Environmental Impacts �of Genetically Modified Plants

Highlights

• Environmental Implications of genetically modified organisms are the fundamental issues to be addressed.
• The unintended effects of genetically modified crops on environment are obvious
• Biosafety concerns of genetically modified organisms need more in depth explorations

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The Story

https://gmo-awareness.com/all-about-gmos/gmo-timeline-a-history-of-genetically-modified-foods/

Questions to be addressed

• Could GM crops outcross to produce weediness?
• Could they harm wildlife and non-target insects?
• Could they help to benefit the environment by providing raw materials?
• Is their environmental impact acceptable or unacceptable?

Am I the audience?

Epidemiologist

Evolutionary Biologist

Ecologist

POLITICIAN

Consumer

Lawyer

Farmer

Environmental Biologist

Toxicologist

Nutritionist

GM seed Company

Genetic Engineer

It matters a lot!

• In general, the effect of agriculture on the environment is obvious whether it is subsistence, organic or intensive.
• GM crops have either positive or negative effect on the environment depending on how and where they are used

(ICSU) (www.nuffieldbioethics.org)

• Issues of baseline environmental impacts are particularly relevant in relation to the release of transgenic commercial crops (Dale et al., 2002)

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Environmental Implications

Gene Flow

• Changes in gene frequencies along with mutation, genetic drift and selection
• Transgene X wild hybridization
• Transgene Stacking
• Horizontal Gene Transfer
• Structure of Genetic Diversity

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Factors affecting Gene Flow

• Distance between cross compatible species
• Synchronization of flowering time
• Ecology of recipient species
• Sexual compatibility
• Biology of the plant
• Amount of pollen produced
• Mating system
• Genetic bridge
• Selective pressure

• Outcrossing rate
• Relative densities of both species
• Types of vectors
• Wind/air turbulence
• Water current
• Temperature
• Humidity
• Light intensity

Transgene X wild hybridization

• Theoretically, for such a hybrid to be developed under natural conditions, a rare hybridization event would be sufficient
• the developed hybrid could have higher fitness compared to its parents
• Brassica rapa × Brassica napus hybrids
• (Hooftman et al., 2014)
• Crop × wild sunflower hybrids
• (Mercer et al., 2007)
• Sugar beet × swiss chard hybrids
• (Ellstrand, 2003)
• Glufosinate resistant feral oilseed rape feral plants
• (Schulze et al., (2014)

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Transgene × wild hybridization

• 44 cultivated plant species can possibly cross with one or more wild relatives
• 28 cultivated species have been witnessed to have hybrids with wild counterparts…(the number was increased when SYMPATRY was considered
• 48 species displayed something more than just morphological intermediates. Ellstrand (2002)

Possibility of hybrid development exist by introgression of GMO with its wild relatives

Transgene × wild hybridization

• Once the hybrid is developed, its persistence will depend on
• Fitness
• Life cycle
• Fecundity
• Seed dormancy
• Genotype X Environment interactions
• Selection pressure
• Regaining of selective fitness by successive back crossing

Transgene × wild hybridization

level of risk probability in 25 different crops

Transgene Stacking

• 43.7 million hectares of stacked biotech traits were planted
• The possibilities
• Presence of mul
• 28 cultivated species have been witnessed to have hybrids with wild counterparts…(the number was increased when SYMPATRY was considered
• 48 species displayed something more than just morphological intermediates. Ellstrand (2002)

Transgene Stacking

• Multiple transgenes will be found in wild
• Nuclear encoded genes may combine with plastid encoded genes (rare case)
• Wide range of resistance in successive generations
• Stack of gene related to single metabolic pathway can escape

• Possible risk scenario (Kok et al., 2013)
• Gene instability
• Changes in the level of gene expression
• Synergistic or antagonistic effect

Transgene Stacking

• Expression of CP4 EPSPS + Cry gene resulted in alterations in the energy/carbohydrate and detoxification pathway in maize (Agapito-Tenfen et al., 2014)
• Stacked transgene oilseed volunteers have already been persisting in Canada (Dietz-Pfeilstetter and Zwerger, 2009)
• Cultivation of stacked GM maize (Cry1 Ab and mCry3A proteins) had no greater differences than respective single maize events (Raybould et al., 2010)

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• ?? Case-by-Case variations

Horizontal gene transfer

• Stable transfer of genes other than parent to offspring (sexual/asexual
• Possible risk scenario (Ho et al., 2000)
• Transfer of antibiotic resistance transgene to pathogens
• Transfer of transgenes to viruses
• Transfer of transgenes to humans

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Horizontal gene transfer

• Important factors for HGT
• Transgene size
• Nuclear/Plastid transgene
• Sequence mosaicism
• Selective pressure
• Copy number
• Genome size of recipient species
• Codon usage
• Type of promotor
• Compatibility of RNA and protein synthetic machinery

(Tepfer et al., 2003; Daniell et al., 2001)

Horizontal gene transfer

• Transfer of nptII gene to Acinetorbactor spp
• Arabidopsis
• Oilseed rape
• Tobacco
• Alfalfa
• Carrot (Tepfer et al., 2003)
• HGT Frequency depends upon donor and recipient species.
• Plants to Prokaryotes is as low as 2 x 10^-17 _Or 10 recombinants per 250 m²
• HGT of Rhodnius prolixus less than 1.14 × 10^-16

HGT is prevalent in nature and should be accounted during assessment

Structure of genetic diversity

• Reduction of differentiation between populations
• Increase in diversity between individuals
• Domestication bottleneck
• Complete extinction of wild populations
• Genetic diversity is mainly disturbed by Pollen mediated gene flow
• Possibility after transgene has been transferred to wild counterpart
• Generation of selective sweeps
• Population decline or local extinction

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• No such report has been recorded.
• Disturbance in genetic diversity?........Farmer can control!

