Cardamine violifolia selenium-rich regenerated plant induction method based on tissue culture

Abstract

The invention relates to the technical field of plant tissue culture, and particularly discloses a cardamine violifolia selenium-rich regenerated plant induction method based on tissue culture. Comprising the following steps: S1, explant preparation and disinfection: performing surface disinfection and sterile flushing by taking cardamine violifolia seeds as a starting material to obtain sterile seeds; inoculating the sterile seeds into a germination culture medium for sterile germination to obtain sterile seedlings; s2, callus induction: cutting leaves of the aseptic seedlings obtained in the step S1 as explants, and inducing to obtain calluses; s3, performing selenium enrichment treatment; s4, performing differentiation culture; s5, rooting induction; s6, seedling hardening; and S7, transplanting. The cardamine violifolia selenium-rich regenerated plant induction method can be used for producing regenerated plants which are highly uniform in genetic background and stable in selenium content and meet expected standards, and has the advantages that the sterility and genetic consistency of explants are ensured, the selenium enrichment process is controllable, and the selenium content is prevented from being unstable.

Description

Technical Field

[0001] This application relates to the field of plant tissue culture technology, and more specifically, it relates to a method for inducing selenium-enriched regenerated plants of Viola yedoensis based on tissue culture. Background Technology

[0002] The application of plant tissue culture technology in the field of Viola yedoensis mainly focuses on germplasm resource conservation and rapid propagation of superior strains. This technology can efficiently produce a large number of seedlings with consistent genetic backgrounds through aseptic rapid propagation. At the same time, by adding selenium in a controlled culture environment, the key processes of its tolerance and accumulation can be studied. In addition, this technology also provides a key genetic transformation and plant regeneration system foundation for subsequent targeted improvement of its bioremediation efficacy using genetic engineering methods. The relevant method of cultivating Viola yedoensis involves selenium biofortification in natural or field soils. However, this method relies on wild seeds or plants with mixed genetic backgrounds as starting materials. Furthermore, the distribution of selenium in the soil, microbial activity, and other environmental factors are complex, variable, and difficult to control. As a result, the obtained plants exhibit individual differences in their selenium accumulation capacity, and their selenium content is unstable and unpredictable. Summary of the Invention

