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The Particle Inflow Gun
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The Particle Inflow Gun
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For information on operation of the particle inflow gun, please refer to the following publications:
Finer JJ, P Vain, MW Jones, MD McMullen (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Reports 11:232-238.
Vain P, N Keen, J Murillo, C Rathus, C Nemes, JJ Finer (1993) Development of the Particle Inflow Gun. Plant Cell Tiss Org Cult 33:237-246.
We have evaluated a number of different target tissues for optimization of particle bombardment. In our early work, leaf tissue of cowpea was the best target tissue that we could find for transient GUS expression. The problem with that system was that plants needed to be maintained in the greenhouses and GUS assays were very slow and costly. Results were obtained 4 days after bombardment and the GUS assay mix (which we used A LOT) contains cyanide. We are now using GFP for all of our transient and optimization work and the lima bean (Phaseolus lunatus) provides a wonderful model for transient expression. Transient expression can be initially observed within 5-6 hours and easily quantified 24 hours post-bombardment. Transient expression can be very high (see our GFP page) and the target tissue can be generated within 16-24 hours without the need to maintain stock plants.
For this work, seeds of lima bean (obtained from any x-mart, grocery or health food store) are imbibed overnight in a Petri dish or other container with moistened paper towels (we add 25 ml water to two pieces of paper towels, holding 4 lima bean seeds in one Magenta GA7 container). If you want to see REALLY consistent and high transient expression, it is best to grow lima bean plants, harvest seeds soon after drying and store the seeds properly. The next day/morning, the seed coat is removed and the seed halves are separated. The seed halves need to be put back together until just prior to bombardment or the target tissue may become too desiccated. The inside surface of the lima bean cotyledons are bombarded and the cots can be placed on any medium for observation the next day. We work under semi-sterile conditions and often, do not surface sterilize the seeds or paper towels. The water that we use to imbibe the seeds along with the particles are sterilized. Lima beans are white, which makes observations of gfp very straightforward.
