Production method of fritillaria pallidiflora seedlings

Abstract

The invention belongs to the technical field of plant cell engineering, and relates to a production method of fritillaria pallidiflora seedlings, which sequentially comprises the following steps: explant sampling and pretreatment, sterile explant acquisition, callus induction, pretreatment before callus redifferentiation, cluster bud proliferation, rooting culture, in-vitro bulb induction, pretreatment before transplanting and transplanting. By adopting the scheme for producing the bulbus fritillariae penneri seedlings, the beneficial effects are as follows: (1) the proliferation rate is high, and the culture period is short; (2) seedlings can be produced all year round, and the efficiency is high; (3) the tissue culture seedlings are good in quality, robust and high in cultivation survival rate; compared with traditional bulb propagation, seed sowing and other modes, the method has the advantages that propagation efficiency is high, culture time is short, seedlings are robust, pathogenic bacteria are not contained, the survival rate is high, cost is low, and the method is suitable for large-scale production of the bulbus fritillariae pennyi seedlings.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cell engineering, and particularly relates to a method for culturing and rapidly propagating Fritillaria davidiana by comprehensive treatment of plant tissue culture to obtain a large number of seedlings. Background Art

[0002] Pengze Fritillaria (Fritillaria monatha Migo) is a new distribution of Fritillaria genus in Jiangxi Province. It is mainly produced in Pengze County and Hukou County of Jiujiang City, Jiangxi Province. It is a new variety of Fritillaria medicinal materials. The total alkaloid content of Pengze Fritillaria is higher than that of Zhejiang Fritillaria and Sichuan Fritillaria. Its antitussive effect is similar to that of Zhejiang Fritillaria, and its acute toxicity is relatively low. It has a high development value. However, due to the small distribution area and predatory excavation of wild Pengze Fritillaria, the resources of Pengze Fritillaria are shrinking. Although there is a small amount of artificial cultivation, the traditional bulb asexual reproduction coefficient is low, and seed reproduction takes a long time. Therefore, the seed source problem seriously restricts its orderly development and utilization, so it is necessary to develop seedling propagation technology for it.

[0003] The rapid propagation of Fritillaria dahurica seedlings by plant tissue culture is a good method for obtaining Fritillaria dahurica seedlings on a large scale. The in vitro culture research of Fritillaria dahurica is currently focused on callus induction, component analysis and screening of high-yield cell lines. Since the in vitro seedling induction of Fritillaria dahurica faces many difficulties, the production method of seedlings by tissue culture has not been reported or publicly applied. Summary of the invention

[0004] The technical problem to be solved by the invention is to provide a method for cultivating Pengze Fritillaria seedlings with short growth cycle, high transplant survival rate, high seedling production efficiency, low cost and simple operation.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: the culture steps sequentially include explant material collection and pretreatment, sterile explant acquisition, callus induction, callus pretreatment before redifferentiation, cluster bud proliferation, rooting culture, in vitro bulb induction, pretreatment before transplantation and transplantation; specifically include the following steps:

[0006] (1) Explant collection and pretreatment: Different explant types require different collection periods. Different explants use different immersion pretreatments and low-temperature pretreatments;

[0007] (2) Obtaining sterile explants: Different types of explants are sterilized using different methods to obtain sterile explants for the next step of culture;

[0008] (3) callus induction: inoculating the sterile explant obtained in step (2) into a liquid shallow callus culture medium for callus induction culture to obtain callus tissue in good condition;

[0009] (4) Low temperature treatment before callus redifferentiation: inoculating the callus tissue obtained in step (3) onto a solid-liquid double-layer redifferentiation medium, and then performing low temperature treatment;

[0010] (5) cluster bud proliferation: inoculating the cluster buds obtained by redifferentiation in step (4) into a solid cluster bud proliferation medium for proliferation culture;

[0011] (6) Rooting culture: cutting the clustered buds obtained in step (5) and inoculating them onto a solid rooting medium for rooting induction to obtain rooted seedlings;

[0012] (7) In vitro bulb induction: in a season that is not suitable for transplanting, the clustered buds obtained in step (5) are cut and inoculated onto a solid in vitro bulb induction medium to induce in vitro bulbs;

[0013] (8) Pretreatment before transplanting and transplanting: Before transplanting, the rooted seedlings obtained in step (6) and the in vitro bulbs obtained in step (7) are subjected to programmed cooling pretreatment, then taken out of the bottle, cleaned, soaked for pretreatment, and then transplanted and cultivated in the substrate for growth to obtain Pengze Fritillaria seedlings.

[0014] By adopting the above technical scheme, a large number of experiments have confirmed that the cultivation method of Akebia seedlings can achieve high proliferation rate, large biological yield, short cultivation cycle; tissue culture seedlings are of good quality, seedlings are strong, and the cultivation survival rate is high.

