Method for inducing loose callus of idesia polycarpa
Through the method of inducing the formation of loose callus tissue by primary rhizomes after sterile germination of mountain tung seeds, the problem of immature breeding and breeding technology of mountain tung seeds is solved, the reproduction coefficient is improved and the growth cycle is shortened, and technical support is provided for the high-quality development of the industry.
Patent Information
- Application Number
- CN202411536025.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-31
- Publication Date
- 2025-04-11
AI Technical Summary
The lack of mountain tung tung seed resource evaluation system and immature breeding technology have led to lag in the development of mountain tung seed industry, seriously affecting its high-quality development.
The primary rhizome obtained after germination of sterile seeds of tung tung seeds was used as explant material, and the formation of compact callus was induced by tissue culture technology, and the formation of loose callus was further induced.
It improves the reproduction coefficient of mountain tung tung seeds, shortens its growth cycle, and lays the foundation for the genetic transformation of important trait genes, and realizes the creation of good varieties and factory seedling cultivation.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of plant tissue culture and rapid propagation, and in particular to a method for inducing loose callus of Castanopsis japonicifolia. Background Art
[0002] Idesia polycarpa Maxim. is a deciduous tree of the genus Idesia in the family Flacourtiaceae. It is also known as mountain sycamore, half-frost red, oil grape, etc. It is rich in nutrients, linoleic acid, linolenic acid, unsaturated fatty acids, phenols, flavonoids, rare elements and other beneficial ingredients. It has the effects of improving human blood lipid metabolism, softening blood vessels, and resisting thrombosis. Idesia polycarpa is a pure natural woody edible oil.
[0003] There are prominent problems such as the lack of a resource evaluation system for Castanopsis chinensis, immature variety selection and breeding technology, difficulty in distinguishing males and females at the seedling stage, unstable seedling traits, and yet to be discovered genes for important traits. These problems have led to the lagging development of the Castanopsis chinensis industry and seriously affected the high-quality development of the industry.
[0004] In the natural state, the reproduction of Castanopsis truncatum mainly relies on bird dissemination. This method has a high chance of seed germination and seedling growth, because the seed coat of Castanopsis truncatum seeds is hard and has a waxy surface, and the dormancy period is not easy to break. How to improve the breeding technology of improved varieties, realize the homologous transformation and functional verification of the gene mining of important traits of Castanopsis truncatum, and use molecular breeding technology to achieve high-quality and efficient breeding of Castanopsis truncatum is imminent. Summary of the invention
[0005] The purpose of the present invention is to provide a method for inducing loose callus of Castanopsis japonici. The present invention has the characteristics of being able to effectively improve the reproduction coefficient of Castanopsis japonici, shorten its growth cycle, and lay a foundation for the genetic transformation of important trait genes of Castanopsis japonici.
[0006] The technical solution of the present invention:
[0007] A method for inducing loose callus of Castanopsis schrenkiana, using primary rhizomes obtained after aseptic germination of Castanopsis schrenkiana seeds as explant materials, then inducing compact callus through tissue culture, and further inducing to form loose callus, specifically comprising the following steps:
[0008] (1) Obtaining primary rhizome explants: After sterilizing, take the seeds of Castanopsis sylvestris and add GA3 to heat them in a water bath. Then, under a sterile environment, sow the seeds in a culture bottle supported by cotton and culture them in a dark room to germinate and obtain primary rhizome explants.
[0009] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 22±3°C, light intensity 2100-2500 lx and light cycle 11-13 h / d. After 8-30 days of culture, the primary rhizome enlarges and the color changes from white-green to light yellow, thereby obtaining compact callus;
[0010] (3) Inducing loose callus tissue: The compacted callus tissue in step (2) is inoculated into a modified MS medium with a pH value of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator are 22±3°C, a light intensity of 2100-2500 lx and a light cycle of 11-13 h / d. After 8-30 days of culture, loose callus tissue is induced to form.
[0011] In the above step (1), the disinfection treatment method is to soak the Castanopsis schrenkiana seeds in concentrated sulfuric acid for 6-10 minutes, with the amount of concentrated sulfuric acid used being preferably enough to submerge the seeds, and then wash with water until no concentrated sulfuric acid remains.
