Tissue culture medium combination and tissue culture rapid propagation method for dysosma versipellis

Abstract

The invention relates to the technical field of tissue culture of dysosma versipellis, in particular to a tissue culture medium combination and a tissue culture rapid propagation method of dysosma versipellis. The invention provides a dysosma versipellis tissue culture medium combination and a tissue culture rapid propagation method.The dysosma versipellis tissue culture medium combination comprises a germination culture medium, a callus induction culture medium, a cluster bud proliferation culture medium and a rooting culture medium, the callus induction rate can be increased, and the tissue culture rapid propagation rate can be increased. The inductivity and the multiplication coefficient of the multiple shoots of the dysosma versipellis are improved, and the rooting rate of tissue culture seedlings of the dysosma versipellis is improved. According to the tissue culture rapid propagation method for the dysosma versipellis, the dysosma versipellis is high in propagation speed, short in production period, simple in equipment and convenient for industrialized production, and guarantee conditions are provided for production of authentic medicinal materials and postpartum processing and selling.

Description

Technical Field

[0001] This invention relates to the field of tissue culture technology for Podophyllum hexandrum, specifically to a tissue culture medium combination and a method for rapid tissue culture propagation of Podophyllum hexandrum. Background Technology

[0002] Star anise Dysosmaversipellis (Hance) M. Cheng is a species of Podophyllum in the family Berberidaceae. Dysosma As a perennial herb with important medicinal value, wild resources of Podophyllum hexandrum are on the verge of extinction. Tissue culture technology has become an important means of protecting and propagating Podophyllum hexandrum.

[0003] However, current tissue culture methods for Podophyllum hexandrum have problems such as low proliferation rate, difficulty in rooting, and low survival rate of transplanted seedlings, which limit the application of Podophyllum hexandrum. Therefore, it is urgent to improve the composition of tissue culture medium and the rapid propagation method of tissue culture for Podophyllum hexandrum in order to improve the proliferation rate, rooting rate and survival rate of transplanted seedlings. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of low proliferation rate of Podophyllum hexandrum in the prior art, thereby providing a tissue culture medium combination for Podophyllum hexandrum.

[0005] Another technical problem this invention aims to solve is overcoming the shortcomings of existing technologies, such as low proliferation rate, difficulty in rooting, and low survival rate of transplanted seedlings. This invention provides a rapid tissue culture propagation method for *Podophyllum hexandrum*. Using this method, after inoculating and culturing callus tissue of *Podophyllum hexandrum*, clustered shoots can be induced in 20-25 days, with an induction rate of 70%-90%. Based on this, proliferation culture allows for a large number of clustered shoots to multiply, approximately once every 25 days, with an average multiplication factor of 4.5 times. The multiplied clustered shoots are then transferred to the rooting medium provided by this invention and cultured for 25-35 days to develop a large number of roots, with a rooting rate of approximately 90%. After rooting culture, further culture for 30-45 days is required before transplanting and hardening off. Rooting culture is considered complete when the number of roots is ≥3 and the number of leaves is ≥3.

[0006] This invention provides a rapid tissue culture propagation method for *Podophyllum hexandrum*, mastering the key techniques for induction and growth of shoot clusters and seedling rooting in tissue culture of *Podophyllum hexandrum*. It successfully solves the difficulties in rooting, low proliferation rate, and low survival rate in *Podophyllum hexandrum* tissue culture, enabling rapid seedling production and industrialized cultivation of this endangered and important medicinal plant. Using this invention also effectively protects this rare wild plant resource, preventing illegal harvesting and establishing a virtuous cycle of sustainable utilization and rational development.

[0007] Therefore, the present invention provides the following technical solution: This invention provides a tissue culture medium combination for Podophyllum hexandrum, comprising: germination medium, callus induction medium, shoot proliferation medium and rooting medium; The germination medium consists of MS medium as the basic medium, and also includes 20-30 g / L sucrose, 6-8 g / L agar, and 0.4-0.6 mg / L banana powder; The callus induction medium comprises: a modified MS medium as the basal medium, and further comprising NAA 0.15~0.25 mg / L, 2,4-D 0.4~0.7 mg / L, sucrose 20~30 g / L, agar 6~8 g / L and banana powder 0.4~0.6 mg / L; preferably, the callus induction medium comprises: a modified MS medium as the basal medium, and further comprising NAA 0.2 mg / L, 2,4-D 0.5 mg / L, sucrose 30 g / L, agar 6 g / L and banana powder 0.5 mg / L; And / or, the composition of the callus induction medium includes: modified MS medium as the basic medium, and further includes 0.15~0.25 mg / L NAA, 0.9~1.1 mg / L KT, 0.5-1.0 mg / L GA3, 20~30 g / L sucrose, 6~8 g / L agar and 0.4~0.6 mg / L banana powder; The composition of the bud proliferation medium includes: modified MS medium as the basic medium, and further includes KT 0.9~1.1 mg / L or 6-BA 1.2~1.8 mg / L, 0.3~0.5 mg / L NAA, sucrose 20~30 g / L, agar 6~8 g / L, and banana powder 0.4~0.6 mg / L; preferably, the composition of the bud proliferation medium includes: modified MS medium as the basic medium, and further includes KT 1.0 mg / L or 6-BA 1.5 mg / L, 0.5 mg / L NAA, sucrose 30 g / L, agar 6 g / L, and banana powder 0.5 mg / L.

