In order to obtain marker-free transgenic rice with improved osmotic stress resistance, expression vector named as pABRII-MTLDL contains mtld gene and lacks plant selectable marker gene ( hph ), was constructed from pCabMTLDII and pTRA132 to be used in co-transformation technique with another plasmid contained hygromycine phosphotransferase gene as plant selectable marker. In co-transformation technique gene of interest and plant selectable marker gene are located on different vectors and introduce into plants genome as unlinked fragments. In this way it would be possible to achieve marker free transgenic plant during the generations of segregation. The digestion of pCabMTLDII(a 7.7 kb plasmid which contains both mtld and hph genes) with the Eco RI and Bam HI enzymes resulted in a 1.8 kb fragment, which contained the entire E. coli coding region for the mt1d structural gene together with nopaline synthease terminator. This 1.8 kilobase fragment was then inserted into the Eco RI and Bam HI restriction sites of the modified pTRA132(a 4.4 kb plasmid which contains only hph gene), in which the hph coding sequence and nopaline synthease terminator had been deleted. The cloning of the fragment into the modified vector pTRA132, resulted in a plasmid designated pABRII-MTLD. The recovery of these 3 kb and 1.8 kb fragment from agarose gel was done by High Pure PCR Product Purification Kit and the ligation of these fragment was done by T 4 ligase enzyme using Rapid DNA Ligation Kit. This resulted plasmid pABRIIMTLD was about 4.9 kb. Mtld gene in the resulted cassette (pABRII-MTLD) conferes elevated tolerance against drought and salinity stresses and it is controlled by mosaic virus 35S promoter. The plasmid was analyzed by digestion with Bam HI ، Eco RI ، Hin dIII و pst I enzymes and PCR analysis in which accuracy of construction were confirmed. Then plasmid pABRIIMTLD with pTRA132(containing hph gene) in ratio of 1:1 introduced into high cell-dividing and regeneration ability embryogenic calli derived from the mature seeds of a rice variety, Hashemi , by biolistic transformation method. About 100 small and globular embryogenic calli were arranged on the center of petridishes and the explant of each petri was shot twice.Then selection of transformed cell was carried out on callus induction and also regeneration medium using 50 mg/1 hygromycin B as antibiotic.Transformed cells which contain plant selectable marker gene can survive on medium containing antibiotic. Putatively transformed cell clusters were identified from the bombarded tissues after 3 rounds selection (every 14-21 days) on callus induction, N6 medium, containing 50 mg/1 hygromycine B. After 6-8 weeks selection, hygromycine B