A microfluidic chip and experimental method for studying the plasticity behavior of plant roots

Abstract

This invention relates to a microfluidic chip and experimental method for studying the plasticity of plant roots. The chip includes a nutrient solution inlet, a seed inlet, a plant root growth channel, and a nutrient solution outlet. By designing a microfluidic chip for studying the plasticity of plant roots, and employing a visual structural design, it can simulate how plant roots adapt to the environment by undergoing physiological and morphological changes when encountering particulate obstacles in the soil. This overcomes the problem that traditional methods cannot image how roots change themselves to avoid particulate obstacles due to the opacity of the soil. The variable two-dimensional structure and clear imaging advantages of this microfluidic chip solve this problem, providing tools and experimental methods for studying plant roots.

Description

Technical Field

[0001] This invention relates to the field of plant root research technology, and in particular to a microfluidic chip and experimental method for studying the plasticity behavior of plant roots. Background Art

[0002] Revealing the growth mechanisms of root systems is of great significance for optimizing agricultural production and improving crop varieties. Currently, there is no satisfactory method to track the plastic behavior characteristics of root systems. Applying microfluidic technology to the field of root research explores the enormous application potential of microfluidic systems in plant root research.

[0003] During plant root growth in the soil, they are subjected to mechanical resistance from the external environment. They must adapt their growth behavior through complex interactions of responses at different times, leading to changes in root morphology. The most obvious changes are in the overall root morphology and growth rate, often accompanied by alterations in cell morphology. Mechanical stress physically restricts root growth, potentially causing roots to shorten, reducing growth potential, and negatively impacting the overall health of the plant.

[0004] To monitor root growth, images must be captured periodically, which can be time-consuming and technically challenging. Traditional methods, such as culturing plant roots on culture media, cannot meet the requirements for real-time, high-resolution dynamic imaging, nor can they quantify plant root growth. Traditional microfluidic chip structures used for studying plant roots are mostly simple straight channels, which reduce the complexity of the root growth environment in the soil and do not take into account the mechanical stress encountered by roots in the soil, which may lead to biased experimental results. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a microfluidic chip and experimental method for studying the plasticity behavior of plant roots, which can further study the growth of roots under different scale channels and the increasingly compact channel as it goes down, as well as the mechanical characteristics of the plasticity behavior of plant roots.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is: a microfluidic chip for the plastic behavior of plant roots, the innovation of which is: including a nutrient solution inlet, a seed inlet, a plant root growth channel and a nutrient solution outlet;

[0007] The nutrient solution inlet is provided with a horizontal inlet pipe at its output end; the plant root growth channel is connected in parallel to the horizontal inlet pipe;

[0008] The plant root growth channels include eight channels, a1 to a8. Channels a1 to a4 are 100 μm, 200 μm, 350 μm, and 1000 μm in diameter, respectively. Channel a5 is a gradually decreasing channel from 550 μm to 50 μm. Channel a6 is a narrow, curved channel with a width of 100 μm. Channel a7 has a width of 1 mm and contains several cylindrical and semi-cylindrical obstacles with cross-sectional dimensions ranging from 300 μm to 600 μm. Channel a8 is a wide, curved channel with widths of 250 μm, 400 μm, and 600 μm.

[0009] The seed inlets are multiple and located on the liquid inlet horizontal tube, and the seed inlets correspond to the ends of the eight plant root growth channels.

[0010] The plant root growth channel is provided with a liquid outlet horizontal pipe at its output end, and the nutrient solution outlet is located on the liquid outlet horizontal pipe.

[0011] An experimental method for studying the plasticity behavior of plant roots using a microfluidic chip, the innovation of which lies in the following: The specific experimental method is as follows:

[0012] S1: Fluid simulation of microfluidic chip: Nutrient solution is filled into the nutrient solution inlet of the microfluidic chip, and the inlet flow rate is 0.01 m / s; the outlet pressure of the nutrient solution is atmospheric pressure; the flow rate of the fluid is monitored at 15 locations on the plant root growth channel.

