A living in situ culture method of plant roots and its application

Abstract

The present application discloses a method for in situ culture of plant roots and its application. The method comprises: step 1, spreading sterilized Arabidopsis seeds flat on the upper edge of a glass slide culture medium; the thickness of the culture medium is 0.9-1.4 mm; step 2, placing the glass slide with seeds in a culture dish equipped with a culture device and culturing vertically under light; the culture device comprises a circular bottom plate, and a fixing device provided on the circular bottom plate for fixing the glass slide with seeds and a humidifying device for increasing the air humidity in the culture dish. After the vertical culture is completed, the present application can directly prepare slides without moving the Arabidopsis roots and observe the arrangement of microtubules in the root cells under a microscope, thereby avoiding damage and displacement of the Arabidopsis roots when preparing slides using traditional methods; specifically, by selecting a specific device during the vertical culture process, the technical problem that the existing culture dish cannot fix the glass slide, causing the root growth to bend and thus affecting the root observation is overcome.

Description

Technical Field

[0001] The present application belongs to the field of plant microscopy technology and relates to a method for in situ cultivation of plant roots and its application. Background Art

[0002] Arabidopsis thaliana, a cruciferous plant, is often used as a model plant for biological research. Relatively few studies have been conducted on Arabidopsis roots, and a well-defined anatomical model of the root has been established. These studies focus on the molecular mechanisms underlying root phototropism and hydrotropism, the growth and development of the taproot, the characteristics of root hair tip growth, the dynamics of the root microtubule cytoskeleton, and the effects of plant hormones on root growth. Arabidopsis thaliana has a short plant stature and extremely thin roots, which can be grown on solid culture media, facilitating phenotypic observation. Therefore, it has long been used in the study of root morphology. To this day, Arabidopsis thaliana remains an effective tool for identifying genes and phenotypes involved in root development.

[0003] The nutritional organs of higher plants primarily consist of roots, stems, and leaves, all of which play a vital role in their normal growth. Roots not only provide support and anchorage, but also absorb the water and minerals necessary for plant growth and development, while also responding specifically to diverse biotic and abiotic signals.

[0004] Root cell morphology is mainly observed by hand sectioning, paraffin sectioning, and frozen sectioning. However, sectioning can damage the integrity of the roots. Therefore, to meet the needs of in situ observation of living organisms, living seedlings are usually used as slides for observation. However, Arabidopsis plants are short and their roots are extremely thin and weak. Even the slightest displacement during slide preparation can cause mechanical damage to the roots and affect the localization of related proteins in the roots (specifically, fluorescently labeled proteins can be used to monitor the in situ culture system, allowing rapid observation of root growth, displacement, and root damage).

[0005] However, there are currently no reports on how to avoid damaging and displacing the Arabidopsis roots during slide preparation, while also ensuring that the coverslip completely covers the roots and that they grow upright normally. Therefore, it is necessary to establish an in situ culture and observation system that can address these issues. Summary of the Invention

[0006] To address the above-mentioned issues, a method for in situ cultivation of plant roots and its application are provided, which utilizes a specific device to culture Arabidopsis thaliana. By selecting a specific device during vertical cultivation, this application overcomes the technical problem of existing culture dishes being unable to fix glass slides, causing root growth to bend and thus affecting root observation. Furthermore, by controlling the specific thickness of the glass slide culture medium and the placement of the Arabidopsis seeds on the glass slide, this method promotes seedling growth while ensuring that the Arabidopsis seedlings can be effectively observed.

[0007] The specific technical solutions of this application are as follows:

[0008] The present application provides a method for in situ cultivation of plant roots, comprising the following steps:

[0009] Step 1: Spread unsolidified culture medium on a glass slide, wait for it to solidify, and then spread sterilized Arabidopsis seeds on the upper edge of the culture medium on the glass slide; the thickness of the culture medium is 0.9-1.4 mm;

[0010] Step 2: Place the glass slide with the seeds in a culture dish equipped with a culture device and culture it vertically under light;

[0011] The culture device includes a circular bottom plate, a fixing device arranged on the circular bottom plate for fixing a glass slide with seeds, and a humidifying device for increasing the humidity of the air in the culture dish. The plant is Arabidopsis thaliana.

[0012] Optionally, in step 1, the sterilized Arabidopsis seeds are spread flat on the upper edge of the culture medium on the glass slide at a position 1 / 10-1 / 3.

