Fritillaria unibiacteata seed rapid propagation seedling raising method and culture medium

Abstract

The invention provides a method and a culture medium for rapid propagation and seedling raising of Fritillaria unibiacteata seeds, and belongs to the technical field of tissue culture. According to the method, fritillaria unibiacteata seeds are used as explants, an antibacterial SH culture medium is used for establishing a sterile system, in-vitro regenerated plants are obtained from the processes of explant material taking, disinfection pretreatment, primary culture, subculture, rooting culture, greenhouse seedling hardening and cultivation domestication, mass propagation of tissue culture seedlings is achieved, the excellent characteristics of varieties can be kept, and the method is suitable for large-scale popularization and application. And the rapid propagation method for the fritillaria unibiacteata tissue culture, which is remarkable in effect, is established. Compared with tissue culture with underground fresh bulbs as explants, the total pollution rate is reduced from 57.9% to 13.3%, the pollution rate is reduced by 44.6%, and the overall benefit is greatly improved. The propagation speed of the fritillaria unibiacteata seed induced callus is high, 36 days is a cycle, the induction rate reaches 85.3%, the proliferation multiple of subculture is more than 660 times, and more stable and excellent cluster 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 species of Sichuan Fritillaria medicinal plants. 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 cough, clearing away heat and resolving phlegm, relieving asthma and relieving cough, dispersing nodules and eliminating carbuncles, and yin deficiency and fatigue cough. It is widely used in the treatment of respiratory diseases such as cough and asthma. It is a precious and rare Sichuan local medicinal material. It grows in wetlands or rock crevices such as forest shrubs, grasslands, river beaches and valleys at an altitude of 2,800 to 4,000 meters. It likes 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, in nature, the dormancy period of dark purple Fritillaria seeds 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 is to collect wild resources, which has led to 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 foreign 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, and the price of commercial-grade raw materials of dark purple Fritillaria has continued to rise, with a price increase of 30%. The sales price of its first-grade products (Baoyue in the Arms) 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] The 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 invention provides a medium for tissue culture of dark purple Fritillaria thunbergii 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 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;

[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 / LAC;

[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 culture 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 niacin+0.5mg / LVB6+5.0mg / LVB1+30g / L sucrose.

[0013] The invention also provides a method for rapid propagation of dark purple Fritillaria seeds into seedlings, which comprises taking the dark purple Fritillaria seeds as explants and adopting 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 callus tissue into the proliferation culture medium for proliferation culture to obtain clustered buds; inoculating clustered buds into the induction culture medium for induction culture to obtain clustered bulbs; inoculating 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 3000Lux-4500Lux, and light 10-12h / d.

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

[0017] Preferably, the rooting culture conditions are: temperature 18-20°C, humidity 60%-70%, light intensity 4500Lux-6000Lux, and light 12-15h / 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, sterilizing them with 75% alcohol for 15s, rinsing with sterile water, and adding HgCl2 mixed solution for sterilization 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-20°C, humidity 60%-70%, and light intensity 6000Lux; the steps of hardening the seedlings in the culture room are: half-open the bottle cap for the first 3 days, open it during the day and cover it at night, and completely open the bottle cap for the next 4 days. During the hardening process in the culture room, for bottle seedlings with wilting, spray 1-2mL of 10-fold diluted SH nutrient mother 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, which has the advantages of less material, low cost, short cycle, high reproduction rate, simple operation, safe production and stable quality and inheritance, and is not adversely affected by changes in the external environment and disastrous climate, and the experimental conditions are controllable, which is extremely beneficial to plant growth, and is convenient for long-term periodic cycle cultivation and cultivation domestication, and can overcome the shortcomings of high dark purple Fritillaria reproduction cost, low proliferation multiples, and potential safety hazards in the production process. The present invention is an effective method for efficiently solving the problems of scale expansion of Sichuan Fritillaria planting industry and scarcity of raw medicinal material resources.

[0023] The present invention uses the seeds in the dark purple Fritillaria seed fruit (Baguachui) as explants, and compared with the tissue culture using the underground fresh bulb as the explant, the overall contamination rate is reduced from 57.9% to 13.3%, and the contamination rate is reduced by 44.6%, which 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, an induction rate of 85.3%, and a proliferation multiple of more than 660 times in subculture, so that more stable and excellent clustered buds can be obtained. Compared with the underground fresh bulb as the explant, the present invention uses the 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 This is the explant treatment diagram;

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

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

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

[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] Fig. 9 The results of callus induction in antibacterial MS medium;

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

[0034] The invention provides a medium for tissue culture of dark purple Fritillaria thunbergii 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 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;

