Acclimatization and transplantation method for bulbus fritillariae cirrhosae tissue culture seedlings
By combining indoor hardening-off and field cultivation with treatment with neomycin sulfate and brassinolide, the problems of slow growth and poor disease resistance of Fritillaria cirrhosa tissue culture seedlings during transplantation were solved, achieving high survival rate and rapid growth, and supporting the large-scale production of Fritillaria cirrhosa.
Patent Information
- Application Number
- CN202511969051.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-03-06
AI Technical Summary
Fritillaria cirrhosa tissue culture seedlings have problems such as slow growth, poor disease resistance and low survival rate during transplantation, especially due to congenital physiological defects, insufficient root development and poor environmental adaptability.
A combination of indoor hardening-off and field cultivation was used, with soil treated with neomycin sulfate and brassinolide solution, along with specific light conditions and sterilization treatment, to promote the growth and disease resistance of tissue culture seedlings and improve their survival rate.
It significantly improved the survival rate and growth rate of Fritillaria cirrhosa tissue culture seedlings, solved the problems of slow growth and weak disease resistance after transplanting, and provided stable technical support for industrial production.
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Figure CN121605910A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant hardening and transplanting technology, specifically relating to a method for hardening and transplanting tissue culture seedlings of Fritillaria cirrhosa. Background Technology
[0002] Hardening and transplanting of Fritillaria cirrhosa tissue culture seedlings is a key technical link between laboratory tissue culture production and large-scale field planting. Its core lies in enabling tissue culture seedlings to gradually adapt to the natural environment through scientific acclimatization and transition, ultimately achieving high survival rates in field planting.
[0003] Currently, this technology system faces several key challenges: First, tissue culture seedlings formed under sterile conditions have inherent physiological defects, with loose stomatal structures and incomplete development of the leaf epidermal wax layer. This makes them highly sensitive to external stimuli such as strong light, drought stress, and pathogen infection. During hardening-off and transplanting, leaf scorching, rapid wilting, and even the death of the entire plant are common, directly affecting the transplant survival rate. Second, the primary roots formed in test tubes are mostly short and thick roots without root hairs. After transplanting, it is necessary to quickly induce the germination and extension of new roots to restore the ability to absorb water and nutrients. However, current technologies have not yet deeply explored the regulatory mechanisms of rhizosphere microbial communities. Rhizosphere microecological imbalance often leads to insufficient root water absorption, further exacerbating the weak growth after transplanting. Finally, tissue culture seedlings generally suffer from insufficient chlorophyll synthesis capacity and low root activity, resulting in slow accumulation of photosynthetic products. At the same time, they have low activity of immune-related enzymes, weak disease resistance, and are susceptible to soil-borne diseases such as damping-off and root rot. In addition, the drastic environmental changes during transplanting can easily induce transplant shock, and coupled with insufficient root development, the initial survival rate is significantly lower than that required for industrial production, which seriously restricts the benefits of large-scale production.
[0004] Achieving a smooth transition and high survival rate for Fritillaria cirrhosa tissue culture seedlings from the laboratory to the field, and thus realizing the large-scale, standardized, and sustainable cultivation of Fritillaria cirrhosa, is one of the technical challenges that need to be addressed in this field. Summary of the Invention
[0005] The problem this invention aims to solve is to provide a method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings, in order to address the issues of slow growth, poor disease resistance, and low survival rate during the transplantation process.
[0006] The technical solution adopted to solve this technical problem is a method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings, which includes the following steps: The tissue culture seedlings of Fritillaria cirrhosa are hardened in the room along with the culture medium, then taken out and sterilized, and transferred to the field soil for field cultivation, thus realizing the hardening and transplanting of the tissue culture seedlings of Fritillaria cirrhosa. Neomycin sulfate solution and brassinolide solution were added to the soil in the field.
[0007] Preferably, the molar concentration of neomycin sulfate solution is 2-4 mmol / L; and the mass concentration of brassinolide solution is 0.5-2 mg / L.
[0008] More preferably, the molar concentration of neomycin sulfate solution is 3 mmol / L; and the mass concentration of brassinolide solution is 1 mg / L.
[0009] More preferably, per 1m 2 Apply 0.8-1.2 L of neomycin sulfate solution and 0.8-1.2 L of brassinolide solution to the soil in the field.
