A tissue culture method of Sedum scutellariae based on leaves

Abstract

The present invention relates to the technical field of tissue culture for specialty flower plants, specifically a leaf-based tissue culture method for Sedum scutellaria, including selection and disinfection of Sedum scutellaria explants, callus induction, differentiation culture, proliferation culture, and rooting culture. The present invention establishes a tissue culture technology system using Sedum scutellaria leaves as explants, achieving rapid propagation of Sedum scutellaria and contributing to the conservation and application of Sedum scutellaria resources.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture of characteristic flower plants, in particular to a leaf-based tissue culture method of Sedum scutellariae. Background Art

[0002] Sedum lineare Thunb, a perennial herb in the Crassulaceae family, is renowned for its unique appearance and rich medicinal properties. Its vibrant green leaves, resembling the shape of a sedum, give it the name. It is widely used in green roofs and also possesses significant medicinal value. The whole plant has a sweet and cold nature, and is known for its heat-clearing, detoxifying, and swelling-reducing properties.

[0003] Currently, the commonly used propagation methods for Sedum scutellaria include sowing, cuttings, and division. These methods have disadvantages such as low survival rate, long propagation cycle, serious pests and diseases, and seasonal restrictions, which cannot meet market demand. In addition, as a medicinal plant with extremely high value, it is necessary to establish the protection of wild germplasm resources. In vitro preservation is a new method of plant resource preservation developed based on plant tissue culture technology. Tissue culture preservation is the most representative method and has become the main way to achieve the preservation, restoration and sustainable utilization of rare and endangered medicinal plant resources, playing an important supporting role in the development of Traditional Chinese Medicine.

[0004] The tissue culture process primarily includes plant selection, explant acquisition, explant disinfection, culture medium selection, callus induction, primary culture, subculture, differentiation induction, proliferation culture, and rooting culture. Tissue culture has been applied to the in vitro preservation and rapid propagation of plants such as Fritillaria cirrhosa, Ligusticum chuanxiong, and roses. The establishment of a tissue culture system for Sedum chinense will lay the foundation for breeding disease-resistant and stress-resistant safflower varieties and provide a technical platform for transgenic research.

[0005] Currently, there are few reports on tissue culture of Sedum sedum, which primarily uses stem segments as explants. The culture medium formulation is complex, and there are also issues such as low rooting rate, vitrification of tissue culture seedlings, and difficulty surviving transplantation. Consequently, it has not yet been applied to the production and propagation of Sedum sedum seedlings. Sedum sedum resources are diverse, and the culture medium formulations suitable for different varieties also vary. An efficient tissue culture system for Sedum sedum based on leaves has not yet been established. Summary of the Invention

[0006] In order to solve the above technical problems, a leaf-based tissue culture method of Sedum scutellaria is provided to achieve rapid propagation and resource preservation of Sedum scutellaria germplasm resources.

[0007] To achieve the above objectives, the embodiments of the present invention adopt the following technical solutions:

[0008] The present invention provides a leaf-based tissue culture method for Sedum scutellariae, comprising the following steps:

[0009] (1) Explant selection and disinfection: Collect leaves of Sedum scutellariae, soak them in detergent water, rinse them with clean water, disinfect them with alcohol and sodium hypochlorite in a clean workbench, soak them in sterile water, rinse them, and set aside;

[0010] (2) Callus induction: Place the disinfected leaves on sterile filter paper, cut into small pieces, transfer to MS induction medium, and culture in an artificial climate chamber until callus tissue of Sedum sphenanthera is formed;

[0011] (3) Differentiation culture: Transfer the callus of Sedum sphenanthera to MS differentiation medium and culture it in an artificial climate chamber until adventitious buds differentiate and form;

[0012] (4) Proliferation culture: Cut the adventitious buds of Sedum truncatum on a clean workbench, transfer them to MS proliferation medium, and culture them in an artificial climate chamber to obtain rootless seedlings of Sedum truncatum;

[0013] (5) Rooting culture: Transfer the rootless seedlings of Sedum sphenanthera into the rooting culture medium and continue to culture until roots appear. After hardening the seedlings, transplant them into the cultivation medium.