[0015] As a further improvement of the present invention, the different explants described in step (1) are divided into three categories: one category is young stem segments and leaves, the second category is bulbs in the early growth stage, and the two categories of explants are generally collected from January to March each year in northern Jiangxi; the third category is dormant bulbs, and the collection period in northern Jiangxi is generally from April to December each year; the use of multiple explants to cultivate Pengze Fritillaria seedlings not only facilitates the acquisition of explants, but also makes full use of materials and avoids waste.

[0016] As a further improvement of the present invention, in the step (1), when the selected explants are young stem segments and leaves and bulbs in the early stage of growth, the pretreatment method is: after the explants are removed from the body, they are first cleaned with detergent and running water. The young stem segments and leaves are wrapped with wet tissues soaked with a 0.2-0.5% concentration of carbendazim solution and then placed at 2-5°C for moisturizing and refrigeration for 0-7 days. The bulbs in the early stage of growth are cut off 0.5mm of the outer skin, wrapped with wet tissues soaked with a 0.2-0.5% concentration of carbendazim solution, and then placed at 2-5°C for moisturizing and refrigeration for 7-14 days. When the selected explants are dormant bulbs, the pretreatment method is: after the dormant bulbs are cut off 0.5mm of the outer skin, wrapped with wet tissues soaked with a 0.5-1% concentration of carbendazim solution, and then placed at 2-5°C for moisturizing and refrigeration for 14-30 days.

[0017] Different explants are pretreated in different ways, and the pretreatment methods used are more suitable for the subsequent culture of each explant. If no corresponding pretreatment is performed, the subsequent culture effect will be poor or even impossible to continue to culture. The pretreatment process is conducive to the subsequent culture and shortens the culture cycle.

[0018] As a further improvement of the present invention, in the step (2), when the selected explants are young stem segments and leaves, the sterilization treatment method is: the pre-treated explants are sterilized with 70-75% alcohol for 10-30 seconds, then sterilized with mercuric chloride for 3-7 minutes, and then rinsed with sterile water for use; when the selected explants are bulbs in the early growth stage, the sterilization treatment method is: the pre-treated explants are sterilized with 70-75% alcohol for 1-2 minutes, then sterilized with mercuric chloride for 10-15 minutes, and then rinsed with sterile water for use. When the selected explants are dormant bulbs, the sterilization treatment method is: the pre-treated explants are sterilized with 70-75% alcohol for 1-2 minutes, then sterilized with mercuric chloride for 10-20 minutes, and then rinsed with sterile water for use. Aseptic treatment is very critical to the subsequent culture process and directly affects the subsequent culture. Through a large number of experiments, it has been verified that this sterilization method is the best for subsequent culture.

[0019] As a further improvement of the present invention, the liquid shallow callus culture medium in step (3) is formulated as MS+0.01-0.5 mg / L 6-BA+1.0-3.0 mg / L NAA+0.01-2.0 mg / L TDZ+0.5-3.0 mg / L 2,4-D+30 g / L sucrose, pH 5.5-6.0; the culture conditions are: light intensity 1500-2000 lux, light 1-5 h / d, temperature 25±1°C; the liquid shallow layer thickness is 0.5-3 cm. Such culture conditions can obtain callus with good growth status, which is conducive to subsequent culture.

[0020] As a further improvement of the present invention, the formula of the liquid culture medium in the solid-liquid double-layer redifferentiation culture medium in the step (4) is MS+0.5-4.0mg / L 6-BA+0.1-1.0mg / L NAA+0.01-1.0mg / L TDZ+0.1-1.0mg / L 2,4-D+30g / L sucrose, pH5.5-6.0, and thickness of 0.2-1.0cm; the lower layer of the solid-liquid double-layer redifferentiation culture medium is a solid culture medium with 6.5g / L agar added, and the rest of the formula is consistent with the upper liquid culture medium, thereby forming a solid-liquid double-layer culture medium; the culture conditions are: light intensity 1500-2000lux, light 5-10h / d; low temperature treatment temperature 5-10℃, treatment time 7-14d. Then the temperature is transferred to 25±1℃ for redifferentiation induction. The use of a solid-liquid double-layer redifferentiation medium can significantly increase the proliferation of callus and reduce the occurrence of browning. The use of low-temperature treatment for a period of time followed by redifferentiation induction can significantly increase the redifferentiation efficiency and is conducive to obtaining more clustered buds.

[0021] As a further improvement of the present invention, the solid bud proliferation culture medium in step (5) is formulated as MS+1.0-3.0 mg / L 6-BA+0.1-0.5 mg / L NAA+0.01-0.1 mg / L 2,4-D+30 g / L sucrose+6.5 g / L agar, pH 5.5-6.0; the culture conditions are: light intensity 3000-5000 lux, light 8-12 h / d, temperature 25±1° C. Such culture conditions can obtain robust buds, which is conducive to subsequent culture.