[0013] Optionally, in step 1, the amount of agar added to the culture medium is (10.5-12) g / L.

[0014] Optionally, in step 2, the circular chassis is annular and hollow in the middle;

[0015] The material of the culture device includes any one of acrylonitrile-butadiene-styrene plastic, polylactic acid, nylon, polycarbonate, polystyrene, polycaprolactone, polyphenylsulfone, thermoplastic polyurethane, and polyetheretherketone; preferably, nylon is selected.

[0016] Optionally, in step 2, the humidifying device is a groove for placing a water sink; the fixing device includes a first clamp and a second clamp, and the first clamp and the second clamp are centrally symmetrical on the circular chassis; the glass slide is placed between the first clamp and the second clamp to fix the glass slide; a humidity detector with a display screen is provided on the side wall of the fixing device; the groove is provided on one side in the width direction of the glass slide.

[0017] Optionally, in step 2, the fixing device includes two stabilizing shafts symmetrically arranged on the circular chassis, and a clamping member connected to the stabilizing shafts; the clamping member includes a fixing block and a concave clamping block arranged at the bottom of the fixing block, a first through hole is provided on a side wall of the concave clamping block, and a thread is provided on an inner wall of the first through hole; the clamping member is connected to the stabilizing shaft by a bolt threadedly connected to the inner wall of the first through hole;

[0018] A slide mounting groove is provided on opposite sides of the fixing blocks on the two stabilizing shafts; a second through hole is provided on the top and the lower part of the side wall of the fixing block, and a thread is provided on the inner wall of the second through hole;

[0019] The humidification device includes a water-carrying container arranged on one side in the width direction of the slide, and the interior of the water-carrying container is hollowed to form a hollow cavity; the top of the water-carrying container is provided with a plurality of water outlet pipes evenly spaced and connected to the hollow cavity; the upper part of the outer wall of the water-carrying container is provided with a water inlet pipe connected to the hollow cavity; the water inlet pipe can extend to the outside of the culture dish; a humidity detector with a display screen is provided on the side wall of the fixing device; preferably, the water outlet pipe is arranged in an arc shape with the arc opening facing downward; the diameter of the water outlet pipe is 0.15-0.35 cm.

[0020] Furthermore, the method for using the culture device in step 2 is:

[0021] Place the glass slide fixture with the seeds in the culture dish, place the two concave blocks of the clamping pieces with their openings facing downward on the stabilizing shafts, move the clamping pieces to place the two ends of the glass slide with the seeds in the glass slide mounting grooves of the two fixing blocks, and when the two ends of the glass slide touch the bottom of the grooves of the fixing blocks, use bolts to thread them at the first through holes; use bolts to thread them at the second through holes on the top and side walls of the fixing blocks to secure the glass slide in the culture device;

[0022] Sterile water is introduced into the water inlet pipe and added to the water container until the water is sprayed out from the outlet pipe and falls on the fixed device, so that the humidity of the culture medium is in the range of 55-80%.

[0023] Apply 40-100kPa pressure to pass sterile water into the water container, and water will flow out of the outlet pipe for 1-4s.

[0024] The present application also provides an application of the above-mentioned in situ culture method for in situ observation, including: after the vertical culture is completed, the slide is removed, the slide is prepared, and the arrangement of microtubules in the root cells is observed under a microscope;

[0025] The conditions for selecting a glass slide are as follows: a glass slide carrying Arabidopsis seedlings with roots cultured to 0.8-1.2 cm was selected, the slide was directly prepared without moving the roots, and the slide was directly observed under a microscope.

[0026] The beneficial effects of this application include but are not limited to:

[0027] 1. The in situ observation method of Arabidopsis roots provided in this application places a specific culture device in the culture dish. On the one hand, this culture device can fix the glass slide in the middle position of the culture dish, avoiding the technical barrier that the traditional round culture dish cannot fix the glass slide, causing the glass slide to shake, resulting in the root growth bending and affecting the observation; on the other hand, the humidification setting ensures the humidity of the culture medium in the culture dish.

[0028] The agar content in the culture medium used is controlled at (1.05-1.2) g / 100 mL. The culture medium under this agar content can not only ensure the growth of Arabidopsis seeds, but also the hardness after solidification can ensure that the Arabidopsis roots will not grow into the culture medium during the culture process, which is conducive to the later microscopic root observation.

