A tissue culture method for Fritillaria cirrhosa D. Don

Abstract

The present invention relates to a tissue culture method for Fritillaria cirrhosa, belonging to the technical field of Fritillaria cirrhosa tissue culture. The tissue culture method for Fritillaria cirrhosa includes: selecting fresh and healthy Fritillaria cirrhosa bulbs as explants, and then successively performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture; the pre-inoculation pretreatment includes the following processes: performing low-temperature plasma treatment on the Fritillaria cirrhosa bulbs, and then cutting and soaking them in an aqueous solution containing polyvinylpyrrolidone and carboxymethyl chitosan. On the basis of the existing tissue culture method for Fritillaria cirrhosa, the present invention proposes a new tissue culture method for Fritillaria cirrhosa based on the synergistic effect of low-temperature plasma pretreatment and chemical induction with cis-3-hexen-1-ol, which can effectively inhibit the browning phenomenon during the culture process, improve the culture efficiency, and has the characteristics of low browning rate and high induction rate, providing a new means for the rapid large-scale propagation of Fritillaria cirrhosa.

Description

Technical Field

[0001] The invention relates to the technical field of Fritillaria cirrhosa tissue culture, in particular to a Fritillaria cirrhosa tissue culture method. Background Art

[0002] As a plant with great medicinal value in the field of traditional Chinese medicine, Fritillaria cirrhosa has a long history of application. Its dried bulbs contain rich bioactive ingredients, such as alkaloids, saponins, polysaccharides, etc. These active ingredients give Fritillaria cirrhosa significant pharmacological effects such as clearing heat and moistening the lungs, resolving phlegm and relieving cough, dispersing nodules and reducing swelling. In clinical practice, Fritillaria cirrhosa is widely used to treat lung heat and dry cough, dry cough with little sputum, yin deficiency and fatigue cough, blood in sputum, scrofula, mastitis, lung abscess and other diseases. It is a key raw material for many Chinese medicine prescriptions and finished medicines (such as Fritillaria cirrhosa loquat paste, snake gall and Fritillaria cirrhosa liquid, etc.), and occupies an important position in the Chinese medicine system.

[0003] However, the natural reproduction ability of Fritillaria cirrhosa is relatively weak, and it faces many reproduction problems. Its seed germination rate is low, the growth cycle is long, and it has strict requirements on the growth environment. These factors have led to the decreasing resources of wild Fritillaria cirrhosa, which is difficult to meet the growing market demand, making the sustainable supply of Fritillaria cirrhosa face severe challenges. Therefore, exploring an efficient and reliable method to achieve the rapid reproduction of Fritillaria cirrhosa and the preservation of excellent varieties has become a key issue that needs to be solved urgently.

[0004] As an effective means of rapid propagation and preservation of excellent varieties, tissue culture technology plays a vital role in the large-scale production of Fritillaria cirrhosa. At present, the tissue culture method of Fritillaria cirrhosa mainly covers the following key steps:

[0005] 1. Explant selection and disinfection: Carefully select healthy, disease-free and insect-free Fritillaria cirrhosa bulbs as explants, and use chemical disinfectants (such as mercuric chloride, sodium hypochlorite, etc.) to disinfect their surfaces to minimize microbial contamination and create good sterile conditions for subsequent culture processes.

[0006] 2. Pre-culture: Place the disinfected explants on a sterile culture medium for a short period of pre-culture. This step is intended to help the explants gradually adapt to the culture environment and prepare for subsequent induced culture.

[0007] 3. Induction culture: By precisely adjusting the hormone ratio in the culture medium (such as the ratio of auxin to cytokinin), the explants are induced to form callus tissue or directly differentiate into adventitious buds. This is a critical link in the tissue culture process and can affect the subsequent reproduction effect.

[0008] 4. Subculture: After the induced callus or adventitious buds are divided, they are transferred to a new culture medium for continuous culture to increase the multiplication coefficient and achieve rapid proliferation of Fritillaria cirrhosa D. Don.

