High-throughput isolation of male gametophyte cells of <i>Solanum lycopersicum</i> var. Micro-Tom by fluorescence-activated cell sorting

Efficient isolation of male gametes has enabled unprecedented advances in omics research, crucial for elucidating the molecular mechanisms governing male gametogenesis and fertilization. In this study, we developed a method for isolating generative and sperm cells from the economically important crop Solanum lycopersicum (tomato). A double fluorescent marker line was generated in the tomato variety Micro-Tom, employing mTurquoise and mScarlet-I fluorescent proteins under the control of promoters exhibiting preferential activity in generative and sperm cells, respectively. Then we developed a protocol that combines male gamete release from pollen grains and pollen tubes of this double fluorescent marker line and SYTOX Red live/dead cell stain to obtain viable cells by fluorescence-activated cell sorting. This allows the isolation of generative cells from mature pollen grains, and of sperm cells from pollen tubes after semi-in vivo growth, in both high quantity and purity. Additionally, an unexpected mScarlet-I signal in the vegetative nucleus, that persists until the sperm cells are formed, allows the sorting of vegetative nuclei. We anticipate that our novel double marker line will accelerate research into tomato male gametogenesis, thereby enhancing efforts to improve the resilience of fertilization processes to climate change.