A method and culture medium for rapid propagation of dark purple Fritillaria seeds

Abstract

The present invention provides a method and culture medium for rapid propagation of dark purple Fritillaria seeds, belonging to the field of tissue culture technology. The present invention uses dark purple Fritillaria seeds as explants and establishes a sterile system with antibacterial SH culture medium. From explant material extraction, disinfection pretreatment to primary culture, subculture, rooting culture, greenhouse seedling hardening and cultivation domestication process, in vitro regeneration plants are obtained, achieving large-scale propagation of tissue culture seedlings while maintaining the excellent characteristics of the variety. A tissue culture rapid propagation method for dark purple Fritillaria with significant effect is established. Compared with tissue culture using underground fresh bulbs as explants, the present invention reduces the overall contamination rate from 57.9% to 13.3%, a decrease of 44.6%, and greatly improves the overall benefit. The dark purple Fritillaria seeds of the present invention have a fast propagation speed of inducing callus tissue, with a cycle of 36 days and an induction rate of 85.3%. The proliferation multiple of subculture is more than 660 times, and more stable and excellent clustered buds can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue culture, and particularly relates to a method for rapid propagation of dark purple fritillaria seed seedlings and a culture medium. Background Art

[0002] Dark Purple Fritillaria (Fritillaria unibracteata Hsiao et K.C.Hsia) is a high-quality variety of the six medicinal plants of Sichuan Fritillaria. It is a perennial herb of the genus Fritillaria in the Liliaceae family. Its underground bulbs are used as medicine. Its main effects include moistening the lungs and relieving coughs, clearing away heat and resolving phlegm, relieving asthma and relieving coughs, dispersing nodules and eliminating carbuncles, and treating yin deficiency and fatigue coughs. It is widely used to treat respiratory diseases such as cough and asthma. It is a precious and rare Sichuan native medicinal material. It grows in wetlands or rock crevices such as forest shrubs, grasslands, riverbanks and valleys at an altitude of 2,800 to 4,000 meters. It prefers a cool climate and is cold-resistant, moisture-loving, and shade-loving.

[0003] The traditional propagation technology of dark purple Fritillaria adopts two methods: seed propagation and bulb propagation. However, due to its special habitat, the seed propagation dormancy period of dark purple Fritillaria in nature is long, the natural germination rate is low, and the emergence rate is poor. Under the traditional artificial cultivation model, the bulb propagation coefficient is also low. The propagation and planting technology system is not yet mature, and the process of specialization and scale is slow. For a long time, the main method has been to collect wild resources, resulting in serious damage to the reserves of dark purple Fritillaria and its seed population in natural habitats.

[0004] With the growing demand for dark purple Fritillaria in the domestic and international Chinese medicinal materials market in the past decade, especially in the past three years, the good results of traditional Chinese medicine in the diagnosis and treatment of lung nodules have been widely recognized. The price of commercial-grade raw medicinal materials of dark purple Fritillaria has continued to rise, with a price increase of 30%. The sales price of its first-grade medicinal products (Bao Zhong Bao Yue) is even higher. This has further exacerbated the current predatory exploitation of wild resources of dark purple Fritillaria, and the species is on the verge of extinction. Therefore, how to use modern bioengineering technology to effectively solve the problem of scarcity of dark purple Fritillaria resources and promote sustainable and high-quality development has become very urgent. Summary of the Invention

[0005] In view of this, one of the objects of the present invention is to provide a culture medium for tissue culture of Fritillaria thunbergii seeds.

[0006] A second object of the present invention is to provide a method for quickly propagating seedlings from dark purple Fritillaria seeds.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a culture medium for tissue culture of dark purple Fritillaria seeds, comprising a primary culture medium, a secondary culture medium and a rooting culture medium, wherein the primary culture medium is composed of: antibacterial SH medium + 1.5-2.5 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D;

[0009] The subculture medium includes a proliferation medium and an induction medium; the proliferation medium is composed of: antibacterial SH medium + 1.5-2.0 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D; the induction medium is composed of: antibacterial SH medium + 0.2-0.5 mg / L KT + 0.1-0.25 mg / LTDZ + 0.5-1.5 mg / L 2,4-D + 0.3 g / L AC;

[0010] The rooting medium is composed of: antibacterial 1 / 2SH medium + 0.5-1.0 mg / LNAA + 1.0-2.0 mg / LIBA + 0.3 g / LAC;

[0011] The antibacterial SH medium is SH medium+0.75mL / LPPM.

[0012] Preferably, the composition of the SH medium is: 2.5g / LKNO3+0.3g / LNH4H2PO4+0.4g / LMgSO4·7H2O+0.2g / L CaCl2+106.2mg / L MnSO4·4H2O+5.0mg / L H3BO3+1.0mg / LZnSO4·7H2O+0.21mg / LNa2MoO4·2H2O+0.2mg / L CuSO4·5H2O+1.0mg / LKI+0.1mg / L CoCl2+20mg / L Na2-EDTA+15mg / L FeSO4·7H2O+100mg / L inositol+2.0mg / L glycine+5.0mg / L nicotinic acid+0.5mg / LVB6+5.0mg / LVB1+30g / L sucrose.

