Plant Molecular Farming Utilizes Transgenic Plants Biology Essay

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The process of plant molecular farming (PMF) utilizes transgenic plants for the production of high‑value recombinant proteins {Twyman, 2003 #1}. Transgenic plant production platforms possess several potential advantages when compared to the alternative production platforms currently being used, namely mammalian and microbial cultures. The main advantage of PMF is the potential for rapid and large-scale production at a very low cost. Additional advantages of using transgenic plant production platforms include their ability to produce proteins which require post-translational modification (complex proteins), ease of purification and low risk of mammalian pathogen contamination {Breyer, 2009 #2}. All transgenic plants intended for PMF field use would be required to undergo rigorous health and environmental risk assessment before they can be used; however the practice of PMF raises multiple questions with regard to biosafety and risk management. The recent focus on PMF biosafety suggests the existence of possible risks. These risks which arise from PMF manifest in two distinct categories. The first and most broad category applies to every transgenic plant (even those not used for PMF) and concerns the risk of transgene pollution to the environment and/or non-specific organisms. The second and more focused category concerns to the risk of product safety with respect to recombinant protein pollution in the environment and through product administration to humans and animals. In this essay, I intend to outline the environmental and health risks associated with transgenic plants and focus on the use of transient expression technology as a potential solution for these risk factors.

Transgene pollution as a result of PMF practices may occur via the spread of a foreign gene(s) to a surrounding environment. The mechanism of gene flow is responsible for the transfer of foreign genes from transgenic to non-transgenic plants, related wild species or microorganisms. The impact of transgenic DNA integration into foreign species could severely affect a wide variety of intertwined natural and agricultural systems. For example, the incorporation of a transgene into a food crop provides the opportunity for its protein product to enter the food chain as well disrupting the genome of that species. The impact of primary transgene (gene encoding the recombinant protein of economic value) transfer on the survival and reproduction of wild species would be difficult to predict. However, it is undesirable for recombinant proteins which are intended for disease treatment or commercial application to be expressed in natural plant populations including non-feed and food crops and microorganisms. Analagous to the former scenario, the transfer of secondary transgenic material (selectable marker DNA, fragments of the DNA vector) could equally impact a natural ecosystem. The risk with respect to secondary transgenic material is focused on the transfer of pesticide and antibiotic resistance genes being transferred not only to other wild plant species, but to soil and animal pathogen microbes. The spread of superfluous genetic information is unwanted due to the negative impact it may create on other organisms and their relationships with one another.

Transgene integration into wild plant and microbial species follows two distinct routes: vertical and horizontal gene transfer. Vertical gene transfer is the process by which a transgenic material flows between plants that are partially sexually compatible (). Vertical gene transfer is the most widespread form of transgene pollution and occurs via the dispersal of transgenic pollen (Eastham and Sweet), which results in a hybrid plant with a transgenic paternal lineage. This method of gene flow has been documented between transgenic and non-transgenic varieties of the same crop species () in addition to sexually compatible wild species. Mikkelsen et al. (1996) observed that secondary transgenic material (herbicide resistance marker) was transferred from transgenic Brassica napus to its naturally growing relative Brassica campestris. Solutions to mitigate vertical gene transfer have proven ineffective as a method of complete physical containment of pollen is extremely difficult to achieve. Additionally, the colonization of a natural environment could occur through a hybrid with a transgenic maternal lineage. Thus seed dispersal could occur during growth, harvesting or transport.

Horizontal gene transfer occurs when transgenic material is integrated into species which are not sexually compatible, such as plant to microbe. This process usually results in the incorporation of secondary transgenic material such as pesticide and antibiotic resistance genes into common soil microbes such as Agrobacterium tumefaciens. Tremendous risk occurs when these benign microbes transfer this secondary transgenic material to other potentially pathogenic ones, or to the digestive tracts of herbivores resulting in potentially new strains of resistance bacteria. However, the risks of horizontal gene transfer are now considered to be highly unlikely due to lack of evidence. Kay et al. (2002) showed the horizontal transfer of antibiotic marker genes occurred only under highly modified and idealized conditions in which the bacteria contained a sequence homologous to the transgenic region of the plant. No gene flow was demonstrated between the plant and wild type strains of the bacteria. Secondly, in the event of secondary gene transfer from a transgenic plant into bacteria, a selective pressure would be needed for the maintenance of the transgene in a bacterial population. Since the majority of natural plants possess antibiotic-resistant forms of bacteria on them, it would seem more likely that secondary transgenic material could move to human pathogens (Smalla 2000). Although it has been demonstrated that DNA can be taken up from saliva by human oral bacteria and that cells lining the gastrointestinal tract can incorporate DNA from the gut, a lack of evidence exists for this mechanism to have incorporated transgenic information into a bacterial population (glypohosate resistant plants reference). Biosafety research has indicated that horizontal gene transfer is unlikely to represent a significant hazard, thus focusing on ways to prevent transgene pollution by vertical gene transfer.

