Tissue culture and rapid propagation of Asparagus cochinchinensis

Abstract

The application discloses a culture medium combination for tissue culture of Asparagus cochinchinensis, which is composed of an explant culture medium, an axillary bud and cluster bud induction culture medium, an axillary bud and cluster bud proliferation culture medium, rooting water and a rooting culture medium. Through the tissue culture of the culture medium combination, the explant contamination rate of Asparagus cochinchinensis can be controlled within 10%, the explant budding rate is higher than 80%, the first rooting rate is higher than 60%, and the second rooting rate is higher than 90%, so that the culture medium combination can be directly used for factory production of Asparagus cochinchinensis tissue culture, and lays a solid foundation for mass propagation of Asparagus cochinchinensis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of plant tissue culture, in particular to a tissue culture and rapid propagation technology of Asparagus cochinchinensis. BACKGROUND

[0002] Asparagus cochinchinensis is a perennial herb of Liliaceae Asparagus, which has excellent quality, high content of polysaccharides, amino acids and extract, and slightly low content of saponin ingredients, sweet entry and no bitter taste, is a native medicinal material in Neijiang, has a long history of consumption, registered geographical indication trademark in 2022, and is included in the top ten geographical indication native medicinal materials in Sichuan, which is very different from Asparagus cochinchinensis in Guangxi and Yunnan in morphology, composition and propagation mode.

[0003] Asparagus cochinchinensis has low seed setting rate and low germination rate, and is traditionally propagated by division, which requires the mother plant to grow for 3-4 years, and only 3-5 plants can be propagated from one plant in the harvesting season, greatly limiting the large-scale planting of Asparagus cochinchinensis. The establishment of tissue culture and rapid propagation technology of Asparagus cochinchinensis can solve the problem of seedling production. However, the same medicinal material has different genetic materials and tissue structures due to different growth environments in different producing areas, which affects the growth and development of plants, and also affects the interaction with the environment. The tissue culture and propagation method of one plant is not suitable for all producing areas. The tissue culture and propagation method of Asparagus cochinchinensis reported in the patent CN106359087A cannot form tissue culture seedlings, especially in the rooting process, which has the problems of no root formation or very low rooting rate. SUMMARY

[0004] The present application provides a culture medium combination for tissue culture and propagation of Asparagus cochinchinensis and a tissue culture and propagation method of Asparagus cochinchinensis to solve the problems of long propagation cycle, low division propagation efficiency, difficult rooting in tissue culture and propagation, and low survival rate after transplanting.

[0005] The culture medium combination for tissue culture and propagation of Asparagus cochinchinensis comprises an explant culture medium, an axillary bud and cluster bud induction culture medium, an axillary bud and cluster bud proliferation culture medium, rooting water and a rooting culture medium.

[0006] The explant culture medium is any one or more of a lower segment explant culture medium, a middle segment explant culture medium and an upper segment explant culture medium.

[0007] The lower segment explant culture medium is a culture medium containing 1.1-1.5 mg / L 6-benzyl purine, 0.03-0.07 mg / L naphthalene acetic acid, 0.1-0.3 mg / L kinetin, 5-6 g / L agar and 20-40 g / L sucrose.

[0008] The middle segment explant culture medium is a culture medium containing 1.1-1.5 mg / L 6-benzyl purine, 0.03-0.07 mg / L naphthalene acetic acid, 0.1-0.3 mg / L kinetin, 5-6 g / L agar and 20-40 g / L sucrose.

[0009] The middle section explant culture medium is MS culture medium + 0.8-1.0 mg / L 6-benzyl purine

[0010] + 0.03-0.07 mg / L naphthalene acetic acid + 0.1-0.3 mg / L kinetin + 5-6 g / L agar + 20-40 g / L sucrose;

[0011] The upper section explant culture medium is MS culture medium + 0.8-1.1 mg / L 6-benzyl purine

[0012] + 0.1-0.3 mg / L naphthalene acetic acid + 0.1-0.3 mg / L kinetin + 5-6 g / L agar + 20-40 g / L sucrose;

[0013] The axillary bud and cluster bud induction culture medium is MS culture medium + 1.2-1.4 mg / L 6-benzyl purine + 0.1-0.3 mg / L naphthalene acetic acid + 0.1-0.3 mg / L kinetin + 20-40 g / L sucrose + 5-6 g / L agar;

[0014] The axillary bud and cluster bud proliferation culture medium is MS culture medium + 1.2-1.4 mg / L 6-benzyl purine + 0.08-0.12 mg / L naphthalene acetic acid + 0.1-0.3 mg / L indole acetic acid + 0.1-0.3 mg / L kinetin

[0015] + 20-40 g / L sucrose + 5-6 g / L agar;

[0016] The rooting water is 1.5-2.5 mg / L naphthalene acetic acid aqueous solution, or an aqueous solution containing 5.0-10.0 mg / L naphthalene acetic acid and 3.0-7.0 mg / L indole acetic acid, or an aqueous solution containing 1.0-5.0 mg / L naphthalene acetic acid and 5.0-15.0 mg / L indole acetic acid;

[0017] The rooting culture medium is 1 / 4MS (1 / 4 iron salt) culture medium + 0.3-0.7 mg / L indole butyric acid + 0.3-0.7 mg / L indole acetic acid + 0.03-0.07 mg / L kinetin + 3.0-5.0 mg / L chlormequat chloride +

[0018] 30-70 g / L banana + 10-30 g / L sucrose + 5-6 g / L agar.

[0019] Further, the lower section explant culture medium is MS culture medium + 1.2 mg / L 6-benzyl purine + 0.05 mg / L naphthalene acetic acid + 0.2 mg / L kinetin + 5.5 g / L agar + 30 g / L sucrose;

[0020] The medium for the middle section explant culture is MS medium+1.0 mg / L 6-benzylpurine+0.05 mg / L naphthalene acetic acid+0.2 mg / L kinetin+5.5 g / L agar+30 g / L sucrose;

[0021] The medium for the upper section explant culture is MS medium+1.0 mg / L 6-benzylpurine+0.2 mg / L naphthalene acetic acid+0.2 mg / L kinetin+5.5 g / L agar+30 g / L sucrose;

[0022] The medium for the axillary bud and cluster bud induction culture is MS medium+1.2 mg / L 6-benzylpurine

[0023] +0.2 mg / L naphthalene acetic acid+0.2 mg / L kinetin+30 g / L sucrose+5.5 g / L agar;

[0024] The medium for the axillary bud and cluster bud proliferation culture is MS medium+1.2 mg / L 6-benzylpurine

[0025] +0.1 mg / L naphthalene acetic acid+0.2 mg / L indole acetic acid+0.2 mg / L kinetin+30 g / L sucrose+5.5 g / L agar;

[0026] The rooting water is a 2.0 mg / L naphthalene acetic acid aqueous solution, or an aqueous solution containing 8.0 mg / L naphthalene acetic acid and 5.0 mg / L indole acetic acid, or an aqueous solution containing 2.0 mg / L naphthalene acetic acid and 10.0 mg / L indole acetic acid;

[0027] The rooting medium is 1 / 4MS (1 / 4 iron salt) medium+0.5 mg / L indole butyric acid+0.5 mg / L indole acetic acid+0.05 mg / L kinetin+4.0 mg / L chlormequat+50 g / L banana+20 g / L sucrose+5.5 g / L agar.