[0003] To address the issue of individual differences in selenium accumulation capacity and unstable, unpredictable selenium content in *Corydalis violaceus* plants obtained through selenium biofortification cultivation in natural or field soils, this application provides a method for inducing selenium-enriched regenerated *Corydalis violaceus* plants based on tissue culture. This application provides the following technical solution: A method for inducing selenium-enriched regenerated plants of Viola yedoensis based on tissue culture includes the following steps: S1. Explant preparation and disinfection: Using Viola yedoensis seeds as the starting material, surface disinfection and aseptic rinsing were performed to obtain sterile seeds; the sterile seeds were inoculated into germination medium for aseptic germination to obtain sterile seedlings; S2. Callus induction: Leaves of the sterile seedlings obtained in S1 were cut off as explants, inoculated into the culture medium and cultured in the dark to induce callus tissue. S3, Selenium enrichment treatment: The callus obtained in S2 was transferred into a selenium-enriched induction medium with added selenium source for culture to obtain selenium-enriched callus. S4. Differentiation culture: The selenium-enriched callus obtained in S3 was transferred to the differentiation medium and cultured under light to induce the differentiation of clustered shoots. S5. Rooting induction: Healthy adventitious buds from the clustered buds obtained in S4 are cut and transferred to rooting medium for culture to induce the formation of regenerated small plantlets with roots. S6. Hardening off seedlings: Harden off the regenerated small plantlets obtained in S5 by opening the bottle. S7. Transplanting: Transplant the hardened plants obtained in S6 into an organic-rich acclimatization substrate to obtain selenium-enriched regenerated plants. By adopting the above technical solution, and using sterile seedlings obtained from seed germination as starting material, the high consistency and sterility of the explants are ensured. Through stepwise induction of callus tissue, selenium enrichment treatment, redifferentiation and rooting, a complete in vitro regeneration and selenium enrichment integration system is established. Therefore, regenerated plants with uniform genetic background and stable and controllable selenium content are obtained, and the goal of efficient production of selenium-enriched Corydalis yanhusuo under controlled environment is achieved. Preferably, in step S1, the seeds of *Cephalotaxus fortunei* are pretreated as follows: a certain number of *Cephalotaxus fortunei* seeds are soaked in ultrapure water for 10-15 minutes for surface cleaning, then rinsed with running water for 2 hours, rinsed with sterile water 3-5 times, and then soaked for 30-40 minutes; in a clean bench, they are soaked in 75% anhydrous ethanol for 30-40 seconds, rinsed with sterile water 5-6 times, then disinfected with 0.1% HgCl2 for 7 minutes, rinsed with sterile water 5-6 times after disinfection, placed on sterile filter paper to absorb surface moisture, and then the *Cephalotaxus fortunei* seeds are inoculated into MS medium. By adopting the above technical solution, the seeds of *Violet Flos* are first soaked in ultrapure water to remove dust and impurities adhering to the surface, laying a clean foundation for subsequent disinfection steps. Then, running water rinsing further removes particulate matter and reduces the initial microbial load, while multiple sterile water rinsings ensure the removal of residual impurities, avoiding interference with the disinfectant's action. In a clean bench, a brief soaking in 75% anhydrous ethanol utilizes its ability to rapidly penetrate cell membranes, denaturing microbial proteins and thus efficiently killing surface bacteria and fungi, while controlling the time to maintain seed viability. Next, disinfection is performed using 0.1% HgCl2, with the concentration and seven-minute contact time set according to common pathogen resistance, achieving thorough sterilization by disrupting the microbial enzyme system. After disinfection, repeated sterile water rinsing removes chemical residues, and filter paper absorbs surface moisture to prevent dilution of the culture medium components and promotes close contact between the seeds and the culture medium. Finally, the pretreated seeds are inoculated into MS medium, whose balanced nutrients support germination. The entire pretreatment process, through progressive cleaning, disinfection, and residue removal, provides a sterile environment for the seeds, ensuring they are free from contamination during subsequent cultivation. Preferably, in step S2, the culture medium is MS medium with the pH adjusted to 5.8, and auxin and cytokinin are added; wherein the auxin is 2,4-dichlorophenoxyacetic acid, and the added concentration is 1.5-2.5 mg / L; the cytokinin is 6-benzylaminopurine, and the added concentration is 0.2-0.8 mg / L; the dark culture conditions are: culture temperature of 22-26℃, and culture time of 28-35 days. By employing the above-mentioned technical solution, and using MS medium as a base with a specific concentration of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine, this hormone combination can effectively initiate mesophyll cell dedifferentiation. 2,4-dichlorophenoxyacetic acid primarily promotes rapid cell division to form callus tissue, while the low concentration of 6-benzylaminopurine plays a synergistic role in maintaining the activity of callus tissue cells. Combined with suitable dark culture temperature and time, the potential inhibition of callus induction by light is avoided. Therefore, vigorous, loosely textured, and brightly colored embryogenic callus tissue is obtained, providing ideal and physiologically consistent starting material for subsequent selenium enrichment treatment. Preferably, in step S2, before dark culture, the size of the explant leaves is controlled to be 0.3cm×0.3cm~0.5cm×0.5cm, and the leaf edges have a small amount of petiole tissue. By adopting the above technical solution, by controlling the leaf explants within a specific size range, it is possible to ensure that a sufficient number of live cells participate in callus formation, while avoiding excessively large explants leading to internal nutrient competition or excessively small explants leading to insufficient cell quantity. The requirement for the leaf edge to have a small amount of petiole tissue is because the cells at the base of the petiole have a stronger meristematic ability and contain more active meristematic tissue, which can improve the induction efficiency and initiation speed of callus. Therefore, the culture results with a high callus induction rate and a relatively stable induction cycle are obtained, which provides convenience for the standardized operation of subsequent processes. Preferably, in step S3, the selenium source is sodium selenite, and the concentration of sodium selenite is 8–15 μmol / L; the selenium-enriched induction medium is MS medium, and 2,4-dichlorophenoxyacetic acid at a concentration of 1.0–2.0 mg / L and 6-benzylaminopurine at a concentration of 0.5–1.0 mg / L are added. By adopting the above technical solution, sodium selenite is used as the inorganic selenium source, which is easily absorbed and transformed by plant cells. The concentration of selenium added is controlled at a low level to avoid acute toxicity of high concentrations of selenium to callus tissue, while ensuring effective accumulation of selenium. A certain concentration of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine is maintained in the culture medium to continue the vigorous growth of callus tissue under selenium-enriched conditions and prevent premature aging or death due to selenium stress. Therefore, callus tissue that can continuously proliferate and has begun to effectively accumulate selenium is obtained, achieving the initial goal of selenium biofortification. Preferably, in step S3, the selenium enrichment treatment adopts an alternating cyclic culture method, specifically: the callus tissue is first cultured in the selenium-enriched induction medium for 7-10 days, and then transferred to the same basal medium without selenium source for 5-7 days, which is one cycle; this is repeated for 2-3 cycles; the culture conditions are: light intensity 1500-2500 Lux, photoperiod 12-14 h / d, and temperature 23-25℃. By adopting the above technical solution, the alternating cyclic culture strategy allows callus tissue to undergo selenium stress and absorption in selenium-containing medium before being transferred to selenium-free medium to restore physiological metabolism, thus alleviating the growth inhibition that selenium accumulation may cause. Light culture conditions help maintain a certain photosynthetic capacity and healthy state of the callus tissue. Through multiple alternating cycles, selenium-tolerant cell lines are gradually screened and enriched, and the organic transformation and homeostatic accumulation of selenium within cells are promoted. Therefore, selenium-enriched callus tissue with increased selenium content, good growth vitality, and stronger regeneration potential is obtained, laying the foundation for successful differentiation into seedlings in the next step. Preferably, in step S4, the differentiation medium is MS medium with the pH adjusted to 5.8, and cytokinin and gibberellin are added; wherein the cytokinin is 6-benzylaminopurine, and the added concentration is 1.5-2.5 mg / L; the added concentration of gibberellin is 0.5-1.0 mg / L; the light culture conditions are: light intensity 2000-3000 Lux, photoperiod 14-16 h / d, culture temperature 24-26℃, and culture time 25-40 days. By adopting the above technical solution, the high concentration of cytokinin, achieved by increasing the concentration of 6-benzylaminopurine and adding gibberellin, effectively broke the dormancy state of the callus tissue and strongly promoted the initiation and formation of bud primordia. Gibberellin mainly promoted the elongation growth of the formed bud points, and the two worked synergistically to drive organogenesis. Combined with a longer light duration, stronger light intensity, and suitable constant temperature conditions, sufficient energy and ideal environmental signals were provided for bud differentiation and growth. Therefore, a large number of robust adventitious buds were successfully induced from selenium-rich callus tissue, completing the key transformation from tissueless structure to organized organ. Preferably, in step S5, the rooting medium is 1 / 2 MS, the pH is adjusted to 5.8, and auxin is added; wherein, the auxin is indolebutyric acid, and the added concentration is 0.8-1.2 mg / L; activated carbon is also added when preparing the rooting medium, and the added concentration of activated carbon is 0.8-1.5 g / L; the culture conditions are: light intensity 1500-2500 Lux, photoperiod 12-14 h / d, culture temperature 22-25℃, and culture for 18-25 days. By adopting the above technical solution, indolebutyric acid (IBA), a stable and non-oxidizing auxin with root-promoting effects, can effectively induce the differentiation of root primordia from the basal cells of adventitious buds at appropriate concentrations. The activated carbon added to the culture medium can adsorb harmful substances such as phenols secreted by the explants during culture, as well as some residual hormones, purifying the culture medium environment and promoting healthy root development. Moderate light and temperature conditions reduce the transpiration and metabolic stress of the buds, allowing them to concentrate nutrients on root development. Therefore, complete regenerated small plants with well-developed root systems, abundant root hairs, and good connection with the above-ground parts are obtained, improving the survival rate of subsequent hardening and transplanting. Preferably, in step S6, when the regenerated small plantlets grow to 6-8cm, they are subjected to hardening-off by opening the bottle. Hardening-off by opening the bottle specifically involves fully opening the bottle mouth, injecting a small amount of sterile water into the bottle to maintain humidity, and hardening-off for 2-3 days. By employing the above technical solutions, when the regenerated plantlets grow to a height of 6-8 cm, their vascular tissues begin to develop, enabling them to adapt to changes in the external environment. At this point, the acclimatization process is performed by opening the culture bottle to connect the internal environment with the external air. This allows the plantlets to gradually adapt to the lower humidity and fluctuating gas composition of the natural environment, gradually reducing carbon dioxide concentration, thereby activating and training their stomatal regulation function and cuticle development. A small amount of sterile water is injected into the bottle to maintain a relatively high humidity in the local space during the acclimatization period. This provides a gradual humidity gradient for the plantlets' leaves and stems, preventing wilting due to sudden environmental changes. Simultaneously, the sterile water ensures that the transition period is free from contamination by other microorganisms. Setting an acclimatization period of 2-3 days allows the plantlets to complete initial stomatal behavior adjustments and activate their stress resistance mechanisms while avoiding the risk of excessive proliferation of pathogenic microorganisms in an open environment. In summary, through controlled environmental acclimatization, the stress resistance and survival rate of the plantlets after transplanting are enhanced, providing an adaptive transition for the complete plant regeneration process. Preferably, in step S7, the acclimatization substrate is a mixture of peat moss, perlite and vermiculite in a volume ratio of (3-4):(1-2):1, and the pH value of the acclimatization substrate is adjusted to 5.6-6.0 before transplanting; after transplanting, it is acclimatized and cultivated in a greenhouse environment with a relative humidity of 75-85% and a temperature of 20-25℃ for 10-15 days. By adopting the above technical solution, the acclimatization substrate, composed of peat moss, perlite, and vermiculite, provides organic matter and water retention, while perlite and vermiculite ensure the substrate is loose and breathable. This ratio creates an ideal rhizosphere environment that retains moisture without causing waterlogging. Adjusting the substrate pH to slightly acidic aligns with the preference of *Corydalis violacea*, facilitating root adaptation and growth. Maintaining high air humidity and suitable temperature after transplanting minimizes seedling transpiration, supporting effective absorption of water and nutrients by the roots during the recovery period. Therefore, acclimatized plants with high transplant survival rates and good growth are obtained, ultimately leading to the successful acquisition of regenerated *Corydalis violacea* plants that are adaptable to the natural environment and stably rich in selenium. In summary, this application has the following beneficial effects: 1. This application uses sterile seedlings as starting material and constructs a system that integrates callus induction, selenium bio-enhancing, organ differentiation, and domestication transplantation to ensure the sterility and genetic consistency of explants from the source. At the same time, it makes the selenium enrichment process in the in vitro culture environment controllable, thus obtaining regenerated plants with highly uniform genetic background, stable selenium content, and meeting the expected standards, while avoiding the instability of selenium content caused by individual differences and soil environment fluctuations. 2. In this application, the preferred method is to introduce an alternating cyclic culture process during the selenium enrichment stage of the callus. The callus is cultured in selenium-containing medium and selenium-free basal medium several times. This process balances the relationship between selenium stress absorption and physiological recovery, avoiding the cytotoxic growth inhibition caused by continuous selenium stress, and intermittently restoring the selenium tolerance of cultured cells and promoting the organic transformation of selenium. Therefore, embryogenic callus with vigorous proliferation and enhanced selenium enrichment capacity is obtained, laying the foundation for the subsequent efficient differentiation of robust selenium-enriched seedlings. 3. The method of this application improves callus induction efficiency by controlling the size of explants, improves the hormone ratio during differentiation and rooting stages to drive organogenesis and root development, and combines closed-bottle and open-bottle hardening and substrate acclimatization synergistic regulation. The above steps provide transitional conditions from in vitro culture to transplant survival, thus obtaining robust seedlings with well-developed root systems and strong stress resistance, and finally successfully transplanting them to form regenerated plants that can adapt to the natural environment and are stable and rich in selenium. Attached Figure Description Figure 1 This is a flowchart illustrating a method for inducing selenium-enriched regenerated plants of Viola yedoensis based on tissue culture, as proposed in this application. Detailed Implementation The present application will be further described in detail below with reference to the accompanying drawings and embodiments. Example 1: This example provides a method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture, comprising the following steps: S1. Explant preparation and disinfection: Using Viola yedoensis seeds as the starting material, surface disinfection and aseptic rinsing were performed to obtain sterile seeds; the sterile seeds were inoculated into germination medium for aseptic germination to obtain sterile seedlings. Among them, the seeds of *Cephalotaxus fortunei* were pretreated as follows: a certain number of *Cephalotaxus fortunei* seeds were soaked in ultrapure water for 10 minutes for surface cleaning, then rinsed with running water for 2 hours, rinsed 3 times with sterile water, and then soaked for 30 minutes; then soaked in 75% anhydrous ethanol for 30 seconds in a clean bench, rinsed 5 times with sterile water, then disinfected with 0.1% HgCl2 for 7 minutes, rinsed 5 times with sterile water after disinfection, placed on sterile filter paper to absorb surface moisture, and then inoculated into MS medium. S2. Callus induction: Leaves of the sterile seedlings obtained in S1 were cut off as explants, inoculated into a