Andrew T Binns, 35Hales Corners, WI

6051757, Apr 18, 2000

Jun 5, 1995

8/464542

Kenneth Allen Barton - Madison WI
Andrew Norton Binns - Philadelphia PA
Antonius J. M. Matzke - Salzburg, AT

Washington University - St. Louis MO

C12N 1582
C12N 1584
A01H 104

800294

Inactivation of the cytokinin autonomy gene of T-DNA in broad host range Ti plasmid produces mutant T-DNA vectors suitable for insertion of foreign genes; insertion of the mutant T-DNA by an in vitro tissue culture technique or any other technique into plant cells produces genetically engineered plant cells that can be regenerated into complete plants with roots. The inactivation of the cytokinin autonomy gene disarms the Ti plasmid and produces a useful gene vector for higher plants. The inactivation of the cytokinin gene may be accomplished by techniques such as point mutation, inversion, deletion, transposition, substitution or insertion. In the case of tobacco, for example, transformation of tobacco stem segments with engineered bacterial strains resulting from the insertion of DNA encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the "rooty locus" produced transformed plant cells that were capable of regeneration into intact, normal tobacco plants. The yeast gene and entire T-DNA were present in the regenerated plants in multiple copies, and nopaline was found in all tissues. The plants were fertile and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA.