Intragenic vectors for plant transformation within gene pools.

Abstract

The design of vectors for plant transformation has recently progressed to the development of intragenic systems. This involves identifying plant-derived DNA sequences similar to important vector components. The most useful approach involves adjoining two fragments from plant genomes to form sequences that have the functional equivalence of vector elements such as: T-DNA borders for Agrobacterium-mediated transformation, bacterial origins of replication and bacterial selectable elements. Such DNA fragments have been identified from a wide range of plant species, suggesting that intragenic vectors can be constructed from the genome of any plant species. Intragenic vectors provide a mechanism for the well-defined genetic improvement of plants with the entire DNA destined for transfer originating from within the gene pool already available to plant breeders. In this manner, genes can be introgressed into elite cultivars in a single step without linkage drag and, most importantly, without the incorporation of foreign DNA. The resulting genetically modified (GM) plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. The genetic makeup of the resulting plants is equivalent to minor rearrangements or micro-translocations that could theoretically arise through natural or induced rearrangements of the endogenous genome. Given the public concerns over the deployment of GM crops in agriculture, especially for food crops, intragenic vector systems offer a more socially acceptable and responsible way forward for genetic engineering. It may especially help us to alleviate some of the ethical issues associated with the transfer of DNA across wide taxonomic boundaries. Plants derived from the use of intragenic vectors raise important issues concerning the definition, regulation and testing of GM plants. This review critically evaluates the progress toward the development and use of intragenic vectors and the implications of their use for the genetic improvement of crops.

Intragenic Agrobacterium-mediated gene transfer mimics micro-translocations without foreign DNA

“…The generation of intragenic plants described in this study is synonymous with micro-translocations occurring within genomes (Conner et al 2007 ; Barrell et al 2010 ). To demonstrate this, the chromosomal locations of the DNA fragments that were conjoined to assemble the intragenic T-DNA and their subsequent integration were used to generate a Circos plot (Krzywinski et al 2009 ) (Fig.…”

“…To construct an intragenic T-DNA region, only short DNA fragments need to be adjoined for assembly of functional T-DNA borders. Such short sequences are known to exist in many species (Conner et al 2007 ; Barrell et al 2010 ). The fragments from the N. tabacum genome used to gain this T-DNA functionality involved much longer sequences (Supplementary Figs S1 , S3 ) that could only be considered as being of N. tabacum origin.…”

“…Using a common 24 bp T-DNA consensus sequence: 5′GRCAGGATATATNNNNNKGTMAWS3′ (where R = G or A, K = T or G, S = G or C, M = C or A, W = A or T and N = any nucleotide) (Conner et al 2007 ; Barrell et al 2010 ), public EST databases were searched for the two motifs, 5′GRCAGGATATAT3′ and 5′KGTMAWS3′, that flank the five-nucleotide variable region. All possible nucleotide configurations of these two motifs were used to search plant EST databases using BLAST (Altschul et al 1997 ), while limiting the results to N. tabacum .…”

“…The intragenic vector concept involves the identification of functional vector elements within plant genomes and their assembly into vectors for gene transfer to plants (Conner et al 2007 ). For the transfer of genes within the gene pools available to plant breeders, this allows genetic modification without the introduction of foreign DNA (Barrell et al 2010 ). It thereby addresses many of the ethical and/or public concerns raised in the genetic engineering debate (Nielsen 2003 ; Dixon 2005 ; Myskja 2006 ).…”

Applications of biotechnology and genomics in potato improvement

“…Gelvin (); Zupan and Zambryski ()). Two strategies that have been developed to achieve the transfer of plant DNA within a species are cisgenic (Schouten et al ., ) and intragenic approaches (Barrell et al ., ; Conner et al ., ). Agrobacterium is used to transfer the intragenic or cisgenic genes and T‐DNA into the plant host.…”