[0028] The application also provides a tissue culture breeding method for Asparagus cochinchinensis.

[0029] a. Taking the explant of Asparagus cochinchinensis, washing and disinfecting, and culturing in the sterile explant culture medium, when the explant bud grows to 2-5 cm, taking out and cutting into single spores, and culturing in the sterile axillary bud and cluster bud induction culture medium, when the axillary bud or cluster adventitious bud grows to 2-5 cm, taking out and cutting into single spores, and culturing in the sterile axillary bud and cluster bud proliferation culture medium, to obtain the proliferation seedlings;

[0030] b. Taking the proliferation seedlings obtained in step a, removing the yellowing leaves, top tender growth points and base brown parts, soaking in the rooting water, and then culturing in the rooting culture medium, to obtain the rooting seedlings;

[0031] c. Take the rooted seedlings obtained in step c, harden them off, and transplant them.

[0032] Further, the Neijiang asparagus explants described in step a are cut into three segments: upper, middle, and lower. The top part is the upper segment, followed by the middle and lower segments, with each segment having 2-3 nodes. The upper segment is inoculated with the upper segment explant culture medium, the middle segment with the middle segment explant culture medium, and the lower segment with the lower segment explant culture medium.

[0033] Furthermore, the explants of *Asparagus neijiangensis* are preferably the tender stems of the clonal *Asparagus neijiangensis*.

[0034] Further, the cleaning described in step a) involves sequentially washing the three-sectioned Neijiang Asparagus explants with a Tween-containing detergent solution, running water, and distilled water, or sequentially washing the intact Neijiang Asparagus explants with a detergent solution, running water, 1% potassium permanganate solution, and sterile water.

[0035] Furthermore, the disinfection described in step a) is carried out separately according to the season and the explant site, specifically as follows:

[0036] In spring and summer, the upper and middle sections of tender stems are disinfected with 75% alcohol for 30-40 seconds, 0.3% sodium hypochlorite for 3 minutes, and 0.1% mercuric chloride solution for 4-5 minutes; the lower section of tender stems is disinfected with 75% alcohol for 60 seconds, 0.3% sodium hypochlorite for 3-4 minutes, and 0.1% mercuric chloride solution for 5 minutes.

[0037] In autumn and winter, the upper tender stems are disinfected with 75% alcohol for 50-60 seconds, 0.3% sodium hypochlorite for 3-3.5 minutes, and 0.1% mercuric chloride solution for 4-4.5 minutes; the lower tender stems are disinfected with 75% alcohol for 1-2 minutes, 0.3% sodium hypochlorite for 4-5 minutes, and 0.1% mercuric chloride solution for 4-5 minutes; and the lower tender stems are disinfected with 75% alcohol for 1-2 minutes, 0.3% sodium hypochlorite for 4-5 minutes, and 0.1% mercuric chloride solution for 5-6 minutes.

[0038] Further, the characteristic is that: the propagation seedling in step b) is a propagation seedling with a stem height of more than 3cm, fully expanded pseudo-leaf, and a basal mass diameter of more than 1cm; the soaking time is 30-60s.

[0039] Further, the rooted seedlings mentioned in step c) are seedlings with 3 or more fleshy roots and a root length greater than 0.5 cm; the hardening-off process involves sealed cultivation for 7 to 10 days under conditions of 2000 lx to 3000 lx light and 23°C to 28°C, followed by cultivation at a relative humidity of over 60% and a temperature of 25°C. + Open culture at 5℃ for 1-2 days.

[0040] Further, the step c) transplanting is washing the culture medium on the tissue culture seedling with 70% carbendazim 1000 times liquid, transplanting to the substrate composed of grass charcoal, vermiculite and perlite, and culturing in the high-arched shed covered with transparent film.

[0041] The mass ratio of the grass charcoal: vermiculite: perlite is 1-2:1:1.

[0042] The clone Neijiang Asparagus of the application is a fine strain (clone) reproduced by asexual reproduction method using Neijiang Asparagus high-quality single plant vegetative body, referred to as clone strain (clone), and simply referred to as clone Neijiang Asparagus.

[0043] The MS culture medium of the application is one of the most commonly used culture media in plant tissues, and the formula composition is as follows:

[0044]

[0045] The 1 / 4 MS culture medium of the application is a culture medium in which the content of the large element component in the MS culture medium formula is reduced by 3 / 4, and the content of the remaining components remains unchanged.

[0046] The 1 / 4 MS (1 / 4 iron salt) of the application is a culture medium in which the content of the large element and iron salt in the MS culture medium formula is reduced by 3 / 4, and the content of the remaining components remains unchanged.

[0047] The culture medium combination for Neijiang Asparagus tissue culture propagation of the application can make the Neijiang Asparagus explant have a high budding rate, good quality and thick and strong bud body by using specific plant growth hormones in different growth stages of Neijiang Asparagus and accurately controlling the amount of the plant growth hormones. The experiments prove that the Neijiang Asparagus explant pollution rate can be controlled within 10% by the Neijiang Asparagus tissue culture propagation method of the application, the Neijiang Asparagus explant budding rate is higher than 80%, the first rooting rate is more than 60%, the second rooting rate is more than 90%, and the Neijiang Asparagus can be directly used for factory production, thereby laying a solid foundation for the mass propagation of Neijiang Asparagus.

[0048] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0049] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the application to the following examples. Any technology realized based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 Actual scene record of Neijiang Asparagus propagation process

[0051] Figure 2 Growth of different proliferated seedlings

[0052] Figure 3 Effects of different cytokinins on growth of Neijing Asparagus DETAILED DESCRIPTION

[0053] The raw materials, reagents and equipment used in the detailed description of the present application are known products; they can be obtained by commercial purchase.

[0054] Example 1 Neijing Asparagus tissue culture breeding method

[0055] 1. Propagation material requirements

[0056] Select a single plant of a healthy, disease-free and high-yield excellent Neijing Asparagus clone, cut the aboveground part from 3-5 cm above the spore, dig out the cut underground part, and transplant it to a clean indoor or greenhouse for culture. Disinfect with a water solution of multi-purpose fungicide or thiophanate-methyl every week for the first month. When the new shoots grow to 15-30 cm and the stipules are not yet unfolded, prepare the material for collection.