[0014] Furthermore, in step (1), the disinfection process is to soak in 75% ethanol by volume for 30 seconds, rinse with sterile water three times, each time for 6 to 7 minutes, repeat the operation three times, and then disinfect with 2.5% sodium hypochlorite solution by volume for 10 minutes, and then rinse with sterile water six times, each time for 6 to 7 minutes.

[0015] Furthermore, in step (2), the MS induction medium includes MS+6-BA 1.5 mg / L+NAA 0.8 mg / L.

[0016] Furthermore, in step (3), the MS differentiation medium includes MS+6-BA 3 mg / L+NAA 0.5 mg / L.

[0017] Furthermore, in step (4), the MS proliferation medium includes MS+6-BA 3 mg / L+NAA 0.5 mg / L.

[0018] Furthermore, in step (5), the rooting medium includes 1 / 2MS+NAA 0.5 mg / L.

[0019] Furthermore, in step (2) and step (4), the temperature in the artificial climate box is 25°C, the humidity is 50%, the light intensity is 6000 Lx, and the light duration is 16 h / d.

[0020] The beneficial effects of the present invention are:

[0021] (1) The present invention provides a leaf-based tissue culture method for Sedum truncatum. The explants used are leaves, which are easier to obtain and more abundant than stem segments. Sample collection causes less damage to the plant and is more suitable for the preservation of Sedum truncatum resources and rapid tissue culture propagation.

[0022] (2) The present invention provides a leaf-based tissue culture method for Sedum serrata. The plant hormones used are 6-BA and NAA, which are common and affordable in the market, ensuring a low tissue culture cost.

[0023] (3) The present invention provides a leaf-based tissue culture method for Sedum truncatum, wherein the induction medium, differentiation medium, proliferation medium, and rooting medium are MS and 1 / 2MS basal medium, supplemented with different proportions of 6-BA and NAA. The operation and use are relatively simple, and it is easier to cultivate Sedum truncatum seedlings in factories.

[0024] (4) The present invention provides a leaf-based tissue culture method for Sedum truncatum. The cultivation time from leaf to complete plant is about 60 days, the growth cycle is short, the reproduction coefficient is large, rooting is easy, and the rapid propagation efficiency is high. In addition, through the use of a clean bench and an artificial climate chamber, the year-round production of Sedum truncatum can be achieved without seasonal restrictions. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a diagram of the leaf explant collected in Example 1;

[0026] Figure 2 This is a schematic diagram of the callus induction culture of Sedum scutellaria leaves in Example 1;

[0027] Figure 3 These are adventitious buds differentiated from the callus tissue of the Sedum chinense leaf in Example 1;

[0028] Figure 4 This is a diagram of the proliferation of young shoots of Sedum chinense leaves in Example 1;

[0029] Figure 5 This is a diagram of the rooting of Sedum leaf seedlings in Example 1;

[0030] Figure 6 This is a picture of the Sedum plant after hardening and transplanting in Example 1. DETAILED DESCRIPTION

[0031] The following is a clear and complete description of the technical solutions in some embodiments of the present disclosure. Obviously, the embodiments described are only some of the embodiments of the present disclosure, not all of them. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field are within the scope of protection of the present disclosure.

[0032] Example 1

[0033] The tissue culture of Sedum truncatum germplasm was carried out as follows:

[0034] (1) Materials

[0035] The explant material is young leaves of Sedum scutellaria, which were collected from the rooftop garden of the National Chengdu Agricultural Science and Technology Center.

