L i s t of abbreviations: GUS, b-glucuronidase; MS m e d i u m , Murashige and Skoog medium; PAT, phosphinothricin acetyl transferase; PPT, phosphinotricin.
AbstractAn optimized procedure for transformation of wheat with the use of a Biolistic Particle Delivery System PD S 1000/He to deliver ~breign DNA is described in detail. The bacterial uidA and bar genes (both driven by plant promoters) were utilized as the reporter and selectable marker genes, respectively. Moderately high gas pressure appeared to be most important to achieve the highest level of transient GUS expression in target tissues. There was, however, no apparent correlation between transient and stable GUS expression. The presence of telomeric DNA sequences in an uidA gene-containing vector did not influence transient GUS expression but, apparently, prevented its stable expression. Mechanical lesions caused by the bombardment (tungsten particles) seemed to be less severe when embryo-derived calli, instead of freshly excised immature embryos, were used as the target tissue. The limited ability of callus cells for regeneration, together with a restricted number of cells that receive the foreign DNA by particle bombardment, result in a low efficiency of wheat stable transformation.
The aim of the study was to characterize DNA lesions caused by microprojectile bombardment and by the post-bombardment presence of tungsten particles in transformed cells. For the sake of simplicity, plasmid DNA was used as a target for bombardment with naked tungsten particles. Unexpectedly extensive DNA degradation was observed under standard bombardment conditions. However, no further DNA fragmentation occurred under post-bombardment conditions, simulated by incubation of plasmid DNA with a suspension of tungsten particles. Instead, relaxation and linearization of supercoiled circular plasmids (pAHC25 and others) took place. It is concluded that the observed linearization (a single site double-strand break in DNA circle) results from the ability of tungsten to catalyse the hydrolysis of phosphodiester bonds in torsionally strained sites of native DNA selectively.
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