[0008] The rooting medium comprises: 1 / 2 MS as the basal medium, and further comprising 20-30 g / L sucrose, 6-8 g / L agar, 0.5-1.0 mg / L activated charcoal, and 0.4-0.6 mg / L IBA. Preferably, the rooting medium comprises: 1 / 2 MS as the basal medium, and further comprising 20 g / L sucrose, 6 g / L agar, 1.0 mg / L activated charcoal, and 0.5 mg / L IBA.

[0009] The modified MS medium consists of: potassium nitrate 19 g / L, ammonium nitrate 16.5 g / L, potassium dihydrogen phosphate 1.7 g / L, magnesium sulfate heptahydrate 3.75 g / L, anhydrous calcium chloride 3.3 g / L, potassium iodide 0.083 g / L, boric acid 0.62 g / L, manganese sulfate 1.69 g / L, zinc sulfate 0.86 g / L, sodium sulfate 0.025 g / L, copper sulfate 0.0025 g / L, cobalt chloride 0.0025 g / L, disodium EDTA 3.73 g / L, ferrous sulfate 2.78 g / L, inositol 15 g / L, glycine 0.3 g / L, thiamine hydrochloride 0.015 g / L, pyridoxine hydrochloride 0.075 g / L, and nicotinic acid 0.075 g / L.

[0010] The tissue samples were placed in culture containers containing the aforementioned callus induction medium and cultured. After 20 days, several callus tissues regenerated from the buds in each container. Each callus tissue was then separated and transferred to a shoot proliferation medium for continuous culture. After 25 days, several new shoots regenerated from the callus tissues in each container. The shoots can be further propagated on the shoot proliferation medium or rooting medium, depending on the specific needs of the tissue culture seedlings. After obtaining the shoots, the *Podophyllum hexandrum* plants requiring rooting were transferred to a rooting medium for rooting culture.

[0011] Preferably, the pH values ​​of the callus induction medium, shoot proliferation medium, and rooting medium are 5.8 to 6.0, respectively.

[0012] This invention provides a method for rapid tissue culture propagation of Podophyllum hexandrum, which utilizes the tissue culture medium combination described in the above technical solution for tissue culture, including: Seeds of *Podophyllum hexandrum* were inoculated into a germination medium for germination culture to obtain germinated seeds. Embryos were prepared from the germinated seeds and inoculated into a callus induction medium for induction culture to obtain callus tissue. This invention preferably uses *Podophyllum hexandrum* seeds as the culture material. The seeds are preferably harvested when the berry peel is light yellow, ideally from late July to early August. After harvesting, germination treatment is performed by soaking the seeds in a solution of 50 mg / L ETH, GA3, and 2,4-D for 48 hours, followed by dark incubation in a laboratory at 15-25°C for 10-15 days to induce germination. Germination treatment promotes later seed maturation.

[0013] Afterwards, the seeds and embryos of *Podophyllum hexandrum* are disinfected: Preferably, the seeds are first soaked in a solution of 0.02% benzalkonium chloride for 5 minutes to remove residue, then soaked in 70% chlorothalonil for 20-30 minutes, rinsed with running water for 30-60 minutes, and then placed in a clean bench for 20-30 minutes of UV irradiation. Next, they are soaked in 75% alcohol for 25-35 seconds and rinsed 2-3 times with sterile water. The seeds are then soaked in 20% sodium hypochlorite for 25-35 minutes and rinsed 2-3 times with sterile water. Afterwards, the outer seed coat is removed to extract the embryo. The embryo is then soaked in 0.05%-0.15% mercuric chloride for 3 minutes and rinsed 5-6 times with sterile water before being inoculated into a culture medium. The disinfection method of this invention achieves a 100% disinfection efficiency for explant seeds. Embryos begin to germinate with pale yellow spots after 20 days, 10 days earlier than other treatments. The germination rate of embryos induced by this invention after 20 days is over 90%, and the callus tissue after 30 days of induction is large and green.

[0014] The callus tissue was inoculated into a shoot proliferation medium for proliferation culture to obtain sterile seedlings; shoots were obtained by proliferation culture using the callus tissue of the present invention, and the proliferation coefficient of the shoots reached 4.46 and the growth was optimal.

[0015] The sterile seedlings were inoculated into a rooting medium for rooting culture to obtain tissue culture seedlings.

[0016] Preferably, the rooting culture temperature is 24~26℃, the light intensity of the rooting culture is 2000~3000 lux, and the rooting culture time is 20~25 days, more preferably 23~25 days; the rooting culture is a light culture, and the light culture time is 12h / d.

[0017] The rooting culture of this invention results in rooting in approximately 25 days. Rooted plants are removed and placed in a natural environment for 5-7 days. After 5-7 days of hardening off, the seedlings are transplanted into a substrate composed of peat moss, crushed dead branches and leaves, and fine sand (volume ratio 1:1:1), or silt, peanut shells (0-10mm), and well-rotted dead branches (10-20mm) (volume ratio 1:2:1). The environment is maintained at a temperature of 15-25℃, humidity of approximately 50%-70%, and light intensity of 3000-6000 lux. Transplanting to the field after one month achieves a survival rate of over 85%. The peanut shells have a particle size of 5-10mm, and the dead branches have a particle size of 10-20mm.

[0018] Preferably, the induction culture temperature is 23~25℃, the light intensity is 2000-3000 lux, and the induction culture time is 20~35 days. The induction culture is a light culture, and the light culture time is 12h / d.

[0019] Preferably, the temperature for the proliferation culture is 23-25℃, the light intensity for the proliferation culture is 2000-3000 lux, and the proliferation culture time is 25-30 days. The proliferation culture is a light culture, and the light culture time is 12h / d.

