Tissue culture and rapid propagation technology of asparagus fern

Abstract

The invention discloses a culture medium combination for tissue culture and breeding of asparagus fern. The culture medium combination consists 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. By means of breeding of the culture medium combination, the pollution rate of the asparagus fern explant can be controlled within 10%, the germination rate of the explant is higher than 80%, the first rooting rate reaches 60% or above, the second rooting rate reaches 90% or above, the culture medium combination can be directly used for factory production of asparagus fern tissue culture, and a solid foundation is laid for mass propagation of asparagus fern.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture, and in particular to a tissue culture and rapid propagation technology of Neijiang asparagus. Background Art

[0002] Neijiang Asparagus is a perennial herb of the genus Asparagus in the Liliaceae family. It has superior quality, high content of polysaccharides, amino acids and extracts, slightly lower content of saponins, sweet taste at the beginning and no bitterness in the aftertaste. It is a local medicinal material in Neijiang with a long history of consumption. It registered a geographical indication certification trademark in 2022 and was shortlisted as one of the "Top Ten Geographical Indication Authentic Medicinal Materials in Sichuan". It is very different in morphology, composition and reproduction methods from the Asparagus in Guangxi, Yunnan and other production areas.

[0003] Neijiang Asparagus has low seed setting rate and germination rate. It is traditionally propagated by division. This method requires the mother plant to grow for 3-4 years. During the harvesting season, one plant can only reproduce 3-5 plants, which greatly limits the scale of planting of Neijiang Asparagus. The establishment of Neijiang Asparagus tissue culture rapid propagation technology can solve the seedling problem in production. However, the same medicinal material has different genetic materials and tissue structures in different production areas due to their different growth environments. Different genetic materials and tissue structures affect the growth and development laws of the plant body and also affect the interaction with the environment. It is not the case that the tissue culture and breeding methods of one plant are suitable for all production areas. Neijiang Asparagus adopts publicly reported methods such as patent CN106359087A for tissue culture propagation, but tissue culture seedlings cannot be formed, especially in the rooting process, and there is a problem that roots cannot be formed or the rooting rate is extremely low. Summary of the invention

[0004] The invention aims to solve the existing problems of long reproduction cycle of Neijiang asparagus, low efficiency of ramet reproduction, difficulty in rooting in tissue culture reproduction and low survival rate of transplantation, and provides a culture medium combination for tissue culture reproduction of Neijiang asparagus and a tissue culture reproduction method of Neijiang asparagus.

[0005] The present invention provides a culture medium combination for tissue culture and propagation of Neijiang asparagus, 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;

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

[0007] The lower explant culture medium is MS medium + 1.1-1.5 mg / L 6-benzylpurine

[0008] + 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 culture medium;

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

[0010] + 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 culture medium;

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

[0012] + 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 culture medium;

[0013] The axillary bud and cluster bud induction medium is a medium of 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;

[0014] The axillary bud and cluster bud proliferation culture medium is MS culture 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

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

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

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

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

[0019] Further, the lower explant culture medium is a culture medium of 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;

[0020] The middle explant culture medium is a culture medium of 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;

[0021] The upper explant culture medium is a culture medium of 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;

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

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

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

[0025] +0.1mg / L naphthaleneacetic acid +0.2mg / L indoleacetic acid +0.2mg / L kinetin +30g / L sucrose +5.5g / L agar medium;

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

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

[0028] The present invention also provides a method for tissue culture and breeding of Neijiang Asparagus cochinchinensis, which utilizes the above-mentioned culture medium combination culture, and the specific steps are as follows:

[0029] a. Take Neijiang Asparagus explants, clean and disinfect them, and culture them in sterile explant medium. When the explant buds grow to 2-5 cm, take them out and cut them into single spores, and culture them in sterile axillary bud and cluster bud induction medium. When the axillary buds or cluster adventitious buds grow to 2-5 cm, take them out and cut them into single spores, and culture them in sterile axillary bud and cluster bud proliferation medium to obtain proliferated seedlings;

[0030] b. Take the proliferated seedlings obtained in step a, remove the yellowed leaves, the tender growth points at the top and the browned parts at the base, soak them in rooting water and then inoculate them into a rooting medium to obtain rooted seedlings;

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

[0032] Furthermore, the Neijiang Asparagus cochinchinensis explant described in step a is cut into three sections, the top part is the upper section, followed by the middle section and the lower section, each section has 2-3 nodes; the upper section is accessed to the upper section explant culture medium, the middle section is accessed to the middle section explant culture medium, and the lower section is accessed to the lower section explant culture medium.

[0033] Furthermore, the Neijiang Asparagus cochinchinensis explant is preferably the tender stem of the asexual Neijiang Asparagus cochinchinensis.

[0034] Furthermore, the cleaning in step a) is to sequentially use a washing powder aqueous solution containing Tween, running water and distilled water to clean the Neijiang Asparagus cochinchinensis explants cut into three sections, or to sequentially use a washing powder aqueous solution, running water, 1% potassium permanganate solution and sterile water to clean the intact Neijiang Asparagus cochinchinensis explants.