[0057] 2. Collection and treatment of explants

[0058] 2.1 Collection time and site

[0059] Reduce watering and keep the air dry one week before collecting the explants. In the morning of the inoculation day, cut the healthy and undamaged tender stems from 5 cm above the ground as explants. If long-distance transportation is required, store the explants in a low-temperature box with clean gauze for moisture retention. Note that the explants should not be directly in contact with ice or other cooling materials.

[0060] 2.2 Material pretreatment

[0061] In a clean room, cut the explants into 2-3 node segments according to the upper, middle and lower sections. The top part is the upper section, followed by the middle section and the lower section. Place them separately. Then prepare an explant cleaning agent by mixing 2L laundry detergent with 2-4 drops of Tween 2. Carefully clean the surface dirt of the explant segments, and then rinse them with running water for 30-60 minutes. Ensure that the explants are rolling up and down in the running water during the rinsing period. After rinsing, wash the explants with distilled water for 2-3 times for standby use. (Alternatively, first clean the surface dirt of the explants with laundry detergent, rinse them with running water for 30-60 minutes, soak them in 1% potassium permanganate solution for 8-10 minutes, and then wash them with sterile water for 2-3 times for standby use).

[0062] 3. Preparation before culture

[0063] 3.1 Disinfection of super-clean bench

[0064] Open the ultraclean workbench UV lamp sterilization for 20-30 min, then open the ultraclean workbench fan blowing 10-20 min, and then use 75% medical alcohol wipe the table.

[0065] 3.2 Medium preparation and sterilization

[0066] According to the various types of medium formula and the volume of the preparation, calculate the amount of various drug mother liquor and water, weigh the required amount of coagulant and carbon source, heat and dissolve with 70%-80% of the final volume of water, add macronutrients, iron salt, trace elements, organic ingredients, plant growth regulator mother liquor, etc. in turn, stir while adding, adjust pH, constant volume, sub-packaging, sealing, labeling. After preparation, use high-pressure steam sterilization on the same day, sterilize at 0.105 MPa, 121℃ for 20-25 min.

[0067] 3.3 Medium preservation

[0068] The sterilized medium is placed at room temperature for 3-5 days, and used within 20 days, and used within one month at 2-4℃.

[0069] 4, Tissue culture

[0070] 4.1 Culture conditions

[0071] The culture temperature is 25±1℃, the light time is 12h / d-14h / d, and the light intensity is 1500lx-3000lx.

[0072] 4.2 Explant disinfection

[0073] The pretreated material is placed on the disinfected ultraclean bench, and the following disinfection methods are used according to the season:

[0074] Spring and summer material disinfection method:

[0075] Upper section: 75% alcohol 30-40s; 0.3% sodium hypochlorite 3min; 0.1% mercury 4-5min;

[0076] Middle section: 75% alcohol 30s; 0.3% sodium hypochlorite 3min; 0.1% mercury 4-5min;

[0077] Lower section: 75% alcohol 1min; 0.3% sodium hypochlorite 3-4min; 0.1% mercury 5min;

[0078] Autumn and winter material disinfection method:

[0079] Upper section: 75% alcohol 50-60s; 0.3% sodium hypochlorite 3-3.5min; 0.1% mercury 4-4.5min;

[0080] Middle section: 75% alcohol 1-2 min; 0.3% sodium hypochlorite 4-5 min; 0.1% mercury 4-5 min;

[0081] Lower section: 75% alcohol 1-2 min; 0.3% sodium hypochlorite 4-5 min; 0.1% mercury 5-6 min;

[0082] 4.3 Inoculation of explants

[0083] On the clean bench, the lower section explants are placed obliquely in the lower section explant medium according to the growth direction of the explants, and the spore base should be close to the medium when obliquely placed (in large-scale production, the explants can be directly placed on the surface of the medium); the middle section explants are obliquely or horizontally inoculated in the middle section explant medium; and the upper section material is horizontally placed in the upper section explant medium. The lower section explant medium has the following formula: MS+6-BA 1.2 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L; the middle section explant medium has the following formula: MS+6-BA 1.0 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L; and the upper section explant medium has the following formula: MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L.

[0084] 4.4 Axillary bud and cluster bud induction

[0085] The explant buds with a length of 2-5 cm are taken out, cut into single spores, and inoculated into the axillary bud and cluster bud induction medium, which has the following formula: MS+6-BA 1.2 mg / L+NAA 0.2 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L.

[0086] 4.5 Axillary bud and cluster bud proliferation

[0087] The axillary buds or cluster adventitious buds with a length of 2-5 cm are taken out, cut into single spores, and inoculated into the proliferation medium, which has the following formula: MS+6-BA 1.2 mg / L+NAA 0.1 mg / L+IAA 0.2 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L.

[0088] 4.6 Rooting culture

[0089] The proliferated seedlings with more than 2 months of growth time, more than 3 cm of stem height, completely unfolded phyllodes, and more than 1 cm of diameter of the base mass are selected, yellowing leaves are removed, the top young growth points are cut off, the base browned parts are removed, and the seedlings are quickly dipped in sterilized rooting water, wherein the rooting water formula is NAA 8.0 mg / L+IAA 5.0 mg / L or NAA 2.0 mg / L+IAA 10.0 mg / L; or the seedlings are soaked in sterilized rooting water for 30-60 s, wherein the rooting water formula is NAA 2.0 mg / L; the seedlings are transferred into a rooting medium: 1 / 4MS (1 / 4 iron salt)+IBA 0.5 mg / L+IAA 0.5 mg / L+KT 0.05 mg / L+CCC 4.0 mg / L+banana 50 g / L+sucrose 20 g / L+agar 5.5 g / L, and the seedlings are cultured for about 90-120 d to start rooting, and the rooting rate is about 60%, and the seedlings without rooting are continuously transferred into a new rooting medium, and all the seedlings can root after 90 d.

