Arabidopsis culture and root micro-morphology research method based on micro-fluidic integrated chip

Abstract

The present application provides a method for culturing Arabidopsis and studying root micro-morphology based on a micro-millifluidic integrated chip, a PMMA mold is manufactured through a numerical control machine tool, and polydimethylsiloxane is configured to manufacture the micro-millifluidic integrated chip; after concentration gradient testing of the micro-millifluidic integrated chip, the Arabidopsis is cultured on the micro-millifluidic chip, and root observation and high-salt stress treatment are performed. The present application makes up for the defect that a traditional soft photolithography chip cannot realize millimeter-level processing, the millimeter-level channel provides sufficient space for the growth of Arabidopsis roots, and the growth of Arabidopsis can be directly observed dynamically, so that the dynamic development process of the Arabidopsis root system can be monitored; and the micrometer-level channel can realize real-time switching and concentration real-time control of the liquid, the micrometer-level concentration gradient formation area is combined with the millimeter-level plant root culture area, not only the growth space of Arabidopsis is expanded, but also a new research strategy is provided for studying the instantaneous stimulation response mechanism of plants in the external environment.

Description

Technical Field

[0001] This invention relates to a method for Arabidopsis thaliana cultivation and root micromorphology research based on microfluidic integrated chips, belonging to the field of biomaterials cultivation technology. Background Technology

[0002] Roots, as the organs through which plants sense changes in the external soil environment and absorb water, inorganic salts, and nutrients, play an irreplaceable role throughout the entire growth stage of a plant. When faced with external biotic and abiotic stresses, plants can initiate defense responses by altering their morphological structure and hormonal signals, and their root system structure will also undergo various morphological changes in response to environmental changes.

[0003] Traditional agar-based and soil-based culture methods struggle to achieve high-resolution, continuous observation of plant roots. However, advancements in nanofluidics and the rise of PDMS materials have broken down these limitations. PDMS-based plant nanofluidics chips enable continuous monitoring of plant roots and control of the rhizosphere environment in a short period. Nevertheless, traditional soft lithography techniques struggle to achieve millimeter-level dimensions or differentiate heights on a single chip, thus hindering improvements in expanding the normal growth space for Arabidopsis thaliana. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems in existing plant root culture and monitoring methods, and to propose an Arabidopsis thaliana culture method based on microfluidic chips.

[0005] The technical solution of this invention: A method for cultivating Arabidopsis thaliana based on a microfluidic chip, specifically including the following steps:

[0006] (1) A PMMA mold was manufactured by CNC machine tool, and polydimethylsiloxane was used to fabricate a microfluidic chip; the microfluidic chip is divided into a plant root culture zone and a concentration gradient formation zone, wherein the size of the plant root culture zone is 4.5cm x 1mm x 1mm, and the height of the concentration gradient formation zone is 250μm; the culture method is vertical culture; the specific fabrication process is as follows:

[0007] Polydimethylsiloxane was prepared at a mass ratio of 10:1 (A glue: B glue). After vacuuming for 30 minutes, it was poured into a PMMA mold and heat-cured at 70°C for 2 hours. It was then cut off and bonded to glass using a plasma bonding machine. After drying for 2 hours, the bonding effect was strengthened. Then, a hole was punched, and half of the seed opening at the top was cut off horizontally with a knife.

[0008] (2) Perform concentration gradient test on the microfluidic chip made in step (1): Use a 1 mL syringe to draw chelated iron colored solution and sterile water respectively, and then place them on the two syringe placement ends of the injection pump. Tighten the button to prevent the syringe from sliding. Connect the stainless steel connector to the two water inlets of the microfluidic chip. Set the pump parameters to flow rates of 5, 10, 20, 30, 40 and 50 μL / min respectively. Count the time required for the solution to reach the water outlet and take a picture.

[0009] (3) Cultivate Arabidopsis thaliana on the microfluidic chip made in step (1): Rinse the microfluidic chip channel 3 times with sterile water using a 1 mL syringe, then rinse 3 times with Hogrange medium, and fill the upper end of the microfluidic chip with sugar-free 1 / 2 MS medium; wash the Arabidopsis thaliana seeds with 75% ethanol for 15 min, then wash 3 times with sterile water for 3 min each time; then use a syringe needle to apply the Arabidopsis thaliana seeds into the medium at the upper end of the microfluidic chip agar, place the microfluidic chip vertically in a glass bottle containing a small amount of sterile water, cover the bottle mouth with sealing film, and place it in a light incubator for cultivation.

