TRANSFORMATION OF POPULUS HYBRIDS TO STUDY AND IMPROVE PEST RESISTANCE

Plant defense mechanisms that have activity against a specified pest c ontribute to resistance of trees to that pest, provided that the defen se mechanism is expressed at adequate levels in the appropriate tissue s. Additionally, the stability and (or) physiological efficiency of th e resistance may be increased by expressing the defense mechanism only when the tree is threatened by pest attack or challenge. In studies a imed at understanding and improving pest resistance of trees, 2 Populu s hybrids, Populus alba L. X P. grandidentata MICHX. and P. X eurameri cana (DODE) GUINIER, were transformed with chimeric plant defense gene constructs based on the potato proteinase inhibitor II (pin2) gene. A n Agrobacterium binary vector system was used to transform these hybri ds with one of the following chimeric genes: 1) a wound-inducible pin2 promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene; 2) a bacterial nopaline synthase (nos) promoter linked to a PIN 2 structural gene; or 3) a cauliflower mosaic virus 35s promoter linke d to a PIN2 structural gene. All of the transgenic poplars also were t ransformed with a selectable marker gene consisting of a nos promoter linked to a neomycin phosphotransferase II (NPT II) structural gene wh ich confers kanamycin resistance. Tissue-specific expression of the no s-NPT II gene construct is being evaluated with enzyme-linked immunoso rbent assays (ELISAs). Transgenic poplar lines from separate transform ation events demonstrate variable levels of nos-NPT II expression. Exp ression of nos-NPT II was detected in leaves, petioles, stems, and roo ts of one transgenic poplar (Tr15). Thus, the nos promoter has the pot ential to regulate chimeric defense genes in poplar when constitutive, whole-plant expression is warranted. Assays of the CAT reporter gene were hampered by a component of wounded poplar leaf extracts that inhi bits CAT enzyme activity. Nevertheless, inducible expression of the pi n2-CAT gene construct was demonstrated by northern hybridization, indi cating that pin2 has the potential to promote expression of introduced defense genes in response to pest attack or challenge. An established field test of transgenic poplars containing pin2-CAT has completed it s third season, and is being evaluated for transgene expression and ef fects of gene insertion on growth. Efforts to confirm transgene expres sion in poplars transformed with nos-PIN2 or 35s-PIN2 currently are un derway. Transgenic poplars expressing PIN2 will be evaluated for resis tance to insects (imported willow leaf beetle, Plagiodera versicolora LAICHARTING, and cottonwood leaf beetle, Chrysomela scripta F.) and fu ngal pathogens (Septoria musiva PECK. and Melampsora medusae THUM.).