501 Production of Virus-Free Plant Propagation Material from Infected Nectarine Trees G.A. Manganaris Dept. of Horticulture Aristotle University 54124 Thessaloniki, Greece manganar@agro.auth.gr A.S. Economou Institute of Agrobiotechnology CERTH 54124 Thermi, Greece aseconom@agro.auth.gr I. Boubourakas and N. Katis Dept. of Plant Protection Aristotle University 54124 Thessaloniki, Greece katis@agro.auth.gr Keywords: Meristem-tip culture, plum pox virus (PPV), polymerase chain reaction (PCR), Prunus persica var. nectarina, sharka, thermotherapy Abstract Plum pox virus (PPV) is one of the major and common diseases in stone-fruit trees. The effect of thermotherapy in PPV-infected nectarine plants (Prunus persica var. nectarina Max, cv. Arm King) and plant regeneration from meristem-tip explants, as well as use of a reliable RT-PCR that detects extremely low virus titre of PPV in in vitro cultures were studied. Three weeks of thermotherapy at a maximum temperature of 35°C gave tender explants that used for meristem-tip culture. Meristem-tip explants first were established on WPM free of growth regulators and then for multiple shoot were subcultured on WPM supplemented with 8 μM BA and 0.8 μM IAA. Individual shoots produced from such meristem-tip cultures were tested for PPV presence with RT-PCR and gave ‘negative’ results (virus-free) at a frequency of 82.8%. Rooted in vitro microcuttings, derived from virus-free cultures, were established in the greenhouse conditions successfully. INTRODUCTION Sharka or plum pox, caused by plum pox virus (PPV), is the most serious disease of Prunus species resulting in reduced fruit quality and quantity and shortening of trees economical life. In addition, it is spread rapidly to neighboring trees through aphids (Herrera et al., 1998). Most cultivated Prunus species are highly susceptible and conventional breeding has not produced highly resistant and commercially acceptable varieties (Ravelonandro et al., 2000), while sources of virus resistance among peach and nectarine cultivars are rather limited (Escalettes et al., 1988). The use of transgenic fruit trees as a resistance strategy for virus epidemics offers challenging interpretations (Ravelonandro et al., 2000), however, further investigations should be conducted in the field of genetic engineering. It is therefore evident that thermotherapy combined with meristem-tip culture is the main way of producing virus-free plants (Walkey, 1980). Certified healthy propagation material for establishing new peach and nectarine orchards is of great importance. Virus testing of in vitro-derived planting material is vital for producing healthy propagation material. However, virus diagnosis in in vitro cultures needs a reliable and sensitive molecular assay that can detect minute amounts of virus titre. The aim of this work was to develop a protocol for obtaining clean from plum pox virus propagation material in nectarine via thermotherapy and meristem-tip culture using highly sensitive diagnostic techniques for virus detection. MATERIALS AND METHODS Plant Material Virus-infected shoots of nectarine cv. Arm King were collected from orchard trees of the Pomology Institute in Naoussa (Northern Greece) that had tested positive for PPV by DAS-ELIZA. The lateral buds of these shoots were excised and used as scions for Proc. 1 st IS on Accl. & Estab. Micropop. Plants Eds. A.S. Economou & P.E. Read Acta Hort. 616, ISHS 2003