Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat.

Abstract

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.