A liquid culture device and method for studying symbiosis of arbuscular mycorrhizal fungi

Abstract

The present invention belongs to the field of plant and microbial symbiosis research, and specifically relates to a liquid culture device and method for studying symbiosis of arbuscular mycorrhizal fungi, comprising a box body for containing liquid culture nutrient solution for studying symbiosis of arbuscular mycorrhizal fungi, wherein a fixing plate is arranged in the box body, a plurality of through holes are arranged in the fixing plate, a hollow tube body is penetrated in the through holes, a rock wool strip is arranged at the bottom of the tube body, and one end of the rock wool strip extends to the inside of the tube body through the bottom of the tube body; the device uses a pipette (centrifuge tube) and rock wool as supports to ensure that the supporting plant grows vertically, and the upper part of the rock wool is separated from the liquid surface to ensure that the spores can smoothly coexist with the plant root system under aerobic conditions; in addition, the rock wool nutrient delivery device in the device is very clever, and trace elements, bottom nutrient liquid and external components can be delivered through the rock wool, so as to study the influence of these components on plants under symbiotic conditions.

Description

Technical Field

[0001] The invention belongs to the research field of plant and microorganism symbiosis, and specifically relates to a liquid culture device and method for studying arbuscular mycorrhizal fungal symbiosis. Background Art

[0002] Arbuscular mycorrhiza (AM) is a symbiotic system widely found between plants and fungi in nature. It colonizes in the root cortex of most terrestrial plants and forms a special arbuscular structure. This symbiotic system can be traced back to 400 million years ago. AM fungi can promote the absorption of water, minerals and nutrients by plants while absorbing nutrients from plants (Maillet et al., 2011). Therefore, understanding the interaction mechanism between plants and AM fungi is of great significance to understanding the signal recognition and regulation mechanism in the process of building AM symbiosis.

[0003] At present, the understanding of the interaction and nutrient absorption mechanisms between plants and AM fungi is not thorough enough. When studying these mechanisms, people often use small leguminous model plants such as Lotus japonicus and alfalfa (M. truncatula) as experimental materials, and culture plants in natural soil or substrates such as vermiculite and perlite. AM fungal isolates are often used as a single variable to study the symbiotic mechanism between plants and AM fungi. The complexity of the farmland soil environment (which may contain a large number of microorganisms) and the difficulty in obtaining a clean root system have an impact on the research. With the advancement of experimental methods, people began to culture hairy roots in culture dishes and then inoculated AM fungal spores on the hairy roots, but this method has great limitations on the research of complete plants; although the liquid culture device can meet the growth needs of complete plants, considering that plants and AM fungi form a symbiotic relationship in the roots and AM fungi are aerobic fungi, it is difficult for hyphae and spores to grow in the traditional liquid culture mode. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the existing system and provide a liquid culture device and method for studying the symbiosis of arbuscular mycorrhizal fungi, so as to solve the technical problem that mycelium and spores are difficult to grow in the traditional liquid culture mode.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A liquid culture device for studying symbiosis of arbuscular mycorrhizal fungi, comprising a box body for containing a liquid culture nutrient solution for studying symbiosis of arbuscular mycorrhizal fungi, wherein a fixing plate is provided in the box body, a plurality of through holes are provided on the fixing plate, a hollow tube body is passed through the through holes, a rock wool strip is provided at the bottom of the tube body, one end of the rock wool strip extends to the inside of the tube body through the bottom of the tube body, and the other end of the rock wool strip can extend into the nutrient solution at least in a culture state.

[0007] As a further optimization solution of the present invention, the box body is a transparent box body with a cover.

[0008] A method for assembling a liquid culture device for studying the symbiosis of arbuscular mycorrhizal fungi as described above, using a pipette box as a box body for containing nutrient solution, using a pipette rack matched with the pipette box as a fixing plate, using a pipette or a centrifuge tube as a tube body, cutting off the tip of the pipette to expose an opening that can accommodate a rock wool strip and can fix the rock wool, making a tube body that is connected up and down, passing the rock wool strip through the opening of the pipette and fixing it, passing the pipette with the rock wool strip fixed through the through hole on the pipette rack, and assembling the liquid culture device.

[0009] An application of the liquid culture device as described above in studying the symbiotic relationship of arbuscular mycorrhizal fungi.

