Culture device for crithmum maritimum callus and application method

Abstract

The invention discloses a culture device for crithmum maritimum callus and an application method, and relates to the technical field of tissue culture. A mode is randomly switched by set environmental factors in a crithmum maritimum callus culture period of 3-4 weeks; the growth environment factors of the crithmum maritimum can randomly generate severe environment factor combinations of high (low) temperature, high (low) PH value, high (low) osmotic pressure and strong (weak) oxygen supply, the severe natural environment of crithmum maritimum growth can be simulated, and the growth capability, metabolic activity and regeneration capability of cultured crithmum maritimum callus are improved. Most importantly, through random switching of severe environments, bacteria can be efficiently killed and reproduction of the bacteria can be restrained, the problem that the bacteria in a severe environment for a long time derives tolerance and can continue reproduction is avoided, the highly sterile environment of callus culture is guaranteed, meanwhile, extra economic cost and energy are not needed to create the sterile environment, and the method is suitable for large-scale popularization and application. Meanwhile, the problem that bacteria derive tolerance can be efficiently avoided.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, in particular to a culture device for sea fennel callus and an application method. Background Art

[0002] The callus culture of sea fennel is a plant tissue culture technology, which is not only related to botanical research, but also to the development of cosmetics and skin care products. Sea fennel is an aromatic plant of the Umbelliferae family that grows on the coast. The callus culture filtrate is mainly derived from stem cells extracted from the roots of the plant. This culture filtrate has important applications in the cosmetics industry, especially in skin protection and repair.

[0003] The callus culture filtrate of sea fennel has multiple functions, including anti-oxidation, soothing and skin repair. Since the natural growth environment of sea fennel is very harsh, it can withstand extreme environmental conditions such as high salinity, high (low) temperature and high ultraviolet radiation, accelerate the metabolism of the epidermis, improve the skin's self-healing ability, and promote the regeneration of cell tissue. These characteristics make the callus culture filtrate of sea fennel used as an effective skin protectant in cosmetics.

[0004] The callus culture of sea fennel is mostly carried out in the form of an incubator. During the callus culture process, if bacteria, fungi or other microorganisms are present, they may compete with the cultured plant tissue for nutrients or even produce harmful substances, affecting the growth and differentiation of plant tissue. In addition, some microorganisms may directly attack plant tissue, causing tissue death. Moreover, in scientific research, the repeatability and reliability of the experiment are very important. If microbial contamination occurs during the callus culture process, the accuracy of the experimental results cannot be guaranteed, thus affecting the scientific value of the research.

[0005] Currently, the way to ensure the sterility of callus tissue culture is to disinfect instruments before cultivation. However, the callus tissue itself may carry some bacteria or fungi, so it is difficult to completely avoid bacterial invasion. In addition, some bacteria may undergo gene mutations (bacteria acquire new traits through gene mutations, which may help them survive under adverse conditions), horizontal gene transfer (acquiring tolerance-related genes from other bacteria through conjugation, transformation or transduction), or phenotypic variation (adjusting their own physiological state to adapt to environmental changes) during the disinfection period, thereby deriving the ability to avoid disinfection by developing tolerance to the current harsh environmental temperature, pH, etc. Experimental personnel have spent a lot of manpower and material resources on this issue and have not been able to find a more suitable sterilization method.

[0006] In view of the above problems, it is urgently necessary to carry out innovative design based on the original culture device and application method for sea fennel callus tissue. Summary of the invention

