Culture method for promoting rapid germination and seedling formation of ice hockey seeds
By using specific culture media and light conditions in the sterile culture of ice hockey seeds, the problems of low germination rate and weak seedling growth of ice hockey seeds are solved, and rapid germination of seeds and rapid differentiation and development of seeds are achieved.
Patent Information
- Application Number
- CN202510053436.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-05-23
AI Technical Summary
The germination rate of ice hockey seeds is low, the germination time is long, and the seedlings grow weakly, so it is difficult for the existing technology to effectively solve these problems.
The sterile culture was carried out using specific culture medium, light intensity and photoperiod conditions, including the use of 1/2MS culture medium, the addition of 6-BA and NAA phytohormones, sucrose 13-17g/L, activated carbon 0.4-0.6g/L, and 2500Lx light culture was carried out after 2W of dark culture.
The germination rate of ice hockey seeds and the rapid differentiation and development of seedlings have been significantly improved, and the effect of rapid germination of ice hockey seeds into seedlings has been achieved.
Smart Images
Figure CN120021554A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of seed biology and the technical field of medicinal plant biotechnology, and specifically involves a sterile culture method for promoting the rapid germination and seedling formation of ice ball seeds by adopting specific culture medium, light intensity and photoperiod conditions. Background Art
[0002] The source plants of the ice ball are Pleione yunnanensis (Rolfe) Rolfe and Pleione bulbocodioides (Franch.) Rolfe of the Orchidaceae genus. The two are plants of the same genus and have similar growth and development habits.
[0003] The propagation methods of ice ball seeds include asexual propagation and sexual propagation. Asexual propagation mainly uses pseudobulbs as materials for division propagation, but it has disadvantages such as low proliferation coefficient, easy degeneration of species, and high production cost; sexual propagation uses seeds as materials, through tissue culture technology, sterile germination, and production of tissue culture seedlings, but it has disadvantages such as low seed germination rate, long germination time, and long production cycle.
[0004] The mature capsule of ice ball contains a large number of seeds, which are a mass of undifferentiated embryonic cells, with underdeveloped or incomplete endosperm. The seeds have poor water and air permeability and almost do not germinate under natural conditions. In a sterile environment, by adding various nutrients and plant hormones, seed germination and differentiation can be promoted under suitable conditions.
[0005] In addition to photosynthesis, plants' responses to light also involve photomorphogenesis, including light-requiring germination of seeds. Currently, research on P. glaucosa seeds mainly focuses on culture medium composition, germination characteristics, and symbiotic germination, but no relevant research has been conducted on the role of sucrose, activated carbon in the culture medium, and light in the germination process.
[0006] The inventors have invented a method for rapidly germinating Pseudomonas aeruginosa seeds by screening the concentrations of sucrose and activated carbon in the culture medium, systematically studying the light intensity and photoperiod, and combining the characteristics of seed germination. Summary of the invention
[0007] The technical problem to be solved by the present invention is to overcome the problems of low germination rate, long germination time and weak seedling growth of ice ball seeds in the existing technology, thereby providing a new method that is different from other methods and can greatly promote the rapid germination and seedling formation of ice ball seeds.
[0008] The technical solution of the present invention:
[0009] A method for promoting the rapid germination of ice ball seeds into seedlings, characterized in that it comprises the following steps:
[0010] (1) Screening of suitable basic medium: Using the mature capsules of Bletilla bulbocodioides var. minor as materials, after disinfection, the seeds are taken out and sown in the basic medium. The basic medium is 1 / 2MS medium supplemented with 0.2 mg / L of 6-BA and 0.1 mg / L of NAA;
[0011] (2) Screening of suitable sucrose concentration: Using the mature capsules of Bletilla bulbocodioides var. minor as materials, after disinfection, the seeds are taken out and sown in the medium with suitable sucrose concentration. The medium with suitable sucrose concentration is 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13 - 17 g / L;
[0012] (3) Screening of suitable activated carbon concentration: Using the mature capsules of Bletilla bulbocodioides var. minor as materials, after disinfection, the seeds are taken out and sown in the medium with suitable activated carbon concentration. The medium with suitable activated carbon concentration is 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13 - 17 g / L + activated carbon 0.4 - 0.6 g / L;
[0013] (4) Screening of suitable light intensity and photoperiod: Using the mature capsules of Bletilla bulbocodioides var. minor as materials, after disinfection, the seeds are taken out and sown in the medium composed of 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13 - 17 g / L + activated carbon 0.4 - 0.6 g / L, and cultured under different light intensity and photoperiod conditions. The light intensity and photoperiod are to culture for 2W under dark culture conditions first, and then culture for 6W under 2500Lx light.
