Virus diseases in portuguese almond trees: Diagnosis, in situ detection and genetic engineering for virus resistance

In this work we have studied the cycle of PDV and PNRSV, along the year in the two almond producing regions (Algarve and Tras-os-Montes) plus in a third one, in middle Portugal. The data were collected always from the same trees in each region ('Boa Casta' and 'Bonita de Sao Bras'). We have also studied virus distribution in tissue, using in situ RT-PCR, and found that in flower buds, PDV appears in all meristematic cells, located mostly around the nucleus and forming a ring-like structure. Interestingly, PDV was also found inside pollen grains, and again around the nuclear region. These data are discussed in terms of virus behaviour in orchards. To introduce virus resistance we have started to prepare and test several constructs. These constructs are based in the coat protein gene of PDV and PNRSV and include several variations in the fragment used (sense, antisense and a partially deleted gene sequence, plus a sense construct flanqued by nuclear matrix attachment regions). The cloned fragments were first transferred to transformation vectors provided by the John Innes Institute (pGREEN), then transferred to Agrobacterium, and are now being used in Nicotiana transformation for testing efficiency.