[0020] Preferably, after the rooting culture, the seedlings are hardened off; the method of hardening off includes: placing them in a natural environment for 5 to 7 days, and then leaving them covered for another 5 to 7 days.

[0021] Preferably, after the seedling hardening process, the process also includes transplanting; the substrate used for transplanting includes: peat moss, crushed dead branches and leaves and fine sand mixed in a volume ratio of (1-2):1:1; and / or, silt, peanut shells and decomposed dead branches mixed in a volume ratio of (1-2):2:1.

[0022] Preferably, the embryo is further subjected to pretreatment and sterilization before inoculation; the pretreatment method includes: soaking the embryo in a 500-fold dilution of 70% chlorothalonil fungicide for 5-6 minutes, and then soaking it in a 50 mg / L gibberellin solution for 48-50 hours.

[0023] Preferably, the seed collection time for preparing *Polygonum aviculare* embryos includes: June 25th to July 10th or July 25th to August 10th.

[0024] The technical solution of this invention has the following advantages: 1. The tissue culture medium composition for *Podophyllum hexandrum* provided by this invention comprises: NAA (Natural Acid) to promote rooting and induce callus formation; 2,4-D to strongly induce callus and promote cell division; sucrose as a carbon source, providing energy and regulating osmotic pressure; banana powder to enhance embryo resistance and germination rate; and KT (Kinetic Toxin) to promote cell division and induce shoot differentiation. Under specific concentrations of NAA, KT, sucrose, agar, and banana powder, the callus induction rate is improved.

[0025] The bud proliferation medium used a modified MS medium as the basal medium, and with the addition of specific concentrations of KT, sucrose, agar, and banana powder, the induction rate and proliferation coefficient of bud clusters were improved. KT was used to promote cell division and induce callus differentiation into buds.

[0026] The rooting medium used 1 / 2 MS as the basic medium. With the help of specific concentrations of sucrose, agar, activated carbon and IBA, the rooting rate of tissue culture seedlings of Podophyllum hexandrum was improved, with a rooting rate of about 90%. Among them, the role of activated carbon is to resist oxidation and prevent browning, and the role of IBA is to promote rooting and root development.

[0027] 2. The rapid tissue culture propagation method for Podophyllum hexandrum provided by the present invention involves inoculating the embryos of Podophyllum hexandrum into a callus induction medium for induction culture to obtain callus tissue; The callus tissue was inoculated into a shoot proliferation medium for proliferation culture to obtain sterile seedlings; the sterile seedlings were inoculated into a rooting medium for rooting culture to obtain tissue culture seedlings. The callus tissue had a high induction rate, shoot proliferation rate and proliferation coefficient, and tissue culture seedlings had a high rooting rate.

[0028] The tissue culture method for rapid propagation of Podophyllum hexandrum of the present invention can be carried out under artificially controlled conditions, without being restricted by factors such as season, climate and soil. It can eliminate the influence of pests and diseases and pesticide residues, and strictly control the quality of Podophyllum hexandrum.

[0029] The tissue culture method for rapid propagation of Podophyllum hexandrum of the present invention has a fast propagation speed, short production cycle, simple equipment, small footprint, and can save manpower and material resources, making it convenient for industrial production.

[0030] The tissue culture rapid propagation method of Podophyllum hexandrum of the present invention performs detoxification treatment during the tissue culture process, which improves the quality of Podophyllum hexandrum and effectively solves the problems of unstable quality, uneven appearance and active ingredients of wild Podophyllum hexandrum, providing a guarantee for the production of authentic medicinal materials and post-harvest processing and sales.

[0031] The tissue culture method for rapid propagation of Podophyllum hexandrum of the present invention contains important technical steps for rapid propagation and stable rooting of Podophyllum hexandrum, achieving technical stability and high propagation coefficient, and can be applied to industrial production.

[0032] Artificial cultivation of *Podophyllum hexandrum* seedlings obtained using the tissue culture rapid propagation technology provided by this invention yields finished *Podophyllum hexandrum* products with minimal individual variation and uniform quality, providing stable raw materials for the production of podophyllotoxin. The tissue culture rapid propagation method of *Podophyllum hexandrum* of this invention preserves the germplasm resources of *Podophyllum hexandrum*, while also contributing to the protection of wild *Podophyllum hexandrum* resources and reducing damage to natural resources. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 An image showing the appearance of the embryo in a Podophyllum oxypetalum seed; Figure 2 Diagram showing the swelling of the embryo in a Podophyllum hexandrum seed; Figure 3This is a diagram of the embryo germination of Podophyllum hexandrum; Figure 4 This is a diagram showing the induction of callus tissue after 30 days of embryo culture of Podophyllum hexandrum in Example 2. Figure 5 This is a diagram of callus induction 30 days after embryo culture of Podophyllum hexandrum in Example 2. Figure 6 This is a diagram of callus induction 30 days after embryo culture of Podophyllum hexandrum in Example 2. Figure 7 This is a diagram illustrating the induction of shoot clusters from callus culture in Example 3 of Podophyllum hexandrum; Figure 8 This is a diagram illustrating the induction of shoot clusters from callus culture in Example 3 of Podophyllum hexandrum; Figure 9 This is a diagram illustrating the induction of shoot clusters from callus culture in Example 3 of Podophyllum hexandrum; Figure 10 This is a picture of robust seedlings with clustered buds after 15 days of cultivation of Podophyllum hexandrum in Example 4; Figure 11 This is a diagram of the rooting of clustered shoots in a bottle after 15 days of cultivation of Podophyllum hexandrum in Example 4. Figure 12 This is a diagram showing the rooting of clustered shoots of Podophyllum hexandrum cultured for 15 days in Example 4. Figure 13 This is a diagram showing the growth of rooted seedlings of Podophyllum hexandrum in Example 6 after 10 days of transplanting with a cover to maintain moisture. Figure 14 This is a diagram showing the growth of rooted seedlings of Podophyllum hexandrum (Example 6) after 10 days of natural growth without a cover and transplanting. Detailed Implementation

[0035] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0036] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0037] In the following examples, late June to early July is June 25 to July 10, and late July to early August is July 25 to August 10.

[0038] Example 1 1. Practical technical solution for seed treatment in the propagation of Podophyllum hexandrum 1.1 Test Materials The experimental material was obtained from the Jinsha River Xiangxi Rare Botanical Garden, and the morphologically mature berries of an eight-year-old Podophyllum hexandrum plant grown indoors.

[0039] 1.2 Seed collection and germination pre-test of Podophyllum hexandrum Collect mature seeds at two stages: Stage A: when the pericarp of the Podocarpus berries turns light yellow, around late June to early July; Stage B: when the pericarp of the Podocarpus berries turns dark brown, around late July to early August (the specific time varies depending on the different growing environment and care).

[0040] After collecting the seeds of *Podophyllum hexandrum* using both methods, a portion was stored in a 4℃ refrigerator for later use, while the other portion was harvested and threshed under natural ventilation and then subjected to germination tests for treatments 1-3. The germination tests were conducted using sterilized germination boxes and germination paper, and the germination conditions were set as follows: Treatment 1: A layer of absorbent cotton was placed in the germination box, the seeds were placed on the absorbent cotton, and then two sheets of germination paper were placed on top of the seeds. Germination was then carried out in a light incubator with a temperature range of 15-25℃. At 25℃, the light intensity was 1500 lux and the light incubation time was 12 hours. At 15℃, the seeds were cultured in darkness for 12 hours. Treatment 2: Line a germination box with a layer of absorbent cotton, place the seeds on the absorbent cotton, and then cover the seeds with two sheets of germination paper. Then, culture them in a constant temperature incubator at a temperature of 24±1℃, a light intensity of 1500 lux, and a photoperiod of 12h. Treatment 3: Line a germination box with a layer of absorbent cotton, place the seeds on the absorbent cotton, and then cover the seeds with two sheets of germination paper. Then, use a constant temperature incubator for cultivation at a temperature of 25℃, a light intensity of 1500 lux, and a photoperiod of 12h. The germination time of each group of seeds was recorded based on the germination time of the first seed. Fifteen days after the first seed germinated, the number of germinating seeds was counted, and the germination rate was calculated. The formula for calculating the germination rate is as follows: Germination rate (%) = number of germinated seeds / number of seeds × 100%.

[0041] The germination rate results are shown in Table 1. It can be seen that the seeds of Podocarpus macrocarpus require a minimum of 15 days to germinate, and the germination rate is about 30% higher than that of other treatments.

[0042] Table 1. Effects of two maturity levels on seed germination under different environmental conditions.

[0043] As shown in Table 1, the seeds of Podophyllum hexandrum were collected at two different times, when the pericarp was light yellow and dark brown. Both times, the seeds were harvested and threshed under natural ventilation, and the germination test was conducted. The results showed no significant difference, indicating that the seeds were physiologically mature when the pericarp was light yellow. Harvesting the seeds at this stage allowed them to separate from the mother plant earlier, shortening the seed morphological maturation time by about 7 days. The highest germination rate of 63.33% was observed when the seeds were collected at the end of July to the beginning of August.

[0044] All culture environments in the following experiments were based on seeds of Podophyllum hexandrum harvested in late July to early August.

[0045] 1.3 Pretreatment and sterilization of Podophyllum hexandrum seeds Treatment P1: Podophyllum hexandrum seeds were first soaked in a 500-fold dilution of 70% chlorothalonil fungicide for 5 minutes, then soaked in a 50 mg / L GA3 (gibberellin 3) solution for 48 hours. After soaking, the seeds were first soaked in a 0.02% (v / v) solution of benzalkonium chloride for 5 minutes to remove residue, then soaked in 70% chlorothalonil for 20-30 minutes, rinsed with running water for 30-60 minutes, then placed in a clean bench for UV irradiation for 20-30 minutes, soaked in 75% alcohol for 25-35 seconds, and rinsed 2-3 times with sterile water. The seeds were then soaked in a 20% sodium hypochlorite solution for 25-35 minutes, rinsed 2-3 times with sterile water, and the embryos were extracted and soaked in 0.05%–0.15% mercuric chloride solution for 3 minutes, then rinsed 5-6 times with sterile water. See the image of the seed embryo for details. Figure 1 The embryos were then inoculated into germination medium and cultured in the dark for 10-15 days at 25℃. The germination medium consisted of MS medium as the basal medium, supplemented with 30 g / L sucrose, 6 g / L agar, and 0.5 g / L banana powder, with the pH adjusted to 5.8-6.0. See the embryo enlargement diagram below. Figure 2 See embryo germination diagram Figure 3 .

[0046] Treatment P2: Same as treatment P1, except that it is first soaked in a 500-fold dilution of 70% chlorothalonil fungicide for 5 minutes, and then soaked in a 50 mg / L ETH (ethylene) solution for 48 hours.

[0047] Treatment P3: Same as treatment P1, except that it is first soaked in a 500-fold dilution of 70% chlorothalonil fungicide for 5 minutes, and then soaked in a 50 mg / L solution of 2,4-D (2,4-dichlorophenoxyacetic acid) for 48 hours.

[0048] After 30 days of cultivation, treatments P1 through P3 were used to statistically analyze the number of contaminants, contamination rate, number of germinations, and germination rate (see Table 2). It can be seen that treatment P1 achieved 100% sterilization efficiency for the explants of *Podophyllum hexandrum* seeds, with a contamination rate of 0%, and germination began after 15 days, 10 days earlier than the other treatments. Furthermore, the germination rate of treatment P1 was higher than that of treatments P2 and P3. Therefore, the optimal treatment and sterilization method for *Podophyllum hexandrum* seeds is to collect them when the berry peel is light yellow and physiologically mature. Under different hormone treatments, the seed morphological maturation time can be shortened by approximately 10 days. After sowing, germination culture was carried out under dark conditions at 25℃, resulting in the highest germination rate of 93.33% for *Podophyllum hexandrum* seeds. The presence of fungal or bacterial growth in the culture medium was considered contamination.

[0049] Table 2. Effects of seed treatment on contamination rate and germination rate

[0050] Example 2 1. Direct callus induction from embryos There are four types of callus induction culture medium P4: P4-1, P4-2, P4-3 and P4-4. The culture medium for P4-1 consisted of modified MS medium as the basic medium, and also contained 0.1 mg / L 2,4-D, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P4-2 consisted of modified MS medium as the basic medium, and also contained 0.3 mg / L 2,4-D, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P4-3 consisted of modified MS medium as the basic medium, and also contained 0.5 mg / L 2,4-D, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P4-4 consisted of modified MS medium as the basic medium, and also contained 0.7 mg / L 2,4-D, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. Preparation of embryos: Seeds of *Podophyllum hexandrum* pretreated with GA3 were used as culture material. They were first soaked in a solution of 0.02% benzalkonium chloride for 5 minutes to remove residue, then soaked in 70% chlorothalonil for 20 minutes, rinsed with running water for 30 minutes, and then irradiated with UV light in a clean bench for 20 minutes. Afterwards, they were soaked in 75% alcohol for 25 seconds and rinsed three times with sterile water. The seeds were then soaked in 20% sodium hypochlorite for 25 minutes and rinsed three times with sterile water. The seed coat was gently cut along the ventral suture with a scalpel to remove the embryos, which were then soaked in 0.05% mercuric chloride for 3 minutes and rinsed with sterile water for 6 minutes. One embryo was inoculated into each of media P4-1, P4-2, P4-3, and P4-4. The embryos were then cultured in a laboratory with a light intensity of 2000-3000 lux, a temperature of 23-25℃, and a photoperiod of 12 h / d. The optimal medium for callus induction was selected. The day of embryo inoculation was designated as day 1 of culture. Results were analyzed after 30 days of culture and are shown in Tables 3 and 4. Figure 4 .

[0051] Table 3. Effects of different concentrations of 2,4-D on callus proliferation and plant growth in modified MS medium.

[0052] There are four types of culture media for P5: P5-1, P5-2, P5-3 and P5-4.

[0053] The culture medium for P5-1 consisted of modified MS medium as the basic medium, and also contained only 0.6 mg / L KT, 0.2 mg / L NAA, 1.0 mg / L GA3, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P5-2 consisted of modified MS medium as the basic medium, and also contained only 0.8 mg / L KT, 0.2 mg / L NAA, 1.0 mg / L GA3, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P5-3 consisted of modified MS medium as the basic medium, and also contained 1.0 mg / L KT, 0.2 mg / L NAA, 1.0 mg / L GA3, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P5-4 consisted of modified MS medium as the basic medium, and also contained 1.2 mg / L KT, 0.2 mg / L NAA, 1.0 mg / L GA3, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. One embryo of *Podophyllum hexandrum* was inoculated into P5-1, P5-2, P5-3, and P5-4 culture media, respectively. The embryos were then induced in a culture room with a light intensity of 2000-3000 lux and a temperature of 23-25℃ for 12 h / d. The optimal culture medium for callus induction was selected, with the day of embryo inoculation designated as day 1 of culture. Callus formation occurred after 20 days of culture, and results were collected after 30 days of culture. The results are shown in Table 4. Figure 5 .

[0054] Table 4. Effects of different concentrations of KT on callus proliferation and plant growth in modified MS basal medium.

[0055] There are four types of culture media for P6: P6-1, P6-2, P6-3 and P6-4; The culture medium for P6-1 consisted of modified MS medium as the basic medium, and also contained 0.5 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P6-2 consisted of modified MS medium as the basic medium, and also contained 1.0 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P6-3 consisted of modified MS medium as the basic medium, and also contained 1.5 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. The culture medium for P6-4 consisted of modified MS medium as the basic medium, and also contained 2.0 mg / L 6-BA, 0.2 mg / L NAA, 30 g / L sucrose, 6 g / L agar and 0.5 mg / L banana powder, with the pH adjusted to 5.8-6.0. One embryo from *Podophyllum hexandrum* was inoculated into P6-1, P6-2, P6-3, and P6-4 media, with each media containing one embryo. Induction culture was then performed in a culture room with a light intensity of 2000-3000 lux and a temperature of 24℃, for 12 h / d. The optimal medium for callus induction was selected. The day of embryo inoculation was designated as day 1 of culture. Results were analyzed after 30 days of culture and are shown in Table 5. Figure 6 .

[0056] Table 5. Effects of different concentrations of 6-BA on callus proliferation and plant growth in modified MS medium.

[0057] Callus induction rate (%) = number of callus produced / number of inoculated embryos × 100%; average proliferation coefficient = number of callus produced per embryo / number of inoculated embryos.

[0058] According to Tables 3-5 and Figures 4-6 It was found that the modified MS medium resulted in the best plant growth, and the addition of 0.5 mg / L 2,4-D led to the optimal callus induction rate and proliferation coefficient. Therefore, the optimal callus induction medium was modified MS + 0.5 mg / L 2,4-D + 30 g / L sucrose + 6 g / L agar + 0.5 mg / L banana powder. When seeds were cultured on the modified MS medium, the callus induction rate reached over 90% after 15 days, and the callus tissue was large and pale yellow after 30 days. Specific elements and their concentrations per liter are as follows: The modified MS medium consisted of: potassium nitrate 19 g / L, ammonium nitrate 16.5 g / L, potassium dihydrogen phosphate 1.7 g / L, magnesium sulfate heptahydrate 3.75 g / L, anhydrous calcium chloride 3.3 g / L, potassium iodide 0.083 g / L, boric acid 0.62 g / L, manganese sulfate 1.69 g / L, zinc sulfate 0.86 g / L, sodium sulfate 0.025 g / L, copper sulfate 0.0025 g / L, cobalt chloride 0.0025 g / L, disodium EDTA 3.73 g / L, ferrous sulfate 2.78 g / L, inositol 15 g / L, glycine 0.3 g / L, thiamine hydrochloride 0.015 g / L, pyridoxine hydrochloride 0.075 g / L, and nicotinic acid 0.075 g / L. The increased amount of organic matter is intended to enhance the supply of carbon, nitrogen, and vitamins, thereby promoting the growth rate, metabolic activity, and differentiation potential of Podophyllum hexandrum.

[0059] The optimal callus induction culture medium is as follows: using modified MS medium as the basic medium, with the following added concentrations: 0.2 mg / L NAA, 0.5 mg / L 2,4-D, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder; adjust the pH to 5.8-6.0.

[0060] or, The callus induction culture medium was prepared as follows: modified MS medium was used as the basic medium, with the following hormone concentrations added: 0.2 mg / L NAA, 1.0 mg / L KT, 1.0 mg / L GA3, 30 g / L sucrose, 6 g / L agar, and 0.5 mg / L banana powder; the pH was adjusted to 5.8-6.0.

[0061] Example 3: Induction of shoot clusters through callus proliferation culture The composition of the modified MS medium is the same as in Example 2.

[0062] The callus tissue obtained from the P4-3 medium in Example 2 was divided and inoculated into P7, P8, and P9 media for shoot proliferation culture. The culture environment was 2000-3000 lux light intensity and 24℃, with a light intensity of 12 h / d. The optimal medium for shoot induction was screened, with the day of inoculation recorded as day 1 of culture. Shoots could be induced after 25-30 days of culture. The results were statistically analyzed after 30 days of culture and are shown in Tables 6-8. Figures 7-9 .

[0063] P7 used modified MS medium as the basic medium, with different concentrations of kinetin KT (0.1, 0.5, 1.0, 1.5) mg / L and NAA 0.5 mg / L added, along with sucrose 30 g / L and agar 6 g / L, and the pH was adjusted to 5.8-6.0.

[0064] P8 uses modified MS medium as the basic medium, with different concentrations of 6-benzylaminopurine 6-BA (0.5, 1.0, 1.5, 2.0) mg / L and NAA 0.5 mg / L added, along with 30 g / L sucrose and 6 g / L agar, and the pH is adjusted to 5.8-6.0.

[0065] P9 was prepared using modified MS medium as the basic medium, with different concentrations of thiamethoxam TDZ (0.05, 0.1, 0.15, 0.2) mg / L and NAA 0.5 mg / L added, along with sucrose 30 g / L and agar 6 g / L, and the pH was adjusted to 5.8-6.0.

[0066] Table 6. Effects of adding different concentrations of KT to modified MS medium on shoot proliferation and plant growth.

[0067] Table 7. Effects of different concentrations of 6-BA added to modified MS medium on shoot proliferation and plant growth.

[0068] Table 8. Effects of different concentrations of TDZ on shoot proliferation and plant growth in MS.

[0069] Tables 6-8 show that the growth of *Podophyllum hexandrum* bud-forming plants was optimal in the modified MS medium. The addition of KT 1.0 mg / L resulted in the highest bud induction rate and proliferation coefficient. Therefore, the optimal bud proliferation medium is modified MS medium + KT 1.0 mg / L + NAA 0.5 mg / L + sucrose 30 g / L + agar 6 g / L + banana powder 0.5 mg / L. The buds can be further propagated on the bud proliferation medium or rooting medium, depending on the specific needs of the tissue culture seedlings.

[0070] Example 4: Seedling Rooting Culture The rooting medium P10 consists of 1 / 2 MS as the basic medium, with the addition of 20 g / L sucrose, 6 g / L agar, and 1.0 mg / L AC (activated carbon), and the pH is adjusted to 5.8-6.0.

[0071] The rooting medium P11 consists of 1 / 2 MS as the basic medium, with the addition of 20 g / L sucrose, 6 g / L agar, 1.0 mg / L AC (activated carbon), and 0.2 mg / L IBA, and the pH is adjusted to 5.8-6.0.

[0072] The rooting medium P12 consists of 1 / 2 MS as the basic medium, with the addition of 20 g / L sucrose, 6 g / L agar, 1.0 mg / L AC (activated carbon), and 0.5 mg / L IBA, and the pH is adjusted to 5.8-6.0.

[0073] Based on the experiment in Example 3, it was found that *Podophyllum hexandrum* directly forms an independent seedling during the induction of clustered shoots. Therefore, the seedlings were transferred to rooting media P10, P11, and P12 for cultivation. The culture environment was 2000-3000 lux light, 24-26℃ temperature, and 12 h / d light / d. Ten bottles were treated per batch, with the day of inoculation recorded as day 1 of cultivation. The results were statistically analyzed after 25 days of cultivation, as shown in Table 9. It can be seen that the rooting time of rooting medium P12 was 5 days later than that of rooting medium P10, but the number and length of roots were superior to those of P11 and P10. Therefore, rooting medium P12 is the optimal culture medium. See the image of robust seedlings of *Podophyllum hexandrum* after 15 days of cultivation. Figure 10-12 .

[0074] Table 9. Effects of different concentrations of IBA on rooting

[0075] Note: Rooting time is calculated based on the rooting time of the first root.

[0076] Example 5: Conventional hardening-off and transplanting 5.1 Acclimatization of Rooted Seedlings (Sterile Seedlings) In Example 4, when the roots of the rooted seedlings reached 3-5 cm in length, they were removed from the culture room for hardening-off. During hardening-off, the culture bottles were first placed in a natural environment for 5-7 days, and then the caps were tightened for 5-7 days to allow them to gradually adapt to the environment outside the culture bottles and culture room, thereby improving the survival rate of the rooted seedlings.

[0077] 5.2 Selection of transplanting substrate The transplanting substrate P13 is a mixture of peat moss (0~10mm) and perlite in a volume ratio of 2:1. The transplanting substrate P14 is a mixture of rice husks (0~10mm) and sawdust in a volume ratio of 2:1. The transplanting substrate P15 is a mixture of peat moss, crushed dead branches and leaves, and fine sand in a volume ratio of 2:1:1. The particle size of the crushed dead branches and leaves is 10-20 mm.

[0078] Spray the transplanting substrates P13, P14, and P15 evenly with a 500-fold dilution of carbendazim and seal them for 24 hours. Transplant the plants after the odor has completely dissipated.

[0079] 5.3 Aseptic Vaccine Cleaning and Sterilization After hardening off, the rooted seedlings from Example 4 were removed from the rooting medium with tweezers, rinsed in clean tap water, and then soaked in a 1000-fold dilution of carbendazim for 10-20 minutes for disinfection. Finally, they were laid flat on clean newspaper and placed in a cool place to absorb surface moisture before transplanting.

[0080] 5.4 Transplanting When transplanting, place 1-2cm thick bark (bark diameter 1-2cm) → 1-2cm thick substrate → 0.2-0.5g slow-release fertilizer → 2-3cm thick substrate from the bottom of the pot to the top to increase soil permeability, aeration and fertilizer efficiency. Finally, plant the rooted seedling from Example 4 in the center of the pot, compact the soil around it, water it thoroughly, and place it under the greenhouse bed frame.

[0081] 5.5 Post-transplanting management After transplanting, the light intensity should be gradually increased from weak to strong (3000-5000 lux). Water the ground frequently to maintain an air humidity of about 50%. For the first two weeks, when the substrate is dry, water the potting soil with a 1000-fold dilution of carbendazim solution. During the first to second weeks after transplanting, spray the foliar fertilizer potassium dihydrogen phosphate at 0.1%-0.2%, applying 10-20 ml per plant. Afterward, drench the roots with a water-soluble NPK fertilizer at a mass ratio of 20:10:20 and a concentration of 800-1000 times, applying 30-50 ml per plant. Alternate watering the potting soil every 7 days. The results are shown in Table 10.

[0082] Table 10. Effects of different substrates on survival rate

[0083] Table 10 shows that when transplanted into substrate P15 and maintained using the above-described maintenance methods, the survival rate of rooted seedlings of Podophyllum hexandrum was significantly higher than that of the other two substrates.

[0084] Example 6: Optimization of Seedling Hardening and Transplanting 6.1 Transplanting substrate Transplanting substrate P16 is prepared by mixing silt and peanut shells (5-10mm) in a volume ratio of 2:1. Transplanting substrate P17 is prepared by mixing silt and well-rotted dead branches (10-20mm) in a volume ratio of 2:1. Transplanting substrate P18 is prepared by mixing silt, peanut shells (5-10mm), and well-rotted dead branches (10-20mm) in a volume ratio of 1:2:1. Before applying the substrate, spray a 500-fold dilution of hymexazol evenly and seal for 24 hours. Transplant the plants after the odor has completely dissipated.

[0085] 6.2 Aseptic Vaccine Cleaning and Sterilization In Example 4, the rooted seedlings with roots 3-5cm long were removed from the culture room and hardened off directly. They were placed in a natural environment for 5-7 days, and the bottle caps were opened for 5-7 days. After hardening off, the bottle caps were unscrewed and the sterile seedlings were taken out directly with tweezers, rinsed in clean tap water, and then soaked in a 1000-fold dilution of carbendazim for 10-20 minutes for disinfection. Finally, they were laid flat on clean newspaper and placed in a cool place to absorb the surface moisture before transplanting.

[0086] 6.3 Transplanting When transplanting, place the following layers from bottom to top in the pot: 1-2cm diameter well-rotted branches → 1-2cm thick substrate → 0.2-0.5g organic fertilizer → 2-3cm thick substrate. This increases soil permeability, aeration, and fertility. Finally, plant the seedling in the center of the pot, firming the soil around it. Water thoroughly and place it in a growing box. Place a 1cm high shelf under the bottom tray and water until the water just covers the bottom of the pot. Cover the growing box, leaving two ventilation holes. Place it in a cool, shady place. Water the roots once with onion water that has been soaked (for 24 hours), keeping water droplets in the growing box. New leaves will begin to sprout from the sterile surface after 7 days. Cover and maintain moisture for 10 days after transplanting. See... Figure 13 After being transplanted and cultured without a lid for 10 days, see Figure 14 .