[0013] S2: Imaging and Quantification of the Plastic Behavior of Arabidopsis thaliana Roots: The microfluidic system was used to align the root with the imaging plane of the microscope, improving the imaging resolution and clarity, so that the plant roots could be imaged while growing; the quantitative results of the experiment were generated by averaging multiple experiments.

[0014] S3: Arabidopsis thaliana seed root experiment: Nutrient solution was injected into the nutrient solution inlet with a syringe until the nutrient solution filled the microfluidic channel. Agar was filled into the seed inlet, and then wild-type Arabidopsis thaliana seeds were placed into the seed inlet. The microfluidic chip with the seeds placed was tilted and placed into a culture bottle with culture medium for rooting. The final experimental results were observed.

[0015] The advantages of this invention are:

[0016] 1) This invention designs a microfluidic chip for studying the plasticity behavior of plant roots, employing a visual structural design. It can simulate how plant roots adapt to the environment by undergoing physiological and morphological changes when encountering particulate obstacles in the soil. This overcomes the problem that the traditional growth process of how roots change themselves to avoid particulate obstacles cannot be imaged due to the opacity of the soil. The variable two-dimensional structure and clear imaging advantages of this microfluidic chip can solve this problem, providing tools and experimental methods for the study of plant roots. Attached Figure Description

[0017] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0018] Figure 1 This is a schematic diagram of the structure of a microfluidic chip for studying the plasticity behavior of plant roots according to the present invention.

[0019] Figure 2 This invention relates to a microfluidic chip for studying the plasticity behavior of plant roots, showing flow rate monitoring points and flow rate variation diagrams.

[0020] Figure 3 These are a set of experimental images used in the study of plant root plasticity behavior according to the present invention.

[0021] Figure 4 This is another experimental imaging image of the plastic behavior of plant roots according to the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0023] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0024] like Figures 1 to 4 The microfluidic chip shown includes a nutrient solution inlet 1, a seed inlet 2, a plant root growth channel 3, and a nutrient solution outlet 4.

[0025] A horizontal inlet pipe is provided at the output end of nutrient solution inlet 1; the plant root growth channel is connected in parallel to the horizontal inlet pipe.

[0026] The plant root growth channel 3 includes eight channels, a1 to a8. Channels a1 to a4 are 100μm, 200μm, 350μm, and 1000μm, respectively; channel a5 is a gradually changing channel from 550μm to 50μm; channel a6 is a narrow curved channel with a width of 100μm; channel a7 is 1mm wide and contains several cylindrical and semi-cylindrical obstacles with cross-sectional dimensions ranging from 300μm to 600μm; channel a8 is a wide curved channel with widths of 250μm, 400μm, and 600μm.

[0027] The seed inlet 2 has several inlets and is located on the liquid inlet horizontal tube, and the seed inlet 2 corresponds to the end of the eight plant root growth channels.

[0028] The plant root growth channel 3 is equipped with a liquid outlet horizontal tube at its output end, and the nutrient solution outlet is located on the liquid outlet horizontal tube.

[0029] An experimental method for a microfluidic chip to study the plastic behavior of plant roots is described below:

