Method for establishing swallow suspension cell culture system

Abstract

The invention provides a method for establishing a swallow suspension cell culture system. Through the steps of fragile callus starting culture, suspension culture establishment, suspension cell proliferation culture, suspension cell updating and the like, massive uniform and consistent swallow flower cells can be rapidly obtained, and the method can be applied to the fields of protoplast separation, plant regeneration, genetic transformation, polyploidy mutagenesis, secondary metabolite production, artificial seed preparation and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for establishing a floribunda suspension cell culture system. Background Art

[0002] As an important branch of modern biotechnology, plant cell culture technology has demonstrated tremendous application potential in the fields of plant secondary metabolite production, variety improvement, and genetic engineering research. Among them, suspension cell culture technology has become one of the core technologies for plant cell engineering research and industrial applications due to its significant advantages, such as good cell dispersion, rapid growth rate, high proliferation coefficient, and ease of scale-up. Compared with traditional solid culture systems, suspension cell culture can achieve efficient delivery of nutrients, gases, and metabolites through a dynamic liquid environment, significantly improving the uniformity and stability of cell populations, and providing key technical support for the construction of standardized experimental systems and industrial production.

[0003] Iris laevigata (Iris laevigata Fisch.), a perennial plant of the Iridaceae family, possesses ornamental, medicinal, and ecological value. Its vibrant colors and strong adaptability make it a valuable resource for waterscape gardening. Furthermore, the active ingredients in its rhizomes possess anti-inflammatory and antioxidant properties, making it a promising resource for development. At present, there are very few research reports on suspension cell culture systems for Iris plants. The only report is that Wang et al. established a suspension cell culture system in Iris germanica 'Skating Party', a bearded iris, and based on this system, they achieved efficient proliferation and differentiation of suspension cells of this variety, and finally formed a rapid propagation system in which 1 gram of suspension cells could produce 4,000 regenerated plants after 3-4 months of culture (Yuexin Wang, Zoran Jeknić et al., Efficient Plant Regeneration From Suspension-Cultured Cells of Tall Bearded Iris [J]. HortScience. 1999, 34(4):730-735).

[0004] However, as a representative species of the monocotyledonous iris family, cell culture research on the iris is still in its infancy, and a suspension cell culture system has yet to be established. Due to the biological characteristics of monocotyledons, such as low callus induction rates and difficulty in dispersion, establishing a suspension cell culture system for these plants has always been a technical challenge in the field of plant cell engineering.

[0005] Therefore, the establishment of a suspension cell culture system for Iris serrata can not only enrich the cell culture technology of bearded iris plants and provide a new experimental model for analyzing the molecular mechanism of dedifferentiation and redifferentiation of monocotyledonous plant cells; it can also lay a key technical foundation for the large-scale production of Iris serrata, the rapid breeding of high-quality germplasm and the application of biotechnologies such as gene editing. It has important theoretical value and practical significance for promoting the diversified development of Iris plant resources and the development of monocotyledonous plant cell engineering technology. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for establishing a suspension cell culture system of Swallow's Soft tissue. Through the steps of initiating culture of fragile callus tissue, establishing suspension culture, suspension cell proliferation culture, and suspension cell renewal, a large number of uniform Swallow's Soft tissue cells can be quickly obtained, which can be used in the fields of protoplast isolation, plant regeneration, genetic transformation, polyploid mutagenesis, secondary metabolite production, and artificial seed preparation.

[0007] In order to achieve the above technology, the technical solution adopted by the present invention is: a method for establishing a suspension cell culture system of Swallow's Bloom, comprising the following steps:

[0008] Step 1: Initiation culture of fragile callus: select callus induced by seeds of Swallow Flower, and perform continuous subculture on fragile callus initiation medium in a dark environment at a culture temperature of 26°C until a sufficient number of fragile callus are produced.

[0009] Step 2: Establish suspension culture. Take the fragile callus obtained in step 1 and start suspension culture at a ratio of 1 g of fragile callus to 100 ml of suspension culture medium. The culture temperature is 24°C and the flow rate is 120 rpm / min. After 20 days, filter through a 30-mesh filter to remove clumping callus tissue and collect cells through a 150-mesh filter. Resuspend at a ratio of 1 g of suspension cells to 100 ml of suspension culture medium and culture under the same conditions for 20 days.

[0010] Step 3: Suspension cell proliferation culture: The Swallow Flower suspension cells obtained in step 2 were collected by filtering with a 150-mesh filter, and resuspended in suspension culture medium at a ratio of 1 g of suspension cells to 150 ml of suspension culture medium for suspension cell proliferation culture. The culture temperature was 24°C, 120 rpm / min, and subcultured every 10 days.

[0011] Step 4: Suspension cell renewal. The suspension cells of the Swallow's Soft Cells obtained in step 3 can be renewed after 6 months of subculture. The Swallow's Soft Cells are transferred to the fragile callus initiation medium. After culturing in a dark environment at 26°C for 30 days, the yellow callus with strong growth and no browning is taken to repeat steps 2 and 3.

[0012] The fragile callus initiation medium described in step 1 is MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.8 g / L agar, with a pH of 5.8-6.0.

[0013] The suspension culture medium described in step 2 is MS+0.5 mg / L 6-BA+0.2 mg / L NAA+0.5 mg / L 2,4-D+30 g / L sucrose, with a pH of 5.8-6.0.

[0014] The technology adopted in the present invention has the following beneficial effects in the process of establishing a culture system of the floribunda suspension cell: the floribunda suspension cell proliferates quickly, the obtained suspension cells have good activity, and can proliferate sustainably. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present invention will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments. The advantages and implementations of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings:

[0016] Attachment Figure 1 This is a picture of the resulting friable callus.

[0017] Attachment Figure 2 This is a diagram of starting a suspension culture.

[0018] Attachment Figure 3 This is the status of suspension cells after 5 proliferations

[0019] Attachment Figure 4 These are dividing suspension cells.

[0020] Attachment Figure 5 These are suspension cells that have been plated on fragile callus initiation medium for 30 days. DETAILED DESCRIPTION

[0021] The present invention is described in further detail below with reference to the embodiments and accompanying drawings to facilitate understanding of the present invention.

[0022] Unless otherwise specified, the experimental methods used in the examples are all conventional methods, and researchers in this field can achieve the results by following conventional tissue culture methods. The materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial channels.

[0023] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for establishing a suspension cell culture system of Flos sylvestris.

[0024] The preparation method of the relevant culture medium in the embodiment of the present invention is as follows:

[0025] The culture medium for initiating friable callus tissue is MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.8 g / L agar, with a pH of 5.8-6.0.

[0026] The suspension culture medium is MS+0.5 mg / L 6-BA+0.2 mg / L NAA+0.5 mg / L 2,4-D+30 g / L sucrose, with a pH of 5.8-6.0.

[0027] The present invention provides a method for establishing a suspension cell culture system of a scutellaria baicalensis. The method comprises the following steps:

[0028] (1) Initiation of culture of fragile callus tissue

[0029] Select callus tissue induced by swallowtail flower seeds, use a sharp scalpel to cut the healthy callus tissue into clumps with a diameter of 1-2 mm, and place them on the fragile callus initiation medium. The inoculation density is 50 clumps per 90 mm culture dish. In a dark environment with a culture temperature of 26 ℃, subculture once every 20 days and continue subculture until a sufficient amount of fragile callus tissue is produced. Fragile callus tissue is milky white, grows faster, and has a loose texture. Figure 1 This is a picture of the resulting friable callus.

[0030] (2) Establishment of suspension culture

[0031] Use a 720 mm diameter sterile glass tissue culture flask as the culture container. The volume of the suspension culture medium should not exceed 30% of the container. Use a scalpel to scrape the fragile callus tissue and place it in the container. Gently shake to resuspend. Start the suspension culture at a ratio of 1 g of fragile callus tissue to 100 ml of suspension culture medium. The culture temperature is 24 ℃, in the dark, and the speed is 120 rpm / min. Figure 2 This is the start of suspension culture. After 20 days, filter the suspended cell solution through a 30-mesh filter into a sterile 50-ml centrifuge tube. Use a 150-mesh filter to collect the cells in the centrifuge tube and resuspend them at a ratio of 1 g of suspended cells to 100 ml of suspension culture medium. Continue culturing under the same conditions for 20 days.

[0032] (3) Suspension cell proliferation culture

[0033] After the establishment of the suspension culture, the cells were collected using a 150-mesh filter, resuspended at a ratio of 1 g of suspended cells to 150 ml of suspension culture medium, and cultured in the dark at 24℃ and 120 rpm / min, with subculturing every 10 days. Figure 3 Figure 5 is a diagram of the state of the suspension cells after 5 rounds of proliferation. Figure 4 Figure 6 is a suspension cell that is dividing.

