This method can help answer key questions in the plant cell biology field, such as how tip-growing plant cells penetrate cell physical barriers. The main advantage of this technique is the ability to acquire high-resolution images that rebuilds a cell's deformation process at the micrometer scale gap under a conventional microscope. To make a device for examination of the growing pollen tubes and moss protonemata, first make approximately 11 grams of a PDMS, and load it into a four-inch mold.
Then, degas the mold for 20 minutes in a vacuum chamber. Next, cure the mold at 65 degrees Celsius for 90 minutes. Once cured, peel off the PDMS layer from the mold, and open the access holes to the channel within the PDMS using a biopsy punch tool.
Next, expose the PDMS and the five-centimeter glass dish to air plasma for 50 seconds. To seal off the microfluidic network, press the PDMS layer onto the glass dish, and cure it for 30 minutes at 65 degrees Celsius. To make a microdevice designed for root hair examination, two molds are loaded and cured.
When punching out the channel holes, use a two-millimeter punch. After exposing both layers to air plasma for 50 seconds, join them together, including a coverslip, under a stereomicroscope. Use the custom-made aligner tool to accomplish this.
Cure the assembly for 30 minutes in the oven to seal off the microfluidic network. After curing, remove the coverslip from the device, and transfer it to a five-centimeter glass dish. Before using the pollen tube microdevice, degas it in a vacuum chamber for 20 minutes.
Add growth medium to the pistil inlet using a fine-tipped micropipette. Load the other wells with the same medium. Allow a few minutes for the channels to fill.
Meanwhile, place a moist paper towel into the dish to help maintain local humidity. Now, collect pollen grains from a T.fournieri blue and white flower. Transfer the grains onto the stigma using a dissecting needle.
Next, cut a one-centimeter long pollinated style lengthwise, and then insert the cut style into the inlet of the microfluidic device. When the cut style is inserted into the inlet of microfluidic device with tweezers, it is important not to hold the style very tightly because it may damage the style. Then, secure a lid onto the dish using tape, and transfer the dish to an incubator at 28 degrees Celsius where it must remain in the dark for five to six hours.
Working under a laminar flow hood, begin by sterilizing transgenic A.thaliana Columbia seeds. Soak them in cleaning solution for five minutes. Then, rinse the seeds in autoclaved water thoroughly.
Once sterilized, store the seeds at four degrees Celsius in darkness for 48 hours. A few days later, degas the microdevice for 20 minutes before using it. Next, load the appropriate growth medium into the wells of the device using a finely tipped pipette, and wait a few minutes for the solution to be pulled through all the channels.
Also, load some moist paper towel onto the dish to maintain humidity. Now, transfer a prepared seed into the device's inlet. Then, secure the lid on using tape, and transfer the dish to a 22 degree Celsius incubator with continuous light.
Before its use, sterilize the microdevice under UV overnight. The following day, degas the microdevice for 20 minutes. Once degassed, load the microwells with the appropriate growth medium.
While the channels gradually fill, surround the microdevice with some autoclaved water to maintain humidity. Transfer a small piece of moss protonemata tissue to the inlet of the microdevice. Now, culture the device at 25 degrees Celsius under continuous light.
After two to three weeks of growth, take bright-field images of the results under a microscope. Tip-growing plant cells encounter a series of physical barriers along their growth paths in vivo, such as in the transmitting tract and at the micropyle, not to mention the path of root hairs in the soil. The presented microfluidic in vitro cell culture platforms enable the examination of tip-growing process in three types of plant cells, pollen tubes, root hairs, and moss protonemata.
Viewing is accomplished through one-micron gaps in the devices. Because micro-gaps of this size are fragile, sometimes they will close, especially after repeated use. Thus, it is important to verify that the gaps are intact before performing the experiments.
For the pollen tube study, live-cell imaging was used to monitor morphological changes in the apical region of pollen tubes, as well as the vegetative nucleus and sperm cells in response to encountering an extremely small space. After its development, this technique paved the way for researchers in the field of plant cell biology to explore the elongation capability of tip-growing plant cells, such as pollen tubes, root hairs, and moss protonemata in an extremely small space.
