High-efficiency tissue culture and rapid propagation method of atractylodes lancea using seeds as explants

Abstract

This invention discloses a highly efficient tissue culture method for rapid propagation of Atractylodes lancea using seeds as explants. The method uses Atractylodes lancea seeds as the starting explant. The seeds are washed with running water and laundry detergent, pre-soaked in 0.05% Tween-80 sterile water, and sterilized with a mixture of 0.1% mercuric chloride and 0.02% AgNO3 to obtain sterile material. Germinating shoots are obtained by sequentially passing the material through a 1 / 2 MS germination medium containing GA3 and a low concentration of cytokinin. Hypocotyls are excised and induced into callus tissue in MS medium supplemented with 2,4-D, 6-BA, zeatin / thiamethoxam, and brassinolide. Following adventitious shoot induction, proliferation, seedling strengthening, and rooting culture, complete regenerated tissue culture seedlings are finally obtained. This invention effectively improves seed germination rate, callus induction efficiency, and adventitious shoot differentiation ability through synergistic optimization of hormone ratios and added components in the culture medium at each stage, resulting in high seedling proliferation coefficients and robust rooting.

Description

Technical Field

[0001] This invention relates to the field of medicinal plant tissue culture technology, specifically to a method for efficient tissue culture and rapid propagation of Atractylodes lancea using seeds as explants. Background Technology

[0002] Atractylodes lancea (Thunb.) DC. is a perennial medicinal plant belonging to the genus Atractylodes in the family Asteraceae. It is a traditional and precious Chinese medicinal herb with high medicinal value and strong market demand. Due to over-harvesting and habitat destruction, wild resources are becoming increasingly scarce, and artificial cultivation has become the main way to supply the medicinal material.

[0003] Traditional seed propagation of Atractylodes lancea suffers from low germination rates and uneven emergence, while division propagation is inefficient, both failing to meet the demands of large-scale seedling production. Tissue culture is an effective means of achieving industrialized seedling production, but existing tissue culture systems still have many problems: explant sterilization struggles to balance contamination and survival rates; seed germination results in severe browning and poor uniformity; callus induction and bud differentiation are inefficient, easily leading to browning, vitrification, and deformed seedlings; and subsequent processes such as proliferation, seedling strengthening, rooting, and transplanting are incomplete, resulting in poor overall seedling cultivation outcomes.

[0004] Therefore, there is an urgent need for a tissue culture rapid propagation system with low browning, high survival rate, and strong stability to provide technical support for the large-scale seedling cultivation of Atractylodes lancea. Summary of the Invention

[0005] The purpose of this invention is to provide a highly efficient tissue culture and rapid propagation method for Atractylodes lancea using seeds as explants, in order to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A highly efficient tissue culture and rapid propagation method for Atractylodes lancea using seeds as explants includes the following steps:

[0008] (1) Using Atractylodes lancea seeds as explants, wash with a little laundry detergent and running water, then pre-soak in 0.05% Tween-80 sterile water, and finally disinfect with a mixture of 0.1% mercuric chloride and 0.02% AgNO3 and rinse clean.

[0009] (2) After disinfection, the seeds were inoculated into the germination medium and cultured for 15-25 days to obtain germination. The formula of the germination medium is: 1 / 2 MS medium, 25 g / L sucrose, 6.5 g / L agar, 1.0~2.0 mg / L GA3, 0.05-0.1 mg / L 6-BA or NAA, 0.5 g / L activated carbon;

[0010] (3) Cut the hypocotyls of the sprouts obtained in step (2) and inoculate them into callus induction medium for culture. The formula of the callus induction medium is: MS medium, 30 g / L sucrose, 7.0 g / L agar, 0.8 mg / L 2,4-dichlorophenoxyacetic acid, 0.1 mg / L 6-BA, 0.2-0.6 mg / L zeatin or thidiazuron, and 0.01 mg / L brassinolide;

[0011] (4) Cut small pieces of callus and inoculate them into adventitious shoot induction medium for culture. The formula of adventitious shoot induction medium is: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 0.5-1.5 mg / L zeatin or thidiazuron, 0.5 mg / L NAA, and 0.05 mg / L brassinolide;

[0012] (5) Adventitious buds are inoculated on proliferation medium and cultured for 25-35 days to obtain clustered buds;

[0013] (6) Separate the clustered buds into single buds and inoculate them on a seedling culture medium to obtain seedlings;

[0014] (7) The seedlings were inoculated on rooting medium to obtain tissue culture seedlings.

