Rafael Narancio, Daniel Isenegger, Rafael Reyno, German Spangenberg, Marco Dalla-Rizza
{"title":"Tissue culture and high-efficiency transformation in an apomictic initial variety of Paspalum notatum Flüggé","authors":"Rafael Narancio, Daniel Isenegger, Rafael Reyno, German Spangenberg, Marco Dalla-Rizza","doi":"10.1007/s11627-024-10434-z","DOIUrl":null,"url":null,"abstract":"<p><i>Paspalum notatum</i> Flüggé is a subtropical grass native from the Americas and one of the main components of the subtropical South American grasslands. Although <i>P. notatum</i> exhibits a high forage productivity and persistence, some limitations are observed in the species, such as forage quality due to high lignin content. The development of efficient tissue culture and plant transformation protocols in cultivars for forage production are necessary to embrace novel genetic improvement through transgenesis and genome editing. Efficient regeneration and transformation systems for many plant species can be hampered by genotype dependency, which can only be determined through extensive laboratory testing and evaluation. In this study, various factors extracted from protocols have been evaluated to establish efficient somatic embryogenesis, plant regeneration, and genetic transformation in apomictic initial variety INIA Sepé. Optimal somatic embryogenesis and regeneration from explant sources, such as mature seeds and meristematic tissue derived from <i>in vitro</i> propagated tillers, ranged between 79 and 88%. Microprojectile bombardment of two plasmids encoding a constitutively regulated reporter gene and <i>nptII</i> selectable marker expression constructs showed an average transformation efficiency of 10.3% from meristematic tissue as an explant source. Furthermore, the constitutive promoters, <i>35S</i> and <i>ZmUbi</i>, were tested in <i>GFP</i> transient expression assays to evaluate optimal cassettes for transgene(s) expression. Translation of a wheat codon–optimized <i>Cas9</i> sequence using <i>Cas9:2A:tuGFP</i> fusion protein was also tested in protoplasts. The information generated on promoter activity and Cas9 translation, and the establishment of an efficient transformation system, provided useful tools for future work in genetic engineering and genome editing in <i>P. notatum</i> cv. INIA Sepé.</p>","PeriodicalId":13293,"journal":{"name":"In Vitro Cellular & Developmental Biology - Plant","volume":"22 1","pages":""},"PeriodicalIF":2.2000,"publicationDate":"2024-06-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"In Vitro Cellular & Developmental Biology - Plant","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s11627-024-10434-z","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Paspalum notatum Flüggé is a subtropical grass native from the Americas and one of the main components of the subtropical South American grasslands. Although P. notatum exhibits a high forage productivity and persistence, some limitations are observed in the species, such as forage quality due to high lignin content. The development of efficient tissue culture and plant transformation protocols in cultivars for forage production are necessary to embrace novel genetic improvement through transgenesis and genome editing. Efficient regeneration and transformation systems for many plant species can be hampered by genotype dependency, which can only be determined through extensive laboratory testing and evaluation. In this study, various factors extracted from protocols have been evaluated to establish efficient somatic embryogenesis, plant regeneration, and genetic transformation in apomictic initial variety INIA Sepé. Optimal somatic embryogenesis and regeneration from explant sources, such as mature seeds and meristematic tissue derived from in vitro propagated tillers, ranged between 79 and 88%. Microprojectile bombardment of two plasmids encoding a constitutively regulated reporter gene and nptII selectable marker expression constructs showed an average transformation efficiency of 10.3% from meristematic tissue as an explant source. Furthermore, the constitutive promoters, 35S and ZmUbi, were tested in GFP transient expression assays to evaluate optimal cassettes for transgene(s) expression. Translation of a wheat codon–optimized Cas9 sequence using Cas9:2A:tuGFP fusion protein was also tested in protoplasts. The information generated on promoter activity and Cas9 translation, and the establishment of an efficient transformation system, provided useful tools for future work in genetic engineering and genome editing in P. notatum cv. INIA Sepé.
期刊介绍:
Founded in 1965, In Vitro Cellular & Developmental Biology - Plant is the only journal devoted solely to worldwide coverage of in vitro biology in plants. Its high-caliber original research and reviews make it required reading for anyone who needs comprehensive coverage of the latest developments and state-of-the-art research in plant cell and tissue culture and biotechnology from around the world.