[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 the art can be used. In the present invention, the function 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, and more preferably 1.9-2.0 mg / L; the final concentration of brassinolide is preferably 0.6-0.8 mg / L, and more preferably 0.7 mg / L; the final concentration of 2,4-D is preferably 0.6-0.9 mg / L, and 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 clustered buds to form clustered bulbs and improve the ability of expansion. In the proliferation medium of the present invention, the final concentration of KT is preferably 1.6-1.9 mg / L, and more preferably 1.7-1.8 mg / L; the final concentration of brassinolide is preferably 0.6-0.9 mg / L, and more preferably 0.7-0.8 mg / L; the final concentration of 2,4-D is preferably 0.6-0.9 mg / L, and 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 function of the rooting medium is to induce the clustered bulbs to generate adventitious roots, form rooted bulbs, and present rapid propagation in the in vitro tissue bottle seedling stage. In the rooting medium of the present invention, the final concentration of NAA is preferably 0.6-0.9 mg / L, more preferably 0.7-0.8 mg / L; the final concentration of IBA is preferably 1.2-1.8 mg / L, more preferably 1.4-1.6 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 antibacterial SH culture medium of the present invention is composed of six major components, namely, agar, macroelement solution (KNO3+NH4H2PO4+MgSO4·7H2O+CaCl2), trace element mother solution (MnSO4·4H2O+H3BO3+ZnSO4·7H2O+Na2MoO4·2H2O+CuSO4·5H2O+KI+CoCl2), iron salt mother solution (Na2-EDTA+FeSO4·7H2O), organic component mother solution (inositol+glycine+nicotinic acid+VB6+VB1) and antibacterial agent PPM. The six major components of the antibacterial SH culture medium should be prepared and used immediately, dissolved one by one in sequence and then added, and should not be mixed and dissolved with each other; calcium chloride is easy to absorb moisture, so it needs to be weighed quickly and dissolved at the end before being added. If necessary, it can be heated appropriately until all are fully dissolved. Due to the addition of plant tissue culture antibacterial agent PPM, Tween20 and pH regulator must not be added. Compared with the MS culture medium mostly used in the art, the present invention provides an antibacterial SH culture medium which is more suitable for the tissue culture of the second-level endangered medicinal plant Fritillaria thunbergii, and has strong specificity. During its preparation process, NH4H2PO4 is used to replace the strictly controlled explosive NH4NO3 in the MS culture medium, making the experimental operation safer.

[0043] The invention also provides a method for rapid propagation of dark purple Fritillaria seeds into seedlings, which comprises taking the dark purple Fritillaria seeds as explants and adopting 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 thunbergii is 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 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 with 75% alcohol for 15s, rinsing with sterile water, and then adding 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 following steps are preferably included: 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 10-12h / d; the temperature of the primary culture is preferably 19°C, humidity is preferably 55%-60%, light intensity is preferably 3500Lux-4000Lux, and light time is preferably 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 10-12h / d; the temperature of 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 time is preferably 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 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] In the method for rapid propagation of dark purple Fritillaria seeds of the present invention, preferably, it also includes the step of hardening the seedlings in a culture room, and the conditions of hardening the seedlings in the culture room are: temperature 18°C ​​to 20°C, humidity 60% to 70%, and light intensity 6000Lux. Preferably, the step of hardening the seedlings in the culture room is: half-open the bottle cap for the first 3 days, open it during the day, cover it at night, and completely open the bottle cap for the next 4 days. During the hardening process in the culture room, for bottle seedlings with wilting, spray 1 to 2 mL of 10-fold diluted SH nutrient mother solution on the plants.

[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 utilizes dark purple Fritillaria seeds as explants, establishes a sterile system with antibacterial SH culture medium, obtains in vitro regenerated plants from explant sampling, disinfection pretreatment to primary culture (callus induction), subculture (proliferation), rooting culture and seedling hardening in a culture room, realizes large-scale reproduction of tissue culture seedlings, and can maintain the excellent characteristics of the variety, thus establishing a significant method for rapid tissue culture reproduction of dark purple Fritillaria.

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

[0051] In the following embodiments, unless otherwise specified, all of them are conventional methods.

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

[0053] In the following embodiments, each link of the inoculation process must strictly implement the principle of "sterile concept, aseptic operation", and the operator's work clothes, gloves, masks, head covers, shoe covers and inoculation equipment must be carried out in accordance with SOP to ensure that the exogenous contamination of the inoculation and transfer clean room Class A area 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): Use 1.0 mol / L NaOH to dissolve and prepare a 2,4-D solution with a concentration of 0.1 mg / mL for later use. Source of raw materials: 2,4-D purity: ≥97%, specification: 25g / bottle, batch number: G2114198, storage temperature: 2-8°C, manufacturer: Shanghai Aladdin Biochemical Technology Co., Ltd. (aladdin).

