ENHANCED NITROGEN-FIXATION CAPABILITIES O F SOYBEAN RHIZOBIA BY INTER-SPECIFIC 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 th e cells facilitated the cellular lysis by incubation for 1 hr in a rea ction solution composed of Tris-HCl buffer (pH 7.5) containing 0.6 M M gSO4 and 5 mg/ml lysozyme. Exchanging of the solution at the half-way point of the reaction led to sufficient protoplast formation. The prep ared protoplasts could regenerate at rates ranging from 3X10(-2) to 6. 4X10(-3). The polyethylene glycol treatment was adopted for inter- and intra-specific fusion of protoplasts. The fused protoplasts between r hizobia with two different auxotrophic markers were regenerated by pla ting in a soft agar layer of a minimum medium containing 0.6 M mannito l at the regeneration rate of 10(-7) levels. After the several dozens of repeated subcultures the intra-specific fusion products between Bro dyrhizobium japonicum and the inter-specific fusion products of B. jap onicum with Sinorhizobium fredii were found capable of forming nodules against the host plant in the pot experiment. The number of nodules p roduced 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 resul ts suggested that production of highly effective nitrogen fixing strai ns by cell-fusion technique is possible.