Induced mutation in gene controlling ice nucleation activity by EMS, in two strains of Pseudomonas viridiflava like bacteria

Ice nucleation active bacteria in high populations are the cause of frost injury to the plants at higher subzero temperatures. Ice nucleation genetic base of bacteria is single chromosomal gene ina. To inducing mutations in ina gene, Pseudomonas viridiflava like bacteria were incubated with the mutagenic material (EMS) in 20 and 25 µl/ml concentrations.To track ina gene in isolates of P. viridiflava like polymerase chain reaction was used by ina gene special primer that was designed of conserved regions of gene in several species of ice nucleation bacteria. Ice nucleation activity was assessed using a drop of freezing. To investigate the loss of ice nucleation activity in suspensions containing 107 cells per ml (0.5 absorbance units at 600 nm) of each mutant was sprayed on orange seedlings and seedlings treated with sterile distilled water were used as negative controls. Seedlings were kept for 7 to 10 hours at 10 °C and then were returned to the greenhouse. Four mutant colonies were not able to create any frost damage on experimental seedlings.