Experiments were conducted to establish the nature and frequency of stable transgene expression in standard (cv. 'Lineker') and spray-type (cv. 'Shuhou-no-chikara') chrysanthemum (Dendranthema grandiflora (Ramat.) Kitamura) when four different gene introduction methods - particle bombardment, Agroinfection, sonication-assisted Agrobacterium transformation (SAAT) or Agrolistics (bombardment + Agroinfection) - were applied. In the latter three, pBI121 or pKT2 (intron-containing novel plasmid construct) - both containing the uidA and nptII genes under the control of a CaMV-35S promoter and harboured in Agrobacterium tumefaciens LBA4404 - were utilized, while in particle bombardment pSKGN1 was utilized. Transformation efficiencies (TEs) were scored, based on the number of putative transformants - harvested off a 25 mgl^-1 kanamycin selective medium - obtained from any of these methods. An analysis of the GUS expression and molecular (PCR) derived TEs indicates that 'Lineker' is the most susceptible cultivar for transformation and shows the following TE values: bombardment (pSKGN1) (20.6 %), SAAT (pKT2) (15.8 %), Agroinfection (pKT2) (5 %), Agrolistics (no plasmid and pBI121) (7.1 %) or (no plasmid and pKT2) (7.7 %), Agrolistics (pSKGN1 and pBI121) (5.6 %) or (pSKGN1 and pKT2) (16.7 %). PCR-based transformants were not obtained for 'Shuhou-no-chikara', but occurred at 9.1 % for pKT2-SAAT. An optimized regeneration system, coupled with a step-wise kanamycin selection process allows for the selection of transformed tissue, while eliminating most escapes. However, cultivar-dependence, chimeric and variable stable TE and loss of stable TE in greenhouse-acclimatized putative transforments (possible gene silencing) were observed, and remain one of the main issues hampering successful chrysanthemum transformation.

Abbreviations: blue staining area (BSA), gene introduction method (GIM), GUS focal point (GFP), sonication-assisted Agrobacterium transformation (SAAT), transformation efficiency (TE), stable transgene expression (STE), transient transgene expression (TTE)