Begin the assay by laying the labeled A.thaliana pollen recipient plant on its side, positioning the emasculated flower on the stage of an inverted microscope to image the stigma. To fix the position of the emasculated recipient flower, immobilize the stem using strips of masking tape. From the pollen donor plant, remove a healthy and freshly-opened flower and place the pollen donor plant under a dissecting microscope.
Gather pollen grains using fine-tipped forceps. Remove excess pollen grains by lightly touching the forceps against the donor petals until the pollen grain's monolayer is formed. Return to the pollen recipient plant and use a low power objective lens to focus the inverted microscope on the stigma.
Holding the forceps along the opening between the arms of the forceps, carefully approach and unpollinated stigmatic papilla cell. Continue approaching the unpollinated stigmatic papilla cell until the suitably positioned pollen grain on the forceps makes light contact with its surface. Once pollen attachment is confirmed, slowly withdraw the forceps.
Orient the pollen grain with its equatorial axis visible and in sharp focus, and immediately switch to a higher power objective lens, like 20x. Capture the first pollen grain image as T-0, and continue capturing images at one-minute intervals for 10 minutes. Adjust the focus to accommodate small movements in the pollen grain or stigma.
Record the room's ambient temperature and relative humidity every two minutes, allowing future comparisons between experimental replicates. Save the images in a lossless format, such as ND2, before repeating the pollination and imaging steps for additional pollen grains to acquire data for control and experimental pollinations. Begin the data measurements for pollen hydration assay using image analysis software.
Use similar parameters of digital zoom and the approach defining the pollen boundary for all measurements in the datasets. Record the semiminor axis values for all time points in the dataset. Once the measurements are complete for a dataset, export the raw semiminor axis values of each individual time point to a spreadsheet.
Present the data in columns per image stack and calculate the percentage semiminor axis change. Repeat the steps and obtain data from at least 15 hydrated pollen grains for each plant line. A few pollen grains may fail to hydrate or may hydrate significantly slower than expected.
They're known as dud grains. Exclude these from the dataset. Calculate the mean values for each time point per plant using the specialized software package GraphPad Prism.
Analyze the hydration data from wild-type and mutant lines at each time point using unpaired T-test and one-way ANOVA to compare the means of the specific time point of interest. To compare the means across all the time points, perform multiple T-tests between wild-type and mutant lines across multiple time points. The pollen hydration time series data for wild-type plants collected on different days showed that the minimum and maximum values for the means between replicates for all time points were within 3%This representative data for wild-type pollinations demonstrated a high degree of consistency for relatively low sample numbers and across different days.