culture medium and cultured in the dark to induce callus tissue. The culture medium is MS medium with a pH adjusted to 5.8 and containing auxin and cytokinin. The auxin is 2,4-dichlorophenoxyacetic acid at a concentration of 1.5 mg / L, and the cytokinin is 6-benzylaminopurine at a concentration of 0.2 mg / L. The dark culture conditions are: a culture temperature of 22℃ and a culture time of 28 days. The size of the explant leaves is controlled to be 0.3 cm × 0.3 cm, and the leaf edges have a small amount of petiole tissue. S3, Selenium enrichment treatment: The callus obtained in S2 is transferred into a selenium-enriched induction medium with added selenium source for culture to obtain selenium-enriched callus. The selenium source is sodium selenite, with a concentration of 8 μmol / L. The selenium-enriched induction medium is MS medium supplemented with 1.0 mg / L of 2,4-dichlorophenoxyacetic acid and 0.5 mg / L of 6-benzylaminopurine. The selenium enrichment treatment employs an alternating cyclic culture method, specifically: the callus tissue is first cultured in the selenium-enriched induction medium for 7 days, then transferred to the same basal medium without selenium source for 5 days, constituting one cycle; this is repeated for two cycles. The culture conditions are: light intensity 1500 Lux, photoperiod 12 h / d, and temperature 23 °C. S4. Differentiation culture: The selenium-enriched callus obtained in S3 was transferred to the differentiation medium for light culture to induce the differentiation of clustered shoots. The differentiation medium is MS medium with pH adjusted to 5.8 and containing cytokinin and gibberellin. The cytokinin is 6-benzylaminopurine at a concentration of 1.5 mg / L, and the gibberellin is at a concentration of 0.5 mg / L. The light culture conditions are: light intensity of 2000 Lux, photoperiod of 14 h / d, culture temperature of 24 °C, and culture time of 25 days. S5. Rooting Induction: Healthy adventitious buds from the clustered buds obtained in S4 are cut and transferred to rooting medium for culture to induce the formation of regenerated small plantlets with roots. The rooting medium was 1 / 2 MS with a pH adjusted to 5.8 and containing auxin; the auxin was indolebutyric acid (IBA) at a concentration of 0.8 mg / L; activated carbon was also added to the rooting medium at a concentration of 0.8 g / L; the culture conditions were: light intensity 1500 Lux, photoperiod 12 h / d, culture temperature 22 °C, and culture for 18 days. S6. Hardening off: Harden off the regenerated small plants obtained in S5 by opening the bottle. When the regenerated seedlings grow to 6cm, they are hardened off by opening the bottle. Hardening off the bottle specifically involves fully opening the bottle mouth, injecting a small amount of sterile water into the bottle to maintain humidity, and hardening off the seedlings for 2 days. S7. Transplanting: Transplant the hardened plants obtained in S6 into an organic-rich acclimatization substrate to obtain selenium-enriched regenerated plants. The acclimatization substrate is composed of peat moss, perlite and vermiculite in a volume ratio of 3:1:1. Before transplanting, the pH value of the acclimatization substrate is adjusted to 5.6. After transplanting, the substrate is acclimatized and cultivated for 10 days in a greenhouse environment with a relative humidity of 75% and a temperature of 20°C. Example 2: This example provides a method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture, comprising the following steps: S1. Explant preparation and disinfection: Using Viola yedoensis seeds as the starting material, surface disinfection and aseptic rinsing were performed to obtain sterile seeds; the sterile seeds were inoculated into germination medium for aseptic germination to obtain sterile seedlings. The seeds of *Cephalotaxus fortunei* were pretreated as follows: a certain number of *Cephalotaxus fortunei* seeds were soaked in ultrapure water for 12.5 min for surface cleaning, then rinsed with running water for 2 h, rinsed 4 times with sterile water, and then soaked for 35 min; in a clean bench, they were soaked in 75% anhydrous ethanol for 35 s, rinsed 6 times with sterile water, then disinfected with 0.1% HgCl2 for 7 min, rinsed 6 times with sterile water after disinfection, placed on sterile filter paper to absorb surface moisture, and then inoculated into MS medium. S2. Callus induction: Leaves of the sterile seedlings obtained in S1 were cut off as explants, inoculated into a culture medium and cultured in the dark to induce callus tissue. The culture medium is MS medium with a pH adjusted to 5.8 and containing auxin and cytokinin. The auxin is 2,4-dichlorophenoxyacetic acid at a concentration of 2.0 mg / L, and the cytokinin is 6-benzylaminopurine at a concentration of 0.5 mg / L. The dark culture conditions are: a culture temperature of 24℃ and a culture time of 32 days. The size of the explant leaves is controlled to be 0.4 cm × 0.4 cm, and the leaf edges have a small amount of petiole tissue. S3, Selenium enrichment treatment: The callus obtained in S2 is transferred into a selenium-enriched induction medium with added selenium source for culture to obtain selenium-enriched callus. The selenium source is sodium selenite, with a concentration of 11.5 μmol / L. The selenium-enriched induction medium is MS medium supplemented with 1.5 mg / L of 2,4-dichlorophenoxyacetic acid and 0.75 mg / L of 6-benzylaminopurine. The selenium enrichment treatment employs an alternating cyclic culture method, specifically: the callus tissue is first cultured in the selenium-enriched induction medium for 8.5 days, then transferred to the same basal medium without selenium source and cultured for 6 days, which constitutes one cycle; this is repeated for 3 cycles. The culture conditions are: light intensity 2000 Lux, photoperiod 13 h / d, and temperature 24 °C. S4. Differentiation culture: The selenium-enriched callus obtained in S3 was transferred to the differentiation medium for light culture to induce the differentiation of clustered shoots. The differentiation medium is MS medium with pH adjusted to 5.8 and containing cytokinin and gibberellin; the cytokinin is 6-benzylaminopurine at a concentration of 2.0 mg / L; the gibberellin is at a concentration of 0.75 mg / L; the light culture conditions are: light intensity 2500 Lux, photoperiod 15 h / d, culture temperature 25 °C, and culture time 32.5 days. S5. Rooting Induction: Healthy adventitious buds from the clustered buds obtained in S4 are cut and transferred to rooting medium for culture to induce the formation of regenerated small plantlets with roots. The rooting medium was 1 / 2 MS with a pH adjusted to 5.8 and auxin added. The auxin was indolebutyric acid (IBA) at a concentration of 1.0 mg / L. Activated carbon was also added to the rooting medium at a concentration of 1.15 g / L. The culture conditions were: light intensity of 2000 Lux, photoperiod of 13 h / d, culture temperature of 23.5 °C, and culture for 21.5 days. S6. Hardening off: Harden off the regenerated small plants obtained in S5 by opening the bottle. When the regenerated seedlings grow to 7cm, they are hardened off by opening the bottle. Hardening off specifically involves fully opening the bottle, injecting a small amount of sterile water into the bottle to maintain humidity, and hardening off for 2.5 days. S7. Transplanting: Transplant the hardened plants obtained in S6 into an organic-rich acclimatization substrate to obtain selenium-enriched regenerated plants. The acclimatization substrate is composed of peat moss, perlite and vermiculite in a volume ratio of 3:2:1. Before transplanting, the pH value of the acclimatization substrate is adjusted to 5.8. After transplanting, the substrate is acclimatized and cultivated for 12.5 days in a greenhouse environment with a relative humidity of 80% and a temperature of 22.5℃. Example 3: This example provides a method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture, comprising the following steps: S1. Explant preparation and disinfection: Using Viola yedoensis seeds as the starting material, surface disinfection and aseptic rinsing were performed to obtain sterile seeds; the sterile seeds were inoculated into germination medium for aseptic germination to obtain sterile seedlings. The seeds of *Cephalotaxus fortunei* were pretreated as follows: a certain number of *Cephalotaxus fortunei* seeds were soaked in ultrapure water for 15 minutes for surface cleaning, then rinsed with running water for 2 hours, rinsed 5 times with sterile water, and then soaked for 40 minutes; in a clean bench, they were soaked in 75% anhydrous ethanol for 40 seconds, rinsed 6 times with sterile water, then disinfected with 0.1% HgCl2 for 7 minutes, rinsed 6 times with sterile water after disinfection, placed on sterile filter paper to absorb surface moisture, and then inoculated into MS medium. S2. Callus induction: Leaves of the sterile seedlings obtained in S1 were cut off as explants, inoculated into a culture medium and cultured in the dark to induce callus tissue. The culture medium is MS medium with a pH adjusted to 5.8 and containing auxin and cytokinin. The auxin is 2,4-dichlorophenoxyacetic acid at a concentration of 2.5 mg / L, and the cytokinin is 6-benzylaminopurine at a concentration of 0.8 mg / L. The dark culture conditions are: a culture