[0015] Furthermore, in step (1), the water rinsing time is 20-60 minutes; the pre-soaking time is 2-4 hours; the mixing and disinfection time is 3-10 minutes, and the rinsing is 5-8 times.

[0016] Furthermore, the cultivation conditions in step (2) are as follows: 12 h of light cultivation at 22℃ and 12 h of dark cultivation at 18℃, with a light intensity of 1500-2000 lx.

[0017] Furthermore, the culture temperature in step (3) is 23-27℃, and after 15 days of dark culture, it is cultured under light for 10 hours, then under dark for 14 hours, with a light intensity of 1500-2000 lx, for a total culture period of 25-35 days.

[0018] Furthermore, the culture temperature in step (4) is 23-27℃, with 14 h of light culture, 10 h of dark culture, 2000-2500 lx of light, and 25-35 days of culture.

[0019] Furthermore, the formulation of the proliferation medium in step (5) is as follows: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 1.0 mg / L zeatin or thidiazuron, 0.5 mg / L NAA, and 0.05 mg / L brassinolide.

[0020] Furthermore, the formula of the seedling culture medium mentioned in step (6) is: MS medium, 35 g / L sucrose, 7 g / L agar, 0.05 mg / L 6-BA, 0.2 mg / L NAA, 0.1~0.2 mg / L GA3, and 100 g / L banana puree.

[0021] Furthermore, the rooting medium formula in step (7) is: 1 / 2 MS medium, 30 g / L sucrose, 7 g / L agar, 0.2 mg / L NAA, 0.5 mg / L IBA, 0.5 g / L activated carbon, and 50 g / L banana puree.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention effectively improves seed germination rate, callus induction efficiency, and adventitious bud differentiation ability by synergistically optimizing the hormone ratio and added components of the culture medium at each stage. The resulting seedlings have a high proliferation coefficient and robust root development. The obtained tissue culture seedlings exhibit uniform growth, well-developed root systems, and high transplant survival rates, providing stable and high-quality seedling supply technical support for the standardized cultivation and germplasm resource protection of Atractylodes lancea. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0025] Unless otherwise specified, all materials used in the examples are commercially available.

[0026] Example 1: Optimization of disinfection treatment for Atractylodes lancea seed explants

[0027] Four disinfection treatment groups were set up, with two gradients in each group. Seeds of *Atractylodes lancea* harvested that year were used as explant material. The seeds were soaked in water for 24 hours before disinfection. The specific details of the four disinfection treatment groups are as follows:

[0028] Treatment A: Routine disinfection

[0029] (1) Add a little laundry detergent + rinse with running water for 30 min + disinfect with 75% ethanol for 30 s + disinfect with 0.1% mercuric chloride for 5 min + rinse with sterile water 5 times.

[0030] (2) Add a little laundry detergent + rinse with running water for 30 min + disinfect with 75% ethanol for 30 s + disinfect with 0.1% mercuric chloride for 10 min + rinse with sterile water 5 times.

[0031] Treatment B: Tween-80 pre-soaking + mercuric chloride disinfection

[0032] (1) Add a little laundry detergent + rinse with running water for 30 min + pre-soak in 0.05% Tween-80 sterile water for 3 h + disinfect with 75% ethanol for 30 s + disinfect with 0.1% mercuric chloride for 8 min + rinse with sterile water 6 times.

[0033] (2) Add a little laundry detergent + rinse with running water for 30 min + pre-soak in 0.05% Tween-80 sterile water for 3 h + disinfect with 75% ethanol for 30 s + disinfect with 0.1% mercuric chloride for 6 min + rinse with sterile water 6 times.