[0030] S1: Fluid simulation of the microfluidic chip: Nutrient solution is introduced into the nutrient solution inlet of the microfluidic chip, with an inlet flow rate of 0.01 m / s; the nutrient solution outlet is at atmospheric pressure; the fluid velocity is monitored at 15 locations along the plant root growth channel; from Figure 2 For each corresponding location, select 15 locations and label them as locations 1-15. Collect the flow velocity changes at each location and obtain... Figure 2 B. Figure 2 C Figure 2 D、 Figure 2 E and Figure 2 F; The magnitude of the flow velocity is inversely proportional to the magnitude of the pressure it receives, and the magnitude of the flow velocity can reflect the magnitude of the pressure received at the corresponding location; Figure 2 B reflects that the larger the channel size, the greater the flow velocity, and therefore the smaller the pressure on the channel; positions 5, 6, and 7 are points from top to bottom in channel a5, respectively. Figure 2 C shows that the flow velocity decreases, therefore the pressure increases further down; position 9 is the point at the bend in channel a6, from... Figure 2 D shows that the flow velocity decreases at the bend of the narrow, curved channel, while the pressure increases at this point; positions 11 and 12 are the midpoint between the cylinder and the wall, and the point closest to the cylinder, respectively. Figure 2 E shows that the closer to the cylindrical obstacle, the lower the material velocity and the higher the pressure; position 14 is the point at the bend in channel a8, from... Figure 2 As can be seen from F, the flow velocity decreases at the bend of the narrow, curved channel, while the pressure increases at this point.

[0031] S2: Imaging and Quantification of the Mechanical Characteristics of Arabidopsis thaliana Roots: The microfluidic system was used to align the roots with the imaging plane of the microscope, improving the imaging resolution and clarity, and enabling the plant roots to be imaged while growing; the quantitative results were generated by averaging multiple experiments.

[0032] S3: Arabidopsis thaliana seed root experiment: Nutrient solution was injected into the nutrient solution inlet with a syringe until the nutrient solution filled the microfluidic channel. Agar was filled into the seed inlet, and then wild-type Arabidopsis thaliana seeds were placed into the seed inlet. The microfluidic chip with the seeds placed was tilted and placed into a culture bottle with culture medium for rooting. The final experimental results were observed.

[0033] Figure 3 A represents the dynamic growth process of roots in microfluidic chip channels with sizes of 100 μm, 200 μm, 350 μm, and 1000 μm. To gain a more comprehensive and intuitive understanding of the impact of different microfluidic channel sizes on root growth, the length of Arabidopsis roots in channels of four different sizes was quantified from day 4 to day 7. The results are as follows: Figure 3 As shown in D; from Figure 3 As can be seen from D, the channel size in the microfluidic chip affects the rate of plant cell proliferation and elongation. The larger the channel size, the less mechanical stress on the Arabidopsis roots, the faster the plant cell proliferation, and the longer the root system. Channels of 100μm and 200μm, due to their smaller size, exert greater mechanical stress on the roots, resulting in slower plant growth and shorter root lengths. Conversely, channels of 350μm and 1000μm, due to their larger size, exert less mechanical stress on the roots, resulting in faster plant growth and longer root lengths. Smaller channels limit cell proliferation, thus restricting root growth; while wider channels provide more space for cell division and elongation, thus not restricting root growth.

[0034] Figure 3 B is a magnified image of the root tips in channels a1-a4 on day 6. The plant cells in the roots are clearly visible, indicating that the microfluidic and microscopic observation system can achieve cellular-level observation precision. Root hairs and walls exhibit good interaction; the larger the channel size, the more vigorous and longer the root hairs. Furthermore, imaging of root growth in channels of different sizes also reveals significant changes in root diameter. Figure 3E is a quantitative diagram of the root maturity zone diameter in channels a1 to a4. The diagram shows that the larger the channel size, the larger the root diameter. In channels of 100 μm, the root system fills the channel width, and due to the mechanical stress exerted by the channel on the root system, the root diameter remains at 100 μm. In channels from 100 μm to 350 μm, the diameter of Arabidopsis roots changes significantly. This is because the channel width within this range exerts considerable mechanical stress on root growth in the width direction, and channels from 100 μm to 350 μm have a significant impact on root diameter, which changes noticeably with the width of the growing channel. In channels from 350 μm to 1000 μm, the root diameter of Arabidopsis remains relatively unchanged. This is because the root diameter of Arabidopsis seedlings is typically less than 350 μm, and in channels from 350 μm to 1000 μm, the root system is not significantly affected by mechanical stress in the width direction, thus having little impact on root diameter.