[0022] As a further improvement of the present invention, the solid rooting medium in step (6) is formulated as 1 / 2MS or MS+0.1-1.0mg / L NAA+0.1-3.0mg / L IBA+30g / L sucrose+0.1-1.0g / L activated carbon+6.5g / L agar, pH5.5-6.0; the culture conditions are: light intensity 3000-5000lux, light 8-12h / d, temperature 25±1°C. Such culture conditions can obtain healthy rooting seedlings, which are beneficial to subsequent transplanting.

[0023] As a further improvement of the present invention, the solid in vitro bulb induction culture medium in step (7) is formulated as 1 / 2MS or MS+0.1-1.0mg / L NAA+0.1-3.0mg / L IBA+1.0-5.0mg / L PP333+50-120g / L sucrose+0.1-1.0g / L activated carbon+6.5g / L agar, pH5.5-6.0; the culture conditions are: light intensity 5000-6000lux, light 12-16h / d, temperature 25±1°C. This culture condition can induce in vitro bulbs with a large enough diameter, and the in vitro bulb seedlings can grow for a long time. When the leaves wither, the in vitro bulbs can be stored in the culture bottle for a long time and transplanted at the appropriate time.

[0024] As a further improvement of the present invention, in the pretreatment before transplanting in the step (8), the rooted seedlings obtained in the step (6) and the in vitro bulbs with bottles obtained in the step (7) are subjected to programmed cooling pretreatment, and the process of programmed cooling treatment is: first, the culture temperature is reduced from 25°C to 20°C, and the treatment is carried out for 3 to 5 days, and then further reduced to 15°C, and the treatment is carried out for 3 to 5 days, and then further reduced to 10°C, and the treatment is carried out for 3 to 5 days, and finally reduced to 5°C, and the treatment is carried out for 3 to 5 days.

[0025] As a further improvement of the present invention, the rooted seedlings or in vitro bulbs treated with programmed cooling in step (8) are cleaned after being taken out of the bottle, and then immersed in a solution of carbendazim with a concentration of 0.2-0.5% for 1-3 hours. The rooted seedlings or in vitro bulbs are then transplanted into a substrate with a ratio of peat:perlite=1-3:1, appropriately shaded, with humidity maintained at 80-90% in the early stage and 70-80% in the later stage, and the light intensity during cultivation is maintained at 2000-3000 lux in the early stage and 3000-5000 lux in the later stage, and the temperature is 5-20°C, thereby obtaining Pengze Fritillaria seedlings.

[0026] The technical scheme for producing Pengze Fritillaria seedlings of the present invention has the following beneficial effects: (1) high proliferation rate and short culture cycle; (2) seedlings can be produced all year round with high efficiency; (3) tissue culture seedlings are of good quality, the seedlings are strong and the cultivation survival rate is high; the present invention utilizes the methods of explant pretreatment, aseptic explant acquisition, callus induction, pretreatment before callus redifferentiation, cluster bud proliferation, rooting culture, in vitro bulb induction, pretreatment before transplanting and transplanting to produce and cultivate Pengze Fritillaria seedlings, which has high propagation efficiency, short culture time, strong seedlings, high cultivation survival rate and low cost compared with traditional bulb propagation and seed sowing propagation and other cultivation methods, and is suitable for large-scale production of Pengze Fritillaria seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The specific implementation process of the present invention is further described in detail below with reference to the accompanying drawings.

[0028] Figure 1 The technical solution flow of a method for producing Pengze Fritillaria seedlings. The culture steps of each stage and the corresponding culture results are shown in the figure. The culture steps sequentially include explant material collection and pretreatment, sterile explant acquisition, callus induction, pretreatment before callus redifferentiation, cluster bud proliferation, rooting culture, in vitro bulb induction, pretreatment before transplantation and transplantation. DETAILED DESCRIPTION

[0029] Embodiment 1:

[0030] A method for culturing and rapidly propagating Fritillaria pengze by using young stem segments and leaves as explants and using plant tissue culture to obtain a large number of seedlings has the following specific steps:

[0031] (1) Explant collection and pretreatment: From January to March each year, young stem segments and leaves were collected and washed with detergent and running water. The young stem segments and leaves were wrapped with a wet tissue soaked in 0.3% carbendazim solution and then stored at 4°C for 5 days to keep them moist.

[0032] (2) Obtaining sterile explants: After pretreatment, the selected young stem segments and leaves were disinfected with 75% alcohol for 30 seconds and then sterilized with mercuric chloride for 5 minutes, and then rinsed with sterile water for later use;

[0033] (3) Callus induction: The obtained sterile explants were inoculated into a liquid shallow callus culture medium for callus induction culture to obtain callus tissue in good condition; wherein the liquid shallow callus culture medium formula is MS+0.3mg / L 6-BA+2.0mg / L NAA+2.0mg / L TDZ+1.0mg / L 2,4-D+30g / L sucrose, pH5.8; the culture conditions are: light intensity 1500lux, light 5h / d, temperature 25±1℃; the thickness of the liquid shallow layer is 2cm. Under this culture condition, callus tissue in good growth condition can be obtained, and the callus induction rate is 75%. Compared with the solid culture medium under the same culture medium ratio, the callus induction rate is 23%.

