Method for converting compact callus of super bamboo reed into loose callus
By adding gibberellin to the culture medium and alternating between solid and liquid culture media, the problem of unstable texture of super reed callus was solved, and the directional transformation of loose callus was achieved, meeting the material requirements for suspension culture and protoplast preparation.
Patent Information
- Application Number
- CN202511043445.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-11-04
AI Technical Summary
In the existing technology, the texture of super reed callus is unstable, and it is impossible to obtain loose callus in a directional manner, which cannot meet the requirements of suspension culture and protoplast preparation.
By adding gibberellin (GA) to the culture medium and combining it with alternating solid and liquid culture, the texture transformation of super reed callus was regulated, achieving the transformation of compact callus to loose callus.
We successfully transformed the compact callus of *Arundo donax* into a loose one, meeting the material requirements for suspension culture and protoplast preparation while maintaining the activity and proliferation capacity of the callus.
Smart Images
Figure CN120883907A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for converting compact callus tissue of *Arundo donax* into loose callus tissue. Background Technology
[0002] Arundodis( Arundo donax (L.) Belongs to the Poaceae family and the Arundo genus. It is a perennial plant with tall stems and strong tillering ability. Super Arundo gracilis, obtained through bio-breeding technology, has a higher biomass and stronger environmental adaptability, and can be planted on marginal lands such as tidal flats, swamps, wastelands, and abandoned tailings. The harvested super Arundo gracilis stems have a wide range of applications. They can replace coal in thermal power generation, and can also be used to produce hydrogen, natural gas, biochar, etc. through thermal conversion technology, thus replacing traditional fossil fuels.
[0003] Under tissue culture conditions, callus tissue is an ideal material for plant regeneration, protoplast preparation, plant genetic transformation, and cell suspension culture. Callus tissue formed from plant cell dedifferentiation varies greatly in texture, color, and redifferentiation capacity. Different textures of callus tissue can meet different applications. Loose callus tissue contains numerous intercellular spaces, with cells arranged randomly, easily dispersing into single cells or small clusters of a few cells, making it the most suitable material for suspension culture and protoplast preparation.
[0004] The texture of callus tissue is affected by factors such as culture medium and culture method. In order to further improve the creation of a stable cell suspension culture system and protoplast preparation system for Super Reed, it is crucial to adjust the culture medium and culture method to change the texture of Super Reed callus tissue from firm to loose, thus meeting the corresponding requirements. Summary of the Invention
[0005] In view of the technical problems existing in the background art, the present invention provides a method for converting super reed shoots into dense callus tissue into loose callus tissue, aiming to solve the technical problem that the callus tissue obtained by the existing callus induction technology is unstable in texture and cannot be directionally obtained into loose callus tissue to meet the requirements for super reed shoots loose callus tissue.
[0006] In a first aspect, the present invention provides a method for converting super-tough callus tissue into loose callus tissue, comprising the following steps: Tight callus tissue of *Arundo donax* was transferred to the first culture medium for subculture and proliferation to obtain loose callus tissue. The first culture medium included MS medium, plant hormones, carbon source and coagulant. The plant hormones included 2,4-D, GA and KT. Alternatively, the compact type of super bamboo callus obtained from the solid medium is transferred to a liquid medium for subculture and proliferation, then the liquid medium is removed, and the callus is transferred to fresh solid medium for continuous culture to obtain loose type callus; the effective components in the liquid medium and the fresh solid medium are the same.
[0007] Preferably, the plant hormones in the first medium include 2-3 mg / L 2,4-D, 1-2 mg / L GA and 0.1-0.5 mg / L KT.
[0008] Preferably, the components of the first medium include MS medium, 2-3 mg / L 2,4-D, 1-2 mg / L GA, 0.1-0.5 mg / L KT, 30 g / L sucrose and 4-6 g / L plant gel.
[0009] Preferably, the culture conditions in the first medium are dark culture at 25±2℃ for at least 30 days.
[0010] Preferably, the components of the liquid medium include MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT and 30 g / L sucrose.
[0011] Preferably, the culture conditions in the liquid medium are dark culture at 25±2℃ for 2 weeks by placing the liquid medium on a constant temperature shaker.