[0013] The present invention also provides a method for quickly propagating seedlings from dark purple Fritillaria seeds, which uses the dark purple Fritillaria seeds as explants and adopts the above culture medium for culturing.

[0014] Preferably, the method comprises the following steps: inoculating dark purple Fritillaria seeds into the primary culture medium for primary culture to obtain callus tissue; inoculating the callus tissue into the proliferation culture medium for proliferation culture to obtain clustered buds; inoculating the clustered buds into the induction culture medium for induction culture to obtain clustered bulbs; inoculating the clustered bulbs into the rooting culture medium for rooting culture to obtain rooted bulbs.

[0015] Preferably, the conditions for the primary culture are: temperature 18-20° C., humidity 50%-65%, light intensity 3000 Lux-4500 Lux, and light 10-12 h / d.

[0016] Preferably, the conditions for the proliferation culture and induction culture are: temperature 18-20° C., humidity 55%-65%, light intensity 3000 Lux-4500 Lux, and light 10-12 h / d.

[0017] Preferably, the rooting culture conditions are: temperature 18-20° C., humidity 60%-70%, light intensity 4500 Lux-6000 Lux, and light 12-15 h / d.

[0018] Preferably, the disinfection method of dark purple Fritillaria seeds includes disinfection outside the clean bench and disinfection inside the clean bench; the disinfection outside the clean bench includes the following steps: soaking the dark purple Fritillaria seeds in 25ppm GA3 for 48h, and then soaking them in a 300-fold solution of 50% carbendazim wettable powder for 30min; the disinfection inside the clean bench includes the following steps: peeling out the seeds in the seeds, disinfecting them with 75% alcohol for 15s, rinsing them with sterile water, and then adding an HgCl2 mixed solution for disinfection for 6min; the mass volume fraction of HgCl2 in the HgCl2 mixed solution is 0.1%, and the HgCl2 mixed solution contains 0.6mL Tween 20.

[0019] Preferably, the method further comprises the step of hardening the seedlings in a culture room.

[0020] Preferably, the conditions for hardening the seedlings in the culture room are: temperature 18° C. to 20° C., humidity 60% to 70%, and light intensity 6000 Lux; the steps for hardening the seedlings in the culture room are: half-opening the bottle cap for the first 3 days, opening it during the day and covering it at night, and fully opening the bottle cap for the next 4 days. During the hardening process in the culture room, for bottle seedlings that wilt, spray 1 to 2 mL of 10-fold diluted SH nutrient solution on the plants.

[0021] Beneficial effects of the present invention:

[0022] The present invention provides a method for rapidly obtaining a large number of regenerated plants using dark purple Fritillaria seeds. The method has the advantages of requiring less material, low cost, short cycle, high reproduction rate, simple operation, safe production, and stable quality and genetics. Furthermore, the method is not adversely affected by changes in the external environment or catastrophic climate, and the experimental conditions are controllable, which is extremely beneficial to plant growth. It facilitates long-term cyclical cultivation and domestication, and can overcome the shortcomings of dark purple Fritillaria such as high reproduction cost, low proliferation multiples, and potential safety hazards in the production process. The present invention is an effective method for efficiently solving the current problems of expanding the scale of the Sichuan Fritillaria cultivation industry and the scarcity of raw medicinal material resources.

[0023] The present invention uses the seeds in the dark purple Fritillaria seed fruit (Baguachui) as explants. Compared with tissue culture using underground fresh bulbs as explants, the overall contamination rate is reduced from 57.9% to 13.3%, and the contamination rate decreases by 44.6%, greatly improving the overall benefit. The dark purple Fritillaria seed-induced callus of the present invention has a fast propagation speed, with a cycle of 36 days and an induction rate of 85.3%. The proliferation multiple of subculture is more than 660 times, and more stable high-quality clustered buds can be obtained. Compared with using underground fresh bulbs as explants, the present invention uses dark purple Fritillaria seeds for tissue culture and rapid propagation, and its raw material cost is reduced by about 46 times, and the overall yield rate is significantly increased. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is antibacterial SH medium;

[0025] Figure 2 Figure 1 is the explant treatment diagram;

[0026] Figure 3 Induce callus tissue for primary culture;

[0027] Figure 4 Induce callus clumps for primary culture;

[0028] Figure 5 The results of proliferation culture medium are shown, from left to right, respectively, as the results of proliferation culture medium induction of the third, fourth, and fifth generations of callus tissue;

[0029] Figure 6 This is the result of culture in induction medium;

[0030] Figure 7 To cultivate fruits for rooting;

[0031] Figure 8 This is the result of primary culture with 0.2 mg / L brassinolide in primary culture medium;

[0032] Figure 9 The results of callus induction on antibacterial MS medium;

[0033] Figure 10 The results of callus induction using antibacterial MS medium. DETAILED DESCRIPTION

[0034] The present invention provides a culture medium for tissue culture of dark purple Fritillaria seeds, comprising a primary culture medium, a secondary culture medium and a rooting culture medium, wherein the primary culture medium is composed of: antibacterial SH medium + 1.5-2.5 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D;

[0035] The subculture medium includes a proliferation medium and an induction medium; the proliferation medium is composed of: antibacterial SH medium + 1.5-2.0 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D; the induction medium is composed of: antibacterial SH medium + 0.2-0.5 mg / L KT + 0.1-0.25 mg / LTDZ + 0.5-1.5 mg / L 2,4-D + 0.3 g / LAC (activated carbon);

[0036] The rooting medium is composed of: antibacterial 1 / 2SH medium + 0.5-1.0 mg / LNAA + 1.0-2.0 mg / LIBA + 0.3 g / LAC;

[0037] The antibacterial SH medium is SH medium+0.75mL / LPPM.