[0035] In real soil environments, soil density generally increases and compaction increases with depth, which can hinder root development and thus affect plant growth and development. The a5 channel simulates this situation, with its width gradually decreasing from 550 μm to 50 μm. The dynamic growth process of Arabidopsis roots during days 4-7 in the a5 channel is shown in the image. Figure 3 As shown in C, the imaging image reveals that root growth is rapid at the beginning of the widest part of the root system, but slows down significantly as the width decreases. To better analyze the Arabidopsis root system in channel a5, the growth elements of the Arabidopsis root system were quantified. Figure 3 F is a quantitative graph showing the growth length of Arabidopsis roots from day 4 to day 7 and the width of the channel corresponding to the root tip each day. Figure 3 G is a quantitative graph of growth changes over 4-7 days, where root growth change = root length on the current day - root length on the previous day; from Figure 3 G shows that the root growth of Arabidopsis thaliana gradually slows down as the number of days increases. Figure 3 In F, it can be observed that the width of the channel corresponding to the root tip becomes smaller and smaller, and the mechanical stress of the channel on the root system becomes greater and greater. The change in root length on days 4-5 is significantly greater than that on days 5-6 and 6-7. After the root system grows to day 6, the width of the channel corresponding to the root tip is less than 150 μm, which poses a great mechanical stress on the root system. Arabidopsis roots adapt to environmental changes by changing their own morphology, reducing their own diameter to fill the channel. The root growth in channel a5 can be used to infer the root growth in real soil environment. Overly compacted soil will restrict root growth to a certain extent, but the root adaptability of plants can help them adapt to different soil conditions to a certain extent.

[0036] An imaging system combining a microfluidic chip and a microscope can provide high-resolution, real-time imaging of the plasticity of roots in response to mechanical obstacles. This study designed an a6 channel with a 100 μm curved narrow channel, an a7 channel containing irregularly arranged cylindrical and semi-cylindrical obstacles with cross-sectional diameters ranging from 300 μm to 600 μm, and an a8 channel with curved wide channels of 250 μm, 400 μm, and 600 μm, respectively, based on the root size of Arabidopsis seedlings, to explore the plasticity of roots under different mechanical obstacles. Figure 4 A is a real-time growth image of Arabidopsis thaliana roots in channel a6 from day 4 to 7. As can be seen from the image, since the channel width is 100 μm and the diameter of Arabidopsis thaliana roots under natural growth is greater than 100 μm, the Arabidopsis thaliana roots fill the channel by regulating their own growth and development. As time goes by, the shape of Arabidopsis thaliana presents the "Z" shape of the channel, adapting to changes in the environment by changing its own shape. Figure 4 B is a comparison of Arabidopsis root length in channel a6 (100μm curved channel) and channel a1 (100μm straight channel) on days 4-7. It can be observed that the root length in the curved channel is shorter than that in the straight channel. This is because the mechanical stress on the roots is different in the two channels, with the mechanical stress in the curved channel being greater than that in the straight channel.

[0037] Channel a7 contains cylindrical and semi-cylindrical obstacles with cross-sectional diameters ranging from 300 μm to 600 μm, used to simulate soil particles encountered by roots in the soil. Imaging results of the roots on days 4-7 are shown below. Figure 4 C. On day 4, the root system bypassed the first and second cylinders. Both cylinders had a cross-sectional diameter of 450 μm and a distance of 200 μm between them. It can be observed that the root system changed its shape to bypass the first cylinder, then passed through the gap between the first and second cylinders, and continued growing around the second cylinder. On day 4, due to the larger gaps between the subsequent cylinders, the root system grew rapidly. The root system continued to grow downwards along the growth direction of day 4 until it encountered the ninth cylinder. Since the distance between the ninth cylinder and its wall was only 100 μm, this gap caused significant mechanical stress to the plant's root growth. The Arabidopsis root system adjusted its shape to pass through the gap between the ninth cylinder and its wall, but this also affected its growth rate. Figure 4D is a comparison of Arabidopsis root lengths in 1mm channels (a7 with cylindrical obstruction and a4 without cylindrical obstruction) from day 4 to day 7. The figure clearly shows that the presence of the cylindrical obstruction in the channel exerts significant mechanical stress on Arabidopsis root growth. The root length in the channel with the cylindrical obstruction is significantly shorter than that in the channel without it. Especially after day 4, in channel a7, the roots have to pass through a 100μm wide gap formed by the cylinder and the wall, hindering root growth. The stress levels on the roots differ between channels a7 and a4, and the difference in root length between the two channels increases progressively.