Fate of naked DNA

• nDNA encoding a resistance or tolerance trait can possibly persist in the natural environment (Barnes and Turner, 2016)

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• Relatively GM DNA amount is quite low. Is it a risk?
• Gene pool for surrounding microbial communities
• Viral pathogens can receive the DNA
• Transgene could move from microbes to intestines of animals….Really?
• These DNA segments may introduce amino acids, indels to bacteria through transposition or homologous recombination

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Fate of naked DNA

• Degraded DNA fragments of 680 bp   were detected within 28 days in maize cob silage, while only 194 bp fragments were observed in whole plant silage up to 35 days (Einspanier et al., 2004) .
• A case study of detecting CP4EPSPS in sheep fed with Round Ready canola resulted in the detection of 527 bp fragments after 2 minutes (Alexander et al., 2004)
• Persistence of nDNA from Bt corn (event MON863) containing Bt3Bb1 and nptII genes and DNA from plasmid Pns1 in water was reported to decrease by two orders of magnitude within >4 days (Zhu, 2006).

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Fate of naked DNA

• Persistence of nDNA in Agricultural ecosystems is almost zero
• The possibility of nDNA transfer to animal intestines is negligible as it would be degraded by nucleases in the gut.
• We eat the DNA every day in the form of fruits, vegetables and other foods.

Weediness

• Establishment of a transgene or transgene × wild hybrid as a weed in other fields or other habitats is referred as weediness

Small genetic modification (GM x Crop hybrid) can cause large ecological alteration (Williamson et al., 1990)

Addition of a single transgene is unlikely to establish a crop/wild as a weed (Luby and McNichol (1995)

• Traits of interest
• Tolerance to herbicide
• Resistance to various stresses

• Resistance to pests
• Traits responsible for enhanced growth

Weediness

Rate of weediness =

frequency of hybridization + net selective effects of transgene

Possible Herbicide resistance mechanisms

Herbicide Detoxification

Reduced herbicide entry

Reduced herbicide translocation

Target site overproduction

Change in intercellular compartmentalization

(Tappeser et al., 2014)

Weediness

B. napus × B. rapa hybrids in Quebec have been witnessed for many years

• 2001-05
• Weedy hybrids
• Intermediate genomes
• ↓ male fertility
• F1 backcross hybrids (Warwick et al., 2008)

Transgenic traits don’t significantly increase the fitness of the plants in semi natural habitats (GM Science Review Panel)

With zero herbicide selection pressure, escaped herbicide resistance transgene from GM soybean to its wild counterpart (i.e. Glycine soja) can still persist in nature (Guan et al., 2015)

Weediness

• Since 1996, 24 glyphosate tolerant weeds have been identified (Gilbert, 2013)
• WeedScience.Org (2014) says that a chronological increase in resistant weeds at global scale has been observed from 1996 to 2011.
• 145 Plant species have become resistant to eight herbicide groups

Toxicity to life

• Crop
• Trait
• Local weed flora
• Local fauna
• Farm management practices
• Climatic conditions
• Alternate host species for insect pest
• Promoter
• Level of expression
• Target tissue of GM plant
• Frequency of herbicide and insecticide application

Toxicity to life

Herbicide Toxicity

General risks affiliated with cultivation of GM crops and application of broad spectrum herbicide

Changes in plant defence

Weakening of plant defence

Modification of N metabolism

Lethality to aquatic animals

↑ Bacterial Biomass

Suppression of Cytochrome P450

Disruption in Amino acid biosynthesis

Disturbance in biology of host plant

Cytotoxicity to human cells

Disturbed biodiversity of weeds

Disturbed symbiotic relationship

Modified Food chains

Toxicity to life

Herbicide Toxicity

• Direct glyphosate-induced plant defence weakening and increased pathogen virulence (Johal and Huber, 2009)
• Application glyphosate reduced nodule formation and activity of N fixing bacteria in soybean (King et al., 2001)
• Suppression of Cytochrome P450 is affiliated risk of glyphosate application
• Glyphosate disrupts gut bacteria in cattle and poultry (Samsel and Seneff, 2013)
• Increased bacterial biomass and enhanced activities of urease and alkaline phosphatase have been observed in rhizosphere of Basta-tolerant oilseed

(Sessitsch et al., 2005)

• Herbicide stratification is directly linked to temperature stratification in ectotherms. (Jones et al., 2016)

Toxicity to life

• Bt toxicity includes possible immediate or delayed harmful environmental implications which threat target and non-target organisms (Yu et al., 2011)
• Toxicity of Bt has been observed in lacewings, earthworms, herbivores, honeybees, human fetus (Saxena and Stotzky, 2000; Agrawal, 2000; Aris and Leblanc, 2011)
• No significant risk were affiliated with larval survival and prepupal weight of honey bee in response to Bt-maize pollen.
• Contrary to GM maize, Heliconia rostrate pollen posed significant toxic effects (Hendriksma et al., 2011)
• Higher mortality, reduced egg production and lower proportion of females reaching maturity was observed in Daphnia manga; a crustacean arthropod, when fed with Cry1Ab maize (Dekalb 818 YG) (Szenasi et al., 2014).