[0038] The present invention does not specifically limit the specific sources of each raw material in each culture medium, and conventional commercial products in this field can be used. In the present invention, the role of the primary culture medium is to induce callus formation, establish a sterile system, and accelerate the progress of in vitro propagation. The final concentration of KT in the primary culture medium is preferably 1.8-2.2 mg / L, more preferably 1.9-2.0 mg / L; the final concentration of brassinolide is preferably 0.6-0.8 mg / L, more preferably 0.7 mg / L; the final concentration of 2,4-D is preferably 0.6-0.9 mg / L, more preferably 0.7-0.8 mg / L.

[0039] In the present invention, the function of the proliferation medium in the subculture medium is to carry out callus proliferation culture and increase the proliferation multiple, and the function of the induction medium is to induce the formation of clustered bulbs from clustered buds and enhance the propagation capacity. In the proliferation medium of the present invention, the final concentration of KT is preferably 1.6-1.9 mg / L, more preferably 1.7-1.8 mg / L; the final concentration of brassinolide is preferably 0.6-0.9 mg / L, more preferably 0.7-0.8 mg / L; and the final concentration of 2,4-D is preferably 0.6-0.9 mg / L, more preferably 0.7-0.8 mg / L. In the induction medium of the present invention, the final concentration of KT is preferably 0.3-0.4 mg / L, more preferably 0.35 mg / L; the final concentration of TDZ is preferably 0.15-0.20 mg / L, more preferably 0.17-0.18 mg / L; the final concentration of 2,4-D is preferably 0.7-1.2 mg / L, more preferably 0.8-1.0 mg / L.

[0040] In the present invention, the rooting medium is used to induce the formation of adventitious roots from the bulblets, thereby forming rooted bulbs and rapidly propagating them to the in vitro tissue flask stage. In the rooting medium of the present invention, the final concentration of NAA is preferably 0.6 to 0.9 mg / L, more preferably 0.7 to 0.8 mg / L; the final concentration of IBA is preferably 1.2 to 1.8 mg / L, more preferably 1.4 to 1.6 mg / L.

[0041] In the present invention, the composition of the SH medium is preferably: 2.5g / L KNO3+0.3g / LNH4H2PO4+0.4g / L MgSO4·7H2O+0.2g / L CaCl2+106.2mg / L MnSO4·4H2O+5.0mg / LH3BO3+1.0mg / L ZnSO4·7H2O+0.21mg / L Na2MoO4·2H2O+0.2mg / LCuSO4·5H2O+1.0mg / L KI+0.1mg / L CoCl2+20mg / LNa2-EDTA+15mg / LFeSO4·7H2O+100mg / L inositol+2.0mg / L glycine+5.0mg / L niacin+0.5mg / LVB6+5.0mg / L VB1+30g / L sucrose. The antibacterial 1 / 2SH culture medium is 1 / 2SH culture medium+0.75mL / LPPM.

[0042] The antimicrobial SH medium described herein is composed of six major components: agar, a macroelement solution (KNO3 + NH4H2PO4 + MgSO4·7H2O + CaCl2), a trace element mother solution (MnSO4·4H2O + H3BO3 + ZnSO4·7H2O + Na2MoO4·2H2O + CuSO4·5H2O + KI + CoCl2), a ferric salt mother solution (Na2-EDTA + FeSO4·7H2O), an organic component mother solution (inositol + glycine + niacin + VB6 + VB1), and the antimicrobial agent PPM. These six major components of the antimicrobial SH medium should be prepared immediately before use and dissolved individually in order. Mixing them together is strictly prohibited. Calcium chloride absorbs moisture easily and should be quickly weighed and dissolved before addition. If necessary, heating can be performed until all components are fully dissolved. Due to the addition of the plant tissue culture antimicrobial agent PPM, Tween 20 and pH adjusters must not be added. Compared with the MS culture medium mostly used in this field, the present invention provides an antibacterial SH culture medium that is more suitable for the tissue culture of the second-level endangered medicinal plant Fritillaria thunbergii. It has strong specificity. During its preparation process, NH4H2PO4 is used to replace the strictly controlled explosive substance NH4NO3 in the MS culture medium, making the experimental operation safer.

[0043] The present invention also provides a method for quickly propagating seedlings from dark purple Fritillaria seeds, which uses the dark purple Fritillaria seeds as explants and adopts the above culture medium for culturing.