[0034] (4) Suspension cell renewal

[0035] Suspension cell renewal can be performed after 6 months of subculturing. The cells were collected using a 150-mesh filter, washed with suspension culture medium, and placed in a sterile 50-ml centrifuge tube. The suspension cells were evenly placed on the friable callus initiation medium, the medium was tilted, and the excess medium was removed. The cells were cultured in the dark at 26℃ for 30 days. Healthy yellow calli without browning were selected and subjected to the suspension culture establishment and suspension cell proliferation culture operations again. Figure 5 Figure 7 is a suspension cell that was placed on the friable callus initiation medium for 30 days.

[0036] The present application provides a method for establishing a suspension cell culture system of Yansiflower. The suspension cell culture system of Yansiflower can be rapidly established through the steps of friable callus initiation culture, suspension culture establishment, suspension cell proliferation culture, and suspension cell renewal. The present application is easy to master, has low cost and high efficiency, and can produce a large number of healthy Yansiflower cells for a long time, thereby laying a foundation for the subsequent establishment of an efficient elite propagation system and a genetic transformation system.

[0037] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-substantial changes and substitutions made by those skilled in the art based on the present application are within the scope of the present application.

Claims

1. A method for establishing a suspension cell culture system of Swallowtail jasmine, characterized in that: The following steps are involved: Step 1: Initiation of friable callus culture: select callus induced by seeds of Hibiscus sabdariffa and perform continuous subculture on friable callus initiation medium until a sufficient number of friable callus are produced; Step 2: Establish suspension culture. Take the fragile callus obtained in step 1 and start suspension culture at a ratio of 1 g of fragile callus to 100 ml of suspension culture medium. After 20 days, filter through a 30-mesh filter to remove clumping callus, and filter through a 150-mesh filter to collect cells. Resuspend at a ratio of 1 g of suspension cells to 100 ml of suspension culture medium, perform subculture, and continue culture for 20 days. Step 3: suspension cell proliferation culture: The Swallow Flower suspension cells obtained in step 2 were collected by filtering with a 150-mesh filter, and resuspended in suspension culture medium at a ratio of 1 g of suspension cells to 150 ml of suspension culture medium. The suspension cells were cultured for proliferation and subcultured every 10 days. Step 4: Suspension cell renewal. The suspension cells of the Swallow's Soft Cells obtained in step 3 can be renewed after 6 months of subculture. The Swallow's Soft Cells are transferred to the fragile callus initiation medium. After culturing in a dark environment at 26°C for 30 days, the yellow callus with strong growth and no browning is taken to repeat steps 2 and 3.

2. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The friable callus initiation medium described in step 1 is MS + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.8 g / agar, with a pH of 5.8-6.

0.

3. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The continuous subculture described in step 1 refers to using a sharp scalpel to cut healthy callus tissue into clumps with a diameter of 1-2 mm, placing them on fragile callus tissue initiation medium, inoculating 50 clumps per 90 mm culture dish, and subculturing them every 20 days in a dark environment at a culture temperature of 26°C.

4. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The fragile callus tissue described in step 1 refers to a callus tissue that is milky white, grows rapidly, and has a loose texture.

5. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The suspension culture medium described in step 2 and step 3 is MS+0.5 mg / L 6-BA+0.2 mg / L NAA+0.5 mg / L 2,4-D+30 g / L sucrose, with a pH of 5.8-6.

0.

6. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The suspension culture described in step 2 is carried out in a dark environment, at a constant temperature of 24° C. and a rotation culture speed of 120 rpm / min.

7. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The suspension cell proliferation culture described in step 3 is cultured in a dark environment, at a constant temperature of 24° C. and a rotation speed of 120 rpm / min.

8. The method for establishing a suspension cell culture system of Swallowtail Herba according to claim 1, characterized in that The Swallow Bloom suspension cells as described in step 4 were transferred to the fragile callus initiation medium. The specific method was to collect the suspension cells using a 150-mesh filter, rinse the cells with suspension culture medium into a sterile 50 ml centrifuge tube, and evenly place the suspension cells on the fragile callus initiation medium. The medium was tilted and excess culture medium was aspirated.

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