[0029] By controlling the position of the seeds on the glass slide culture medium and combining the circular bottom plate of the culture device with a ring shape (hollow in the middle), it is ensured that the germination and emergence process of Arabidopsis seeds can be observed.

[0030] This application is the first to combine a humidifying device and a fixing device for use in the culture medium cultivation process in a culture dish.

[0031] 2. The fixing device used to secure the seed-carrying glass slide in the culture device of this application is adjustable, ensuring that the slide is secure during the culture process and can be easily placed and removed without breakage. Furthermore, it is possible to culture and observe roots on glass slides of different specifications (thickness, length, and width).

[0032] The humidification device can make the culture medium reach a specific humidity: this is achieved by specific applied pressure of 40-100kPa, water outlet time of 1-3s, outlet pipe position and outlet pipe diameter of 0.15-0.35cm, and arc outlet pipe shape limitation.

[0033] The fixing device is made of nylon material. The humidifying device can ensure that the culture medium meets the specific humidity while allowing the fixing device made of nylon material to absorb water and expand to further enhance the fixing effect.

[0034] 3. The in situ observation method of Arabidopsis roots provided in this application is to select a glass slide containing Arabidopsis seedlings with roots cultured to 0.8-1.2 cm after the vertical culture is completed. The slide can be directly prepared without moving the Arabidopsis roots and the arrangement of microtubules in the root cells can be observed under a microscope, avoiding damage and displacement of the Arabidopsis roots when preparing slides using traditional methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0036] Figure 1 This is a schematic diagram of the structure of the culture device in Example 1 of the present application;

[0037] Figure 2 3D picture of the sink;

[0038] Figure 3 This is a schematic diagram of the structure of the culture device in Example 2 of the present application;

[0039] Figure 4 This is a schematic structural diagram of the culture device with a glass slide mounted thereon according to Example 2 of the present application;

[0040] Figure 5 This is a partial component diagram of the culture device in Example 2 of the present application;

[0041] Figure 6 This is another partial component diagram of the culture device in Example 2 of the present application;

[0042] Figure 7 This is a structural diagram of a concave block, a local component of the culture device in Example 2 of the present application;

[0043] Figure 8 This is a schematic diagram of the vertical cross-section structure of a local component clamp of the culture device in Example 2 of the present application;

[0044] Figure 9 This is a physical picture of the culture dish when the culture device of Example 1 of this application is installed;

[0045] Figure 10 For this application, the vertical culture diagram is in the culture device;

[0046] Figure 11 This is a microtubule morphology image of the root of the Arabidopsis seedlings cultured in Example 6-3 observed under a microscope in Example 6 of the present application (Bar=10 μm);

[0047] Figure 12 This is a microtubule morphology diagram of the roots of Arabidopsis seedlings cultured in Example 6-1 under microscope observation in Example 6 of this application;

[0048] Figure 13 This is a growth comparison diagram of the present application; (a) shows the growth on the third day without illumination using a 3D culture device, and (b) shows the growth on the third day using a 3D culture device.

[0049] List of parts and reference numerals:

[0050] 1 Culture device, 201 First clamp, 202 Second clamp, 3 Groove, 4 Circular bottom plate, 5 Temperature measuring instrument, 6 Stabilizing shaft, 701 Fixing block, 7011 Glass slide mounting groove, 7012 Second through hole, 702 Concave clamp, 8 Bolts, 9 Water container, 901 Water outlet pipe, 902 Water inlet pipe, 10 Glass slide. DETAILED DESCRIPTION

[0051] In order to more clearly illustrate the overall concept of the present application, the following is described in detail by way of example. In the description below, a large number of specific details are provided to provide a more thorough understanding of the present application. However, it is obvious to those skilled in the art that the present application can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present application, some technical features well known in the art are not described. Consumables not otherwise specified below are all commercially available.

[0052] In the examples of this specification, the Arabidopsis thaliana L. mCherry-TUA5 strain was selected for culture and observation; the culture device was a 3D culture device, which was designed using a 3D printing website and printed using a 3D printer. The 3D culture device was made of materials including acrylonitrile butadiene styrene (ABS), polylactic acid (PLA), nylon (PA), polycarbonate (PC), polystyrene (PS), polycaprolactone (PCL), polyphenylsulfone (PPSF), thermoplastic polyurethane (TPU), and polyetheretherketone (PEEK).

[0053] Fluorescence observation experiments were performed using an ultra-high resolution confocal microscope Leica SP8. The culture medium used in the examples of the present invention is as follows:

[0054] 1 / 2MS medium: MS (Duchefa) 2.165 g / L, sucrose 10 g / L, agar 11 g / L, MES 0.5 g / L, pH: 5.8-6.2.

[0055] Materials preparation:

[0056] Seed pretreatment:

[0057] Before sowing, Arabidopsis mCherry-TUA5 seeds need to be divided into small centrifuge tubes. First, rinse the seeds with a mixture of 75% ethanol and Triton X-100 for 1 minute 30 seconds; then rinse the seeds twice with 70% ethanol, each time for 1 minute 30 seconds; finally, spread the seeds and ethanol on sterile filter paper.

[0058] Before use, the 3D culture device was soaked in 75% ethanol and sterilized by UV light.

[0059] The circular bottom plate of the 3D culture device in the following examples has an outer circumference of 8.5 cm and an inner circumference of 7 cm. The glass slide has a length of 7.62 cm and a width of 2.54 cm.

[0060] Example 1 Figure 1-2 As shown, a 3D culture device includes a circular base (in the shape of a ring, hollow in the middle, so that the roots of the seedlings can be clearly observed when they grow), a fixing device arranged on the circular base for fixing the slide to the center position of the culture dish, and a humidifying device for maintaining the air humidity in the culture dish.

[0061] The fixing device includes a first clamping member and a second clamping member, and the first clamping member and the second clamping member are centrally symmetrical on the circular chassis (such as Figure 1-2 shown).

[0062] Specifically, the first clamp and the second clamp are composed of two symmetrically arranged L-shaped baffles of exactly the same size and shape. Four right angles are reserved between the four L-shaped baffles, and the humidification device is a groove for placing a water tank arranged on one side of the width direction of the slide. A humidity detector with a display screen is provided on the side wall of the fixing device (movably connected to the fixing device, and the present application adopts a card connection: a third groove is provided on the side wall, and the hook above the temperature detector is embedded in the third groove to achieve connection; the same is true for Example 2 below. The temperature detector can also be directly bonded to the side wall). The culture device is made of nylon material.

[0063] The sink measures 4cm long, 1cm wide, and 0.5cm high. The height of the L-shaped baffle is approximately 1.7cm.

[0064] Example 2 Figure 3-8 As shown, a 3D culture device is different from Example 1 in that the fixing device includes two stabilizing shafts symmetrically arranged on a circular base, and a clamping member connected to the stabilizing shafts; the clamping member includes a fixing block and a concave clamping block arranged at the bottom of the fixing block, a first through hole is provided on a side wall of the concave clamping block, and a thread is provided on the inner wall of the first through hole; the concave clamping block is placed on the stabilizing shaft with its opening facing downward, and the clamping member is connected to the stabilizing shaft by a bolt threadedly connected to the clamping member through the through hole;

[0065] A slide mounting groove is provided on opposite sides of the fixing blocks on the two stabilizing shafts; a second through hole is provided on the top and the lower part of the side wall of the fixing block, and a thread is provided on the inner wall of the second through hole;

[0066] The width and height of the glass slide mounting groove are respectively larger than the width and height of the glass slide (both are about 1.2 times larger); the distance between the two clamping parts can be flexibly adjusted by moving the concave clamping block.

[0067] The humidification device includes a water container arranged on one side in the width direction of the slide, and the interior of the water container is hollowed to form a hollow cavity; the top of the water container is provided with a plurality of evenly spaced water outlet pipes connected to the hollow cavity (the pipe openings are facing the fixing device, and the diameter (inner wall distance) is 0.15-0.3 cm); the upper portion of the outer wall of the water container is provided with a water inlet pipe connected to the hollow cavity, and the water inlet pipe can extend to the outside of the culture dish (when the culture dish cover is closed, the pressure between the cover and the culture dish body will not affect the water supply of the water inlet pipe); one end of the water inlet pipe located outside the culture dish is connected to a pressure water supply device.

[0068] A humidity detector with a display screen is provided on the side wall of the fixing device. The culture device is made of nylon.

[0069] As a preferred embodiment of the device of Example 2, Figure 3 As shown, the water outlet pipe is arranged in an arc shape with the arc opening facing downwards, so as to ensure that the water is sprayed in the direction of the water spraying onto the culture medium and the fixing device.