[0035] Furthermore, the disinfection in step a) is carried out according to the season and the explant position, specifically:

[0036] In spring and summer, the upper and middle 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 tender stems are 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 upper 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; 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] Furthermore, it is characterized in that: the proliferated seedlings in step b) are proliferated seedlings with stem height of more than 3 cm, fully expanded pseudo-leaves, and base mass diameter greater than 1 cm; and the soaking is for 30-60 seconds.

[0039] Furthermore, the rooted seedlings in step c) are rooted seedlings with more than 3 fleshy roots and roots longer than 0.5 cm; the seedling hardening is carried out by sealing and culturing for 7 to 10 days under the conditions of 2000 lx to 3000 lx of light and 23°C to 28°C of temperature, and then in an airtight manner with a relative humidity of more than 60% and a temperature of 25 + Incubate at 5°C for 1-2 days.

[0040] Furthermore, the transplanting in step c) is to wash away the culture medium on the tissue culture seedlings with 1000 times 70% carbendazim solution, move them to a matrix composed of peat soil, vermiculite and perlite, and culture them in a high arch shed covered with a transparent film;

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

[0042] The asexual Neijiang Asparagus cochinchinensis described in the present invention is an excellent strain (line) propagated by asexual reproduction using high-quality single plant vegetative bodies of Neijiang Asparagus cochinchinensis as materials, and is called an asexual strain (line), or asexual Neijiang Asparagus cochinchinensis for short.

[0043] The MS medium of the present invention is one of the most commonly used mediums in plant tissues, and its formula consists of:

[0044]

[0045] The 1 / 4MS culture medium of the present invention is a culture medium in which the content of the macroelements in the MS culture medium formula is reduced by 3 / 4, while the contents of the remaining components remain unchanged.

[0046] The 1 / 4MS (1 / 4 iron salt) described in the present invention is a culture medium in which the content of both the macroelements and the iron salt in the MS culture medium formula is reduced by 3 / 4, while the content of the other components remains unchanged.

[0047] The culture medium combination for tissue culture and propagation of Neijiang asparagus of the present invention uses a specific plant growth hormone at different growth stages of Neijiang asparagus and accurately controls the dosage thereof, so that the bud formation rate of Neijiang asparagus explants is high, the bud formation quality is good, and the buds are thick. Experiments have proved that through the tissue culture and propagation method of Neijiang asparagus of the present invention, 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 reaches more than 60%, and the second rooting rate reaches more than 90%. It can be directly used for the factory production of Neijiang asparagus, laying a solid foundation for the large-scale propagation of Neijiang asparagus.

[0048] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0049] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Real-life record of the breeding process of Neijiang Asparagus cochinchinensis

[0051] Figure 2 Growth of different proliferated seedlings

[0052] Figure 3 Effects of Different Cytokinins on the Growth of Asparagus cochinchinensis DETAILED DESCRIPTION

[0053] The raw materials, reagents and equipment used in the specific embodiments of the present invention are all known products and can be purchased commercially.

[0054] Embodiment 1 Neijiang Asparagus cochinchinensis tissue culture breeding method

[0055] 1. Requirements for propagation materials

[0056] Select high-quality Neijiang Asparagus cochinchinensis asexual plants that are healthy, free of pests and diseases, and have high yields. Cut off the above-ground part of the plant 3-5 cm above the spore, dig out the entire plant that has been cut off from the underground part, and transplant it indoors or in a greenhouse with a cleaner environment for cultivation. Disinfect it with an aqueous solution of carbendazim or methyl thiophanate once a week for the first month, and prepare for sampling when the new shoots grow to 15-30 cm and the pseudo-leaves have not yet unfolded.

[0057] 2. Collection and processing of explants

[0058] 2.1 Collection time and location

[0059] Reduce watering one week before collecting explants and keep the air dry. On the morning of the inoculation day, cut strong, undamaged young stems 5 cm from the ground as explants and use them immediately. If long-distance transportation is required, place them in a low-temperature box and add clean gauze to store and keep them moist. Note that the explants cannot directly contact ice or other cooling materials.

[0060] 2.2 Material pretreatment

[0061] In a clean room, cut the explant into 2-3 sections according to the upper, middle and lower sections, with the top section being the upper section, followed by the middle section and the lower section, and place them separately. Then, use 2L of washing powder water and 2-4 drops of Tween to prepare an explant cleaning agent, carefully clean the dirt on the surface of the explant section, and then rinse with running water for 30-60 minutes. During the rinsing period, ensure that the explant rolls up and down in the running water. After the rinsing is completed, wash it with distilled water for 2-3 times for use. (Or first use washing powder water to carefully clean the dirt on the surface of the explant, rinse with running water for 30-60 minutes, soak in 1% potassium permanganate solution for 8-10 minutes, and rinse with sterile water for 2-3 times for use).

[0062] 3. Preparation before cultivation

[0063] 3.1 Clean bench disinfection

[0064] Turn on the clean bench under ultraviolet light for 20 to 30 minutes, then turn on the clean bench fan for 10 to 20 minutes, and then wipe the table surface with 75% medical alcohol.

[0065] 3.2 Culture medium preparation and sterilization

[0066] According to the formula of various culture media and the volume to be prepared, calculate the amount of various drug mother solutions and water required, weigh the required amount of coagulant and carbon source, heat and dissolve with 70% to 80% of the final volume of water, add macroelements, iron salts, trace elements, organic ingredients, plant growth regulator mother solutions, etc. in turn, stir while adding, adjust pH, constant volume, package, seal, and mark. After preparation, sterilize with high-pressure steam on the same day, sterilize at 0.105MPa and 121℃ for 20-25min.

