| Tomato spotted wilt virus (TSWV) causes devastating losses in greenhouse ornamentals, including chrysanthemum, Dendranthema grandiflora. TSWV resistance in tobacco, tomato, and lettuce has been obtained via transformation of these crops with the TSWV Nucleocapsid (N) gene. Efforts were directed at developing an efficient transformation system for chrysanthemum to allow placement of the TSWV N gene into this crop.; We developed the first Agrobacterium-mediated transformation system to work for multiple chrysanthemum cultivars: Iridon, Hekla, and Polaris. Regeneration of transformed shoots was accomplished from leaf explants under a constant selection pressure of 50 mg/L paromomycin. Explants were first cultured on Mum B medium (Murashige and Skoog (MS) basal medium supplemented with 2.0 mg/L indole-acetic acid (IAA) and 0.23 mg/L benzlyadenine (BA)) augmented with 0.5 mg/L 2,4-dichlorophenoxy-acetic acid (2,4-D). After callus formation, explants were transferred to Mum B medium lacking 2,4-D. Shoot elongation occurred following transfer of shoot primordia onto MS medium containing 2.0 mg/L kinetin (Kin), 0.02 mg/L naphthalene-acetic acid (NAA), and 10 mg/L gibberellic acid (GA{dollar}sb3{dollar}). Shoots were rooted on 25% MS medium supplemented with 0.1 mg/L NAA.; Explants of 'Polaris' were transformed with TSWV N-gene constructs containing either a full length N gene (nucleotides 1983-2767)(pTSWVN+), a full length N gene encoding a truncated N protein (pTSWVNt), or an antisense version of the N gene (pTSWVN-). Resistance screens were conducted on 236 transformed lines. One pTSWVNt and two pTSWVN-transformed lines were found to have high levels of resistance to TSWV infection. We identified six other lines exhibiting symptom expression delay and a decreased incidence of necrotic symptoms. Molecular analysis of the highly-resistant lines indicated that they had at least three transgene insertion sites, no detectable N-protein production, and low transgene message accumulation. This study is the first report of TSWV resistance in chrysanthemum, and represents the first time a major ornamental crop has been genetically engineered for resistance to pathogen infection. |
