedly with deionized water until the pH of the filtrate is neutral, vacuum dry at 60℃ for 12h to obtain modified chitosan.

[0028] The method for preparing the modified polyvinylpyrrolidone includes the following steps: S21. Dissolve 10g of polyvinylpyrrolidone in 100mL of deionized water and stir until completely dissolved. Purge with nitrogen for 30min to remove dissolved oxygen from the solution. Add 5g of L-ascorbic acid and 0.2g of potassium persulfate and stir to dissolve. Under nitrogen protection, heat in a water bath at 65℃ and stir magnetically for 4h. After the reaction is complete, cool to room temperature, dialyze the reaction solution to purify it, and freeze-dry to obtain ascorbic acid-polyvinylpyrrolidone. S22. Dissolve 5g of ascorbic acid-polyvinylpyrrolidone in 50mL of PBS buffer (pH 7.0, 0.1 M), stir until completely dissolved, purge with nitrogen for 20min to remove oxygen, add 0.5g of 3-acrylamidophenylboronic acid and 0.05g of potassium persulfate, stir to dissolve, heat in a 65℃ water bath under nitrogen protection, and magnetically stir for 6h. After the reaction is complete, cool to room temperature, dialyze to purify the reaction solution, and freeze-dry to obtain modified polyvinylpyrrolidone.

[0029] Example 2: This example is based on Example 1, but differs from Example 1 in that the additives in this example include: MES at a concentration of 15 mM, modified chitosan at a concentration of 1.2 g / L, and modified polyvinylpyrrolidone at a concentration of 2.0 g / L. The other components and preparation methods are the same as in Example 1.

[0030] Example 3: This example is based on Example 1, but differs from Example 1 in that the additives in this example include: MES at a concentration of 5 mM, modified chitosan at a concentration of 0.5 g / L, and modified polyvinylpyrrolidone at a concentration of 1.0 g / L. The other components and preparation methods are the same as in Example 1.

[0031] Comparative Example 1: This comparative example is based on Example 1, but differs from Example 1 in that the modified chitosan in this comparative example is ungrafted caffeic acid.

[0032] The preparation method of the modified chitosan includes the following steps: S11. Soak 10g of granular activated carbon in 5% dilute hydrochloric acid for 2 hours, filter, wash repeatedly with deionized water until the pH of the filtrate is neutral, and dry at 80℃ to obtain pretreated activated carbon. S12. Dissolve 2g of chitosan in 200mL of 1% acetic acid aqueous solution and stir until completely dissolved. Add pretreated activated carbon, ultrasonically disperse for 10min, and magnetically stir and impregnate at room temperature for 12h. Slowly add 10% NaOH solution while stirring to adjust the pH to 8.5-9.0, continue stirring for 1h, filter, wash repeatedly with deionized water until the pH of the filtrate is neutral, and vacuum dry at 60℃ for 12h to obtain modified chitosan.

[0033] The other components and preparation methods are the same as in Example 1.

[0034] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that the modified chitosan in this comparative example is not coated with activated carbon.

[0035] The preparation method of the modified chitosan includes the following steps: S11. Dissolve 2g of chitosan in 200mL of 1% acetic acid aqueous solution and stir until completely dissolved. Then add 1g of caffeic acid, 0.8g of EDC·HCl and 0.4g of NHS in sequence and stir to dissolve. Adjust the pH to 5.5 with 0.1M NaOH. Under nitrogen protection, stir and react at 25℃ for 16 hours. Dilute the reaction solution by dialysis and freeze-dry to obtain modified chitosan.

[0036] The other components and preparation methods are the same as in Example 1.

[0037] Comparative Example 3: This comparative example is based on Example 1, but differs from Example 1 in that the modified polyvinylpyrrolidone described in this comparative example is not grafted with phenylboronic acid.

[0038] The method for preparing the modified polyvinylpyrrolidone includes the following steps: S21. Dissolve 10g of polyvinylpyrrolidone in 100mL of deionized water and stir until completely dissolved. Purge with nitrogen for 30min to remove dissolved oxygen from the solution. Add 5g of L-ascorbic acid and 0.2g of potassium persulfate and stir to dissolve. Under nitrogen protection, heat in a water bath at 65℃ and stir magnetically for 4h. After the reaction is complete, cool to room temperature, dialyze the reaction solution to purify it, and freeze-dry to obtain modified polyvinylpyrrolidone.

[0039] The other components and preparation methods are the same as in Example 1.

[0040] Comparative Example 4: This comparative example is based on Example 1, but differs from Example 1 in that the modified polyvinylpyrrolidone in this comparative example is not grafted with ascorbic acid.