[0067] 3.3 Culture medium storage

[0068] The sterilized culture medium should be kept at room temperature for 3-5 days and used up within 20 days. If kept at 2℃~4℃, it should be used up within one month.

[0069] 4. Tissue culture

[0070] 4.1 Culture conditions

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

[0072] 4.2 Explant disinfection

[0073] Place the pre-treated materials on a sterilized clean bench and sterilize them using the following methods depending on the season:

[0074] Methods for disinfecting materials in spring and summer:

[0075] Upper section: 75% alcohol for 30-40 seconds; 0.3% sodium hypochlorite for 3 minutes; 0.1% mercuric chloride for 4-5 minutes;

[0076] Middle section: 75% alcohol for 30 seconds; 0.3% sodium hypochlorite for 3 minutes; 0.1% mercuric chloride for 4-5 minutes;

[0077] Lower section: 75% alcohol for 1 minute; 0.3% sodium hypochlorite for 3-4 minutes; 0.1% mercuric chloride for 5 minutes;

[0078] Materials disinfection methods in autumn and winter:

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

[0080] Middle section: 75% alcohol for 1-2 minutes; 0.3% sodium hypochlorite for 4-5 minutes; 0.1% mercuric chloride for 4-5 minutes;

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

[0082] 4.3 Explant inoculation

[0083] On the clean bench, place the lower explant obliquely in the lower explant culture medium according to the explant growth direction. When placing it obliquely, the base of the spores should be close to the culture medium (it can be placed directly flat on the culture medium surface in large-scale production); the middle explant is placed obliquely or flatly inoculated in the middle explant culture medium; the upper material is placed flat in the upper explant culture medium. Among them, the formula of the lower explant culture medium is: MS+6-BA1.2mg / L+NAA0.05mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L; the formula of the middle explant culture medium is: MS+6-BA1.0 mg / L+NAA0.05mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L; the formula of the upper explant culture medium is: MS+6-BA1.0 mg / L+NAA 0.2mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L.

[0084] 4.4 Axillary bud and cluster bud induction

[0085] The explant buds that have grown to 2-5 cm are removed, cut into single spores, and inoculated into axillary bud and cluster bud induction medium. The medium formula is: MS+6-BA1.2 mg / L+NAA0.2mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L.

[0086] 4.5 Axillary bud and cluster bud proliferation

[0087] The axillary buds or clustered adventitious buds that have grown to 2-5 cm are removed, cut into single spores, and inoculated into the proliferation medium. The culture medium formula is: MS+6-BA1.2 mg / L+NAA0.1mg / L+IAA0.2 mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L.

[0088] 4.6 Rooting culture

[0089] For the proliferation seedlings with a growth time of more than 2 months, a stem height of more than 3cm, fully expanded pseudo-leaves, and a base mass diameter greater than 1cm, remove the yellowed leaves, cut off the tender growth point at the top, remove the browned part at the base, and quickly dip them in sterile rooting water, where the rooting water formula is: NAA 8.0mg / L+IAA5.0mg / L or NAA2.0mg / L+IAA 10.0mg / L; or soak them in sterile rooting water for 30-60s, where the rooting water formula is: NAA2.0mg / L; transfer them to the rooting medium: 1 / 4MS (1 / 4 iron salt) + IBA 0.5mg / L + IAA0.5mg / L + KT 0.05mg / L + CCC 4.0mg / L+50g / L banana, 20g / L sucrose, 5.5g / L agar, rooting begins around 90-120d of culture, with a rooting rate of around 60%, and the unrooted materials are transferred to new rooting medium, all of them can take root after 90d.

[0090] 5. Tempering and domestication of seedlings

[0091] Select rooted seedlings with strong roots, more than one fleshy root, and roots longer than 0.5 cm, and move them to a greenhouse with a light intensity of 2000lx-3000lx and a temperature of 23℃-28℃ for 7-10 days of closed bottle hardening. Loosen the bottle cap or sealing film, and then open the bottle for 1-2 days of hardening. During this period, keep the relative humidity of the air above 60% and the temperature at 25 + 5℃, avoid direct sunlight.

[0092] 6. Transplanting

[0093] After the seedlings are opened and hardened, wash off the culture medium with 1000 times 70% carbendazim solution (i.e., a solution containing 70% carbendazim diluted 1000 times with water), and transplant to a matrix of peat soil: vermiculite: perlite = 1:1:1 or 2:1:1, and cultivate in a high arch shed covered with a transparent film. Spray water 2-3 times a week within 1 month after transplanting to ensure that the air humidity is above 80%. One month after transplanting, the asparagus tissue culture seedlings begin to grow young new branches, and the films are gradually removed, that is, first peel off the films at both ends, and after 3-4 days, open the bottom of the film for ventilation for 3-4 days, and then peel off all the films. The acclimation survival rate is above 95%. After 80% of the tissue culture seedlings grow new branches, spray foliar fertilizer once a week, with concentrations from low to high, sterilize once a month, and transplant to the field about half a year later. See the real-life record of the breeding process for details. Figure 1 .