[0014] Further, in the steps (1), (2), (3), and (4), the mature capsules of Bletilla bulbocodioides var. minor are unopened capsules 150 - 180 days after pollination.
[0015] Further, the disinfection in the steps (1), (2), (3), and (4) means putting the mature unopened capsules into a mixed solution of dishwashing liquid and 84 disinfectant and soaking for 10 minutes, then using a soft brush dipped in dishwashing liquid water to brush the surface, especially the wrinkled parts of the capsule surface need to be specially cleaned, then washing with tap water and then washing once with pure water, and loading into a sterile bottle; placing the washed capsules on the ultra-clean workbench, first soaking in 75% ethanol solution for 30 seconds, washing 2 times with sterile water, then adding 2 drops of surfactant Tween 80 to 2% sodium hypochlorite solution and sterilizing for 8 - 10 minutes, and finally washing 5 times with sterile water.
[0016] Further, the taking out of the seeds in the steps (1), (2), (3), and (4) means sucking the surface moisture of the disinfected Bletilla bulbocodioides var. minor capsules with sterile filter paper, cutting the pericarp with a blade, taking out the seeds inside the capsule, and evenly sowing them on the medium.
[0017] Furthermore, in step (1), step (2) and step (3), the lighting conditions are: 2500Lx of light and 12 hours of light per day.
[0018] Furthermore, in the steps (1), (2), (3) and (4), the temperature of the culture chamber is 23-25° C. and the humidity is 70-80%.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] 1. The present invention uses the mature capsule of Psoralea corylifolia as material, and through cultivation in a specific culture medium and specific lighting conditions, improves the germination rate, promotes rapid differentiation and development of seedlings, and achieves the effect of rapid germination of Psoralea corylifolia seeds into seedlings.
[0021] 2. Take sucrose concentration as an important factor in seedling growth and development. As an important carbon source, sucrose plays an important role in the germination and growth of ice ball seeds. The determination of the appropriate concentration provides a decisive material basis for the growth of leaves and the expansion of protocorms.
[0022] 3. Use the activated carbon concentration as an important factor in the growth and development of seedlings. Activated carbon generally plays the role of adsorbing brown substances in the culture medium and providing a dark environment for the growth and development of the root system. Screening out the appropriate activated carbon concentration can effectively promote the development of the root system of ice ball seedlings and the growth of the aboveground part.
[0023] 4. Take light intensity and photoperiod as important factors for seedling growth and development. Light intensity and photoperiod play an important role in seed germination and organ development. Screening out light conditions suitable for the germination and differentiation of ice ball seeds is important for promoting rapid seedling formation. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] In order to clearly present the purpose, technical solution and beneficial effects of the present invention, the present invention provides the following drawings for illustration:
[0025] Figure 1 The growth conditions of the ice ball seeds in Example 1 after being cultured in different basic culture media for 120 days.
[0026] Figure 2 This is the growth condition of the ice ball seeds in Example 4 after being cultured for 60 days under different light intensities and light cycles.
[0027] Figure 3 This is the growth condition of the ice ball seeds in Example 4 when cultured for 120 days under different light intensities and light cycles. DETAILED DESCRIPTION
[0028] The present invention will be described in detail below with reference to embodiments.
[0029] Example 1: Screening of suitable basic culture medium
[0030] The mature unopened capsules of ice balls were used as materials. After sterilization, the seeds were inoculated on the following basic culture medium for cultivation, and a certain amount of activated carbon and sucrose were added. 10 bottles were used for each treatment, and repeated 3 times. After inoculation, they were placed in a culture room with a temperature of 23-25℃, a light of 2500Lx, a light of 12h per day, and a humidity of 70-80%. The seed germination and the growth of the seedlings were observed on the 25th, 60th and 120th day after sowing, and the results were statistically analyzed (Table 1).