[0010] A method for studying arbuscular mycorrhizal fungal symbiosis comprises the following steps:

[0011] (1) sterilizing and germinating the seeds of the plants of the arbuscular mycorrhizal fungal symbiotic system to be studied to obtain plant seedlings;

[0012] (2) adding a nutrient solution for liquid culture used to study the symbiosis of arbuscular mycorrhizal fungi into the above-mentioned liquid culture device, sterilizing it and cooling it for later use;

[0013] (3) Under sterile conditions, sterile spores of arbuscular mycorrhizal fungi are inoculated on the rock wool strips inside the tube body of the liquid culture device, the roots of the plant seedlings obtained in step (1) are rolled up and wrapped with the rock wool, and the wrapped seedlings are transplanted into the tube body, and the rock wool at the roots of the seedlings is closely contacted with the rock wool strips inoculated with sterile spores of the liquid culture device;

[0014] (4) placing the liquid culture device of the inoculated plants in step (3) in a constant temperature greenhouse, and cultivating them according to growth conditions suitable for the inoculated plants to obtain an arbuscular mycorrhizal fungal symbiosis system for arbuscular mycorrhizal fungal symbiosis research.

[0015] As a further optimization scheme of the present invention, the symbiotic study of arbuscular mycorrhizal fungi includes determining the infection rate and infection intensity of AM fungi on the obtained symbiotic system of arbuscular mycorrhizal fungi.

[0016] As a further optimization scheme of the present invention, the plants of the symbiotic system of arbuscular mycorrhizal fungi to be studied are Lotus japonicus, alfalfa, rice or corn.

[0017] As a further optimization scheme of the present invention, the plant of the arbuscular mycorrhizal fungal symbiotic system to be studied is Lotus japonicus, and the nutrient solution is Hoagland's nutrient solution containing 0.05 mmol / L phosphorus concentration. The suitable growth conditions for Lotus japonicus are: 26°C during the day, 22°C at night, 16 hours of light exposure, and 70% humidity.

[0018] As a further optimized solution of the present invention, the method for sterilizing and germinating the seeds in step (1) comprises:

[0019] The method of aseptic disinfection is: first disinfect the plant seeds with 75% alcohol and 12% bleach in turn, then wash them with 75% alcohol for more than 3 times, and finally wash them with sterile water for 3-5 times.

[0020] The germination method is as follows: under sterile conditions, plant seeds are spread on a water agar medium with a mass concentration of 1.2%, and cultured in a dark sealed medium at 4°C for more than 12 hours for vernalization. After the vernalization, they are cultured at 28°C until germinated plant seedlings are obtained.

[0021] The beneficial effects of the present invention are:

[0022] 1) In this device, pipettes (centrifuge tubes) and rock wool are used as supports to ensure that the plants grow vertically. The upper part of the rock wool is separated from the liquid surface to ensure that the spores can coexist smoothly with the plant roots under aerobic conditions. In addition, the rock wool nutrient delivery device in the device is very clever. Trace elements, bottom nutrient liquid and external components can be delivered through the rock wool to facilitate the study of the effects of these components on plants under symbiotic conditions.

[0023] 2) The present invention utilizes rock wool to replace the traditional soil matrix. The upper part of the rock wool is separated from the liquid surface to ensure that the spores can smoothly carry out aerobic respiration and symbiosis with the plant roots. The lower part of the rock wool transports nutrients, elements and external components from the bottom nutrient solution to facilitate the study of the effects of these components on plants under symbiotic conditions. The materials required for the present invention are all taken from laboratory waste materials, and have the remarkable characteristics of being green, environmentally friendly, easy to obtain, and low in cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the overall structure of the pipette box of the present invention;

[0025] Figure 2 It is a schematic structural diagram of the pipette of the present invention;

[0026] Figure 3 It is a structural schematic diagram of the pipette stand of the present invention;

[0027] Figure 4 It is a schematic diagram of the structure of the rock wool of the present invention;

[0028] Figure 5 It is a schematic structural diagram of the pipette box of the present invention;

[0029] Figure 6 Figure A is a disassembled diagram of the hydroponic device, Figure B is an assembly diagram, Figure C is a planting diagram, and Figure D is a diagram of the hydroponic device in a greenhouse; Figure E is an infection effect diagram.

