Production of fertile transgenic wheat plants via tissue electroporation

Electroporation has been used effectively to deliver DNA into the tissue of intact wheat immature embryos. Transformed plantlets have been recovered a fter electroporation using held strengths of 275 and 750 V/cm, 960-mu F cap acitor and 50 mu g/ml of linear plasmid DNA, containing bar and uidA genes. The field strength of 750 V/cm proved to be more effective for DNA deliver y (estimated by transient GUS expression) and for recovery of transformed p lants (two transgenic plants were recovered with an efficiency of 0.4%). Af ter application of a field strength of 275 V/cm there was no visual evidenc e of transient GUS expression, but one transgenic plant was recovered with an efficiency of 0.2%, based on the number of electroporated embryos. This indicates that the amount of DNA delivered into the cells was too low for v isual identification of transient GUS expression and that GUS expression ma y not provide an appropriate assessment of the efficiency of DNA delivery. Southern blot hybridisation has revealed a low copy number of transgene int egration with some rearrangements in integrated loci. None of the transgeni c plants has shown any visual GUS expression, although we could amplify the transcript of the uidA gene in T-0 progeny using RT-PCR. This may indicate that suppression of uidA expression occurred at the post-transcriptional l evel. The efficiency of tissue electroporation is still dependent on the qu ality of the plant material which is used but the transformation events wer e reproducible from one group of experiments to another. At present, this t echnique is dependent on a combination of factors including pretreatments o f the recipient tissue, quality of tissue culture and optimisation of elect roporation conditions. (C) 2000 Elsevier Science Ireland Ltd. All rights re served.