A method for rapidly propagating tissue culture seedlings by using panax notoginseng anther callus

Abstract

This invention discloses a method for rapidly propagating tissue culture seedlings using callus tissue from Panax notoginseng flowers. Using three-year-old Panax notoginseng flowers as material, the method involves explant disinfection, inoculation, induction of embryogenic callus, proliferation of embryogenic callus, and differentiation of embryogenic callus into seedlings to achieve rapid propagation of Panax notoginseng tissue. The tissue culture method of this invention results in a high callus induction rate and good callus growth. Furthermore, the callus tissue can rapidly root. Therefore, the tissue culture method of this invention is simple to operate, low in cost, and has a short breeding period, effectively providing technical support for the further preservation and optimization of germplasm resources and promoting the development of the Panax notoginseng industry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for rapidly propagating tissue culture seedlings using callus tissue from Panax notoginseng flowers. Background Technology

[0002] Panax notoginseng (Burk) FHChen, also known as Tianqi, is a plant belonging to the genus Panax in the Araliaceae family. It is primarily used to treat coronary heart disease and cardiovascular diseases, playing important roles in hemostasis, antihypertensive effects, antithrombotic effects, and neuroprotection. It has high medicinal value in traditional medicine. In recent years, Panax notoginseng has been widely used in pharmaceuticals, food, and cosmetics. In China, the demand for Panax notoginseng is approximately 5000 tons. However, Panax notoginseng has demanding growth requirements; it can only be grown in Wenshan Prefecture of Yunnan Province and parts of Guangxi Province. Furthermore, its long growth cycle, severe pests and diseases, continuous cropping obstacles, pesticide residues, and germplasm degradation make it difficult for Panax notoginseng resources to meet market demand.

[0003] Tissue culture technology can rapidly propagate Panax notoginseng seedlings, shorten the breeding period, and optimize germplasm resources, which is one of the effective ways to solve the above problems.

[0004] Plant tissue culture refers to the technique of placing plant tissues and organs such as roots, stems, leaves, flowers, anthers, and reproductive cells under sterile conditions in artificially prepared culture media to obtain complete plant bodies or other biological products through artificially created conditions. In existing reports on Panax notoginseng tissue culture, stems and leaves are generally used as explants. Chinese invention patent application CN116711636.A discloses a method for rapidly propagating tissue culture seedlings using stems or leaves of Panax notoginseng seedlings after seed germination as explants through embryogenic callus. Chinese invention patent application CN201711451016.4 discloses a method for inducing anther callus in Panax notoginseng; however, methods for inducing germination and rooting of anther callus have not been reported. Improving the germination and rooting rates of anther callus is crucial for the rapid division of anther callus into tissue culture seedlings. Summary of the Invention

[0005] This invention provides a method for rapidly propagating tissue culture seedlings using callus tissue from Panax notoginseng anthers, solving the technical problem of rapid propagation of Panax notoginseng from seeds to seedlings. Using the tissue culture method of this invention to culture the anthers of Panax notoginseng plants, the callus induction rate is high, the browning degree is low, and the callus differentiation effect is good and can achieve rapid rooting. Therefore, the tissue culture method of this invention is simple to operate and low in cost, and can provide technical support for the rapid propagation of Panax notoginseng seedlings.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] 1. Aseptic treatment of explants: Cut off unopened inflorescences and rinse them with tap water under slow flow for 1 hour. Under aseptic conditions, soak them in 75% alcohol for 30-60 seconds, then rinse them 3 times with sterile water. Next, soak them in 10% sodium hypochlorite solution for 4-8 minutes, shaking them 3-5 times during the process. Finally, rinse them 5 times with sterile water, blot dry with sterile filter paper, and use sterile tweezers to remove the anthers from the Panax notoginseng flowers for later use.