[0070] As a preferred embodiment of the device of Example 2, the water outlet pipe includes a first tube body and a second tube body arranged in the inner cavity of the first tube body, which can be movably connected to the inner wall of the first tube body through the outer wall of the second tube body; the extension and contraction of the water outlet pipe body is achieved by the forward and backward movement of the second tube body in the inner cavity of the first tube body, so that the gravity strength of sterile water falling into the culture medium can be controlled by adjusting the length of the water pipe body, thereby better ensuring that the seedlings will not be impacted when spraying water.

[0071] The fixing device in this embodiment can adjust the fixed position of the slide, which can realize the cultivation and observation of roots on slide culture media of different specifications (thickness, length, width). At the same time, the structure is relatively simple and the parts are all detachable, which is convenient to carry and replace.

[0072] Example 3 A method for in situ cultivation of Arabidopsis roots, comprising:

[0073] This embodiment uses the device of Example 1 as a 3D culture device.

[0074] Step 1: Culture medium preparation and sterile seeding

[0075] In a clean bench, use a pipette to place unsolidified 1 / 2 MS culture medium on a glass slide (sterilized at high temperature and high pressure). The volume of culture medium is 1.2 mL, and the thickness of the culture medium is as uniform as possible, about 1.0 mm. Sterilized Arabidopsis seeds are spread flat on the 1 / 4 position of the upper edge of the culture medium on the glass slide (30 seeds are spread on each glass slide), so that the Arabidopsis seeds are distributed on the edge of the glass slide.

[0076] Step 2: Vertical Cultivation

[0077] 1) Vernalize the seeds in a dark and low-temperature environment before vertical culture: Wrap the culture medium with tin foil and vernalize the seeds in a sterile and dark environment at 4°C for 48 hours.

[0078] 2) Place the slide fixture into a 9 cm diameter circular glass culture dish (sterilized at high temperature and high pressure), secure it by placing the water tank into the reserved groove, and secure the slide with the seeds between the first clamp and the second clamp (i.e., within the right angle reserved between the clamps) to secure the slide; finally, inject sterile water into the water tank, and vertically place the circular culture dish in a constant temperature and humidity climate chamber for vertical culture. The vertical culture conditions are: temperature 20 ± 2 ° C, relative humidity 80%, light conditions for 16 hours, and light intensity 150 μmol·m -2 ·s -1 , light / 8 hours dark; vertical culture for 4 days. During this period, the humidity near the fixture inside the culture dish (equivalent to the culture medium) was monitored daily using a humidity meter. After the vertical culture was completed, a slide containing an Arabidopsis seedling with roots cultured to 1 cm was prepared without removing the roots. The arrangement of microtubules in the root cells was observed under a microscope.

[0079] Example 4 A method for in situ cultivation of Arabidopsis roots, comprising:

[0080] This example uses Example 2 as the 3D culture device.

[0081] Step 1: Same as Example 3;

[0082] Step 2: Place the slide fixing device into a circular glass culture dish with a diameter of 9 cm (sterilized at high temperature and high pressure), place the openings of the two concave blocks of the two clamps facing downward, and place them on the stable shaft respectively. Then, by moving the clamps, place / extend the two ends of the slide covered with seeds into the slide mounting grooves of the two fixing blocks respectively. When the two ends of the slide touch the bottom of the groove of the fixing blocks respectively (at the same time, adjust the position of the slide and the fixing block to ensure that the slide is placed in the center of the culture dish), use a bolt to make a threaded connection at the first through hole. As the bolt is gradually rotated, it extends into the concave block and fixes the concave block on the stable shaft; in this way, the slide is fixed on the culture device.

[0083] Furthermore, bolts are used to make threaded connections at the second through holes on the top and side walls of the fixing block. As the bolts are gradually rotated, they extend into the slide mounting grooves and contact / fit with the top and side walls of the slide respectively, thereby further fixing the slide in the culture device.

[0084] A circular culture dish was placed vertically for vertical culture. The vertical culture conditions were the same as those in Example 3. During the vertical culture period, a pressure water delivery device located outside the culture dish was activated to add sterile water to a water container through a water inlet pipe (the pressure applied during water delivery was controlled to be approximately 55 kPa, and one end of the water inlet pipe located outside the culture dish was connected to the pressure water delivery device) until water was sprayed out through a water outlet pipe (with a diameter (internal wall distance) of 0.2 cm) and landed on a fixture. The humidity near the fixture (i.e., the culture medium position) was observed by observing the humidity on the humidity meter display to ensure that the humidity was between 55% and 80%. Observations were made once daily, both during the day and at night.

