Rocker-type liquid culture device and culture method

Abstract

This invention discloses a rocker-type liquid culture device, comprising: a rocker-type shaker; a culture box disposed on the rocker-type shaker, the culture box having multiple partitions that divide the culture box into multiple culture spaces; and a culture bottle placed correspondingly within the culture spaces of the culture box. Each culture bottle includes an outer bottle and an inner bottle. The outer bottle holds the liquid culture medium, and the inner bottle is placed inside the outer bottle. The bottom of the inner bottle has a support portion and is spaced apart from the bottom of the outer bottle. The bottom of the inner bottle has multiple water-permeable holes, and its inner surface has at least one partition forming a culture zone. In this rocker-type liquid culture device, the culture box is placed on the rocker-type shaker, which shakes regularly. Because the partitions prevent the culture material from being continuously immersed in the culture medium, it still receives periodic nutrient supply, allowing the tissue culture material to grow rapidly.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a rocker-type liquid culture device and culture method. Background Technology

[0002] In the industrialization of plant tissue culture, common cultivation methods are solid-state culture and liquid-state culture. Solid-state culture is currently more widely used, while liquid-state culture has not been widely adopted due to its tendency to restrict plant respiration and hinder growth. However, liquid-state culture offers faster propagation, allows for better nutrient access to the material, and enables more precise control of solution temperature and nutrient concentration. To address the issue of respiration restriction in liquid-state culture, various solutions have been proposed in production, among which intermittent immersion culture is a frequently reported method. This method involves periodically immersing tissue culture materials in a culture medium. It requires an air pump, a timing device, a conduction device, and a filter membrane, among other complex equipment. Crucially, a good seal is essential; any air leakage will prevent its implementation. This drawback hinders the widespread application of intermittent immersion culture in production.

[0003] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] The purpose of this invention is to provide a rocker-type liquid culture device, thereby overcoming the disadvantages of liquid culture devices such as complex structure and easy air leakage.

[0005] Another object of the present invention is to provide a method for culturing using a rocker-type liquid culture device.

[0006] To achieve the above objectives, the present invention provides a rocker-type liquid culture device, comprising: a rocker-type shaker; a culture chamber disposed on the rocker-type shaker, the culture chamber having multiple partitions that divide the culture chamber into multiple culture spaces; and a culture bottle correspondingly placed within the culture spaces of the culture chamber, the culture bottle comprising an outer bottle and an inner bottle, the outer bottle for holding liquid culture medium, the inner bottle being placed inside the outer bottle, the bottom of the inner bottle having a support portion and being spaced apart from the bottom of the outer bottle, the bottom of the inner bottle having multiple water-permeable holes, and the inner surface having at least one partition forming a culture zone.

[0007] Preferably, in the above technical solution, the incubator is a sealed structure with an air inlet and a vent, and a vent filter membrane is provided at the vent.

[0008] Preferably, in the above technical solution, the partitions inside the incubator are arranged in a crisscross pattern.

[0009] Preferably, in the above technical solution, the inner bottle has multiple water-permeable holes on the surrounding walls near the bottom of the bottle.

[0010] Preferably, in the above technical solution, the bottle wall around the inner bottle is in the form of a fence.

[0011] A rocker-type liquid culture method, using the above-mentioned culture device, includes the following steps:

[0012] (1) Prepare nutrient solution: Dispense the prepared nutrient solution into the inner bottle of the culture bottle. The amount of culture solution added should just cover the bottom of the inner bottle. Then place it in the incubator, seal the cap, and sterilize.

[0013] (2) Inoculation: Select seedlings, put the seedlings into the inner bottle of the culture bottle, then put the culture bottle into the culture space of the incubator and cover the lid of the incubator.

[0014] (3) Cultivation: Place the incubator on the rocker-type shaker, connect the air inlet of the incubator to the air inlet pipe, introduce CO2, start the shaker, and carry out cultivation.

[0015] Preferably, in the above technical solution, the cultivation conditions in step (3) are: cultivation temperature 23-27℃, light intensity 2400-3200lx, light cycle 10-12h, and cultivation for 50-70 days.