[0090] 5. Seedling hardening

[0091] The rooted seedlings with strong seedlings, thick roots, more than one fleshy root, and more than 0.5 cm of root length are selected, and the seedlings are closed-bottle hardened in a greenhouse with illumination of 2000 lx-3000 lx and temperature of 23 ℃-28 ℃ for 7-10 d, the bottle cap or sealing film is loosened, and then the seedlings are opened-bottle hardened for 1-2 d, during which the relative air humidity is kept to be more than 60%, and the temperature is kept to be 25 +

[0092] 6. Transplanting

[0093] After the opened-bottle hardening is finished, the medium is washed away with 70% carbendazim 1000 times liquid (i.e. a solution obtained by diluting 70% carbendazim with water by 1000 times), and the seedlings are transplanted into a substrate with a ratio of grass charcoal: vermiculite: perlite = 1:1:1 or 2:1:1, and the seedlings are cultured in a high-arched greenhouse covered with transparent film, the seedlings are sprayed with water 2-3 times per week within 1 month after the transplanting to ensure that the air humidity is more than 80%, and after 1 month of the transplanting, the wintergreen tissue culture seedlings start to grow young new branches, and the film is gradually removed, i.e. the film at two ends is first removed, the film at the bottom is opened for ventilation after 3-4 days, and then the film is completely removed, and the hardening survival rate is more than 95%. After 80% of the tissue culture seedlings grow new branches, foliar fertilizer is sprayed once per week, the concentration is gradually increased, and sterilization is performed once per month, and the seedlings are transplanted to the field after about half a year. Figure 1 .

[0094] The beneficial effects of the present application are illustrated by the following test examples:

[0095] In the early experimental process, the tissue culture of Neijiang wintergreen was carried out according to the medium provided in the following table 1, and the Neijiang wintergreen could not root. Therefore, the tissue culture of Neijiang wintergreen was further researched.

[0096] ​Table 1 Test situation of asparagus germplasm rooting formula applied to Neijiang asparagus

[0097]

[0098] Note: 【1】Yu Wehua, Shi Qian, Zhang Xiangjun, et al. Establishment of "Gui Dong 1" asparagus regeneration system [J]. Northern Horticulture, 2020(20): 7.

[0099] 【2】Quan Miaohua, Oulijun, He Anna, et al. Study on tissue culture and rapid propagation of asparagus [J]. Chinese Traditional and Herbal Drugs, 2012, 043(008): 1599-1603.

[0100] 【3】Li Lin. Asparagus cluster bud rapid induction and test tube seedling rooting technology research [D]. Guizhou University.

[0101] 【4】Yang Pingfei, Luo Ming, Liu Hai, et al. Asparagus tissue culture research [J]. Seed, 2019, 38(5): 3.

[0102] 【5】Pan Limei, Ma Xiaojun, Weishugen, Wang Xiaofeng, Ke Fang. Optimization of asparagus tissue culture conditions [J]. Hubei Agricultural Sciences, 2012, 51(19): 4396-4398

[0103] 【6】Wu Shangjiang. Asparagus tissue culture and in vitro rapid propagation system construction [D]. Guizhou University.

[0104] 【7】Huang Dongxiang. "A method for asparagus tissue culture and rapid propagation of seedlings", CN201610728870.X. 2019.

[0105] 【8】Zhou Jinsong, et al. "A method for asparagus tissue culture and rapid propagation", CN201710141841.8. 2019.

[0106] 【9】He Quanhui, et al. "Asparagus tissue culture and rapid propagation method", CN102487830B. 2016.

[0107]

[10] Cha Ping, Li Haibin. A method for improving the induction rate of asparagus fleshy tuberous roots: 202211715956. 2022.

[0108]

[11] Yu Wehua, Shi Qian, Fan Yongsheng, et al. A method for asparagus in-vitro rooting and tuber formation: 202310396246[P][2024-11-04].

[0109]

[12] Yang Yaogong, Ma Heping, Lan Xiaozhong, et al. Study on tissue culture and rapid propagation system of asparagus fern [J]. Forestry Practical Technology, 2008, 000(012): 16-18.

[0110] Test Example 1 Innei Jiang Asparagus Tissue Culture Breeding Culture Research

[0111] 1. Explant part screening

[0112] The spores, upper end of tender stem, middle part of tender stem, lower part of tender stem, tuber, and young phyllode of the single plant of Innei Jiang asparagus clone cultured in the greenhouse were selected as the tissue culture explant materials. The 75% alcohol was used for disinfection for 60s or 2min (the spores, upper end of tender stem, middle part of tender stem, lower part of tender stem, and young phyllode were disinfected for 60s, and the tuber was disinfected for 2min), the sterile water was used for washing for 3 times, the 0.1% mercury was used for disinfection for 10min, and the sterile water was used for washing for 3 times, and then the materials were prepared. The explant inoculation medium was MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+KT 0.2 mg / L, sucrose 30g / L, and agar 5.5g / L. One explant was inoculated in each bottle, 20 bottles were inoculated with the spores, and 40 bottles were inoculated with other explant materials. After the culture under the same conditions (temperature 25±1℃, light time 12h / d-14h / d, and light intensity 1500lx-3000lx) for 1 month, the number of contaminations and the number of differentiated new shoots were counted, and the seedling rate was evaluated according to the differentiation of new shoots.

[0113] The different explants had great differences in the difficulty of obtaining and the contamination rate in the field. The statistics of contamination and seedling rate in the process of explant screening are shown in Table 2.

[0114] Table 2 Explant part screening test

[0115]

[0116]

[0117] As shown in Table 1, the spores were the most difficult to obtain in the field, and the contamination rate was extremely high, which was not suitable for explant; the tuber and phyllode had low contamination rate, but it was difficult to form seedlings, which was not suitable for explant material, and the different parts of tender stem could all differentiate seedlings, and had low contamination rate, which was the ideal material for asparagus explant.

[0118] 2. Explant cleaning and disinfection parameter screening

[0119] The previous experiment only used 75% alcohol and 0.1% mercury to disinfect explants, and the contamination rate was extremely high during the tissue culture process. Therefore, the tender stems were used as explants, first washed with laundry detergent water to clean the surface dirt, then rinsed with running water for 30 min, and then disinfected with 75% alcohol, 0.3% sodium hypochlorite and 0.1% mercury. The disinfected explants were inoculated in the medium (MS+6-BA 1.0 mg / L+NAA 0.1 mg / L+KT 0.2 mg / L, sucrose 30 g / L, agar 5.5 g / L) and cultured under the same conditions (temperature 25±1℃, light time 12h / d-14h / d, light intensity 1500lx-3000lx). The browning rate of the explants was observed and counted.

[0120] The specific cleaning and disinfection parameters and their corresponding contamination rate, browning rate and normal rate during the breeding process are shown in Table 3 and Table 4.

[0121] Table 3 Disinfection treatment method ①

[0122]

[0123]

[0124]

[0125] As can be seen from Table 4, the explant contamination rate is relatively low when disinfected with alcohol for 2 min and mercury for 10-20 min, but the spore germination is weak and slow, the growth is poor, and the browning rate is high. By segmenting the tender stems and shortening the alcohol disinfection time, the disinfection treatment in Table 5 is increased, and the disinfection method with better spore growth is selected.

