Robust and reproducible agrobacterium-mediated transformation system of the C4 genetic model species setaria viridis

<i>Setaria viridis</i> (green foxtail) has been identified as a potential experimental model system to genetically and molecularly characterise the C4 monocotyledonous grasses due to its small physical size, short generation time and prolific seed production, together with a sequenced and annotated genome. <i>Setaria viridis</i> is the wild ancestor of the cropping species, foxtail millet <i>(Setaria italica)</i>, with both <i>Setaria</i> species sharing a close evolutionary relationship with the agronomically important species, maize, sorghum, and sugarcane, as well as the bioenergy feedstocks, switchgrass, and <i>Miscanthus</i>. However, an efficient and reproducible transformation protocol is required to further advance the use of <i>S. viridis</i> to study the molecular genetics of C4 monocotyledonous grasses. An efficient and reproducible protocol was established for <i>Agrobacterium tumefaciens</i>-mediated transformation of <i>S. viridis</i> (Accession A10) regenerable callus material derived from mature seeds, a protocol that returned an average transformation efficiency of 6.3%. The efficiency of this protocol was the result of the: (i) use of mature embryo derived callus material; (ii) age of the seed used to induce callus formation; (iii) composition of the callus induction media, including the addition of the ethylene inhibitor, silver nitrate; (iv) use of a co-cultivation approach, and; (v) concentration of the selective agent. Our protocol furthers the use of <i>S. viridis</i> as an experimental model system to study the molecular genetics of C4 monocotyledonous grasses for the potential future development of improved C4 cropping species.