[0041] The method for preparing the modified polyvinylpyrrolidone includes the following steps: S21. Dissolve 5g of polyvinylpyrrolidone in 50mL of PBS buffer (pH 7.0, 0.1M), stir until completely dissolved, purge with nitrogen for 20min to remove oxygen, add 0.5g of 3-acrylamidophenylboronic acid and 0.05g of potassium persulfate, stir to dissolve, heat in a 65℃ water bath under nitrogen protection, and magnetically stir for 6h. After the reaction is complete, cool to room temperature, dialyze the reaction solution to purify it, and freeze-dry to obtain modified polyvinylpyrrolidone.

[0042] The other components and preparation methods are the same as in Example 1.

[0043] Comparative Example 5: This comparative example is based on Example 1, but differs from Example 1 in that the callus induction culture medium described in this comparative example does not contain modified chitosan.

[0044] Comparative Example 6: This comparative example is based on Example 1, but differs from Example 1 in that the callus induction culture medium described in this comparative example does not contain modified polyvinylpyrrolidone.

[0045] Comparative Example 7: This comparative example is based on Example 1, but differs from Example 1 in that the callus induction culture medium described in this comparative example does not contain MES.

[0046] The other components and preparation methods are the same as in Example 1.

[0047] Comparative Example 8: This comparative example is based on Example 1, but differs from Example 1 in that the callus induction culture medium described in this comparative example does not contain modified chitosan, modified polyvinylpyrrolidone, and MES.

[0048] Experimental verification: Experiment 1: Healthy, disease-free *Limonium bicolor* plants were selected, and young leaves were collected to prepare sterile explants. All groups underwent callus induction culture under the same conditions (temperature 26℃, light intensity 2000 Lux, light duration 12 hours / day) for 20 days. Each group had 5 replicates, with 4 explants inoculated per bottle, for a total of 20 explants. Browning of each explant was recorded on days 3, 7, and 20 of culture. Browning rate (%) = number of explants showing obvious browning / total number of inoculated explants × 100%. Callus induction rate (%) = number of explants inducing callus / total number of inoculated explants × 100%, calculated on day 20 of culture.

[0049] Table 1: Browning status of explants and callus induction results: Table 1 shows the browning of explants and the results of callus induction. In Comparative Example 8, when all three additives were absent, the browning rate was over 80% on day 20, the callus induction rate was the lowest, and a large number of explants died, confirming that the browning problem of *Limonium bicolor* explants was extremely serious. In Examples 1-3, the browning rate was controlled below 14% on day 20, and the callus induction rate was over 90%.

[0050] Experiment 2: (1) Select the well-growing portion of the callus tissue induced in each group and cut it into pieces of approximately 0.5cm. 3 Small pieces were uniformly inoculated onto adventitious bud proliferation medium and cultured at 26℃, 2000 Lux, 12 hours / day for 30 days. The adventitious bud differentiation rate was then calculated.

[0051] (2) Select 20 adventitious buds with a height of 2-3cm and strong growth from each group of proliferation culture, cut them off individually and inoculate them on rooting medium, and culture them at 26℃, 2000Lux, 12 hours / day for 35 days, and calculate the rooting rate.

[0052] (3) Select tissue culture seedlings with intact root systems (≥2 roots and ≥1.5cm in length) from groups with a rooting rate ≥68%, and carry out hardening treatment. After hardening, gently wash off the agar from the roots, transplant them into nutrient pots containing substrate, and place them in a greenhouse (temperature 22-28℃, relative humidity 60%-75%, natural light) for cultivation. Investigate the survival status every 5 days, and calculate the survival rate on the 45th day, the plant height (cm) and the maximum number of leaves (pieces) 45 days after transplanting.

[0053] Table 2: Results of adventitious shoot differentiation rate, rooting rate and survival rate of callus tissue: Table 2 shows the results of adventitious bud differentiation rate, rooting rate, and survival rate of the callus tissue. Example 1 showed the best results, with its high-quality callus tissue further ensuring the adventitious bud proliferation coefficient, rooting rate, and final transplant survival rate. Figure 1 The figure shows a comparison of the growth status of the two-color Limonium sinense tissues of the present invention. a) shows the rooting status of the two-color Limonium sinense tissues cultivated by callus induction culture in Example 1, and b) shows the rooting status of the two-color Limonium sinense tissues cultivated by callus induction culture in Comparative Example 8. It can be clearly seen from the figure that the rooting status of Example 1 is better, the leaf growth is better, and the growth is more excellent.

