Rapid methylation of transgenes upon plant transformation – a methodology

JG Philips1, KJ Dudley2, PM Waterhouse1 and RP Hellens1

  1. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, Brisbane, Queensland, Australia
  2. Institute for Future Environments, Central Analytical Research Facility, Queensland University of Technology, Brisbane, Queensland, Australia

Genetic Modification, whereby foreign DNA (transgenes) is introduced into plants, serves multiple purposes in plant biology research and can improve or introduce favourable traits in many crop species. However, transgene expression is highly variable due to multiple and interacting factors, one of these is DNA methylation which results in transcriptional inhibition or 'gene silencing'. As a result, large numbers of plants must be screened to find stable transformants with consistent transgene expression. Finding ways to mitigate transgene methylation will be greatly beneficial to the plant transformation community. Here we present a methodology of an assay to rapidly assess transgene methylation. With this assay, variables such as intron inclusion within the transgene and 'knock-outs' of genes involved in the transcriptional gene silencing pathways can be rapidly tested within a transient plant transformation system. Transient co-infiltration in Nicotiana benthamiana was carried out using the eGFP reporter gene under the constitutive expression of the 35S promoter and a hairpin RNA targeting the eGFP transgene. Using this assay, we demonstrate accumulation of methylation over a relatively short time course with 50% methylation of all cytosine nucleotides at five days post infiltration in a region encompassing a 418 bp junction of the 35S promoter and eGFP transgene.