A simple method to enrich an Agrobacterium‐transformed population for plants containing only T‐DNA sequences

Abstract

SummaryA simple modi®cation to standard binary vector design has been utilized to enrich an Agrobacteriumtransformed population for plants containing only T-DNA sequences. A lethal gene was incorporated into the non-T-DNA portion of a binary vector, along with a screenable marker. The resulting class of vectors is designated as NTL T-DNA vectors (non-T-DNA lethal gene-containing T-DNA vectors). The lethal gene used here is a CaMV 35S-barnase gene with an intron in the coding sequence (barnase-INT); the screenable marker is a pMAS-luciferase gene with an intron in the coding sequence (LUC-int). To evaluate the utility of this vector design, tobacco plants were transformed with either the NTL T-DNA vector or a control vector from which most of the barnase-INT gene was deleted. Populations of 50 transgenic plants were scored for LUC expression. The results indicated a dramatic reduction in the presence of non-T-DNA sequences in the transgenic population using the NTL T-DNA vector. Only one transgenic plant was found to be LUC+ using the NTL vector, compared with 42 of 50 plants using the control vector. Importantly, the ef®ciency with which transformed tobacco plants was obtained was reduced by no more than 30%. The reduction in LUC+ transgenics was partially reversed when a barstar-expressing tobacco line was transformed, indicating that barnase expression was responsible for the reduced frequency of incorporating non-T-DNA sequences. Similar transformation results were obtained with tomato and grape. The incorporation of a barnase-INT gene outside the left border appears to provide a generally applicable tool for enriching an Agrobacterium-transformed population for plants containing only T-DNA sequences.

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

“…Hanson et al (132) showed that the incorporation of a toxic "killer" gene into the binary vector backbone sequences could severely reduce the percentage of transgenic plants containing such extra sequences. Remarkably, the transformation frequency of tobacco, tomato, and grape plants infected using this modified binary vector did not substantially differ from that of plants infected using a binary vector lacking the killer gene.…”

Intragenic vectors for gene transfer without foreign DNA

“…by PCR) or by targeted selection strategies. Inserting the barnase suicide gene into the vector backbone eliminates the recovery of plants expressing this gene and markedly reduces the frequency of transformed plants with unwanted vector backbone sequences (Hanson et al 1999). Negative selection markers such as the cytosine deaminase (codA) gene (Stougaard 1993) could accomplish the same result.…”

Current Status of Binary Vectors and Superbinary Vectors

“…This is a useful indication for the reduction of the transfer of vector backbone, although the effect of such a modification may be different depending on the types of vectors, strains, plant species, and other factors. Another method was to place a killer gene, whose product is harmful to plant cells, in the vector backbone so that the cells that acquired the backbone are eliminated (Hanson et al, 1999).…”