[0094] The beneficial effects of the present invention are described below by means of test examples:

[0095] In the early stage of the experiment, the tissue culture of Neijiang Asparagus cochinchinensis was carried out according to the culture medium provided in the literature as shown in Table 1, but no rooting occurred. Therefore, the tissue culture of Neijiang Asparagus cochinchinensis was studied in depth.

[0096] Table 1 Experimental results of applying the rooting formula of Asparagus genus to Neijiang Asparagus

[0097]

[0098] Note: [1] Yu Weihua, Shi Qian, Zhang Xiangjun, et al. "Establishment of the Regeneration System of Asparagus cochinchinensis No. 1"[J]. Northern Horticulture, 2020(20):7.

[0099] 【2】Quan Miaohua, Ou Lijun, He Anna, et al. Study on tissue culture rapid propagation system of Asparagus cochinchinensis[J]. Chinese Herbal Medicine, 2012, 043(008): 1599-1603.

[0100] 【3】Li Lin. Research on the rapid induction of asparagus shoots and rooting technology of test tube seedlings[D]. Guizhou University.

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

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

[0103] 【6】Wu Shangjiao. Construction of tissue culture and in vitro rapid propagation system of Asparagus cochinchinensis[D]. Guizhou University.

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

[0105] 【8】Zhou Jinsong et al. "A tissue culture rapid propagation method for Asparagus cochinchinensis.", CN201710141841.8.2019.

[0106] 【9】He Quanhui et al. "Tissue culture propagation method of Asparagus cochinchinensis.", CN102487830B.2016.

[0107]

[10] Zha Ping, Li Haibin. A method for improving the induction rate of Asparagus cochinchinensis fleshy roots: 202211715956.2022.

[0108]

[11] Yu Weihua, Shi Qian, Fan Yongsheng, et al. A method for growing roots and tubers of Asparagus cochinchinensis in a bottle: 202310396246[P][2024-11-04].

[0109]

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

[0110] Test Example 1 Study on the tissue culture and breeding of Neijiang Asparagus cochinchinensis of the present invention①

[0111] 1. Explant site screening

[0112] The spores, the upper end of the tender stem, the middle part of the tender stem, the lower part of the tender stem, the tuber, and the young and tender pseudo-leaves of the Neijiang Asparagus asexual line single plant cultured in the greenhouse were selected as tissue culture explant materials. Use 75% alcohol to disinfect for 60s or 2min (the spores, the upper end of the tender stem, the middle part of the tender stem, the lower part of the tender stem, and the young and tender pseudo-leaves are disinfected for 60s, and the tuber is disinfected for 2min), washed 3 times with sterile water, disinfected with 0.1% mercuric chloride for 10min, and washed 3 times with sterile water for standby use. The explant inoculation medium is: MS+6-BA1.0 mg / L+NAA0.1mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L. One explant is inoculated in each bottle, of which spores are inoculated in 20 bottles, and other explant materials are inoculated in 40 bottles. After one month of cultivation under the same conditions (temperature of 25±1℃, light hours of 12h / d~14h / d, light intensity of 1500lx~3000lx), the number of contaminants and the number of differentiated new buds were counted, and the seedling rate was evaluated based on the differentiation of new buds.

[0113] The difficulty of obtaining different explants in the field and the contamination rate vary greatly. The statistical results of contamination and seedling formation during the explant screening process are shown in Table 2

[0114] Table 2 Explant site screening test

[0115]

[0116]

[0117] As can be seen from Table 1, spores are the most difficult to obtain when collected in the field, and the contamination rate is extremely high, so they are not suitable as explants; although tubers and pseudo-leaves have lower contamination rates, they are difficult to grow into seedlings and are not suitable as explant materials. Different parts of the tender stems can differentiate into seedlings and have lower contamination rates, making them ideal materials for Asparagus cochinchinensis explants.

[0118] 2. Screening of explant cleaning and disinfection parameters

[0119] In the early experiments, only 75% alcohol and 0.1% mercuric chloride were used to disinfect the explants, and the contamination rate was extremely high during tissue culture. Therefore, the tender stems were used as explants, and the surface dirt was first cleaned with detergent water, and then rinsed with running water for 30 minutes, and then disinfected with 75% alcohol, 0.3% sodium hypochlorite and 0.1% mercuric chloride. The disinfected explants were inoculated in the culture medium (MS+6-BA1.0 mg / L+NAA0.1mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L), and then cultured under the same conditions (temperature 25±1℃, light hours 12h / d~14h / d, light intensity 1500lx~3000lx), and the browning rate of the explants was observed and counted.

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

[0121] Table 3 Disinfection treatment method ①

[0122]

[0123]

[0124]

[0125] From Table 4, we can see that alcohol disinfection for 2 minutes and mercuric chloride disinfection for 10-20 minutes have a relatively low explant contamination rate, but the spores are weak, grow slowly, grow poorly, and the browning rate is at a high level. By segmenting the tender stems and shortening the alcohol disinfection time, adding the disinfection treatments in Table 5, we can screen the disinfection method with better spore growth.

