Transformation of persimmon with a pear fruit polygalacturonase inhibiting protein (PGIP) gene

Abstract

Persimmon ( Diospyros kaki cv. Jiro) was transformed with the gene encoding the pear fruit ( Pyrus communis) polygalacturonase inhibiting protein (PGIP) using Agrobacterium tumefaciens EHA101. Two plasmid constructions were used for transformation; pDU94.0928 containing chimeric genes of PGIP, GUS and NPTII in its T-DNA region, and pYS95.091 containing only the GUS and NPTII sequences (control transformations). Among 165 callus lines obtained from infection by A. tumefaciens with pDU94.0928, 15 showed GUS activity. A PGIP gene fragment of 132 bp was amplified by PCR in nine of 15 callus lines. Shoots were regenerated from three of the nine PGIP-containing callus lines. The transformation of the regenerated shoots was confirmed by PCR and Southern blot analyses using NTPII, GUS and pear PGIP probes. PGIP expression in shoot tissues was analyzed by western blot analysis. The protein expression was observed in all three shoot lines regenerated from transformed calli with the PGIP gene. Fungal PG activity was inhibited by crude extracts from shoots transformed with the PGIP gene substantially more than by those from the control transformants obtained from the transformation with pYS95.091.