[0044] In the present invention, the dark purple Fritillaria seeds are preferably seeds in fresh dark purple Fritillaria seeds. Preferably, the Fritillaria cirrhosa seeds need to be disinfected before inoculation. The disinfection method preferably includes disinfection outside the clean bench and disinfection inside the clean bench; the disinfection outside the clean bench preferably includes the following steps: soaking the dark purple Fritillaria seeds in 25ppm GA3 for 48h, and then soaking them in a 300-fold solution of 50% carbendazim wettable powder for 30min; the disinfection inside the clean bench preferably includes the following steps: peeling out the seeds in the seeds, disinfecting them with 75% alcohol for 15s, rinsing them with sterile water, and then adding an HgCl2 mixed solution for disinfection for 6min; the mass volume fraction of HgCl2 in the HgCl2 mixed solution is 0.1%, and the HgCl2 mixed solution contains 0.6mL Tween 20.

[0045] In the method for rapid propagation of seedlings from dark purple Fritillaria seeds of the present invention, the method preferably includes the following steps: inoculating the dark purple Fritillaria seeds into the primary culture medium for primary culture to obtain callus tissue; inoculating the callus tissue into the proliferation culture medium for proliferation culture to obtain clustered buds; inoculating the clustered buds into the induction culture medium for induction culture to obtain clustered bulbs; inoculating the clustered bulbs into the rooting culture medium for rooting culture to obtain rooted bulbs.

[0046] In the present invention, the conditions for the primary culture are preferably: temperature 18-20°C, humidity 50%-65%, light intensity 3000Lux-4500Lux, and light illumination for 10-12h / d; the temperature for the primary culture is preferably 19°C, humidity is preferably 55%-60%, light intensity is preferably 3500Lux-4000Lux, and light illumination for 11h / d. In the present invention, the conditions for the proliferation culture and induction culture are preferably: temperature 18-20°C, humidity 55%-65%, light intensity 3000Lux-4500Lux, and light illumination for 10-12h / d; the temperature for the proliferation culture and induction culture is preferably 19°C, humidity is preferably 58%-62%, more preferably 59%-60%, light intensity is preferably 3500Lux-4000Lux, and light illumination for 11h / d. In the present invention, the conditions for rooting culture are preferably: temperature 18-20°C, humidity 60%-70%, light intensity 4500Lux-6000Lux, and illumination time 12-15h / d; the temperature for rooting culture is preferably 19°C, the humidity is preferably 62%-68%, the light intensity is preferably 4800Lux-5400Lux, and the illumination time is preferably 13-14h / d.

[0047] The method for rapid propagation of dark purple Fritillaria seed seedlings of the present invention preferably also includes a step of hardening the seedlings in a culture room. The conditions for hardening the seedlings in the culture room are: temperature 18°C ​​to 20°C, humidity 60% to 70%, and light intensity 6000 Lux. Preferably, the hardening step in the culture room is as follows: for the first three days, the bottle cap is half-opened, opened during the day and covered at night, and the bottle cap is fully opened for the last four days. During the hardening process, any seedlings that wilt are sprayed with 1 to 2 mL of a 10-fold diluted SH nutrient solution.

[0048] In the present invention, the SH nutrient mother solution is preferably composed of a macroelement solution (KNO3+NH4H2PO4+MgSO4·7H2O+CaCl2), a trace element mother solution (MnSO4·4H2O+H3BO3+ZnSO4·7H2O+Na2MoO4·2H2O+CuSO4·5H2O+KI+CoCl2), an iron salt mother solution (Na2-EDTA+FeSO4·7H2O) and an organic component mother solution (inositol+glycine+niacin+VB6+VB1).

[0049] The present invention innovatively uses dark purple Fritillaria seeds as explants and establishes a sterile system with antibacterial SH culture medium. From explant collection, disinfection pretreatment to primary culture (callus induction), subculture (proliferation), rooting culture and culture room seedling hardening, in vitro regenerated plants are obtained, achieving large-scale reproduction of tissue culture seedlings while maintaining the excellent characteristics of the variety, and establishing a significantly effective dark purple Fritillaria tissue culture rapid propagation method.

[0050] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] In the following examples, unless otherwise specified, all methods are conventional.

[0052] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0053] In the following examples, every step of the inoculation process must strictly implement the principle of "sterile concept and aseptic operation". The operator's work clothes, gloves, masks, head covers, shoe covers and inoculation equipment must be carried out in accordance with the SOP to ensure that the exogenous contamination in the Class A area of ​​the inoculation and transfer clean room is controlled within 1%.

[0054] The sources and preparations of some of the raw materials in the following examples are as follows:

[0055] PPM (Piant Preservative Mixture) purity: ≥99.8%, specification: 25mL / bottle, batch number: PTC341815100, storage temperature: 2-8°C, manufacturer: Beijing Coolaibo Technology Co., Ltd.

[0056] Auxin 2,4-D (2,4-dichlorophenoxyacetic acid): Prepare a 0.1 mg / mL 2,4-D solution using 1.0 mol / L NaOH as solubilizer. Source: 2,4-D purity: ≥97%, Specification: 25 g / bottle, Batch number: G2114198, Storage temperature: 2-8°C. Manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd. (aladdin).

[0057] Auxin NAA (α-naphthylacetic acid): Prepare a 0.1 mg / mL NAA solution using 95% ethanol as a solubilizer. Source: NAA purity: ≥98.0%, Specification: 25 g / bottle, Batch Number: 201905310, Storage Temperature: 2-8°C, Manufacturer: Chengdu Kelong Chemicals Co., Ltd.