[0126] Table 5 Disinfection treatment test ②

[0127]

[0128] As can be seen from Table 5, the upper and middle tender stems were disinfected with 75% alcohol for 30s, 0.3% sodium hypochlorite for 3min, and 0.1% mercury solution for 4-5min, which had the best disinfection effect, and the new shoots grew strong; the lower tender stems were disinfected with 75% alcohol for 60s, 0.3% sodium hypochlorite for 3-4min, and 0.1% mercury solution for 5min, which had the best disinfection effect, and the new shoots grew extremely strong.

[0129] Asparagus is in the growing season in spring and summer, the cell metabolism of explants is active, and the tissue is relatively delicate. If the disinfection time is too long, the disinfectant may cause great damage to the cells; Asparagus grows slowly in autumn and winter, and the physiological activity of explants is weakened, and its stress resistance is relatively strong. At this time, the disinfection time can be appropriately extended, and the explants can withstand it without causing too much damage. It can also more effectively kill microorganisms, reduce the pollution rate, and improve the success rate of culture. Therefore, according to the actual experience accumulation, the specific disinfection method of Neijiang asparagus explants is:

[0130] Spring and summer material disinfection method:

[0131] Upper segment: 75% alcohol 30-40s; 0.3% sodium hypochlorite 3min; 0.1% mercury 4-5min;

[0132] Middle segment: 75% alcohol 30s; 0.3% sodium hypochlorite 3min; 0.1% mercury 4-5min;

[0133] Lower segment: 75% alcohol 1min; 0.3% sodium hypochlorite 3-4min; 0.1% mercury 5min;

[0134] Autumn and winter material disinfection method:

[0135] Upper segment: 75% alcohol 50-60s; 0.3% sodium hypochlorite 3-3.5min; 0.1% mercury 4-4.5min;

[0136] Middle segment: 75% alcohol 1-2min; 0.3% sodium hypochlorite 4-5min; 0.1% mercury 4-5min;

[0137] Lower segment: 75% alcohol 1-2min; 0.3% sodium hypochlorite 4-5min; 0.1% mercury 5-6min.

[0138] 3. Explant culture medium screening

[0139] The early experiment found that different explant parts had great differences in growth on different hormone ratio culture media. Therefore, the explant culture medium was screened for different explant parts. Based on the previous culture medium screening, it was found that different hormones such as 6-BA, NAA, KT had different effects on different explant parts. Therefore, the effects of 6-BA, NAA, KT and other hormones on the formation of axillary buds of different explant parts were mainly investigated. The hormone ratio in the explant culture medium and the growth during the corresponding tissue culture breeding process are shown in Table 6,

[0140] Table 6 Explant culture medium screening test

[0141]

[0142]

[0143] From Table 6, it can be seen that the upper, middle and lower sections of the stems can all form new shoots, with the highest shoot formation rate in the lower section and the highest callus formation rate in the upper section. Different sections require different hormone ratios for shoot formation. The optimal hormone ratio for shoot formation in the upper section is MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+KT 0.2 mg / L, with a shoot formation rate of 87.5%; the optimal hormone ratio for shoot formation in the middle section is MS+6-BA 1.0 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, with a shoot formation rate of 95%; and the optimal hormone ratio for shoot formation in the lower section is MS+6-BA 1.2 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, with a shoot formation rate of 100%. Although the shoot formation rate in the upper section is slightly lower, the upper section has more bud primordia per node, and thus more shoots can be formed from the same node, and is also suitable for use as an explant.

[0144] In the tissue culture medium, sucrose and agar perform multiple important functions, such as providing energy, maintaining osmotic pressure, preventing microbial contamination and solidifying the medium. In order to reduce production costs, the amount of sucrose and agar in the formula was adjusted based on the above-mentioned hormone dosage. The final formula for the explant culture medium is as follows: the optimal medium formula for the upper section of the stem is MS+6-BA 1.0 mg / L+NAA 0.2 mg / L+KT 0.2 mg / L, with sucrose 30 g / L and agar 5.5 g / L; the optimal medium formula for the middle section is MS+6-BA 1.0 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, with sucrose 30 g / L and agar 5.5 g / L; and the optimal medium formula for the lower section is MS+6-BA 1.2 mg / L+NAA 0.05 mg / L+KT 0.2 mg / L, with sucrose 30 g / L and agar 5.5 g / L.

[0145] 4. Selection of proliferation medium

[0146] Axillary shoots or multiple shoots formed from the explants need to be transferred again before being suitable for proliferation culture. The growth is good when transferred to the original explant culture medium or the axillary shoot induction medium. In order to facilitate production, simplify operations and facilitate storage in the industrial production of Asparagus cochinchinensis, the material after explant culture is uniformly transferred to the axillary shoot and multiple shoot induction medium (MS+6-BA 1.2 mg / L+NAA 0.2 mg / L+KT 0.2 mg / L, sucrose 30 g / L, agar 5.5 g / L), and then induced and subjected to proliferation culture.

[0147] On the basis of axillary bud and cluster bud induction medium, the most suitable proliferation medium for Asparagus cochinchinensis L. was screened by using different basal media, different types and concentrations of auxins and cytokinins, and their combinations. The specific method was as follows: the 2-5 cm long buds obtained by culturing on the most suitable medium in the item of "3, screening of explant medium" were cut into small segments with buds, and then inoculated into the axillary bud and cluster bud induction medium to obtain 2-5 cm axillary buds or cluster adventitious buds, which were cut into single buds, and finally inoculated into different proliferation media for culture, and the growth state was observed and counted. Figures 2-3

[0148]

[0149]

[0150] The growth state was greatly improved; (4) the use of trace TDZ (0.1-0.2 mg / L) would form more short and thick buds, and the growth and development would be limited in the later stage, therefore, the most suitable medium for the proliferation of Neijiang Asparagus cochinchinensis was MS+6-BA 1.2 mg / L+NAA 0.1 mg / L+IAA 0.2 mg / L+KT 0.2 mg / L, sucrose 30 g / L, and agar 5.5 g / L, and the average proliferation rate was 5.37.

[0151] 5. Screening of rooting medium

[0152] It was found in the early experiment that the rooting rate of Neijiang Asparagus cochinchinensis was low, and the basal medium had a certain influence on the rooting of Neijiang Asparagus cochinchinensis, especially the combination of 1 / 4MS, 1 / 4MS (1 / 4 iron salt), 1 / 2MS and NAA, IAA, IBA had a greater influence on the rooting of Neijiang Asparagus cochinchinensis. The influence of the types and concentrations of basal medium, 6-BA, NAA, IAA, IBA, KT, 2,4-D, GA and organic matter and their combinations on the rooting of Neijiang Asparagus cochinchinensis was investigated. The most suitable medium for proliferation of Neijiang Asparagus cochinchinensis was used to screen the rooting medium, and the Neijiang Asparagus cochinchinensis proliferation seedlings with higher lignification and dense mass at the base were used as the material.