[0126] Table 5 Disinfection treatment test ②

[0127]

[0128] As can be seen from Table 5, the best disinfection effect was achieved for the upper and middle tender stems when using 75% alcohol for 30 seconds, 0.3% sodium hypochlorite for 3 minutes, and 0.1% mercuric chloride solution for 4-5 minutes, and the new shoots grew strongly; the best disinfection effect was achieved for the lower tender stems when using 75% alcohol for 60 seconds, 0.3% sodium hypochlorite for 3-4 minutes, and 0.1% mercuric chloride solution for 5 minutes, and the new shoots grew extremely strongly.

[0129] Asparagus is in a period of vigorous growth in spring and summer, the cell metabolism of the explant is active, and the tissue is relatively delicate. If the disinfection time is too long, the disinfectant may cause greater damage to the cells; in autumn and winter, asparagus grows slowly, the physiological activity of the explant weakens, and its stress resistance is relatively strong. At this time, the disinfection time can be appropriately extended, and the explant can withstand it without causing too much damage to it. It can also kill microorganisms more effectively, reduce the contamination rate, and increase the success rate of cultivation. Therefore, based on actual experience, the specific disinfection method for Neijiang Asparagus explants is determined as follows:

[0130] Methods for disinfecting materials in spring and summer:

[0131] Upper section: 75% alcohol for 30-40 seconds; 0.3% sodium hypochlorite for 3 minutes; 0.1% mercuric chloride for 4-5 minutes;

[0132] Middle section: 75% alcohol for 30 seconds; 0.3% sodium hypochlorite for 3 minutes; 0.1% mercuric chloride for 4-5 minutes;

[0133] Lower section: 75% alcohol for 1 minute; 0.3% sodium hypochlorite for 3-4 minutes; 0.1% mercuric chloride for 5 minutes;

[0134] Disinfection methods for materials in autumn and winter:

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

[0136] Middle section: 75% alcohol for 1-2 minutes; 0.3% sodium hypochlorite for 4-5 minutes; 0.1% mercuric chloride for 4-5 minutes;

[0137] Lower section: 75% alcohol for 1-2 minutes; 0.3% sodium hypochlorite for 4-5 minutes; 0.1% mercuric chloride for 5-6 minutes.

[0138] 3. Explant culture medium screening

[0139] Preliminary experiments found that different explant parts had different growth rates on culture media with different hormone ratios, so explant culture media were screened for different explant parts. Based on the preliminary culture media screening, it was found that different hormones such as 6-BA, NAA, and KT had different effects on callus tissues in different explant parts, so the main focus was on the effects of hormone combinations such as 6-BA, NAA, and KT on axillary bud formation in different explant parts. The specific hormone ratios in the explant culture medium and the corresponding growth conditions during tissue culture are shown in Table 6.

[0140] Table 6 Explant culture medium screening test

[0141]

[0142]

[0143] As shown in Table 6, new buds can be formed in the upper, middle and lower sections of the stem, among which the lower section has the highest budding rate and the upper section has the highest callus rate. Different parts require different hormone ratios for budding. The best budding hormone ratio for the upper section is: MS+6-BA1.0 mg / L+NAA 0.2mg / L+KT 0.2mg / L, with a budding rate of 87.5%; the best budding hormone ratio for the middle section is: MS+6-BA1.0 mg / L+NAA0.05mg / L+KT 0.2mg / L, with a budding rate of 95%; the best budding hormone ratio for the lower section is: MS+6-BA1.2 mg / L+NAA 0.05mg / L+KT 0.2mg / L, with a budding rate of 100%. Although the budding rate of the upper section is slightly lower, because the upper section stem nodes have denser spores, more spores can be formed in the same node, which is also suitable for explants.

[0144] Sucrose and agar in tissue culture medium respectively bear multiple important functions such as providing energy, maintaining osmotic pressure, preventing microbial contamination and solidifying medium. Based on the above hormone dosage, the dosage of sucrose and agar in the formula is adjusted to reduce production costs. The final formula of explant culture medium is determined as follows: the most suitable medium formula for stem upper explant culture is: MS+6-BA1.0 mg / L+NAA 0.2mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L. The most suitable medium formula for middle explant is: MS+6-BA1.0 mg / L+NAA0.05mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L. The most suitable culture medium formula for lower segment explants is: MS+6-BA1.2 mg / L+NAA 0.05mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L.

[0145] 4. Screening of proliferation medium

[0146] The axillary buds or cluster buds formed by the explants need to be transferred again before they are suitable for proliferation culture, and they grow better when transferred to the original explant culture medium or the axillary bud induction medium. In the factory production of Asparagus, in order to facilitate production, simplify operation and easy storage, the materials after explant culture are uniformly transferred to the axillary bud and cluster bud induction medium (MS+6-BA1.2mg / L+NAA0.2mg / L+KT 0.2mg / L, sucrose 30g / L, agar 5.5g / L), and then proliferation culture is carried out after induction.

[0147] On the basis of the axillary bud and cluster bud induction medium, the most suitable axillary bud and cluster bud proliferation medium formula for rapid propagation of Asparagus cochinchinensis was investigated by using different basal medium, different auxin types and concentrations, different cytokinin types and concentrations and their matching. The specific method is: the 2-5 cm long buds obtained by culture in the most suitable medium under the above "3. Screening of explant culture medium" are cut into small pieces with spores, and then inoculated into 2-5 cm axillary buds or cluster adventitious buds obtained by culture in the axillary bud and cluster bud induction medium, cut into single spores, and finally inoculated into different proliferation mediums for culture, and the growth trend is observed and counted ( Figures 2-3 ).