[0008] 2. Induction of embryogenic callus from Panax notoginseng: Aseptic anthers were inoculated onto callus induction medium and cultured in the dark at 25°C for 30 days to obtain embryogenic callus from Panax notoginseng. The callus induction medium was MS basal medium supplemented with 1-3 mg / L 2,4-D (2,4-phenoxyacetic acid), 1.5 mg / L 6-BA (6-benzylaminopurine), 0.5 g / L activated carbon, 0.5 g / L PVP (polyvinylpyrrolidone), and 30 g / L sucrose, with a pH of 5.4-5.8.

[0009] 3. Proliferation of Panax notoginseng embryogenic callus: Panax notoginseng embryogenic callus was inoculated onto proliferation medium and cultured in the dark at 25°C for 30–60 days to obtain a large amount of Panax notoginseng embryogenic callus. The proliferation medium was MS basal medium supplemented with 2.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4–5.8.

[0010] 4. Induction of embryogenic callus from Panax notoginseng into shoots: The embryogenic callus obtained above was inoculated on shoot induction medium and cultured under light for 12 h / d until the embryogenic callus differentiated into shoots. The shoot induction medium was MS basal medium supplemented with 1.0 mg / L KT (synthetic cytokinin), 0.5-1.0 mg / L IBA (indolebutyric acid), 0.5-1.0 mg / L BR (brassinolide), 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.8.

[0011] 5. Rooting induction of Panax notoginseng embryogenic callus: The embryogenic callus with shoots obtained above was transferred to a rooting medium and cultured under light for 30 days. The rooting medium was MS basal medium supplemented with 1.0-3.0 mg / L 2,4-D, 0.5-1.5 mg / L 6-BA, 0.8 mg / L KT, 0.3 g / L activated carbon, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.8.

[0012] 6. Culture of Panax notoginseng embryogenic callus into seedlings: The tissues obtained above were transferred to seedling culture medium and cultured under light to form complete Panax notoginseng tissue culture seedlings. The seedling culture medium was 1 / 2 MS basal medium supplemented with 2.0 mg / L KT, 0.3 g / L activated carbon, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.8.

[0013] The amount of agar added to the MS medium is 8 g / L.

[0014] The above-mentioned bud induction, rooting induction, and shoot induction culture temperature was 23±2℃, the light duration was 12h / d, and the light intensity was 1600-2000LX.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention selects unopened inflorescences, pre-treats and sterilizes the inflorescences before extracting the anthers, which not only effectively reduces the contamination rate of the explants, but also significantly reduces the damage of ethanol and sodium hypochlorite to the anthers, thus resulting in a better callus induction rate.

[0017] The callus induction medium of this invention effectively promotes callus formation and achieves a high success rate in inducing callus. Furthermore, the invention utilizes two anti-browning agents, resulting in callus with low browning levels and good growth. Finally, the rooting medium of this invention induces rapid rooting of the callus. In summary, the tissue culture method of this invention for culturing explants of Panax notoginseng plants yields a high callus induction rate, good callus growth, and rapid rooting. Therefore, the tissue culture method of this invention is simple to operate, low in cost, and has a short breeding period, effectively providing technical support for the preservation and optimization of germplasm resources and promoting the development of the Panax notoginseng industry. Attached Figure Description

[0018] Figure 1 Schematic diagram of the induction results of embryogenic callus tissue from Panax notoginseng flower;

[0019] Figure 2 Schematic diagram of the results of anther embryogenic callus proliferation culture;

[0020] Figure 3 Schematic diagram of the results of anther embryogenic callus shoot induction culture;

[0021] Figure 4 A schematic diagram showing the results of rooting induction culture of callus with anther embryogenic structure;

[0022] Figure 5 This is a schematic diagram of anther embryogenic callus seedling culture. Detailed Implementation

[0023] To better explain the present invention, the principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical solutions involved in the embodiments of the present invention are conventional solutions in the art; unless otherwise specified, the reagents or materials mentioned are all from commercial channels.

