Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

Abstract

Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.

Fluorescence chromosome banding and FISH mapping in perennial ryegrass, Lolium perenne L.

“…Transgenesis is often used by agricultural biotechnologists to introduce traits of agronomic interest into crops [49–54] but instability in the expression of the introduced transgene is common [55–57]. The transgenes may unpredictably integrate at different locations in the host genome and be subject to “position effect” variation in expression patterns [51, 58].…”

Genetic transformation of barley: limiting factors

“…Sha et al (2004) examined T-DNA flanking regions in rice and they detected preferential insertion into the coding areas of the genome. Choi et al (2002) used fluorescence in situ hybridization for raw mapping of transgenes and screening for homozygous transgenic barley prepared by microprojectile bombardment. No preferential integration sites among the chromosomes were found; however, within a chromosome a distal preference for transgene integration was observed.…”

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

“…If molecular cytogenetic data is available in addition to the molecular evidence, it would give more accurate information for the GM plant and that will be helpful to explain the safety problems such as uncertian or unstable gene functions. FISH has been used to complement molecular data, especially to visualize single copy transgenes on chromosomes (Abranches et al, 2000;Dong et al, 2001;Jackson et al, 2001;Salvo-Garrido et al, 2001, 2004Choi et al, 2002;Svitashev et al, 2000;Romano et al 2003;Harwood et al, 2005;Travella et al, 2005;Santos et al, 2006). This technique makes it possible to map transgenes on a specific chromosome accurately.…”