{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T13:30:20Z","timestamp":1789047020319,"version":"build-2803163510"},"reference-count":8,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"},{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32325035"],"award-info":[{"award-number":["32325035"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Plant Biotechnology Journal"],"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Alfalfa (\n                    <jats:italic>Medicago sativa<\/jats:italic>\n                    ) is the most widely cultivated forage crop worldwide, owing to its high protein content, balanced amino acid profile, and abundance of vitamins and minerals. However, weed infestation severely threatens alfalfa yield and quality. The lack of herbicide\u2011resistant cultivars, combined with the diversity of weed species, has made herbicide tolerance a key breeding objective. To address this challenge, we developed a highly optimized prime editing system and, for the first time, achieved prime editing in alfalfa by enhancing the expression efficiency of its components. Specifically, we introduced mutations in three target genes\n                    <jats:italic>MsALS1<\/jats:italic>\n                    ,\n                    <jats:italic>MsALS2<\/jats:italic>\n                    and\n                    <jats:italic>MsACC1<\/jats:italic>\n                    to confer herbicide resistance: mutations in\n                    <jats:italic>MsALS1<\/jats:italic>\n                    and\n                    <jats:italic>MsALS2<\/jats:italic>\n                    were designed to confer resistance to ALS\u2011inhibiting herbicides (based on evidence from their rice homologs), while those in\n                    <jats:italic>MsACC1<\/jats:italic>\n                    were targeted to confer resistance to aryloxyphenoxypropionate (APP) herbicides. Using the Csy4 nuclease system, we performed simultaneous prime editing of all three genes, ultimately generating alfalfa lines resistant to nicosulfuron and haloxyfop\u2011P\u2011methyl. This study represents the first successful multi\u2011gene prime editing in alfalfa, providing a novel tool and a valuable approach for alfalfa molecular breeding.\n                  <\/jats:p>","DOI":"10.1111\/pbi.70752","type":"journal-article","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T12:59:59Z","timestamp":1789045199000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Generation of Herbicide\u2010Resistant Alfalfa Germplasm via Prime Editing"],"prefix":"10.1111","author":[{"given":"Shihao","family":"Li","sequence":"first","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuzhi","family":"Xia","sequence":"additional","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"},{"name":"Food Crops Research Institute, Yunnan Academy of Agricultural Sciences  Kunming China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhengzheng","family":"Wei","sequence":"additional","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Na","family":"Wang","sequence":"additional","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4717-8335","authenticated-orcid":false,"given":"Hao","family":"Lin","sequence":"additional","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lifang","family":"Niu","sequence":"additional","affiliation":[{"name":"Biotechnology Research Institute Chinese Academy of Agricultural Sciences  Beijing China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,10]]},"reference":[{"key":"e_1_2_7_2_1","doi-asserted-by":"crossref","first-page":"721","DOI":"10.1111\/pbi.13239","article-title":"Generation of a Multiplex Mutagenesis Population via Pooled CRISPR\u2010Cas9 in Soya Bean","volume":"18","author":"Bai M.","year":"2020","journal-title":"Plant Biotechnology Journal"},{"key":"e_1_2_7_3_1","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1007\/s00299-021-02827-w","article-title":"Generation of a Multi\u2010Herbicide\u2010Tolerant Alfalfa by Using Base Editing","volume":"41","author":"Bottero E.","year":"2022","journal-title":"Plant Cell Reports"},{"key":"e_1_2_7_4_1","doi-asserted-by":"crossref","first-page":"750","DOI":"10.1016\/j.cj.2020.11.003","article-title":"Trp548Met Mutation of Acetolactate Synthase in Rice Confers Resistance to a Broad Spectrum of ALS\u2010Inhibiting Herbicides","volume":"9","author":"Chen L.","year":"2021","journal-title":"Crop Journal"},{"key":"e_1_2_7_5_1","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1038\/s41576-024-00720-2","article-title":"Targeted Genome\u2010Modification Tools and Their Advanced Applications in Crop Breeding","volume":"25","author":"Li B.","year":"2024","journal-title":"Nature Reviews Genetics"},{"key":"e_1_2_7_6_1","doi-asserted-by":"crossref","first-page":"875","DOI":"10.1038\/s41587-019-0393-7","article-title":"Targeted, Random Mutagenesis of Plant Genes With Dual Cytosine and Adenine Base Editors","volume":"38","author":"Li C.","year":"2020","journal-title":"Nature Biotechnology"},{"key":"e_1_2_7_7_1","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1111\/pbi.13497","article-title":"Precise Genome Modification in Tomato Using an Improved Prime Editing System","volume":"19","author":"Lu Y.","year":"2021","journal-title":"Plant Biotechnology Journal"},{"key":"e_1_2_7_8_1","doi-asserted-by":"crossref","first-page":"156","DOI":"10.1186\/s13059-023-02990-1","article-title":"Efficient and Versatile Multiplex Prime Editing in Hexaploid Wheat","volume":"24","author":"Ni P.","year":"2023","journal-title":"Genome Biology"},{"key":"e_1_2_7_9_1","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.molp.2024.12.015","article-title":"Medicago2035: Genomes, Functional Genomics, and Molecular Breeding","volume":"18","author":"Ye Q.","year":"2025","journal-title":"Molecular Plant"}],"container-title":["Plant Biotechnology Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/pbi.70752","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/pbi.70752","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/pbi.70752","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T13:00:00Z","timestamp":1789045200000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/pbi.70752"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,10]]},"references-count":8,"alternative-id":["10.1111\/pbi.70752"],"URL":"https:\/\/doi.org\/10.1111\/pbi.70752","archive":["Portico"],"relation":{},"ISSN":["1467-7644","1467-7652"],"issn-type":[{"value":"1467-7644","type":"print"},{"value":"1467-7652","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,10]]},"assertion":[{"value":"2026-04-29","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-08-19","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-09-10","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"pbi.70752"}}