Genetic pollution

10/04/2021 Department of Genetics and plant breeding 1

MASTER’s SEMINAR-I
on
GENETIC POLLUTION- “a multiplying nightmare’’
PRESENTED BY:
Priyanka
PGS19AGR8245
M. Sc. (Agri.)
10/04/2021 2
Department of Genetics and plant breeding

1) INTRODUCTION
2) HOW GENETIC
POLLUTION
OCCUR??
3) IMPACT OF
GENETIC
POLLUTION ALONG
WITH CASE STUDIES
4) STRATAGIES TO
MITIGATE GENETIC
POLLUTION
5) BIOSAFETY
MEASURES
6) CONCLUSION
SEMINAR OUTLINE
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What is GMO???
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Current status GM Crops
Crops Area (Mha) In Percentage(%)
Soyabean 95.9 50
Maize 58.9 31
Cotton 24.9 13
Canola 10.1 5.30
Others 1.9 <1
SOURCE: (ISAAA 2018)
In the last 22 years ,the global area of transgenic crops has increased
significantly from 1.7 million hectare in 1996 to 191.7mha in 2018

Gene flow
• Gene flow occurs when individuals join new populations and
reproduce.
• Migrants change the distribution of genetic diversity among
populations by modifying allele frequencies
• High rates of gene flow can reduce the genetic diffrentiation
between two groups.

Transgene flow/Escape
• It is a process where the transgene inserted to a GM
crops has been escaped to its wild Species/neighbour
crops .
• The principle concern about the transgene flow is the
loss of potentially useful crop genetic diversity in
recipient population.

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Types
• Vertical gene flow: Gene flow within species
• Horizontal gene flow: Gene flow between the species
• Diagonal gene flow: Gene flow between closely
related species
After 22 years of GM crops cultivation we failed to
control gene flow in a systematic manner( Ryffel, 2014)

VERTICAL GENE TRANSFER HORIZONTAL GENE TRANSFER

Genetic pollution
The dispersal of contaminated or altered genes
from genetically engineered organism to natural
organism
"Uncontrolled spread of genetic information
(frequently referring to transgenes) into the
genomes of organisms in which such genes are
not present in nature”
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Causes of genetic pollution
Cross-breeding of GM
crops with the wild
varieties by cross
pollination
Consumption of
GM foods
Improper disposal
of unsuccessful
GM crops
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Genetic contamination may arise in these
situations:
• Wild, related flora growing nearby are pollinated by a GE
crop.
• Non-GE or organic crops in neighbouring fields are
pollinated by the GE crop.
• A semi-wild, weed or ‘feral’ population of GE plants
develops if the GE crop survives in the agricultural or
natural environment.
• Micro-organisms in the soil or the intestines of animals
eating the GE crop acquire the foreign genes
(www.greenpeace.org).
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(Rizwan et al., 2019)

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(Rizwan et al., 2019)

Impacts of genetic pollution
1) Direct effects on non target organisms
2) Genetically modified organisms might lead the non-GM organisms to extinction
3) Impacts of transgenic crops on parasitoids
4) Unknown health consequences are a common objection GMO organisms
5) Transgene escape from these crops may lead to the development of super weeds
6) Cross pollination with the cultivated and wild type with GM species may
lead to genetic contamination of the cultivated wild type which could alter local
ecosystem

Direct effects on non-target organisms
• Transgenic pollen harms
monarch larvae
• In May 1999, it was reported that
pollen from Bacillus thuringiensis
(Bt) insect resistant corn had a
negative impact on Monarch
butterfly larvae
• This report raised concerns and
questions about potential risks to
Monarchs and perhaps other
non-target organisms
(Losey et al,1999, New York)

Table 2. Impacts of transgenic crops on
parasitoids
(Gatehouse et al., 2011)
Protein Transgenic
plant
Natural enemy Pest Effects on
natural eneny
Bt (cry1AB) Corn Diaraetiella rapae Chillo partellus Reduced survival
owing to host
mortality
Bt (cry1AC) Cotton Microplitis
mediator
Helicoverpa
armigera
Wasp survival and
development
negatively affected
Bt (cry1AC) Broccoli Diadegma insulare P.xylostella No affect on
parasitoid when
exposed to BT-
resistant host
CpTI Potato Eulophus
pennicornis
Lacanobia oleracea Fewer hosts
parasitized,no
effects on
parasitoids
GNA Sugarcane P.pyralophagus E .loftini Reduced size and
longevity of adult
wasps

Increased weediness
Weediness
 There are apprehensions about GM crops becoming weeds
Development of super weeds

s

Yook et al. (2021)

• To quantify the gene flow from glufosinate ammonium resistant soyabean
to its wild relative
• To assess the potential weed risk of hybrids resulting from the gene flow
during their entire life cycle under field condition
Materials and methods
• Glufosinate – ammonium resistant soyabean (Glycine max L. cv.
Kwangakong 2n=40)used as pollen donor
• Wild soyabean (Glycine soja Sieb. And Zucc.,IT 182932, 2N=40) used as
pollen reciepient
• Two year field experiment conducted in authorized LMO field
• In the first year (2013) the planting distance between pollen donor and
pollen receipient were 0.5,1,2,4, 6 and in (2014) 0,0.25,0.5,1,2,4,6 & 8
Objectives

Experimental field design (A) and GR soybean and wild soybean planted in the LMO field .
(B) to evaluate gene flow from GR soybean (G. max) to wild soybean (G. soja) in Suwon,
Korea. Planting distances between pollen donor (GR soybean) and pollen recipient (wild
soybean) were 0.5, 1, 2, 4 and 6 m in 2013 and 0, 0.25, 0.5, 1, 2, 4, 6, and 8 m in 2014.
Yook et al. (2021)

Vegetative growth characteristics in canopy height (A), stem length (B), stem
diameter (C), and leaf area (D) of GR soybean , wild soybean , F1 hybrid , and F2
hybrid measured by 70 days after sowing. Leaf area was measured at 70 days after
sowing.

Phenotypic performances on reproductive traits and flowering phenology for parental
soybeans and hybrids
Parameter GR soybean Wild soybean F1 hybrid F2 hybrid
First flowering 25 July 7 August 2 August 2 August
End of
flowering
28 August 10 September 3 September 10 September
Duration of
flowering
32.2 ± 0.60́b 31.8 ± 0.87b 33.0 ± 0.00b 38.0 ± 0.89a
Pollen no. 434 ± 236.1a 300 ± 64.14a 460 ± 65.13a 267.7± 22.05a
Pollen viability
(%)
98.1 ± 0.77a 91.5 ± 2.57b 84.5 ± 2.16c 93.2 ± 1.87b
Flower no. 369.8 ± 25.35b 1110.5 ±124.58a 1144.0 ± 13.06a 1161.0 ± 52.60a
Table 2
Yook et al. (2021)

Number of seeds tested, survivors after glufosinate-ammonium treatment, hybrids confirmed by
PCR analysis, and gene flow percentage from GR soybean to wild soybean in 2013 and 2014.
Distance(m) 2013 2014
Tested
seed no
Survivors no. Hybrids
no.
% Gene flow rate Tested
seed no
Survivors
no.
Hybrids no. % Gene flow rate
0 - - - - 2367 8 7 0.296
0.25 - - - - 2315 6 6 0.259
0.5 4174 8 8 0.192 2479 6 5 0.202
1 3887 8 8 0.206 2257 5 4 0.177
2 3544 3 3 0.085 2456 5 5 0.204
4 3484 2 2 0.059 4160 6 4 0.096
6 3384 2 2 0.057 4436 3 2 0.045
8 - - - - 3987 2 1 0.025
Yook et al. (2021)
Gene flow rate % = No:of survived soybean × No:of soybean with bar−specificband 100
Total seeds tested No of survived soybean tested for PCR

Potential gene flow rate (%) from GR soybean to wild soybean in 2013 (○) and 2014 (●) (A) and
using pooled data (■) (B). The points and the vertical bars represent the mean values and the
standard errors of observed gene flow rates, respectively

