Abstract
Setaria viridis (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. Setaria viridis is the wild ancestor of the cropping species, foxtail millet (Setaria italica), with both Setaria species sharing a close evolutionary relationship with the agronomically important species, maize, sorghum, and sugarcane, as well as the bioenergy feedstocks, switchgrass, and Miscanthus. However, an efficient and reproducible transformation protocol is required to further advance the use of S. viridis to study the molecular genetics of C4 monocotyledonous grasses. An efficient and reproducible protocol was established for Agrobacterium tumefaciens-mediated transformation of S. viridis (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 S. viridis as an experimental model system to study the molecular genetics of C4 monocotyledonous grasses for the potential future development of improved C4 cropping species.
Highlights
The C4 monocotyledonous grasses contribute a large proportion of global food production for both human and animal consumption, as well as biomass for biofuel production (Vogel, 2008; Kellogg, 2013; OECD-FAO, 2017)
Freshly harvested (0 years of storage at 4◦C), and 1, 2, 3, and 4-year-old seeds that had been harvested from mature S. viridis wild-type plants, with or without their seed husk, were cultured on either callus induction medium (CIM) or N6D medium plates to assess these multiple factors in parallel to determine which combination returned the highest efficiency of callus induction
The age of the dehusked seeds was demonstrated to strongly influence the rate of callus induction, with the highest induction rate achieved when 1-year-old dehusked seeds were used as the starting material on both CIM (58.3%) and N6D (51.4%) media
Summary
The C4 monocotyledonous (monocot) grasses contribute a large proportion of global food production for both human and animal consumption, as well as biomass for biofuel production (Vogel, 2008; Kellogg, 2013; OECD-FAO, 2017). S. viridis is supported with a sequenced and annotated genome (Li and Brutnell, 2011; Bennetzen et al, 2012; Sebastian et al, 2014), and a comprehensive transcriptome of an elongating internode (Martin et al, 2016) and an expanding leaf (Studer et al, 2016) The availability of these genetic tools for S. viridis has facilitated studies on the complex C4 gene networks, studies that resulted in the identification and characterisation of several gene families involved in cell wall biosynthesis, such as Cellulose synthase genes (CesA and Csl; Ermawar et al, 2015; Muthamilarasan et al, 2015), and monolignol synthesis genes (Ferreira et al, 2019). The potential use of S. viridis as an experimental model has led to the required development of an efficient and reproducible transformation protocol to enable the continued molecular investigation into the complex gene networks that control C4 photosynthesis and cell wall development
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