[0087] 6.5 Post-transplanting management After transplanting, gradually increase the light intensity to 3000-5000 lux, frequently water the ground, and maintain the humidity in the cultivation box at around 50%. For the first two weeks, when the soil is dry, water the potting soil with tap water containing citric acid (pH 6.0). Afterward, use water-soluble humic acid fertilizers with different concentrations. The mass ratio of nitrogen, phosphorus, and potassium in the water-soluble humic acid fertilizers is 20:10:20, and the concentration is 1000 times. Spray 10 ml per plant and drench the roots of each plant with 30 ml per plant, alternating between watering the potting soil every 7 days. Use organic fertilizer with a concentration of 1000 times, spray 10 ml per plant and drench the roots of each plant with 30 ml per plant, alternating between watering the potting soil every 7 days (results are shown in Table 11).

[0088] Table 11. Effects of different substrates on survival rate

[0089] Table 11 shows that when transplanted into the P18 mixed substrate of silt: peanut shells: decomposed dead branches = 1:2:1 and maintained using the above-described maintenance methods, the survival rate was significantly higher than that of the other two substrates.

[0090] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A tissue culture medium composition for Podophyllum hexandrum, characterized in that, include: Germination medium, callus induction medium, shoot proliferation medium, and rooting medium; The germination medium consists of MS medium as the basic medium, and also includes 20-30 g / L sucrose, 6-8 g / L agar, and 0.4-0.6 mg / L banana powder; The callus induction medium consists of: modified MS medium as the basic medium, and also includes NAA 0.15~0.25 mg / L, 2,4-D 0.4~0.7 mg / L, sucrose 20~30 g / L, agar 6~8 g / L, and banana powder 0.4~0.6 mg / L; And / or, the composition of the callus induction medium includes: modified MS medium as the basic medium, and further includes 0.15~0.25 mg / L NAA, 0.9~1.1 mg / L KT, 0.5-1.0 mg / L GA3, 20~30 g / L sucrose, 6~8 g / L agar, and 0.4~0.6 mg / L banana powder; The composition of the bud proliferation medium includes: modified MS medium as the basic medium, and also includes KT 0.9~1.1mg / L or 6-BA 1.2~1.8mg / L, 0.3~0.5mg / L NAA, sucrose 20~30g / L, agar 6~8g / L, and banana powder 0.4~0.6mg / L; The rooting medium consists of 1 / 2 MS as the basic medium, and also includes 20-30 g / L sucrose, 6-8 g / L agar, 0.5-1.0 mg / L activated carbon and 0.4-0.6 mg / L IBA.

2. The tissue culture medium combination according to claim 1, characterized in that, The pH values ​​of the germination medium, callus induction medium, shoot proliferation medium, and rooting medium are 5.8 to 6.0, respectively.

3. A rapid tissue culture propagation method for Podophyllum hexandrum, characterized in that, Tissue culture using the tissue culture medium combination according to claim 1 or 2 includes: The seeds of Podophyllum hexandrum were inoculated into a germination medium for germination culture to obtain germinated seeds; Seed embryos were prepared from germinated Podophyllum hexandrum seeds, and the seed embryos were inoculated into callus induction medium for induction culture to obtain callus tissue. The callus tissue was inoculated into a shoot proliferation medium for proliferation culture to obtain sterile seedlings; The sterile seedlings were inoculated into a rooting medium for rooting culture to obtain tissue culture seedlings.

4. The rapid tissue culture propagation method according to claim 3, characterized in that, The germination culture is conducted at a temperature of 15-25℃ for 10-15 days in the dark. The induction culture is conducted at a temperature of 23-25℃ with a light intensity of 2000-3000 lux for 20-35 days.

5. The rapid tissue culture propagation method according to claim 3, characterized in that, The temperature for the proliferation culture is 23~25℃, the light intensity for the proliferation culture is 2000-3000 lux, and the proliferation culture time is 25~30 days.

6. The rapid tissue culture propagation method according to claim 3, characterized in that, The rooting culture temperature is 24~26℃, the light intensity is 2000-3000 lux, and the rooting culture time is 20~25 days.

7. The rapid tissue culture propagation method according to claim 3, characterized in that, After the rooting culture, the process also includes hardening off the seedlings; the hardening off method includes: placing the seedlings in a natural environment for 5 to 7 days, and then leaving them covered for another 5 to 7 days.

8. The rapid propagation method by tissue culture according to claim 7, characterized in that, After the seedling hardening process, the process also includes transplanting; the substrate used for transplanting includes: peat moss, crushed dead branches and leaves and fine sand mixed in a volume ratio of (1-2):1:1; and / or, silt, peanut shells and decomposed dead branches mixed in a volume ratio of (1-2):2:

1.

9. The rapid tissue culture propagation method according to claim 3, characterized in that, The embryos are further subjected to pretreatment and sterilization before inoculation; the pretreatment method includes: soaking the embryos in a 500-fold dilution of 70% chlorothalonil fungicide for 5-6 minutes, and then soaking them in a 50 mg / L gibberellin solution for 48-50 hours.

10. The rapid propagation method for tissue culture according to claim 3, characterized in that, The seed collection period for preparing *Podophyllum hexandrum* embryos includes: June 25th to July 10th or July 25th to August 10th.

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