Insecticide Toxicity

Toxicity to life

• Bt doses could then possibly affect tri-trophic interactions (i.e plant-herbivores-their natural enemies) in synergistic, additive, or antagonistic ways.
• Presence of Bt toxins in aphid (Myzus persicae) samples detected by double sandwich enzyme-linked immunosorbent assay confirmed possible consequences of these toxins on food chains and trophic levels of herbivore-natural enemies (Burgio et al., 2007)
• Non-inertness of combined effect of Cry1Ab and Cry1Acas well as in response to 1 to 200,000 ppm was confirmed.
• Cry1Ab concentration of 100 ppm resulted in death of human embryonic kidney cells (Mesnage et al., 2012).

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Insecticide Toxicity

Indirect Impacts of GM

Effect on soil and water

Effect on biodiversity

Reduced efficiency of pest, disease and weed control

Evolution of insecticide and pesticide resistance

Evolution of herbicide resistance

Indirect Impacts of GM

Effect on biodiversity

• Application of broad spectrum herbicides reduces weed diversity in farmlands.
• Diversity, density and biomass of the seed bank in farmland was evidently lesser in GM systems contrary to conventional systems (Bohan et al., 2005)
• UK Farm Scale Evaluations (FSE) reported 20-36% reduction in weed seed bank (Andow, 2003)
• Disturbed tri-trophic interactions and symbiotic associations
• Knock-on effects on higher trophic levels
• Foraging behavior can also be modified
• Shift in food web /Shift in soil biota
• Emigration of agrobiont wolf spider = application of Baccaneer® Plus (glyphosate) (Wrinn et al., 2012)

Indirect Impacts of GM

• GM crops resistant to herbicides invite broad-spectrum herbicide application (Benbrook, 2012)
• Addition of glyphosate in farmland water and ultimately to the aquatic ecosystems and its impact on aquatic life is apparent
• transfer of Bt toxins from GM crops to soil and water have many possible routes including pollen deposition during anthesis, root exudates and GM plant residues (Yu et al., 2011)
• Bt toxins bind to the clay and humic substances, rendering the biodegradable proteins (Zwahlen et al., 2003; Clark et al., 2005; Viktorov, 2008; Saxena et al., 2010)
• Persistence of Bt toxins in the soil is largely dependent on type of toxin and soil type not the no. of expressed transgenes (Marutescu, 2012)

Effect on soil and water

Indirect Impacts of GM

• Western corn rootworm resistant to Cry3Bb1 and mCry3A
• European corn borer, pink bollworm, cotton bollworms resistant to Bt toxins

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Reduced efficiency of pest, disease and weed control

Evolution of insecticide and pesticide resistance

Evolution of herbicide resistance

• Horseweed resistant to diclofop-methyl
• Asiatic dayflower, buckwheat and common lambsquarters resistant to glyphosate
• Glyphosate resistant annual ryegrass in Australia
• 467 unique cases of herbicide resistant weeds have been recorded (WeedScience.org)

Indirect Impacts of GM

• Target site overproduction
• Modification in intracellular herbicide compartmentation
• Minimal herbicide absorbance and translocation
• Herbicide detoxification
• Insensitivity to target site
• Intensity of selection
• Mating behavior of insect pest
• Seasonal changes
• Population regulation of refuges

Resistance development mechanisms

Science and politics in EU regulation of GMOs

Article 3(11) of Regulation 178/2002

“A scientifically based process consisting of four steps: hazard identification, hazard characterization, exposure assessment and risk characterization”

Article 6(2) of Regulation 178/2002

“ Risk assessment shall be based on the available scientific evidence and undertaken in an independent, objective and transparent manner”

(www.eur-lex.europa.eu)

Science and politics in EU regulation of GMOs

Limitations

• Pervaded by key uncertainties
• Dose response curve
• Application of animal/in vitro study results to humans
• Difference in laboratory and field applications

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EFSA says “Comparative assessment”

Why only chemical similarity ?

What about anti-nutritional factors ?

Who is the real counter part of GM ?

Let’s talk about FUTURE

Transgene and its spatio-temporal expression

Cheaper Gene stacks?

Consider possible routes to bacteria and viruses

Consider food webs/chains

Adopt multicrop culture

Consider insertional mutagenic effects

Pre-release quantification of hybridization

Bioinformatics prediction tools for toxic and allergenic effects of foods

Theoretical verse-case scenario

Discover pathways involved in DNA release

After effects of dsRNA silencing