[0085] Specifically, in the culture device, the water outlet time during the day is 2 seconds, and the humidity on the humidity meter display screen shows about 60% after the water outlet is finished. Spraying once can ensure that the humidity of the culture medium during the day can be within the range of 55%-60%; near dusk, under the same spraying conditions, the water outlet pipe discharges water for 1-2 seconds, and the humidity on the humidity meter display screen shows about 80% after the water outlet is finished; the night time is shorter, and the humidity of the culture medium is always about 80% after spraying once.

[0086] The above Examples 3-4 were repeated 20 times respectively to compare the fixation degree of the glass slide, the moisturizing effect of the culture medium, and the effect on the growth of the seedlings of different culture devices.

[0087] Table 1 Comparison of effects of different culture devices

[0088]

[0089] According to the data in Table 1, Device 1 experienced breakage three times during the slide installation or removal process. This is because the four L-plates in Device 1 are too tight. While the slide is securely mounted, the tightness results in excessive force when removing or placing the slide, leading to breakage. This can cause the seedlings to move due to the breakage, potentially damaging their fragile roots. Furthermore, if the slide breaks during removal, it cannot be fully removed and placed under a microscope for observation, thus wasting the previous culturing process. If the four L-plates are slightly loose, the seedlings may shift when the culture dish is moved for observation.

[0090] Regarding the length of the seedling rhizomes, the root lengths of Examples 3 and 4 met the observation requirements after 3 days of illumination. However, in Example 3, the rhizomes of some seedlings were less than 0.8 cm in length after 3 days of illumination. This may be due to inappropriate humidity near the culture medium in Example 3. When selecting, select those with better growth.

[0091] Furthermore, during the experiment, sterile water was added to the water container at a pressure of 40-100 kPa and then continuously sprayed from the water outlet pipe (0.2 cm diameter) for approximately 1-4 seconds. This controlled the humidity of the air near the culture medium within a range of 55-80%. At this pressure, the water did not impact the seeds / sprouts / seedlings on the culture medium. The seedlings' rhizomes grew fastest and best at this point, reaching a length of 0.83-1.82 cm after 2-3 days of illumination. There was no yellowing, mold, or dehydration. Furthermore, this pressure did not impact the water outlet pipe, ensuring its stability during the water discharge process and preventing displacement or deformation of the pipe opening.

[0092] Furthermore, the applicant found that when the culture device made of nylon PA was used in Example 4, the distance between the fixing plates on both sides of the slide became narrower after water spraying (the distance between the side walls of the fixing blocks on both sides of the slide was reduced from 2.55 cm to 2.54 cm). This shows that nylon PA has a certain degree of water absorption and expansion. After absorbing a certain amount of water, it can expand appropriately and enhance the fixation effect on the slide.

[0093] Example 5 A method for in situ cultivation of Arabidopsis roots, comprising:

[0094] Step 1: Culture medium preparation and sterile seeding

[0095] In a clean bench, use a pipette to place unsolidified 1 / 2 MS culture medium on four glass slides (sterilized at high temperature and high pressure), and take 0.8 mL, 1.2 mL, 1.5 mL, and 2.0 mL of culture medium, respectively, to make the culture medium thickness as uniform as possible, with thicknesses of <0.9 mm, 0.9-1.2 mm, 1.0-1.4 mm, and 1.3-1.5 mm, respectively; Sterilized Arabidopsis seeds are spread flat on the upper edge of the culture medium on the glass slide at 1 / 4 (30 seeds per glass slide), so that the Arabidopsis seeds are distributed at the edge of the glass slide;

[0096] Step 2: Vertical Cultivation

[0097] 1) Vernalize the seeds in a dark and low-temperature environment before vertical culture: Wrap the culture medium with tin foil and vernalize the seeds in a sterile and dark environment at 4°C for 48 hours.

[0098] 2) Two more groups were set up: Example 5-1 and Example 5-2, which adopted the culture methods of Example 3 and Example 4 respectively.

[0099] The effects of different culture medium volumes (thicknesses) on Arabidopsis seed germination and fluorescence in situ observation were investigated. The specific results are shown in Table 2 below.