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for tissue culture of *Hypericum perforatum*, characterized in that, Includes the following steps: S1. Preparation and disinfection of explants: Take leaves of Limonium bicolor, and wash and disinfect them in sequence to obtain sterile explants; S2. Callus induction culture: The sterile explants obtained in step S1 are inoculated onto the callus induction culture medium and cultured to induce callus tissue. S3. Adventitious bud proliferation culture: The callus tissue obtained in step S2 is inoculated onto adventitious bud proliferation medium and cultured to differentiate and proliferate adventitious buds; S4. Rooting culture: The adventitious buds obtained in step S3 are divided and inoculated onto rooting medium for culture to induce rooting and obtain tissue culture seedlings; S5. Hardening and Transplanting: After hardening the tissue culture seedlings obtained in step S4, transplant them into the substrate; The callus induction culture medium includes a basic culture medium and additives; The basic culture medium includes: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-benzylaminopurine and 0.2 mg / L naphthaleneacetic acid; The additives include: MES at a concentration of 5-15 mM, modified chitosan at a concentration of 0.5-1.2 g / L, and modified polyvinylpyrrolidone at a concentration of 1.0-2.0 g / L. The modified chitosan is a caffeic acid-grafted chitosan-coated activated carbon composite material. The modified polyvinylpyrrolidone is phenylboronic acid-modified ascorbic acid-grafted polyvinylpyrrolidone.

2. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The preparation method of the modified chitosan includes the following steps: S11. Dissolve chitosan in an aqueous acetic acid solution and stir until completely dissolved. Add caffeic acid, EDC·HCl and NHS in sequence and stir to dissolve. Adjust the pH with NaOH and stir the reaction under nitrogen protection. Purify the reaction solution by dialyzing and freeze-dry to obtain caffeic acid-chitosan. S12. Add granular activated carbon to dilute hydrochloric acid for soaking, filter, wash with deionized water, and dry at 80°C to obtain pretreated activated carbon. S13. Dissolve caffeic acid-chitosan in an aqueous acetic acid solution and stir until completely dissolved. Add pretreated activated carbon, disperse ultrasonically, and impregnate with magnetic stirring. Slowly add NaOH solution while stirring to adjust the pH. Continue stirring, filter, wash with deionized water, and vacuum dry to obtain modified chitosan.

3. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The method for preparing the modified polyvinylpyrrolidone includes the following steps: S21. Dissolve polyvinylpyrrolidone in deionized water and stir until completely dissolved. Purge with nitrogen to remove oxygen. Add L-ascorbic acid and potassium persulfate and stir to dissolve. Under nitrogen protection, heat in a water bath and stir magnetically. After the reaction is complete, cool to room temperature, dialysis to purify the reaction solution, and freeze-dry to obtain ascorbic acid-polyvinylpyrrolidone. S22. Dissolve ascorbic acid-polyvinylpyrrolidone in PBS buffer, stir until completely dissolved, purge with nitrogen to remove oxygen, add 3-acrylamidophenylboronic acid and potassium persulfate, stir to dissolve, heat in a water bath under nitrogen protection, and stir magnetically to react. After the reaction is complete, cool to room temperature, dialysis to purify the reaction solution, and freeze-dry to obtain modified polyvinylpyrrolidone.

4. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The method for preparing the callus induction culture medium includes the following steps: S31. Modified chitosan and modified polyvinylpyrrolidone were dissolved in PBS buffer, magnetically stirred, and filtered through a 0.22 μm filter membrane for sterilization to obtain the composite material; S32. Take MS basal medium, sucrose, and agar, add 6-benzylaminopurine and naphthaleneacetic acid, then add MES, adjust the pH, autoclave, cool, add the composite material, mix thoroughly, dispense into culture flasks, and let solidify for later use.

5. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The adventitious bud proliferation medium in S3 consists of: 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-benzylaminopurine and 0.1 mg / L naphthaleneacetic acid.

6. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The rooting medium in S4 consists of 4.74 g / L MS basal medium, 30 g / L sucrose, 7 g / L agar, and 0.2 mg / L indolebutyric acid.

7. The method for tissue culture of *Limonium sibiricum* according to claim 1, characterized in that, The hardening-off treatment in S5 includes low-light hardening, weak-light hardening, normal-light hardening, and strong-light hardening, performed sequentially. Low-light hardening involves placing the tissue culture seedlings under a light intensity of 3000 Lux for 12 hours / day in closed bottles for 2 days. Weak-light hardening involves placing the tissue culture seedlings under a light intensity of 5000 Lux for 12 hours / day in closed bottles for 2 days. Normal-light hardening involves placing the tissue culture seedlings under a light intensity of 15000 Lux for 12 hours / day in open bottles for 3 days. Strong-light hardening involves placing the tissue culture seedlings under a light intensity of 20000 Lux for 12 hours / day in open bottles for 4 days.

Images