HIGH-FREQUENCY SHOOT REGENERATION AND AGROBACTERIUM-MEDIATED TRANSFORMATION OF CHRYSANTHEMUM (DENDRANTHEMA-GRANDIFLORA)

An efficient, high-frequency transformation protocol was developed for the commercial chrysanthemum cultivar 'Iridon'. Regeneration protocols were also developed for two other commercial cultivars, 'Hekla' and 'Polaris'. All protocols utilized embryogenesis media composed of the basal medium of Murashige and Skoog (MS) supplemented with 11.5 mu M indoleacetic acid and 0.5 or 1.0 mu M benzyladenine. Regeneration of shoots from cultivar 'Iridon' was obtained with continuous culture on these media until shoots were transferred to rooting medium at approximately 6 weeks. By contrast, shoot regeneration from 'Hekla' explants required transfer to hormone-free medium 2 weeks after initial culturing. 'Polaris' regeneration required transfer of explants to hormone-free medium when shoot primordia first developed as well as continuous culture of explants in the absence of blue wavelengths of light. Shoots from all cultivars were rooted on 25% MS medium with 0.5 mu M naphthaleneacetic acid. Three wild type strains of Agrobacterium tumefaciens (Ach5, A281, Chry5) were evaluated for tumor production on the three cultivars. Chry5 and A281 were significantly more virulent on all three cultivars than was Ach5. Transformed plants of 'Iridon' were obtained using Agrobacterium strain EHA105, a disarmed version of A281. Explants were transformed with two plasmids, pBI121, which encodes kanamycin resistance and beta-glucuronidase (GUS) activity, and pBI121. containing the nucleocapsid gene of a highly virulent isolate of tomato spotted wilt virus (TSWV). Transformed shoots regenerated and rooted on medium containing 50 mu g/ml kanamycin. Vegetatively propagated progeny of transformed plants were identified which expressed GUS activity and which contained multiple copies of the TSWV nucleocapsid gene.