[0058] Cytokinin KT (Kinetin): Prepare a 0.1 mg / mL KT solution using 1.0 mol / L HCl as a solubilizer. Source: KT purity: ≥98.0%, Specification: 250 mg / bottle, Batch Number: 705N021, Storage Temperature: 2-8°C. Manufacturer: Beijing Solarbio Technology Co., Ltd.

[0059] Cytokinin BR (Brassinolide): Prepare a 1.0 mg / mL brassinolide solution using anhydrous ethanol as solubilizer. Source: BR purity: ≥90%, Specification: 50 mg / bottle, Batch Number: C15566732, Storage Temperature: 2-8°C. Manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd.

[0060] Cytokinin TDZ (thiabendazole, 2,4-dichlorobenzoic acid): Prepare a 0.1 mg / mL TDZ solution using 1.0 mol / L NaOH as solubilizer. Source: TDZ purity: ≥96%, Specification: 25 g / bottle, Batch number: C15793064, Storage temperature: 2-8°C. Manufacturer: Shanghai MacLean Biochemical Technology Co., Ltd.

[0061] Rooting Agent IBA (Indolebutyric Acid): Prepare a 0.1 mg / mL IBA solution using 1.0 mol / L NaOH as a solubilizer. Source: IBA purity: ≥98%, Specification: 5 g / bottle, Batch No.: 302O031, Storage Temperature: 2-8°C, Manufacturer: Beijing Solarbio Technology Co., Ltd.

[0062] Example 1

[0063] Preparation of antibacterial SH culture medium

[0064] (1) Take laboratory ultrapure water, bring it to a boil, and evenly add agar to a final concentration of 7.5 g / L. Stir rapidly to prevent it from clumping on the surface of the boiling water. Continue boiling over medium heat until the agar is completely melted (approximately 45 minutes).

[0065] (2) Use freshly boiled ultrapure water to replenish the evaporated water; boil again for 5 minutes until the agar is fully melted.

[0066] (3) Keep heating on low heat, then add the freshly prepared macroelement solution (2.5g / L KNO3+0.3g / L NH4H2PO4+0.4g / LMgSO4·7H2O+0.2g / L CaCl2) to the agar solution slowly, evenly, and in sequence, and bring to a boil; then add the trace element mother solution (106.2mg / LMnSO4·4H2O+5.0mg / L H3BO3+1.0mg / L ZnSO4·7H2O+0.21mg / LNa2MoO4·2H2O+0.2mg / L CuSO4·5H2O+1.0mg / L KI+0.1mg / L CoCl2), iron salt mother solution (20mg / LNa2-EDTA+15mg / L FeSO4·7H2O), organic component mother solution (100mg / L inositol + 2.0mg / L glycine + 5.0mg / L nicotinic acid + 0.5mg / LVB6 + 5.0mg / LVB1) in this order, and add them slowly, evenly and separately in sufficient amounts under stirring.

[0067] (4) Add 30 g / L sucrose and 0.75 mL / L PPM, dissolve thoroughly and mix well.

[0068] (5) Finally, the culture medium is dispensed into clean tissue culture bottles and enters the culture medium sterilization procedure. The culture medium sterilization condition is 121℃ for 20 minutes. After the sterilization time is reached, the air is manually released and the culture medium is safely and quickly removed from the autoclave and placed in a clean area (class 100) for natural cooling (the low-temperature air conditioning in the clean area can be turned on on hot days) to maintain the most suitable solidification prototype of the culture medium.

[0069] The antibacterial SH culture medium of the present invention is as Figure 1 shown.

[0070] Example 2

[0071] 1. Source of explants: The "Fritillaria cirrhosa Modern Agricultural Planting Demonstration Zone" of Sichuan Songzan Xuebei Biotechnology Co., Ltd. is located in Hanpan Village, Shuijing Township, Chuanzhusi Town, Songpan County, Aba Prefecture, at an altitude of 3289.28 meters.

[0072] Explant material: seeds from the seed fruit of a five-year-old dark purple Fritillaria plant

[0073] Explant disinfection treatment (see Figure 2 ):

[0074] (1) Freshly picked dark purple Fritillaria cirrhosa seeds were rinsed with laboratory ultrapure water for 4 h, filtered, and soaked in 25 ppm gibberellin GA3 for 48 h. After filtering again, the surface moisture was dried, and then soaked in a 300-fold diluted solution of 50% carbendazim wettable powder for 30 min. After filtering, the seeds were washed with sterile ultrapure water for 6 times, each time for 1 min.

[0075] (2) Place the above-mentioned preliminarily processed seeds and fruits in the transfer window, turn on the ultraviolet light and fan for 5 minutes, and then transfer them to the semi-sterile control room in the clean area in time; disinfect them with 75% alcohol for 60 seconds, filter them, and wash them with sterilized ultrapure water 3 times, each time for 1 minute. After drying the surface moisture of the seeds and fruits, place them in a sterilized container and immediately transfer them to the Class 100 operating table in the clean area.