[0100] Table 2 Effects of different culture medium thicknesses on subsequent culture observations

[0101]

[0102] in conclusion:

[0103] 1) Table 2 shows that different culture medium volumes significantly impact the growth and in situ observation of Arabidopsis seedlings. Compared to Example 1, where the culture medium volume was 0.8 ml, the Arabidopsis seedlings in Examples 2-4 exhibited relatively good growth and development. While the Arabidopsis seedlings in Example 4 grew well, the roots were obscured during in situ microscopic observation, likely due to excessively thick culture medium. This suggests that a culture medium volume between 1.2 ml and 1.5 ml (approximately 0.9-1.4 mm thick) did not affect the germination and growth of Arabidopsis seeds or subsequent in situ observations during in situ root observation.

[0104] 2) The effect of culture medium volume of 1.2-1.5ml (thickness of about 0.9-1.4mm) on the germination and growth of Arabidopsis seeds and in situ observation was not significantly different. Therefore, the sequence number 2 groups in Examples 5-1 and 5-2 in Example 5 were respectively used as preferred examples. 30 Arabidopsis seeds were selected and planted in groups of 10 seeds each. After vernalization at low temperature and dark protection, and vertical culture was carried out to explore the effect of the planting position on the growth of Arabidopsis seeds. The experimental process found that the sterilized Arabidopsis seeds were spread flat on the upper edge of the slide culture medium at 1 / 4, and the Arabidopsis seeds were distributed as much as possible on the edge of the slide (such as Figure 10 As shown, they are distributed along the edge of the slide in the width direction, which makes it easier to observe all the seedling roots later). This is most beneficial for the growth of seedling roots and the later observation of the arrangement of microtubules in the seedling root cells using a microscope.

[0105] If you place the Arabidopsis seeds in the center of the slide, the roots will grow beyond the width of the slide, preventing the coverslip from fully covering the roots during slide preparation. This will hinder and affect subsequent slide preparation and microscopic observation. Place the Arabidopsis seeds flat on the top 1 / 4 of the slide's culture medium, ensuring that the seeds are distributed along the edge of the slide to ensure effective observation of the Arabidopsis seedlings.

[0106] Furthermore, the agar content in the culture medium is controlled at (1.05-1.2) g / 100 mL. The applicant has found that the culture medium with this agar content can not only ensure the growth of Arabidopsis seeds, but also the hardness after solidification can ensure that the Arabidopsis roots will not grow into the culture medium during the culture process, which is beneficial to the later microscopic root observation.

[0107] Example 6

[0108] Based on the optimal screening results described above and based on Example 4, two groups were set up, namely Example 6-1 and Example 6-2. Twenty Arabidopsis seeds were selected and planted, with 10 seeds per group, before being vernalized at low temperature and in the dark. Example 6-1 cultured Arabidopsis without a 3D culture device, Example 6-2 cultured Arabidopsis using the 3D culture device of Example 1, and Example 6-3 cultured Arabidopsis using the 3D culture device of Example 2. The experimental procedures for Example 6-2 were the same as those for Example 3; the experimental procedures for Example 6-3 were the same as those for Example 4. The effects of different Arabidopsis culture devices on Arabidopsis cultivation were investigated. The experimental results are shown in Table 3.

[0109] Table 3 Effects of different Arabidopsis culture devices on Arabidopsis culture

[0110]

[0111]

[0112] According to Table 3, Example 6-1 has the problem that the culture medium loses water easily during the growth of the seedlings, resulting in poor growth of the Arabidopsis seedlings and a long growth cycle. At the same time, there is also the problem that the slide is not fixed stably and the Arabidopsis roots cannot remain in situ, resulting in erroneous results during microscopy observation, such as Figure 12 (Bar=10um) shows the microtubule morphology of the root: the microtubules are disordered and fragmented, with only a few bundles of shaped microtubules. During the implementation of Examples 6-2 and 6-3, the humidification device can effectively ensure that the culture medium remains in a good moist state, providing suitable moisture for the Arabidopsis seedlings. The Arabidopsis seedlings grow well (as shown in the growth potential comparison chart of Examples 6-1 and 6-3). The roots can always remain in situ during the culture and preparation process, especially in Example 6-3, the rhizomes grow faster and longer. Figure 11 As shown (Bar=10 μm), the arrangement of microtubules in the root cells of 6-3 Arabidopsis seedlings can be successfully observed, and the microtubule morphology is clear and orderly.

[0113] like Figure 13 As shown, (a) shows the growth of Example 6-1 on the third day without the 3D culture device, and (b) shows the growth of Example 6-2 on the third day with the culture device. It can be seen that after using the specific culture device, the seedlings grew straighter and longer.