[0034] Treatment C: Tween-80 pre-soaking + synergistic disinfection with mercuric chloride and silver nitrate

[0035] (1) Add a little laundry detergent and rinse with running water for 30 min, pre-soak in 0.05% Tween-80 sterile water for 3 h, disinfect with 75% ethanol for 30 s, disinfect with 0.1% mercuric chloride and 0.02% AgNO3 mixture for 5 min, and rinse with sterile water 6 times.

[0036] (2) Add a little laundry detergent and rinse with running water for 30 min, pre-soak in 0.05% Tween-80 sterile water for 3 h, disinfect with 75% ethanol for 30 s, disinfect with 0.1% mercuric chloride and 0.02% AgNO3 mixture for 7 min, and rinse with sterile water 6 times.

[0037] Treatment D: Combined disinfection with NaClO and H2O2

[0038] (1) Add a little laundry detergent and rinse with running water for 30 min, disinfect with 75% ethanol for 30 s, disinfect with 2% NaClO-3% H2O2 mixture for 15 min, and rinse with sterile water 5 times.

[0039] (2) Add a little laundry detergent and rinse with running water for 30 min, disinfect with 75% ethanol for 30 s, disinfect with 2% NaClO-3% H2O2 mixture for 20 min, and rinse with sterile water 5 times.

[0040] After disinfection, the seeds were inoculated into 1 / 2 MS basal medium and cultured under the following conditions: temperature 20-25℃, 12h light / 12h dark culture, and light intensity 1500-2000 lx. On day 10, the contamination rate (number of contaminated seeds * 100% / number of inoculated seeds), survival rate (number of uncontaminated and surviving seeds * 100% / number of inoculated seeds), and seed appearance were statistically analyzed. The results are shown in Table 1.

[0041] Table 1. Analysis of the results of disinfection treatment of Atractylodes lancea seeds

[0042]

[0043] The results showed that different disinfection methods had significant differences in the control of contamination and the maintenance of viability in Atractylodes lancea seeds. Conventional disinfection was incomplete and caused severe phytotoxicity; the effect of single mercuric chloride disinfection was improved after pre-soaking, but the problem of incomplete disinfection still existed; combined disinfection with oxidants was extremely damaging to seeds and was not suitable for disinfection of Atractylodes lancea seeds. Considering all indicators, pre-soaking combined with synergistic disinfection of mercuric chloride and silver nitrate was the best treatment scheme (treatment C), which can effectively control microbial contamination, minimize chemical phytotoxicity, and significantly improve seed survival rate. It can be regarded as the preferred process for disinfection of explants in Atractylodes lancea tissue culture.

[0044] Example 2 Optimization of Seed Germination Promotion Culture

[0045] Seeds of *Atractylodes lancea* were disinfected using the C2 disinfection method, and then inoculated into a germination-promoting medium. The culture conditions were: 22℃ light for 12 h, followed by 18℃ darkness for 12 h, with a light intensity of 1500–2000 lx. After 20 days, germination rate (number of germinating seeds * 100% / number of inoculated seeds), browning rate (number of browned buds * 100% / number of germinating seeds), and germination uniformity were calculated. (For treatments with a final germination rate higher than 50%, uniformity was defined as the number of days required for the cumulative germination of seeds to reach 50% of the total inoculated number; for treatments with a final germination rate lower than 50%, uniformity was defined as the number of days required for the final germination of its own seeds to reach 50%).

[0046] Six germination-promoting culture media were prepared, based on 1 / 2 MS, with 25 g / L sucrose, 6.5 g / L agar, and different concentrations of growth regulators added. The pH ranged from 5.8 to 6.0. The specific growth regulators and their concentrations are as follows:

[0047] Formula A (control): 1 / 2 MS;

[0048] Formula B: 1 / 2 MS + 0.5 g / L activated carbon;

[0049] Formula C: 1 / 2 MS + GA3 1.0 mg / L;

[0050] Formula D: 1 / 2 MS + GA3 2.0 mg / L + activated charcoal 0.5 g / L;

[0051] Formula E: 1 / 2 MS + GA3 1.0 mg / L + 6-BA 0.05 mg / L + activated charcoal 0.5 g / L;

[0052] Formula F: 1 / 2 MS + GA3 2.0 mg / L + NAA 0.05 mg / L + activated charcoal 0.5 g / L;

[0053] GA3 is gibberellin, 6-BA is 6-benzylaminopurine, and NAA is naphthaleneacetic acid.

