{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T04:17:21Z","timestamp":1791260241199,"version":"4.1.0"},"reference-count":65,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T00:00:00Z","timestamp":1784160000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Plant Sci."],"abstract":"<jats:p>\n                    Traditional genetic transformation approaches relying on\n                    <jats:italic>Agrobacterium tumefaciens<\/jats:italic>\n                    for the delivery of CRISPR\/Cas9 reagents usually provide plants that stably integrate the gene construct in their genome. To meet the EU commission\u2019s proposal for a new legislation on plants obtained by new genomic techniques (NGTs), it is important to develop new protocols that produce transgene-free genome edited plants (NGT category 1). Protoplasts are a promising platform, since delivery of CRISPR\/Cas9 reagents as ribonucleoproteins (RNPs) is effective in cells lacking their wall. This allows genetic modifications from a transient application, leaving no traces in the recipient genome apart from the desired targeted mutations. With the aim of implementing transgene-free editing of eggplant (\n                    <jats:italic>Solanum melongena<\/jats:italic>\n                    L.), we adapted and improved a protocol previously established in potato and tomato for the isolation of protoplasts from cotyledonary leaves and subsequent CRISPR\/Cas9 reagents delivery. Isolated protoplasts were subjected to\n                    <jats:italic>in vitro<\/jats:italic>\n                    culture and regeneration, and the first shoots were regenerated from calli approximately 4\u20135 months after isolation. Alongside, two transfection protocols were tested for the delivery of RNPs into eggplant protoplasts, one using polyethylene glycol (PEG) in two concentrations (25% and 40%) and one exploiting two formulations of lipofectamines (Lipofectamine CRISPRMAX\u2122 and Lipofectamine\u2122 3000), all targeting\n                    <jats:italic>SmChl_H<\/jats:italic>\n                    gene, whose inactivation can cause a chlorotic phenotype. Efficient callus regeneration from transfected protoplasts was obtained and the editing efficiency (calculated as the percentage of edited calli on the total of calli that underwent sequencing) was evaluated. 25% PEG treatment provided the highest editing efficiency, and fully edited biallelic calli were retrieved, showing the expected chlorotic phenotype. Even if the efficiency of\n                    <jats:italic>in vitro<\/jats:italic>\n                    regeneration of plants from calli still needs improvement, edited plants were regenerated from protoplasts, representing the first report of RNP mediated genome editing in eggplant protoplasts.\n                  <\/jats:p>","DOI":"10.3389\/fpls.2026.1846175","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T05:37:25Z","timestamp":1784180245000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Tiny but powerful: protoplast isolation in Solanum melongena L. and RNPs mediated genome editing"],"prefix":"10.3389","volume":"17","author":[{"given":"Martina","family":"Ferrero","sequence":"first","affiliation":[{"name":"Department of Agricultural, Forest and Food Sciences (DISAFA), University of Torino","place":["Grugliasco, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mat\u00edas Nicol\u00e1s","family":"Gonz\u00e1lez","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Swedish University of Agricultural Sciences","place":["Lomma, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Irene","family":"Perrone","sequence":"additional","affiliation":[{"name":"Institute for Sustainable Plant Protection, National Research Council (CNR-IPSP)","place":["Torino, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Per","family":"Hofvander","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Swedish University of Agricultural Sciences","place":["Lomma, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariette","family":"Andersson","sequence":"additional","affiliation":[{"name":"Department of Plant Breeding, Swedish University of Agricultural Sciences","place":["Lomma, Sweden"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrea","family":"Moglia","sequence":"additional","affiliation":[{"name":"Department of Agricultural, Forest and Food Sciences (DISAFA), University of Torino","place":["Grugliasco, Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2026,7,16]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.3390\/horticulturae7040078","article-title":"Genetic engineering of eggplant (Solanum melongena L.): Progress, controversy and potential","volume":"7","author":"Alam","year":"2021","journal-title":"Horticulturae"},{"key":"B2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1111\/pbr.70023","article-title":"Current trends in CRISPR\u2010Cas system based genome editing in tomato (Solanum lycopersicum)","author":"Anand","year":"2025","journal-title":"Plant Breed."},{"key":"B3","doi-asserted-by":"publisher","first-page":"378","DOI":"10.1111\/ppl.12731","article-title":"Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery","volume":"164","author":"Andersson","year":"2018","journal-title":"Physiol. 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