Genetic transformation of wheat via particle bombardment.
Since its first invention in the late 1980s the particle gun has evolved from a basic gunpowder driven machine firing tungsten particles to one more refined which uses helium gas as the propellant to launch alternative heavy metal particles such as gold and silver. The simple principle is that DNA-coated microscopic particles (microcarriers) are accelerated at high speed by helium gas within a vacuum and travel at such a velocity as to penetrate target cells. However, the process itself involves a range of parameters which are open to variation: microparticle type and size, gun settings (rupture pressure, target distance, vacuum drawn, etc.), preparation of components (e.g., gold coating), and preparation of plant tissues. Here is presented a method optimized for transformation of wheat immature embryos using the Bio-Rad PDS-1000/He particle gun to deliver gold particles coated with a gene of interest and the selectable marker gene bar at 650 psi rupture pressure. Following bombardment, various tissue culture phases are used to encourage embryogenic callus formation and regeneration of plantlets and subsequent selection using glufosinate ammonium causes suppression of non-transformed tissues, thus assisting the detection of transformed plants. This protocol has been used successfully to generate transgenic plants for a wide range of wheat varieties, both spring and winter bread wheats (T. aestivum L.) and durum wheats (T. turgidum L.).
Plant Biology and Crop Science Department, Rothamsted Research, Harpenden, Hertfordshire, UK.
Caroline A
CA
Sparks
Huw D
HD
Jones
D019470:Biolistics / Q000379:methods*; D018014:Gene Transfer Techniques; D006046:Gold; D030821:Plants, Genetically Modified; D012639:Seeds / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014908:Triticum / Q000235:genetics* / Q000378:metabolism
D006046:Gold