[0148]

[0149]

[0150] The growth status was greatly improved; (4) The use of trace amounts of TDZ (0.1-0.2 mg / L) would form more short and thick buds, which would restrict the later growth and development. Therefore, after comprehensive analysis, the most suitable culture medium formula for the proliferation of Neijiang Asparagus cochinchinensis is: MS+6-BA1.2 mg / L+NAA0.1 mg / L+IAA0.2 mg / L+KT0.2 mg / L, sucrose 30 g / L, agar 5.5 g / L, and the average proliferation rate is 5.37.

[0151] 5. Rooting medium screening test

[0152] Preliminary experiments found that the rooting rate of Neijiang Asparagus is low, and the basal culture medium has a certain effect on the rooting of Neijiang Asparagus, especially the combination of basal culture medium such as 1 / 4MS, 1 / 4MS (1 / 4 iron salt), 1 / 2MS and hormones such as NAA, IAA, IBA, etc. has a greater effect on the rooting of Neijiang Asparagus. The experiment was designed to investigate the effects of the types and concentrations of basal culture medium, 6-BA, NAA, IAA, IBA, KT, 2,4-D, GA and organic matter and their combination on the rooting of Neijiang Asparagus. Specifically, the Neijiang Asparagus proliferating seedlings with a high degree of lignification and dense clumps at the base obtained from the most suitable proliferating culture medium formula in "4. Proliferation culture medium screening" were used as materials for rooting culture medium screening.

[0153] Table 8 Experiment on the effect of main hormone treatments on rooting of Neijiang Asparagus cochinchinensis

[0154]

[0155]

[0156] From the observation results, it can be seen that due to the large number of treatment hormones and the high overall hormone concentration, most of the treatments grew very poorly, and most of them did not take root. Even if they took root, the rooting rate was low. The addition of 6-BA had a greater effect on the rooting of Neijiang Asparagus, and Neijiang Asparagus with different concentrations of 6-BA did not take root. The combination of NAA, IBA, and IAA had a certain effect on the rooting of Neijiang Asparagus, and the addition of KT had a certain promoting effect on rooting. The addition of GA and 2,4-D had little effect on rooting. The addition of organic matter such as bananas and coconut powder had a slight promoting effect on the rooting of Neijiang Asparagus, but the overall rooting rate of Neijiang Asparagus was low, and further experimental optimization is needed.

[0157] Although some Neijiang Asparagus rooted in the treatment with higher NAA content, the rooting rate was low. Therefore, we considered using NAA, IBA, and IAA as the main hormones and continued to optimize the rooting formula.

[0158] 1 / 4MS (1 / 4Fe) was used as the basic culture medium, the KT concentration was fixed at 0.2m / L, and different concentrations and types of hormones, sucrose 20g / L, and agar 5.5-6g / L were added. The experimental treatment methods and results are shown in Table 9.

[0159] Table 9 Effects of different hormones on rooting of Asparagus cochinchinensis

[0160]

[0161]

[0162] As shown in Table 9, with the increase of NAA concentration, the growth of the aboveground part of Neijiang Asparagus gradually worsened. When the NAA concentration was high, the aboveground part of Neijiang Asparagus grew poorly until it died. The concentration of auxin NAA should not exceed 1 mg / L during rooting. The combination of low concentration IBA and IAA can promote the rooting of Neijiang Asparagus, while high concentration will inhibit 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 low, with the highest rooting rate being only 12%. The best rooting medium formula in 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 Screening of the first step of rooting water in the two-step rooting method

[0166] In conventional tests, the rooting rate of Neijiang Asparagus is very low. After multiple tests, it was found that the two-step rooting method can improve the rooting rate of Neijiang Asparagus. The Neijiang Asparagus seedlings with a high degree of lignification, quasi-leaf expansion, and dense clumps at the base obtained from the most suitable proliferation medium formula in "4. Proliferation medium screening" were used as materials to screen rooting water and rooting medium, and cultured using the two-step rooting method. The first step of the two-step rooting method is to dip the base of the Neijiang Asparagus mass to be rooted in rooting water for a few seconds and then dry it slightly. The second step is to inoculate the Neijiang Asparagus dipped in rooting water into conventional rooting medium. The conventional rooting medium is 1 / 4MS (1 / 4Fe) + IAA 1.0mg / L + IBA 1.0mg / L + KT 0.2mg / L, sucrose 20g / L, agar 6g / L, and the formula of rooting water is shown in Table 10.

[0167] Table 10 Rooting water screening test and results 1

[0168]

[0169] After instantaneous treatment of the base mass of Neijiang Asparagus with different types and concentrations of auxins, the rooting rate of Neijiang Asparagus was different. High concentration of NAA (above 10mg / L) easily caused the aboveground part of Neijiang Asparagus to turn yellow, but it would stimulate the formation of root primordia; low concentration of IBA or IAA instantaneous stimulation had little effect on the rooting of Neijiang Asparagus, and medium and high concentrations of NAA combined with IAA instantaneous stimulation of the base mass of Neijiang Asparagus was conducive to the formation of root primordia. The best formula for instantaneous treatment rooting water was: NAA8.0mg / L+IAA5mg / L.