[0009] 5. Proliferation culture: By further optimizing culture conditions such as light, temperature, and nutrient components, the proliferation rate of callus or adventitious buds is further increased to improve the propagation efficiency.

[0010] 6. Rooting culture: The proliferated shoots without roots are transferred to a rooting medium to promote root formation, and finally complete plants are formed to complete the entire tissue culture process.

[0011] Although certain progress has been made in the tissue culture of Fritillaria cirrhosa D. Don in the prior art, such as CN110100730A, CN103621406A, CN112970589A, CN114847159A, etc., there are still many deficiencies. Among them, explant browning is a common problem that seriously affects the culture effect. The browning phenomenon not only hinders the normal growth and differentiation of explants, but also significantly reduces the induction rate and multiplication coefficient, and even leads to the failure of the entire culture process. The main reason for explant browning is that phenolic substances are oxidized into quinone substances under the catalysis of polyphenol oxidase, and these quinone substances further polymerize to form brown substances, resulting in explant browning. In addition, although traditional chemical disinfection methods can effectively kill microorganisms, they often cause a certain degree of damage to explants, thereby affecting their growth and differentiation ability and resulting in poor culture effects. Moreover, the existing technology has a long culture period and cannot meet the needs of large-scale production, restricting the industrial development of Fritillaria cirrhosa D. Don.

[0012] Therefore, aiming at the deficiencies of the existing technology, it is of great practical significance and application value to develop a new tissue culture method for Fritillaria cirrhosa D. Don. Summary of the Invention

[0013] To solve the above problems, the present invention provides a tissue culture method for Fritillaria cirrhosa D. Don.

[0014] The present invention provides a tissue culture method for Fritillaria cirrhosa D. Don, and the tissue culture method for Fritillaria cirrhosa D. Don includes: selecting fresh and healthy Fritillaria cirrhosa D. Don bulbs as explants, and then successively performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture, and rooting culture;

[0015] The pre-inoculation pretreatment includes the following process: subjecting the Fritillaria cirrhosa D. Don bulbs to low-temperature plasma treatment, then cutting them and soaking them in an aqueous solution containing polyvinylpyrrolidone and carboxymethyl chitosan;

[0016] The working condition parameters of the low-temperature plasma treatment include: using a mixed gas composed of argon and oxygen with a flow rate of 100-150 ml / min as the working medium, a discharge power of 20-30 W, and a treatment time of 30-50 seconds;

[0017] Both the culture medium used during the pre-culture and the induction culture contain cis-3-hexen-1-ol.

[0018] Furthermore, the volume ratio of the argon to the oxygen is (5-8):1, the discharge power is 26 W, and the treatment time is 40 seconds.

[0019] Furthermore, the concentration of the polyvinylpyrrolidone is 1.5-2.5 g / L.

[0020] Furthermore, the concentration of the carboxymethyl chitosan is 3-5 g / L.

[0021] Furthermore, the working condition parameters of the pre-culture include:

[0022] Inoculated into the following culture medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 5.5-6.5 g / L agar + 0.5-1.0 mg / L 6-BA + 0.8-1.5 mg / L NAA + 0.2-0.6 mg / L 2,4-D + 10-14 mg / L cis-3-hexen-1-ol;

[0023] The culture conditions are: temperature 21-23 °C, relative air humidity 70-80%, light culture for 7-10 days, light time 12 hours / day, and light intensity 700-900 Lux.

[0024] Furthermore, the working condition parameters of the induction culture include:

[0025] Inoculated into the following culture medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 0.8-1.5 mg / L NAA + 0.4-0.8 mg / L 2,4-D + 15-18 mg / L cis-3-hexen-1-ol;

[0026] The culture conditions are: temperature 21-23 °C, relative air humidity 70-80%, light time 12 hours / day, light intensity 1950-2050 Lux, and culture time 12-15 days.

[0027] Furthermore, the working condition parameters of the subculture include:

[0028] Inoculated on the following medium: MS with a pH of 5.5 - 6.0 + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 1.0 - 1.5 mg / L KT + 0.1 - 0.3 g / L activated carbon;

[0029] Cultured at a temperature of 21 - 23 °C, relative air humidity of 70 - 80%, light duration of 12 hours per day, light intensity of 1950 - 2050 Lux, and culture time of 14 - 17 days.

