Transgenic tobacco plants constitutively expressing acidic chitinase from cucumber

Transgenic tobacco plants (Nicotiana tabacum L., cv. Petit Havana) were pro duced by inoculation of leaf discs with Agrobacterium tumefaciens LBA 4404 harbouring a disarmed binary vector pBIC1 containing a class III cucumber c hitinase cDNA. Tobacco cell colonies resistant to 100 mg/L kanamycin were s elected and regenerated into complete, fertile plants. Molecular analysis b y polymerase chain reaction and Southern hybridisation confirmed the integr ation of various numbers of T-DNAs into the plant genome. A single copy of T-DNA was found in six plants (50%), the remaining six plants contained mul tiple copies of T-DNAs either at one locus as tandem or inverted copies, or as independently integrated copies at different loci. Integration of incom plete T-DNA was found only in one plant. Constitutive expression of the chi tinase in transformed leaves and correct post-translational processing of t he mature enzyme leading to extracellular localisation of the enzyme in tra nsgenic plants were detected by Western blot analysis. The level of transge ne expression was not in correlation with the observed copy number of the i ntegrated chitinase gene. Using the OBR (Ostazin brilliant red)-chitin plat e assay, a substantially higher chitinase activity was detected in some tra nsgenic tobacco plants than in control, non-transformed plants.