New Binary Ti Vectors with Co-directional Replicons for Agrobacterium Tumefaciens-mediated Transformation of Higher Plants

Abstract

Small, high-yielding binary Ti vectors of Agrobacterium tumefaciens were constructed. All four basic components of the vector, ColE1 replicon (715 bp) for Escherichia coli, VS1 replicon (2,659 bp) for A. tumefaciens, a bacterial kanamycin resistance gene (999 bp), and the T-DNA region (170 bp), were modified to reduce the vector size to 4,566 bp and to introduce a number of mutations to increase the copy number and other functionality. The transcriptional direction of VS1 replicon can be the same as that of ColE1 replicon (co-directional transcription), or opposite (head-on transcription) as in the case of widely used vectors (pPZP or pCambia). The new binary vectors with co-directional transcription yielded up to four-fold higher E. coli transformation frequency, and 1.2 to 1.7-fold higher in A. tumefaciens than the head-on transcription. Compared to the pCambia vector, the new vectors have over ten-fold higher transformation frequencies in A. tumefaciens and five-fold greater DNA yields in E. coli. The proper functions of the new T-DNA borders and new plant selection marker genes were confirmed after tobacco leaf discs transformation. Genetic analysis of kanamycin resistance trait among T1 progeny indicates that the kanamycin resistance genes were integrated stably into a locus or closely linked loci of the nuclear chromosomal DNA of the primary transgenic plants and inherited to the second generation. A tetracycline-based binary Ti vectors was also constructed to facilitate efficient cloning afforded by the Gateway Technology for Agrobacterium-mediated transformation of higher plants. Because the Gateway Technology entry/donor vectors are kanamycin-based, tetracycline-based destination and expression vectors are easily selected for the antibiotic resistance in the E. coli. The Gateway expression vector with a gus gene was constructed. The appendices include a manuscript by Su G, Park S, Lee S, and Murai N entitled “Low co-cultivatin temperature at 20°C improved Agrobacterium tumefacciens-mediated transformation of tobacco leaf discs”. Provisional Patent (application number 61328250) entitled “Binary Ti vectors with Co-directional replicons for transformation of eukaryotes and prokaryotes” was applied.