[0024] Example 1:

[0025] 1. Selection and sterilization of explants: One group of explants were selected from the unflowered inflorescences of three-year-old Panax notoginseng. They were rinsed with tap water under a slow flow for 1 hour, then soaked in 75% alcohol for 30-60 seconds in a sterile room, rinsed three times with sterile water, then soaked in 5%-10% sodium hypochlorite solution for 4-8 minutes, and finally rinsed five times with sterile water. After drying with sterile filter paper, the anthers were removed with sterile forceps and kept for later use. The other group of explants were selected from the unflowered inflorescences of three-year-old Panax notoginseng. They were rinsed with tap water under a slow flow for 1 hour, then the anthers were removed with sterile forceps, soaked in 75% alcohol for 30-60 seconds, then rinsed three times with sterile water, then soaked in 5%-10% sodium hypochlorite solution for 4-8 minutes, and finally rinsed five times with sterile water. After drying with sterile filter paper, the explants were kept for later use.

[0026]

[0027] Table 1. Effects of different disinfection methods on aseptic treatment of Panax notoginseng flowers.

[0028]

[0029] As can be seen from the results in Table 1, considering both the contamination rate and the induction rate, soaking the unflowered Panax notoginseng flowers in 10% sodium hypochlorite solution for 4 minutes can significantly reduce the contamination rate and result in a high survival rate, making it the optimal treatment method and time.

[0030] 2. Induction of embryogenic callus from Panax notoginseng anthers: Sterile anthers were inoculated onto callus induction medium and cultured in the dark at 25°C for 30 days to obtain pale yellow embryogenic callus from Panax notoginseng. The callus induction medium was based on MS medium, supplemented with 1-3 mg / L 2,4-D, 1.5 mg / L 6-BA, 0.5 g / L activated charcoal, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.8.

[0031] Table 2. Effects of different disinfection methods on the induction rate of embryogenic callus from Panax notoginseng flowers.

[0032]

[0033] Table 2 shows that the induction rate of anther-embryonic callus varied considerably. The optimal culture medium composition was MS as the basal medium, supplemented with 2 mg / L 2,4-D, 1.5 mg / L 6-BA, 0.5 g / L activated charcoal, 0.5 g / L PVP, and 30 g / L [unclear - possibly a specific compound or solution]. Furthermore, the anther-embryonic callus formation of Panax notoginseng was observed... Figure 1 ;

[0034] 3. Proliferation of Panax notoginseng flower embryogenic callus: Panax notoginseng embryogenic callus was inoculated onto proliferation medium and cultured in the dark at 25℃ for 30–60 days to obtain a large number of loosely structured Panax notoginseng embryogenic callus. The proliferation medium was MS medium as the basal medium, supplemented with 2.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4–5.8. The proliferation results are shown in the figure. Figure 2 ;

[0035] 4. Induction of embryogenic callus from Panax notoginseng flowers into shoots: The embryogenic callus obtained above was inoculated on shoot induction medium and cultured under light for 12 h / d (temperature 23±2℃, light intensity 1600-2000X) until the embryogenic callus differentiated into shoots. The shoot induction medium was MS basal medium supplemented with 1.0 mg / L KT, 0-1.0 mg / L IBA, 0-1.0 mg / L BR, 0.5 g / L PVP, 30 g / L sucrose, and pH 5.4-5.8.

[0036] Table 3. Effects of different hormone ratios on the induction rate of embryogenic callus from Panax notoginseng flowers.

[0037]

[0038]

[0039] The results in Table 3 show that the addition of BR and KT significantly promotes the induction of embryogenic callus buds in Panax notoginseng. The synergistic effect of BR, KT, and IBA further enhances the induction of embryogenic callus buds. The optimal culture medium composition for inducing anther-embryonic callus buds was MS as the basal medium, supplemented with 1.0 mg / L KT, 1.0 mg / L IBA, and 1.0 mg / L BR. The induction results are shown in Table 3. Figure 3 ;

[0040] 5. Rooting induction of Panax notoginseng embryogenic callus: The embryogenic callus with shoots obtained above was transferred to a rooting medium and cultured under 12h / d light for 30d (temperature 23±2℃, light intensity 1600-2000LX). The rooting medium was MS medium as the basal medium, supplemented with 1.0-3.0mg / L 2,4-D, 0.5-1.5mg / L 6-BA, 0.8mg / L KT, 0.3g / L activated carbon, 0.5g / L PVP, and 30g / L sucrose, with a pH of 5.4-5.8.

