Conditions for Agrobacterium-mediated transformation of
wheat (Triticum aestivum L.) were defined using wheat
suspension cells as a model system and green fluorescent protein (GFP) as a
visual marker. Different strains of
Agrobacterium tumefaciens were compared using
established wheat cell suspension cultures, where the frequency of cell
clusters showing transient activity of GFP ranged from 2 to 52%. High
levels of transient GFP activity and stable transformed callus lines were
obtained with plasmid pTO134 containing a gfp gene with
an enhanced CaMV 35S promoter and a bar gene with a 35S
promoter in combination with Agrobacterium strain AGL0.
These results suggest that the important variables in
Agrobacterium-mediated transformation of wheat cells
include media composition, Agrobacterium strain, plasmid
vector and the addition of virulence-inducing agents such as acetosyringone.
The conditions deemed optimal for transformation of wheat suspension cell
lines were applied to scutella isolated from immature embryos and
scutella-derived calli. Transient GFP expression in these tissues ranged from
10 to 75% and, while quite variable among and within cultivars, stably
transformed scutellum-derived callus was obtained. Further studies with
scutellum-derived calli suggested that variables such as duration of
pre-inoculation culture and co-cultivation, as well as co-cultivation
temperature, were also important. Optimisation of these variables resulted in
the recovery of transformed wheat plants at a transformation frequency of
1.8%, which is comparable with other reports.
Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter