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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.<\/jats:p>","DOI":"10.1093\/jxb\/eraf322","type":"journal-article","created":{"date-parts":[[2025,7,16]],"date-time":"2025-07-16T14:29:31Z","timestamp":1752676171000},"page":"6347-6354","source":"Crossref","is-referenced-by-count":1,"title":["High-throughput isolation of male gametophyte cells of\n                    <i>Solanum lycopersicum<\/i>\n                    var. Micro-Tom by fluorescence-activated cell sorting"],"prefix":"10.1093","volume":"76","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9296-0070","authenticated-orcid":false,"given":"Mar\u00eda","family":"Flores-Tornero","sequence":"first","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa , Av. da Rep\u00fablica, Oeiras 2780-157 ,","place":["Portugal"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4177-3398","authenticated-orcid":false,"given":"Helena","family":"Sapeta","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa , Av. da Rep\u00fablica, Oeiras 2780-157 ,","place":["Portugal"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2589-6216","authenticated-orcid":false,"given":"Maria","family":"Lobato-G\u00f3mez","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), Universidad Polit\u00e9cnica de Valencia (UPV) , Valencia ,","place":["Spain"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5273-2903","authenticated-orcid":false,"given":"Beatriz","family":"Teixeira","sequence":"additional","affiliation":[{"name":"Instituto Gulbenkian de Ci\u00eancia , Rua da Quinta Grande 6, Oeiras 2780-156 ,","place":["Portugal"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0327-0917","authenticated-orcid":false,"given":"Marta","family":"Monteiro","sequence":"additional","affiliation":[{"name":"Instituto Gulbenkian de Ci\u00eancia , Rua da Quinta Grande 6, Oeiras 2780-156 ,","place":["Portugal"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4266-9581","authenticated-orcid":false,"given":"Antonio","family":"Granell","sequence":"additional","affiliation":[{"name":"Instituto de Biolog\u00eda Molecular y Celular de Plantas (IBMCP), Consejo Superior de Investigaciones Cient\u00edficas (CSIC), Universidad Polit\u00e9cnica de Valencia (UPV) , Valencia ,","place":["Spain"]}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6845-6122","authenticated-orcid":false,"given":"J\u00f6rg D","family":"Becker","sequence":"additional","affiliation":[{"name":"Instituto de Tecnologia Qu\u00edmica e Biol\u00f3gica, Universidade Nova de Lisboa , Av. da Rep\u00fablica, Oeiras 2780-157 ,","place":["Portugal"]}]}],"member":"286","published-online":{"date-parts":[[2025,7,16]]},"reference":[{"key":"2025112512082070800_eraf322-B1","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1387\/ijdb.052019lb","article-title":"The making of gametes in higher plants","volume":"49","author":"Boavida","year":"2005","journal-title":"International Journal of Developmental Biology"},{"key":"2025112512082070800_eraf322-B2","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/1746-4811-8-44","article-title":"FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei","volume":"8","author":"Borges","year":"2012","journal-title":"Plant Methods"},{"key":"2025112512082070800_eraf322-B3","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.1104\/pp.108.125229","article-title":"Comparative transcriptomics of Arabidopsis sperm cells","volume":"148","author":"Borges","year":"2008","journal-title":"Plant Physiology"},{"key":"2025112512082070800_eraf322-B4","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1017\/S096719940000294X","article-title":"Isolation of viable sperm cells from tobacco (Nicotiana tabacum)","volume":"4","author":"Cao","year":"1996","journal-title":"Zygote"},{"key":"2025112512082070800_eraf322-B5","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1046\/j.1365-313X.2003.01761.x","article-title":"Sperm cells of Zea mays have a complex complement of mRNAs","volume":"34","author":"Engel","year":"2003","journal-title":"The Plant Journal"},{"key":"2025112512082070800_eraf322-B6","doi-asserted-by":"crossref","first-page":"3449","DOI":"10.1093\/jxb\/erad117","article-title":"Fifty years of sperm cell isolations: from structural to omic studies","volume":"74","author":"Flores-Tornero","year":"2023","journal-title":"Journal of Experimental Botany"},{"key":"2025112512082070800_eraf322-B7","unstructured":"Sapeta H, Flores-Tornero M, Becker JD. \u00a02025. 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