Last modified July 31, 1997
Background:
KYRT1 (Torisky et al., 1997) is a disarmed version of A. tumefaciens strain Chry 5 (Bush and Pueppke, 1991). The wild type strain Chry 5 (Kovacs and Pueppke, 1993; Kovacs and Pueppke, 1994) has been shown to have high virulence on many solanaceous and non-solanaceous crops. We have recently determined this strain to be very effective for the transformation of soybean (unpublished data).
Derivation and Utilization:
KYRT1 has a Chry 5 chromosomal background and was selected for resistance to Rifampicin at 100 mg/L. A double crossover recombination event was forced that replaced a significant portion of the T-DNA along with the right border, effectively disarming this strain. The double crossover DNA sequence that was used to disarm the Ti plasmid contains a Beta-lactamase gene (selected for with Carbenicillin at 100 mg/L). The loss of the plasmid carrying the double crossover fragment of T-DNA was forced by introducing the pHIJ1 incompatibility plasmid of Hirsch and Berenger (1984). This pHIJ1 plasmid contains genes for resistance to Chloramphenicol (CAT), Streptomycin/Spectinomycin (Str), and Gentamycin (Gmr). A pBin 19 type binary plasmid can be introduced and seledcted for with Kanamycin at 200 mg/L. Cells harboring the binary plasmid will lose the incP plasmid as both have RK2 origins of replication, only one of which will be maintained by the host KYRT1. We have successfully used tri-parental, freeze-thaw, and electroporation based protocols to move plasmids of interest into this strain.
Growth and Transformation:
KYRT1 is a relatively rapid growing strain that may exhibit a tendency to clump when grown to high densities. Optimal transformation occurs when cells are harvested in early to mid log phase. After co-cultivation with plant tissue KYRT1 can be killed with Cefoxatime (250-500 mg/L), Vancomycin (100 mg/L), Timentin (50-100 mg/L), or Tetracycline (15-50 mg/L).
Request KYRT1:
KYRT1 is currently available for research purposes. To enquire about obtaining this strain, please contact:
Dr. G.B. Collins,
KYRT1 Request
Plant Sciences Building
1405 Veterans Drive
University of Kentucky
Lexington, KY 40546-0312
References:
Bush, A. L., and S.G. Pueppke. 1991. Characterization of an unusual new Agrobacterium tumefaciens strain from Chrysanthemum moriflorium Ram. Appl. Environm. Microbiol. 57: 2468-2472.
Hirsch, P.R., and J.E. Berenger. 1984. A physical map of pPH1JI and pJB4JI. Plasmid 12:139-141.
Kovacs, L., and S.G. Pueppke. 1993. The chromosomal background of mapping and genetic organization of Agrobacterium tumefaciens Chry 5 conditions high virulence on soybean. Mol. Plant-Microbe Interact. 6:601-608.
Kovacs, L., and S.G. Pueppke. 1994. Mapping and genetic organization of pTiChry5, a novel Ti plasmid from a highly virulent Agrobacterium tumefaciens strain. Mol. Gen. Genet. 242:327-336.
Torisky, R.S., L. Kovacs, S. Avdiushko, J.D. Newman, A.G. Hunt, and G.B. Collins. 1997. Development of a binary vector system for plant transformation based on the supervirulent Agrobacterium tumefaciens strain Chry 5. Plant Cell Reports. 17:102-108.