{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T10:11:33Z","timestamp":1788862293428,"version":"build-2803163510"},"reference-count":20,"publisher":"MDPI AG","issue":"18","license":[{"start":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T00:00:00Z","timestamp":1788825600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100002888","name":"R&D Program of Beijing Municipal Education Commission","doi-asserted-by":"publisher","award":["KM202410020004"],"award-info":[{"award-number":["KM202410020004"]}],"id":[{"id":"10.13039\/501100002888","id-type":"DOI","asserted-by":"publisher"}]},{"award":["KM202410020004"],"award-info":[{"award-number":["KM202410020004"]}],"id":[{"id":"https:\/\/ror.org\/04bpn6s66","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003352","name":"Improving Scientific Research and Innovation Capacity of Young Teachers in Beijing University of Agriculture","doi-asserted-by":"publisher","award":["QJKC-20230048"],"award-info":[{"award-number":["QJKC-20230048"]}],"id":[{"id":"10.13039\/501100003352","id-type":"DOI","asserted-by":"publisher"}]},{"award":["QJKC-20230048"],"award-info":[{"award-number":["QJKC-20230048"]}],"id":[{"id":"https:\/\/ror.org\/03t9adt98","id-type":"ROR","asserted-by":"publisher"}]},{"name":"the Fundamental Research Funds for the Central Universities","award":["BFUKF202605"],"award-info":[{"award-number":["BFUKF202605"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Plants"],"abstract":"<jats:p>Stable transformation in rose is slow and genotype-dependent, motivating rapid cellular assays for candidate-gene testing. We systematically evaluated mesophyll protoplast isolation and PEG-mediated transient expression in Rosa chinensis \u2018Old Blush\u2019. Sequential experiments compared enzyme preparations, digestion time, D-mannitol concentration, cultivar, and donor cultivation regime. Under the tested conditions, Yakult preparations containing 3.0% Cellulase R-10 and 2.0% Macerozyme R-10, 16 h digestion, and 0.4 mol\/L D-mannitol yielded 4.641 \u00b1 0.102 \u00d7 106 protoplasts\/g fresh weight with 94.26 \u00b1 0.15% FDA viability. Recovery varied significantly among donor regimes and between the two cultivars tested. In a 27-combination factorial experiment with three plants as blocks, 25% PEG 4000 working solution (12.5% nominal final concentration), 8 min transfection, and 20 h dark incubation gave the highest observed mGFP6-positive fraction (7.14 \u00b1 0.22%). This efficiency supports imaging of individually transfected cells under the tested conditions, but broader population-level or high-throughput applications require further validation. Protoplast division, plant regeneration, stable transformation, cross-date reproducibility, and transfer to other genotypes were not assessed.<\/jats:p>","DOI":"10.3390\/plants15182739","type":"journal-article","created":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T09:02:24Z","timestamp":1788858144000},"page":"2739","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Systematic Evaluation of Mesophyll Protoplast Isolation and PEG-Mediated Transient Expression in Rosa chinensis \u2018Old Blush\u2019"],"prefix":"10.3390","volume":"15","author":[{"given":"Yiyang","family":"Zhao","sequence":"first","affiliation":[{"name":"College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Yin","family":"Zhong","sequence":"additional","affiliation":[{"name":"College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"given":"Fangmei","family":"Hou","sequence":"additional","affiliation":[{"name":"College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4887-8977","authenticated-orcid":false,"given":"Yousry A.","family":"El-Kassaby","sequence":"additional","affiliation":[{"name":"Department of Forest and Conservation Sciences, Faculty of Forestry, Forest Sciences Centre, University of British Columbia, Vancouver, BC V6T 1Z4, Canada"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8849-2366","authenticated-orcid":false,"given":"Deqiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"College of Landscape Architecture, Beijing University of Agriculture, Beijing 102206, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,8]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"772","DOI":"10.1038\/s41588-018-0110-3","article-title":"The Rosa genome provides new insights into the domestication of modern roses","volume":"50","author":"Raymond","year":"2018","journal-title":"Nat. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1007\/s11240-009-9621-z","article-title":"Somatic embryogenesis and transformation of the diploid Rosa chinensis cv Old Blush","volume":"100","author":"Vergne","year":"2010","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"962","DOI":"10.1038\/187962a0","article-title":"A method for the isolation of plant protoplasts and vacuoles","volume":"187","author":"Cocking","year":"1960","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1565","DOI":"10.1038\/nprot.2007.199","article-title":"Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis","volume":"2","author":"Yoo","year":"2007","journal-title":"Nat. Protoc."