[0012] Preferably, the rotation speed of the constant temperature shaker is 50-100 rpm.
[0013] Preferably, sterile filter paper is used to absorb the callus during the process of transferring the callus to fresh solid medium.
[0014] Preferably, the components of the fresh solid medium include MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT, 30 g / L sucrose and 4-6 g / L plant gel.
[0015] Preferably, the culture conditions in the fresh solid medium are dark culture at 25±2℃ for at least 30 days.
[0016] Compared with the prior art, the present application has the following beneficial effects: The present application is based on the fact that during the induction and proliferation of super bamboo callus, the medium is optimized by adding gibberellin, and the culture mode is switched to solid-liquid medium alternation culture, so that the compact type of callus is converted into loose type callus, thereby meeting the demand for loose type callus texture in super bamboo protoplast isolation and cell suspension culture, and building a technical platform for creating better new varieties of super bamboo. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 The texture of the callus finally obtained in Example 1 and Comparative Examples 1-2 of the present application; Figure 2 The texture of the callus finally obtained in Example 2 and Comparative Example 3 of the present application. DETAILED DESCRIPTION
[0018] The embodiments of the technical solutions of the present application will be described in detail below with reference to the accompanying drawings. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.
[0019] In order to solve the technical problem that the callus obtained by the existing callus induction technology has unstable texture and cannot be directly obtained in loose type to meet the demand for loose type callus of bamboo cane, the present application provides a method for converting compact type callus of super bamboo cane into loose type callus, wherein the conversion of the texture of the callus of super bamboo cane is regulated by changing the type of hormone and the mode of subculture in the culture medium, and the loose type callus of super bamboo cane is directly obtained.
[0020] In the first aspect, the embodiments of the present application provide a method for converting compact type callus of super bamboo cane into loose type callus, comprising the following steps: The compact type callus of super bamboo cane is transferred to a first culture medium for subculture and proliferation culture to obtain loose type callus; the first culture medium comprises MS culture medium, plant hormone, carbon source and coagulant; the plant hormone comprises 2,4-D, GA and KT; Alternatively, the compact type callus of super bamboo cane obtained from solid culture medium is transferred to liquid culture medium for subculture and proliferation culture, then the liquid culture medium is removed, and the callus is transferred to fresh solid culture medium for continuous culture to obtain loose type callus; the effective components in the liquid culture medium and the fresh solid culture medium are the same.
[0021] In the technical solution of the embodiments of the present application, gibberellin (GA) is added to the callus subculture medium. As a plant growth regulator, gibberellin plays a role in plant cell elongation, but for callus, it can convert compact type callus into loose type without affecting the activity of callus, and the obtained material can be used for cell suspension culture and preparation of protoplast. In addition, solid culture and liquid culture are alternately performed to convert compact type callus into loose type callus. Long-term solid culture causes unevenness in the absorption of nutrients by callus clumps, while liquid culture can solve this drawback. Therefore, liquid culture and solid culture are alternately used, and slow shaking is combined to promote the dispersibility of callus, so that super bamboo cane callus with strong vitality and good dispersibility can be obtained.
[0022] Further, in some embodiments, the plant hormones in the first medium include 2-3 mg / L 2,4-D, 1-2 mg / L GA, and 0.1-0.5 mg / L KT.
[0023] Further, in some embodiments, the components of the first medium include MS medium, 2-3 mg / L 2,4-D, 1-2 mg / L GA, 0.1-0.5 mg / L KT, 30 g / L sucrose, and 4-6 g / L phytagel.
[0024] Further, in some embodiments, the culture conditions in the first medium are dark culture at 25±2℃ for at least 30 days.
[0025] Further, in some embodiments, the components of the liquid medium include MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT, and 30 g / L sucrose.
[0026] Further, in some embodiments, the culture conditions in the liquid medium are dark culture at 25±2℃ for 2 weeks by placing the liquid medium on a constant temperature shaker.
[0027] Further, in some embodiments, the rotation speed of the constant temperature shaker is 50-100 rpm.
[0028] In the technical solution of the embodiments of the present application, the lower rotation speed of the constant temperature shaker can avoid excessive damage, but is sufficient to loosen the edges of the callus.