[0054] Table 2 Results of Atractylodes lancea seed germination culture

[0055]

[0056] Table 2 shows that adding activated carbon alone can only alleviate browning and has limited effect on promoting germination; applying GA3 alone can significantly accelerate the seed germination process, but cannot effectively control browning; Formula D is the optimal culture medium formula for the germination of Atractylodes lancea seeds, which can simultaneously achieve high germination rate, low browning rate and high germination uniformity, and the seedling growth state is optimal, making it the first choice for aseptic sowing and germination of Atractylodes lancea; adding 6-BA and NAA (Formulas E and F) to the optimal formula will not weaken the germination and browning inhibition effects, and the effect is also good.

[0057] Example 3: Optimization of Callus Induction

[0058] Seeds of *Atractylodes lancea* were cultured for 20 days using formula D to promote germination. Hypocotyls of germinating shoots were then inoculated onto callus induction medium under the following conditions: 25±2℃, 15 days of dark culture, followed by 5 days of 10h light / 14h dark culture, with a light intensity of 1500–2000 lx. The callus induction rate (number of explants forming callus × 100% / number of inoculated explants) and callus browning rate (number of browned explants × 100% / number of inoculated explants) were calculated. The appearance of the callus tissue was observed, and the results are shown in Table 3.

[0059] Eight groups of callus induction media were set up, using MS as the basal medium, with 30 g / L sucrose and 7.0 g / L agar added, along with different concentrations of growth regulators. The pH ranged from 5.8 to 6.0. The growth regulators included 6-BA (6-benzylaminopurine), ZT (zeatin), BR (brassinolide), TDZ (thiazidazole), and 2,4-D (2,4-dichlorophenoxyacetic acid). The specific additions and concentrations of the growth regulators are as follows:

[0060] Culture medium 1: MS + 2,4-D 0.8 mg / L + 6-BA 0.6 mg / L;

[0061] Culture medium 2: MS + 2,4-D 0.8 mg / L + 6-BA 0.6 mg / L + BR 0.01 mg / L;

[0062] Culture medium 3: MS + 2,4-D 0.8 mg / L + ZT 0.6 mg / L;

[0063] Culture medium 4: MS + 2,4-D 0.8 mg / L + 6-BA 0.1 mg / L + ZT 0.6 mg / L;

[0064] Culture medium 5: MS + 2,4-D 0.8 mg / L + 6-BA 0.1 mg / L + ZT 0.6 mg / L + BR 0.01 mg / L;

[0065] Culture medium 6: MS + 2,4-D 0.8 mg / L + TDZ 0.2 mg / L;

[0066] Culture medium 7: MS + 2,4-D 0.8 mg / L + 6-BA 0.1 mg / L + TDZ 0.2 mg / L;

[0067] Culture medium 8: MS + 2,4-D 0.8 mg / L + 6-BA 0.1 mg / L + TDZ 0.2 mg / L + BR 0.01 mg / L.

[0068] Table 3 Results of callus induction culture of Atractylodes lancea

[0069]

[0070] Culture medium 1 showed an induction rate of only 46.5% and a browning rate as high as 38.4%, with few callus tissues, a hard texture, and severe browning, failing to meet experimental requirements. Adding 0.01 mg / L BR to culture medium 2 slightly improved the induction rate and reduced the browning rate to 30.8%, but the overall induction efficiency remained low. Culture medium 3 showed an induction rate of 53.4% ​​and a browning rate of 36.1%, with brittle callus tissues, significant browning, and poor stability. Culture medium 4 increased the induction rate to 59.7%, but the browning rate also rose to 39.2%, with callus centers prone to necrosis. Culture medium 6 showed an induction rate of 73.5% and a browning rate of 32.4%, with uneven callus formation and a generally poor condition. Culture medium 7 achieved an induction rate of 90.2%, but the browning rate remained at 29.5%, and some callus tissues exhibited slight vitrification, indicating significant defects.