[0034] (4) Low temperature treatment before callus redifferentiation: The callus tissue obtained in step (3) is inoculated onto a solid-liquid double-layer redifferentiation medium, and then subjected to low temperature treatment; the liquid culture medium in the solid-liquid double-layer redifferentiation medium has a formula of MS+3.0mg / L 6-BA+0.5mg / L NAA+1.0mg / L TDZ+1.0mg / L 2,4-D+30g / L sucrose, pH 5.8, and a thickness of 0.5cm; the lower layer of the solid-liquid double-layer redifferentiation medium is a solid culture medium with 6.5g / L agar added, and the rest of the formula is the same as the upper liquid culture medium, thereby forming a solid-liquid double-layer culture medium; the culture conditions are: light intensity 2000lux, light 10h / d; low temperature treatment temperature 5°C, treatment time 10d. Then, the culture medium is transferred to a temperature of 25±1°C for redifferentiation induction. The callus induction rate was 80% when using solid-liquid double-layer redifferentiation medium, which was higher than 68% of solid culture; and the browning rate was 12.5% ​​when using solid-liquid double-layer culture, which was lower than 36% of solid culture. In addition, the redifferentiation induction after low temperature treatment for a period of time can significantly improve the redifferentiation efficiency, and the cluster bud induction rate reached 87%, while the cluster bud induction rate without low temperature treatment was only 14%.

[0035] (5) Cluster bud proliferation: The cluster buds obtained by redifferentiation are inoculated into a solid cluster bud proliferation medium for proliferation culture; the solid cluster bud proliferation medium is formulated as MS+3.0mg / L 6-BA+0.2mg / L NAA+0.05mg / L 2,4-D+30g / L sucrose+6.5g / L agar, pH 5.8; the culture conditions are: light intensity 5000lux, light 12h / d, temperature 25±1°C. This culture condition can obtain robust cluster buds, with a proliferation multiple of 6.85, which is conducive to subsequent culture.

[0036] (6) Rooting culture: The obtained clustered buds were cut and inoculated on a solid rooting medium for rooting induction to obtain rooted seedlings; the solid rooting medium formula was MS + 0.3 mg / L NAA + 0.3 mg / L IBA + 30 g / L sucrose + 0.5 g / L activated carbon + 6.5 g / L agar, pH 5.8; the culture conditions were: light intensity 5000 lux, light 12 h / d, temperature 25 ± 1 ° C. Under this culture condition, the rooting rate was 87%, and strong rooted seedlings were obtained, which was conducive to subsequent transplanting.

[0037] (7) In vitro bulb induction: in a season not suitable for transplanting, the clustered buds obtained in the step are cut and inoculated on a solid in vitro bulb induction medium to induce in vitro bulbs; the solid in vitro bulb induction medium is formulated as MS+1.0mg / L NAA+2.0mg / L IBA+3.0mg / L PP333+60g / L sucrose+0.5g / L activated carbon+6.5g / L agar, pH 5.8; the culture conditions are: light intensity 5000lux, light 12h / d, temperature 25±1°C. Under these culture conditions, in vitro bulbs with a diameter of about 1.5cm can be induced, which can be stored in a culture bottle for a long time and transplanted at an appropriate time.

[0038] (8) Pretreatment before transplanting and transplanting: Before transplanting, the rooted seedlings or in vitro bulbs are pretreated with programmed cooling, then taken out of the bottle for cleaning, and then soaked for pretreatment before being transplanted into the substrate for growth to obtain Pengze Fritillaria seedlings.

[0039] The rooted seedlings or in vitro bulbs in bottles were pretreated with programmed cooling. The process of programmed cooling treatment was as follows: first, the culture temperature was lowered from 25°C to 20°C for 3 days, then further lowered to 15°C for 3 days, then further lowered to 10°C for 3 days, and finally lowered to 5°C for 3 days.

[0040] After the rooted seedlings or in vitro bulbs treated with programmed cooling are taken out of the bottle, they are cleaned and then soaked in a 0.3% carbendazim solution for 2 hours. Then the rooted seedlings or in vitro bulbs are transplanted into a substrate with a peat:perlite ratio of 1:1, shaded appropriately, with humidity maintained at 80-90% in the early stage and 70-80% in the later stage. During cultivation, the light intensity is maintained at 2000-3000 lux in the early stage and 3000-5000 lux in the later stage, and the temperature is 5-20°C, and Pengze Fritillaria seedlings can be obtained.

