Application of cobalt chloride in promoting Korean pine somatic embryogenesis
By treating Korean pine callus tissue with cobalt chloride, the problems of decreased mesoembryogenic callus differentiation capacity and synchronization difficulties in Korean pine seroembryo development were solved, resulting in a significant improvement in the efficiency and quality of Korean pine seroembryo development.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-19
- Publication Date
- 2026-03-27
AI Technical Summary
The embryogenesis of Korean pine involves problems such as decreased embryogenic callus differentiation ability, difficulty in synchronization, and a high proportion of deformed embryos, resulting in the inability of embryo yield and quality to meet the needs of large-scale production.
Red pine callus was treated with cobalt chloride (CoCl2) at a specific concentration, and somatic embryogenesis was promoted by dark culture and suspension culture. The specific steps included dark culture at 25°C for 8 weeks, and culture using proliferation and suspension culture media containing cobalt chloride.
It significantly improved the proliferation rate and somatic embryogenesis efficiency of Korean pine callus, with the proliferation rate increasing by 146%~167%, the number of somatic embryos increasing by 1.37~2.61 times, and the normal embryo rate increasing by 20.25 times.
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Figure CN121730199A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue regeneration technology, specifically relating to the application of cobalt chloride in promoting embryogenesis in Korean pine. Background Technology
[0002] Korean pine is a precious native tree species in my country and the dominant species in the climax community of Korean pine broad-leaved forests in temperate humid regions, possessing significant ecological, economic, and social value. Artificial cultivation of Korean pine began relatively late, and breeding work started even later. Currently, there is a severe shortage of artificially sourced germplasm, and the availability of superior germplasm resources for resource expansion is very limited. The existing seed propagation methods are too long and have insufficient grafting coefficients, failing to meet the needs of large-scale afforestation. Therefore, establishing an efficient somatic embryogenesis technology system is of great significance for improving the large-scale propagation and application of high-quality Korean pine germplasm and solving the problem of high-quality germplasm scarcity.
[0003] Somatic embryogenesis is one of the important means to achieve efficient and large-scale propagation of superior tree varieties and preservation of excellent traits in forest tree reproduction and genetic breeding. In recent years, in the process of developing the somatic embryogenesis system of Korean pine, problems have been found, such as the rapid decline in embryogenic callus differentiation ability with increasing subgeneration, difficulty in somatic embryo synchronization, and a high proportion of deformed embryos. As a result, the yield and quality of somatic embryos cannot meet the needs of mass production of somatic embryo seedlings. This will become one of the key technical bottlenecks for the promotion and application of the somatic embryogenesis pathway in Korean pine. Summary of the Invention
[0004] The purpose of this invention is to improve the efficiency and quality of somatic embryogenesis in red pine callus tissue.
[0005] This invention provides the application of cobalt chloride in promoting embryogenesis in red pine, wherein the concentration of cobalt chloride is 50 μM / L.
[0006] To further define, promoting somatic embryogenesis refers to increasing the proliferation rate of embryogenic callus and improving the efficiency of somatic embryogenesis.
[0007] This invention provides a method for promoting the embryogenesis of Korean pine. The specific steps of the method are as follows: Korean pine callus tissue is added to a proliferation medium containing cobalt chloride, cultured in the dark, and then cultured to mature, thereby inducing the embryogenesis of Korean pine.
[0008] Further specified, the concentration of cobalt chloride is 50 μM / L cobalt chloride.
[0009] Further specifying the conditions for dark culture, the culture time is 25°C for 8 weeks.
[0010] Further, the culture period is 14 days.
[0011] Further specifying, the proliferation medium formulation is as follows: based on mLV, supplemented with 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 25 g / L sucrose, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L L-glutamine, 4 g / L-1 gellan gum and 50 μM / L cobalt chloride.
[0012] Further, after dark culture, the red pine callus tissue was added to the suspension culture medium to suspend and disperse the cells. The dispersed cells were then filtered through a Buchner funnel and placed in the maturation culture medium for maturation culture. The suspended cells were cultured in the maturation culture medium at 25°C in the dark for 60 days.
[0013] Further specifying the composition of the suspension medium: based on mLV, with the addition of 25 g / L sucrose, 0.5 g / L acid-hydrolyzed casein and 0.5 g / L L-glutamine.
[0014] Further defining the composition of the mature culture medium: based on mLV, with the addition of 8 g / L gellan gum, 68 g / L sucrose, 1 g / L activated carbon and 0.5 g / L acid-hydrolyzed casein, pH=5.8, 21 mg / L ABA and 0.5 g / L L-glutamine.
[0015] Beneficial Effects: Treatment with different concentrations of cobalt chloride (CoCl2) revealed that callus proliferation rate was significantly increased after treatment with 20 μM / L–50 μM / L CoCl2, with increases of 146%–167% compared to the control (Table 1). After pre-culturing callus tissue at CoCl2 concentrations of 20 μM·L⁻¹, 50 μM·L⁻¹, and 100 μM / L for 14 days, the number of somatic embryos increased by 1.37, 2.61, and 2.35 times, respectively, compared to the control group. Simultaneously, the rate of normal embryos significantly increased after CoCl2 treatment, with the 50 μM / L treatment showing the most significant effect, increasing by 20.25 times compared to the control group, and the normal embryo rate reached 61%. Attached Figure Description
[0016] Figure 1 Figure showing the effect of different concentrations of CoCl2 treatment on somatic embryogenesis; Figure 2 Figure 1 shows the phenotypic results of the effects of different concentrations of CoCl2 treatment on somatic embryogenesis; a. ck (control); b. 5 μM / L CoCl2; c. 10 μM / L CoCl2; d. 20 μM / L CoCl2; e. 50 μM / L CoCl2; f. 100 μM / L CoCl2. Detailed Implementation
[0017] 1. Preparation of callus proliferation medium: After sterilization, add 0, 5, 10, 20, 50, and 100 μM / L cobalt chloride respectively. The proliferation medium formula is: mLV supplemented with 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 25 g / L sucrose, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L L-glutamine, and 4 g / L gellan gum. Adjust the pH to approximately 5.8. Note: Add L-glutamine after autoclaving at 121℃.