[0010] More preferably, per 1m 2 Apply 1L of neomycin sulfate solution and 1L of brassinolide solution to the soil in the field.
[0011] Preferably, the indoor hardening conditions are: temperature of 14~18℃, humidity of 80~90%, and alternating 16h light and 8h dark culture with white light of 1200~1800 lux for 7~9 days.
[0012] More preferably, the indoor hardening conditions are: a temperature of 16℃, a humidity of 85%, and alternating 16h light and 8h dark culture with white light of 1500 lux for 7 days.
[0013] Preferably, sterilization is performed by soaking in a carbendazim solution for 10-15 minutes.
[0014] More preferably, sterilization is performed by soaking in a carbendazim solution for 12 minutes.
[0015] The preferred field cultivation conditions are: in addition to natural light, red and white light with an intensity of 1200~1500 lux is added for 10~14 hours per day for 10~20 days of cultivation.
[0016] More preferably, the field cultivation conditions are as follows: in addition to natural light, red and white light with an intensity of 1500 lux is added for 12 hours a day for 15 days.
[0017] More preferably, the ratio of white light to red light in the red-white light is 5:1.
[0018] The present invention has the following beneficial effects: The present invention provides a method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings. First, the tissue culture seedlings, along with the culture medium, are hardened indoors. This allows the seedlings to smoothly transition after being removed from the culture container, relying on their original nutrients and water, significantly mitigating the stress response caused by sudden environmental changes and thus greatly improving the survival rate in the early stages of transplanting. Next, the seedlings are sterilized and transplanted into soil containing neomycin sulfate and brassinolide for further cultivation. This achieves the technical effects of disease resistance, antibacterial activity, stimulation of bulb enlargement, and promotion of plant growth. Ultimately, this method effectively solves the problems of slow growth, weak disease resistance, and low survival rate of Fritillaria cirrhosa tissue culture seedlings after transplanting, providing stable and reliable technical support for industrial production. Attached Figure Description
[0019] Figure 1 This is a diagram showing the state of Fritillaria cirrhosa tissue culture seedlings after indoor hardening-off; Figure 2 This is a diagram showing the state of Fritillaria cirrhosa tissue culture seedlings after indoor hardening-off and their transfer to field soil. Figure 3 A diagram showing the state of Fritillaria cirrhosa tissue culture seedlings being cultivated in the field; Figure 4 A diagram showing the state of Fritillaria bulbs harvested after 110 days of cultivation. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of this invention, and not all of them.
[0021] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.
[0022] The features and performance of the present invention will be further described in detail below with reference to embodiments.
[0023] In both the examples and experimental cases, the field cultivation was carried out at the Fritillaria cirrhosa planting base in Linchuan Township, Huzhu Tu Autonomous County, Qinghai Lvkang Biological Development Co., Ltd.
[0024] Example 1 A method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings includes the following steps: (1) The root length of Fritillaria cirrhosa tissue culture seedlings that have sprouted and have 1-2 cm in length, together with the culture medium, were hardened off indoors at a temperature of 16℃ and a humidity of 85% under white light with an intensity of 1500 lux. The seedlings were cultured for 7 days with alternating light and darkness for 16 hours and 8 hours. (2) Remove the Fritillaria cirrhosa tissue culture seedlings that have been hardened off indoors from the culture medium, soak them in carbendazim solution for 12 minutes, then blot dry the surface moisture with filter paper, and transplant them into the field soil, spacing them per 1m. 2 1L of neomycin sulfate solution with a molar concentration of 3mmol / L and 1L of brassinolide solution with a mass concentration of 1mg / L were applied to the soil in the field. In addition to natural light, red and white light with an intensity of 1500 lux was added for 12 hours of daily irradiation. After 15 days of field cultivation, the tissue culture seedlings of Fritillaria cirrhosa were hardened off and transplanted. The ratio of white light to red light in the red and white light was 5:1.
[0025] The condition of Fritillaria cirrhosa tissue culture seedlings after indoor hardening is shown in the figure. Figure 1 The state of Fritillaria cirrhosa tissue culture seedlings after indoor hardening-off and transfer to field soil is shown in the following figure. Figure 2 See the image showing the condition of Fritillaria cirrhosa tissue culture seedlings in the field. Figure 3 .