[0041] Embodiment 2:

[0042] A method for culturing and rapidly propagating Fritillaria dasyphylla by using bulbs in the growth period as explants and using plant tissue culture to obtain a large number of seedlings has the following specific steps:

[0043] (1) Explant collection and pretreatment: Different explant types have different collection periods. Different explants are treated with different soaking pretreatments and low-temperature pretreatments. From January to March each year, bulbs in the growing period are dug up, cleaned, and 0.5 mm of the outer skin of the bulbs is cut off. Then, the bulbs are wrapped with wet wipes soaked in 0.3% carbendazim solution and placed in a moisturizing refrigerated storage at 4°C for 10 days.

[0044] (2) Obtaining sterile explants: After pretreatment of the growing bulbs, disinfect them with 75% alcohol for 1 min, sterilize them with mercuric chloride for 12 min, and then rinse them with sterile water for later use.

[0045] (3) Callus induction: The obtained sterile explants were inoculated into a liquid shallow callus culture medium for callus induction culture to obtain callus tissue in good condition; wherein the liquid shallow callus culture medium formula is MS+0.3mg / L 6-BA+2.5mg / L NAA+2.0mg / L TDZ+2.0mg / L 2,4-D+30g / L sucrose, pH5.8; the culture conditions are: light intensity 1500lux, light 5h / d, temperature 25±1℃; the thickness of the liquid shallow layer is 2cm. Under this culture condition, callus tissue in good growth condition can be obtained, and the callus induction rate is 87%. Compared with the solid culture medium under the same culture medium ratio, the callus induction rate is 35%.

[0046] (4) Low temperature treatment before callus redifferentiation: The callus tissue was inoculated onto a solid-liquid double-layer redifferentiation medium, and then subjected to low temperature treatment; the liquid medium in the solid-liquid double-layer redifferentiation medium was formulated as MS+2.0mg / L 6-BA+1.0mg / L NAA+0.5mg / L TDZ+0.2mg / L 2,4-D+30g / L sucrose, pH 5.5-6.0, and a thickness of 1.0cm; the lower layer of the solid-liquid double-layer redifferentiation medium was a solid medium with 6.5g / L agar added, and the rest of the formula was the same as the upper liquid medium, thereby forming a solid-liquid double-layer medium; the culture conditions were: light intensity 2000lux, light 8h / d; low temperature treatment temperature 8°C, treatment time 10d. Then the culture was transferred to a temperature of 25±1°C for redifferentiation induction. The callus induction rate was 88% when using solid-liquid double-layer redifferentiation medium, which was higher than 65% of solid culture; and the browning rate was 10.5% when using solid-liquid double-layer culture, which was lower than 39% of solid culture. In addition, the redifferentiation induction after low temperature treatment for a period of time can significantly improve the redifferentiation efficiency, and the cluster bud induction rate reached 92%, while the cluster bud induction rate without low temperature treatment was only 18%.

[0047] (5) Cluster bud proliferation: The cluster buds obtained by redifferentiation are inoculated into a solid cluster bud proliferation medium for proliferation culture; the solid cluster bud proliferation medium is formulated as MS+2.0mg / L 6-BA+0.1mg / L NAA+0.05mg / L 2,4-D+30g / L sucrose+6.5g / L agar, pH 5.8; the culture conditions are: light intensity 3000lux, light 10h / d, temperature 25±1°C. This culture condition can obtain robust cluster buds, with a proliferation multiple of 7.35, which is conducive to subsequent culture.

[0048] (6) Rooting culture: The obtained clustered buds were cut and inoculated on a solid rooting medium for rooting induction to obtain rooted seedlings; the solid rooting medium formula was MS + 0.2 mg / L NAA + 0.2 mg / L IBA + 30 g / L sucrose + 0.5 g / L activated carbon + 6.5 g / L agar, pH 5.8; the culture conditions were: light intensity 5000 lux, light 12 h / d, temperature 25 ± 1 ° C. Under this culture condition, the rooting rate was 82%, and strong rooted seedlings were obtained, which was conducive to subsequent transplanting.

[0049] (7) In vitro bulb induction: in a season not suitable for transplanting, the clustered buds obtained in the step are cut and inoculated on a solid in vitro bulb induction medium to induce in vitro bulbs; the solid in vitro bulb induction medium is formulated as MS+1.0mg / L NAA+1.0mg / L IBA+4.0mg / L PP333+80g / L sucrose+0.5g / L activated carbon+6.5g / L agar, pH 5.8; the culture conditions are: light intensity 5000lux, light 12h / d, temperature 25±1°C. Under these culture conditions, in vitro bulbs with a diameter of about 1.8cm can be induced, which can be stored in a culture bottle for a long time and transplanted at an appropriate time.

[0050] (8) Pretreatment before transplanting and transplanting: Before transplanting, the rooted seedlings or in vitro bulbs are pretreated with programmed cooling, then taken out of the bottle for cleaning, and then soaked for pretreatment before being transplanted into the substrate for growth to obtain Pengze Fritillaria seedlings.

