Genetic approaches for studying transgene inheritance and genetic recombination in three successive generations of transformed tobacco

Abstract

Transgene integration into plant genomes is a complex process accompanied by molecular rearrangements. Classic methods that are normally used to study transgenic population genetics are generally inadequate for assessing such integration. Two major characteristics of transgenic populations are that a transgenic genome may harbor many copies of the transgene and that molecular rearrangements can create an unstable transgenic locus. In this work, we examined the segregation of T1, T2 and T3 transgenic tobacco progenies. Since transfer DNA (T-DNA) contains the NptII selectable marker gene that confers resistance to kanamycin, we used this characteristic in developing a method to estimate the number of functional inserts integrated into the genome. This approach was based on calculation of the theoretical segregation ratios in successive generations. Mendelian ratios of 3:1, 15:1 and 63:1 were confirmed for five transformation events whereas six transformation events yielded non-segregating progenies, a finding that raised questions about causal factors. A second approach based on a maximum likelihood method was performed to estimate recombination frequencies between linked inserts. Recombination estimates varied among transformation events and over generations. Some transgenic loci were unstable and evolved continuously to segregate independently in the T3 generation. Recombination and amplification of the transgene and filler DNA yielded additional transformed genotypes.

Randomly Detected Genetically Modified (GM) Maize (Zea mays L.) near a Transport Route Revealed a Fragile 45S rDNA Phenotype

“…However, a recent study on barley ( Hordeum vulgare L.), maize ( Zea mays L.), rice ( Oryza sativa L.), ryegrass ( Lolium perenne L.), and sorghum ( Sorghum bicolor L.) have shown that chemically induced replication and transcription stress caused epigenetic alterations leading to instability of the 45S rDNA cluster; thus, linking fragile site phenotype expression of the 45S rDNA locus to transcriptional defects and epigenetic alterations [33]. Furthermore, plant transformation is often associated with complex genome rearrangements and epigenetic alterations [38–40]. Taking these results in the context of genetic engineering, whether or not epigenetic changes caused by genetic modification could affect cytogenetic features of the 45S rDNA cluster is an intriguing subject for investigation.…”

Genetically transformed tobacco plants expressing synthetic EPSPS gene confer tolerance against glyphosate herbicide

“…Usually preferences are given to transgene events with single copy number and Agrobacterium mediated transformation is considered a method of choice for gene transformation because it offers low copy insertions, simple and precise integration patterns, and higher transgene expression. However, correlation between copy number and gene expression has also been declined in literature (Tizaoui and Kchouk 2012). The qRT-PCR analysis confirmed that CP4-EPSPS gene was expressing at elevated levels in some of the tested transgenic lines, while low in others.…”

Studies on Trans-Generational Transcriptional Silencing of <i>cry</i>1<i>Ac</i> Gene in Tobacco Transgenics

“…What we observe in our study, could also be a similar case. Studies have shown that level of expression among plants transformed with the same construct can differ considerably, and in many cases this has nothing to do with copy number [13] [14].…”