[0030] Furthermore, the working condition parameters for the proliferation culture include:

[0031] Inoculated on the following medium: MS with a pH of 5.5 - 6.0 + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 0.8 - 1.5 mg / L NAA + 0.5 - 1.0 mg / L 2,4 - D;

[0032] Cultured at a temperature of 21 - 23 °C, relative air humidity of 70 - 80%, light duration of 12 hours per day, light intensity of 1600 - 1800 Lux, and culture time of 15 - 18 days.

[0033] Furthermore, the working condition parameters for the rooting culture include:

[0034] Inoculated on the following medium: MS with a pH of 5.5 - 6.0 + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 1.5 - 2.0 mg / L KT + 1.5 - 2.0 mg / L NAA;

[0035] Cultured at a temperature of 21 - 23 °C, relative air humidity of 70 - 80%, light duration of 12 hours per day, light intensity of 2400 - 2600 Lux, and when the Fritillaria cirrhosa D. Don small bulbs grow to 5 - 6 cm in height and 5 - 10 roots emerge, acclimatization and transplantation are carried out.

[0036] The above - mentioned technical solution provided by the embodiments of the present invention has at least the following advantages compared with the prior art:

[0037] The embodiments of the present invention provide a tissue culture method for Fritillaria cirrhosa D. Don. Based on the existing tissue culture method for Fritillaria cirrhosa D. Don, the present invention proposes a new tissue culture method for Fritillaria cirrhosa D. Don based on the synergistic effect of low - temperature plasma pretreatment and cis - 3 - hexen - 1 - ol chemical induction, which can effectively inhibit the browning phenomenon during the culture process, improve the culture efficiency, and has the characteristics of low browning rate and high induction rate, providing a new means for the rapid large - scale propagation of Fritillaria cirrhosa D. Don. Specifically:

[0038] On the one hand, the present invention performs low-temperature plasma treatment on the Fritillaria cirrhosa bulb explants before inoculation and culture, controls key parameters such as discharge power and treatment time, thereby achieving efficient sterilization and activity stimulation of the Fritillaria cirrhosa bulb explants, while also avoiding damage to the Fritillaria cirrhosa bulb explants caused by excessively high discharge power or excessively long treatment time, as well as toxic damage to plant tissue cells caused by traditional chemical disinfection agents containing mercury, etc., which is beneficial to promoting the formation of subsequent callus tissue and reducing the overflow of phenolic substances, thereby inhibiting browning and improving the induction rate.

[0039] On the other hand, the present invention finds that cis-3-hexen-1-ol can significantly inhibit the activity of polyphenol oxidase, and further applies it to the tissue culture of Fritillaria cirrhosa, thereby inhibiting the browning of the tissue during the culture process by inhibiting the activity of polyphenol oxidase, thereby further reducing the browning rate and increasing the induction rate. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. For example, in the following examples and comparative examples, polyvinyl pyrrolidone (CAS No.: 9003-39-8), carboxymethyl chitosan (CAS No.: 83512-85-0), cis-3-hexene-1-ol (CAS No.: 928-96-1), MS culture medium, 6-BA (6-benzylaminopurine), NAA (naphthaleneacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indolebutyric acid), KT (kinetin), etc. can all be commercially available products; at the same time, the steps and parameters involved, if not specifically limited or specifically described, can be carried out according to the existing tissue culture method steps and condition parameters of Fritillaria cirrhosa or directly carried out using existing equipment. For example, in the low-temperature plasma treatment, the discharge mode can be dielectric barrier discharge, the discharge frequency can be 5-20kHz (such as 15Hz), the discharge voltage can be 15-30kV (such as 20kV), and other parameters can be set with reference to the conventional parameters of the existing low-temperature plasma treatment, and the present invention document will not repeat them one by one.