[0012] Specifically, in the above step (1), the disinfection treatment method is to soak the Castanopsis chinensis seeds in concentrated sulfuric acid for 8 minutes, with the amount of concentrated sulfuric acid used being preferably enough to submerge the seeds, and then wash with water until no concentrated sulfuric acid remains.
[0013] In the above step (1), the concentration of GA3 is 180-220 mg / L, the water bath temperature is 35-45°C, the water bath is heated for 1.5-2.5 hours, and the amount of GA3 used is preferably enough to submerge the seeds.
[0014] Specifically, in the aforementioned step (1), the concentration of GA3 is 200 mg / L, the water bath temperature is 40° C., the water bath is heated for 2 h, and the amount of GA3 used is preferably enough to submerge the seeds.
[0015] In the aforementioned step (2), loose callus tissue is induced: the compacted callus tissue in step (2) is inoculated into a modified MS medium, pH = 5.2-5.6, and placed in a light incubator for light culture. The culture conditions in the light incubator are 22±2°C, light intensity 2500lx and light cycle 12h / d. After 10 days of culture, loose callus tissue is induced.
[0016] In the above step (2), the culture medium for inducing compacted callus tissue consists of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, with a pH of 5.8.
[0017] In the above step (3), loose callus tissue is induced: the compacted callus tissue in step (2) is inoculated into a modified MS medium, pH = 5.2-5.6, and placed in a light incubator for light culture. The cultivation conditions in the light incubator are 22±2°C, light intensity 2500lx and light cycle 12h / d. After 10 days of culture, loose callus tissue is induced.
[0018] In the above step (3), the improved MS medium is composed of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 10-25 g / L sucrose + 5 g / L agar, pH = 5.4.
[0019] Specifically, in the aforementioned step (3), the improved MS medium is composed of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 15 g / L sucrose + 5 g / L agar, pH = 5.4.
[0020] Compared with the prior art, the present invention has the following beneficial effects:
[0021] Since Castanopsis truncatum is a perennial tall tree, it is difficult to select and create improved varieties, the seeds are dormant, the chance is very high, the vegetative reproduction species are degraded, the pests and diseases are serious, the growth cycle is long, the reproduction coefficient is low, and the market demand cannot be met. The present invention induces loose callus tissue through primary rhizomes. In the loose callus tissue induction stage, the loose callus tissue induction rate is as high as 81.48%, and the value-added multiple reaches 5.8; this method can provide a large amount of test materials for subsequent seedling breeding, and at the same time lays a good foundation for molecular breeding and homology verification of important trait gene functions, aiming to provide technical support for the creation of Castanopsis truncatum improved varieties, factory seedling cultivation and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 :Effects of concentrated sulfuric acid treatment on germination of Castanopsis chinensis seeds;
[0023] Figure 2 :Effects of different sucrose concentrations on the induction and proliferation of loose callus;
[0024] Figure 3 : Seed germination external stage;
[0025] Figure 4 : Callus compaction stage;
[0026] Figure 5 : Loose callus stage. DETAILED DESCRIPTION
[0027] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.
[0028] Embodiment 1:
[0029] (1) Obtaining primary rhizome explants: Take the seeds of Castanopsis chinensis and soak them in concentrated sulfuric acid for 8 minutes. The amount of concentrated sulfuric acid used should be enough to submerge the seeds. After soaking, wash them with water until there is no residual concentrated sulfuric acid. Then, add 200 mg / L GA3 and heat them in a water bath at 40°C for 2 hours. The amount of GA3 used should be enough to submerge the seeds. Then, sow the seeds in a culture bottle with cotton as a support under a sterile environment, place them in a dark room for culture, and germinate to obtain primary rhizome explants.
[0030] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 22° C., a light intensity of 2500 lx, and a light cycle of 12 h / d. After 10 days of culture, the primary rhizome increases in size and changes in color from white-green to light yellow, thereby obtaining compact callus;
[0031] The medium for inducing compact callus consisted of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH = 5.8;
[0032] (3) Inducing loose callus: The compacted callus in step (2) was inoculated into a modified MS medium with a pH of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator were 22±2°C, a light intensity of 2500 lx and a light cycle of 12 h / d. After 10 days of culture, loose callus was induced to form.
