Decapitation Crosses to Test Pollen Fertility Mutations for Defects in Stigma-Style Penetration

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 stigma-style (stigma decapitation) to test whether transferring pollen directly onto an exposed ovary surface significantly improves the transmission efficiency (TE) of a mutant allele. To illustrate this technique, we applied stigma decapitation to investigate a loss-of-function mutation in Arabidopsis OFT1, a gene encoding a putative o-fucosyl transferase functioning in the secretory pathway. oft1-3 mutant pollen showed a significant decrease in transmission efficiency compared to wild type. Decapitation crosses (described here) indicated that the removal of the stigma-style barrier alleviated the transmission deficiency from 858-fold to a 2.6-fold, providing evidence that most, but not all, oft1 pollen deficiencies can be attributed to a reduced ability to penetrate through the stigma-style barrier. This method outlines a genetic strategy to quantify a mutation's impact on the ability of pollen to navigate through the stigma-style barrier on its journey to the ovule.