Preparation of Intact Tissue for Microscopic Analysis of the Endosperm Cell Layer in Developing and Mature Arabidopsis Seeds

One of our research scopes is seed science. We aim to answer the question of how endosperm contribute to seed germination. An in vitro assay called seed growth inhibition assay has revealed physiological importance of the endosperm in seed germination. The Arabidopsis seeds are quite small, and the location of the endosperm cellular layer is beneath the testa, which are the cells. It is challenging to prepare endosperm samples without chemically fixing. Previous analysis of the analysis of the endosperm have used chemically fixed samples, so they were unable to observe the movement of organism proteins. Our protocol enables five cell imaging of the dynamics of endosperm cells. Our protocol can be performed using standard laboratory equipment and enables deeper insight into the intercellular event and molecule mechanisms and the regular endosperm function.

[Narrator] To begin grow Arabidopsis plants on soil or rock wool until the flowering stage. Mark fully opened flowers with colored threads to indicate zero days after flowering. select siliques from 12 to 16 days after flowering for the next procedure. Using fine scissors, cut off the marked developing siliques at the pedicel and collect them in 1.5 milliliter tubes. Prepare a wet filter paper for dissection to prevent desiccation and place the sample under a stereo microscope. Using two forceps, one with thick tips for holding and the other with sharp tips for tearing. Split a valve from the replum. Then using the forceps with tips closed, gently pick up developing seeds from the siliques to avoid damaging them. In employing a 27 gauge injection needle, create a scar approximately 0.1 to 0.2 millimeters in size on the testa and endosperm surrounding the embryo. Hold the seed with forceps without crushing it and make the scar at the junction of the cotyledons and radical. Now, push out the embryo by pinching the seed with forceps, taking care not to crush the seed envelope and maintaining its round shape. Insert the injection needle into the empty seed envelope at the scar and pierce it from the inside out. Next, keep the needle in place and use closed tip forceps to scratch the surface of the Testa. Then cut one side of the seed envelope to allow it to open. Now using sharp tip forceps, open the seed envelope into a sheet. The isolated sample should now be a bilayer sheet composed of endosperm and Testa. Place dry Arabidopsis seeds in a 1.5 milliliter tube and add one milliliter of double distilled water to it. Keep the seeds imbibed for at least 40 minutes at room temperature. Prepare a wet filter paper on the stage of the stereo microscope for dissection and using a 1000 microliter micro pipette, transfer the imbibed seeds onto the wet filter paper. Now place the sample under a stereo microscope. Using an injection needle, make a scar approximately 0.2 millimeters in size on the testa and endosperm surrounding the embryo. Hold the seed with forceps without crushing it, and target the junction between the cotyledon and radical for the scar. Now push out the embryo after pinching the seed with forceps. Avoid crushing the empty seed envelope and preserve its round shape. Using an injection needle, cut both the upper and lower sides of the empty seed envelope to shape it into a cylindrical form. Next, cut the cylindrical seed envelope along its central axis to split it into two pieces. The results should be bilayer sheets made of endosperm and testa. Place the isolated endosperm samples as sheets on a glass slide and mount them in water. Finally, gently place a cover slip over the sample ensuring that the endosperm layer is facing upward toward the cover slip. Use nail polish or grease to seal the edges of the cover slip and prevent desiccation before observing the sample under a microscope. Numerous endosperm cells and their intracellular structures were successfully visualized from seeds harvested at 14 days after flowering using the established preparation protocol. Photo bodies were detected inside the nucleus of endosperm cells, expressing PHYB-GFP under white light, confirming their presence and nuclear localization. Endosperm cells showed reversible photo body formation upon alternating far red and red light exposure confirming PHYB photo reversibility and biological activity up to 4.5 hours after dissection. Fluorescent time-lapse imaging of mitochondria in intact endosperm cells after three hours of seed inhibition was performed and mitochondrial movement was detected. More dynamic mitochondrial motility was observed in endosperm cells after one day of seed inhibition, indicating increasing activity over time.