[0033] The improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+15 g / L sucrose+5 g / L agar, pH=5.4.
[0034] Embodiment 2:
[0035] (1) Obtaining primary rhizome explants: Take the seeds of Castanopsis chinensis and soak them in concentrated sulfuric acid for 6 minutes. The amount of concentrated sulfuric acid used should be enough to submerge the seeds. After soaking, wash them with water until there is no residual concentrated sulfuric acid. Then add 180 mg / L GA3 and heat them in a water bath at 35°C for 1.5 hours. The amount of GA3 used should be enough to submerge the seeds. Then, sow the seeds in a culture bottle with cotton as a support under a sterile environment, place them in a dark room for culture, and germinate to obtain primary rhizome explants.
[0036] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 25° C., a light intensity of 2100 lx, and a light cycle of 11 h / d. After 8 days of culture, the primary rhizome increases in size and changes in color from white-green to light yellow, thereby obtaining compact callus;
[0037] The medium for inducing compact callus consisted of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH = 5.8;
[0038] (3) Inducing loose callus: The compacted callus in step (2) was inoculated into a modified MS medium with a pH of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator were 22±3°C, a light intensity of 2100 lx and a light cycle of 11 h / d. After 8 days of culture, loose callus was induced to form.
[0039] The improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+10 g / L sucrose+5 g / L agar, pH=5.4.
[0040] Embodiment 3:
[0041] (1) Obtaining primary rhizome explants: Take the seeds of Castanopsis chinensis and soak them in concentrated sulfuric acid for 10 minutes. The amount of concentrated sulfuric acid used should be enough to submerge the seeds. After soaking, wash them with water until there is no residual concentrated sulfuric acid. Then add 220 mg / L GA3 and heat them in a water bath at 45°C for 2.5 hours. The amount of GA3 used should be enough to submerge the seeds. Then, sow the seeds in a culture bottle supported by cotton in a sterile environment and culture them in a dark room to obtain primary rhizome explants after germination.
[0042] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 19° C., a light intensity of 2400 lx, and a light cycle of 13 h / d. After 30 days of culture, the primary rhizome increases in size and changes in color from white-green to light yellow, thereby obtaining compact callus;
[0043] The medium for inducing compact callus consisted of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH = 5.8;
[0044] (3) Inducing loose callus: The compacted callus in step (2) was inoculated into a modified MS medium with a pH of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator were 19° C., a light intensity of 2100 lx, and a light cycle of 11 h / d. After 30 days of culture, loose callus was induced to form.
[0045] The improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+25 g / L sucrose+5 g / L agar, pH=5.4.
[0046] Embodiment 4:
[0047] (1) Obtaining primary rhizome explants: Take the seeds of Castanopsis chinensis and soak them in concentrated sulfuric acid for 7 minutes. The amount of concentrated sulfuric acid used should be enough to submerge the seeds. After soaking, wash them with water until there is no residual concentrated sulfuric acid. Then add 210 mg / L GA3 and heat them in a water bath at 42°C for 2.2 hours. The amount of GA3 used should be enough to submerge the seeds. Then, sow the seeds in a culture bottle supported by cotton in a sterile environment and culture them in a dark room to obtain primary rhizome explants after germination.
[0048] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 20° C., a light intensity of 2300 lx, and a light cycle of 12 h / d. After 15 days of culture, the primary rhizome increases in size and changes in color from white-green to light yellow, thereby obtaining compact callus;
[0049] The medium for inducing compact callus consisted of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH = 5.8;
[0050] (3) Inducing loose callus: The compacted callus in step (2) was inoculated into a modified MS medium with a pH of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator were 20° C., a light intensity of 2300 lx, and a light cycle of 12 h / d. After 15 days of culture, loose callus was induced to form.
[0051] The improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+15 g / L sucrose+5 g / L agar, pH=5.4.
