Enhanced Nitrogen Fixation Capabilities of Soybean Rhizobia by Inter- and Intra-specific Cell Fusion

A procedure for protoplast isolation and regeneration in rhizobia has been established. The use of 1% N-laurylsarcosine in pre-washing of the cells facilitated the cellular lysis by incubation for 1 hr in a reaction solution composed of Tris-HCl buffer (pH 7.5) containing 0.6 M MgSO4 and 5 mg/ml lysozyme. Exchanging of the solution at the half-way point of the reaction led to sufficient protoplast formation. The prepared protoplasts could regenerate at rates ranging from 3×10-2 to 6.4×10-3. The polyethylene glycol treatment was adopted for inter- and intra-specific fusion of protoplasts. The fused protoplasts between rhizobia with two different auxotrophic markers were regenerated by plating in a soft agar layer of a minimum medium containing 0.6 M mannitol at the regeneration rate of 10-7 levels. After the several dozens of repeated subcultures the intra-specific fusion products between Bradyrhizobium japonicum and the inter-specific fusion products of B. japonicum with Sinorhizobium fredii were found capable of forming nodules against the host plant in the pot experiment. The number of nodules produced by some of the intra-specific fusion products, after repeated subcultures, was 1.5 times higher than in the parental stocks. Besides, more than twice the nitrogenase activity was detected in the nodules of some of the intra- and inter-specific fusion products. These results suggested that production of highly effective nitrogen fixing strains by cell-fusion technique is possible.