[0071] Culture medium 5 significantly increased the induction rate to 88.6% and the browning rate to a sharp drop to 13.1%. The callus tissue was uniform and loose in texture, bright yellow in color, and showed no browning or vitrification, demonstrating excellent performance. Culture medium 8 further increased the induction rate to 91.6% and reduced the browning rate to 10.2%. The callus tissue was plump, bright in color, and showed no browning or vitrification, making it the optimal formulation for this experiment.

[0072] In summary, medium 8 showed the best callus induction effect, followed by medium 5. Medium 8 had a high callus induction rate, low browning rate, and excellent overall appearance of the callus, making it a better callus induction medium formulation.

[0073] Example 4: Optimization of Adventitious Bud Induction

[0074] After culturing on callus induction medium for 30 days, uniform callus pieces with a diameter of approximately 0.5 cm were excised and inoculated onto adventitious shoot induction medium. Culture conditions: temperature 25±2℃, 14 h light / 10 h dark, light intensity 2000-2500 lx. The adventitious shoot differentiation rate (number of callus tissues differentiating into adventitious shoots × 100% / number of inoculated callus tissues), adventitious shoot differentiation coefficient (number of induced adventitious shoots / number of inoculated callus tissues), and the appearance of the young shoots were statistically analyzed.

[0075] Four groups of adventitious shoot induction media were set up, with MS-based formulations, supplemented with 30 g / L sucrose, 7 g / L agar, and different concentrations of growth regulators. The pH ranged from 5.8 to 6.0. The specific growth regulators and their concentrations are as follows:

[0076] Adventitious bud induction formula 1: MS + ZT 2.5 mg / L + NAA 0.5 mg / L + BR 0.01 mg / L;

[0077] Adventitious bud induction formula 2: MS + 6-BA 1.0 mg / L + ZT 1.5 mg / L + NAA 0.5 mg / L + BR 0.05 mg / L;

[0078] Adventitious bud induction formula 3: MS + TDZ 1.5 mg / L + NAA 0.5 mg / L + BR 0.01 mg / L;

[0079] Adventitious bud induction formula 4: MS + 6-BA 1.0 mg / L + TDZ 0.5 mg / L + NAA 0.5 mg / L + BR 0.05 mg / L.

[0080] Table 4. Induction and culture of adventitious buds of Atractylodes lancea

[0081]

[0082] Different hormone ratios showed significant differences in their effects on inducing adventitious shoots from Atractylodes lancea callus (Table 4). Formula 1, a combination of high-concentration ZT and low-concentration BR, had the lowest adventitious shoot differentiation rate (63.33%) and differentiation coefficient (1.7), with weak shoots and prominent browning and vitrification. Formula 2 had an adventitious shoot differentiation rate of 87.78% and a differentiation coefficient of 4.32, with robust and uniform shoots and significantly reduced browning and vitrification. Formula 3 had a moderate induction effect, easily causing deformed shoots and vitrification, resulting in poor shoot quality. Formula 4 showed the best results in all aspects, with dense and regular adventitious shoots and no obvious browning or vitrification.

[0083] In summary, the combined use of cytokinins showed significantly better results than their single-use formulations. Formula 4 was the suitable culture medium for inducing adventitious shoots in Atractylodes lancea. Formula 2 was the next best, although its adventitious shoot differentiation rate and coefficient were slightly lower than those of Formula 4, and its adventitious shoots performed well.

[0084] Example 5 Optimization of proliferation culture

[0085] After culturing with adventitious bud induction formula 4 for 30 days, the adventitious buds were inoculated onto proliferation medium. Culture conditions: temperature 25±2℃, 14 h light / 10 h dark culture, light intensity 2000-2500 lx. The proliferation coefficient of the clustered buds (number of induced buds / number of inoculated adventitious buds) was calculated, and the appearance of the clustered buds was observed.

