Investigation of biolistics as an alternative transformation method for Narrow-leafed lupin (Lupinus angustifolius)

The potential for lupins as an agricultural crop has been recognized worldwide. Despite work going on in several different laboratories around the world, the efficiency of stable gene transfer by Agrobacterium remains at approximately 1 % for modern varieties. Here we investigate particle bombardment as an alternative method of gene transfer. Preliminary studies were carried out by using tungsten particles ( ~ 1 μm in diameter) coated carrying DNA encoding the gus reporter gene and the bar gene for selection of transformants. Particles were accelerated into embryonic axes by helium inflow. A number of parameters were studied including plasmid preparation methods, pre-treatment of target tissue, particle density, helium pressure, DNA concentration, and the distance between the injector and target tissue (target distance). Expression as determined by blue spots of GUS staining has been increased significantly, but stable transformation, as determined by survival of shoots in selection medium (containing 10 mg/ml PPT), has not been achieved. Analysis of GUS expression seven days post-bombardment, and penetration depth of the tungsten particles in target tissue have suggested that the particles did not penetrate to the LII and LIII tissue layers of embryogenic axes, which give rise to organs including inflorescence of grown plants