[0038] Figure 4 E is a dynamic growth imaging diagram of Arabidopsis thaliana roots in the a8 channel with a curved and wide channel from day 4 to day 7. It can be observed from the figure that on day 6 and day 7, when the roots encounter the inflection point of the channel bend, they adjust their own shape to cope with the change in the shape of the channel. After the roots hit the wall, they bend more to adapt to the channel environment. The shape of the roots shows plasticity as the channel changes. Figure 4 F compares the root length of Arabidopsis thaliana in the narrow curved channel a6 and the wide curved channel a7 on days 4-7. It can be observed that the root length of Arabidopsis thaliana in the narrow curved channel is greater than that in the wide curved channel. This is because the mechanical stress on the roots in the narrow curved channel is greater than that in the wide curved channel.

[0039] Those skilled in the art should understand that this invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to this invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip for studying the plasticity behavior of plant roots, characterized in that: This includes the nutrient solution inlet, seed inlet, plant root growth channel, and nutrient solution outlet; The nutrient solution inlet is provided with a horizontal inlet pipe at its output end; the plant root growth channel is connected in parallel to the horizontal inlet pipe; The plant root growth channel includes eight channels, a1 to a8. Channels a1 to a4 are 100μm, 200μm, 350μm, and 1000μm in diameter, respectively. Channel a5 is a gradually decreasing channel from 550μm to 50μm. Channel a6 is a narrow, curved channel with a width of 100μm. Channel a7 has a width of 1mm and contains several cylindrical and semi-cylindrical obstacles with cross-sectional dimensions ranging from 300μm to 600μm. Channel a8 is a wide, curved channel with a width of 250μm, 400μm, or 600μm. The seed inlets are multiple and are located on the liquid inlet horizontal tube, and the seed inlets correspond to the ends of the eight plant root growth channels. The plant root growth channel is provided with a liquid outlet horizontal pipe at its output end, and the nutrient solution outlet is located on the liquid outlet horizontal pipe.

2. An experimental method for studying the plastic behavior of plant roots using a microfluidic chip as described in claim 1, characterized in that: The specific experimental method is as follows: S1: Fluid simulation of microfluidic chip: Nutrient solution is filled into the nutrient solution inlet of the microfluidic chip, and the inlet flow rate is 0.01 m / s; the outlet pressure of the nutrient solution is atmospheric pressure; the flow rate of the fluid is monitored at 15 locations on the plant root growth channel. S2: Imaging and Quantification of the Mechanical Characteristics of Arabidopsis thaliana Roots: The microfluidic system was used to align the roots with the imaging plane of the microscope, improving the imaging resolution and clarity, and enabling the plant roots to be imaged while growing; the quantitative results were generated by averaging multiple experiments. S3: Arabidopsis thaliana seed root experiment: Nutrient solution was injected into the nutrient solution inlet with a syringe until the nutrient solution filled the microfluidic channel. Agar was filled into the seed inlet, and then wild-type Arabidopsis thaliana seeds were placed into the seed inlet. The microfluidic chip with the seeds placed was tilted and placed into a culture bottle with culture medium for rooting. The final experimental results were observed.

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