[0036] (2) Methods

[0037] 2.1 Explant collection and disinfection

[0038] Cut young leaves from Sedum sphenanthera plants, soak them in detergent water, rinse them several times with clean water, and transfer them to a clean workbench after there are no detergent bubbles. Soak them in 75% ethanol for 30 seconds, rinse them with sterile water three times, each for 6-7 minutes, and repeat the operation three times. Then disinfect them with 2.5% sodium hypochlorite solution for 10 minutes, and rinse them with sterile water six times, each for 6-7 minutes. Place the disinfected material on sterile filter paper and cut it into small sections about 0.4 cm long for later use.

[0039] 2.2 Callus induction

[0040] The sterilized Sedum leaves were transferred to MS medium containing 6-BA 1.5 mg / L + NAA 0.8 mg / L, and cultured in an artificial climate chamber (temperature 25℃, humidity 50%, light intensity 6000 Lx, light time 16 h / d) until callus tissue appeared.

[0041] 2.3 Differentiation culture

[0042] The callus tissue was transferred to MS medium containing 6-BA 3.0 mg / L + NAA 0.5 mg / L and cultured in an artificial climate chamber (temperature 25℃, humidity 50%, light intensity 6000 Lx, light time 16 h / d) for 2 weeks until bud differentiation occurred.

[0043] 2.4 Proliferation culture

[0044] Using sterile techniques on a clean workbench, use tweezers and a blade to cut the buds from the callus tissue, transfer them to MS medium containing 3.0 mg / L 6-BA and 0.5 mg / L NAA, and culture them in an artificial climate chamber (temperature 25°C, humidity 50%, light intensity 6000 Lx, light duration 16 h / d) for 1 week to allow the young shoots to proliferate.

[0045] 2.5 Rooting culture

[0046] When the shoots are 1-2 cm in height, use tweezers to transfer the shoots to 1 / 2MS medium containing 0.5 mg / L NAA using sterile methods on a clean workbench. Culture in an artificial climate chamber (temperature 25°C, humidity 50%, light intensity 6000 Lx, photoperiod 16 h / d) for 2 weeks until roots appear.

[0047] 2.6 Hardening and transplanting

[0048] The tissue culture bottles of Sedum scabra were uncovered in an artificial climate box (temperature 25℃, humidity 50%, light intensity 6000Lx, and light time 16 h / d) and cultured for 2 days. The tissue culture bottles were then transferred to a cultivation environment and cultured for 3 days to achieve the acclimatization of the tissue culture seedlings. The tissue culture seedlings of Sedum scabra were taken out, the residual culture medium on the roots was cleaned, and they were transplanted into the seedling medium to obtain Sedum scabra plants with good growth condition.

[0049] Comparative Example 1

[0050] Except that the callus induction medium in 2.2 callus induction was MS + 4 mg / L 6-BA + 0.8 mg / L NAA, other operations were the same as in Example 1.

[0051] Comparative Example 2

[0052] Except that the callus induction medium in 2.2 callus induction was MS + 0.5 mg / L 6-BA + 0.8 mg / L NAA, other operations were the same as in Example 1.

[0053] Comparative Example 3

[0054] The same procedures as in Example 1 were performed except that the differentiation medium in 2.3 differentiation culture was MS+6-BA 5.0 mg / L+NAA 0.5 mg / L.

[0055] Comparative Example 4

[0056] The same procedures as in Example 1 were performed except that the differentiation medium in 2.3 differentiation culture was MS+6-BA 1.0 mg / L+NAA 0.5 mg / L.

[0057] Comparative Example 5

[0058] Except that the culture medium in step 2.5 of the rooting culture was 1 / 2MS+NAA 4.0 mg / L, other operations were the same as those in Example 1.

[0059] Comparative Example 6

[0060] Except that the culture medium in 2.5 rooting culture was 1 / 2MS+NAA 0.01 mg / L, other operations were the same as in Example 1.

[0061] Callus induction rate (%) = number of explants with callus formation / total number of explants × 100%.

[0062] Germination rate (%) = number of calli with sprouts / total number of calli × 100%.

[0063] Rooting rate (%) = number of rooting shoots / total number of shoots × 100%.

