Marker-Free Transgenic Plants
Dr. S. C. Kaushik
The production of genetically modified plants is a routine procedure for most species and essential for the commercialization of transgenic crops. Genes of interest are introduced commonly by Agrobacterium-mediated transformation, and become integrated at random positions in the genome. The co-introduction of selectable marker genes, especially antibiotic resistance genes is required for the initial selection of plant cells that are complemented with a new trait. Selectable marker genes (SMGs) have been extraordinarily useful in enabling plant transformation because of the low efficiency of transgene integration. The most used SMGs encode proteins resistant to antibiotics or herbicides and use negative selection, i.e., by killing nontransgenic tissue. However, there are perceived risks in wide-scale deployment of SMG-transgenic plants. The possible ecological risks formed by spread of these markers in the natural environment represent a major issue of debate. Commercialization of products from plant biotechnology is hampered largely by public concerns about possible risks related to the introduction of genetically modified (GM) plants. The presence of selectable marker genes, which include genes coding for antibiotic resistance and which are essential for the initial selection of transgenic plants is seen by regulatory agencies in various countries as undesirable. Government regulatory agencies of certain countries no longer allow the commercialization of crops with such markers. Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. Therefore, there is need for the development of techniques for the efficient production of "clean", marker-free transgenic plants.
A second issue of concern relates to the fact that transgenes integrate at random positions in the genome leading to possible unwanted side-effects (mutation) and unpredictable expression patterns. The lack of control over the position of the introduced DNA (resulting in unpredictable gene expression and potentially undesirable mutagenesis of important genes) makes the transformation procedures inefficient and subsequent molecular analysis, which is required for approval on the market, laborious and time-consuming. Thus the development of efficient techniques for the removal of selection markers, as well as the directed integration of transgenes at safe locations in the genome is of great interest to biotech companies. Furthermore, removal of selectable marker genes will also have a technical advantage, since the number of available selectable marker genes is limiting, and stacking of transgenes will become more and more desirable in the near future. This technique involves the development of novel approaches for the efficient production of marker-free transgenic plants harboring transgenes at predetermined genomic locations. This is important for the acceptance and application of GM plants and therefore research has recently been performed to develop marker-free systems. Most efficient selectable markers are antibiotic and herbicide resistance genes, which have perceived biosafety concerns.
There are many developments for effective transformation systems for generating marker-free transgenic plants, without the need for repeated transformation or sexual crossing. The development of marker-free GM products is likely to be advantageous for public acceptance and compliance with regulatory requirements. Generally, three approaches have been employed to generate marker-free transgenic plants. One approach involves removal of a selectable marker gene from transgenic plants (marker removal). A second method is to overexpress specific genes that are capable of promoting transgenic plant regeneration thus allowing their preferential regeneration compared with untransformed plants (marker-free transformation). The third method was developed from a combination of these two methods. One common method is site-specific DNA recombinase-based systems for marker removal. This system consists of a site-specific DNA recombinase (e.g. Cre, Flp or R) and its corresponding recognition sites (lox, FRT or RS, respectively). The recombinase specifically recognizes its target site, cleaves the double-stranded DNA and then specifically rejoins certain double DNA ends to complete the entire recombination process. Most of the current marker excision methods are based on this type of site-specific DNA recombination system. Recently marker-free strawberry transgenic plants using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events were developed. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies. This involved an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. This novel method of transient introduction of the recombinase proteins by Agrobacterium will contribute to development of a highly efficient method for stable directed integration of transgenes and targeted integration would make analysis of the regulation of gene expression more precise and less labor-intensive.
So, public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers.
About Author / Additional Info:
Dr. Suresh Kaushik
A Biotechnology Professional from India
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