研究目的
To develop a system for real-time imaging of Brachypodium roots under osmotic stress to study growth dynamics and stress responses at cellular and tissue levels.
研究成果
The developed microfluidic system successfully enables real-time imaging of Brachypodium root growth and osmotic stress responses. It revealed growth cessation under stress, morphological changes in cells, and validated gene expression patterns, including the first experimental verification of BdDi19 upregulation under drought stress. This setup is promising for high-precision phenotyping of monocot plants.
研究不足
The system allows imaging of only a few channels simultaneously due to the size of monocot seeds. Long-term maintenance beyond 48 hours is challenging. Gene expression analysis at short-term stress (2 hours) did not show clear trends, requiring extended periods for definitive results.
1：Experimental Design and Method Selection:
The study involved designing a PDMS-based microfluidic device to grow Brachypodium seeds, enabling real-time imaging of root growth and stress responses. Light and fluorescence microscopy were used for visualization.
2：Sample Selection and Data Sources:
Brachypodium distachyon seeds (line Bd21-3) were used, sterilized and germinated under controlled conditions. Samples were grown in microfluidic channels with Murashige and Skoog media.
3：List of Experimental Equipment and Materials:
PDMS devices, 3D printed molds, microscopes (Nikon SMZ 1500 stereomicroscope, Zeiss Axio Vert.A1 inverted microscope), neutral red dye, PEG-6000 for osmotic stress, and various lab equipment for fabrication and analysis.
4：Experimental Procedures and Operational Workflow:
Seeds were vernalized, germinated, and inserted into PDMS devices. Top and bottom imaging setups were used for time-lapse growth recording and fluorescence imaging. Osmotic stress was applied using 20% PEG in MS media. Gene expression was analyzed via qRT-PCR.
5：Data Analysis Methods:
Growth rates were measured from time-lapse videos. Fluorescence images were analyzed for morphological changes. Gene expression data were normalized and analyzed using the ΔΔCt method.
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Zeiss Axio Vert.A1 inverted microscope
Axio Vert.A1
Zeiss
Used for fluorescent imaging of roots under stress conditions.
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Nikon SMZ 1500 stereomicroscope
SMZ 1500
Nikon
Used for top imaging of root growth in time-lapse studies.
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PDMS
Material for fabricating microfluidic devices to grow and image plant roots.
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PEG-6000
Used to induce osmotic stress in the growth media.
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Neutral Red dye
Vital stain for fluorescent imaging of live and dead cells in roots.
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Light Cycler 480 II Instrument
480 II
Roche Diagnostics
Used for quantitative reverse transcription polymerase chain reaction (qRT-PCR) to analyze gene expression.
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Epson perfection v700 photo scanner
v700
Epson
Used to visualize the full length of seedlings grown in devices.
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WinRHIZO software
Regent Instruments
Used to analyze root and shoot scan images for growth measurements.
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