Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker

Abstract

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.

Stable chloroplast transformation of immature scutella and inflorescences in wheat (<italic>Triticum aestivum</italic> L.)

“…Nuclear transformation in wheat has been reported by many researchers [34][35][36]. The transformation and regeneration conditions were well established [37,38].…”

Comparison of Agrobacterium -mediated transformation of four barley cultivars using the GFP and GUS reporter genes

“…In contrast, a synthetic sgfp gene similar to that used here, driven by the maize Ubi1 promoter, has been used successfully to transform other monocots such as maize (van der Geest and Petolino 1998), sugarcane , oats (Kaeppler et al 2000) and wheat (Jordan 2000;Weir et al 2001) with no mention of deleterious effects. In other studies with barley, when either the Actin1 or enhanced CaMV 35S promoter has been used to drive GFP expression, there is no mention of reduced transformation efficiencies (Ahlandsberg et al 1999;Carlson et al 2001;.…”

The procedure providing enhanced Agrobacterium-mediated transformation of wheat

“…Dutt & Grosser (2009) found that divers citrus cultivars differently responded to the addition of acetosyringone to the co-cultivation medium. In wheat, it has also been considered as an important factor for successful Agrobacterium-mediated transformation at all (Cheng et al 1997;Weir et al 2001;Wu et al 2003). The addition of acetosyringone was not conclusive for transient GUS expression in FIIE, while it was important for PCIE.…”

“…For decades wheat has been considered as the most recalcitrant cereal species for regeneration and transformation compared to rice and maize mainly due to low efficiency of plant regeneration under tissue culture conditions. A lot of investigations were carried out on the optimization of conditions for the improved Agrobacterium-mediated genetic transformation in this major crop (Weir et al 2001;Wu et al 2003;Patnaik et al 2006;Yao et al 2007) but the transformation efficiency remained low (mostly around 1%) even the model cultivars were used (Weeks et al 1993;Nehra et al 1994;Cheng et al 1997). Increased frequencies were achieved by introduction of high responsive wheat genotypes such as model cultivar Bobwhite (9.3%) (Zhang et al 2000) and generally transformation protocols were further optimized using model cultivars with a relatively high embryogenic capacity (Janakiraman et al 2002).…”