Stratagies to reduce genetic pollution
Physical containment
 Biological containment
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Physical containment
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• Preventing seeds and pollen dispersal (Linder et al., 1998)
• Using various physical barriers in addition to careful
processing of seeds (Arriola, 1997)
• Using pollen barriers (stopping insect flow in crops)
• Careful transportation of seeds from GM plants
• Isolation of cultivars having sophisticated genes with more
sensitive markers
• Growing trap crops, fences, under ground cultivation (Ingram,
2000)

Biological containment
 Two approaches of Transgene
containment
Keeping gene in original GMO
Mitigating the effects of
transgene
• Seed lethal system
• Cleistogamy
• Apomixis
• Maternal effects
• GURT
• Transgene mitigation
(Daniel et al., 2002)

Seed lethal system
Schernthaner et al. (2003), provides a single
repressor containment system based on the
simultaneous insertion at the same locus on
homologous chromosomes of a seed lethal gene
linked to a novel trait (SL-NT) and a repressor
gene (R).c
• When the parental lines are crossed, the offspring will present viable
seeds with the genotype SL-NT/R.
• Upon out crossing, the two alleles will be separated and when
gametes carrying the SL-NT allele are introduced into a non-GM
plant, in the absence of the R element, the seed lethality gene is
activated in the seed embryo and thus any seed containing the novel
trait will not germinate.
Schernthaner et al.(2003)

Method
• Tight repression by R- locus
• Activity of seed specific
promoter
• SL construct should be
lethal to plants

Cleistogamy
• A modification of flower structure to promote self-pollination
and is an effective means against transgene flow ( Husken et
al., 2010)
• It can be induced by mutation or genetic engineering
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CROP GENES FOR CLEISTOGAMY REFERENCES
Rice OsMADS 2, OsMADS 1,
OsMADS 3 and
SUPERWOMAN 1
Lee et al., 2003; Xiao et al.,
2003; Prasad et al., 2005;
Yadav et al., 2007
Barley Cly1 and Cly 2 Wang et al., 2013

Apomixis
• Modification in floral structure and can be propagated by
asexual means (Gressel, 2015; Kwit et al., 2011)
• The over expression of various genes namely (OsLEC1 and
OsLEC2), enhances the production of apomictic embryo
• Use of apomixis for containment of transgene has proven in
GM bahia grass where transgene flow is limited to 0.2%
(Sandhu et al., 2010)
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Objectives:
 The present study was conducted to determine the
potential for PGF from GM cotton to susceptible
plants in typical agricultural settings
 To access pollinator activity and pest population
dyanamics
Yan et al. (2020)

• Non GM Cotton- Zhongmiansuo 49-pollen receptor
• GM Cotton- Zhongmiansuo 79- pollen donor
• Intercrops – Sunflower cultivar DW567
Buckwheat cultivar Kuqiao
Soybean cultivar Zhonghuang2
• Four field were planted in late April in rectangular plots
Yan et al. (2020)

• Field layout for determining the impacts of intercrops on pollen mediated
gene flow from GM cotton. Each field contained two rows of one type of
intercrop alternating with two rows of non-GM cotton, or non-GM cotton
alone as control.
•The GM cotton was planted in a 16 × 20 m rectangle at the south end of each
field.
•Intercropped plots consisted of two rows of non-GM cotton with two rows of
the intercrop.

Yan et al. (2020)

Mean (±SE) numbers of pest insects per non-GM cotton plant in three intercropped treatments,
plus control (no intercrop). Count data were summed across the two sampling
dates on which each category of pest was most abundant in each of the two years.
Year Sunflower Buckwheat Soybean No intercrop F df P
Aphis
gossypii
2017 6.0 0 0.8b 57.7 + 11.4a 44.0 + 9.7a 12.51 3380 <0.001
2018 6.9 + 1.3c 6.1 + 0.8c 21.0 + 5.0b 35.7 + 5.8a 12.68 3188 <0.001
Bemisia
tabaci 2017 4.4 + 0.5c 5.4 + 0.7c 7.8 + 0.6b 14.5 + 1.0a 41.01 3380 <0.001
2018 4.4 + 0.4c 5.0 + 0.5c 8.4 + 0.7b 12.5 + 1.1a 26.48 3188 <0.001
Tetranychus
2017 22.1 + 4.7b 38.5 + 4.0a 10.1 + 2.7c 3.0 + 0.7c 20.72 3380 <0.001
2018 7.8 + 1.6b 11.7 + 1.6a 3.7 + 1.0c 1.8 + 0.5c 12.62 3188 <0.001
Nysius ericae
2017 8.2: + 0.9c 14.9 + 1.2b 13.0 + 1.4b 24.6 + 1.3a 33.03 3380 <0.001
2018 8.0 + 1.1b 10.6 + 0.9b 11.0 1.9b 19.5 + 1.7a 11.76 3188 <0.001
Miridae 2017 0.2a 1.7 + 0.2a 2.0 + 0.2a 2.5 + 0.2a 2.25 3380 0.082
2018 3.6 + 0.3a 2.1 + 0.2b 2.9 + 0.3ab 3.3 + 0.4a 4.49 3188 0.005

Distance (mSunflower Buckwheat Soybean No intercro
2017 0
6.67 * 4.41 10.00 * 5.00 23.33 * 3.33 25.00 * 2.89
3.2 6.67 * 1.67 6.67 * 4.41 20.00 * 2.89 16.67 * 1.67
6.4 5.00 * 2.89 3.33 * 1.67 13.33 * 4.41 13.33 * 3.33
12.8 3.33 * 3.33 1.67 * 1.67 6.67 * 1.67 10.00 * 2.89
51.2 0.00 * 0.00 0.00 * 0.00 1.67 * 1.67 1.67 * 1.67
Average 5.00 * 1.45 4.29 * 1.35 13.10 * 3.14 14.76 * 3.79
2018
0.8 5.00 * 2.89 5.00 * 2.89 20.00 * 5.00 18.33 * 4.41
3.2 5.00 * 2.89 5.00 * 2.89 13.33 * 3.33 13.33 * 3.33
6.4 1.67 * 1.67 3.33 * 1.67 6.67 * 3.33 6.67 * 4.41
12.8 1.67 * 1.67 1.67 * 1.67 5.00 * 2.89 6.67 * 1.67
51.2 1.67 * 1.67 3.33 * 1.67 6.67 * 1.67 3.33 * 1.67
Average 3.33 * 1.09 3.10 * 0.67 11.67 * 2.57 10.00 * 3.02
1.6 11.67 * 1.67 6.67 * 1.67 20.00 * 5.00 30.00 * 5.00
25.6 1.67 * 1.67 1.67 * 1.67 6.67 * 1.67 6.67 * 1.67
1.6 8.33 * 1.67 3.33 * 1.67 21.67 * 1.67 21.67 * 4.41
25.6 0.00 * 0.00 0.00 * 0.00 8.33 * 4.41 0.00 * 0.00
Pollen mediated gene flow under different intercropping

Genetic use restriction technology(GURT)
• It refered as terminator technologies that are experimental forms
of genetic engineering technology that provide the means to either
restrict the use of a plant variety or the expression of a trait in a
plant variety by turning a genetic switch on or off.
• There are currently two types of GURT’s under research
I. Variety specific ( V- GURT)
II. Trait specific (T- GURT
10/04/2021 42

• Genetic use restriction technologies could be used for the environmental
containment of transgenic seeds (V-GURT) or transgenes (T-GURT),
thus solving or marginalizing one of the greatest concerns associated with
GM crops (Collins and Krueger, 2003; FAO, 2001b).
• V-GURTs may generally prevent unwanted gene flow from transgenic to
non transgenic varieties (including wild relatives) because pollen carries
the dominant allele of the lethal/inhibiting protein.
• As an indirect effect, the technology could reduce or remove the need for
buffer zones for gene containment and prevent volunteer seeds from
germinating (V-GURTs) or from expressing the GM trait (T-GURTs).
• Additionally, according to Budd (2004), V-GURTs would be useful to
effectively reduce the risk of creating ‘superweeds’ by reducing the
presence of the GM crop in subsequent years.

Components
4.Inducing substance (Inducer)
• Mostly of chemical origin
• Biodegradable
• Nontoxic for the ecosystem
• Directly applicable in the field or in seeds
It is similar for both T- and V-GURTs
1. a repressor gene (the gene switch) that is responsive to an external stimulus
2. a recombinase gene (the trait activator gene), the expression of which is
blocked by the repressor;
3. a target gene

• Site specific mutagenesis and Recombinase
• Zing finger nucleases
• TALENs
• CRISPER – Cas and EcoR1 restriction endonucleases
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Transgene mitigation

10/04/2021 47
Objectives
• To excise the transgene from the pollen using CinH R-S2
recombination system or a codon optimized serine resolvase CinH
recombinase
• CinH and CinH Drec vectors are constructed
• Plasmids containing the CinH recombinase optimized for codon usage
in plants and the CinH recombination sites (RS2) were constructed
• Agro bacterium strain were used for plant transformation
Moon et al. (2011)

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Schematic illustration of CinH recombinase mediated transgene excision in pollen

a.CinH and CinH_Drec vector constructs a CinH recombinase is under the control of
pollen-specific LAT52 promoter.
Enhanced GFP gene is driven by pollen-specific LAT59 promoter.
Bar gene confers resistance to herbicide glufosinate ammonium.
b. CinH_Drec vector was constructed from the CinH vector by removing CinH
recombinase cassette. LAT52 pollen-specific LAT52 promoter, cinH codon optimized
CinH recombinase gene, 35S T 35S terminator, LAT59 pollen-specific LAT59
promoter, eGFP enhanced GFP gene, NOS P nopaline synthase promoter, bar herbicide
resistant bar gene, NOS T nopaline synthase terminator, RS2 CinH recombinase recog
nition site, LB left border, RB right border

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51
Events Total germi Total transg (
Total non-ti g (
Observed ra
CinH-5 258 222 36 6.2:1
CinH-7 295 280 15 18.7:1
CinH-11 338 333 5 66.6:1
CinH-12 311 290 21 13.8:1
CinH-13 312 229 83 2.8:1
CinH-14 317 236 81 2.9:1
CinH-15 337 315 22 14.3:1
CinH-16 325 317 8 39.6:1
CinH-18 282 204 78 2.6:1
CinH-22 250 243 7 34.7:1
CinH-2 370 250 120 2.1:1
CinH-3 224 150 74 2.0:1
CinH-4 387 250 137 1.8:1
CinH-6 190 151 39 3.9:1
CinH-9 412 289 123 2.3:1
CinH-10 294 213 81 2.6:1
CinH-17 329 248 81 3.1:1
CinH-19 729 716 13 55.1:1
CinH-20 226 139 87 1.6:1
CinH-21 472 288 184 1.6:1
Seggregation analysis of T1 progeny

•Microscopic images of pollen
grains.
Pollen grains from non transgenic
tobacco (Xanthi),
•CinH_Drec event, and 2 CinH
events were collected and screened
under the FITC filtered epi
fluorescent microscopy.
•Left panel images were taken
under white fluorescent light with
1.67 ms exposure time. Right panel
images were taken under blue light
with 3 s exposure time. All images
were taken at 9200 magnifification

Percentage of GFP positive pollen in single transgene copy integrated CinH
transgenic events. Loss of GFP expression served as an effective indicator for
transgene excision

BIOSAFETY
• Protecting human & animal health and
environment from the possible adverse effects of
the products of modern biotechnology.
• Only one crop approved
• 14 crops under various stages of contained field trials
• Include brinjal, cotton, cabbage, groundnut, pigeon pea,
mustard, potato, sorghum, tomato, tobacco, rice, okra and
cauliflower
• Traits include insect resistance, herbicide tolerance,
virus resistance, nutritional enhancement, salt tolerance,
fungal resistance
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• There are six competent authorities as per the rules:
• Recombinant DNA Advisory Committee (RDAC)
• Review Committee on Genetic Manipulation
• (RCGM)
• Genetic Engineering Approval Committee (GEAC)
• Institutional Biosafety Committees (IBSC)
• State Biosafety Co ordination Committees (SBCC)
• District Level Committees (DLC)
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Protocol for release of transgenic crops
10/04/2021 57

”

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