Agrobacterium - mediated Transformation of Chrysanthemum (Dendranthema grandiflora) Plants with a Disease Resistance Gene (pac1)

Abstract

We constructed a binary vector to improve the transformation efficiency and transgene expression in chrysanthemum plants in an Agrobacterium-mediated system. Most of the sequences unnecessary for maintaining plasmids in bacteria were removed from the pBI121 vector, and an expression cassette of the neomycin phosphotransferase II gene, in which a point mutation was repaired, and an expression cassette of β-glucuronidase (GUS) with a short 5’ un-translated sequence of β-glucanase from soybean, were set within a transferred DNA. This vector was used to transform leaf discs of chrysanthemum plants, resulting in the transformation efficiency increasing several times compared with pBI121 and the activity of GUS increasing prominently. We also introduced a double-stranded RNA-specific ribonuclease (pac1) from Schizosaccharomyces pombe by using this novel vector to confer simultaneous tolerance against virus and viroid diseases. Using this technique we obtained more than 80 transgenic chrysanthemum plants, and found that half of them expressed a moderate amount of pac1 protein as detected by Western blot analysis.