T-DNA integration into the barley genome from single and double cassette vectors

Abstract

Stable genetic transformation of barley is routinely carried out by co-cultivation of immature zygotic embryos with Agrobacterium tumefaciens carrying binary vectors (1, 2). The target gene and the selection marker are located between the left and right 25-bp direct repeats of the T-DNA borders either within a single or in two adjacent tandemly arranged cassettes (3). The transfer of the T-DNA is polar; an intact right border is required for the transfer, whereas mutations in the left border have little effect on transfer efficiency (4). A single-stranded copy of the lower T-DNA strand is generated from endonucleolytic cleavage sites between the third and fourth bases of the border repeats. The strand is covered with the single-stranded DNA binding protein VirE2, covalently bound to the VirD2 protein at the 5Ј end and exported from the bacterium (4, 5). The VirE1 protein assists in secretion of virE2, which appears to form a voltage gated channel through the plasma membrane of the plant cell (6). The DNA is transported through the channel and guided by several proteins into the cell nucleus. The single-strand T-DNA then probably invades the DNA of the plant chromosome and is integrated by illegitimate recombination followed by secondstrand synthesis. Alternatively a double-stranded form might be generated first and then integrated into the chromosome (5).Details of integration have been primarily investigated in tobacco and Arabidopsis (7-10). Small deletions, base substitutions, duplicated border, and genomic sequences are found around the T-DNA͞plant DNA junctions. Co-integration into a chromosome locus after extrachromosomal second strand synthesis and ligation of the different T-DNA molecules, followed by docking and insertion via double-stranded breaks and repair synthesis is supported by analysis of co-integration of two or more T-DNAs in the same locus (11). All ten possible combinations-i.e., inverted as well as tandem arrangementsoccurred with regard to the left and right borders. The junction regions of direct repeats of T-DNA were investigated by selecting these with a vector containing a promoterless kanamycin phosphotransferase gene near the right border and a 35S CaMV promoter near the left border (12). Transformants from two T-DNA strands linking the promoter with the gene were selected on media containing kanamycin and revealed integrated T-DNA strands with a precise junction as well as integrations with 8 to 293 bp of filler DNA. Binary vectors carrying a neomycin phosphotransferase gene driven by a nopalinsynthase promoter within the T-DNA and a ␤-glucuronidase (gus) reporter gene under the control of a mannopine synthase promoter outside the left or right border resulted in 75% of transgenic tobacco plants containing the uidA (gus) gene unlinked or linked to the left or right border of the integrated T-DNA (13). Such incorporation of vector backbone sequences can be avoided by cotransformation of the target gene with the virD1, virD2, and virE2 virulence genes within the left and right border ...

Genetically engineered stem rust resistance in barley using the Rpg1 gene

“…Plant H228.40, with two putatively linked transgene copies segregating 3:1 for resistant and susceptible progeny, yielded 74 bp genomic DNA unidentifiable as a specific gene region in the BLAST search. In plant H228.19 with five transgene copies at four independent locations, a left border in tandem with a right border was isolated, indicating that two of the copies are inserted in a tandem direct repeat configuration, as is the rule in barley (24). Plant H228.1, with nine inserted transgene copies and a seedling lethal phenotype, yielded a 60-bp unidentifiable genomic flanking sequence and three highly scrambled flanking sequences.…”

“…DNA sequences adjacent to the left border of the integrated T-DNA from plasmid pNRG040 were isolated and cloned into SmaI restricted vector PUC18 as described (24). The DNA insert was sequenced with the universal M13 forward sequencing primer by using the BigDye Terminator system on an ABI Prism 377 DNA sequencer (Applied Biosystems) at Amplicon (Pullman, WA).…”

Integration and characterization of T-DNA insertion in upland cotton

“…In a report on aspen, 16 of 20 LB regions were deleted, ranging from 2 to 24 bp (Kumar & Fladung 2000). In a study of barley, 36 out of 39 LB regions were deleted, ranging from 1 to 95 bp from the cleavage site (Stahl et al 2002). These results suggest that T-DNA integration mechanisms are similar between monocotyledonous and dicotyledonous species.…”

Genetic Transformation of Triticeae Cereals for Molecular Farming

“…The earliest published account of the use of cereal grain to express human genes concerned the five proteins antithrombin III, 1-antitrypsin, lysozyme, serum albumin and lactoferrin (Stahl et al, 2002). Here, the concern was not the quantity or quality of the recombinant proteins, but rather the detection of the T-DNA integration sites in the barley genome.…”