Role of endophytic rhizobial plasmids in legume nodulation and plant growth promotion in rice

The plasmid curing experiment was carried out using the strains RREM36 and RREM25 conferring resistance to Chloramphenicol (75 μg/ml) and for Kanamycin (65 μg/ml) respectively. 0.1 ml aliquot of exponentially growing cultures (approximately 10⁶cells/ml) was spread on TY plates and incubated at 35 and 37°C. From these plates 100 colonies were picked up and streaked on TY and TY + Kanamycin (65 μg/ml) for RREM25 and TY + Chloramphenicol (75 μg/ml) for RREM36 and incubated at 28°C for 3 days. Number of cells in TY alone and TY plates supplemented with appropriate antibiotics was counted. TY broth with graded concentration of acrydine orange (0, 5, 10, 40, 45, 50, 55 μg/ml) was prepared and 0.1 ml of exponentially growing rhizobial cells were inoculated. After 7 days of growth cell suspension (100 μl) was spread on TY plates and incubated at 28°C for 3 days. 100 colonies from each type of plate were selected and re-streaked in TY and TY plate with respective antibiotic. It was observed that plasmids of cells of RREM 25 and RREM 36 were lost at 37°C. Gradual loss of plasmids starting from 35 to 60 per cent was observed with increase in incubation period and 85 per cent loss was recorded after 7 days of incubation. Gradual decrease in growth of these two strains was observed with increase in concentration of acrydine orange 45 μg/ml and at 55 μg/ml the plasmids were lost completely in RREM25 and RREM36.