[0153] Table 8 Influence of main hormone treatment on the rooting of Neijiang Asparagus cochinchinensis

[0154]

[0155]

[0156] ​From the observation results, it can be seen that due to the treatment of more hormones, the overall hormone concentration is higher, most of the treatments have poor growth, and most of them do not root, even if they root, the rooting rate is also low. The addition of 6-BA has a greater effect on the rooting of Neijiang Asparagus, and Neijiang Asparagus treated with different concentrations of 6-BA does not root. NAA, IBA, IAA combination has a certain effect on the rooting of Neijiang Asparagus, KT addition has a certain promoting effect on rooting, GA and 2,4-D addition has little effect on rooting, and organic matter such as banana and coconut powder has a slight promoting effect on the rooting of Neijiang Asparagus, but the overall rooting rate of Neijiang Asparagus is still low, and further optimization tests are needed.

[0157] The treatment with high NAA content has some Neijiang Asparagus rooting, but the rooting rate is low, so NAA, IBA, and IAA are considered as the main hormones to continue to optimize the rooting formula.

[0158] Take 1 / 4MS (1 / 4Fe) as the basic medium, fix the KT concentration at 0.2m / L, add different concentrations and different types of hormones, sucrose 20g / L, agar 5.5-6g / L. The test treatment method and results are shown in Table 9.

[0159] Table 9 Effect of different hormones on the rooting of Neijiang Asparagus

[0160]

[0161]

[0162] As can be seen from Table 9, with the increase of NAA concentration, the growth of Neijiang Asparagus above ground gradually deteriorates, when the NAA concentration is high, the growth of Neijiang Asparagus above ground is poor and even dies, the concentration of auxin NAA should not exceed 1mg / L when rooting; low concentration of IBA and IAA can promote the rooting of Neijiang Asparagus, high concentration can inhibit the rooting, when the concentration of IBA and IAA is balanced, the rooting rate of Neijiang Asparagus is the strongest, but the overall rooting rate is still low, the highest rooting rate is only 12%. The best rooting medium formula of the preliminary experiment is: 1 / 4MS (1 / 4Fe) + IAA 1.0mg / L + IBA 1.0mg / L + KT 0.2mg / L, sucrose 20g / L, agar 6g / L.

[0163] 6. Rooting water screening test

[0164] 6.1 Method 1

[0165] 6.1.1 First step rooting water screening in two-step rooting method

[0166] The rooting rate of N. jingdongensis is very low by using the conventional test. The two-step rooting method can improve the rooting rate of N. jingdongensis. The proliferated seedlings of N. jingdongensis with higher lignification degree, leaf-like expansion and dense mass at the base obtained from the optimal proliferated medium in the step 4 are used as the material to carry out the rooting water and rooting medium screening. The two-step rooting method is used for the culture. In the first step, the base of the N. jingdongensis mass to be rooted is dipped in the rooting water for a few seconds, and then is slightly dried. In the second step, the N. jingdongensis dipped in the rooting water is inoculated into the conventional rooting medium. The conventional rooting medium is 1 / 4MS (1 / 4Fe) + IAA 1.0 mg / L + IBA 1.0 mg / L + KT 0.2 mg / L, sucrose 20 g / L and agar 6 g / L. The formula of the rooting water is shown in Table 10.

[0167] Table 10 Screening test of rooting water and results

[0168]

[0169] After the base mass of N. jingdongensis is treated instantaneously with different types and concentrations of auxin, the rooting rate of N. jingdongensis is different. High concentration of NAA (higher than 10 mg / L) can easily cause the aboveground part of N. jingdongensis to turn yellow, but can stimulate the formation of root primordia. Low concentration of IBA or IAA has little effect on the rooting of N. jingdongensis. Instantaneous stimulation of N. jingdongensis base mass with medium-high concentration of NAA and IAA is beneficial to the formation of root primordia. The optimal instantaneous treatment rooting water formula is NAA 8.0 mg / L + IAA 5 mg / L.

[0170] 6.1.2 Optimization of the second step medium in the two-step rooting method

[0171] The proliferated seedlings of N. jingdongensis with higher lignification degree, leaf-like expansion and dense mass at the base obtained from the optimal proliferated medium in the step 4 are used as the material. In the first step, the base mass of N. jingdongensis is treated instantaneously with NAA 8.0 mg / L + IAA 5 mg / L. The treated mass is inoculated into the second step medium. The second step medium is treated with 1 / 4MS (1 / 4Fe) + IAA 1.0 mg / L + IBA 1.0 mg / L, and different concentrations of kinetin are added, as shown in Table 11. Through the test of the effect of different concentrations of KT on the rooting of N. jingdongensis, it is shown that when the concentration of KT is 0.05 mg / L, the rooting rate of N. jingdongensis is the highest, which is 32%.

[0172] Table 11 Effect of different concentrations of kinetin on the rooting of N. jingdongensis

[0173]

[0174] The second step treatment medium is 1 / 4MS (1 / 4Fe) + IAA 1.0 mg / L + IBA 1.0 mg / L + KT 0.05 mg / L, and different concentrations and types of hormones are added, as shown in Table 12. The results show that the addition of CCC (chlormequat) promotes the rooting of Neijing Asparagus, and the roots are thick and the rooting time is shortened, which is beneficial to seedling transplanting. The optimal CCC concentration is 4 mg / L. The addition of ABA and activated carbon does not promote the rooting of Neijing Asparagus. Low concentration of TIBA has a slight promoting effect on the rooting of Neijing Asparagus. Considering the cost of different hormones, it is recommended to add 4 mg / L of CCC to the second step rooting medium.

[0175] Table 12 Effect of different hormones on the rooting of Neijing Asparagus

[0176]

[0177] The basic medium is fixed as 1 / 4MS (1 / 4Fe), the concentration of KT is 0.05 mg / L or 2 mg / L, the concentration of CCC is CCC 4.0 mg / L, sucrose is 20 g / L, agar is 5.5 g / L, and different concentrations of IAA, IBA and organic matter banana are added. The proliferated seedlings of Neijing Asparagus with high lignification degree, leaf-like expansion and dense mass at the base obtained from the optimal proliferation medium in "4. Proliferation medium screening" are used as materials. In the first step, NAA 8.0 mg / L + IAA 5 mg / L is used to stimulate the base mass of Neijing Asparagus instantaneously, and the treated mass is inoculated into the second step medium (Table 13),

[0178] Table 13 Effect of banana addition and hormone combination on the rooting of Neijing Asparagus

[0179]

[0180] As shown in Table 13, the addition of organic matter banana is beneficial to improve the rooting rate of Neijing Asparagus and promote the normal growth of the rooted seedlings. The optimal rooting medium formula is 1 / 4MS (1 / 4Fe) + IBA 0.5 mg / L + IAA 0.5 mg / L + KT 0.05 mg / L + CCC 4.0 mg / L + banana 50 g / L, sucrose 20 g / L, agar 5.5 g / L.