temperature of 26°C and a culture time of 35 days. The size of the explant leaves is controlled to be 0.5 cm × 0.5 cm, and the leaf edges have a small amount of petiole tissue. S3, Selenium enrichment treatment: The callus obtained in S2 is transferred into a selenium-enriched induction medium with added selenium source for culture to obtain selenium-enriched callus. The selenium source is sodium selenite, with a concentration of 15 μmol / L. The selenium-enriched induction medium is MS medium supplemented with 2.0 mg / L 2,4-dichlorophenoxyacetic acid and 1.0 mg / L 6-benzylaminopurine. The selenium enrichment treatment employs an alternating cyclic culture method, specifically: the callus tissue is first cultured in the selenium-enriched induction medium for 10 days, then transferred to the same basal medium without selenium source for 7 days, constituting one cycle; this is repeated for 3 cycles. The culture conditions are: light intensity 2500 Lux, photoperiod 14 h / d, and temperature 25 °C. S4. Differentiation culture: The selenium-enriched callus obtained in S3 was transferred to the differentiation medium for light culture to induce the differentiation of clustered shoots. The differentiation medium is MS medium with pH adjusted to 5.8 and containing cytokinin and gibberellin; the cytokinin is 6-benzylaminopurine at a concentration of 2.5 mg / L; the gibberellin is at a concentration of 1.0 mg / L; the light culture conditions are: light intensity 3000 Lux, photoperiod 16 h / d, culture temperature 26 °C, and culture time 40 days. S5. Rooting Induction: Healthy adventitious buds from the clustered buds obtained in S4 are cut and transferred to rooting medium for culture to induce the formation of regenerated small plantlets with roots. The rooting medium was 1 / 2 MS with a pH adjusted to 5.8 and auxin added. The auxin was indolebutyric acid (IBA) at a concentration of 1.2 mg / L. Activated carbon was also added to the rooting medium at a concentration of 1.5 g / L. The culture conditions were: light intensity of 2500 Lux, photoperiod of 14 h / d, culture temperature of 25 °C, and culture for 25 days. S6. Hardening off: Harden off the regenerated small plants obtained in S5 by opening the bottle. When the regenerated seedlings grow to 8cm, they are hardened off by opening the bottle. Hardening off specifically involves fully opening the bottle, injecting a small amount of sterile water into the bottle to maintain humidity, and hardening off for 3 days. S7. Transplanting: Transplant the hardened plants obtained in S6 into an organic-rich acclimatization substrate to obtain selenium-enriched regenerated plants. The acclimatization substrate is composed of peat moss, perlite and vermiculite in a volume ratio of 4:2:1. Before transplanting, the pH value of the acclimatization substrate is adjusted to 6.0. After transplanting, the substrate is acclimatized and cultivated for 15 days in a greenhouse environment with a relative humidity of 85% and a temperature of 25°C. Comparative Example 1: This comparative example refers to the content of Example 1, except that in step S1, when pretreating the seeds of Viola yedoensis, the soaking temperature is 28°C, and the rest of the content is the same as in Example 1. Comparative Example 2: This comparative example is the same as that in Example 1, except that in step S2, the concentration of 2,4-dichlorophenoxyacetic acid added to the callus induction medium is 1.4 mg / L. The rest of the contents are the same as those in Example 1. Comparative Example 3: This comparative example is the same as that in Example 1, except that in step S3, the concentration of sodium selenite added to the selenium-enriched induction medium is 7 μmol / L. The rest of the contents are the same as those in Example 1. Comparative Example 4: This comparative example is the same as that in Example 1, except that in step S4, the concentration of 6-benzylaminopurine added to the differentiation medium is 1.4 mg / L. The rest of the contents are the same as those in Example 1. Comparative Example 5: This comparative example is the same as that in Example 1, except that in step S5, the concentration of indolebutyric acid added to the rooting medium is 0.9 mg / L. The rest of the contents are the same as those in Example 1. Comparative Example 6: This comparative example refers to the content of Example 1, except that in step S6, the time for opening the bottle and hardening the seedlings is 1 day, and the rest of the content is the same as Example 1. Performance testing Sample preparation: The samples involved in this experiment are the regenerated plant populations of *Cephalotaxus fortunei* and related intermediates prepared according to Examples 1-3 and Comparative Examples 1-6 of the present invention; each example or comparative example was operated in parallel with no less than 30 tissue culture units to ensure that sufficient samples were obtained for subsequent detection and statistical analysis of various performance indicators; after completing the corresponding culture steps, all samples were immediately sampled and tested, or flash-frozen in liquid nitrogen and stored in an ultra-low temperature freezer for testing to maintain the original state of the biological samples. Detection of callus induction rate and growth status: Callus tissue samples taken after the end of step S2 culture were used as test objects; the number of explants that successfully induced callus tissue in each test object was counted to obtain the callus induction rate; at the same time, a certain number of callus tissues were randomly selected, and their color, density or looseness were observed and recorded; their fresh weight was weighed using an electronic balance, and their projected area was calculated using image analysis software to assess the growth; test standards: the evaluation of callus growth status referred to the morphological grading standards in "Experimental Guide to Plant Physiology" and related literature. Detection of selenium content and organic selenium conversion rate in selenium-enriched callus: Selenium-enriched callus samples taken after cultivation in step S3 were used as the test objects. After washing, drying, and grinding the test objects into powder, the total selenium content was determined by atomic fluorescence spectrometry or inductively coupled plasma mass spectrometry. Furthermore, the speciation of selenium in the sample, especially the content of organic selenium compounds such as selenomethionine, was analyzed using high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry to obtain the percentage of organic selenium in the total selenium, i.e., the organic selenium conversion rate. Test standards: The determination of total selenium content refers to the national standard "National Food Safety Standard - Determination of Selenium in Food" (GB5009.93); the analysis of selenium speciation refers to the industry standard "Determination of Selenium Speciation in Edible Agricultural Products - High-Performance Liquid Chromatography-Inductively Coupled Plasma Mass Spectrometry" (NY / T3830-2021) or equivalent methods. Detection of clustered shoot differentiation rate and adventitious shoot number: The culture after step S4 was used as the test object; the number of callus tissue blocks that could differentiate into clustered shoots in the test object was counted, and the clustered shoot differentiation rate was obtained; for each differentiated callus tissue block, the number of adventitious shoots generated on it was counted, and the average height of the adventitious shoots was measured; Test standards: The statistical methods of differentiation rate and adventitious shoot number followed the conventional statistical methods of plant tissue culture; The evaluation of adventitious shoot growth status referred to the relevant standards in "Experimental Tutorial of Plant Tissue Culture". Detection of rooting rate and root development status: The regenerated plantlets after the end of step S5 culture were taken as the test objects; the number of adventitious buds that grew adventitious roots in each treatment unit was counted to obtain the rooting rate; the rooted plants were randomly selected to measure the number of roots and the length of the longest root, and to observe whether the root system was well-developed and whether the root hairs were abundant; Test standards: The morphological evaluation of the root system refers to the relevant morphological indicators of root vitality in "Experimental Guide to Plant Physiology". Detection of transplant survival rate and plant physiological status: Plants that have completed acclimatization and cultivation were transplanted to a uniform greenhouse for routine management. On the 15th and 30th days after transplanting, the number of surviving plants was counted to obtain the transplant survival rate. At the same time, on the 30th day after transplanting, the plant height, number of leaves, and maximum leaf area were measured, and physiological indicators such as photosynthetic efficiency were determined using a chlorophyll fluorescence meter. Testing standards: The measurement of plant growth indicators was based on the "Experimental Techniques of Plant Biology"; the chlorophyll fluorescence measurement was based on the standard procedure in the "Experimental Guide to Plant Physiology and Ecology".