[0018] 2. Callus proliferation culture method: The prepared culture medium was poured into 90mm petri dishes and cooled and solidified. After solidification, the red pine embryogenic callus was transferred to fresh proliferation culture medium using sterilized forceps. Three dishes were used for each treatment, with three 0.1g clusters of fresh callus tissue placed in each dish. On day 14 after inoculation, the fresh weight of the callus tissue was weighed, and the callus proliferation rate was calculated. Callus proliferation rate = (fresh weight of proliferated callus tissue - fresh weight of initial inoculated callus tissue) / fresh weight of initial inoculated callus tissue × 100%.
[0019] 3. The embryonic maturation procedure is as follows: Callus tissues cultured for 14 days with different concentrations of cobalt chloride were selected. 0.35g of embryogenic callus tissue was added to a 50ml centrifuge tube containing 15ml of liquid culture medium for suspension and dispersal (suspension medium: based on mLV, supplemented with 25 g·L⁻¹). -1 Sucrose, 0.5 g·L -1 Acid-hydrolyzed casein and 0.5 g·L -1 L-glutamine).
[0020] 4. After breaking up the callus tissue with the pipette tip, use a pipette to draw 3ml of liquid into a Buchner funnel containing filter paper. Use a vacuum pump to remove the excess liquid from the filter paper and continue vacuuming until no more water drips.
[0021] 5. Use tweezers to transfer the filter paper into the maturation culture medium. The maturation culture medium formula is: mLV + 8 g / L gellan gum + 68 g / L sucrose + 1 g / L activated carbon + 0.5 g / L acid-hydrolyzed casein, pH=5.8. After high temperature and high pressure sterilization, place it in a laminar flow hood and lower the temperature to about 45°C. Then add 21 mg / L of filtered ABA and 0.5 g / L L-glutamine.
[0022] 6. After culturing the culture dish in the dark at (25±2)℃ for 8 weeks, count the number of somatic embryos generated from the embryogenic callus under different treatments. The number of somatic embryos generated per gram of embryogenic callus = the number of somatic embryos generated / the fresh weight of the embryogenic callus.
[0023] Results: Eight weeks after somatic embryogenesis, the total number of somatic embryos and the number of normal embryos in callus tissue supplemented with CoCl2 were counted, and the somatic embryogenesis efficiency was calculated. Figure 1 As shown, compared with the control group without added CoCl2, the number and quality of somatic embryogenesis were significantly improved after adding 50 μM / L and 100 μM / L CoCl2. Figure 2 Further visual demonstration shows that adding a higher concentration of CoCl2 significantly increases the number of somatic embryos and noticeably improves the synchronized development of embryos. This indicates that this method not only increases the number of somatic embryos but also effectively improves the quality of embryo development, laying a solid foundation for subsequent plant regeneration.
[0024] Table 1. Effects of different concentrations of CoCl2 on the proliferation rate of pine embryogenic callus.
Claims
1. The application of cobalt chloride in promoting embryogenesis in Korean pine, characterized in that, The concentration of cobalt chloride is 50 μM / L.
2. The application according to claim 1, characterized in that, Promoting somatic embryogenesis refers to increasing the proliferation rate of embryogenic callus, improving the efficiency of somatic embryogenesis, and enhancing the quality of somatic embryos.
3. A method for promoting embryogenesis in Korean pine, characterized in that, The specific steps of the method are as follows: add the Korean pine callus tissue into a proliferation medium containing cobalt chloride, culture it in the dark, and then culture it to mature, thereby inducing Korean pine embryogenesis.
4. The method according to claim 3, characterized in that, The concentration of cobalt chloride is 50 μM / L.
5. The method according to claim 3, characterized in that, The conditions for dark culture were 25°C for 8 weeks.
6. The method according to claim 3, characterized in that, Proliferation culture for 14 days.
7. The method according to claim 3, characterized in that, The proliferation medium was formulated as follows: based on mLV, with the addition of 2 mg / L 2,4-D, 0.5 mg / L 6-BA, 25 g / L sucrose, 0.5 g / L acid-hydrolyzed casein, 0.5 g / L L-glutamine, 4 g / L-1 gellan gum and 50 μM / L cobalt chloride.
8. The method according to claim 3, characterized in that, After dark culture, the red pine callus tissue was added to the suspension culture medium to suspend and disperse the cells. The dispersed cells were then filtered through a Buchner funnel and placed in the maturation culture medium for maturation culture. The cells were cultured in the maturation culture medium in the dark at 25°C for 60 days.
9. The method according to claim 8, characterized in that, The suspension medium consisted of mLV as the basal medium, with the addition of 25 g / L sucrose, 0.5 g / L acid-hydrolyzed casein and 0.5 g / L L-glutamine.
10. The method according to claim 9, characterized in that, Composition of the maturation medium: based on mLV, with the addition of 8 g / L gellan gum, 68 g / L sucrose, 1 g / L activated carbon and 0.5 g / L acid-hydrolyzed casein, pH=5.8, 21 mg / L ABA and 0.5 g / L L-glutamine.