Abstract

An efficient shoot regeneration system has been developed involving organogenesis from stem tissue of the strawberry [Fragaria ananassa] 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 -&smallcap;D-glucuronide (X-Gluc) and PCR using specific primers permitted the identification of transformants. Inoculations were then carried out using the cowpea [Vigna unguiculata] protease [proteinase] 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 a-N-benzoyl-&smallcap;D&smallcap;L-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. PCR was used to confirm the presence of the gene sequence.