[0010] Furthermore, the formulation of the sugar-free 1 / 2 MS medium is as follows: take 0.55 g MS, 0.125 g MES, 2.5 g agar, add KOH to adjust the pH to 5.8, bring the volume to 250 mL, autoclave, and pour into plates.

[0011] (4) Root observation and high salt treatment were carried out on the Arabidopsis thaliana grown in step (2).

[0012] Furthermore, the specific operation of Arabidopsis root observation in step (4) is as follows: place the microfluidic chip containing Arabidopsis under a microscope and take pictures of the Arabidopsis root system using time delay and fixed point.

[0013] Furthermore, the specific operation of the high-salt treatment of Arabidopsis thaliana in step (4) is as follows: two 1mL syringes are used to draw sterile Hoagland medium and Hoagland medium containing 0.5M NaCl respectively. Then, a plastic tube with a stainless steel connector with an outer diameter of 0.75mm is connected to the syringe and the piston is pushed to expel the air. The two syringes are placed in the syringe pump and fixed. Then, the syringe pump is turned on and the culture medium is pumped out at a flow rate of 20μL / min. The microfluidic chip is placed on the stage of the microscope. Then, the stainless steel connector is connected to the sample inlet of the chip. The flow rate of the syringe pump is changed to 10μL / min. The pump is used for 10min and photographed with a microscope.

[0014] Furthermore, the injection pump in steps (2) and (4) is a two-way 1mL injection pump.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] This invention overcomes the limitation of traditional soft lithography chips in achieving millimeter-level processing. Mold fabrication based on a microfluidic system can increase the size of the microfluidic chip. Millimeter-level channels are more conducive to the growth of Arabidopsis thaliana and allow for direct observation of Arabidopsis thaliana growth, facilitating the monitoring of the dynamic development process of Arabidopsis thaliana roots. When constructing the microfluidic chip, the combination of a micrometer-level concentration gradient formation zone and a millimeter-level plant root culture zone expands the growth space of Arabidopsis thaliana. It has the advantages of simple operation, high resolution, and real-time observation, providing a new research strategy for studying the active response mechanism of plants under instantaneous external influences, and has important theoretical and practical significance. Attached Figure Description

[0017] Appendix Figure 1 This is a flowchart of the fabrication process of the microfluidic chip and a flowchart of the Arabidopsis thaliana cultivation process in this embodiment of the invention.

[0018] Appendix Figure 2 A is a concentration gradient formation diagram in an embodiment of the present invention. Figure 2 B and 2C are the formation time and gradient formation diagrams at different flow rates, respectively.

[0019] Appendix Figure 3 These are growth diagrams of Arabidopsis thaliana and phenotypic diagrams of Arabidopsis thaliana under high-salt treatment in embodiments of the present invention. Detailed Implementation

[0020] The technical solutions of the present invention are further described below with reference to several embodiments. In the description of this specification, the content of each embodiment refers to specific technical features described herein that are included in at least one implementation of the present invention. In this specification, illustrative representations of embodiments do not necessarily refer to the same implementation or example. Furthermore, the specific technical features described may be combined in any suitable manner in one or more implementations or examples. Example 1

[0021] The fabrication method of the microfluidic chip, and its construction flowchart are as follows: Figure 1 As shown, the specific steps include:

[0022] 1) The structure of the microfluidic chip was drawn using AutoCAD software. The gradient forming module has a height and width of 250μm; the culture module has a height and width of 1mm and a length of 4.5cm; the outlet and inlet are circular with a diameter of 2.5mm and a height of 5mm. Then, we used a CNC machine tool to process the PMMA male mold.

