Agrohacterium tumefaciens strains were isolated from 11 different ecological and temporal regions Punjab, Pakistan. From these ecological and temporal regions 62 A. tumefaciens strains were isolated from 33 plants belonging to 18 different families. These strains were characterized morphologically, biochemically and physiologically. All stains belong to biotype I as they produced ketolactose from lactose. Strains Shared morphological, biochemical and physiological characters, however they exhibited minor differences in colony and cell sizes, in some biochemical and physiological characters. Optimum temperature for the growth of these strains was 28 C. Irrespective of temperature, i.e. 28° or 37°C, all strains showed specific affinity for pH 6. Metallic salts profiles revealed that all strains showed resistance against Ba (except Pg 1), Mn, Pb and Zn salts, while all were sensitive to Cr salt. or other metallic salts tested various responses were observed. Antibiotic resistance/ sensitivity test of the strains showed that strains have differences in the antibiotic spectrum. However most of them could tolerate ampicillin, carbenicillin and kanamycin, while some strains exhibited resistance against streptomycin and tetracycline. Host range and host specificity experiments depicted that, except MA2, PR 1, ST3 and Pg I, all strains have wide host range causing tumorigenesis on atleast 8 host plants (out of 15 plants) tested in the experiment. Some of the strains induced tumors on monocots plants, either on Triticum aestivum (5 strains) or Zea mays (19 strains), while four of these strains, CC2, DE I, FV 1 and PP 1, caused tumorigenesis both on T aestivum and Z. mays. However on other monocots, Allium cepa and Oryza sativa none of the strain induced tumorigenesis. Out 62 strains 21 strains (AMI, CF3, CD I, EI1, EI2, ED, Pg2, MAS, MA6, Ma4, MI2, MD, MI4, PR" ST3, ST4, TCl, BPI, BP2, MAl and MA3) were sensitive to carbenicillin and ka1amycin and 17 strains (AM I, CF3, CD1, EI1, EI2, ED, Pg2, MA5, MA6, Ma4, MI2, MI3, MI4, PRI, ST3, ST4 and TCI) were utilized in the transformation experiments by co-cultivating calluses (of Brassica oleracea L.).and out of these 17 strains 7 strains (AM1, EI1, EI2, E13, MI2, MI3 and MI4), which exhibited better callus proliferation response were used in the co-cultivation experiments with explants (of Capsicum annuum L., Linum usitatissimum L, Lycopersicon esculentum Mill., Medicago falcata L., Raphamus sativus L. and Spinacea oleracea L.) and expression of T-DNA was monitored on auxin free M,S, medium, while 4 strains (which were obtained from the tumors of plants growing in saline patches) could resist 1M NaC1 in the growth medium and with these strains transformation of B oleracea calluses was carried out via co-cultivation and expression of T-DNA genes was observed on auxin free M,S, medium supplemented with 1M NaC1. In callus co-cultivation experiment all strains demonstrated T-DNA transfer and expression. However 7 strains (AMI, EI1, EI2, EI3, MI2, MI3 and MI4) showed better callus proliferation response. Three strains AM I, EI2 and ED gave efficient callus proliferation response accompanied by rhizogenesis and, only AM1 co-cultivated calli exhibit, caulogenesis. Auxin content of transformed calluses was higher than the control calluses (calluses growing on M.S. medium supplemented with 2,4-D and coconut mil). Flax leaf discs transformation was carried out with 7 A. tumefaciens strains (AI, EI1, EI2, E13, MI2, MI3 and MI4). AMI, EI2 and EI3 were successful in transforming flax leaf discs co-cultivated for 2-3 minutes duration at 28°C. The leaf discs co-cultivated with these strains exhibited rhizogenic, caulogenic and callogepic responses. Maximum variability was showed by the leaf discs co-cultivated with AMI: Auxin content of transformed leaf discs were higher than the control (fresh tissues). The transformed tissues also showed additional protein bands on the S S-P AGE. Transformation of calluses, with 17 strains (AM 1, CF3, CD I, EI1, EI2, 13, Pg2, MAS, MA6, Ma4, MI2, M13, MI4, PRI, ST3, ST4 and TCI), and explants with 7 strains (AMI, EI1, EI2, EI3, MI2, MI3 and MI4), carried out for three different time durations (1,3 and 10 minutes) at three different temperatures (24°, 280° and 37°C) reflected that for maximum T-DNA transfer and expression, from these trains, requires co-cultivation for 3 minutes at 28°C.
The genetical analysis of salt tolerant strains, BP1, BP2, MAl and MA3, revealed that salt tolerant genes were plasmid born as transformants were scored in three E. coli K 12 strains C600, NEM259 and TG 1 with all the strains. While transconjugants were stored with BP 1, BP2 and MA 1 by using E. coli K 12 strain HB 101 and CSR603 as host, both at 28° and 37°C. However better conjugation frequency was obtaine4 in E. coli K 12 strain HB 101 at 28°C. Plasmids pTiSH6, pTiSH7, pTiSH29 and pTiSH31 present in BP1, BP2, MA1 and MA3 respectively were successfully cured with pH (8) treatment both at 28° and 37°C. With the loss pf plasmid salt tolerant property of the strain was also lost, confirming that salt tolerance in these strains was encoded by plasmid. The plasmid free strains obtained at 37°C lost viability, while plasmid free derivatives obtained at 28°C revealed that their tumorigenic ability was lost as they were unable to form tumors on C. annuum and L. esculentum. Transformation of salt sensitive genome of B. oleracea with these four strains demonstrated that calluses proliferated on auxin free M. S. medium supplemented with 1M NaCl, hence confirming that salt tolerance could be transferred to plant genome. The auxin content of the transformed calli was higher that control calluses (rowing on hormone supplemented M. S. medium). The transformed calli also exhibited extra proteins bands.