[0041] Table 4. Effects of different hormone ratios on the rooting induction rate of Panax notoginseng flower embryogenic callus.

[0042]

[0043] As shown in Table 4, the optimal culture medium composition for rooting induction of anther-embryonic callus was MS medium supplemented with 2.0 mg / L 2,4-D, 1.0 mg / L 6-BA, 0.8 mg / L KT, 0.3 g / L activated charcoal, and 0.5 g / L PVP. The induction results are shown in Table 4. Figure 4 ;

[0044] 6. Culture of Panax notoginseng embryogenic callus into seedlings: The tissues obtained above were transferred to a seedling culture medium and cultured under light to form complete Panax notoginseng tissue culture seedlings. The seedling culture medium was based on 1 / 2 MS medium supplemented with 2.0 mg / L KT, 0.3 g / L activated carbon, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.8. The results of seedling formation are shown in [the table below]. Figure 5 .

[0045] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for rapidly propagating tissue culture seedlings using Panax notoginseng flower callus, characterized in that, The steps are as follows: (1) Cut off the unflowered Panax notoginseng inflorescences, rinse them with tap water, disinfect them with disinfectant alcohol and sodium hypochlorite, and collect sterile anthers; inoculate the sterile anthers onto the callus induction medium and culture them in the dark at 25°C to obtain Panax notoginseng embryogenic callus. (2) Inoculate the embryogenic callus of Panax notoginseng onto the proliferation medium and culture it in the dark at 25°C for 30-60 days to promote the proliferation of the embryogenic callus of Panax notoginseng. (3) Inoculate the embryogenic callus of Panax notoginseng on the bud induction medium and culture it under light until the embryogenic callus differentiates into buds; after the embryogenic callus with buds is transferred to the rooting medium and cultured under light for 30 days, it is transferred to the seedling medium and cultured under light to obtain Panax notoginseng tissue culture seedlings. The specific process for obtaining sterile anthers is as follows: cut off unopened inflorescences and rinse them with tap water under slow flow for 1 hour. Then, under sterile conditions, soak them in 75% alcohol for 30-60 seconds. Rinse them three times with sterile water, then soak them in 5%-10% sodium hypochlorite solution for 4-8 minutes. Finally, rinse them five times with sterile water, absorb the water with sterile filter paper, and use sterile tweezers to remove the anthers from the Panax notoginseng flowers for later use. The callus induction medium consisted of MS basal medium + 1-3 mg / L 2,4-D + 1.5 mg / L 6-BA + 0.5 g / L activated charcoal + 0.5 g / L PVP + 30 g / L sucrose, with a pH of 5.4-5.

8. The proliferation medium consisted of MS basal medium + 2.0 mg / L 2,4-D + 0.5 mg / L 6-BA + 0.5 g / L PVP + 30 g / L sucrose, with a pH of 5.4-5.

8. The bud induction medium was MS basal medium + 1.0 mg / L KT + 0.5~1.0 mg / L IBA + 0.5~1.0 mg / L BR + 0.5 g / L PVP + 30 g / L sucrose, with a pH of 5.4-5.8; The rooting medium was MS basal medium containing 1.0-3.0 mg / L 2,4-D, 0.5-1.5 mg / L 6-BA, 0.8 mg / L KT, 0.3 g / L activated carbon, 0.5 g / L PVP, and 30 g / L sucrose, with a pH of 5.4-5.

8. The seedling culture medium consisted of 1 / 2 MS basal medium + 2.0 mg / L KT + 0.3 g / L activated carbon + 0.5 g / L PVP + 30 g / L sucrose, with a pH of 5.4-5.

8.

2. The method for rapidly propagating tissue culture seedlings using Panax notoginseng flower callus according to claim 1, characterized in that: In step (3), the light culture temperature is 23±2℃, the light time is 12h / d, and the light intensity is 1600-2000LX.

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