[0114] In summary, the present invention uses a specific 3D culture device and a culture medium volume of 1.2-1.5 ml (approximately 0.9-1.4 mm thick) to culture Arabidopsis seedlings. Sterilized Arabidopsis seeds are spread flat on a glass slide at a position 1 / 10-1 / 3 (preferably 1 / 4) of the upper edge of the culture medium to ensure normal slide preparation and in situ observation of the seedlings. This invention establishes a method for in situ culture and observation of Arabidopsis roots, providing technical support for monitoring the real-time dynamic behavior of fluorescent proteins in roots and providing an excellent platform for studying root developmental mechanisms.

[0115] The foregoing is merely an embodiment of the present application, and the scope of protection of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, the present application may have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc. made within the technical ideas and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method for in situ cultivation of plant roots, characterized in that: The steps include: Step 1: Spread unsolidified culture medium on a glass slide, wait for it to solidify, and then spread sterilized Arabidopsis seeds on the upper edge of the culture medium on the glass slide; the thickness of the culture medium is 0.9-1.4 mm; Step 2: Place the glass slide with the seeds in a culture dish equipped with a culture device and culture it vertically under light; The culture device includes a circular bottom plate, a fixing device arranged on the circular bottom plate for fixing a glass slide with seeds, and a humidifying device for increasing the humidity of the air in the culture dish; The amount of agar added to the culture medium is (10.5-12) g / L, and the material of the culture device is nylon; In step 2, the fixing device includes two stabilizing shafts symmetrically arranged on a circular chassis and a clamp connected to the stabilizing shafts; the clamp includes a fixing block and a concave clamping block arranged at the bottom of the fixing block, a first through hole is provided on a side wall of the concave clamping block, and a thread is provided on the inner wall of the first through hole; the clamp is connected to the stabilizing shaft by a bolt threadedly connected to the inner wall of the first through hole; A slide mounting groove is provided on opposite sides of the fixing blocks on the two stabilizing shafts; a second through hole is provided on the top and the lower part of the side wall of the fixing block, and a thread is provided on the inner wall of the second through hole; The humidification device includes a water container disposed on one side of the slide in the width direction, the interior of the water container being hollowed to form a hollow cavity; a plurality of evenly spaced water outlet pipes connected to the hollow cavity are disposed on the top of the water container; a water inlet pipe connected to the hollow cavity is disposed on the upper portion of the outer wall of the water container; the water inlet pipe can extend outside the culture dish; the water outlet pipe is arranged in an arc shape with the arc opening facing downward; the diameter of the water outlet pipe is 0.15-0.35 cm; sterile water is introduced into the water container under a pressure of 40-100 kPa, and water is discharged from the water outlet pipe for 1-4 seconds; A humidity detector with a display screen is arranged on the side wall of the fixing device.

2. The in situ culture method according to claim 1, characterized in that: In step 1, the sterilized Arabidopsis seeds are spread flat on the upper edge of the glass slide culture medium at 1 / 10-1 / 3.

3. The in situ culture method according to claim 1, characterized in that: The circular chassis is ring-shaped and hollow in the middle.

4. The in situ culture method according to claim 1, characterized in that: The method of using the culture device in step 2 is: Place the glass slide fixture with the seeds in the culture dish, place the two concave clamping blocks with their openings facing downward on the stabilizing shafts, move the clamping blocks to place the two ends of the glass slide with the seeds in the glass slide mounting grooves of the two fixing blocks, and when the two ends of the glass slide touch the bottom of the grooves of the fixing blocks, use bolts to thread them at the first through holes; Using bolts to thread the second through holes on the top and side walls of the fixing block to fix the glass slide in the culture device; Sterile water is introduced into the water inlet pipe and added to the water container until the water is sprayed out from the outlet pipe and falls on the fixed device, so that the humidity of the culture medium is in the range of 55-80%.

5. The in situ culture method according to any one of claims 1 to 4, characterized in that: The plant is Arabidopsis thaliana.

6. Use of the in vivo in situ culture method according to any one of claims 1 to 4 in in situ observation, characterized in that: include: After the vertical culture is completed, the slides are removed and sections are prepared. The arrangement of microtubules in the root cells is observed under a microscope; The conditions for selecting a glass slide are as follows: a glass slide carrying Arabidopsis seedlings with roots cultured to 0.8-1.2 cm was selected, the slide was directly prepared without moving the roots, and the slide was directly observed under a microscope.

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