[0026] Example 2 A method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings includes the following steps: (1) The root length of Fritillaria cirrhosa tissue culture seedlings that have sprouted and have 1-2 cm in length, together with the culture medium, were hardened off indoors at a temperature of 14℃ and a humidity of 90% under white light with an intensity of 1200 lux. The seedlings were cultured for 7 days with alternating light and darkness for 16 hours and 8 hours. (2) Remove the Fritillaria cirrhosa tissue culture seedlings that have been hardened off indoors from the culture medium, soak them in carbendazim solution for 10 minutes, then blot off the surface moisture with filter paper, and transplant them into the field soil, spacing them per 1m. 2 0.8L of 2mmol / L neomycin sulfate solution and 0.8L of 0.5mg / L brassinolide solution were applied to the field soil. In addition to natural light, red and white light with an intensity of 1200 lux was added for 14 hours daily. After 10 days of field cultivation, the tissue culture seedlings of Fritillaria cirrhosa were hardened off and transplanted. The ratio of white light to red light in the red and white light was 5:1.
[0027] Example 3 A method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings includes the following steps: (1) The root length of Fritillaria cirrhosa tissue culture seedlings that have sprouted and have 1-2 cm in length, together with the culture medium, were hardened off indoors at a temperature of 18℃ and a humidity of 80% under white light with an intensity of 1800 lux. The seedlings were cultured alternately for 7 days with 16 hours of light and 8 hours of darkness. (2) Remove the Fritillaria cirrhosa tissue culture seedlings that have been hardened off indoors from the culture medium, soak them in carbendazim solution for 15 minutes, then blot off the surface moisture with filter paper, and transplant them into the field soil, spacing them per 1m. 2 1.2 L of 4 mmol / L neomycin sulfate solution and 1.2 L of 2 mg / L brassinolide solution were applied to the soil in the field. In addition to natural light, red and white light with an intensity of 1500 lux was added for 14 hours every day. After 20 days of field cultivation, the tissue culture seedlings of Fritillaria cirrhosa were hardened off and transplanted. The ratio of white light to red light in the red and white light was 5:1.
[0028] Experimental Example 1. Screening of indoor hardening-off time Tissue culture seedlings of Fritillaria cirrhosa with roots 1-2 cm in length and already emerged were hardened off indoors at 16℃ and 85% humidity under 1500 lux of white light, alternating between 16 hours of light and 8 hours of darkness. Four different hardening times were set: treatment groups 1-4 had hardening times of 3, 5, 7, and 9 days, respectively. After hardening, the seedlings were transferred to field soil for 15 days of field culture. The growth status and survival rate of the tissue culture seedlings were recorded, and the results are shown in Table 1.
[0029] Table 1. Effects of different hardening times on the growth status and survival rate of Fritillaria cirrhosa tissue culture seedlings.
[0030] As shown in Table 1, the Fritillaria cirrhosa tissue culture seedlings with a hardening period of 7 days showed good growth and the highest survival rate, reaching 75.3%; the seedlings transplanted with a hardening period of too short a period showed poor growth and were more difficult to survive.
[0031] 2. Screening of field cultivation light sources After indoor hardening-off, the tissue culture seedlings were removed and soaked in a 1:1000 carbendazim solution for 12 minutes. The surface moisture was then absorbed with filter paper, and the seedlings were transplanted into field soil for field cultivation. In addition to natural light, five different light source conditions were added, each with a light intensity of 1500 lux and a daily light duration of 12 hours. Treatment groups 1-6 were natural light, natural light + white light, natural light + white light:red light = 5:1, natural light + red light:blue light = 5:1, natural light + red light:blue light = 1:1, and natural light + red light:blue light = 1:5, respectively. After 15 days of cultivation, the survival rate of the Fritillaria cirrhosa tissue culture seedlings was recorded. The survival rates of the Fritillaria cirrhosa tissue culture seedlings under different light sources are shown in Table 2.
[0032] Table 2. Effects of different light sources on the survival rate of Fritillaria cirrhosa tissue culture seedlings
[0033] As shown in Table 2, the survival rate of treatment group 3 (natural light + white light: red light = 5:1) was the highest, reaching 82.3% compared to natural light. This indicates that an appropriate amount of red light supplementation can enhance photosynthetic efficiency and promote seedling growth. However, an excessively high proportion of red light will lead to a decrease in the survival rate, as excessive red light promotes stem elongation while neglecting root development, thus affecting overall health.