[0057] Auxin NAA (α-naphthylacetic acid): Dissolve with 95% ethanol to prepare a 0.1 mg / mL NAA solution for later use. Source of raw materials: NAA purity: ≥98.0%, specification: 25g / bottle, batch number: 201905310, storage temperature: 2-8°C, manufacturer: Chengdu Kelong Chemicals Co., Ltd.

[0058] Cytokinin KT (kineticin): Use 1.0 mol / L HCl to dissolve and prepare a 0.1 mg / mL KT solution for later use. Source of raw materials: KT purity: ≥98.0%, specification: 250 mg / bottle, batch number: 705N021, storage temperature: 2-8°C, manufacturer: Beijing Solarbio Technology Co., Ltd. (Solarbio).

[0059] Cytokinin BR (Brassinolide): Use anhydrous ethanol to dissolve and prepare a 1.0 mg / mL brassinolide solution for later use. Source of raw materials: 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): Dissolve with 1.0 mol / L NaOH to prepare a TDZ solution with a concentration of 0.1 mg / mL for later use. Source of raw materials: 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): Use 1.0 mol / L NaOH to dissolve and prepare a 0.1 mg / mL IBA solution for later use. Source of raw materials: IBA purity: ≥98%, specification: 5g / bottle, batch number: 302O031, storage temperature: 2-8°C, manufacturer: Beijing Solarbio Technology Co., Ltd. (Solarbio).

[0062] Example 1

[0063] Preparation of antibacterial SH 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 the agar from clumping on the surface of the boiling water. Continue to boil 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 at 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 sequentially, 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), and add them slowly, evenly and separately in sufficient amount under stirring.

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

[0068] (5) Finally, the culture medium is packaged 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 taken out of the sterilizer and placed in a clean area (class 100) for natural cooling (the low-temperature air conditioner 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, with an altitude of 3289.28 meters.

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

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

[0074] (1) Freshly picked dark purple Fritillaria thunbergii fruits were rinsed with laboratory ultrapure water for 4 h, filtered, soaked in 25 ppm gibberellin GA3 for 48 h, filtered again, dried on the surface, soaked in a 300-fold diluted solution of 50% carbendazim wettable powder for 30 min, filtered, and washed 6 times with sterile ultrapure water, 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 promptly transfer them to the semi-sterile control room in the clean area; sterilize them with 75% alcohol for 60 seconds, filter them, and wash them with sterilized ultrapure water for 3 times, each time for 1 minute. After the surface moisture of the seeds and fruits is dried, 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 promptly transfer them to a completely sterilized tissue culture bottle. 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 six 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 (bottle height of 10 cm, bottle bottom diameter of 7 cm) was selected for inoculation. The tissue culture bottle was filled with the antibacterial SH medium obtained in Example 1, and 30 seeds were inoculated in each bottle.

[0079] The inoculated tissue culture bottle was quickly transferred to the culture room. The environmental conditions in 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 11h.

[0080] The control group used the two-year-old dark purple Fritillaria thunbergii lower fresh bulbs as explants to perform the above operation. The results showed that the contamination rate of the dark purple Fritillaria thunbergii seeds as explants was 13.3% on average, while the contamination rate of the two-year-old plant underground fresh bulbs as explants was 57.9% on average. It can be seen that the overall contamination rate of the tissue culture rapid propagation using seeds as explants of the present invention is reduced by 44.6%, and the material collection method of the present invention saves considerable manpower, financial and material costs, and greatly improves the overall benefit.

[0081] Example 3

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

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

[0084] Cultivation 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, hard, clear, delicate and shiny, without obvious fibrous substances, and has a pure and lasting smell. The overall induction rate is 85.3%. After another 30 days of cultivation, 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 with a diameter of 4 cm to 5 cm obtained in Example 3 was inoculated into a culture bottle containing a proliferation 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 10 h / d. Subculture was carried out at a cycle of 4 weeks / generation, and 5 generations were continuously transferred to form more callus tissue, completing the first stage of subculture. Figure 5At this stage, the callus tissue induced by the primary culture is divided into 4 pieces, and the culture cycle is 4 weeks, and the culture is repeated until the fifth generation, and the number of proliferation reaches more than 128 bottles. The proliferation culture medium consists 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 culture 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 h / d. The culturing cycle was 4 weeks, and the clustered buds were cultured into clustered bulbs, completing the second stage of subculture. Figure 6 The induction medium is composed of antibacterial SH medium, 0.2 mg / L KT, 0.1 mg / LTDZ, 1.0 mg / L 2,4-D, 30 g / L sucrose and 0.3 g / LAC. The operation at this stage is carried out in a sequential round culture manner according to the progress of the clustered buds formed in the first stage above, until all callus tissues complete the proliferation culture from clustered buds to clustered bulbs.

[0089] After the above two stages of subculture, each bottle of callus tissue reached 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, its proliferation multiple was 660 times, with significant effect.