[0016] Preferably, in the above technical solution, the rocker-type rocking bed has a rocking frequency of 40-70 times / min.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The rocker-type liquid culture device of the present invention is used for liquid culture. Compared with solid culture, liquid culture allows for more precise control of solution temperature and nutrient concentration, resulting in faster reproduction. This device places the culture chamber on a rocker-type shaker. The shaker shakes regularly, and because the culture material is blocked by partitions, it is not continuously immersed in the culture solution, but still receives periodic nutrient supply, enabling rapid growth of the tissue culture material. The periodic immersion culture method solves the problem of limited respiration in liquid culture, providing nutrients while ensuring normal respiration of the culture material.

[0019] (2) In the liquid culture method of this invention, CO2 is used as the carbon source, and CO2 is introduced into the incubator. Conventional solid culture media require the addition of sugar as a carbon source to provide to the culture material. Therefore, tissue culture seedlings grown in solid culture generally have weak respiration capacity, and the sugar content in the culture medium can easily lead to contamination. This invention uses CO2 as the carbon source, allowing the material to perform photosynthesis autonomously, and the respiration capacity is stronger, resulting in a high survival rate after transplanting. The contamination rate is also reduced after sugar is not added to the nutrient solution. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the incubator structure in the rocker-type liquid culture device according to the present invention;

[0021] Figure 2 This is a schematic diagram of the structure of the culture flask in the rocker-type liquid culture device according to the present invention. Detailed Implementation

[0022] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.

[0023] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0024] like Figures 1 to 2 As shown, a rocker-type liquid culture device according to a specific embodiment of the present invention includes a rocker-type shaker, an incubator 1, and a culture bottle 2. In use, the seedlings to be cultured and the liquid culture solution are placed in the culture bottle 2, the culture bottle 2 is placed in the incubator 1, and the incubator 1 is placed on the rocker-type shaker. The shaker oscillates regularly to carry out the culture.

[0025] The specific structure is as follows: The incubator 1 is placed on a rocker-type shaker (not shown). The incubator 1 contains multiple partitions 11; in this embodiment, two partitions 11 are arranged perpendicularly to each other, dividing the incubator into four culture spaces. The incubator 1 is a sealed structure with an air inlet 12 and a vent 13. A breathable filter membrane is installed at the vent. The air inlet 12 is connected to an air inlet pipe 14, allowing CO2 gas to be introduced into the incubator 1. Culture bottles 2 are placed within the culture spaces of the incubator. Culture bottles 2 include an inner bottle 21 and an outer bottle 22. The outer bottle 22 is an open structure used to hold liquid culture medium. Both the inner and outer bottles of culture bottle 2 are made of high-temperature resistant plastic. The outer bottle 22 is cylindrical, sized to fit perfectly into the small cubic culture space area of ​​the incubator. The bottom of the inner bottle 21 has a support part 23, which is a support rod. Four support rods are located at the bottom of the inner bottle 21, which is placed inside the outer bottle 22. The inner bottle 21 is 0.5 cm smaller in diameter than the outer bottle 22. Due to the support rod at the bottom of the inner bottle, there is a 1 cm gap between the bottom of the inner bottle 21 and the bottom of the outer bottle 22. The bottom of the inner bottle 21 has multiple permeable holes, allowing liquid culture medium to enter. In this embodiment, a permeable filter screen is laid at the bottom of the inner bottle 21. Preferably, the inner bottle 21 has multiple permeable holes on its surrounding walls near the bottom. The walls of the inner bottle are fence-like, facilitating the entry of liquid culture medium. At least one partition 24 is provided on the inner surface of the bottom of the inner bottle 21; in this embodiment, one partition is provided. The partition 24 divides the inner bottle into two culture zones.

[0026] The method of using the rocker-type liquid culture device of the present invention is as follows: During culture, first pour the prepared nutrient solution into the inner bottle 21, the volume of which should just submerge the bottom of the inner bottle 21. Then place it in the incubator 1, cover it, and sterilize it at high temperature. In the inoculation chamber, place the inoculation material in two areas of the inner bottle 21. After inoculation, directly place the culture bottle 2 into the incubator 1, rotate and adjust the culture bottle 2 so that the partition 24 is perpendicular to the shaker, ensuring that the liquid can sway from side to side. Then cover the incubator 1 and connect the CO2 vent pipe. Place the incubator on the rocker-type shaker, start the shaker, and shake at a frequency of 50 times / min. The culture solution will sway from side to side like ocean waves. Because the partition is blocked, the culture material will not be continuously immersed in the culture solution, but can still receive nutrient supply periodically, which can make the tissue culture material grow rapidly.