[0034] 3. Screening of soil additives After the indoor hardening-off period, the tissue culture seedlings were taken out and soaked in a 1:1000 carbendazim solution for 12 minutes. The surface moisture of the tissue culture seedlings was then absorbed with filter paper, and they were transplanted into field soil for field cultivation. A white light:red light ratio of 5:1 with an intensity of 1500 lux was added for cultivation, with a daily light duration of 12 hours. After 15 days of cultivation, the survival rate of the Fritillaria cirrhosa tissue culture seedlings was recorded.
[0035] Four treatment groups were set up. Treatment groups 1-4 were respectively treated with tap water, 3 mmol / L neomycin sulfate solution, 1 mg / L brassinolide solution, and 3 mmol / L neomycin sulfate solution + 1 mg / L brassinolide solution. The treatments were applied per 1 m³ of water. 2 1 L of 3 mmol / L neomycin sulfate solution and 1 L of 1 mg / L brassinolide solution were applied to the field soil. After 15 days of culture, the survival rate of Fritillaria cirrhosa tissue culture seedlings was recorded. The survival rates of the four treatment groups are shown in Table 3. Mature bulbs were harvested at 110 days. Figure 4 As shown.
[0036] Table 3. Effects of different soil substrate additives on the survival rate of Fritillaria cirrhosa tissue culture seedlings
[0037] Table 3 shows that adding brassinolide alone did not significantly improve the survival rate of Fritillaria cirrhosa tissue culture seedlings, while adding neomycin sulfate alone improved the survival rate. The combined use of both resulted in the highest survival rate, reaching 95.0%. The mechanism is that neomycin sulfate reduces phospholipase activity, thus inhibiting bacterial growth and improving the disease resistance of the seedlings. On this basis, brassinolide accelerates the growth rate of the seedlings, enabling them to complete bulb renewal more quickly. The combined use of both resulted in a high survival rate for the Fritillaria cirrhosa tissue culture seedlings.
[0038] The present invention has been described according to the above embodiments. It should be understood that the above embodiments do not limit the present invention in any way. All technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of the present invention.
Claims
1. A method for hardening off and transplanting tissue culture seedlings of Fritillaria cirrhosa, characterized in that, The method comprises the following steps: The Fritillaria delavayi Hance tissue culture seedlings are acclimatized in a room together with the culture medium, then taken out for sterilization and transferred to the field soil for field culture, so as to realize the acclimatization and transplanting of the Fritillaria delavayi Hance tissue culture seedlings; The neomycin sulfate solution and the brassinolide solution are added to the field soil.
2. The method for transplanting the Fritillaria cirrhosa meristem seedlings after acclimatization according to claim 1, characterized in that, The molar concentration of the neomycin sulfate solution is 2-4 mmol / L, and the mass concentration of the brassinolide solution is 0.5-2 mg / L.
3. The method for transplanting the Fritillaria cirrhosa meristem seedlings of claim 2, characterized in that, The per 1 m 2 0.8-1.2 L of a solution of neomycin sulfate and 0.8-1.2 L of a solution of brassinolide are applied to the field soil.
4. The method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings as described in claim 1, characterized in that, The acclimatization conditions in the room are as follows: the temperature is 14-18 DEG C, the humidity is 80-90%, and the white light with an intensity of 1200-1800 lux is used for 16h light and 8h darkness alternating culture for 7-9d.
5. The method for transplanting the Fritillaria cirrhosa meristem seedlings after acclimatization according to claim 1, characterized in that, The sterilization is performed by immersing the Fritillaria delavayi Hance tissue culture seedlings in a carbendazim solution for 10-15 min.
6. The method for transplanting the Fritillaria cirrhosa meristem seedlings after acclimatization according to claim 1, characterized in that, The field culture conditions are as follows: on the basis of natural light, the red and white light with an intensity of 1200-1500 lux is additionally used for 10-14h daily illumination, and the culture is performed for 10-20d.
7. The method for hardening and transplanting Fritillaria cirrhosa tissue culture seedlings as described in claim 6, characterized in that, The ratio of the white light to the red light in the red and white light is 5:1.