Establishment of an efficient genetic transformation system for Tanacetum cinerariifolium

Abstract The Dalmatian Daisy Tanacetum cinerariifolium is an Asteraceae plant species that produces the natural insecticide “pyrethrum”, which is effective against mosquito disease vectors and household pests. To enhance the content of pyrethrum in flowers, a more detailed understanding of the mechanisms underlying pyrethrum biosynthesis is needed. Even though gene transformation and genome editing techniques are vital for investigating pyrethrin biosynthesis, limited information is available on the transformation of T. cinerariifolium. Furthermore, each seedling possesses a distinct genotype with large variations by self-incompatibility. We herein employed T. cinerariifolium line #14 with weak self-incompatibility to establish a protocol of efficient regeneration from leaf segments and transformation. Leaf segments formed calli on 1/2 Murashige and Skoog’s basal medium (MS) with naphthalene acetic acid 1 mg L-1 and 6-benzylaminopurine (BAP) 2 mg L-1, regenerated shoots from calli on 1/2 MS with BAP 0.5 mg L-1 and GA3 0.2 mg L-1, and elongated shoot stems on 1/2 MS with indole-3-butyric acid 0.5 mg L-1 and BAP 0.5 mg L-1. To establish genetic transformation, Rhizobium radiobacter strain EHA105 with the highest infectivity and the mas1'-2' bidirectional promoter with the highest expression were used, and the antibiotic G418 was added to medium at a concentration of 10 to 20 mg L-1 to select transformed cells. By using established regeneration techniques, we successfully obtained transformants that highly expressed the transgene gusA. This technique allowed us to elucidate the mechanisms underlying pyrethrum biosynthesis in T. cinerariifolium and was useful for creating transgenic T. cinerariifolium with increased pyrethrum biosynthesis.