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

[0171] The Neijiang Asparagus cochinchinensis seedlings with high degree of lignification, quasi-leaf expansion, and dense clumps at the base obtained from the most suitable proliferation medium formula in "4. Proliferation medium screening" were used as materials. In the first step, NAA8.0mg / L+IAA5mg / L hormones were used to instantly stimulate the base clumps of Neijiang Asparagus cochinchinensis, and the treated clumps were inoculated into the second step culture medium. The second step culture medium was treated with 1 / 4MS (1 / 4Fe) + IAA1.0mg / L + IBA1.0mg / L, and different concentrations of kinetin were added as shown in Table 11. The effect of different KT concentrations on the rooting of Neijiang Asparagus cochinchinensis showed that when the KT concentration was 0.05mg / L, the rooting rate of Neijiang Asparagus cochinchinensis was the highest, at 32%.

[0172] Table 11 Effects of different concentrations of kinetin on the rooting of Asparagus neijiangensis

[0173]

[0174] 1 / 4MS (1 / 4Fe) + IAA 1.0mg / L + IBA 1.0mg / L + KT 0.05mg / L was used as the basal medium for the second step treatment, and different concentrations and types of hormones were added, as shown in Table 12. The results showed that the addition of CCC (chlormequat) promoted the rooting of Neijiang Asparagus cochinchinensis, and its root strips were thick, the rooting time was shortened, and it was conducive to seedling hardening and transplanting. The optimal CCC concentration was 4mg / L. The addition of ABA and activated carbon had no promoting effect on the rooting of Neijiang Asparagus cochinchinensis, and low concentrations of TIBA had a slight promoting effect on the rooting of Neijiang Asparagus cochinchinensis. Considering the cost of different hormones, it is recommended to add 4mg / L of CCC to the rooting medium in the second step.

[0175] Table 12 Effects of different hormone additions on rooting of Neijiang Asparagus cochinchinensis

[0176]

[0177] The formula of the fixed basic culture medium is 1 / 4MS (1 / 4Fe), the concentration of KT is 0.05mg / L or 2mg / L, the concentration of CCC is CCC4.0mg / L, sucrose is 20g / L, agar is 5.5g / L, different concentrations of IAA, IBA and organic banana are added, and the Neijiang asparagus seedlings with high lignification, quasi-leaf expansion, and dense clumps at the base obtained in the most suitable proliferation culture medium formula in "4. Proliferation culture medium screening" are used as materials. In the first step, NAA8.0mg / L+IAA5mg / L hormones are used to instantly stimulate the base clumps of Neijiang asparagus, and the treated clumps are inoculated into the second step culture medium (Table 13).

[0178] Table 13 Effects of banana addition and hormone combination on rooting of Neijiang Asparagus cochinchinensis

[0179]

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

[0181] 6.2 Method 2

[0182] The first step is to use the Neijiang Asparagus cochinchinensis seedlings with a high degree of lignification, quasi-leaf expansion, and dense clumps at the base obtained from the most suitable proliferation medium formula in "4. Proliferation medium screening" as materials, and use different hormones alone or in combination for different periods of time; the second step is to inoculate the Neijiang Asparagus cochinchinensis dipped in rooting water into a conventional rooting medium, which is 1 / 4MS (1 / 4Fe) + IAA 1.0mg / L + IBA 1.0mg / L + KT 0.2mg / L, sucrose 20g / L, agar 6g / L. The rooting water screening results are shown in Table 14.

[0183] Table 14 Rooting water test results

[0184]

[0185] In Table 14, USD05012, Y010060, N00522 and other treatments can greatly improve the rooting rate of Neijiang Asparagus cochinchinensis. Among them, the USD05012 combination treatment is: the base is soaked in a sterile aqueous solution of IBA with a concentration of 50 mg / L for 120 seconds and then inoculated on the rooting medium; the Y010060 combination treatment is: the base is soaked in a sterile aqueous solution of IBA 10.0 mg / L + IAA 10.0 mg / L for 60 seconds and then inoculated on the rooting medium; the N00522 combination treatment is treated with a sterile solution of NAA with a concentration of 5.0 mg / L for 120 minutes. Due to the high production cost of 50 mg / L IBA, this concentration is not recommended for production. Based on the test of formula Y010060 and N00522, after reducing the concentration of NAA for root dipping to 2.0 mg / L, the rooting rate remained unchanged, and the production cost of this reagent was the lowest, so it is recommended to use 2.0 mg / L NAA reagent for root dipping.

[0186] By screening rooting water and rooting hormones, combined with production cost and difficulty of operation, NAA2.0mg / L, NAA 8.0mg / L+IAA 5mg / L or NAA 2.0mg / L+IAA 10.0mg / L were selected as the rooting water for the first step. When the rooting water is NAA2.0mg / L, dip it quickly; when the rooting water is NAA 8.0mg / L+IAA 5mg / L or NAA 2.0mg / L+IAA10.0mg / L, soak it for 30-60s.

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

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

[0189] In summary, the present invention uses specific plant growth hormones at different growth stages of Neijiang Asparagus and accurately controls the dosage, so that the bud formation rate of Neijiang Asparagus explants is high, the bud formation quality is good, and the buds are thick. Experiments have proved that through the Neijiang Asparagus tissue culture breeding method of the present invention, 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 reaches more than 60%, and the second rooting rate reaches more than 90%. It can be directly used for the factory production of Neijiang Asparagus, laying a solid foundation for the large-scale expansion and propagation of Neijiang Asparagus.

