Towards genetic based insect resistance in strawberry using the Cowpea trypsin inhibitor gene

An efficient shoot regeneration system has been developed involving organogenesis from stem tissue of the strawberry cultivars Melody, Rhapsody and Symphony. Regeneration also occurred in the presence of antibiotics. The system was shown to be suitable for transformation using the GUS:Intron marker gene construct. Histological analysis using the substrate 5-bromo-4-chloro-3-indolyl B-D-glucuronide (X-Gluc) and the polymerase chain reaction (PCR) using specific primers permitted the identification of transformants. Inoculations were then carried out using the Cowpea protease trypsin inhibitor (CpTi) gene construct. A simple visual assay technique was developed to identify those plants expressing CpTi, allowing them to be selected for further analysis. Trypsin acts on the substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) to produce p-nitroaniline (p-na). Trypsin inhibition by CpTi was easily detected as a reduction in or elimination of yellow colour development which occurred as the hydrolysis of BAPNA to p-na proceeded (Preiser, Schmitz, Maestracci & Crane, 1975). The polymerase chain reaction (PCR) was used to confirm the presence of the gene sequence.