[0086] Two groups of proliferation media were set up, with MS-based formulations, supplemented with 30 g / L sucrose, 7 g / L agar, and different concentrations of growth regulators. The pH ranged from 5.8 to 6.0. The specific growth regulators and their concentrations are as follows:

[0087] Proliferation medium formulation 1: MS + 6-BA 1.0 mg / L + ZT 1.0 mg / L + NAA 0.5 mg / L + BR 0.05 mg / L;

[0088] Proliferation medium formula 2: MS + 6-BA 1.0 mg / L + TDZ 0.5 mg / L + NAA 0.5 mg / L + BR 0.05 mg / L.

[0089] Table 5. Induction of shoot clusters in Atractylodes lancea propagation culture

[0090]

[0091] Table 5 shows that the proliferation coefficients of the clustered shoots in Formula 1 and Formula 2 were 5.47 and 5.60, respectively, with little difference between the two, indicating that both hormone combinations can effectively promote the proliferation and expansion of clustered shoots. In terms of the appearance of the clustered shoots, the shoots under both formulas showed good growth and healthy condition, without any adverse effects such as excessive growth, yellowing, or malformation, indicating good shoot quality. Considering both the proliferation coefficient and the appearance of the shoots, both proliferation culture medium formulas are suitable for the proliferation culture of clustered shoots in Atractylodes lancea, achieving efficient and stable proliferation of clustered shoots with good shoot quality. These formulas can be considered excellent for the rapid propagation of clustered shoots in Atractylodes lancea tissue culture.

[0092] Example 6 Optimization of robust seedling cultivation

[0093] After 30 days of proliferation culture using proliferation medium formula 1, the clustered shoots were separated into single shoots and inoculated onto seedling strengthening medium. Culture conditions: temperature 25±2℃, 15 h light / 9 h dark culture, light intensity 2500-3000 lx. Seedling height was measured after 40 days, and the percentage of abnormal seedlings (vitrified, yellowed, and malformed seedlings) was recorded. The appearance of the seedlings was also observed.

[0094] Four groups of seedling-strengthening culture media were prepared, with MS-based formulations, supplemented with 35 g / L sucrose, 7 g / L agar, and different concentrations of growth regulators. The pH ranged from 5.8 to 6.0. The specific growth regulators and their concentrations are as follows:

[0095] Seedling strengthening medium formula 1: MS + 6-BA 0.05 mg / L + NAA 0.2 mg / L;

[0096] Seedling growth medium formula 2: MS + 6-BA 0.05 mg / L + NAA 0.2 mg / L + GA3 0.1 mg / L + banana puree 100g / L;

[0097] (3) Seedling strengthening culture medium formula 2: MS + 6-BA 0.05 mg / L + NAA 0.2 mg / L + GA3 0.2 mg / L + banana puree 100g / L;

[0098] Seedling growth medium formula 4: MS + 6-BA 0.05 mg / L + NAA 0.2 mg / L + GA3 0.5 mg / L + banana puree 100g / L.

[0099] Table 6. Cultivation of robust seedlings of Atractylodes lancea

[0100]

[0101] Based on three indicators—seedling growth, abnormal seedling rate, and appearance—the four formulations showed significant differences in their seedling-strengthening effects. Formulations 1 and 4 resulted in stunted and weak seedlings and excessive, abnormal growth, respectively, with poor seedling-strengthening effects. Formulations 2 and 3 performed exceptionally well, with abnormal seedling rates ranging from only 5.33% to 6.67%. The plants were robust, with dark green leaves and no obvious physiological abnormalities, making them the optimal formulation combinations for cultivating robust Atractylodes lancea seedlings. Among them, formulations 2 and 3 showed excellent effects in both inhibiting abnormal seedling occurrence and promoting seedling growth, resulting in seedlings of good overall condition.