[0090] Example 5

[0091] Rooting culture

[0092] The clumped bulbs obtained in Example 4 were inoculated into a large tissue culture bottle (bottle height 14 cm, bottle bottom diameter 9.5 cm) filled with rooting medium with a 4-week culture cycle. The temperature was regulated to 20°C, the humidity to 60% to 70%, the light intensity to 6000 Lux, and the light intensity to 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 / LIBA and 0.3 g / LAC.

[0093] Cultivation effect: According to the above-mentioned combination and ratio of growth hormones in the culture medium, adventitious roots of clustered bulbs are induced. After 60 days of cultivation, rooted bulb bottle seedlings are formed, and the rooting rate and seedling rate are more than 96%. The bottle seedlings have straight stems, tall seedlings, green leaves, and a strong and developed root system. There are no root rot and lesions. The dryness of old leaves is less than 3%, showing an excellent overall rooting culture effect, completing the rapid propagation of dark purple Fritillaria tissue culture seedlings in the bottle 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, and 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 mass began to brown. As time went on, the browning became more and more serious, which was not conducive to the acquisition 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 callus tissue was induced in the 48th day. The induced callus tissue was loose in texture, with obvious fibrous substances and browning. Fig. 9 At the same time, nearly half of the seeds were directly induced to germinate. Fig.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 Method for hardening seedlings in a culture room: The healthy rooted bottle seedlings obtained in Example 5 were placed in the above-mentioned culture room environment conditions. For the first 3 days, the bottle caps were half-opened, opened during the day, and covered at night. For the next 4 days, the bottle caps were completely opened to fully adapt to the external environment. During the hardening process at this stage, for bottle seedlings with wilting, 1 mL of SH nutrient mother solution (diluted at 1:10) was sprayed on the plants for real-time regulation. The SH nutrient mother solution consisted of a large amount of element 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+nicotinic acid+VB6+VB1).

[0106] Example 7

[0107] Comparison of the raw material cost of tissue culture rapid propagation using dark purple Fritillaria seeds as explants and fresh bulbs as explants

[0108] The cost of raw materials for tissue culture and rapid propagation of dark purple Fritillaria seeds as explants is calculated as follows: the average weight of mature seeds of five-year-old dark purple Fritillaria plants is 1.2g / 1000 seeds, so 1kg is 833,000 seeds. At present, the sales price of dark purple Fritillaria seeds in its place of origin (Songpan, Sichuan) is 3,600 yuan / kg, so each seed is worth 0.004 yuan.

[0109] The raw material cost accounting for tissue culture and rapid propagation using fresh bulbs of Dark Purple Fritillaria as explants is 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 two-year-old fresh bulbs from the origin of Dark Purple Fritillaria (Songpan, Sichuan) is 4,000 yuan / kg, and each fresh bulb is worth 0.92 yuan.

[0110] Comparative analysis of raw material costs for tissue culture and 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A medium for tissue culture of dark purple Fritillaria seeds, characterized in that: It includes a primary culture medium, a secondary culture medium and a rooting culture medium, wherein the primary culture medium is composed of: 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 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; 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 / LAC; The antibacterial SH medium is SH medium+0.75mL / LPPM.

2. The culture medium according to claim 1, characterized in that The composition of the SH culture medium is: 2.5g / L KNO3+0.3g / L NH4H2PO4+0.4g / L MgSO4·7H2O+0.2g / L CaCl2+106.2mg / LMnSO4·4H2O+5.0mg / LH3BO3+1.0mg / L ZnSO4·7H2O+0.21mg / LNa2MoO4·2H2O+0.2mg / L CuSO4·5H2O+1.0mg / L KI+0.1mg / L CoCl2+20mg / LNa2-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.

3. A method for rapid propagation of dark purple Fritillaria seeds, characterized in that: Dark purple Fritillaria seeds are used as explants and cultured using the culture medium described in claim 1 or 2.

4. The method according to claim 3, characterized in that 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.

5. The method according to claim 4, 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 10-12 h / d.

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

7. The method according to claim 4, characterized in that The rooting culture conditions are: temperature 18-20° C., humidity 60%-70%, light intensity 4500 Lux-6000 Lux, and light 12-15 h / d.

8. The method according to claim 3, characterized in that 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 seed fruit in 25ppm GA3 for 48h, and then soaking it 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 seed fruit, disinfecting them with 75% alcohol for 15s, rinsing them with sterile water, and adding 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.

9. The method according to claim 3, characterized in that: It also includes the step of hardening the seedlings in the culture room.

10. The method according to claim 9, characterized in that The conditions for hardening the seedlings in the culture room are: temperature 18°C-20°C, humidity 60%-70%, and light intensity 6000Lux. 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 completely opening the bottle cap for the next 4 days. During the hardening process in the culture room, for bottle seedlings with wilting phenomenon, spraying 1-2mL of 10-fold diluted SH nutrient mother solution on the plants.

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