[0030] In the figure: 1. Pipette; 2. Pipette rack; 3. Rock wool; 4. Pipette box. DETAILED DESCRIPTION

[0031] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0032] Example 1

[0033] The present embodiment provides a liquid culture device for studying the symbiosis of arbuscular mycorrhizal fungi, comprising a box body for containing a liquid culture nutrient solution for studying the symbiosis of arbuscular mycorrhizal fungi, a fixing plate is provided in the box body, a plurality of through holes are provided on the fixing plate, a hollow tube body is passed through the through holes, a rock wool strip is provided at the bottom of the tube body, one end of the rock wool strip extends to the inside of the tube body through the bottom of the tube body, and the other end of the rock wool strip can extend into the nutrient solution at least in the culture state. Further preferably, the box body is a transparent box body with a cover.

[0034] like Figure 1-6 As shown, the device can be modified using common tools in a biological laboratory, including a pipette 1, a pipette rack 2, rock wool 3, and a pipette box 4. The pipette box 4 is used as a box body for containing a nutrient solution, the pipette rack 2 matched with the pipette box 4 is used as a fixing plate, and the pipette 1 is used as a tube body. During assembly, the tip of the pipette 1 is cut off to expose an opening that can accommodate a rock wool strip and fix the rock wool 3, so as to form a tube body that is connected up and down, the rock wool strip is passed through the opening of the pipette 1 and fixed, and the pipette 1 fixed with the rock wool strip is passed through the through hole on the pipette rack 2, so as to assemble the liquid culture device.

[0035] It should be noted that the pipette 1 described in the present invention refers to a disposable gun tip commonly used in biological laboratories for pipetting small or trace amounts of liquid, and the pipette rack 2 and the pipette box 4 are gun boxes matching the pipette. Specifically, the rock wool 3 is cut into strips of 10 cm in length and 1 cm in width, and uniformly sealed in a high temperature and humidity resistant sterilization bag to obtain sterilized rock wool strips, and then the pipette 1 is cut to a length of 6.5 cm, and then the rock wool strip is installed in the bottom hole of each pipette 1.

[0036] Example 2

[0037] This embodiment takes Lotus japonicus as an example, uses the existing 5 ml pipette box to modify and culture Lotus japonicus, uses rock wool strips to provide constant water and nutrients for the culture matrix, and constructs a stable and sterile system for studying the symbiotic relationship between Lotus japonicus and arbuscular mycorrhizal fungi.

[0038] The specific steps include:

[0039] (1) Seed disinfection and germination

[0040] The seeds of Lotus japonicus are sterilized and germinated to obtain plant seedlings; wherein:

[0041] The specific steps of seed disinfection and germination are as follows: take an appropriate amount of lotus seeds and put them in a mortar, and grind them evenly with sandpaper; in a clean bench, put the lotus seeds into a 50mL sterilized centrifuge tube, add 30mL of 75% alcohol, shake up and down for 30 seconds, pour out the liquid and add 30mL of 12% Kao bleach, shake and wash for 10 minutes, and then pour out the liquid; add 30mL of 75% alcohol and shake for 1-2 minutes, repeat the washing 3 times; then add 30mL of sterile water and wash 3-5 times, each time for 3-5 minutes, and use after the disinfection is completed;

[0042] Germination: In a clean bench, evenly spread the seeds of Lotus japonicus on a 1.2% square water agar plate, seal the culture dish with a sealing film, and place it flat in a dark refrigerator at 4°C for 12 hours for vernalization. After vernalization, grow it in a 28°C incubator.

[0043] (2) Assembly and disinfection of liquid culture equipment

[0044] Preparation of special nutrient solution for studying symbiosis of arbuscular mycorrhizal fungi: During the planting of Lotus japonicus, Hoagland's nutrient solution with a phosphorus concentration of 0.05mmol / L was used to ensure the nutrients required for the growth of Lotus japonicus. The composition and content of low-phosphorus Hoagland's nutrient solution were: 5mmol / L KNO3, 5mmol / L Ca(NO3)2·4H2O, 2mmol / L MgSO4·7H2O, 20μmol / L FeEDTA, 0.95mmol / L KCl, 0.05mmol / L KH2PO4, 1μmol / L H3BO3 and trace elements 2μmol / L MnCl2·4H2O, 2μmol / L ZnSO4, 0.2μmol / L CuSO4·5H2O, 0.2μmol / L (NH4)6Mo7O 24 .