[0102] Example 7 Optimization of Rooting Culture

[0103] After culturing seedlings in seedling-strengthening medium formula 2 for 40 days, the seedlings were inoculated onto rooting medium. Culture conditions: temperature 25±2℃, 12h light / 12h dark culture, light intensity 2000-2500 lx. Root quantity, root length, and rooting rate were recorded after 30 days.

[0104] Three groups of rooting media were prepared, with the formula based on 1 / 2 MS, supplemented with 30 g / L sucrose, 7 g / L agar, and different concentrations of growth regulators. The pH ranged from 5.8 to 6.0. The specific details of the different growth regulators and their concentrations are as follows:

[0105] Rooting medium formula 1: 1 / 2 MS + NAA 0.2 mg / L + activated charcoal 0.5 g / L;

[0106] Rooting medium formula 2: 1 / 2 MS + NAA 0.2 mg / L + activated charcoal 0.5 g / L + banana puree 50 g / L;

[0107] Rooting medium formula 3: 1 / 2MS + NAA 0.2 mg / L + IBA 0.5 mg / L + activated charcoal 0.5 g / L + banana puree 50 g / L.

[0108] Table 7. Rooting culture of Atractylodes lancea.

[0109]

[0110] The rooting rate of all three Atractylodes lancea rooting formulas was 100%, but the number and quality of roots differed significantly: the single NAA formula had fewer roots, and the roots were thin, weak, and prone to browning; the root quality improved after adding banana puree; while the rooting medium 3 had the best effect, with an average of 6.20 roots per plant and a root length of 4.79 cm. The roots were robust and there was no browning, making it the optimal formula for Atractylodes lancea rooting.

[0111] Example 8: Optimization of seedling hardening and transplanting

[0112] (1) Hardening off seedlings

[0113] After the tissue culture seedlings were cultured in the rooting medium for 30 days (with other conditions being the optimal formulations of Examples 1-7), the bottle caps were loosened in a sterile culture room and left for 4-5 days. Then, they were transferred to a hardening-off greenhouse, the bottle caps were opened, and a small amount of water was sprayed into the bottle. They were then left for another 4-5 days to complete the hardening-off process. After hardening-off, the tissue culture seedlings were removed, and the roots were rinsed with clean water to remove any remaining culture medium. They were then ready for use.

[0114] (2) Substrate preparation and disinfection

[0115] The cultivation substrate should be thoroughly mixed with perlite, vermiculite and peat moss in a volume ratio of 1:2:2. Expose it to the sun for 2-3 days under sunny and high temperature conditions, then spray it with a 1000-fold dilution of 50% carbendazim wettable powder for disinfection. After the substrate has dried, spread it evenly into the seedling trays for later use.

[0116] (3) Transplanting operation

[0117] Before transplanting, lightly prune the roots of the tissue culture seedlings, retaining a root length of 3-4 cm. Then, quickly dip the roots in a 1000-fold dilution of 50% carbendazim for disinfection, and transplant them into seedling trays filled with disinfected substrate at a spacing of 6 cm × 8 cm. After transplanting, immediately water thoroughly to ensure that the roots are in full contact with the substrate.

[0118] (4) Post-planting management

[0119] The temperature was controlled at 25±2℃ during the day and 20±2℃ at night throughout the process, and the light, humidity, ventilation and water and fertilizer conditions were adjusted in stages.

[0120] Week 1: Use shade nets with a shading rate of 70%-80% to provide shade, and maintain the relative humidity of the air at 85%-90%; spray water once in the morning and once in the evening every day to keep the substrate moist (not white or waterlogged), and ventilate for 15 minutes at noon every day.

[0121] Weeks 2-3: Replace with shade netting with a shading rate of 40%-50%, and reduce the relative humidity of the air to 65%-75%; spray water once every evening to keep the substrate moist, and ventilate for 20-30 minutes every noon.

[0122] Week 4 and beyond: Enter the regular management period, spray irrigation once every 2-3 days, remove the shade net, ventilate for about 1 hour at noon every day; in conjunction with watering, apply a 1500-fold dilution of water-soluble fertilizer with an N:P:K ratio of 18-18-18 every 7-10 days.