[0027] Example 1

[0028] Dendrobium orchids were cultured using the aforementioned rocker-type liquid culture device. The culture method included the following steps:

[0029] (1) Nutrient solution preparation: MS basic culture medium + NAA 1.5mg / L + banana juice 30g / L, pH 5.6, sugar-free and agar-free. Mix well and dispense into culture bottles, 60ml per bottle, just enough to submerge the bottom of the inner bottle. After dispensing, place in an incubator, 4 bottles per box, cover the incubator, and sterilize in an autoclave. Cool before use.

[0030] (2) Inoculation: Select a healthy, uncontaminated Dendrobium officinale subculture bottle. In the ultra-clean workbench, take out the seedlings, divide them into individual plants and put them into the inner bottle of the culture bottle. Place 20 plants on each side. After inoculating 4 bottles, put them directly into the incubator. Rotate and adjust the culture bottle so that the partition is perpendicular to the shaker to ensure that the liquid can be shaken from side to side. Then cover the incubator with the lid.

[0031] (3) Cultivation: Take the incubator to the cultivation chamber, connect the CO2 vent pipe to the incubator's input port, and then place the incubator on a rocker-type shaker. Start the shaker and shake at a frequency of 50 times / min. The cultivation chamber temperature is 23℃~27℃, the light intensity is 2400lx~3200lx, and the photoperiod is 10h~12h. After 60 days of cultivation, the seedlings will grow taller and stronger, develop roots, and can then be transplanted.

[0032] Example 2

[0033] Ornamental ferns were cultured using the aforementioned rocker-type liquid culture device. The culture method included the following steps:

[0034] (1) Nutrient solution preparation: 1 / 2 MS basic culture medium + IBA 2.0 mg / L + NAA 0.1 mg / L + activated carbon 1.5 g / L, pH 5.6, sugar-free and agar-free. Mix well and dispense into culture flasks, 60 ml per flask, just enough to submerge the bottom of the inner flask. After dispensing, place in an incubator, 4 flasks per box, cover the incubator, and sterilize in an autoclave. Cool before use.

[0035] (2) Inoculation: Select a healthy, uncontaminated ornamental fern subculture seedling. In a clean bench, take out the seedling, divide it into individual plants and put them into the inner bottle of the culture bottle, 20 plants on each side. After inoculating 4 bottles, put them directly into the incubator. Rotate and adjust the culture bottle so that the partition is perpendicular to the shaker, ensuring that the liquid can be shaken from side to side. Then cover the incubator with the lid.

[0036] (3) Cultivation: Take the incubator to the cultivation chamber, connect the CO2 vent pipe to the incubator's input port, and then place the incubator on a rocker-type shaker. Start the shaker and shake at a frequency of 50 times / min. The cultivation chamber temperature is 23℃~27℃, the light intensity is 2400lx~3200lx, and the photoperiod is 10h~12h. After 60 days of cultivation, the seedlings will grow taller and stronger, develop roots, and can then be transplanted.

[0037] Example 3

[0038] The above-mentioned rocker-type liquid culture device was used to cultivate jasmine. The cultivation method included the following steps:

[0039] (1) Nutrient solution preparation: 1 / 2 MS basic culture medium + IBA 0.8 mg / L, pH 5.6, sugar-free and agar-free. Mix well and dispense into culture bottles, 60 ml per bottle, just enough to submerge the bottom of the inner bottle. After dispensing, place in an incubator, 4 bottles per box, cover the incubator, and sterilize in an autoclave. Cool before use.

[0040] (2) Inoculation: Select a healthy, uncontaminated jasmine subculture seedling. In a clean bench, take out the seedling, cut off the lower half, leaving 3-5cm of the upper half, and place it vertically on the edge of the inner bottle of the culture bottle, 10 seedlings on each side. After inoculating 4 bottles, place them directly into the incubator, rotate and adjust the culture bottle so that the partition is perpendicular to the shaker, ensuring that the liquid can be shaken from side to side, and then cover the incubator lid.

