Bt Brinjal The scope and adequacy of the GEAC environmental risk assessment

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16 Bt Brinjal: The GEAC environmental risk assessmentInserted TransgeneFinding 8. The description of the transgene in Bt brinjal EE-1 is inadequate to support risk assessment. It isnot clearly known that there is only one transgene incorporated into the brinjal genome, that the inserted transgeneexpresses the intended gene product or that the transgene does not interrupt a functional plant gene.(A) Number of inserts. The Dossier indicates that one transgene insert remains in Bt brinjal EE-1. Based on the evidenceprovided, the conclusion is not fully justified. There are several sources of uncertainty that weaken the conclusion, and three ofthese sources of uncertainty may affect risk. Two may increase risk and one may increase or decrease risk.• Uncertainty arises because the size of the digoxigenin (DIG)-labelled Bt probe is not specified. If it is too large, it may notdetect independently inserted fragments;if it is too small, it may not detectBox 1independently inserted fragments notBackcrossing and Transgene Characterizationassociated with the probe.Backcrossing is one way to create crop varieties that are similar to previous elite• Uncertainty also arises because probes varieties with a few new traits added. It is also a useful way to clean up “dirty”transformation events that generate transgenic plants.for other parts of the plasmid, such asAt the time Bt brinjal event EE-1 was created, all know methods for creatingthe nptII gene and several others, are not transgenic events created “dirty” transgenic events, and EE-1 is no exception. Aused. If fragments are inserted in such “dirty” event is one in which there are multiple transgene insertions, often withinversions of the transgene, interspersion of plant DNA, artificially createda way that a new open reading frame isintrons, and transgene fragments. All of the “dirty” elements of the event couldcreated that is associated with a plant create serious risks to the environment.promoter, novel gene products may beThe multiple insertions typically end up on different plant chromosomes or somedistance apart on the same chromosome. Recurrent backcrossing can help toexpressed at high rates.clean up these dirty events.• The third source of uncertainty isOnce a transgenic plant cell is created, it must be regrown into a whole plant.Backcrossing involves crossing the newly created transgenic plant with an eliteassociated with the breeding program.variety or line that the breeder wants the transgenic plant to resemble. RecurrentAlthough the file makes it clear that the backcrossing involves multiple generations of crossing offspring back to the sametransformed plant is backcrossed to elite elite variety or line as diagrammed in the figure below.brinjal germplasm, it is not clear howRecurrent Backcrossing to Elite Line(B 1is the first backcross generation, etc.)many backcross generations were done.B 1B 2B 3B 4Each backcross generation reduces theEliteLineprobability that an additional integrationevent is retained into the final brinjalX X X Xproduct. In other words, the moreTransformedPlantbackcross generations, the less likely anTransgeneundetected integration event is retained inthe final brinjal product (Box 1). Because Proportion ofthe number of backcross generations is Transformed Plant (½) 1 (½) 2 (½) 3Remaining in Offspringnot specified for each of the Bt brinjalproducts, it is uncertain how well the For brinjal’s 12 linkage groups (Doganlar et al. 2002), about 10 backcrossinserted transgene has been characterised. generations are necessary to eliminate all but the chromosome with the transgenewith 99% probability. Ten BC generations would also eliminate all but 0.1% (on(B) Sequence of the inserted transgene. average) of the genetic material associated with the transgene. If only 5 backcrossUnder the assumption that there is only generations were used, the probability that all other chromosomes have beeneliminated is only 62%, and about 3% of the chromosome associated with theone transgene inserted into Bt brinjal EE-1transgene remains. If only 5 backcross generations were used, a significantas asserted by the file, the sequence ofamount of germplasm that potentially contains unintended transgenes remains inthis one transgene was not reported. The the final variety. This creates unnecessary risks to the environment that couldhave been easily reduced with a bit more breeding effort. Hence the number ofsequence of the inserted transgene is typicallybackcross generations is an essential element to characterizing a transgene.required by regulatory authorities around the
Characterisation of Transgene 17world. Although the sequence of the Bt gene, and all of the marker genes was known (but not provided), this is the sequenceof the plasmid, not the sequence of the inserted gene. There are typically differences between the two. The sequence of theinserted transgene is necessary to substantiate the logic behind the reasoning that the marker genes pose no risk, and that theCry1Ac protein is virtually identical to other Cry1Ac proteins that have been studied previously. The sequence will also enableidentification of unintended open reading frames (ORFs), which could result in unintended protein expression.(C) Flanking regions. Information on flanking regions is not provided. Although there are limitations on the extent ofgenetic (DNA) analysis that can be done on brinjal because the entire brinjal genome has not yet been sequenced, there remainseveral significant and important elements that were not done. Typically an inserted transgene will be flanked by a mixtureof plasmid and plant DNA, which is further flanked by only plant DNA associated with the original plant chromosome.Sequencing through the mixed flanking regions to the plant flanking regions is now routinely required by many regulatoryauthorities around the world. The flanking sequences are needed to determine if the transformation process has inadvertentlycreated novel open reading frames and to determine if a functioning plant gene was interrupted by the insertion event. Lackingthe full brinjal sequence, it is not possible to determine unambiguously if a plant gene was or was not interrupted, but becauseof the high correspondence of the brinjal and tomato genomes, the flanking region sequences can be blasted against the tomatogenome until such time that a full brinjal sequence is available.Needed Experiments and Observations1. Probe sensitivity. Provide the size and sequence of the DIG-labelled Bt probe and identify the part of the transgene to whichit is expected to bind.2. Transgene number. Use additional probes that provide complete coverage of the original plasmid to explore for incorporatedtransgene fragments associated with other parts of the transgene. Report the size of these probes and the detection limits ofthe Southern blots.3. Transgene sequence. Sequence the inserted transgene and any other inserted fragments identified in 1) and 2).4. Flanking regions. Sequence the regions flanking the inserted transgene (and transgene fragments) until continuous brinjalDNA is encountered.5. Gene disruption. Identify the brinjal DNA in the flanking regions and determine if it is associated with a potentiallyfunctioning brinjal gene. As long as the brinjal genome has not been sequenced, the tomato and potato genomes can beused for this purpose. If it is associated with a potentially functioning brinjal gene, determine the function and its potentialeffects on the plant.
Page 1 and 2: Table of Contents 1Bt Brinjal Event
Page 3 and 4: Table of Contents iBt Brinjal:The s
Page 5 and 6: Summary and Conclusion 1Summary an
Page 7 and 8: Summary and Conclusion 3The EE-1 t
Page 9 and 10: Summary and Conclusion 5Event EE-1
Page 11 and 12: Summary and Conclusion 7Conclusion
Page 13 and 14: Context and Need 9standards for ER
Page 15 and 16: Context and Need 11and seek to incr
Page 17 and 18: Context and Need 13the reported yie
Page 19: Characterisation of Transgene 15Cha
Page 23 and 24: Characterisation of Transgene 19In
Page 25 and 26: Characterisation of Transgene 21Sci
Page 27 and 28: Environmental Risk Assessment 23Gen
Page 29: Environmental Risk Assessment 25thi
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Bt Brinjal The scope and adequacy of the GEAC environmental risk assessment

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