[0007] The technical solution of the present invention aims at the technical problem that the existing technical solutions are too single, and provides a solution that is significantly different from the existing technologies. Specifically, the purpose of the present invention is to provide a culture device for sea fennel callus tissue, so as to solve the above-mentioned background technology. The current way to ensure the sterility of callus tissue culture is to disinfect the instruments before cultivation, but the callus tissue itself may carry some bacteria or fungi, so it is difficult to completely avoid bacterial invasion, and some bacteria will undergo gene mutations during the disinfection period (bacteria obtain new traits through gene mutations, and these traits may help them survive under adverse conditions), horizontal gene transfer (obtaining tolerance-related genes from other bacteria through conjugation, transformation or transduction), or phenotypic variation (adjusting their own physiological state to adapt to environmental changes), etc., thereby deriving the ability to avoid disinfection due to poor tolerance to the current environmental temperature, pH, etc. The experimenters have spent a lot of manpower and material resources on this issue, and have not been able to find a more suitable sterilization method.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solutions: a culture device and application method for sea fennel callus tissue, comprising a control cabinet, a culture bracket is arranged inside the culture chamber of the control cabinet, and a temperature control module, a pH value control module, an osmotic pressure control module and an oxygen supply module are respectively installed on the inner wall of the culture chamber, and the control cabinet is used to regulate the temperature control module, the pH value control module, the osmotic pressure control module and the oxygen supply module.

[0009] The present invention also discloses an application method of sea fennel callus tissue, which uses the above-mentioned culture device to be applied to the culture process of sea fennel callus tissue, comprising the following steps: S1: Use healthy leaves, stems or shoots of sea fennel as explants, wipe the explants with 70% alcohol, soak them in 5% sodium hypochlorite solution for about 10 minutes, and finally rinse them with sterile water for 3-5 times. Use MS culture medium; S2: Add appropriate amount of plant hormones such as 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (naphthaleneacetic acid) to the culture medium to induce callus formation, and appropriate amount of cytokinin such as BAP (6-benzylaminopurine) to promote the division of sea fennel callus cells; S3: Under sterile conditions, the sterilized explants are cut into small pieces, usually 0.5-1.0 cm in length, and the cut explants are inoculated onto MS medium containing hormones; S4: placing the callus tissue culture medium prepared according to S1-S3 on the culture bracket of the culture chamber in the control cabinet, and setting the initial environment of the culture chamber through the control cabinet to control the temperature control module, pH value control module, osmotic pressure control module and oxygen supply module, the initial temperature is set to 20°C, the initial pH value is set to 6 mol / L (the pH range between 5.8 mol / L and 6.2 mol / L is considered to be the optimal condition for most plant tissue culture), the initial osmotic pressure is set to 200 mM NaCl, and the initial oxygen supply is set to 10 (±1)% by volume; S5: During the 3-4 week Crimson Fennel callus culture period, the temperature, pH value, osmotic pressure and oxygen supply were switched synchronously every two hours through program setting.

[0010] The key difference between the present method and other callus tissue cultivations is that: according to step S5, the temperature, pH value, osmotic pressure and oxygen supply are synchronously switched every two hours; within the same switching period, the temperature is randomly selected between 15°C and 35°C, the pH value is randomly selected between 5.0 mol / L and 7.0 mol / L, the osmotic pressure is randomly selected between 50 mM NaCl and 400 mM NaCl, and the oxygen supply is randomly selected between 5 (±1)% and 25 (±1)%. The key point is that all environmental factors (temperature, pH value, osmotic pressure and oxygen supply, and other environmental factors can also be appropriately added) will be randomly selected and combined.

[0011] According to the mode set in step S5, the environmental factors in the 3-4 week sea fennel callus culture period are random and generally will not be the same. Finally, during the sea fennel callus culture period, the control system is used to set a random combination mode of harsh environmental factors such as high (low) temperature, high (low) pH value, high (low) osmotic pressure and strong (weak) oxygen supply to simulate the harsh natural environment for the growth of sea fennel.