[0052] Embodiment 5:
[0053] (1) Obtaining primary rhizome explants: Take the seeds of Castanopsis chinensis and soak them in concentrated sulfuric acid for 9 minutes. The amount of concentrated sulfuric acid used should be enough to submerge the seeds. After soaking, wash them with water until there is no residual concentrated sulfuric acid. Then, add 190 mg / L GA3 and heat them in a water bath at 38°C for 1.8 hours. The amount of GA3 used should be enough to submerge the seeds. Then, sow the seeds in a culture bottle with cotton as a support under a sterile environment, place them in a dark room for culture, and germinate to obtain primary rhizome explants.
[0054] (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 24° C., a light intensity of 2200 lx, and a light cycle of 12 h / d. After 12 days of culture, the primary rhizome increases in size and changes in color from white-green to light yellow, thereby obtaining compact callus;
[0055] The medium for inducing compact callus consisted of MS + 1.0 mg / L 6-BA + 1.0 mg / L NAA + 30 g / L sucrose + 7 g / L agar, pH = 5.8;
[0056] (3) Inducing loose callus: The compacted callus in step (2) was inoculated into a modified MS medium with a pH of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator were 24° C., a light intensity of 2200 lx, and a light cycle of 12 h / d. After 12 days of culture, loose callus was induced to form;
[0057] The improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+20 g / L sucrose+5 g / L agar, pH=5.4.
[0058] The inventors conducted a large number of experiments, and the following are some of the experimental studies
[0059] Experimental Example 1: Sterile Seed Germination
[0060] The plant material Idesia polycarpa Maxim. in the present invention was collected from Xingyi City, Guizhou Province and used as the material for aseptic germination.
[0061] The mature ash seeds were cleaned and dried, and then soaked in concentrated sulfuric acid. The effects of different soaking times on breaking the dormancy of ash seeds were compared. After 30 days, the germination rate of the seeds was calculated (germination rate = (number of germinated seeds / total number of tests) × 100%). Conclusion: Soaking ash seeds in concentrated sulfuric acid can promote their germination. The optimal soaking time is 8 minutes, and the germination rate is high, reaching 58.0% ( Figure 1 ), the primary rhizomes grow vigorously.
[0062] Experimental Example 2: Induction of compacted callus
[0063] The primary rhizomes formed by seed germination in Experimental Example 1 were used as explants and inoculated into MS medium supplemented with plant growth regulators of different types and concentrations. A two-factor combination test of 6-BA and NAA was adopted, sucrose 30 g / L, agar 7 g / L, pH = 5.8, the culture period was 30 days, the growth conditions were observed and recorded every 5 days, and the induction rate of compact callus was calculated after 30 days (wherein, induction rate = (number of callus formed / total number of inoculated explants) × 100%) to examine the effects of different plant growth regulators on the callus induction of Castanopsis sylvestris. Conclusion: The compact callus induction medium with MS+6-BA 1.0mg / L+NAA 1.0mg / L+sucrose 30g / L+agar 7g / L had the best effect, and the compact callus induction rate reached 74.07% (Table 1). After about 10 days, the color of the rhizome changed from white-green to light yellow, and a circle of compact callus was formed at the base of the primary rhizome. After 30 days, the compact callus increased and turned light yellow.
[0064] Table 1 Effects of different combinations of plant growth regulators on callus induction in Castanopsis sylvestris
[0065]
[0066] Experimental Example 3: Loose callus induction
[0067] Using MS as the basic culture medium, the compact callus induced in Experimental Example 2 was inoculated on a culture medium supplemented with the best combination of plant growth regulators selected in Experimental Example 2, agar 5g / L, pH=5.4, and different sucrose contents as test factors, and a single factor experiment was conducted. The culture conditions were the same as in Example 2. After 15 days, the induction rate of loose callus was counted (loose callus induction rate=(number of loose callus formed / number of seedlings inoculated)×100%), and after 40 days, the proliferation multiples of loose callus were counted (proliferation multiples=(harvest amount-inoculation amount) / inoculation amount, where the harvest amount and inoculation amount represent the weight of the callus at the end and beginning of the culture, respectively), to examine the effects of different sucrose concentrations on the induction and proliferation of loose callus of Castanopsis affine. Conclusion: MS+6-BA 1.0mg / L+NAA1.0mg / L+sucrose 15g / L+agar 5g / L, pH=5.4 is a suitable culture medium for the induction of loose callus of Castanopsis affine ( Figure 2 ), in this culture medium, the induction rate of loose callus was high (the induction rate was 81.48%), the proliferation multiple was large (the proliferation multiple reached 5.8), and the formed loose callus was light yellow and had good growth.
