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ECONEXUS
based on SBSTTA 10 Submission February 2005
Any biological containment system setting out to prevent gene flow of transgenes via pol en and seed must be 100% reliable and effective. V-GURTs is likely to only be as good as its weakest link. There are a number of known events which can interfere with At present there are a number of molecular containment reliable performance of any of the 4 components employed by strategies that aim to restrict gene flow either via pol en, seed V-GURTs. Further research is required to address the or sprouting of vegetative organs (e.g. tubers). Such strategies include male sterility, maternal inheritance, seed sterility, prevention of sprouting, apomixis and temporal and tissue Gene Silencing and epigenetic changes to DNA:
None of these containment systems claims to have the Gene silencing and epigenetic changes to transgenes capacity to prevent gene flow both for pollen and seed except have been observed repeatedly in transgenic plants, for V-GURTs, also known as Terminator Technology. especial y under stress conditions (Broer 1996, Meza et V-GURTs is a genetic use restriction technology that – in its al. 2001). RNA-mediated silencing and DNA methylation design - uses complex inducible gene expression systems to are considered to have evolved as part of a host defense make plants produce sterile seeds under specific conditions, mechanism, active against viruses and parasitic DNA which are active against transgenes (e.g. Riddihough and Pennisi 2001). As no field trials or green house trials, nor any details of a complete and ful y functional V-GURTs plant have been Recently, Srivastava and Ow (2003) for example, found reported in the peer-reviewed literature, there is no scientific that the site specific recombination system Cre/lox did not data available to analyse the performance of this technology perform as expected and the authors suggest that the Cre and its reliability as a whole. Consequently a performance and gene underwent a genetic or epigenetic change. risk analysis for V-GURTs can only be carried out in part, Whilst gene silencing of the repressor gene would lead to focussing on the different individual genetic constructs and sterile seeds, the silencing of either the recombinase or components or the different expression systems for which the toxin gene would result in viable seeds irrespective of whether the inducer was applied or not. The potential The basic design of V-GURTs as detailed in US patent
silencing of the late embryonic abundance (LEA) 5,723,765 by the USDA and Delta & Pine Land is composed promoter, which drives the toxin gene, is regarded by Daniel (2002) as a crucial drawback of the terminator design as put forward by the USDA and Delta & Pine • a cel toxin expressed in the late embryonic stage that wil result in sterile seed. The toxin gene is inactivated by a spacer (short sequence of DNA), which can be removed Reversibility of gene silencing (e.g. Mittelsten Scheid et by a recombinase enzyme. To this effect, the spacer is al. 1998) may further add to unpredictabilities, e.g. seed framed by recombinase recognition sites (e.g. lox sites for • a recombinase enzyme that can activate the toxin gene, • a repressor protein that blocks the recombinase gene Mutations:
Mutations frequently occur. Mutations of, for example, the Once the inducer (e.g. tetracycline) has been applied, it wil introduced recognition sites (e.g. lox sites) would result in remove the repressor protein from the promoter of the a permanently inactive toxin gene, leading to permanently recombinase gene, thus recombinase is produced, which in viable seeds, i.e. in this and al subsequent generations. turn wil remove the spacer from the toxin gene. This now Equal y, mutations to the recombinase promoter or gene al ows the expression of the toxin in the late embryonic stage would result in permanently viable seeds. Gene flow of of the seed, kil ing the cel s and thus the growing seed. There are other variants that use activators rather than Loss of promoter activity:
repressors, but fol ow the same general principles outlined above. Loss or reduction of promoter activity over time has been observed in a number of genetical y engineered systems. The main components of V-GURTs are thus a) an inactive The observed loss of promoter activity in the tetracycline- gene coding for a cel toxin, b) an inducible site specific inactivatable tTA expression system for example is recombination system, c) an inducible expression system presumed to be due to gene silencing (Tang et al. 2004). using external inducers and d) an external inducer. Leaking promoter systems:
disadvantage selective pressure wil favour genetic or epigenetic changes that lead to viable seeds or gene flow via Many of the promoters in the inducible expression pol en and capacity for reproduction, especial y for example systems tested show a low level basal activity rather than for transgenic trees. Effectiveness of V-GURTs applications zero basal activity. For example, leakiness of the may thus decrease over time and generations. tetracycline-inducible promoter system was reported by De Veylder et al. (2000). A further drawback of V-GURTs is that farmers growing conventional or traditional crops of the same species as the V- Insufficient induction of promoter systems by inducing
GURTs variety in neighbouring fields wil still find their crops contaminated by cross pol ination. Whilst this is a problem for If the inducing agent, e.g. tetracycline, does not reach the marketing their crops as GM free, especial y if the GM crops in target cel s in sufficient quantity, the system wil only be question were pharma crops, it may severely impact on food partial y activated, resulting in viable seeds or in pol en security. Farmers who save their traditional or conventional capable of giving rise to viable seeds in neighbouring seeds for replanting may find a significant percentage do not crops. As stated by Daniel (2002), “it wil be difficult to germinate as a result of cross pol ination, which in turn may ascertain whether al the seeds treated with the tetracycline inducer have triggered the gene switch (i.e. whether tetracycline has penetrated al the seeds.)” Unspecific or unintended induction of promoter system:
Many inducible promoters can be induced by more than Broer I (1996). Stress inactivation of foreign genes in one agent or by plant endogenous chemical agents. The transgenic plants. Field Crops Research 45(1-3): 19-25 AlcR based ethanol inducible system for example can be Daniel H (2002). Molecular strategies for gene containment in inappropriately triggered by endogenously produced transgenic crops. Nature Biotechnology 20:581-586 – see ethanol (due to anoxia) - (in Padidam 2003). also Research Errata, Nature Biotechnology 20:843 Segregation of the different genetic components during
De Veylder L, Beeckman T, van Montagu M and Inze D reproduction:
(2000). Increased leakiness of the tetracycline-inducible Triple-Op promoter in dividing cells renders it unsuitable It is crucial that functional components of V-GURTs and for high inducible levels of a dominant negative CDC2aAt the introduced GM trait remain linked during reproduction. gene. Journal of Experimental Botany 51(351):1647-1653 For example, if separation resulting from segregation Meza TJ et al. (2001). The frequency of silencing in should occur between the GM trait gene and the V-GURT Arabidopsis thaliana varies highly between progeny of genes, the GM trait may be passed on through seed and siblings and can be influenced by environmental factors. pol en to crops and weeds. Equal y, if any of the genes involved in the V-GURTs system were segregated from Mittelsten Scheid O et al. (1998). Release of epigenic gene the others, the system would no longer function as silencing by trans-acting mutations in Arabidopsis. required. To avoid segregation, V-GURTs requires that all Proceedings of the National Academy of Sciences, USA the introduced genes be placed in very close proximity on the same chromosome (strand of DNA) to create a linkage that wil reduce the likelihood of segregation as Padidam M (2003). Chemical y regulated gene expression in much as possible. If segregation occurs, any resulting plants. Current Opinion in Plant Biology 6:169-177 gene flow of the transgenic trait (e.g. gene for Bt- Riddihough G and Pennisi E (2001). The Evolution of endotoxin, pharmaceutical component or lignin reduction) Epigenetics. Science Magazine 293(5532) Srivastava V to related cultivated, wild or weed relatives wil occur and and Ow DW (2003). Rare instances of Cre-mediated may be difficult to pick up in time to prevent it spreading deletion product maintained in transgenic wheat. Plant Srivastava V and Ow DW (2003). Rare instances of Cre- mediated deletion product maintained in transgenic wheat. Plant Molecular Biology 52:661-668 Tang W; Luo XY; Samuels V (2004). Regulated gene To date, no functional and complete V-GURTs application has expression with promoters responding to inducers. Plant been detailed in the scientific literature. The evaluation of the capacity of V-GURTs as a gene containment system presented here has thus relied on the evaluation of its envisaged components. A system can be only as good as its weakest parts. At present, none of the components tested for any of the possible V-GURTs systems are 100% reliable or effective. Given that at this stage the individual components of V-GURTs offer less than 100% efficiency or reliability, the combination of these components in one organism wil amount to still less. For example, if each of the 4 components performs to 95%, in combination their performance could reduce efficiency or reliability to as little as 81%. Equal y, future evolution of V-GURTs must be taken into account. Because V-GURTs confer an evolutionary

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