[0123] By following the above cultivation and management procedures, the survival rate of transplanted Atractylodes lancea tissue culture seedlings can reach over 95%, with robust seedling growth and no root rot.

[0124] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for efficient tissue culture and rapid propagation of Atractylodes lancea using seeds as explants, characterized in that: Specifically, the steps include the following: (1) Using Atractylodes lancea seeds as explants, wash with a little laundry detergent and running water, then pre-soak in 0.05% Tween-80 sterile water, and finally disinfect with a mixture of 0.1% mercuric chloride and 0.02% AgNO3 and rinse clean. (2) After disinfection, the seeds were inoculated into the germination medium and cultured for 15-25 days to obtain germination. The formula of the germination medium is: 1 / 2 MS medium, 25 g / L sucrose, 6.5 g / L agar, 1.0~2.0 mg / L GA3, 0.05-0.1 mg / L 6-BA or NAA, and 0.5 g / L activated carbon; (3) Cut the hypocotyls of the sprouts obtained in step (2) and inoculate them into callus induction medium for culture. The formula of the callus induction medium is: MS medium, 30 g / L sucrose, 7.0 g / L agar, 0.8 mg / L 2,4-dichlorophenoxyacetic acid, 0.1 mg / L 6-BA, 0.2-0.6 mg / L zeatin or thidiazuron, and 0.01 mg / L brassinolide; (4) Cut small pieces of callus and inoculate them into adventitious shoot induction medium for culture. The formula of adventitious shoot induction medium is: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 0.5-1.5 mg / L zeatin or thidiazuron, 0.5 mg / L NAA, and 0.05 mg / L brassinolide; (5) Adventitious buds are inoculated on proliferation medium and cultured for 25-35 days to obtain clustered buds; (6) Separate the clustered buds into single buds and inoculate them on a seedling culture medium to obtain seedlings; (7) The seedlings were inoculated on rooting medium to obtain tissue culture seedlings.

2. The method for efficient tissue culture and rapid propagation of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: In step (1), the running water rinsing time is 20-60 minutes; the pre-soaking time is 2-4 hours; the mixing and disinfection time is 3-10 minutes, and rinsing is performed 5-8 times.

3. The efficient tissue culture and rapid propagation method of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The cultivation conditions in step (2) are: 12 h of light cultivation at 22℃ and 12 h of darkness cultivation at 18℃, with a light intensity of 1500-2000 lx.

4. The efficient tissue culture and rapid propagation method of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The culture temperature in step (3) is 23-27℃. After 15 days of dark culture, the culture is carried out under light for 10 hours and under dark for 14 hours. The light intensity is 1500-2000 lx, and the culture lasts for a total of 25-35 days.

5. The efficient tissue culture and rapid propagation method of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The culture temperature in step (4) is 23-27℃, with 14 h of light culture, 10 h of dark culture, 2000-2500 lx of light, and 25-35 days of culture.

6. The efficient tissue culture and rapid propagation method of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The formulation of the proliferation medium in step (5) is as follows: MS medium, 30 g / L sucrose, 7 g / L agar, 1.0 mg / L 6-BA, 1.0 mg / L zeatin or thidiazuron, 0.5 mg / L NAA, and 0.05 mg / L brassinolide.

7. The efficient tissue culture and rapid propagation method for Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The formula for the seedling culture medium mentioned in step (6) is: MS medium, 35 g / L sucrose, 7 g / L agar, 0.05 mg / L 6-BA, 0.2 mg / L NAA, 0.1~0.2 mg / L GA3, and 100 g / L banana puree.

8. The efficient tissue culture and rapid propagation method of Atractylodes lancea using seeds as explants according to claim 1, characterized in that: The rooting medium formula in step (7) is: 1 / 2 MS medium, 30 g / L sucrose, 7 g / L agar, 0.2 mg / L NAA, 0.5 mg / L IBA, 0.5 g / L activated carbon, and 50 g / L banana puree.

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