( rticles. Starting with the b...)Optimization of Biolistic Transformation Using the Helium-Driven PDS-1000/He System

rticles. Starting with the biological conditions reported by this group, we have optimized microparticle accelerator parameters for the helium-powered unit. In the case of transient expression in plants, earlier work with the gunpowder unit (Klein, et al., 1988c) had shown that high levels of transient expression in maize could be achieved using 1.2 mm tungsten microparticles, a target distance of 6 cm, and a chamber vacuum of 28 inches Hg. These conditions formed the basis for the starting conditions used in these assays, and, in fact, are the optimal conditions which we found for transient expression in cauliflower stem sections. We recommend using a similar strategy of starting with previously reported conditions when optimizing parameters for other cell types of interest.

When extrapolating from transient to stable expression, it is important to remember that the optimum transformation conditions may be different in the two cases. For example, Biolistic transformation may damage cells such that they score positive in transient expression assays, but can not divide, and therefore would not produce a stable transformant. As a result, conditions which are found to be optimum for transient expression may be too harsh to produce the maximum number of stable transformants. Therefore, when optimal conditions for transient expression are known, it may be necessary to use more gentle conditions to achieve maximum stable expression. The transformation process may be made less harsh by reducing the velocity of the microcarriers. This may be achieved by reducing the helium pressure, the macrocarrier travel distance, and the size and number of microparticles, and/or by increasing the target distance and the gap
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