[0023] 2) The PDMS prepolymer was mixed with adhesive A and adhesive B in a 10:1 ratio and stirred until homogeneous. The mixture was then vacuum-pumped for 30 minutes to remove air bubbles. The clear, bubble-free PDMS was poured into a PMMA mold and allowed to stand at 4°C. It was then placed in a 70°C oven for 2 hours to allow the PDMS to thermoset. The PDMS adhesive was cut along the sides of the mold wall with a blade and holes were punched using a 0.65 mm punch to create injection ports. A cut was made at the top along the center to create the Arabidopsis seeding port. The glass and PDMS surfaces were treated with a PLASMA plasma cleaner, and the two surfaces were quickly bonded together, compacted, and then placed in a 70°C oven for 1 hour to ensure a tight bond. For subsequent aseptic processing, the PDMS chip was wrapped in newspaper and autoclaved at 121°C, then dried in an oven. Example 2

[0024] The verification of the concentration gradient function of the microfluidic chip includes the following steps:

[0025] Using 1 mL syringes, colored chelated iron (EDDHA-Fe) solution and sterile water were drawn up and placed in the two syringe holders of the syringe pump. The button was tightened to prevent the syringe from slipping. The stainless steel connectors were connected to the two inlets of the chip. The pump parameters were set at flow rates of 5, 10, 20, 30, 40, and 50 μL / min. The time required for the solution to reach the outlet was recorded and photographed. Each concentration was repeated 5 times. The verification results are as follows: Figure 2 As shown. Example 3

[0026] The cultivation process of Arabidopsis thaliana on a nanofluidic chip includes the following steps:

[0027] 1) Sugar-free 1 / 2 MS formula: Weigh 0.55g MS, 0.125g MES, and 2.5g agar. Add KOH to adjust the pH to 5.8, bring the volume to 250mL, autoclave, and pour into plates.

[0028] 2) Arabidopsis seeds were first washed with 75% ethanol for 15 minutes, then rinsed three times with sterile water for 3 minutes each time. The seeds were then placed at 4°C for 2 days for low-temperature treatment. The sterilized PDMS chip was placed in a round dish containing sterile water and submerged. Sterile water was injected into the PDMS chip using a 1 mL syringe, and the chip was rinsed three times to remove air bubbles from the channels. The chip was then rinsed three times with 1 mL of Hoagland medium. Half of the sugar-free MS solid medium was then filled into the planting opening of the Arabidopsis seedlings using a 1 mL syringe and compacted. Finally, sterile seeds were sown on the surface of the medium. The microfluidic chip was held with tweezers and placed in a glass bottle containing sterile water, held vertically, and covered with a breathable membrane. The glass bottle was then placed in a light incubator. Culture conditions: 16 h daytime / 8 h nighttime, temperature 26°C / 24°C, cultured for approximately 7 days.

[0029] The Hoagland medium formula is as follows: Weigh 10.1g KNO3, 11.8g Ca(NO3)2·4H2O, 0.75g EDDHA-Fe, 24.65g MgSO4·7H2O, 4g NH4NO3, 0.143g H3BO3, 0.0905g MnCl2·4H2O, 0.011g ZnSO4·7H2O, 0.00255g CuSO4·5H2O, 0.0045g H3MoO4·H2O, and 6.8g KH2PO4, and add 50 mL of purified water to prepare stock solutions; separately, pipette 2.5 mL of KNO3, 1.5 mL of Ca(NO3)2·4H2O, 1.5 mL of EDDHA-Fe, and 1 mL of... Add MgSO4·7H2O, NH4NO3, H3BO3, MnCl2·4H2O, ZnSO4·7H2O, CuSO4·5H2O, H3MoO4·H2O, 0.5mL KH2PO4, and dilute to 1000mL with purified water. Example 4

[0030] The observation of Arabidopsis root system and high-salt treatment included the following steps:

[0031] 1) Place the nanofluidic chip containing Arabidopsis thaliana under a microscope and use time-delay fixed-point imaging to photograph the root system of Arabidopsis thaliana.

[0032] 2) Draw sterile Hoagland medium and Hoagland medium containing 0.5M NaCl into two 1mL syringes, respectively. Connect the plastic tubing with a 0.75mm outer diameter stainless steel connector to the syringes and push the stopcock to expel air. Place both syringes on the syringe pump and secure them. Then turn on the syringe pump to pump out the culture medium at a flow rate of 20μL / min. Place the nanofluidic chip on the microscope stage and connect the stainless steel connector to the chip's injection port. Change the flow rate of the syringe pump to 10μL / min and use it for 10 minutes, then photograph it using a microscope. The photographic results are as follows. Figure 3 As shown.