[0076] (3) On a Class 100 operating table in a clean area, peel out the seeds from the seed fruit and transfer them to a completely sterilized tissue culture bottle in time. Disinfect with 75% alcohol for 15 seconds, filter, rinse with sterile water three times, 1 minute each time, then add a 0.1% (g / mL) HgCl2 mixture containing 0.6 mL of Tween 20, shake and disinfect for 6 minutes, then quickly filter, rinse with sterile water 6 times, 2 minutes each time, and finally take out the sterilized seeds from the tissue culture bottle and place them in a stainless steel sterile container covered with sterile filter paper. After the surface moisture of the seeds is dried, cover the stainless steel sterile container with the seeds and place it near the alcohol lamp in the front third of the operating table, ready to enter the next stage of the sterile inoculation procedure.

[0077] 2. Explant Inoculation

[0078] A medium-sized tissue culture bottle (10 cm in height and 7 cm in bottom diameter) was selected for inoculation. The antibacterial SH medium obtained in Example 1 was contained in the bottle, and 30 seeds were inoculated per bottle.

[0079] The inoculated tissue culture flask was quickly transferred to the culture room. The environmental conditions of the culture room were a temperature of 18°C ​​to 20°C, a humidity of 50% to 70%, a light intensity of 3000Lux to 6000Lux (LED light source), and a lighting time of 11 hours.

[0080] The control group used the above operation with the fresh underground bulbs of two-year-old dark purple Fritillaria as explants. The results showed that the contamination rate of dark purple Fritillaria seeds as explants averaged 13.3%, while the contamination rate of two-year-old plant underground fresh bulbs as explants averaged 57.9%. Therefore, the overall contamination rate of tissue culture rapid propagation using seeds as explants of the present invention was reduced by 44.6%. In addition, the material collection method of the present invention saves considerable manpower, financial and material costs, significantly improving the overall efficiency.

[0081] Example 3

[0082] The source, material collection and disinfection of explants are the same as those in Example 2. The dark purple Fritillaria seeds were inoculated into a medium-sized tissue culture bottle (the bottle height is 10 cm and the bottle bottom diameter is 7 cm) filled with primary culture medium. For the first 15 days, the temperature was regulated to 18°C, the humidity was 50% to 60%, the light intensity was 3000Lux to 4500Lux, and the light was 10h / d; for the subsequent 21 days, the temperature was regulated to 20°C, the humidity was 55% to 65%, the light intensity was 3000Lux to 4500Lux, and the daytime was 12h / d. After 36 days (i.e., one culture cycle), the callus tissue (i.e., the thin-walled cell mass) of the seeds was induced. Figure 3 .

[0083] The primary culture medium consists of antibacterial SH medium, 2.0 mg / L KT, 1.0 mg / L brassinolide and 0.5 mg / L 2,4-D.

[0084] Culture effect: According to the above culture medium growth hormone combination and ratio, the callus induced by dark purple Fritillaria seeds as explants is light yellow or light green, with a hard texture, clear texture, fine and shiny, no obvious fibrous material, and a pure and long-lasting smell. The overall induction rate is 85.3%. After another 30 days of culture, when the diameter of the callus mass formed by one dark purple Fritillaria seed reaches 4cm to 5cm (see Figure 4 ), ready to enter subculture proliferation culture.

[0085] Example 4

[0086] Subculture

[0087] (1) The callus mass obtained in Example 3 with a diameter of 4 to 5 cm was inoculated into a culture bottle containing a proliferation medium. The temperature was controlled at 20°C, the humidity was 55% to 65%, the light intensity was 3000 Lux to 4500 Lux, and the illumination was 10 hours per day. Subculture was performed at a cycle of 4 weeks per generation, and 5 generations were continuously transferred to form more callus tissue, completing the first stage of subculture. Figure 5At this stage, the callus induced by the primary culture was divided into four pieces and cultured in a cycle of four weeks until the fifth generation, when the number of proliferation reached more than 128 bottles. The proliferation medium consisted of antibacterial SH medium, 1.5 mg / L KT, 1.0 mg / L brassinolide, and 0.5 mg / L 2,4-D.

[0088] (2) The clustered buds partially induced during the fifth generation of subculture proliferation culture in step (1) were inoculated into a large tissue culture bottle (bottle height 14 cm, bottle bottom diameter 9.5 cm) filled with induction medium. During this process, the temperature was controlled at 20°C, the humidity was 55% to 65%, the light intensity was 3000 Lux to 4500 Lux, and the light intensity was 12 hours / day. The clustered buds were cultured into clustered bulbs with a culture cycle of 4 weeks, completing the second stage of subculture. Figure 6 The induction medium consists of antibacterial SH medium, 0.2 mg / L KT, 0.1 mg / L LTDZ, 1.0 mg / L 2,4-D, 30 g / L sucrose, and 0.3 g / L AC. The operation in this stage is based on the progress of the cluster buds formed in the first stage above, and is carried out in a rotational culture until all callus tissues have completed the proliferation culture from cluster buds to cluster bulbs.

[0089] After the above two stages of subculture, each bottle of callus tissue produced an average of 55 bottles of high-quality clustered buds, and each bottle had 12 high-quality clustered buds (average value). Based on this calculation, the proliferation multiple was 660 times, with significant effect.

