Steve Wylie

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.

Transgenic plants of narrow-leafed lupin (Lupinus angustifolius) and yellow lupin (L. luteus) were generated using meristem inoculation with Agrobacterium tumefaciens. Plants with increased tolerance of the herbicide Basta (glufosinate) were produced, as were yellow lupin plants with improved tolerance of bean yellow mosaic virus. This technology is being extended for the introduction of other useful traits such as resistance to cucumber mosaic virus and fungal pathogens. Commercial molecular diagnostic tests for cucumber mosaic virus (RT-PCR and real time fluorescence PCR) and anthracnose (caused by Colletotrichum acutatum rather than C. gloeosporioides [Glomerella cingulata]; PCR) in lupin seeds were developed and are in routine use. Gene mapping of lupins is in progress, with the first molecular marker (for early flowering) developed as a routine PCR test.

We have developed a number of resistance genes to confer resistance to bean yellow mosaic virus and cucumber mosaic virus. The bean yellow mosaic virus genes have been transferred to Lupinus angustifolius and L. luteus and the CMV genes are presently being introduced to L. angustifolius. In the 1997 season eight transgenic L. luteus lines containing BYMV resistance genes were challenged with the virus. Two lines were shown to exhibit reduced disease symptoms, and delayed accumulation of BYMV particles after manual and aphid inoculation of the virus. Virus challenge was carried out in glasshouse trials at Murdoch University and in a screen house at the UWA Field Station. Inoculation trials will proceed again with other transgenic lines in the next growing season. We began a search for the CMV resistance gene present in L. luteus Wodjil in order to isolate and characterise it. It is hoped that in the future this gene may be transferred to other CMV susceptible legumes.