[0042] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0043] Example 1

[0044] This example measures the inhibitory effect of cis-3-hexene-1-ol on polyphenol oxidase, comprising the following steps:

[0045] 1. Reagent preparation

[0046] Different concentrations of cis-3-hexen-1-ol inhibitor sample solutions: diluted with phosphate buffer solution (0.10 mol / L, pH = 6.8) to concentrations of 5 mg / L, 10 mg / L, 15 mg / L, and 30 mg / L;

[0047] Polyphenol oxidase solution: Prepare 200 U / mL polyphenol oxidase solution with phosphate buffer solution (0.10 mol / L, pH = 6.8) and store on ice.

[0048] Levodopa solution: Prepare 5 mmol / L Levodopa solution with phosphate buffer (0.10 mol / L, pH = 6.8);

[0049] Stop solution: 2% HCl solution.

[0050] 2. Add sample

[0051] In a 96-well ELISA plate, 50 μL of different concentrations of cis-3-hexene-1-ol inhibitor sample solution and 50 μL of 200 U / mL polyphenol oxidase solution were added to each well as sample groups; at the same time, a positive control group was set up: phosphate buffer (50 μL) + polyphenol oxidase solution (50 μL); a negative control group was set up: phosphate buffer (100 μL); and an inhibitor solution blank group was set up: cis-3-hexene-1-ol inhibitor sample solution (50 μL) + phosphate buffer (50 μL). Three parallel wells were set up for each sample.

[0052] 3. Oscillation combination

[0053] The ELISA plate was placed on a shaker and shaken at 300 rpm for 5 min to allow cis-3-hexen-1-ol and polyphenol oxidase to fully combine.

[0054] 4. Incubate at 25°C

[0055] Place the enzyme-linked immunosorbent assay (ELISA) plate in an aluminum lunch box and incubate it in a 25°C water bath shaker (rotating speed 100 rpm) for 10 min.

[0056] 5. Add the L-DOPA solution

[0057] Add 50 μL of 5 mmol / L L-DOPA solution to each well.

[0058] 6. Incubate at 37°C

[0059] Continue to place the ELISA plate in a 37°C water bath shaker (rotating speed 100 rpm) and incubate for 10 min.

[0060] 7. OD value determination

[0061] Add the termination solution, take out the ELISA plate, dry the moisture at the bottom with filter paper, quickly place it in an ELISA reader, and measure the OD value at 475 nm using a phosphate buffer solution (0.10 mol / L, pH = 6.8) as the blank control.

[0062] 8. Inhibition rate calculation

[0063] Inhibition rate (%) = [1 - (OD of sample group - OD of inhibitor solution blank group) / (OD of positive control group - OD of negative control group)] × 100%, and calculate the average inhibition rate of each sample.

[0064] The test results show that when the working concentration of cis-3-hexen-1-ol is 5 - 30 mg / L, the inhibition rate (%) of polyphenol oxidase remains above 70.4%; and when the working concentration is 15 mg / L, the inhibition rate (%) of polyphenol oxidase is 92.7% ± 1.1%, and when the working concentration is 30 mg / L, the inhibition rate (%) of polyphenol oxidase > 99%. In addition, in this example, cis-3-hexenal with a concentration of 15 mg / L was used as the inhibitor sample solution for testing, and it was found that its inhibitory effect on polyphenol oxidase was not significant.

[0065] Example 2

[0066] On the basis of Example 1, in this example, cis-3-hexen-1-ol was further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Select fresh and healthy Fritillaria cirrhosa bulbs in March as explants, and after cleaning, perform pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture, and rooting culture in sequence;

[0067] Among them, the pre-inoculation pretreatment includes the following process: subject the Fritillaria cirrhosa bulb to low-temperature plasma treatment, using a mixed gas composed of argon and oxygen with a volume ratio of 7:1 and a flow rate of 120 ml / min as the working medium, with a discharge power of 26 W and a treatment time of 40 seconds; then cut it into blocks with a particle size of 0.5 - 0.8 cm and soak them in an aqueous solution containing 2.0 g / L polyvinylpyrrolidone and 4 g / L carboxymethyl chitosan for 15 min;