[0012] Compared with the prior art, the present invention has the following beneficial effects: The key difference between the present invention and other callus tissue cultivations is that the temperature, pH value, osmotic pressure and oxygen supply are synchronously switched once every two hours. Within the same switching period, the temperature is randomly selected between 15°C and 35°C, the pH value is randomly selected between 5.0 mol / L and 7.0 mol / L, the osmotic pressure is randomly selected between 50 mM NaCl and 400 mM NaCl, and the oxygen supply is randomly selected between 5 (±1)% and 25 (±1)%. The key point is that all environmental factors (temperature, pH value, osmotic pressure and oxygen supply, and other environmental factors can also be appropriately added) are randomly selected and combined; According to the above-mentioned model, the environmental factors will not be the same during the 3-4 week sea fennel callus culture cycle. A combination of harsh environmental factors such as high (low) temperature, high (low) pH value, high (low) osmotic pressure and strong (weak) oxygen supply will appear randomly, which can simulate the harsh natural environment for the growth of sea fennel and enhance the growth ability, metabolic activity and regeneration ability of the sea fennel callus after cultivation. In addition, the most important point is that by switching between random harsh environments, bacteria can be effectively killed and their reproduction can be curbed, avoiding the problem of bacteria developing tolerance and continuing to reproduce in a harsh environment for a long time, ensuring a highly sterile environment for callus culture, without requiring additional economic costs and energy to create a sterile environment, and at the same time effectively avoiding the problem of bacteria developing tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is a schematic diagram of the overall structure of the control cabinet of the present invention; Figure 2 This is a schematic diagram of the internal structure of the cultivation chamber of the present invention; Figure 3 It is a structural schematic diagram of the culture bracket of the present invention; Figure 4 It is a schematic diagram of the first position structure of the control module of the present invention; Figure 5 It is a schematic diagram of the second position structure of the control module of the present invention.

[0014] In the figure: 1. control cabinet; 2. culture tray; 3. temperature control module; 4. pH value control module; 5. osmotic pressure control module; 6. oxygen supply module. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] See also Figure 1-5 The present invention provides a technical solution: a culture device for sea fennel callus tissue, comprising a control cabinet 1, wherein a culture bracket 2 is arranged inside a culture chamber of the control cabinet 1, and a temperature control module 3, a pH value control module 4, an osmotic pressure control module 5 and an oxygen supply module 6 are respectively installed on the inner wall of the culture chamber, and the control cabinet 1 is used to regulate the temperature control module 3, the pH value control module 4, the osmotic pressure control module 5 and the oxygen supply module 6.

[0017] A method for applying sea fennel callus tissue, using the above-mentioned culture device to apply to the culture process of sea fennel callus tissue, comprising the following steps: S1: Use healthy leaves, stems or shoots of sea fennel as explants, wipe the explants with 70% alcohol, soak them in 5% sodium hypochlorite solution for about 10 minutes, and finally rinse them with sterile water for 3-5 times. Use MS culture medium; S2: Add appropriate amount of plant hormones such as 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (naphthaleneacetic acid) to the culture medium to induce callus formation, and appropriate amount of cytokinin such as BAP (6-benzylaminopurine) to promote the division of sea fennel callus cells; S3: Under sterile conditions, the sterilized explants are cut into small pieces, usually 0.5-1.0 cm in length, and the cut explants are inoculated onto MS medium containing hormones; S4: placing the callus culture medium prepared according to S1-S3 on the culture bracket 2 of the culture chamber in the control cabinet 1, and setting the initial environment of the culture chamber through the control cabinet 1 by controlling the temperature control module 3, the pH value control module 4, the osmotic pressure control module 5 and the oxygen supply module 6, the initial temperature is set to 20°C, the initial pH value is set to 6 mol / L (the pH range between 5.8 mol / L and 6.2 mol / L is considered to be the optimal condition for most plant tissue culture), the initial osmotic pressure is set to 200 mM NaCl, and the initial oxygen supply is set to a volume ratio of 10 (±1)%; S5: During the 3-4 week Crimson Fennel callus culture period, the temperature, pH value, osmotic pressure and oxygen supply were switched synchronously every two hours through program setting.