[0051] The rooted seedlings or in vitro bulbs in bottles were pretreated with programmed cooling. The process of programmed cooling treatment was as follows: first, the culture temperature was lowered from 25°C to 20°C for 3 days, then further lowered to 15°C for 3 days, then further lowered to 10°C for 3 days, and finally lowered to 5°C for 3 days.

[0052] After the rooted seedlings or in vitro bulbs treated with programmed cooling are taken out of the bottle, they are cleaned and then soaked in a 0.3% carbendazim solution for 2 hours. Then the rooted seedlings or in vitro bulbs are transplanted into a substrate with a peat:perlite ratio of 1:1, shaded appropriately, with humidity maintained at 80-90% in the early stage and 70-80% in the later stage. During cultivation, the light intensity is maintained at 2000-3000 lux in the early stage and 3000-5000 lux in the later stage, and the temperature is 5-20°C, and Pengze Fritillaria seedlings can be obtained.

[0053] Embodiment 3:

[0054] A method for using dormant bulbs as explants to culture and propagate Pengze Fritillaria thunbergii by comprehensive treatment in a plant tissue culture method to obtain a large number of seedlings. The specific steps are as shown in Example 2, except that the dormant bulbs are generally collected from April to December each year in northern Jiangxi; and the dormant bulbs are dug out and cleaned, and then 0.5mm of the outer skin is cut off, and then wrapped with a wet towel soaked in a 1% concentration of carbendazim solution and placed at 4°C for moisturizing and refrigeration for 30 days. Then disinfected with 75% alcohol for 2 minutes and sterilized with mercuric chloride for 15 minutes, and then rinsed with sterile water and inoculated. The rest of the operations are as in Example 2. The callus induction rate obtained with dormant bulbs as explants is 82%; the cluster bud induction rate is 89%; the rooting rate is 78%; an in vitro bulb with a diameter of about 1.6 cm is obtained, and transplanted after programmed cooling pretreatment, and Pengze Fritillaria thunbergii seedlings in good condition can also be obtained.

[0055] Embodiment 4:

[0056] A method for culturing and rapidly propagating Fritillaria dahurica by using young and tender stem segments and leaves as explants and comprehensively treating them by plant tissue culture to obtain a large number of seedlings, wherein the specific steps are as shown in Example 1, except that the formula of the liquid shallow callus culture medium is MS+0.05mg / L 6-BA+2.0mg / L NAA+0.05mg / L TDZ+1.5mg / L 2,4-D+30g / L sucrose, pH5.5; the formula of the redifferentiation culture medium is MS+0.5mg / L 6-BA+0.1mg / L NAA+0.01mg / L TDZ+0.1mg / L 2,4-D+30g / L sucrose, pH5.8, and the thickness is 0.5cm; the redifferentiation low-temperature treatment temperature is 6°C, and the treatment time is 12d. The formula of solid cluster bud proliferation medium is MS+1.0mg / L 6-BA+0.1mg / L NAA+0.01mg / L 2,4-D+30g / L sucrose+6.5g / L agar, pH5.5-6.0; the rooting medium is 1 / 2MS+0.1mg / L NAA+0.1mg / L IBA+30g / L sucrose+0.1g / L activated carbon+6.5g / L agar, pH5.5-6.0; the in vitro bulb induction medium is 1 / 2MS+0.1mg / L NAA+0.1mg / L IBA+1.0mg / L PP333+50g / L sucrose+0.1g / L activated carbon+6.5g / L agar, pH5.5; the callus induction rate was 68%; the cluster bud induction rate was 80%; the rooting rate was 65%; in vitro bulbs with a diameter of about 1.2cm were obtained, and after programmed cooling pretreatment and transplanting, Pengze Fritillaria seedlings in good condition were obtained.

[0057] Embodiment 5:

[0058] A method for culturing and rapidly propagating Fritillaria dasyphylla by using bulbs in the growth period as explants and comprehensively treating them by plant tissue culture to obtain a large number of seedlings, wherein the specific steps are as shown in Example 2, except that the formula of the liquid shallow callus culture medium is MS+0.02mg / L 6-BA+1.0mg / L NAA+0.01mg / L TDZ+1.5mg / L 2,4-D+30g / L sucrose, pH5.5; the formula of the redifferentiation culture medium is MS+1.5mg / L 6-BA+0.1mg / L NAA+0.05mg / L TDZ+0.5mg / L 2,4-D+30g / L sucrose, pH5.8, and the thickness is 1.0cm; the redifferentiation low-temperature treatment temperature is 5°C, and the treatment time is 7d. The formula of solid cluster bud proliferation medium is MS+2.0mg / L 6-BA+0.2mg / L NAA+0.05mg / L 2,4-D+30g / L sucrose+6.5g / L agar, pH5.5-6.0; the rooting medium is 1 / 2MS+0.5mg / L NAA+0.5mg / L IBA+30g / L sucrose+0.1g / L activated carbon+6.5g / L agar, pH5.5-6.0; the in vitro bulb induction medium is 1 / 2MS+0.1mg / L NAA+0.1mg / L IBA+3.0mg / L PP333+50g / L sucrose+0.1g / L activated carbon+6.5g / L agar, pH5.5; the callus induction rate was 66%; the cluster bud induction rate was 78%; the rooting rate was 67%; in vitro bulbs with a diameter of about 1.5cm were obtained, and after programmed cooling pretreatment and transplanting, Pengze Fritillaria seedlings in good condition were obtained.