Claims

1. A culture medium combination for tissue culture and propagation of Neijiang Asparagus cochinchinensis, characterized in that: The culture medium combination consists 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; The explant culture medium is any one or more of a lower explant culture medium, a middle explant culture medium, and an upper explant culture medium; The lower explant culture medium is a culture medium of MS culture 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 middle explant culture medium is a culture medium of MS culture 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 upper explant culture medium is a culture medium of MS culture 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 a medium of 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 culture medium is a culture medium of MS culture 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 water is a 1.5-2.5 mg / L naphthylacetic acid aqueous solution, or an aqueous solution containing 5.0-10.0 mg / L naphthylacetic acid and 3.0-7.0 mg / L indoleacetic acid, or an aqueous solution containing 1.0-5.0 mg / L naphthylacetic acid and 5.0-15.0 mg / L indoleacetic acid; The rooting medium is 1 / 4MS (1 / 4 iron salt) medium + 0.3-0.7mg / L indolebutyric acid + 0.3-0.7mg / L indoleacetic acid + 0.03-0.07mg / L kinetin + 3.0-5.0mg / L chlormequat + The culture medium is 30-70g / L banana + 10-30g / L sucrose + 5-6g / L agar.

2. The culture medium combination according to claim 1, characterized in that: The lower explant culture medium is a culture medium of 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 middle explant culture medium is a culture medium of 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 upper explant culture medium is a culture medium of 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 a medium of 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 culture medium is a culture medium of MS culture 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 water is a 2.0 mg / L naphthylacetic acid aqueous solution, or an aqueous solution containing 8.0 mg / L naphthylacetic acid and 5.0 mg / L indoleacetic acid, or an aqueous solution containing 2.0 mg / L naphthylacetic acid and 10.0 mg / L indoleacetic acid; The rooting medium is a medium of 1 / 4MS (1 / 4 iron salt) medium+0.5mg / L indolebutyric acid+0.5mg / L indoleacetic acid+0.05mg / L kinetin+4.0mg / L chlormequat+50g / L banana+20g / L sucrose+5.5g / L agar.

3. A method for tissue culture and breeding of Neijiang Asparagus cochinchinensis, characterized in that: It utilizes the culture medium combination culture of claim 1 or 2, and the specific steps are as follows: a. Take Neijiang Asparagus explants, clean and disinfect them, and culture them in sterile explant medium. When the explant buds grow to 2-5 cm, take them out and cut them into single spores, and culture them in sterile axillary bud and cluster bud induction medium. When the axillary buds or cluster adventitious buds grow to 2-5 cm, take them out and cut them into single spores, and culture them in sterile axillary bud and cluster bud proliferation medium to obtain proliferated seedlings; b. Take the proliferated seedlings obtained in step a, remove the yellowed leaves, the tender growth points at the top and the browned parts at the base, soak them in rooting water and then inoculate them into a rooting medium to obtain rooted seedlings; c. Take the rooted seedlings obtained in step c, harden them, and transplant them.

4. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: In step a, the Neijiang Asparagus cochinchinensis explant is cut into three sections: upper, middle and lower. The top part is the upper section, followed by the middle section and the lower section, each section has 2-3 nodes; the upper section is inoculated with an upper section explant culture medium, the middle section is inoculated with a middle section explant culture medium, and the lower section is inoculated with a lower section explant culture medium.

5. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3 or 4, characterized in that: The explants of Neijiang Asparagus cochinchinensis are preferably tender stems of the asexual Neijiang Asparagus cochinchinensis.

6. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: The cleaning in step a) is to sequentially use a washing powder aqueous solution containing Tween, running water and distilled water to clean the Neijiang Asparagus cochinchinensis explants cut into three sections, or sequentially use a washing powder aqueous solution, running water, 1% potassium permanganate solution and sterile water to clean the intact Neijiang Asparagus cochinchinensis explants.

7. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: The disinfection in step a) is carried out according to the season and the explant position, specifically: In spring and summer, the upper and middle 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 tender stems are 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 upper 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; 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.

8. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: Step b) the proliferated seedlings are proliferated seedlings with stem height of more than 3 cm, fully expanded pseudo-leaves, and base mass diameter greater than 1 cm; the soaking is for 30-60 seconds.

9. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: Step c) the rooted seedlings are rooted seedlings with more than 3 fleshy roots and roots longer than 0.5 cm; the seedling hardening is carried out by sealing and culturing for 7 days to 10 days under the conditions of 2000lx to 3000lx of light and 23°C to 28°C of temperature, and then in an airtight manner with a relative humidity of more than 60% and a temperature of 25 + Incubate at 5°C for 1-2 days.

10. The method for tissue culture and propagation of Neijiang Asparagus cochinchinensis according to claim 3, characterized in that: Step c) The transplanting is performed by washing the culture medium on the tissue culture seedlings with 1000 times 70% carbendazim solution, transferring the culture medium to a matrix composed of peat soil, vermiculite and perlite, and culturing the seedlings in a high arch shed covered with a transparent film; The mass ratio of the peat soil: vermiculite:perlite is 1-2:1:1.

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