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ISIS Report 10/01/07

GM Grapevines & Toxic Wines

Synthetic genes, toxins and cyanide gas are some of what to expect from wines on your dinner table Prof. Joe Cummins and Dr. Mae-Wan Ho

A fully referenced version of this article is posted on ISIS members’ website. Details here

Many field trials & commercial approval imminent

A number of genetically modified (GM) grapes have been created, though none is yet commercialised. There were 25 field test releases in USA between 1999 and 2005, and a small deluge of commercial releases is expected any time now.

The bulk of the test releases were of GM grapes resistant to diseases including powdery mildew, Botrytis , Agrobacterium , Clostridium , Xylella , nepovirus and closterovirus. There was one application for improved fruit quality, but the transgene was designated confidential business information.

The disease resistance genes included synthetic antimicrobial peptides encoded by synthetic genes. The majority of the applications for release permits were from Cornell, California and New York State Universities, the rest were from vintners or wine research companies [1].

In Europe, Italy conducted trials of grape modified with a gene regulating the plant hormone auxin. Germany tested grapes resisting fungal diseases , and France tested grapes resisting nepovirus [2].

Australia has field-tested grapes modified for fruit colour or quality [3], most of them carrying antibiotic resistance genes as selectable markers, which would very likely spread to other organisms during wine making. There has been a hiatus in the commercial approval of GM crops recently despite a very large number of field trials. Bureaucrats may regard the low frequency of commercial approvals to be a “log jam” and facilitate a flood of new approvals without warning. It is something that the concerned public should be prepared for.

GM grapes potential hazards not addressed

GM grapes carry all the potential hazards of other GMOs [4-6] ( Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering ; ISIS Report; GM Food Animals Coming , SiS 32), but because GM grape juice and GM wine come as a clear liquids, many people may assume it is safe to drink; not so.

DNA from a GM grape persisted for over a year after wine fermentation, contradicting claims that wine fermentation eliminates DNA [6]. GM DNA in wine carries all the risks of horizontal gene transfer and recombination: creating new viruses and bacteria that cause diseases, triggering cancer in the case of GM DNA with strong promoters jumping into the genome of human cells Other potential hazards from GM grapes are toxins and allergens from the transgene products, or from unexpected metabolic disturbances to the host plant.

Toxic synthetic antimicrobial peptides

Chardonnay grapes have been modified with genes for magainin and peptidyl-glycine-leucine carboxyamide, both synthetic antibiotic peptides originally from frog skin, and neomycin resistance and GUS were included as selectable markers. The GM grape was found to be more resistant to bacteria than to fungi [7, 8]. The toxicity of the GM grape to mammals has not yet been investigated.

Patent applications have been made for producing transgenic grapes with a synthetic version of a cecropin-like toxin, shiva 1. The transgenic grapes resisted bunch rot, powdery mildew and downy mildew [9, 10]. Shiva 1 is an experimental synthetic antibiotic peptide for treating mammals, and treatment of rabbits revealed a narrow range between effective and toxic doses [11]. Great caution is needed in evaluating GM grape with genes for synthetic toxins that could endanger domestic and wildlife, as well as human beings.

Grapevine fan leaf virus resistance was achieved by transforming grape with a coat protein gene from the fan leaf nepovirus [12,13]. The coat protein gene conferred resistance most likely through an RNAi suppression of virus replication [14] ( Subverting the Genetic Text , SiS 24).

Disease resistance with bacteria and viral genes

Grapevines are susceptible to infections with Agrobacterium causing crown gall (tumour) disease (the same Agrobacterium in a disarmed form that's commonly used in genetic modification of plants). The plant tumours are initiated when the bacterium injects plant cells with a DNA Ti (tumour-inducing) plasmid carrying genes for plant cell proliferation. A portion of the Ti plasmid is transferred into the plant cell genome, and depends on the forming a DNA single strand intermediate that is protected from degradation in the plant cell by a coating of vir gene protein produced by the bacterium. The protected single strand of DNA integrates into the plant chromosome and begins activating genes that initiate tumour formation. Agrobacterium -resistant grape vine was made by inserting a gene for a mutant form of the vir gene into the plant genome. The transgenic grape produces the mutant vir protein continuously, which attaches to the infecting part of the Ti plasmid, causing it to be inactivated and destroyed rather than being integrating into the chromosome [15].