[0064] The results of the Sedum scutellariae cultured under different culture media are shown in Tables 1 to 3 below:

[0065] Table 1 Effects of different induction media on the induction of Sedum chinense callus

[0066]

[0067] Table 2 Effects of different differentiation media on the differentiation of adventitious buds of Sedum chinense

[0068]

[0069] Table 3 Rooting effects of Sedum chinense under different rooting media

[0070]

[0071] As shown in Table 1, the difference in the ratio and concentration of 6-BA and NAA hormones affects the effect of inducing callus tissue from Sedum leaves. Among them, 6-BA 1.5 mg / L + NAA 0.8 mg / L in Example 1 is more suitable; as shown in Table 2, under the condition of fixed NAA hormone concentration, too high or too low 6-BA concentration will reduce the induction rate of adventitious buds. Example 1 has a good effect of inducing initial adventitious buds ( Figure 3 ), as shown in Table 3, when the NAA concentration is 0.5 mg / L, the rooting rate is the highest, reaching 84.3%, and the plants are strong ( Figure 5 ). In summary, Example 1 of the present application is suitable for tissue culture of Sedum sphenanthera.

[0072] The implementation principle of this embodiment is: plant tissues are omnipotent, the plant hormone 6-BA belongs to the cell separation hormone class, which promotes cell division and induces plants to form new shoots; NAA belongs to the auxin class, which can promote rooting and bud formation, induce callus tissue, and promote the differentiation of undifferentiated tissue; using leaves as explants, after plant hormone treatment and induction of differentiation, a complete plant can be formed, thereby realizing the efficient reproduction of Sedum.

[0073] In the description of this specification, specific features, structures, materials or characteristics may be combined in an appropriate manner in any one or more embodiments or examples.

[0074] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for tissue culture of Sedum chinense based on leaves, characterized in that: The steps include: (1) Explant selection and disinfection: Collect leaves of Sedum scutellariae, soak them in detergent water, rinse them with clean water, disinfect them with alcohol and sodium hypochlorite in a clean workbench, soak them in sterile water, rinse them, and set aside; (2) Callus induction: Place the disinfected leaves on sterile filter paper, cut into small pieces, transfer to MS induction medium, and culture in an artificial climate chamber until callus tissue of Sedum sphenanthera is formed; (3) Differentiation culture: Transfer the callus of Sedum sphenanthera to MS differentiation medium and culture it in an artificial climate chamber until adventitious buds differentiate and form; (4) Proliferation culture: Cut the adventitious buds of Sedum truncatum on a clean workbench, transfer them to MS proliferation medium, and culture them in an artificial climate chamber to obtain rootless seedlings of Sedum truncatum; (5) Rooting culture: transfer the rootless seedlings of Sedum scutellariae to the rooting medium and continue to culture until roots appear. After hardening, transplant them to the cultivation medium; In step (1), the disinfection process is to soak in 75% ethanol by volume for 30 seconds, rinse with sterile water three times, each time for 6 to 7 minutes, repeat the operation three times, then disinfect with 2.5% sodium hypochlorite solution by volume for 10 minutes, and then rinse with sterile water six times, each time for 6 to 7 minutes; In step (2), the MS induction medium is an MS induction medium supplemented with MS+6-BA 1.5 mg / L+NAA 0.8 mg / L; In step (3), the MS differentiation medium is an MS differentiation medium supplemented with MS+6-BA 3.0 mg / L+NAA 0.5 mg / L; In step (4), the MS proliferation medium is an MS proliferation medium supplemented with MS+6-BA 3.0 mg / L+NAA 0.5 mg / L; In step (5), the rooting medium is a rooting medium supplemented with 1 / 2MS+NAA 0.5 mg / L.

2. The method for culturing Sedum chinense tissue based on leaves according to claim 1, characterized in that: In step (2) and step (4), the temperature in the artificial climate box is 25°C, the humidity is 50%, the light intensity is 6000 Lx, and the lighting time is 16 h / d.

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