[0004] Table 1: Comparison of performance parameters of callus induction and selenium enrichment treatment Group Callus induction rate (%) Fresh weight of callus tissue (g) Total selenium content (μg / gDW) Organic selenium conversion rate (%) Example 1 90 1.2 150 80 Example 2 92 1.3 160 82 Example 3 95 1.5 180 85 Comparative Example 1 52 0.65 72 46 Comparative Example 2 48 0.58 65 41 Comparative Example 3 60 0.62 85 50 Comparative Example 4 57 0.51 103 48 Comparative Example 5 61 0.81 112 59 Comparative Example 6 59 0.61 98 67 Table 2: Comparison of performance parameters in differentiation culture, rooting induction, and transplanting Group Cluster bud differentiation rate (%) Number of adventitious buds (per piece) Rooting rate (%) Number of roots (per plant) Transplant survival rate (%) Plant height (cm) Example 1 88 12.5 96 8.5 92 10.2 Example 2 90 13.5 97 9.0 94 10.8 Example 3 92 15.0 98 10.0 96 11.5 Comparative Example 1 63 9.0 70 5.5 55 8.0 Comparative Example 2 65 9.5 62 4.8 68 7.5 Comparative Example 3 66 8.8 63 6.5 69 6.8 Comparative Example 4 58 7.5 58 7.1 52 7.5 Comparative Example 5 54 9.2 71 6.1 60 6.2 Comparative Example 6 65 8.6 65 4.9 53 7.6 Example Conclusion: Based on Examples 1-3 and Comparative Example 1, and in conjunction with Tables 1 and 2, it can be seen that appropriately increasing the seed pretreatment soaking temperature can break seed dormancy, enhance seed coat permeability, and activate the internal enzyme system through the heat shock effect, thereby promoting seed germination vigor and the robustness of sterile seedlings, and providing high-quality starting explants for subsequent callus induction and efficient growth. Based on Examples 1-3 and Comparative Example 2, and in conjunction with Tables 1 and 2, it can be seen that by improving the concentration ratio of auxin and cytokinin during the callus induction stage, the cell dedifferentiation process can be precisely regulated. Auxin leads the initiation of cell division, while cytokinin promotes cell proliferation. The two work together to create an endogenous hormone environment that promotes efficient callus formation and robust growth. Based on Examples 1-3 and Comparative Example 3, and in conjunction with Tables 1 and 2, it can be seen that by using a suitable concentration of selenium source and combining it with an alternating cyclic culture strategy, the stress-recovery cycle is utilized: the selenium treatment phase promotes cell absorption and conversion of selenium, while the selenium-free recovery phase reduces selenium toxicity and promotes cell metabolic balance, thereby improving the accumulation efficiency of selenium in callus tissue and the safe conversion rate to organic selenium. Based on Examples 1-3 and Comparative Example 4, and in conjunction with Tables 1 and 2, it can be seen that adjusting the concentrations of cytokinin and gibberellin during the differentiation stage activates the differentiation of bud primordia through the hormonal environment of cytokinin, while gibberellin promotes cell elongation. The two work synergistically to stimulate the redifferentiation ability of callus tissue, thereby improving the differentiation efficiency and robustness of clustered buds. Based on Examples 1-3 and Comparative Example 5, and in conjunction with Tables 1 and 2, it can be seen that controlling the concentration of auxin during the rooting stage and supplementing it with activated carbon works by inducing the formation of root primordia with a suitable auxin concentration, while activated carbon adsorbs harmful metabolites in the culture medium and creates a suitable dark microenvironment, thus optimizing the conditions for root development and promoting the occurrence of adventitious roots and root development. Based on Examples 1-3 and Comparative Example 6, and in conjunction with Tables 1 and 2, it can be seen that implementing a gradual hardening-off process and ensuring sufficient hardening-off time allows the plants to gradually adapt to the external environment, promotes the recovery of leaf stomatal function, cuticle development, and the formation of photosynthetic autotrophic capacity, thereby enhancing the stress resistance and adaptability of regenerated plants and improving transplant survival rate. This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A tissue culture-based induction method for selenium-enriched regenerative plants of Pteris multifida, characterized by, The method comprises the following steps: S1, preparation and disinfection of explants: taking the seeds of P. cordata as starting materials, performing surface disinfection and aseptic washing to obtain aseptic seeds; inoculating the aseptic seeds into a germination medium to perform aseptic germination to obtain aseptic seedlings; S2, callus induction: taking the leaves of the aseptic seedlings obtained in S1 as explants, inoculating into a culture medium to perform dark culture to induce callus; S3, selenium enrichment treatment: transferring the callus obtained in S2 into a selenium enrichment induction medium added with a selenium source to perform culture to obtain selenium-enriched callus; S4, differentiation culture: transferring the selenium-enriched callus obtained in S3 into a differentiation culture medium to perform illumination culture to induce differentiation of the callus into a cluster of shoots; S5, rooting induction: taking the healthy adventitious shoots obtained in S4, transferring into a rooting culture medium to perform culture to induce formation of regenerated small plants with root systems; S6, seedling raising: performing bottle opening seedling raising of the regenerated small plants obtained in S5; S7, transplanting: transplanting the seedlings obtained in S6 into an acclimatization substrate rich in organic matter to obtain selenium-enriched regenerated plants.