[0090] Example 5

[0091] Rooting culture

[0092] The bulbs obtained in Example 4 were inoculated into a large tissue culture bottle (14 cm in height and 9.5 cm in bottom diameter) filled with rooting medium, with a culture cycle of 4 weeks. The temperature was controlled at 20°C, the humidity was 60% to 70%, the light intensity was 6000 Lux, and the light intensity was 15 h / d to form rooted bulbs. Figure 7 The rooting medium consists of 1 / 2SH medium + 0.75 mL / LPPM, 0.5 mg / L NAA, 1.0 mg / L IBA and 0.3 g / L AC.

[0093] Culture Results: Using the aforementioned growth hormone combination and ratio in the culture medium, adventitious roots were induced from the bulbs. After 60 days of culture, rooted bulb seedlings were formed, with rooting and seedling rates exceeding 96%. The seedlings had straight stems, tall shoots, green leaves, and robust root systems, with no root rot or lesions. Less than 3% of the old leaves dried out, demonstrating excellent overall rooting results and completing the rapid propagation of dark purple Fritillaria tissue culture seedlings in the seedling stage.

[0094] Comparative Example 1

[0095] The difference from Example 3 is that the concentrations of brassinolide in the primary culture medium are 0.2 mg / L and 1.5 mg / L, respectively. The rest are the same as Example 3.

[0096] The results showed that when the amount of brassinolide in the primary culture medium was less than 0.5 mg / L, the time for inducing callus was as long as 66 days (the time for inducing callus in Example 3 was 36 days), the induction rate was obviously too slow, and the callus clumps were relatively loose. Figure 8 When the dosage of brassinolide in the primary culture medium was higher than 1.0 mg / L, it took 48 days to induce callus tissue, and the induction rate was also slow. The callus tissue was average, but after 10 days of continuous culture, the callus tissue clumps began to brown. As time went on, the browning became more and more serious, which was not conducive to the collection of materials for subculture proliferation culture and the acquisition of proliferation multiples.

[0097] Comparative Example 2

[0098] The difference from Example 3 is that the antibacterial SH medium in the primary culture medium is replaced with an antibacterial MS medium (the specific composition of the antibacterial MS medium is: 1.9g / L KNO3+1.65g / L NH4NO3+0.37g / LMgSO4·7H2O+0.44g / LCaCl2·2H2O+0.17g / L KH2PO4·H2O+22.3mg / LMnSO4·4H2O+6.2mg / L H3BO3+8.6mg / LZnSO4·7H2O+0.25mg / LNa2MoO4·2H2O+0.025mg / L CuSO4·5H2O+0.83mg / L KI+0.025mg / LCoCl2+37.3mg / L Na2-EDTA+27.8mg / L FeSO4·7H2O+100mg / L inositol+2.0mg / L glycine+0.5mg / L nicotinic acid+0.1mg / LVB6+0.5mg / LVB1+30g / L sucrose. The antibacterial MS medium is MS medium + 0.75mL / LPPM), and the rest is the same as in Example 3.

[0099] The results showed that the callus tissue of the seeds was induced on day 48. The induced callus tissue was loose in texture, with obvious fibrous material and browning. Figure 9 At the same time, nearly half of the seeds were directly induced to germinate. Figure 10 , which is not conducive to the formation of callus tissue and subsequent subculture proliferation culture. The overall induction rate is 68.5%, which is lower than the induction rate of 16.8% in Example 3.

[0100] It can be seen that the antibacterial SH medium of the present invention is significantly superior to the antibacterial MS medium in terms of callus induction speed, quality and induction rate. The antibacterial SH medium has strong specificity and is more suitable for tissue culture and rapid propagation of dark purple Fritillaria seeds.

[0101] Example 6

[0102] Example 5: Hardening of the rooted bulbs (rooted bottle seedlings) in the culture room

[0103] 1.1 Environmental conditions for seedling hardening in the culture room:

[0104] The temperature is 18° C. to 20° C., the humidity is 60% to 70% (the humidity is relative humidity, RH), and the light intensity is 6000 Lux.

[0105] 1.2 Hardening in a Cultivation Room: The healthy, rooted seedlings obtained in Example 5 were placed in the aforementioned cultivation room environment. For the first three days, the bottle caps were half-opened, opened during the day and covered at night. The caps were then fully opened for the next four days to allow them to fully adapt to the external environment. During this hardening phase, any seedlings that wilted were sprayed with 1 mL of SH nutrient solution (diluted 1:10) for real-time regulation. The SH nutrient solution consisted of a macroelement solution (KNO3 + NH4H2PO4 + MgSO4·7H2O + CaCl2), a trace element solution (MnSO4·4H2O + H3BO3 + ZnSO4·7H2O + Na2MoO4·2H2O + CuSO4·5H2O + KI + CoCl2), an iron salt solution (Na2-EDTA + FeSO4·7H2O), and an organic component solution (inositol + glycine + niacin + VB6 + VB1).

[0106] Example 7

[0107] Comparison of raw material costs in tissue culture rapid propagation using dark purple Fritillaria seeds as explants compared with fresh bulbs as explants

[0108] The raw material cost calculation for tissue culture rapid propagation using dark purple Fritillaria seeds as explants is as follows: Based on an average weight of 1.2g / 1000 mature seeds from a five-year-old dark purple Fritillaria plant, 1kg contains 833,000 seeds. Currently, the price of dark purple Fritillaria seeds in its native region (Songpan, Sichuan) is 3,600 yuan / kg, meaning each seed is worth 0.004 yuan.

