For confocal microscopy of mature pollen grains, pollen was placed into a 5-μl drop of auramine O solution (0.001%; diluted in water from the 0.1% stock prepared in 50 mM Tris-HCl), allowed to hydrate, covered with a #1.5 coverslip, and sealed with nail polish. Pollen then was visualized with a 100x oil-immersion objective (NA = 1.4) on Olympus Fluoview1000 or Nikon A1+ confocal microscopes using FITC excitation/emission settings and 3x confocal zoom. To count apertures, images from the front and back view of the pollen grains were taken. If some apertures were present on sides of a pollen grain not directly visible by focusing on the front and on the back, then z-stacks were taken (step size = 300 nm) and 3D images were reconstructed using NIS Elements software v.4.20 (Nikon) and used for aperture counting. To determine the size of pollen grains in plants of different ploidy and genotypes, we used the surface areas of “front-view” pollen images (the surface closest to the microscope objective) as a proxy for cell size and measured these areas using NIS Elements and ImageJ.
For imaging tetrads and dyads, anthers were dissected out of stage-9 flower buds [56 (link)] and placed into the Vectashield anti-fade solution (Vector Labs, Burlingame, CA) supplemented with membrane stain CellMask Deep Red (Molecular Probes, Eugene, OR) (5 μg/ml) and calcofluor white (0.02%). Tetrads were released by covering anthers with a coverslip and applying gentle pressure on a coverslip. For imaging apertures at the tetrad and dyad stage, Vectashield was supplemented with DAPI (1 μg/ml). Tetrads and dyads were imaged on confocal Nikon A1+ with 100x oil-immersion objective (NA = 1.4) and 5x confocal zoom. YFP was excited with 514-nm laser and emission was collected at 522–555 nm, DAPI and calcofluor white were excited with 405-nm laser and collected at 424–475 nm, and CellMask Deep Red dye was excited with 640-nm laser and collected at 663–738 nm. Z-stacks of tetrads were obtained with a step size of 300 or 500 nm and 3D-reconstructed using NIS Elements v.4.20 (Nikon) or IMARIS (Bitplane) software.
To measure levels of YFP fluorescence, the INP1pr:INP1-YFP and DMC1pr:INP1-YFP tetrads were prepared simultaneously and imaged on the same day under identical acquisition conditions on confocal Nikon A1+. The mean YFP signal intensity of diffuse cytoplasmic INP1-YFP fluorescence was determined with the help of NIS Elements v.4.20 (Nikon) for one microspore per tetrad (n≥15 tetrads), using a single optical section with diffused fluorescence for each microspore. The optical sections containing assembled puncta of INP1-YFP at the periphery of microspores were excluded from the analysis.
To determine karyotypes of elongated cells in anther filaments [57 (link)], flowers were fixed in a 3:1 mixture of ethanol: acetic acid for 5 min and neutralized in 50 mM potassium phosphate buffer, pH 7.0. Anther filaments were dissected and placed in a drop of DAPI (10 μg/ml) in 80% glycerol. Confocal z-stacks of elongated cells were obtained and chromosomes counted using maximum intensity projections. Microspore mother cells undergoing meiosis were prepared and visualized as described [58 (link)].
For imaging tetrads and dyads, anthers were dissected out of stage-9 flower buds [56 (link)] and placed into the Vectashield anti-fade solution (Vector Labs, Burlingame, CA) supplemented with membrane stain CellMask Deep Red (Molecular Probes, Eugene, OR) (5 μg/ml) and calcofluor white (0.02%). Tetrads were released by covering anthers with a coverslip and applying gentle pressure on a coverslip. For imaging apertures at the tetrad and dyad stage, Vectashield was supplemented with DAPI (1 μg/ml). Tetrads and dyads were imaged on confocal Nikon A1+ with 100x oil-immersion objective (NA = 1.4) and 5x confocal zoom. YFP was excited with 514-nm laser and emission was collected at 522–555 nm, DAPI and calcofluor white were excited with 405-nm laser and collected at 424–475 nm, and CellMask Deep Red dye was excited with 640-nm laser and collected at 663–738 nm. Z-stacks of tetrads were obtained with a step size of 300 or 500 nm and 3D-reconstructed using NIS Elements v.4.20 (Nikon) or IMARIS (Bitplane) software.
To measure levels of YFP fluorescence, the INP1pr:INP1-YFP and DMC1pr:INP1-YFP tetrads were prepared simultaneously and imaged on the same day under identical acquisition conditions on confocal Nikon A1+. The mean YFP signal intensity of diffuse cytoplasmic INP1-YFP fluorescence was determined with the help of NIS Elements v.4.20 (Nikon) for one microspore per tetrad (n≥15 tetrads), using a single optical section with diffused fluorescence for each microspore. The optical sections containing assembled puncta of INP1-YFP at the periphery of microspores were excluded from the analysis.
To determine karyotypes of elongated cells in anther filaments [57 (link)], flowers were fixed in a 3:1 mixture of ethanol: acetic acid for 5 min and neutralized in 50 mM potassium phosphate buffer, pH 7.0. Anther filaments were dissected and placed in a drop of DAPI (10 μg/ml) in 80% glycerol. Confocal z-stacks of elongated cells were obtained and chromosomes counted using maximum intensity projections. Microspore mother cells undergoing meiosis were prepared and visualized as described [58 (link)].
Free full text: Click here