[0045] Add the above nutrient solution into the liquid culture device, sterilize and cool for use; the specific process is: cut the rock wool into strips of 10 cm in length and 1 cm in width, and seal them in a high temperature and wet heat sterilization bag for sterilization to obtain sterilized rock wool strips. Cut the pipette to a length of 6.5 cm and sterilize it in the same way to obtain a sterilized pipette. Install the rock wool strips in the holes at the bottom of each pipette in turn. Finally, in the clean bench, add 500 ml of nutrient solution to the bottom of the pipette box, sterilize at 121°C for 30 minutes, and cool it for use.

[0046] (3) Vaccination

[0047] Under sterile conditions, sterile spores of arbuscular mycorrhizal fungi are inoculated on the rock wool strips inside the tube body of the liquid culture device, the roots of the plant seedlings obtained in step (1) are rolled up and wrapped with the rock wool, and the wrapped seedlings are transplanted into the tube body, and the rock wool at the roots of the seedlings is closely contacted with the rock wool strips inoculated with sterile spores of the liquid culture device;

[0048] Specifically, use tweezers to carefully pick up the Lotus seedlings, place them flat in the rock wool and roll them up, gently wrap the roots, use a pipette to inoculate sterile spores on the top of the original rock wool strip, and transplant the wrapped seedlings into the pipette, making sure that the roots of the seedlings are directly above the sterile spores, and gently press them with tweezers to make them in close contact, to avoid stratification of the rock wool strips so that the seedlings cannot absorb enough water.

[0049] (4) Transplantation

[0050] The liquid culture device of the inoculated plants in step (3) is placed in a constant temperature greenhouse and cultured according to growth conditions suitable for the inoculated plants, wherein the culture conditions are 26° C. during the day and 22° C. at night, the light duration is 16 h, and the humidity is 70%; an arbuscular mycorrhizal fungal symbiosis system is obtained for the study of arbuscular mycorrhizal fungal symbiosis.

[0051] (5) Research on symbiosis of arbuscular mycorrhizal fungi

[0052] The AM fungal infection rate and infection intensity of the obtained symbiotic system of arbuscular mycorrhizal fungi were measured. Specifically, the roots of the six-week-old Lotus japonicus were cleaned, cut to the length of the slide, and FAA fixative was added in a fume hood to immerse the roots for 4 hours. After washing with sterile water, 10% KOH was added for decolorization, and the roots were boiled in a water bath at 90°C for 5-10 minutes until they were translucent. After cooling, they were rinsed with sterile water. 5% lactic acid was added for acidification for 5 minutes, and after being transparent, they were stained with triphenyl blue for 24 hours, and decolorized with lactic acid glycerol on a shaker until they no longer turned blue.

[0053] Infection rate determination:

[0054] F% (Frequency of mycorrhiza in the root system) reflects the AM fungal infection of the root segments of the host plant: the calculation method is F% = (number of root segments infected by AM fungi in the sample / total number of root segments in the sample) * 100%;

[0055] Determination of infection intensity:

[0056] M% (Intensity of the mycorrhizal colonisation in the root system) is a weighted average value that comprehensively reflects the AM fungal infection rate and infection intensity in all root segments: the calculation method is M% = (95*n5+70*n4+30*n3+5*n2+n1) / (total number of root segments in the sample); n5 represents the number of root segments with an AM fungal infection intensity of more than 90%, n4 represents the number of root segments with an AM fungal infection intensity of 70%, n3 represents the number of root segments with an AM fungal infection intensity of 50%, n2 represents the number of root segments with an AM fungal infection intensity of 10%, and n1 represents the number of root segments with an AM fungal infection intensity of 1%.

[0057] It should be noted that the specific planting of plants of different sizes can be adjusted according to the root-to-stem ratio of the plant and with reference to the model of this device. For example, small model plants such as Lotus japonicus and alfalfa can use 2ml and 5ml pipettes, and large plants such as rice and corn can use 10ml and 50ml centrifuge tubes. The rock wool core can also be adjusted according to the different sizes of pipettes (centrifuge tubes).