[0068] The working condition parameters for the pre-culture include: inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.2 g / L agar + 0.8 mg / L 6 - BA + 1.0 mg / L NAA + 0.4 mg / L 2,4 - D + 12 mg / L cis - 3 - hexen - 1 - ol; culturing at a temperature of 22°C, an air relative humidity of 75%, under light for 8 days, with a light time of 12 hours / day and a light intensity of 800 Lux;

[0069] The working condition parameters for the induction culture include: inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L NAA + 0.6 mg / L 2,4 - D + 16 mg / L cis - 3 - hexen - 1 - ol; culturing at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2000 Lux, and a culture time of 13 days;

[0070] The working condition parameters for the subculture include: inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L KT + 0.2 g / L activated carbon; culturing at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2000 Lux, and a culture time of 16 days;

[0071] The working condition parameters for the proliferation culture include: inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L NAA + 0.8 mg / L 2,4 - D; culturing at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 1700 Lux, and a culture time of 17 days;

[0072] The working condition parameters for rooting culture include: inoculation on the following medium: MS + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.8 mg / L KT + 1.6 mg / L NAA with a pH of 5.8; culturing at a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, and a light intensity of 2500 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and develop 5 - 10 roots, acclimatization and transplanting are carried out.

[0073] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is calculated to be 9.8%, and the induction rate is 71.3%.

[0074] Example 3

[0075] On the basis of Example 1, in this example, cis - 3 - hexen - 1 - ol is further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Selecting fresh and healthy Fritillaria cirrhosa bulbs in March as explants, after cleaning them thoroughly, performing pre - inoculation pretreatment, pre - culture, induction culture, sub - culture, proliferation culture, and rooting culture in sequence;

[0076] Among them, the pre - inoculation pretreatment includes the following process: Treating the Fritillaria cirrhosa bulbs with low - temperature plasma, using a mixed gas composed of argon and oxygen with a volume ratio of 5:1 and a flow rate of 100 ml / min as the working medium, a discharge power of 20 W, and a treatment time of 50 seconds; then cutting them into cubes with a particle size of 0.5 - 0.8 cm and soaking them in an aqueous solution containing 1.5 g / L polyvinylpyrrolidone and 3 g / L carboxymethyl chitosan for 15 min;

[0077] The working condition parameters for the pre - culture include: inoculation on the following medium: MS + 25 g / L sucrose + 5.5 g / L agar + 0.5 mg / L 6 - BA + 0.8 mg / L NAA + 0.2 mg / L 2,4 - D + 10 mg / L cis - 3 - hexen - 1 - ol; culturing at a temperature of 22 °C, an air relative humidity of 75%, light - culturing for 10 days, a light duration of 12 hours per day, and a light intensity of 700 Lux;

[0078] The working condition parameters for induction culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 0.8 mg / L NAA + 0.4 mg / L 2,4-D + 15 mg / L cis-3-hexen-1-ol; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1950 Lux, and a culture time of 15 days;

[0079] The working condition parameters for subculture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 1.0 mg / L KT + 0.1 g / L activated carbon; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1900 Lux, and a culture time of 17 days;

[0080] The working condition parameters for proliferation culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 0.8 mg / L NAA + 0.5 mg / L 2,4-D; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1600 Lux, and a culture time of 18 days;

[0081] The working condition parameters for rooting culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 1.5 mg / L KT + 1.5 mg / L NAA; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 2400 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and 5 - 10 roots grow out, hardening-off and transplanting are carried out.

[0082] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 17.1% and the induction rate is 59.7%.