[0033] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No markings in the claims should be construed as limiting the scope of the claims.

[0034] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for cultivating Arabidopsis thaliana based on a microfluidic chip, specifically comprising the following steps: (1) PMMA molds are manufactured by CNC machine tools, and polydimethylsiloxane is used to fabricate microfluidic chips; (2) Perform concentration gradient testing on the microfluidic chip fabricated in step (1); (3) Arabidopsis thaliana is cultured on the microfluidic chip fabricated in step (1); (4) Root observation and high salt treatment were carried out on the Arabidopsis thaliana grown in step (2); Its features are: The nanofluidic chip is divided into a millimeter-scale plant root culture zone and a micrometer-scale concentration gradient formation zone. The plant root culture zone measures 4.5 cm x 1 mm x 1 mm, and the concentration gradient formation zone has a height of 250 μm. The culture method is vertical culture. The specific steps for culturing Arabidopsis thaliana in step (3) are as follows: Rinse the microfluidic chip channels three times with sterile water using a 1 mL syringe, then rinse three times with Hoagland medium. Fill the upper part of the microfluidic chip with 1 / 2 sugar-free MS medium. Wash Arabidopsis seeds with 75% ethanol for 15 min, then wash three times with sterile water for 3 min each time. Then, use a syringe needle to apply Arabidopsis seeds into the medium at the upper part of the microfluidic chip agar. Place the microfluidic chip vertically in a glass bottle containing a small amount of sterile water, cover the bottle opening with sealing film, and place it in a light incubator for cultivation. The specific operation of the high-salt treatment of Arabidopsis thaliana in step (4) is as follows: two 1mL syringes are used to draw sterile Hoagland medium and Hoagland medium containing 0.5M NaCl respectively. Then, a plastic tube with a stainless steel connector with an outer diameter of 0.75mm is connected to the syringe and the piston is pushed to expel the air. The two syringes are placed in the syringe pump and fixed. Then, the syringe pump is turned on and the culture medium is pumped out at a flow rate of 20μL / min. The microfluidic chip is placed on the stage of the microscope. Then, the stainless steel connector is connected to the sample inlet of the chip. The flow rate of the syringe pump is changed to 10μL / min. The pump is used for 10min and photographed with a microscope.

2. The Arabidopsis thaliana cultivation method based on a microfluidic chip according to claim 1, characterized in that: The fabrication process of the microfluidic chip in step (1) is as follows: Polydimethylsiloxane is prepared in a mass ratio of A glue: B glue of 10:

1. After vacuuming for 30 minutes, it is poured into a PMMA mold, thermoset at 70°C for 2 hours, cut off, and bonded to glass by a plasma machine. After drying for 2 hours, the bonding effect is strengthened. Then, a hole is punched with a punch, and half of the seed opening at the top is cut off horizontally with a knife.

3. The Arabidopsis thaliana cultivation method based on a microfluidic chip according to claim 1, characterized in that: The specific steps of the concentration gradient test in step (2) are as follows: Using 1 mL syringes, draw up colored chelated iron solution and sterile water respectively, then place them on the two syringe holders of the syringe pump. Tighten the button to prevent the syringe from slipping. Connect the stainless steel connector to the two inlets of the microfluidic chip. Set the pump parameters to flow rates of 5, 10, 20, 30, 40, and 50 μL / min respectively. Count the time required for the solution to reach the outlet and take a picture.

4. The Arabidopsis thaliana cultivation method based on a microfluidic chip according to claim 1, characterized in that: The formula for the sugar-free 1 / 2 MS medium is as follows: Take 0.55 g MS, 0.125 g MES, and 2.5 g agar, add KOH to adjust the pH to 5.8, bring the volume to 250 mL, autoclave, and pour into plates.

5. The Arabidopsis thaliana cultivation method based on a microfluidic chip according to claim 1, characterized in that: The specific operation for observing Arabidopsis root system in step (4) is as follows: place the microfluidic chip containing Arabidopsis under a microscope and take pictures of Arabidopsis root system by time delay and fixed point.

6. The Arabidopsis thaliana cultivation method based on a microfluidic chip according to claim 1, characterized in that: The syringe pumps used in steps (2) and (4) are two-way 1mL syringe pumps.

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