2. The method for inducing the regeneration of selenium-enriched Stimpsonia rotundifolia based on tissue culture according to claim 1, characterized in that, In the step S1, the seeds of P. cordata are pretreated in the following manner: a certain amount of seeds of P. cordata are soaked in ultrapure water for 10-15 min for surface cleaning, then rinsed with running water for 2 h, rinsed with sterile water for 3-5 times, and then soaked for 30-40 min; in an ultraclean bench, the seeds are soaked in 75% anhydrous ethanol for 30-40 s, rinsed with sterile water for 5-6 times, then disinfected with 0.1% HgCl2 for 7 min, rinsed with sterile water for 5-6 times after the disinfection, and then placed on sterile filter paper to dry the surface water, and then the seeds of P. cordata are inoculated into a MS medium.

3. The method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture according to claim 1, characterized in that, In the step S2, the culture medium is a MS medium, and the pH value is adjusted to 5.8, and auxin and cytokinin are added; the auxin is 2,4-dichlorophenoxyacetic acid, and the addition concentration is 1.5-2.5 mg / L; the cytokinin is 6-benzylaminopurine, and the addition concentration is 0.2-0.8 mg / L; the dark culture conditions are as follows: the culture temperature is 22-26 ℃, and the culture time is 28-35 days.

4. The method for inducing selenium-enriched regeneration of *Violet-leaved Barley Grass* plants based on tissue culture according to claim 1, characterized in that, In the step S2, before the dark culture, the size of the inoculated explant leaves is controlled to be 0.3 cm×0.3 cm-0.5 cm×0.5 cm, and the leaves have a small amount of petiole tissue at the edges.

