PHYSICAL TRAUMA AND TUNGSTEN TOXICITY REDUCE THE EFFICIENCY OF BIOLISTIC TRANSFORMATION

A cell suspension culture of tobacco (Nicotiana tabacum L.) was used a s a model to study injury to cells during biolistic transformation. La wns of cells were bombarded with tungsten particles that were coated w ith a plasmid containing the beta-glucuronidase and the neomycin phosp hotransferase II genes. When a gunpowder-driven biolistic device was u sed, numerous transiently expressing cells were focused around the epi center of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the bl ast epicenter were injured and could not be recovered as stable transf ormants. The injury was primarily caused by physical trauma to the cel ls from gas blast and acoustic shock generated by the device. Postlaun ch baffles or meshes placed in the gunpowder device reduced cell injur y and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was mor e gentle to the cells and also increased the number of transformants. Cell injury could be further moderated by using a mesh and a prelaunch baffle in the helium device. Toxicity of the tungsten microprojectile s also contributed to cell injury. Gold microprojectiles were not toxi c and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used.