In this study, we compared interactions of two Melampsora foliar rust species with poplar, which resulted in either limited or abundant pathogen proliferation. In the pathosystem exhibiting limited pathogen growth, a defence response was observed after invasion of poplar leaf tissues by the biotroph, with late and clear production of reactive oxygen species (ROS) and other products. Characterisation of the histological, biochemical and transcriptional events occurring in both pathosystems showed striking similarity with components of plant defence reactions observed during qualitative resistance. Key components associated with development of an active defence response, such as up-regulation of pathogenesis-related (PR) genes, were observed during infection. Moreover, the time course and strength of gene induction appear to be critical determinants for the outcome of the tree-pathogen interaction. This work provides basic biochemical characterisation and expression data for the study of so-called partial resistance in the poplar-rust pathosystem, which is also applicable to other plant-pathogen interactions resulting in quantitative disease resistance.

To elucidate heterologous promoter function in gymnosperms, we introduced the bean phenylalanine ammonia-lyase-beta-glucuronidase (PAL2-GUS) gene fusion into white pine (Pinus strobus L.). Over 15 lines were produced and integration of Agrobacterium T-DNA was confirmed by Southern analysis. Induction of the reporter gene was detected in all of the lines tested following UV illumination. In contrast, a weak but constant induction was seen in only a few lines following treatment with salicylic acid (SA) or jasmonic acid (JA). However, pretreatment of suspension cultures with SA or JA enhanced the induction of PAL2-GUS expression by UV irradiation. This specific enhancement or potentiation was reduced by 50% by treating the cells with indomethacin, an inhibitor of phospholipase activity, suggesting that the observed potentiation of UV induction involves the octadecanoid pathway. The UV induction was completely abolished by treating the cells with okadaic acid, an inhibitor of phosphatase activity. Thus, the induction of the heterologous PAL2 promoter from bean is consistent with the induction of phenylalanine ammonia-lyase (PAL) in angiosperms. Furthermore, our findings suggest that conifers, although phylogenetically distant to angiosperms, share some conserved promoter elements and some signal transduction mechanisms for UV-light perception.

The gene coding for green fluorescent protein (GFP) from the jellyfish Aequorea victoria was successfully used as a vital marker for the transformation of three woody plant species, black spruce (Picea mariana (Mill.) BSP), white pine (Pinus strobus L.) and poplar (Populus spp.). The gfp gene and the gene conferring resistance to kanamycin (nptII) were introduced by microprojectile bombardment or Agrobacterium tumefaciens-mediated technology. Screening by fluorescence microscopy of the transformed plant material, under the selection of kanamycin, identified five to eight cell lines from each tree species that clearly expressed GFP. Expression of GFP was observed in somatic embryonal cells of the coniferous species and in stem sections of poplar. For all species, GFP transgene expression was stable over multiple subcultures. Stable integration of the gfp gene into plant genomes was confirmed by Southern hybridization or polymerase chain reaction (PCR) analysis. We conclude that GFP can be used as a vital marker and reporter protein in transformation experiments with gymnosperms and deciduous trees.

A genetic transformation procedure for white pine has been developed after cocultivation of embryogenic tissues with Agrobacterium tumefaciens. This efficient transformation procedure led to an average of four independent transformed lines per gram of cocultivated embryogenic tissue and up to 50 transformed lines can be obtained in a routine experiment. Constructs bearing the uidA gene or the green fluorescent protein (GFP) gene were introduced and β-glucuronidase (GUS) activity was followed over time. The expression of the uidA gene was lowest with a 35S-gus-intron construct and was 20-fold higher with a 35S-35S-AMVgus::nptII construct. The addition of scaffold attachment region (SAR) sequences surrounding the gus::nptII fusion did not significantly enhance the GUS activity. Transformed mature somatic embryos have been germinated and plantlets are presently being acclimatized.

Agrobacterium tumefaciens mediated gene transformation and whole plant regeneration from transformed tissue were established for a conifer species. the hybrid larch Larix kaempferi x L. decidua [1]. A nptll gene under the control of the tandem repeat of the 35S CaMV promoter allowed us to regularly obtain transgenic masses. Using somatic embryogenesis, we are able to easily regenerate numerous plantlets from each of these embryogenic masses (EM). Southern hybridization. using the nptll coding sequence as a probe. were performed on needles collected from greenhouse-grown plants. 14 different plants, each originating from independant kanamycin resistant-EM. have: been analysed and all of them appeared to be positive.This procedure has been optimized in order to increase: the yield of antibiotic-resistant EM per experiments and to shorten the time between the cocultivation step and the availability of transgenic material in the greenhouse. These improvements now allow us to regularly obtain an average of 5 masses resistant to the selective agent per petri dish, i.e. 24 kanamycin-resistant EMper gram fresh weight followed by the transfer of plants in the greenhouse within 7 months after cocultivation.

A transformation procedure was developed for hybrid larch embryogenic tissue using Agrobacterium tumefaciens. The cocultivation procedure yielded one to two transformation events per 100 cocultivated masses. The addition of 100 µM coniferyl alcohol increased the yield. This improved procedure was successfully applied to three other genotypes. After 3 months on selective medium, the transgenic tissue remained embryogenic, which allowed production of transgenic plants in the greenhouse. Stable integration of the transgene was confirmed by PCR and Southern hybridisation on transformed tissues and acclimatised plants.