In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

The overall goal of this procedure is to enable live-cell imaging of Arabidopsis zygotes and embryos for visualizing the dynamics of their embryogenesis. This method can help answer key questions in the plant development field about how zygote assymetry and embryo morphology are generated. The main advantage of this technique is that we can trace the living dynamics of zygote polarization and embryo patterning.

Visual demonstration of this method is critical as preparation with micropillar device and the ovules are difficult to explain by text only. To prepare PDMS micropillar array device, first cut the device so that it fits into a 35 millimeter glass-bottom dish and sterilize the top and bottom sides of the device with one 15-minute UV light exposure per side. When all of the sides have been sterilized, transfer the PDMS device into a new 35 millimeter culture dish and add five to seven milliliters of fresh N5T medium to the dish, until the device is completely submerged.

Then place the dish into a vacuum chamber and reduce the pressure leaving the device in the vacuum until all of the air in the device had been replaced with medium. When the device has been degassed, use square tip tweezers to dab the device on a piece of paper towel to remove any excess medium from the sides and bottom. Then place the device onto a 76 by 26 millimeter slide glass and cover it with a 35 millimeter culture dish lid to keep the device hydrated.

Before beginning the dissection, sterilize a 0.40 millimeter gauge needle and fine tweezers with 70 percent ethanol. Next, check the inflorescence of the plant to allow selection of the appropriate siliques for the experiment. The approximately five millimeter siliques contain zygotes and the eight to 10 millimeter siliques include young globular embryos.

Using tweezers, transfer three to four siliques of interest onto a piece of double-sided tape on a 76 by 26 millimeter slide glass. Then transfer the slide under a stereo microscope and use the needle and tweezers to open the ovary walls. Transfer the open siliques onto the micropillar array of the PDMS device and release the ovules into the medium.

Then place and 18 by 18 millimeter cover glass onto the device to push the ovules into the spaces of the micropillar array. When the ovules are in place, use tweezers or fingers to pull the cover glass horizontally off of the device to remove any excess medium, and place the device upside down into a new 35 millimeter glass-bottom dish. Use the tip of the square tweezers to slightly press the device into the bottom of the dish and pour fresh N5T medium into the dish until the device is completely submerged.

Then, seal the dish with parafilm. For time lapse imaging of the ovules, transfer the dish onto the stage of an inverted microscope and use the marker fluorescence to select the ovules for imaging. When the optimal ovules have been located, begin live-imaging the samples according to the manufacturer's recommendations.

This ovule cultivation system allows tracing of the living dynamics of zygote polarization and embryo patterning. For example, here at the accumulation of microtubules as a transverse ring, followed by their formation into a spindle, to be observed. The embryonic cells then quarternately divide to form a radially symmetrical globular shape.

Treatment of the cultured ovules with an actin polymerization inhibitor, however, inhibits polar nuclear migration, resulting in a more symmetric division of the zygote and suggesting a role for actin filaments in zygote polarization. Once mastered, this technique can be completed in two hours if it is performed properly. Following this procedure laser disruption can be performed to answer additional questions about the role of cell to cell communication in embryo patterning.

After watching this video, you should have a good understanding of how to visualize tight events during embryogenesis.