Chrysanthemum Transformation via Agrobacterium

Agrobacterium tumefaciens strain AGL0 carrying plasmid pNK497 was grown in flask on a shaker overnight at 28 °C in LB medium supplemented with 25 mg l⁻¹ rifampicin and 50 mg l⁻¹ kanamycin. Bacterial cultures were diluted in liquid MS medium to an optical density of 0.6 at 600 nm. Leaf explants used for transformation experiments were cut from 4-week-old chrysanthemum plants (Chrysanthemum morifolium ‘Snowdon White’) were first placed in MS with 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid, 1 mg l⁻¹ 6-benzylaminopurine, 1 mg l⁻¹ kinetin and 5 mg l⁻¹ 6-(γ,γ-dimethylallylamino)purine for 3 h and then incubated with bacterial culture for 20 min. Leaf explants were then placed onto filter paper overlaid on MS medium (MS salts, Quoirin and Lepoivre medium, 30 g l⁻¹ sucrose and 8 g l⁻¹ agar, pH 5.8) supplemented with 200 µg l⁻¹ 6-benzylaminopurine and 50 µg l⁻¹ 1-naphthaleneacetic acid. Two days after inoculation, explants were transferred to the same medium supplemented with 500 mg l⁻¹ cefotaxime and 35 mg l⁻¹ kanamycin. Regeneration shoots were cut and grown on MS medium with antibiotics.