[0181] 6.2 Method 2

[0182] The first step is to use the inner Jiang Asparagus proliferous seedlings with higher lignification degree, leaf-like expansion and dense mass at the base obtained from the optimal proliferous medium formula in "4. Proliferous medium screening" as the material, and treat them with different hormones alone or in combination for different time. The second step is to inoculate the inner Jiang Asparagus seedlings soaked with rooting water into the conventional rooting medium, and the conventional rooting medium is 1 / 4MS (1 / 4 Fe) + IAA 1.0 mg / L + IBA 1.0 mg / L + KT 0.2 mg / L + sucrose 20 g / L + agar 6 g / L. The rooting water screening results are shown in Table 14.

[0183] Table 14. Rooting water test results

[0184]

[0185] In Table 14, the treatments of USD05012, Y010060 and N00522 can greatly improve the rooting rate of inner Jiang Asparagus. The USD05012 combination treatment is to soak the base in a sterile IBA solution with a concentration of 50 mg / L for 120 s and then inoculate it into the rooting medium. The Y010060 combination treatment is to soak the base in a sterile solution with a concentration of IBA 10.0 mg / L + IAA 10.0 mg / L for 60 s and then inoculate it into the rooting medium. The N00522 combination treatment is to treat the base with a sterile NAA solution with a concentration of 5.0 mg / L for 120 min. Since the production cost of 50 mg / L IBA is relatively high, this concentration is not recommended for production. Based on the Y010060 and N00522 formula tests, the concentration of NAA for root soaking is reduced to 2.0 mg / L, and the rooting rate remains unchanged. The production cost of this reagent is the lowest, so it is recommended to use 2.0 mg / L NAA for root soaking.

[0186] Through the screening of rooting water and rooting hormones, combined with the production cost and the difficulty of operation, NAA 2.0 mg / L, NAA 8.0 mg / L + IAA 5 mg / L or NAA 2.0 mg / L + IAA 10.0 mg / L are selected as the first step of rooting water. When the rooting water is NAA 2.0 mg / L, it is quickly dipped. When the rooting water is NAA 8.0 mg / L + IAA 5 mg / L or NAA 2.0 mg / L + IAA 10.0 mg / L, it is soaked for 30-60 s.

[0187] Then inoculate it into the second step of rooting medium, and the formula is 1 / 4MS (1 / 4 iron salt) + IBA 0.5 mg / L + IAA 0.5 mg / L + KT 0.05 mg / L + CCC 4.0 mg / L + banana 50 g / L + sucrose 20 g / L + agar 5.5 g / L.

[0188] The rooting rate of Neijiang Asparagus after two-step rooting culture is about 60%, and the rooted materials are selected for seedling acclimatization and transplanting in production, and the unrooted seedlings are inoculated again into rooting culture medium (without rooting water) : 1 / 4MS (1 / 4 iron salt) + IBA 0.5 mg / L + IAA 0.5 mg / L + KT 0.05 mg / L + CCC 4.0 mg / L + banana 50 g / L + sucrose 20 g / L + agar 5.5 g / L, and the rooting rate of the re-cultured seedlings can reach 90%.

[0189] In conclusion, by using specific plant growth hormones at different growth stages of Neijiang Asparagus and accurately controlling the dosage, the bud formation rate of Neijiang Asparagus explants is high, the bud formation quality is good, and the bud body is thick and strong. Experimental results show that, by using the tissue culture propagation method of Neijiang Asparagus, the contamination rate of Neijiang Asparagus explants can be controlled within 10%, the bud formation rate of Neijiang Asparagus explants is higher than 80%, the first rooting rate is higher than 60%, and the second rooting rate is higher than 90%, which can be directly used for industrial production of Neijiang Asparagus, and lays a solid foundation for mass propagation of Neijiang Asparagus.

