Optimised expression cassettes of hpt marker gene for biolistic transformation of Miscanthus sacchariflorus

Miscanthus species, including M. sacchariflorus, are important bioenergy crops, used also as industry feedstocks and in environmental projects. M. sacchariflorus is also utilised as a donor in interspecific crossings to generate new forms of M. × giganteus. Research on Miscanthus species and their application potential may be considerably extended using methods of genetic modification. However, to date there are scare reports on miscanthus transformation. To improve transformation efficiency, new expression cassettes of a selectable marker gene were constructed and used for biolistic transformation of M. sacchariflorus. The sequence of the bacterial hpt gene encoding hygromycin phosphotransferase in the initial pCAMBIA vector was optimised according to codon usage characteristic of Zea mays, which is closely related to Miscanthus sp., and placed under control of 35S CaMV or the Zea mays ubiquitin promoter, resulting in the pCA35Sohpt and pCAUBQohpt vectors. Transformation rate using optimised vectors significantly increased in comparison to pCAMBIA, and reached 4.2–5.0% vs 2.5%. In respective transgenic plants, significantly higher transcription of optimised hpt was determined while the activity of the reporter GUS enzyme was similar. These results constitute basis for specific miscanthus transformation in a variety of research and practical applications. 1.The hpt marker gene was optimised for transformation of Miscanthus sacchariflorus.2.Codon usage in the optimised gene (ohpt) was altered specifically for grasses.3.Transformation efficiency with ohpt 4.2–5% vs. hpt 2.5% significantly increased.4.Promoter 35S or ubiquitin (Zea mays), did not impact activity of the ohpt gene.5.Optimised hpt expression cassettes may be used in research and practical projects. Vectors carrying optimised expression cassettes of the hpt marker gene significantly increased rates of Miscanthus sacchariflorus biolistic transformation and may be applied in research and practical projects.