[0083] Example 4

[0084] On the basis of Example 1, in this example, cis-3-hexen-1-ol is further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Select fresh and healthy Fritillaria cirrhosa bulbs in March as explants. After cleaning them thoroughly, perform pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture, and rooting culture in sequence;

[0085] Among them, the pre-inoculation pretreatment includes the following process: subject the Fritillaria cirrhosa bulb to low-temperature plasma treatment, using a mixed gas composed of argon and oxygen with a volume ratio of 8:1 and a flow rate of 150 ml / min as the working medium, with a discharge power of 30 W and a treatment time of 30 seconds; then cut it into blocks with a particle size of 0.5 - 0.8 cm and soak them in an aqueous solution containing 2.5 g / L polyvinylpyrrolidone and 4 g / L carboxymethyl chitosan for 15 min;

[0086] The working condition parameters for the pre-culture include: inoculate on the following medium: MS with a pH of 5.8 + 35 g / L sucrose + 6.5 g / L agar + 1.0 mg / L 6 - BA + 1.5 mg / L NAA + 0.6 mg / L 2,4 - D + 14 mg / L cis - 3 - hexen - 1 - ol; culture at a temperature of 22 °C, an air relative humidity of 75%, under light culture for 7 days, with a light time of 12 hours / day and a light intensity of 900 Lux;

[0087] The working condition parameters for the induction culture include: inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L NAA + 0.8 mg / L 2,4 - D + 18 mg / L cis - 3 - hexen - 1 - ol; culture at a temperature of 22 °C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2050 Lux, and a culture time of 12 days;

[0088] The working condition parameters for the subculture include: inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L KT + 0.3 g / L activated carbon; culture at a temperature of 22 °C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2050 Lux, and a culture time of 14 days;

[0089] The working condition parameters for the proliferation culture include: inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L NAA + 1.0 mg / L 2,4 - D; culture at a temperature of 22 °C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 1800 Lux, and a culture time of 15 days;

[0090] The working condition parameters for rooting culture include: inoculation on the following medium: MS + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 2.0 mg / L KT + 2.0 mg / L NAA with a pH of 5.8; the culture is carried out at a temperature of 22°C, a relative air humidity of 75%, a light duration of 12 hours per day, and a light intensity of 2600 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and have 5 - 10 roots, acclimatization and transplantation are carried out.

[0091] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, the browning rate of the tissue culture method of Fritillaria cirrhosa in this example is calculated to be 14.3%, and the induction rate is 63.6%.

[0092] Comparative Example 1

[0093] This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: 1) the low - temperature plasma treatment in the pre - inoculation pretreatment is adjusted to traditional disinfection treatment (soaking in 75% ethanol for 10 s, rinsing twice with sterile water, soaking in 0.1% mercuric chloride solution for 7 min, and rinsing twice with sterile water, 2 minutes each time); 2) cis - 3 - hexen - 1 - ol is not added to the medium used during pre - culture and induction culture; the remaining steps and parameters are the same.

[0094] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, the browning rate of the tissue culture method of Fritillaria cirrhosa in this example is calculated to be 69.1%, and the induction rate is 16.5%.

[0095] Comparative Example 2

[0096] This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: the low - temperature plasma treatment in the pre - inoculation pretreatment is adjusted to traditional disinfection treatment (soaking in 75% ethanol for 10 s, rinsing twice with sterile water, soaking in 0.1% mercuric chloride solution for 7 min, and rinsing twice with sterile water, 2 minutes each time); the remaining steps and parameters are the same.

[0097] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, the browning rate of the tissue culture method of Fritillaria cirrhosa in this example is calculated to be 57.8%, and the induction rate is 29.1%.

[0098] Comparative Example 3

[0099] This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: cis-3-hexen-1-ol is not added to the culture medium used during pre-culture and induction culture; the remaining steps and parameters are the same.

[0100] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 42.4%, and the induction rate is 38.7%.

[0101] Comparative Example 4

[0102] This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: during the pre-treatment before inoculation, the discharge power in the low-temperature plasma treatment is adjusted to 150 W, and the treatment time is adjusted to 2 minutes; the remaining steps and parameters are the same.

[0103] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 54.6%, and the induction rate is 33.8%.

[0104] In summary, the embodiments of the present invention provide a tissue culture method for Fritillaria cirrhosa. Based on the existing tissue culture method for Fritillaria cirrhosa, the present invention proposes a new tissue culture method for Fritillaria cirrhosa based on the synergistic effect of low-temperature plasma pre-treatment and chemical induction with cis-3-hexen-1-ol, which can effectively inhibit the browning phenomenon during the culture process, improve the culture efficiency, and has the characteristics of a low browning rate and a high induction rate, providing a new means for the rapid large-scale propagation of Fritillaria cirrhosa.

