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The overall duration for this protocol is approximately 8 hours spanning 4 days (an average of 2 h/day per two workers) excluding microscopy and LC-MS/MS analysis.During sexual reproduction in flowering plants, pollen grains germinate on the stigma surface and grow through the stigma-style tissue to reach the ovary and deliver sperm cells for fertilization. Here, we outline a method to test whether a pollen fertility mutation specifically disrupts pollen penetration through the stigma-style barrier. This method surgically removes the s