Grape bunch rot ( Botrytis ) is caused by the fungus Botrytis cinerea , while the fatal Pierce's disease is caused by the bacterium Xylella . Both pathogens use enzymes that degrade the plant cell wall to invade the grape tissue. A gene for an inhibitor of the pathogen wall-digesting enzyme (polygalacturonase inhibiting protein) from pear fruit was used to transform grape vines. The transgenic DNA included the CaMV promoter, a TMV enhancer, the pear gene, and octopine synthase terminator, accompanied by the GUS gene and a neomycin resistance gene as selectable markers. The transgenic grape was reported to resist both the bacterial gene and the fungal gene [16].

Cyanide-producing grape

Grape has also been genetically modified to resist insects by making them produce hydrogen cyanide when attacked by insects. Cyanogenic plants are characterized by the liberation of HCN in the course of tissue injury, due to the hydrolysis of cyanogenic glucosides. Most of our knowledge of cyanogenicity comes from Sorghum bicolor , which contains large quantities of the cyanogenic glucoside, dhurrin. Prussic acid, a derivative of cyanide, is also a serious potential problem. Crop species most commonly associated with prussic acid poisoning are sorghum, Johnsongrass, and Sudangrass. Grain sorghum typically has more potential for toxic levels of prussic acid than forage sorghum or Sudangrass. Young, rapidly growing plants are the most likely to contain high levels of prussic acid. Cyanide is more concentrated in young leaves than in older leaves or stems. New sorghum growth following drought or frost is dangerously high in cyanide. Generally, any stress condition that retards normal plant growth may increase prussic acid content. Hydrogen cyanide is released when plant leaves are damaged by trampling, cutting, crushing, chewing, or wilting. Drought-stunted plants accumulate cyanide and can possess toxic levels at maturity. Prussic acid poisoning is most commonly associated with regrowth following a drought-ending rain, or the first fall frost. New growth from frosted or drought-stressed plants is palatable, but dangerously high in cyanide. After a killing frost, at least four days should pass before grazing to allow released hydrogen cyanide to dissipate.

A multigenic trait responsible for biosynthesis of the secondary metabolite, dhurrin cyanogenic glucoside was engineered in grapevine with three genes sequences from sorghum ( Sorghum bicolor ): two cytochrome P450s (CYP79A1 and CYP71E1) and a UDPG-glucosyltransferase (sbHMNGT). The grapevine was modified using a two-step process involving whole plant transformation followed by hairy root transformation. The two step process make sure that the whole plant could be transformed with the dhurrin pathway, while the secondary transformation of the hairy root culture allowed fuller study of the dhurrin produced in roots which had been challenged with the root pathogenic insect Phyloxera .

One dhurrin-positive line was tested and found to release cyanide upon maceration. Co-culture of a cyanogenic hairy root line or a non-cyanogenic line with the specialist rootsucking, gall-forming, aphid-like insect, grapevine phylloxera ( Daktulosphaira vitifoliae ) gave no evidence that the cyanogenic plant tissue was protected from insect infestation. Consistently high levels of dhurrin accumulation may be required for that to occur [17]. We are not sure at all about the ultimate purpose of the modified grape but will certainly avoid drinking juice and wine made from it, provided that it is labeled as such. If it is not labeled, we may all have no choice over cyanide poisoning.

For crying out loud

The modification of grapevines has gone beyond the humdrum of Bt and herbicide tolerance of most GM food crops. The new emphasis is on synthetic genes and proteins, and virus resistance using coat protein gene modifications. The introduction of a cyanogenic toxin into grapevine may be a sign that genetic engineers are growing ever more daring in the recognition that regulators are standing with them against the public. Clearly these new developments are crying out for GM labelling at the very least, and a clean sweep of the regulatory regimes would not come amiss.

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