Claims

1. A culture medium composition for tissue culture propagation of Asparagus cochinchinensis in Neijiang, characterized in that: The culture medium combination consists of explant culture medium, axillary bud and cluster bud induction culture medium, axillary bud and cluster bud proliferation culture medium, rooting water and rooting culture medium; The explant culture medium is any one or more of the following: lower explant culture medium, middle explant culture medium, and upper explant culture medium; The lower explant culture medium is MS medium + 1.1~1.5 mg / L 6-benzylpurine + 0.03~0.07 mg / L naphthaleneacetic acid + 0.1~0.3 mg / L kinetin + 5~6 g / L agar + 20~40 g / L sucrose. The medium for the mid-section explant is MS medium + 0.8~1.0 mg / L 6-benzylpurine + 0.03~0.07 mg / L naphthaleneacetic acid + 0.1~0.3 mg / L kinetin + 5~6 g / L agar + 20~40 g / L sucrose. The culture medium for the upper explant segment is MS medium + 0.8~1.1 mg / L 6-benzylpurine + 0.1~0.3 mg / L naphthaleneacetic acid + 0.1~0.3 mg / L kinetin + 5~6 g / L agar + 20~40 g / L sucrose. The axillary bud and cluster bud induction medium is MS medium + 1.2~1.4 mg / L 6-benzylpurine + 0.1~0.3 mg / L naphthaleneacetic acid + 0.1~0.3 mg / L kinetin + 20~40 g / L sucrose + 5~6 g / L agar. The axillary bud and cluster bud proliferation medium is MS medium + 1.2~1.4 mg / L 6-benzylpurine + 0.08~0.12 mg / L naphthaleneacetic acid + 0.1~0.3 mg / L indoleacetic acid + 0.1~0.3 mg / L kinetin + 20~40 g / L sucrose + 5~6 g / L agar. The rooting solution is an aqueous solution of 1.5~2.5 mg / L naphthaleneacetic acid, or an aqueous solution of 5.0~10.0 mg / L naphthaleneacetic acid and 3.0~7.0 mg / L indoleacetic acid, or an aqueous solution of 1.0~5.0 mg / L naphthaleneacetic acid and 5.0~15.0 mg / L indoleacetic acid; The rooting medium is a medium consisting of 1 / 4 MS (1 / 4 iron salt) medium + 0.3~0.7 mg / L indolebutyric acid + 0.3~0.7 mg / L indoleacetic acid + 0.03~0.07 mg / L kinetin + 3.0~5.0 mg / L chlormequat chloride + 30~70 g / L banana + 10~30 g / L sucrose + 5~6 g / L agar. The explants were the tender stems of the clonal Asparagus neijiangensis; The 1 / 4MS (1 / 4 iron salt) is a medium in which the macroelements and iron salts in the MS medium formulation are reduced by 3 / 4, while the contents of the remaining components remain unchanged; The MS culture medium has the following composition: 。 2. The culture medium combination according to claim 1, characterized in that: The lower explant culture medium is MS medium + 1.2 mg / L 6-benzylpurine + 0.05 mg / L naphthaleneacetic acid + 0.2 mg / L kinetin + 5.5 g / L agar + 30 g / L sucrose. The medium for the mid-section explants was MS medium + 1.0 mg / L 6-benzylpurine + 0.05 mg / L naphthaleneacetic acid + 0.2 mg / L kinetin + 5.5 g / L agar + 30 g / L sucrose. The culture medium for the upper explant segment was MS medium + 1.0 mg / L 6-benzylpurine + 0.2 mg / L naphthaleneacetic acid + 0.2 mg / L kinetin + 5.5 g / L agar + 30 g / L sucrose. The axillary bud and cluster bud induction medium is MS medium + 1.2 mg / L 6-benzylpurine + 0.2 mg / L naphthaleneacetic acid + 0.2 mg / L kinetin + 30 g / L sucrose + 5.5 g / L agar. The axillary bud and cluster bud proliferation medium is MS medium + 1.2 mg / L 6-benzylpurine + 0.1 mg / L naphthaleneacetic acid + 0.2 mg / L indoleacetic acid + 0.2 mg / L kinetin + 30 g / L sucrose + 5.5 g / L agar. The rooting solution is an aqueous solution of 2.0 mg / L naphthaleneacetic acid, or an aqueous solution of 8.0 mg / L naphthaleneacetic acid and 5.0 mg / L indoleacetic acid, or an aqueous solution of 2.0 mg / L naphthaleneacetic acid and 10.0 mg / L indoleacetic acid; The rooting medium is a medium consisting of 1 / 4 MS (1 / 4 iron salt) medium + 0.5 mg / L indolebutyric acid + 0.5 mg / L indoleacetic acid + 0.05 mg / L kinetin + 4.0 mg / L chlormequat chloride + 50 g / L banana + 20 g / L sucrose + 5.5 g / L agar.

3. A method for tissue culture propagation of Asparagus cochinchinensis in Neijiang, characterized in that: It is cultured using the culture medium combination described in claim 1 or 2, and the specific steps are as follows: a. Take explants of *Asparagus officinalis* from Neijiang, clean and disinfect them, and inoculate them into a sterile explant culture medium. When the explant buds grow to 2-5 cm, remove them and cut them into individual spores. Inoculate them into a sterile axillary bud and clustered bud induction culture medium. When the axillary buds or clustered adventitious buds grow to 2-5 cm, remove them and cut them into individual spores again. Inoculate them into a sterile axillary bud and clustered bud proliferation culture medium to obtain proliferated seedlings. The *Asparagus officinalis* explants from Neijiang are cut into three sections: the top part is the top section, followed by the middle and bottom sections, each section having 2-3 nodes. The top section is inoculated into the top section explant culture medium, the middle section into the middle section explant culture medium, and the bottom section into the bottom section explant culture medium. b. Take the propagated seedlings obtained in step a, remove the yellowed leaves, tender growing points at the top and browned parts at the base, soak them in rooting water and then inoculate them into rooting culture medium to obtain rooted seedlings; c. Take the rooted seedlings obtained in step b, harden them off, and transplant them. The explants of Neijiang Asparagus were the tender stems of the clonal Neijiang Asparagus.

4. The method for tissue culture propagation of Asparagus cochinchinensis in Neijiang according to claim 3, characterized in that: Step a) The cleaning process involves sequentially washing the three-sectioned Neijiang Asparagus explants with a Tween-containing detergent solution, running water, and distilled water, or sequentially washing the intact Neijiang Asparagus explants with a detergent solution, running water, 1% potassium permanganate solution, and sterile water.

5. The method for tissue culture propagation of Asparagus cochinchinensis in Neijiang according to claim 3, characterized in that: The disinfection described in step a) is carried out according to the season and the location of the explant, specifically as follows: In spring and summer, the upper and middle sections of tender stems are disinfected with 75% alcohol for 30-40 seconds, 0.3% sodium hypochlorite for 3 minutes, and 0.1% mercuric chloride solution for 4-5 minutes; the lower section of tender stems is disinfected with 75% alcohol for 60 seconds, 0.3% sodium hypochlorite for 3-4 minutes, and 0.1% mercuric chloride solution for 5 minutes. In autumn and winter, the upper tender stems are disinfected with 75% alcohol for 50-60 seconds, 0.3% sodium hypochlorite for 3-3.5 minutes, and 0.1% mercuric chloride solution for 4-4.5 minutes; the lower tender stems are disinfected with 75% alcohol for 1-2 minutes, 0.3% sodium hypochlorite for 4-5 minutes, and 0.1% mercuric chloride solution for 4-5 minutes; and the lower tender stems are disinfected with 75% alcohol for 1-2 minutes, 0.3% sodium hypochlorite for 4-5 minutes, and 0.1% mercuric chloride solution for 5-6 minutes.

6. The method for tissue culture propagation of Asparagus cochinchinensis in Neijiang according to claim 3, characterized in that: Step b) The propagation seedlings are those with a stem height of more than 3cm, fully expanded pseudo-leafs, and a basal mass diameter of more than 1cm; the soaking time is 30-60s.

7. The method for tissue culture propagation of Asparagus cochinchinensis in Neijiang according to claim 3, characterized in that: Step c) refers to rooted seedlings with 3 or more fleshy roots, each root longer than 0.5 cm. Hardening off involves closed cultivation for 7-10 days under conditions of 2000-3000 lx light and 23-28°C, followed by cultivation at a relative humidity of over 60% and a temperature of 25°C. + Open culture at 5℃ for 1-2 days.

8. The method for tissue culture propagation of Asparagus cochinchinensis in Neijiang according to claim 3, characterized in that: Step c) The transplanting involves washing the culture medium off the tissue culture seedlings with a 1000-fold dilution of 70% carbendazim, then transferring them to a substrate composed of peat moss, vermiculite, and perlite, and cultivating them in a high arched greenhouse covered with a transparent film. The mass ratio of peat moss:vermiculite:perlite is 1~2:1:1.

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