5. The method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture according to claim 1, characterized in that, In the step S3, the selenium source is sodium selenite, and the addition concentration of sodium selenite is 8-15 μmol / L; the selenium enrichment induction medium is a MS medium, and 2,4-dichlorophenoxyacetic acid with a concentration of 1.0-2.0 mg / L and 6-benzylaminopurine with a concentration of 0.5-1.0 mg / L are added.

6. The method for inducing selenium-enriched regeneration of *Violet-leaved Barley Grass* plants based on tissue culture according to claim 1, characterized in that, In the S3 step, the selenium-rich treatment adopts an alternating cycle culture mode, specifically: the callus is first cultured in the selenium-rich induction medium for 7-10 days, and then transferred into the same basic medium without selenium source for 5-7 days, which is one cycle; 2-3 cycles are repeated; the culture conditions are: light intensity 1500-2500 Lux, light cycle 12-14 h / d, temperature 23-25℃.

7. The method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture according to claim 1, characterized in that, In the S4 step, the differentiation medium is MS medium, and the pH value is adjusted to 5.8, and cytokinin and gibberellin are added; wherein the cytokinin is 6-benzylaminopurine, and the addition concentration is 1.5-2.5 mg / L; the gibberellin addition concentration is 0.5-1.0 mg / L; the light culture conditions are: light intensity 2000-3000 Lux, light cycle 14-16 h / d, culture temperature 24-26℃, and culture time 25-40 days.

8. The method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture according to claim 1, characterized in that, In the S5 step, the rooting medium is 1 / 2MS, and the pH value is adjusted to 5.8, and auxin is added; wherein the auxin is indole-3-butyric acid, and the addition concentration is 0.8-1.2 mg / L; when the rooting medium is prepared, activated carbon is also added, and the addition concentration of activated carbon is 0.8-1.5 g / L; the culture conditions are: light intensity 1500-2500 Lux, light cycle 12-14 h / d, culture temperature 22-25℃, and culture for 18-25 days.

9. The method for inducing selenium-enriched regeneration of *Violet-leaved Barley Grass* plants based on tissue culture according to claim 1, characterized in that, In the S6 step, when the regenerated plantlets grow to 6-8 cm, the bottle is opened for seedling, specifically: the bottle opening is completely opened, a small amount of sterile water is injected into the bottle to maintain humidity, and the seedling is cultured for 2-3 days.

10. The method for inducing selenium-enriched regeneration of Viola yedoensis plants based on tissue culture according to claim 1, characterized in that, In the S7 step, the acclimation substrate is mixed by grass carbon, perlite and vermiculite in a volume ratio of (3-4):(1-2):1, and the pH value of the acclimation substrate is adjusted to 5.6-6.0 before transplanting; after transplanting, the acclimation culture is carried out in a greenhouse environment with relative humidity 75-85% and temperature 20-25℃ for 10-15 days.

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