PHYSICAL TRAUMA AND TUNGSTEN TOXICITY REDUCE THE EFFICIENCY OF BIOLISTIC TRANSFORMATION

A cell suspension culture of tobacco (Nicotiana tabacum L.) was used as a model to study injury to cells during biolistic transformation. Lawns of cells were bombarded with tungsten particles that were coated with a plasmid containing the beta-glucuronidase and the neomycin phosphotransferase II genes. When a gunpowder-driven biolistic device was used, numerous transiently expressing cells were focused around the epicenter of the blast which was manifested by a hole blown in the filter paper supporting the cells. However, transformed cells nearest the blast epicenter were injured and could not be recovered as stable transformants. The injury was primarily caused by physical trauma to the cells from gas blast and acoustic shock generated by the device. Postlaunch baffles or meshes placed in the gunpowder device reduced cell injury and increased the recovery of kanamycin-resistant colonies 3.5- and 2.5-fold, respectively. A newly developed helium-driven device was more 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 microprojectiles also contributed to cell injury. Gold microprojectiles were not toxic and resulted in fourfold more kanamycin-resistant colonies than when similar quantities of similarly sized tungsten particles were used.