[0029] Further, in some embodiments, sterile filter paper is used to absorb the callus during the process of transferring the callus to the fresh solid medium.
[0030] Further, in some embodiments, the components of the fresh solid medium include MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT, 30 g / L sucrose, and 4-6 g / L phytagel.
[0031] Further, in some embodiments, the culture conditions in the fresh solid medium are dark culture at 25±2℃ for at least 30 days.
[0032] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are used to explain the present application, and cannot be understood as a limitation of the present application. If a specific technology or condition is not specified in the embodiments, the technology or condition described in the literature in the art or according to the product manual is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be obtained by purchase.
[0033] Example 1 A method for transforming compact type callus of super bamboo into loose type callus, comprising the following steps: The compact type callus of super bamboo is taken for subculture proliferation culture, the culture medium formula is MS+2,4-D 2mg / L+GA 2mg / L+KT 0.1mg / L+sucrose 30g / L+plant gel 4g / L, and dark culture is carried out at 25±2 DEG C for 30 days, so that loose type callus is obtained (the final obtained callus texture condition is shown as Figure 1 B).
[0034] Comparative Example 1 The compact type callus of super bamboo is taken for subculture proliferation culture, the culture medium formula is MS+2,4-D 2mg / L+KT 0.1mg / L+sucrose 30g / L+plant gel 4g / L, and dark culture is carried out at 25±2 DEG C for 30 days, so that callus is collected (the final obtained callus texture condition is shown as Figure 1 A).
[0035] The difference between the present comparative example and Example 1 lies in that gibberellin (GA) is not added in the subculture proliferation culture medium.
[0036] Comparative Example 2 The compact type callus of super bamboo is taken for subculture proliferation culture, the culture medium formula is MS+2,4-D 2mg / L+BA 2mg / L+KT 0.1mg / L+sucrose 30g / L+plant gel 4g / L, and dark culture is carried out at 25±2 DEG C for 30 days, so that callus is collected (the final obtained callus texture condition is shown as Figure 1 C).
[0037] The difference between the present comparative example and Example 1 lies in that gibberellin (GA) is replaced by 6-benzylaminopurine (BA) in the subculture proliferation culture medium.
[0038] Example 2 A method for transforming compact type callus of super bamboo into loose type callus, comprising the following steps: The compact type callus of super bamboo obtained from solid culture medium is transferred to liquid culture medium for subculture proliferation culture, the liquid culture medium formula is MS+2,4-D 2mg / L+KT 0.1mg / L+sucrose 30g / L, and dark culture is carried out at 25±2 DEG C in a constant temperature shaker at 50rpm for 2 weeks, then the liquid culture medium is removed, the callus is dried with sterile filter paper, and then transferred back to solid culture medium MS+2,4-D 2mg / L+KT 0.1mg / L+sucrose 30g / L+plant gel 4g / L, and dark culture is carried out at 25±2 DEG C for 30 days, so that loose type callus is obtained (the final obtained callus texture condition is shown as Figure 2 A).
[0039] Comparative Example 3 The compact type of super Arundo avenacea callus obtained from the solid medium was transferred to the solid medium for subculture and proliferation culture. The formula of the solid medium was MS + 2,4-D 2 mg / L + KT 0.1 mg / L + sucrose 30 g / L + plant gel 4 g / L, and the callus was collected after dark culture at 25 ± 2 °C for 44 days (the texture of the callus obtained finally is shown in Figure 2 B).
[0040] The difference between this comparative example and Example 2 is that the compact type of super Arundo avenacea callus was not subjected to solid-liquid alternating culture but was continuously cultured with the solid medium during subculture and proliferation culture.
[0041] The callus obtained after subculture of Example 1, Example 2, Comparative Examples 1-3 was collected, and the texture of the callus was observed.
