Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

We assessed the effect of four different virulence (vir) gene combinations on plant transformation efficiency and transgene behaviour in rice using the pGreen/pSoup dual binary vector system. Transformation experiments were conducted using a pGreen vector containing the bar and gusA expression units with, or without, the virG542, virGN54D, virGwt or the virG/B/C genes added to the backbone. Additonal vir gene(s) significantly altered plant transformation efficiency and the integration of vector backbone sequences. However, no differences in transgene copy number, percentage of expressing lines and expression levels could be detected. Addition of virGwt was the most beneficial, doubling the overall performance of the pGreen/pSoup vector system based on transformation frequency, absence of backbone sequence integration and expression of unselected transgenes. In 39% of the plant lines, the additional vir genes were integrated into the rice genome. The contribution of 'super dual binary' pGreen/pSoup vectors to the development of efficient rice transformation systems and to the production of plants free of selectable marker genes are discussed.

Transgenic lines of the spring barley variety Golden Promise containing the firefly luciferase gene were produced by particle bombardment of immature embryos. Non-destructive analysis of luciferase gene expression was used to monitor the transformation process. This revealed that transformation efficiency, in terms of the percentage of bombarded immature embryos giving rise to transformed callus lines, was very high, up to 40%. Following the expression of the luciferase gene provided a method for the sensitive, non-destructive, realtime monitoring of gene expression throughout the transformation process. Luciferase expression could also be used to easily identify transgenic plants and to identify homozygous transgenic plants at an early stage. The production of transgenic barley by selecting for luciferasepositive material, without an additional selection system, was possible but technically difficult.