The second risk category of product safety and protein pollution represent an additional environmental hazard. Insects, herbivorous animals and microorganisms that rely on a transgenic plant as a source of energy will then ingest transgenic plant material which contains recombinant protein products resulting in protein pollution. These recombinant proteins may prove to be toxic to the primary consumer of the plant, or could result in its bioaccumulation throughout a food chain, thereby affecting animals which do not interact with the transgenic plant at all. The majority of recombinant proteins expressed in plants are directed to the secretory pathway (for accurate folding) (ref) and accumulate in the space between cell wall and membrane, thus can leak into the immediate environment or leaf surface. Furthermore, decaying transgenic plant material and its potential consumption by detritivores remains a major biosafety risk. The lasting effects of protein pollution entering soil and drainage water have not been investigated thus providing the potential for multiple unseen hazards. Ultimately, in an anthropocentric society, the impact of recombinant proteins on human and livestock health is of utmost importance in the field of PMF. The majority of recombinant proteins produced by transgenic plants are identical to their native counterparts, however once purified, the recombinant protein may be contaminated with toxic metabolites native to the plant, or the product itself may prove harmful. Complex recombinant proteins require post-translational modifications which can differ from the same protein derived from its native source. The process of glycosylation consisting of the enzyme‑mediated addition of a carbohydrate unit to a recombinant protein surface is one type of post-translational modification which can differ between plants and animals. The structures of the glycan chains produced in plants lack terminal sialic acid residues and instead possess the plant specific residues α-1,3-fucose and β-1,2-xylose (Cabanes-Macheteau 1999). These differences in post-translational modification may lead to recombinant proteins derived from PMF sources being inactive or potentially immunogenic. Human Erythropoietin (EPO) possesses a half-life of approximately 6-8 hours attributed to its glycosylation. However, plant-derived EPO which lacks the terminal sialic acid residue only possesses a half-life of five minutes or less (Fukada 1989). Chargelegue et al. (2000) demonstrated that recombinant human antibodies derived from transgenic plants were not immunogenic in a mouse model of testing. Recently, it has been proven that by creating a transgenic plant with both the foreign gene and human glycosyltransferase, it is possible to modify the glycosylation pattern of the recombinant protein thereby mimicking its native form (Bakker 2001). Currently, the required method used to minimize human illness from a recombinant protein is to conduct biological testing to ensure the function and low-immunogenicity of the recombinant protein.

Transgenic plant material (DNA) or its recombinant protein products carry multiple risks including the potential to spread between related crop species, from non-food to feed crops, enter the food chain of humans and animals, leach into the environment due to poor waste management and through exposure to non-target organisms and unintentional mixing during harvesting and transport. The aforementioned risks stem from the creation of stable transgenic plants harbouring copies of the transgene permanently integrated in its nuclear genome. This permanent incorporation of foreign genetic material allows for a transgenic plant to pass it on through reproduction while allowing the constant interaction of a gene product with the environment. These spatial and temporal behaviours of stable transgenic plants prevent them from being contained both physically and biologically. A solution to these problems is to use the method of Agrobacterium-mediated transient expression for the production of valuable foreign proteins (). Transient gene expression in plants allows for the accumulation of large quantities of recombinant protein in a rapid timescale, without the need for a stably transformed plant. Fraley et al. (1984) described this method of expression however it has only recently been applied as a viable solution in PMF.

The soil phytopathogenic bacterium Agrobacterium tumefaciens is a natural genetic engineer whose internal DNA transport system has been greatly studied by researchers. The result was a series of attenuated laboratory strains which were able to provide a vector to shuttle transgenes to plants (Lacroix 2006). Once A. tumefaciens begins interacting with the plant cell, it attaches to its surface. Following this, the bacterium is able to transfer a specialized portion of its DNA (T-DNA) into the plant cell and stably integrate it into an area of the plant's chromosome. This results in a stable transgenic plant. However, recent studies have shown that this transformation process also yields numerous copies of single-stranded T-DNA which exist for several days in the host cell stabilized in a bacterial protein complex.