[0042] As shown in Figure 1 , Figure 1 B is the texture display of the callus collected in Example 1 (with the addition of gibberellin), Figure 1 A is the texture display of the callus collected in Comparative Example 1 (without the addition of gibberellin), Figure 1 C is the texture display of the callus collected in Comparative Example 2 (with the addition of benzylaminopurine). It was found by observation that in the medium with the addition of gibberellin (GA), the color of the super Arundo avenacea callus changed from light yellow to milk white, and the dispersibility was good; the callus obtained in the medium without the addition of gibberellin (GA) and the addition of cell division factor 6-benzylaminopurine (BA) was still compact and in the form of light yellow clumps. The results showed that gibberellin helped to change the compact type of super Arundo avenacea callus into loose type, which was used for cell suspension culture or preparation and separation of protoplasts.
[0043] As shown in Figure 2 , Figure 2 A is the texture display of the callus collected in Example 2 (solid-liquid alternating culture), Figure 2 B is the texture display of the callus collected in Comparative Example 3 (continuous solid culture). It was found by observation that after two cycles of solid-liquid alternating culture, the compact callus became loose, and the granularity was good, and the proliferation ability was strong. The above results showed that the method of solid-liquid alternating culture helped to change the compact type of callus into loose type, and maintained good proliferation ability, so that the ideal loose type of super Arundo avenacea callus could be obtained as the material for subsequent experiments.
[0044] Note that the present application is not limited to the above-described embodiments. The above-described embodiments are merely examples, and embodiments having substantially the same configuration as the technical idea and achieving the same effects within the scope of the technical idea of the present application are included in the technical scope of the present application. Furthermore, other modes constructed by applying various modifications that can be thought of by those skilled in the art to the embodiments or by combining part of the constituent elements of the embodiments are also included in the scope of the present application without departing from the spirit of the present application.
Claims
1. A method for transforming super-firm callus tissue of *Prunus armeniaca* into loose callus tissue, characterized in that, Includes the following steps: Tight callus tissue of *Arundo donax* was transferred to a first culture medium for subculture to obtain loose callus tissue. The first culture medium included MS medium, plant hormones, carbon source and coagulant. The plant hormones included 2,4-D, GA and KT. Alternatively, the super-firm callus obtained from solid culture medium is transferred to liquid culture medium for subculture and proliferation. Then, the liquid culture medium is removed, and the callus is transferred to fresh solid culture medium for further culture to obtain loose callus. The liquid culture medium and the fresh solid culture medium contain the same active ingredients.
2. The method for converting super reed shoots into loose callus tissue according to claim 1, characterized in that, The plant hormones in the first culture medium include 2-3 mg / L 2,4-D, 1-2 mg / L GA and 0.1-0.5 mg / L KT.
3. The method for converting super reed shoots into loose callus tissue according to claim 2, characterized in that, The first culture medium consists of MS medium, 2-3 mg / L 2,4-D, 1-2 mg / L GA, 0.1-0.5 mg / L KT, 30 g / L sucrose, and 4-6 g / L plant gel.
4. The method for converting super-tough callus tissue into loose callus tissue according to claim 1, characterized in that, The culture conditions in the first culture medium are: dark culture at 25±2℃ for at least 30 days.
5. The method for converting super reed (Prunus armeniaca) compact callus into loose callus according to claim 1, characterized in that, The liquid culture medium comprises MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT, and 30 g / L sucrose.
6. The method for converting super reed shoots into loose callus tissue according to claim 1, characterized in that, The culture conditions in the liquid culture medium are as follows: the liquid culture medium is placed on a constant temperature shaker and cultured in the dark at 25±2℃ for 2 weeks.
7. The method for converting super reed shoots into loose callus tissue according to claim 6, characterized in that, The rotation speed of the constant temperature shaker is 50~100 rpm.
8. The method for converting super-firm callus tissue into loose callus tissue according to claim 1, characterized in that, During the process of transferring the callus tissue back to fresh solid culture medium, sterile filter paper is used to blot the callus tissue dry.
9. The method for converting super reed shoots into loose callus tissue according to claim 1, characterized in that, The fresh solid culture medium comprises MS medium, 2-3 mg / L 2,4-D, 0.1-0.5 mg / L KT, 30 g / L sucrose, and 4-6 g / L plant gel.
10. The method for converting super-tough callus tissue into loose callus tissue according to claim 1, characterized in that, The culture conditions in the fresh solid culture medium are: dark culture at 25±2℃ for at least 30 days.