[0031] Table 1 Screening of suitable basic culture medium for ice ball
[0032]
[0033] From the statistical results, it can be seen that when the ice ball seeds were cultured to 25 days, they could germinate in all culture media. In the treatment with KC medium as the basic culture medium, the seeds differentiated more root hairs, while in the treatment with 1 / 2MS medium as the basic culture medium, the root hair differentiation was less. When cultured to 60 days, treatments 2 and 3 were relatively good, and basically differentiated leaves; when cultured to 120 days, treatment 3 continued to maintain vigorous growth and differentiated protocorms, and the overall growth was better than other treatments ( Figure 1 ). Therefore, in this scheme, the most suitable basic culture medium for P. glaucoma seeds is 1 / 2MS medium + 6-BA0.2 mg / L + NAA0.1 mg / L.
[0034] Example 2: Screening for suitable sucrose concentration
[0035] The mature unopened capsules of ice balls were used as materials. After sterilization, the seeds were inoculated on a medium containing different concentrations of sucrose for cultivation. The medium was 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + activated carbon 0.5g / L. Ten bottles were used for each treatment, and repeated 3 times. After inoculation, they were placed in a culture room with a temperature of 23-25℃, a light of 2500Lx, a light of 12h per day, and a humidity of 70-80%. The seed germination and the growth of the seedlings were observed 40 days after sowing, and the results were statistically analyzed (Table 2).
[0036] Table 2 Screening of suitable sucrose concentration for ice ball
[0037]
[0038] From the statistical results, it can be seen that the germination can be achieved within the range of sucrose concentration from 11g / L to 19g / L, with a slight difference in germination rate. When the sucrose concentration is 13g / L to 17g / L, the tissue culture seedlings differentiate into leaves and protocorms. Therefore, in this scheme, the optimal sucrose concentration for ice ball seeds is 13g / L to 17g / L.
[0039] Example 3: Screening of suitable activated carbon concentration
[0040] The mature unopened capsules of ice balls were used as materials. After sterilization, the seeds were inoculated on a culture medium containing different concentrations of activated carbon for cultivation. The culture medium was 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13-17g / L. Ten bottles were used for each treatment, and repeated 3 times. After inoculation, they were placed in a culture room with a temperature of 23-25℃, a light of 2500Lx, a light of 12h per day, and a humidity of 70-80%. The seed germination and the growth of the seedlings were observed 40 days after sowing, and the results were statistically analyzed (Table 3).
[0041] Table 3 Screening of suitable activated carbon concentration of ice ball
[0042]
[0043] From the statistical results, it can be seen that in treatment 1 without adding activated carbon, the seeds germinated very little, and the germinated seeds did not grow or differentiate. As the activated carbon concentration increased, the seed germination rate increased. The seed germination rates of treatments 3, 4 and 5 were similar, but the leaf growth and root growth of treatments 3 and 4 were more advantageous. Therefore, in this scheme, the most suitable activated carbon concentration for ice ball seeds is 0.4-0.6g / L.
[0044] Example 4: Screening for suitable light intensity and photoperiod
[0045] The mature unopened capsules of ice balls were used as materials. After sterilization, the seeds were inoculated into a medium composed of 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13-17 g / L + activated carbon 0.4-0.6 g / L. They were cultured under different light intensities and photoperiods. Ten bottles were used for each treatment and repeated three times. After inoculation, they were placed in a culture room with a temperature of 23-25°C and a humidity of 70-80%. The seed germination and the growth of the seedlings were observed at 60d and 120d after sowing, and the results were statistically analyzed (Table 4).
[0046] Table 4 Suitable light intensity and photoperiod for screening of ice ball
[0047]
[0048] From the statistical results, it can be seen that the ice ball seeds cultured in full darkness can germinate, but the germinated and grown are white transparent particles, which can differentiate into white buds; the ice ball seeds cultured with 24h / d light have less germination and weak seedling development under 1500Lx and 2500Lx light conditions; the germination and growth of seeds cultured with 12h / d light and 24h / d light are very different; under 2500Lx light conditions, the germination and development of ice ball seeds cultured in dark and light alternating treatments are good, among which the germination and seedling growth of ice ball seeds cultured in light for 6W after 2W dark culture are the best. At 60d of culture, green protocorms grow out, and root hairs, stems and leaves are differentiated. At 120d, the leaves are large and the seedlings grow well ( Figure 2 , 3 ). Therefore, in this scheme, the most suitable light conditions for the germination and growth of P. glacialis seeds are: after 2 weeks of dark culture, culture under a light intensity of 2500Lx, with a culture cycle of 12h / d and a culture period of 6 weeks.