[0018] The key difference between this method and other callus tissue cultivation is that: according to step S5, the temperature, pH value, osmotic pressure and oxygen supply are switched synchronously every two hours; within the same switching period, the temperature is randomly selected between 15°C and 35°C, the pH value is randomly selected between 5.0 mol / L and 7.0 mol / L, the osmotic pressure is randomly selected between 50 mM NaCl and 400 mM NaCl, and the oxygen supply is randomly selected between 5 (±1)% and 25 (±1)%. The key point is that all environmental factors (temperature, pH value, osmotic pressure and oxygen supply, and other environmental factors can also be appropriately added) are randomly selected and combined.

[0019] According to the mode set in step S5, the environmental factors will not be the same during the 3-4 week sea fennel callus culture period. Finally, during the sea fennel callus culture period, the control system is used to set a random combination mode of harsh environmental factors such as high (low) temperature, high (low) pH value, high (low) osmotic pressure and strong (weak) oxygen supply to simulate the harsh natural environment for the growth of sea fennel.

[0020] In this embodiment, the environmental factors set in the pattern of the method steps during the 3-4 week sea fennel callus culture period will not be the same, and a combination of harsh environmental factors such as high (low) temperature, high (low) pH value, high (low) osmotic pressure and strong (weak) oxygen supply will appear randomly, which can simulate the harsh natural environment for the growth of sea fennel and improve the growth ability, metabolic activity and regeneration ability of the sea fennel callus after cultivation. In addition, the most important point is that by switching between random harsh environments, bacteria can be efficiently killed and their reproduction can be curbed, avoiding the problem of bacteria developing tolerance and continuing to reproduce in a harsh environment for a long time, ensuring a highly sterile environment for callus culture, without requiring additional economic costs and energy to create a sterile environment, and at the same time, the derivation of bacterial tolerance can be efficiently avoided.

[0021] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A device for cultivating callus tissue of Crimson Fennel, comprising a control cabinet (1), characterized in that: A culture bracket (2) is arranged inside the culture chamber of the control cabinet (1), and a temperature control module (3), a pH value control module (4), an osmotic pressure control module (5) and an oxygen supply module (6) are respectively installed on the inner wall of the culture chamber, and the control cabinet (1) is used to control the temperature control module (3), the pH value control module (4), the osmotic pressure control module (5) and the oxygen supply module (6).

2. A method for applying sea fennel callus, characterized in that The culture device as claimed in claim 1 is applied to the culture process of sea fennel callus tissue, comprising the following steps: S1: Use healthy leaves, stem segments or young shoots of sea fennel as explants, wipe the explants with 70% alcohol, then soak them in 5% sodium hypochlorite solution for about 10 minutes, and finally rinse them with sterile water 3-5 times; S2: Adding appropriate amount of plant hormones to the MS medium to induce callus formation, and appropriate amount of cytokinin to promote the division of sea fennel callus cells; S3: Under sterile conditions, the sterilized explants were cut into pieces and inoculated onto the MS medium treated with S2 to obtain callus culture medium; S4: placing the callus tissue culture medium prepared according to S1-S3 on a culture bracket (2) of a culture chamber in a control cabinet (1), and setting the initial environment of the culture chamber by controlling a temperature control module (3), a pH value control module (4), an osmotic pressure control module (5) and an oxygen supply module (6) through the control cabinet (1), wherein the initial temperature is set to 20°C, the initial pH value is set to 6 mol / L, the initial osmotic pressure is set to 200 mM NaCl, and the initial oxygen supply is set to a volume ratio of 10 (±1)%; S5: During the 3-4 week Crimson Fennel callus culture period, the temperature, pH value, osmotic pressure and oxygen supply are switched synchronously every two hours through program setting; Among them, the environmental factors that are switched synchronously every two hours are randomly selected and determined. Within the same switching period, the temperature is randomly selected between 15℃-35℃, the pH value is randomly selected between 5.0mol / L-7.0mol / L, the osmotic pressure is randomly selected between 50mM NaCl-400mMNaCl, and the oxygen supply is randomly selected between 5±1%-25±1%.

3. The application method of the sea fennel callus according to claim 2, characterized in that The plant hormone used in S2 is selected from 2,4-dichlorophenoxyacetic acid or naphthylacetic acid.

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