[0105] The various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and individual values within that range. For example, it should be considered that the description of the range from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and individual numbers within that range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0106] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather will be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A tissue culture method for Fritillaria cirrhosa D. Don, characterized in that, The tissue culture method of Fritillaria cirrhosa D. Don includes: selecting fresh and healthy Fritillaria cirrhosa D. Don bulbs as explants, and then successively performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture; The pre-inoculation pretreatment includes the following process: subjecting the Fritillaria cirrhosa D. Don bulbs to low-temperature plasma treatment, then cutting and soaking them in an aqueous solution containing polyvinylpyrrolidone and carboxymethyl chitosan; The working condition parameters of the low-temperature plasma treatment include: using a mixed gas composed of argon and oxygen with a flow rate of 100-150 ml / min as the working medium, a discharge power of 20-30 W, and a treatment time of 30-50 seconds; Both the medium used during pre-culture and the medium used during induction culture contain cis-3-hexen-1-ol; During pre-culture, inoculate on the following medium with a pH of 5.5-6.0: MS + 25-35 g / L sucrose + 5.5-6.5 g / L agar + 0.5-1.0 mg / L 6-BA + 0.8-1.5 mg / L NAA + 0.2-0.6 mg / L 2,4-D + 10-14 mg / L cis-3-hexen-1-ol; During induction culture, inoculate on the following medium with a pH of 5.5-6.0: MS + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 0.8-1.5 mg / L NAA + 0.4-0.8 mg / L 2,4-D + 15-18 mg / L cis-3-hexen-1-ol.

2. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, wherein The volume ratio of the argon to the oxygen is (5-8):1, the discharge power is 26 W, and the treatment time is 40 seconds.

3. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, characterized in that, The concentration of the polyvinylpyrrolidone is 1.5-2.5 g / L.

4. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, wherein The concentration of the carboxymethyl chitosan is 3-5 g / L.

5. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, wherein, The working condition parameters of the pre-culture include: The culture temperature is 21-23 °C, the relative air humidity is 70-80%, the light culture is for 7-10 days, the light time is 12 hours / day, and the light intensity is 700-900 Lux.

6. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, characterized in that, The working condition parameters of the induction culture include: The culture temperature is 21-23 °C, the relative air humidity is 70-80%, the light time is 12 hours / day, the light intensity is 1950-2050 Lux, and the culture time is 12-15 days.

7. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, characterized in that, The working condition parameters of the subculture include: Inoculate on the following medium with a pH of 5.5-6.0: MS + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 1.0-1.5 mg / L KT + 0.1-0.3 g / L activated carbon; The culture temperature is 21-23 °C, the relative air humidity is 70-80%, the light time is 12 hours / day, the light intensity is 1950-2050 Lux, and the culture time is 14-17 days.

8. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, characterized in that, The working condition parameters of the proliferation culture include: Inoculated on the following medium with a pH of 5.5 - 6.0: MS + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 0.8 - 1.5 mg / L NAA + 0.5 - 1.0 mg / L 2,4-D; The culture temperature is 21 - 23 °C, the relative air humidity is 70 - 80%, the light duration is 12 hours per day, the light intensity is 1600 - 1800 Lux, and the culture time is 15 - 18 days.

9. The tissue culture method of Fritillaria cirrhosa D. Don according to claim 1, characterized in that, The working condition parameters of the rooting culture include: Inoculated on the following medium with a pH of 5.5 - 6.0: MS + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 1.5 - 2.0 mg / L KT + 1.5 - 2.0 mg / L NAA; The culture temperature is 21 - 23 °C, the relative air humidity is 70 - 80%, the light duration is 12 hours per day, the light intensity is 2400 - 2600 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and 5 - 10 roots grow out, acclimatization and transplanting are carried out.