[0109] The cost of raw materials for tissue culture and rapid propagation using fresh bulbs of dark purple Fritillaria as explants is calculated as follows: the average weight of two-year-old fresh underground bulbs of dark purple Fritillaria is 0.23g. Based on the sales price of 4,000 yuan / kg for the two-year-old fresh bulbs from the origin of dark purple Fritillaria (Songpan, Sichuan), each fresh bulb is worth 0.92 yuan.

[0110] Comparative analysis of raw material costs for tissue culture rapid propagation using two different parts as explants: assuming that the callus induction rate of primary culture of each fresh bulb is 5 times that of each seed, the experimental value of each seed increases to 0.02 yuan, that is, the raw material cost is reduced by 46 times, and the overall rate of return is significantly increased.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A culture medium for tissue culture of dark purple Fritillaria seeds, characterized in that: The invention comprises a primary culture medium, a secondary culture medium and a rooting culture medium, wherein the composition of the primary culture medium is: antibacterial SH culture medium + 1.5-2.5 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D; The subculture medium includes a proliferation medium and an induction medium; the composition of the proliferation medium is: antibacterial SH medium + 1.5-2.0 mg / L KT + 0.5-1.0 mg / L brassinolide + 0.5-1.0 mg / L 2,4-D； The induction medium is composed of: antibacterial SH medium + 0.2-0.5 mg / L KT + 0.1-0.25 mg / LTDZ + 0.5-1.5 mg / L 2,4-D + 0.3 g / L AC; The rooting medium is composed of: antibacterial 1 / 2SH medium + 0.5-1.0 mg / L NAA + 1.0-2.0 mg / L IBA + 0.3 g / L AC; The antibacterial SH medium is SH medium + 0.75mL / L PPM; The composition of the SH medium is: 2.5g / L KNO3+0.3g / L NH4H2PO4+0.4g / L MgSO4·7H2O+0.2g / L CaCl2+106.2mg / L MnSO4·4H2O+5.0mg / L H3BO3+1.0mg / LZnSO4·7H2O+0.21mg / L Na2MoO4·2H2O+0.2mg / L CuSO4·5H2O+1.0mg / L KI+0.1mg / LCoCl2+20mg / LNa2-EDTA+15mg / L FeSO4·7H2O+100mg / L inositol+2.0mg / L glycine+5.0mg / L niacin+0.5mg / L VB6+5.0mg / L VB1+30g / L sucrose.

2. A method for rapid propagation of dark purple Fritillaria seeds, characterized in that: Using dark purple Fritillaria seeds as explants and culturing them using the culture medium according to claim 1; The method comprises the following steps: inoculating dark purple fritillaria seeds into the primary culture medium for primary culture to obtain callus tissue; inoculating the callus tissue into the proliferation culture medium for proliferation culture to obtain clustered buds; inoculating the clustered buds into the induction culture medium for induction culture to obtain clustered bulbs; and inoculating the clustered bulbs into the rooting culture medium for rooting culture to obtain rooted bulbs.

3. The method according to claim 2, characterized in that The conditions for the primary culture are: temperature 18-20° C., humidity 50%-65%, light intensity 3000 Lux-4500 Lux, and light intensity 10-12 h / d.

4. The method according to claim 2, characterized in that The conditions for the proliferation culture and induction culture are: temperature 18-20° C., humidity 55%-65%, light intensity 3000 Lux-4500 Lux, and light intensity 10-12 h / d.

5. The method according to claim 2, characterized in that The rooting culture conditions are: temperature 18-20° C., humidity 60%-70%, light intensity 4500 Lux-6000 Lux, and light intensity 12-15 h / d.

6. The method according to claim 2, characterized in that The disinfection method for dark purple Fritillaria seeds includes disinfection outside a clean bench and disinfection inside a clean bench; the disinfection outside the clean bench includes the following steps: soaking dark purple Fritillaria seeds in 25ppm GA3 for 48 hours, and then soaking in a 300-fold solution of 50% carbendazim wettable powder for 30 minutes; the disinfection inside the clean bench includes the following steps: peeling out the seeds in the seeds, disinfecting them with 75% alcohol for 15 seconds, rinsing with sterile water, and then adding an HgCl2 mixed solution for disinfection for 6 minutes; the mass volume fraction of HgCl2 in the HgCl2 mixed solution is 0.1%, and the HgCl2 mixed solution contains 0.6mL of Tween 20.

7. The method according to claim 2, characterized in that It also includes the step of hardening the seedlings in the culture room.

8. The method according to claim 7, characterized in that The conditions for hardening the seedlings in the culture room are: temperature 18°C ​​to 20°C, humidity 60% to 70%, and light intensity 6000 Lux. The steps for hardening the seedlings in the culture room are: half-opening the bottle cap for the first three days, opening it during the day and covering it at night, and fully opening the bottle cap for the next four days. During the hardening process in the culture room, for bottle seedlings that wilt, spray 1 to 2 mL of a 10-fold diluted SH nutrient solution on the plants.

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