[0059] Embodiment 6:

[0060] A method for culturing and rapidly propagating Fritillaria dahurica by using dormant bulbs as explants and using plant tissue culture to obtain a large number of seedlings. The specific steps are as shown in Example 3, except that after the dormant bulbs are treated, they are wrapped with wet wipes soaked in a 1% carbendazim solution and placed at 4°C for 10 days of moisturizing and refrigeration. The callus induction rate obtained by using dormant bulbs as explants is 59%; the cluster bud induction rate is 86%; the rooting rate is 77%; and the in vitro bulbs with a diameter of about 1.3 cm are obtained. After being pre-treated with programmed cooling, they are transplanted to obtain Fritillaria dahurica seedlings in good condition.

[0061] Embodiment 7:

[0062] A method for culturing and rapidly propagating Fritillaria dasyphylla by using bulbs in the growth period as explants and comprehensively treating them by plant tissue culture to obtain a large number of seedlings. The specific steps are as shown in Example 2, except that the rooting medium is 1 / 2MS+0.1mg / L NAA+0.1mg / L IBA+30g / L sucrose+0.1g / L activated carbon+6.5g / L agar, with a pH of 5.5-6.0; the in vitro bulb induction medium is 1 / 2MS+0.1mg / L NAA+0.1mg / L IBA+1.0mg / L PP333+50g / L sucrose+0.1g / L activated carbon+6.5g / L agar, with a pH of 5.5; the rooting rate is 83%; and the in vitro bulbs with a diameter of about 1.8cm are obtained, which are transplanted after programmed cooling pretreatment to obtain Fritillaria dasyphylla dasyphylla seedlings in good condition.

[0063] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for producing Pengze Fritillaria seedlings, the cultivation steps sequentially comprising explant material collection and pretreatment, sterile explant acquisition, callus induction, callus pretreatment before redifferentiation, cluster bud proliferation, rooting culture, in vitro bulb induction, pretreatment before transplantation and transplantation; characterized in that, The specific steps include: (1) Explant collection and pretreatment: Different explant types require different collection periods. Different explants use different immersion pretreatments and low-temperature pretreatments; (2) Obtaining sterile explants: Different types of explants are sterilized using different methods to obtain sterile explants for the next step of culture; (3) callus induction: inoculating the sterile explant obtained in step (2) into a liquid shallow callus culture medium for callus induction culture to obtain callus tissue in good condition; (4) Low temperature treatment before callus redifferentiation: inoculating the callus tissue obtained in step (3) onto a solid-liquid double-layer redifferentiation medium, and then performing low temperature treatment; (5) cluster bud proliferation: inoculating the cluster buds obtained by redifferentiation in step (4) into a solid cluster bud proliferation medium for proliferation culture; (6) Rooting culture: cutting the clustered buds obtained in step (5) and inoculating them onto a solid rooting medium for rooting induction to obtain rooted seedlings; (7) In vitro bulb induction: in a season that is not suitable for transplanting, the clustered buds obtained in step (5) are cut and inoculated onto a solid in vitro bulb induction medium to induce in vitro bulbs; (8) Pretreatment before transplanting and transplanting: Before transplanting, the rooted seedlings obtained in step (6) and the in vitro bulbs obtained in step (7) are subjected to programmed cooling pretreatment, then taken out of the bottle, cleaned, soaked for pretreatment, and then transplanted and cultivated in the substrate for growth to obtain Pengze Fritillaria seedlings.

2. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (1), the different explants are divided into three categories: one category is young stem segments and leaves, the second category is bulbs in the early growth stage, and the two categories of explants are generally collected from January to March each year in northern Jiangxi; the third category is dormant bulbs, and the collection period in northern Jiangxi is generally from April to December each year.

3. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (1), when the selected explants are young stem segments and leaves and bulbs in the early growth stage, the pretreatment method is: after the explants are removed from the body, first clean them with detergent and running water. The young stem segments and leaves are wrapped with wet wipes soaked with a 0.2-0.5% carbendazim solution and then placed at 2-5°C for moisturizing and refrigeration for 0-7 days. After the bulbs in the early growth stage are cut off 0.5mm of the outer skin, they are wrapped with wet wipes soaked with a 0.2-0.5% carbendazim solution and then placed at 2-5°C for moisturizing and refrigeration for 7-14 days. When the selected explants are dormant bulbs, the pretreatment method is: after the dormant bulbs are cut off 0.5mm of the outer skin, they are wrapped with wet wipes soaked with a 0.5-1% carbendazim solution and then placed at 2-5°C for moisturizing and refrigeration for 14-30 days.

4. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (2), when the selected explants are young stem segments and leaves, the sterilization treatment method is: the pre-treated explants are disinfected with 70-75% alcohol for 10-30 seconds, then sterilized with mercuric chloride for 3-7 minutes, and then rinsed with sterile water for use; when the selected explants are bulbs in the early growth stage, the sterilization treatment method is: the pre-treated explants are disinfected with 70-75% alcohol for 1-2 minutes, then sterilized with mercuric chloride for 10-15 minutes, and then rinsed with sterile water for use. When the selected explants are dormant bulbs, the sterilization treatment method is: the pre-treated explants are disinfected with 70-75% alcohol for 1-2 minutes, then sterilized with mercuric chloride for 10-20 minutes, and then rinsed with sterile water for use.

5. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (3), the liquid shallow callus tissue culture medium formula is MS+0.01-0.5 mg / L 6-BA+1.0-3.0 mg / L NAA+0.01-2.0 mg / L TDZ+0.5-3.0 mg / L 2,4-D+30 g / L sucrose, pH 5.5-6.0; the culture conditions are: light intensity 1500-2000 lux, light 1-5 h / d, temperature 25±1°C; the liquid shallow layer thickness is 0.5-3 cm.

6. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (4), the formula of the liquid culture medium in the solid-liquid double-layer redifferentiation culture medium is MS+0.5-4.0 mg / L6-BA+0.1-1.0 mg / LNAA+0.01-1.0 mg / L TDZ+0.1-1.0 mg / L 2,4-D+30 g / L sucrose, pH 5.5-6.0, and thickness of 0.2-1.0 cm; the lower layer of the solid-liquid double-layer redifferentiation culture medium is a solid culture medium with 6.5 g / L agar added, and the rest of the formula is consistent with the upper liquid culture medium, thereby forming a solid-liquid double-layer culture medium; the culture conditions are: light intensity 1500-2000 lux, light 5-10 h / d; low temperature treatment temperature 5-10 ° C, treatment time 7-14 days. Then transfer to the temperature of 25±1 ° C for redifferentiation induction.

7. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (5), the solid cluster bud proliferation culture medium formula is MS+1.0~3.0mg / L 6-BA+0.1~0.5mg / L NAA+0.01~0.1mg / L 2,4-D+30g / L sucrose+6.5g / L agar, pH5.5~6.0; the culture conditions are: light intensity 3000~5000lux, light 8~12h / d, temperature 25±1℃.

8. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (6), the solid rooting culture medium formula is 1 / 2MS or MS+0.1-1.0mg / L NAA+0.1-3.0mg / LIBA+30g / L sucrose+0.1-1.0g / L activated carbon+6.5g / L agar, pH5.5-6.0; the culture conditions are: light intensity 3000-5000lux, light 8-12h / d, temperature 25±1°C.

9. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (7), the solid in vitro bulb induction culture medium is formulated as 1 / 2MS or MS+0.1-1.0mg / LNAA+0.1-3.0mg / L IBA+1.0-5.0mg / L PP333+50-120g / L sucrose+0.1-1.0g / L activated carbon+6.5g / L agar, pH 5.5-6.0; the culture conditions are: light intensity 5000-6000lux, light 12-16h / d, temperature 25±1°C.

10. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (8), in the pretreatment before transplanting, the rooted seedlings obtained in step (6) and the in vitro bulbs with bottles obtained in step (7) are subjected to programmed cooling pretreatment, and the process of programmed cooling treatment is: first, the culture temperature is reduced from 25°C to 20°C, and the treatment is carried out for 3 to 5 days, and then further reduced to 15°C, and the treatment is carried out for 3 to 5 days, and then further reduced to 10°C, and the treatment is carried out for 3 to 5 days, and finally reduced to 5°C, and the treatment is carried out for 3 to 5 days.

11. The method for producing Pengze Fritillaria seedlings according to claim 1, characterized in that: In step (8), the rooted seedlings or in vitro bulbs treated with programmed cooling are cleaned after being taken out of the bottle, and then immersed in a solution of carbendazim with a concentration of 0.2-0.5% for 1-3 hours. The rooted seedlings or in vitro bulbs are then transplanted into a substrate with a ratio of peat:perlite=1-3:1, appropriately shaded, with humidity maintained at 80-90% in the early stage and 70-80% in the later stage, and the light intensity during cultivation is maintained at 2000-3000 lux in the early stage and 3000-5000 lux in the later stage, and the temperature is 5-20°C, thereby obtaining Pengze Fritillaria seedlings.

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