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 protocol s 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 i ndoleacetic acid and 0.5 or 1.0 mu M benzyladenine. Regeneration of sh oots from cultivar 'Iridon' was obtained with continuous culture on th ese media until shoots were transferred to rooting medium at approxima tely 6 weeks. By contrast, shoot regeneration from 'Hekla' explants re quired 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 cul ture of explants in the absence of blue wavelengths of light. Shoots f rom all cultivars were rooted on 25% MS medium with 0.5 mu M naphthale neacetic acid. Three wild type strains of Agrobacterium tumefaciens (A ch5, A281, Chry5) were evaluated for tumor production on the three cul tivars. Chry5 and A281 were significantly more virulent on all three c ultivars than was Ach5. Transformed plants of 'Iridon' were obtained u sing Agrobacterium strain EHA105, a disarmed version of A281. Explants were transformed with two plasmids, pBI121, which encodes kanamycin r esistance and beta-glucuronidase (GUS) activity, and pBI121. containin g the nucleocapsid gene of a highly virulent isolate of tomato spotted wilt virus (TSWV). Transformed shoots regenerated and rooted on mediu m containing 50 mu g/ml kanamycin. Vegetatively propagated progeny of transformed plants were identified which expressed GUS activity and wh ich contained multiple copies of the TSWV nucleocapsid gene.