Agrobacterium-mediated transformation and regeneration of Freesia×hybrida

Abstract

A method for Agrobacterium-mediated transformation of Freesia×hybrida is described. Cormlet-derived calli of two cultivars, ‘Mosera’ and ‘Ishikawa f3’ were co-cultivated with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pIG121-Hm, which included hygromycin phosphotransferase gene and an intron-containing β-glucuronidase gene in the T-DNA region. Callus pieces were co-cultivated with A. tumefaciens on the callus proliferation medium [Murashige and Skoog (MS) medium containing 1 mg l⁻¹ thidiazuron, 1 mg l⁻¹ dicamba, 20 mg l⁻¹ 3′,5′-dimethoxy-4′-hydroxyacetophenone, 1% (w/v) glucose, 3% (w/v) sucrose, and 0.2% (w/v) Gelrite]. Then, they were cultured on the callus proliferation medium containing 300 mg l⁻¹ cefatoxime and 10 mg l⁻¹ hygromycin B. Hygromycin-resistant lines of both cultivars regenerated into plantlets after transfer onto MS medium containing 2 mg l⁻¹ 3-indoleacetic acid and 3 mg l⁻¹ 6-benzyl aminopurine and/or plant growth regulator-free MS medium. Transgenic plants were identified by β-glucuronidase assay and verified by Southern blot analysis. Two transgenic plant lines were obtained from 475 callus pieces of ‘Mosera’, and one transgenic plant line was obtained from 290 callus pieces of ‘Ishikawa f3’. This is the first report of the genetic transformation of Freesia. This method will allow the genetic improvement of this horticulturally important flower.