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3821","DOI":"10.1093\/jxb\/erad172","article-title":"Plant protoplasts in the age of synthetic biology","volume":"74","author":"Burgess","year":"2023","journal-title":"J. Exp. Bot."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"110043","DOI":"10.1016\/j.scienta.2021.110043","article-title":"Protoplast technology in ornamental plants: Current progress and potential applications on genetic improvement","volume":"283","author":"Naing","year":"2021","journal-title":"Sci. Hortic."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1023\/A:1005910002625","article-title":"Isolation and culture of mesophyll protoplasts from Rosa hybrida","volume":"50","author":"Marchant","year":"1997","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1079\/ejhs.2001\/5628","article-title":"Factors affecting protoplast isolation and plant regeneration in Rosa spp.","volume":"66","author":"Schum","year":"2001","journal-title":"Eur. J. Hortic. Sci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1023\/A:1022693605436","article-title":"Plant regeneration of rose (Rosa hybrida) from embryogenic cell-derived protoplasts","volume":"73","author":"Kim","year":"2003","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"189","DOI":"10.3109\/10520297209116483","article-title":"The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells","volume":"47","author":"Widholm","year":"1972","journal-title":"Stain Technol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1016\/0304-4211(84)90015-4","article-title":"The measurement of isotonicity and maintenance of osmotic balance in plant protoplast manipulations","volume":"33","author":"Smith","year":"1984","journal-title":"Plant Sci. Lett."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Sivanandhan, G., Bae, S., Sung, C., Choi, S.R., Lee, G.J., and Lim, Y.P. (2021). Optimization of protoplast isolation from leaf mesophylls of Chinese cabbage (Brassica rapa ssp. pekinensis) and subsequent transfection with a binary vector. Plants, 10.","DOI":"10.3390\/plants10122636"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"113360","DOI":"10.1016\/j.indcrop.2021.113360","article-title":"Protoplast isolation, transient transformation, and flow-cytometric analysis of reporter-gene activation in Cannabis sativa L.","volume":"164","author":"Beard","year":"2021","journal-title":"Ind. Crops Prod."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1007\/s11816-022-00786-6","article-title":"Establishment of efficient Cannabis (Cannabis sativa L.) protoplast isolation and transient expression condition","volume":"16","author":"Kim","year":"2022","journal-title":"Plant Biotechnol. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"107885","DOI":"10.1016\/j.plaphy.2023.107885","article-title":"Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia oleifera","volume":"201","author":"Lin","year":"2023","journal-title":"Plant Physiol. Biochem."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Shao, Y., Mu, D., Pan, L., Wilson, I.W., Zheng, Y., Zhu, L., Lu, Z., Wan, L., Fu, J., and Wei, S. (2023). Optimization of isolation and transformation of protoplasts from Uncaria rhynchophylla and its application to transient gene expression analysis. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24043633"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Guo, J., Morrell-Falvey, J.L., Labb\u00e9, J.L., Muchero, W., Kalluri, U.C., Tuskan, G.A., and Chen, J.G. (2012). Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0044908"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Huo, A., Chen, Z., Wang, P., Yang, L., Wang, G., Wang, D., Liao, S., Cheng, T., Chen, J., and Shi, J. (2017). Establishment of transient gene expression systems in protoplasts from Liriodendron hybrid mesophyll cells. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0172475"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"109684","DOI":"10.1016\/j.scienta.2020.109684","article-title":"Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei","volume":"274","author":"Ma","year":"2020","journal-title":"Sci. Hortic."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1104\/pp.103.027979","article-title":"A Gateway cloning vector set for high-throughput functional analysis of genes in planta","volume":"133","author":"Curtis","year":"2003","journal-title":"Plant Physiol."}],"container-title":["Plants"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/18\/2739\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T09:23:17Z","timestamp":1788859397000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2223-7747\/15\/18\/2739"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,8]]},"references-count":20,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["plants15182739"],"URL":"https:\/\/doi.org\/10.3390\/plants15182739","relation":{},"ISSN":["2223-7747"],"issn-type":[{"value":"2223-7747","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,8]]}}}