[0058] The present invention can solve the problem of bacteria contamination in traditional soil culture or matrix culture, because liquid culture can provide clean and uncontaminated roots, maintain highly controlled nutritional conditions and other advantages, but in the traditional liquid culture mode, aerobic AM fungi are difficult to grow, so the present invention uses a pipette (centrifuge tube) and rock wool as supports, and uses rock wool to transport trace elements, nutrient solution and other nutrients required for plant growth, so as to study the effects of these components on plants under symbiotic conditions, and the upper part of the rock wool is separated from the liquid surface to ensure that the spores can smoothly coexist with the plant root system under aerobic conditions. The entire hydroponic device is recyclable (except rock wool), and has significant characteristics such as green environmental protection, easy material acquisition, and low cost, thereby saving the cost of the experiment.

[0059] In addition, the present invention establishes a liquid culture device based on a pipette (or centrifuge tube) and a rock wool core, which can be used to grow small model plants such as lotus and alfalfa or large plants such as rice and corn, and to study the symbiotic relationship between plants and arbuscular mycorrhizal fungi by inoculating arbuscular mycorrhizal fungi.

[0060] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for studying symbiosis of arbuscular mycorrhizal fungi, characterized in that: The following steps are involved: (1) sterilizing and germinating the seeds of the plants of the arbuscular mycorrhizal fungal symbiotic system to be studied to obtain plant seedlings; (2) Add the nutrient solution for liquid culture used to study the symbiosis of arbuscular mycorrhizal fungi into the liquid culture device, sterilize it, and cool it for later use; The liquid culture device comprises: a box body for containing a liquid culture nutrient solution for studying the symbiosis of arbuscular mycorrhizal fungi, a fixing plate is provided in the box body, a plurality of through holes are provided on the fixing plate, a hollow tube body is penetrated in the through holes, a rock wool strip is provided at the bottom of the tube body, one end of the rock wool strip extends to the inside of the tube body through the bottom of the tube body, and the other end of the rock wool strip can extend into the nutrient solution at least in the culture state; (3) Under sterile conditions, sterile spores of arbuscular mycorrhizal fungi are inoculated on the rock wool strips inside the tube of the liquid culture device, the roots of the plant seedlings obtained in step (1) are rolled up and wrapped with the rock wool, and the wrapped seedlings are transplanted into the tube, and the rock wool at the roots of the seedlings is closely contacted with the rock wool strips inoculated with sterile spores in the liquid culture device; (4) Placing the inoculated plant liquid culture device in step (3) in a constant temperature greenhouse and cultivating the inoculated plants under growth conditions suitable for the inoculated plants to obtain an arbuscular mycorrhizal fungal symbiosis system for use in arbuscular mycorrhizal fungal symbiosis research.

2. A method for studying symbiosis of arbuscular mycorrhizal fungi according to claim 1, characterized in that: The symbiotic research on arbuscular mycorrhizal fungi includes determining the infection rate and infection intensity of AM fungi in the obtained symbiotic system of arbuscular mycorrhizal fungi.

3. A method for studying symbiosis of arbuscular mycorrhizal fungi according to claim 1, characterized in that: The plants of the symbiotic system of arbuscular mycorrhizal fungi to be studied are lotus roots, alfalfa, rice or corn.

4. A method for studying symbiosis of arbuscular mycorrhizal fungi according to claim 3, characterized in that: The plant of the symbiotic system of arbuscular mycorrhizal fungi to be studied is Lotus japonicus, and the nutrient solution is Hoagland's nutrient solution containing 0.05 mmol / L phosphorus concentration. The suitable growth conditions for Lotus japonicus are: 26°C during the day, 22°C at night, 16 h of light duration, and 70% humidity.

5. A method for studying symbiosis of arbuscular mycorrhizal fungi according to claim 1, characterized in that: The method for sterilizing and germinating the seeds in step (1) comprises: The method of sterilization is: first sterilize the plant seeds with 75% alcohol and 12% bleach solution in turn, then wash with 75% alcohol for more than 3 times, and finally wash with sterile water for 3-5 times; The germination method is as follows: under sterile conditions, plant seeds are spread on a water agar medium with a mass concentration of 1.2%, and cultured in a dark sealed culture medium at 4°C for more than 12 hours for vernalization. After the vernalization, they are cultured at 28°C until germinated plant seedlings are obtained.

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