New Agrobacterium helper plasmids for gene transfer to plants Elizabeth E. Hood, Stanton B. Gelvin, Leo S. Melchers, and Andre Hoekema We describe the construction of new helper Ti plasmids for Agrobacterium-mediated plant transformation. These plasmids are derived from three different Agrobacterium tumefaciens Ti plasmids: the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying the vir genes that will complement binary vectors in trans. Data are included that demonstrate strain utility. The advantages of Agrobacterium strains harbouring these 'disarmed' Ti plasmids for plant transformation via Agrobacterium are discussed. Keywords: Agrobacterium; plant transformation; helper plasmids; host range; Ti plasmid, T-DNA Introduction A number of methods have been developed for plant genetic transformation, including particle acceleration, electroporation and polyethylene glycol permeabilization of protoplasts, and Agrobacterium-mediated DNA transfer. With the discovery of the natural ability of A. tumefaciens to transfer and integrate part of its DNA stably into the genome of plants, various strategies for the exploitation of A. tumefaciens for gene transfer to plants were developed. The method involves incubating tissue from a selected plant with an A. tumefaciens strain harbouring a Ti plasmid containing the genes of interest in the T-DNA, allowing transfer to occur, then regenerating transformed plants. All the Agrobacterium-mediated transformation methods are based on the observation that the DNA segment transferred by the bacterium can be replaced with foreign DNA, provided transfer signals are present. In many crop species the production of transgenic plants via Agrobacterium-mediated transformation has been achieved. However, a major limitation of Agrobacterium-mediated gene transfer is the host range of A. tumefaciens on plants of interest at either the species or cultivar level. Several A. tumefaciens strains have been identified that harbour broad host range Ti plasmids. These strains each infect a large variety of plants and, although their host ranges overlap, they are not identical. In spite of this, only a few of these oncogenic strains have been disarmed for use in plant genetic engineering. One approach to circumventing the host range limitation of Agrobacterium-mediated gene transfer is to develop several non-oncogenic or disarmed strains useful for gene transfer to plants. Most of the wild-type A. tumefaciens strains that have been isolated from tumours on a wide variety of plants may be classified as octopine,New Agrobacterium helper plasmids for gene transfer to plants Elizabeth E. Hood, Stanton B. Gelvin, Leo S. Melchers, and Andre Hoekema We describe the construction of new helper Ti plasmids for Agrobacterium-mediated plant transformation. These plasmids are derived from three different Agrobacterium tumefaciens Ti plasmids: the octopine plasmid pTiB6, the nopaline plasmid pTiC58, and the L,L-succinamopine plasmid pTiBo542. The T-DNA regions of these plasmids were deleted using site-directed mutagenesis to yield replicons carrying the vir genes that will complement binary vectors in trans. Data are included that demonstrate strain utility. The advantages of Agrobacterium strains harbouring these 'disarmed' Ti plasmids for plant transformation via Agrobacterium are discussed. Keywords: Agrobacterium; plant transformation; helper plasmids; host range; Ti plasmid, T-DNA Introduction A number of methods have been developed for plant genetic transformation, including particle acceleration, electroporation and polyethylene glycol permeabilization of protoplasts, and Agrobacterium-mediated DNA transfer. With the discovery of the natural ability of A. tumefaciens to transfer and integrate part of its DNA stably into the genome of plants, various strategies for the exploitation of A. tumefaciens for gene transfer to plants were developed. The method involves incubating tissue from a selected plant with an A. tumefaciens strain harbouring a Ti plasmid containing the genes of interest in the T-DNA, allowing transfer to occur, then regenerating transformed plants. All the Agrobacterium-mediated transformation methods are based on the observation that the DNA segment transferred by the bacterium can be replaced with foreign DNA, provided transfer signals are present. In many crop species the production of transgenic plants via Agrobacterium-mediated transformation has been achieved. However, a major limitation of Agrobacterium-mediated gene transfer is the host range of A. tumefaciens on plants of interest at either the species or cultivar level. Several A. tumefaciens strains have been identified that harbour broad host range Ti plasmids. These strains each infect a large variety of plants and, although their host ranges overlap, they are not identical. In spite of this, only a few of these oncogenic strains have been disarmed for use in plant genetic engineering. One approach to circumventing the host range limitation of Agrobacterium-mediated gene transfer is to develop several non-oncogenic or disarmed strains useful for gene transfer to plants. Most of the wild-type A. tumefaciens strains that have been isolated from tumours on a wide variety of plants may be classified as octopine,