Production of Ice Nucleation Deficient (Ice-) Mutants of the Epiphytic Strains of Erwinia herbicola

To mutate the Ice Nucleation Active (INA) gene in Erwinia herbicola strains, Tn-5 transposon carried by Psup2021 plasmid was used. This plasmid was transferred to the bacterial cells by electroporation. Electrotransformation was carried out for 2.5 ms at 1800 v and 1 mm distance between the electrodes. Polymerase chain reaction was used for determination of presence or loss of INA gene, using a pair of primers designed for inaZ gene. The ice nucleation activity was tested by the freezing assay technique. The ice- phenotype of the putative mutants were assayed through spraying sunflower seedlings, grown under aseptic condition, with 107 to 108 colony forming units per milliliter of the parent and transformant strains. The treated seedlings were kept at -8°C to -10°C for 8-10 hours and then transferred back to ambient (laboratory) condition for recovery or expression of freezing damage syndrome. Three out of ten electroporated strains yielded ice- mutants that failed to freeze water droplets at -8°C and produce no freezing injury on sunflower seedlings.