Agrobacterium-mediated genetic transformation of Viola cornuta L. "Lutea Splendens" with capsanthin-capsorubin synthase gene

Abstract

Viola cornuta L. ‘Lutea Splendens’ is a perennial ornamental plant with small yellow flowers that naturally grows in the Pyrenees in Spain and France. To develop novel cultivars with orange and red flower colors, we performed Agrobacterium tumefaciens (LBA4404)-mediated transformation with the binary vector pWBVec10a/P35S::Llccs::TNos that harbored capsanthin-capsorubin synthase (Llccs) gene from Lilium lancifolium under the control of CaMV35S constitutive promoter and the nopaline synthase (Nos) terminator. Capsanthin-capsorubin synthase catalyzes the conversion of anteraxanthin and violaxanthin, two yellow ubiquitous 5-6-epoxy-xanthophylls, into capsanthin and capsorubin, two red xanthophylls, respectively. Starting with hypocotyl explants, we developed a transformation protocol with 0.3% shoot regeneration efficiency. Histochemical assay for β-glucuronidase (GUS) activity showed uidA reporter gene expression in all putative Llccs-transgenic shoots. The presence of Llccs transgene, hygromycin phosphotransferase (hpt) selectable marker gene and uidA (GUS) reporter gene in all putative Llccs-transgenic lines were confirmed by PCR analysis. This is the first report on Agrobacterium-mediated genetic transformation of V. cornuta L. with the aim to introduce desirable traits into this species.