[0049] In the above embodiments, the culture medium is 1 / 2MS culture medium + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13-17 g / L + activated carbon 0.4-0.6 g / L, and the culture lighting conditions are first cultured under dark culture conditions for 2 weeks, and then cultured under a light intensity of 2500 Lx for 6 weeks, and the culture cycle is 12 h / d.
[0050] The inventors have conducted in-depth research on the physiological characteristics and germination characteristics of ice ball seeds, and have developed a unique method that has not been discovered by others to promote the germination of ice ball seeds, which has produced unexpected technical effects. The apparatus used in the invention is simple, easy to operate, and easy to carry out factory-scale ice ball seedling production.
[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the above embodiments have described the method of the present invention in detail, those skilled in the art should understand that different changes can be made in form and details without departing from the contents defined in the claims of the present invention.
Claims
1. A method for promoting the rapid germination of ice ball seeds into seedlings, characterized in that: The following steps are involved: (1) Screening of suitable basic culture medium: The mature capsules of Psoralea corylifolia were used as materials. After disinfection, the seeds were taken out and sown in a basic culture medium. The basic culture medium was 1 / 2MS culture medium supplemented with 0.2 mg / L 6-BA and 0.1 mg / L NAA. (2) Screening for suitable sucrose concentration: The mature capsules of Psoralea corylifolia were used as materials. After disinfection, the seeds were taken out and sown in a medium with a suitable sucrose concentration. The medium with a suitable sucrose concentration was 1 / 2MS + 0.2 mg / L 6-BA + 0.1 mg / L NAA + 13-17 g / L sucrose. (3) Screening for suitable activated carbon concentration: The mature capsules of Psoralea corylifolia were used as materials. After disinfection, the seeds were taken out and sown in a medium with suitable activated carbon concentration. The medium with suitable activated carbon concentration was 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13-17 g / L + activated carbon 0.4-0.6 g / L. (4) Screening for suitable light intensity and photoperiod: The mature capsules of P. glacialis were used as materials. After disinfection, the seeds were taken out and sown in a culture medium composed of 1 / 2MS + 6-BA 0.2 mg / L + NAA 0.1 mg / L + sucrose 13-17 g / L + activated carbon 0.4-0.6 g / L. The seeds were cultured under suitable light intensity and photoperiod conditions. The suitable light intensity and photoperiod were first cultured in the dark for 2 weeks and then cultured under 2500 Lx light for 6 weeks.
2. A method for promoting rapid germination of ice ball seeds into seedlings according to claim 1, characterized in that: The mature capsules of the ice ball fruit in the steps (1), (2), (3) and (4) are unopened capsules 150 to 180 days after pollination.
3. The method for promoting rapid germination of ice ball seeds into seedlings according to claim 1, characterized in that: The disinfection in step (1), step (2), step (3) and step (4) refers to soaking the mature unopened capsule in a mixed diluted solution of detergent and 84 disinfectant for 10 minutes, then brushing the surface with a soft brush dipped in detergent water, especially the wrinkles on the surface of the capsule, and then washing it with tap water and then with pure water, and putting it into a sterile bottle; placing the washed capsule on a clean workbench, first soaking it in 75% ethanol solution for 30 seconds, washing it twice with sterile water, then adding 2 drops of surfactant Tween 80 to 2% sodium hypochlorite solution, sterilizing it for 8-10 minutes, and finally washing it with sterile water for 5 times.
4. A method for promoting rapid germination of ice ball seeds into seedlings according to claim 1, characterized in that: The steps (1), (2), (3) and (4) of taking out the seeds refer to absorbing the moisture on the surface of the sterilized ice ball capsule with sterile filter paper, cutting the peel with a blade, taking out the seeds in the capsule, and evenly sowing them on the culture medium.
5. The method for promoting rapid germination of ice ball seeds into seedlings according to claim 1, characterized in that: In the steps (1), (2) and (3), the lighting conditions are: 2500 Lx of light and 12 hours of light per day.
6. A method for promoting rapid germination of ice ball seeds into seedlings according to claim 1, characterized in that: In the steps (1), (2), (3) and (4), the temperature of the culture chamber is 23-25° C. and the humidity is 70-80%.