[0041] (3) Cultivation: Take the incubator to the cultivation chamber, connect the CO2 vent pipe to the incubator's input port, and then place the incubator on a rocker-type shaker. Start the shaker and shake at a frequency of 50 times / min. The cultivation chamber temperature is 23℃~27℃, the light intensity is 2400lx~3200lx, and the photoperiod is 10h~12h. After 60 days of cultivation, the seedlings will grow taller and stronger, develop roots, and can then be transplanted.

[0042] control group

[0043] Dendrobium, ornamental fern, and jasmine were cultured using the traditional solid-state culture method used in the control group. The control group used traditional solid-state rooting culture, inoculating seedlings into their respective agar and sugar-containing media. The medium for Dendrobium was: MS + NAA 1.5 mg / L + banana juice 30 g / L + sugar 30 g / L + agar 4.2 g / L. The medium for ornamental fern was: 1 / 2 MS + IBA 2.0 mg / L + NAA 0.1 mg / L + activated carbon 1.5 g / L + sugar 30 g / L + agar 4.2 g / L. The medium for jasmine was: 1 / 2 MS + IBA 0.8 mg / L + sugar 30 g / L + agar 4.2 g / L. The culture chamber temperature was 23℃–27℃, the light intensity was 2400 lx–3200 lx, the photoperiod was 10 h–12 h, and the seedlings were transplanted after 60 days of culture.

[0044] The survival rate of seedlings after transplanting following the present invention and traditional rooting culture is compared in Table 1.

[0045] Table 1

[0046]

[0047] As shown in Table 1, the transplant survival rate of Dendrobium, ornamental fern and jasmine using the rocker-type liquid culture of the present invention is higher than that of traditional solid rooting culture.

[0048] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A rocker-type liquid culture method, characterized in that, The cultivation using a rocker-type liquid culture device includes the following steps: (1) Prepare nutrient solution: Dispense the prepared nutrient solution into the inner bottle of the culture bottle. The amount of culture solution added should just cover the bottom of the inner bottle. Then place it in the incubator, seal the cap, and sterilize. (2) Inoculation: Select seedlings, put the seedlings into the inner bottle of the culture bottle, then put the culture bottle into the culture space of the incubator and cover the lid of the incubator; (3) Cultivation: Place the incubator on the rocker shaker, connect the air inlet of the incubator to the air inlet pipe, introduce CO2, start the shaker, and carry out cultivation; A rocker-type liquid culture device, comprising: rocker-type shaking bed; An incubator, disposed on the rocker-type shaker, wherein the incubator is provided with multiple partitions, the multiple partitions dividing the incubator into multiple culture spaces; and A culture bottle is placed in the culture space of the incubator. The culture bottle includes an outer bottle and an inner bottle. The outer bottle is used to hold liquid culture medium. The inner bottle is placed inside the outer bottle. The bottom of the inner bottle is provided with a support part and is spaced apart from the bottom of the outer bottle. The bottom of the inner bottle is provided with multiple water-permeable holes, and the inner surface is provided with at least one partition to form a culture zone.

2. The rocker-type liquid culture method according to claim 1, characterized in that, The incubator is a sealed structure with an air inlet and a vent, and a breathable filter membrane is provided at the vent.

3. The rocker-type liquid culture method according to claim 1, characterized in that, The partitions inside the incubator are arranged in a crisscross pattern.

4. The rocker-type liquid culture method according to claim 1, characterized in that, The inner bottle has multiple water-permeable holes on its four sides near the bottom.

5. The rocker-type liquid culture method according to claim 1, characterized in that, The inner bottle has a fence-like structure around its walls.

6. The rocker-type liquid culture method according to claim 1, characterized in that, The cultivation conditions for step (3) are: cultivation temperature 23-27℃, light intensity 2400-3200 lx, light cycle 10-12h, and cultivation for 50-70 days.

7. The rocker-type liquid culture method according to claim 1, characterized in that, The rocker-type rocking bed shakes at a frequency of 40-70 times / min.

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