Claims
1. A method for inducing loose callus of Castanopsis sylvestris, characterized in that: The primary rhizomes obtained after aseptic germination of Castanopsis chinensis seeds are used as explant materials, and then compact callus tissue is obtained by inducing tissue culture, and loose callus tissue is further induced. Specifically, the following steps are included: (1) Obtaining primary rhizome explants: After sterilizing, take the seeds of Castanopsis sylvestris and add GA3 to heat them in a water bath. Then, under a sterile environment, sow the seeds in a culture bottle supported by cotton and culture them in a dark room to germinate and obtain primary rhizome explants. (2) Inducing compact callus: inoculating the primary rhizome explants in step (1) into a compact callus induction medium, placing them in a light incubator for light culture, wherein the culture conditions in the light incubator are 22±3°C, light intensity 2100-2500 lx and light cycle 11-13 h / d. After 8-30 days of culture, the primary rhizome enlarges and the color changes from white-green to light yellow, thereby obtaining compact callus; (3) Inducing loose callus tissue: The compacted callus tissue in step (2) is inoculated into a modified MS medium with a pH value of 5.2-5.6 and placed in a light incubator for light culture. The culture conditions in the light incubator are 22±3°C, a light intensity of 2100-2500 lx and a light cycle of 11-13 h / d. After 8-30 days of culture, loose callus tissue is induced to form.
2. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (1), the disinfection treatment method is to soak the Castanopsis schrenkiana seeds in concentrated sulfuric acid for 6-10 minutes, with the amount of concentrated sulfuric acid used being preferably enough to submerge the seeds, and then wash with water until no concentrated sulfuric acid remains after soaking.
3. The method for inducing loose callus of Castanopsis affine according to claim 1 or 2, characterized in that: In the step (1), the disinfection treatment method is to soak the Castanopsis schrenkiana seeds in concentrated sulfuric acid for 8 minutes, with the amount of concentrated sulfuric acid used being preferably enough to submerge the seeds, and then wash with water until no concentrated sulfuric acid remains after soaking.
4. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (1), the concentration of GA3 is 180-220 mg / L, the water bath temperature is 35-45° C., the water bath is heated for 1.5-2.5 hours, and the amount of GA3 used is preferably enough to submerge the seeds.
5. The method for inducing loose callus of Castanopsis affine according to claim 1 or 4, characterized in that: In the step (1), the concentration of GA3 is 200 mg / L, the water bath temperature is 40° C., the water bath is heated for 2 hours, and the amount of GA3 used is preferably enough to submerge the seeds.
6. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (2), loose callus tissue is induced: the compacted callus tissue in step (2) is inoculated into a modified MS culture medium with a pH of 5.2-5.6, and placed in a light incubator for light culture. The culture conditions in the light incubator are 22±2° C., a light intensity of 2500 lx, and a light cycle of 12 h / d. After 10 days of culture, loose callus tissue is induced.
7. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (2), the culture medium for inducing compacted callus tissue consists of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+30 g / L sucrose+7 g / L agar, with a pH of 5.
8.
8. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (3), loose callus tissue is induced: the compacted callus tissue in step (2) is inoculated into a modified MS culture medium with a pH of 5.2-5.6, and placed in a light incubator for light culture. The culture conditions in the light incubator are 22±2°C, a light intensity of 2500lx and a light cycle of 12h / d. After 10 days of culture, loose callus tissue is induced to form.
9. The method for inducing loose callus of Castanopsis affine according to claim 1, characterized in that: In the step (3), the improved MS culture medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+10-25 g / L sucrose+5 g / L agar, and the pH is 5.
4.
10. The method for inducing loose callus of Castanopsis affine according to claim 1 or 9, characterized in that: In the step (3), the improved MS medium is composed of MS+1.0 mg / L 6-BA+1.0 mg / L NAA+15 g / L sucrose+5 g / L agar, and the pH is 5.4.