Genetic pollution

The term genetic pollution was popularized by environmentalist Jeremy Rifkin in his book “The Biotech
Century”(1998).Genetic pollution accounts to the uncontrolled spread of genetic information (frequently
referring to transgenes) into the genomes of organisms in which such genes are not present in nature.
Genetically engineered (GE) plants contains genes which have been transferred from unrelated species. These
may come from bacteria, viruses,other plants or even animals. If these ‘foreign’ gene are the n transferred into
other organisms,this causes genetic contamination or pollution of the natural genepool.
Genetic pollution is usually associated with the gene flow from a genetically engineered
(GE)organism(orgeneticallymodifiedorganism-GMO)toanon-GMorganism."Geneticpollution"andcollateral
damage from GE field crops already have begun to wreak environmental havoc. Wind, rain, birds,bees, and
insect pollinators have begun carrying genetically-altered pollen into adjoining fields, polluting the
DNAofcrops oforganicand non-GEfarmers’
DEFINITION
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”
Gene flow
Gene flow is the movement of genes from one population to another, conferring new traits – the biophysical
characteristics of the organism – to individuals of the recipient population. This happens by cross-pollination
(also called hybridisation), that is, the pollination of members of one population or genetic pool with that of
another.
Transgene flow
Transgene flow is the loss of potentially useful crop genetic diversity in the recipient population (whether
other crops, landraces or wild relatives). Outbreeding depression (the reduction of fitness from hybridisation)
can lead to a decrease in allelic diversity by extinction of members of a diverse gene pool that are less adapted
to survive because of the particular introgressedtransgenic trait .

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

2. 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

3. SEMINAR OUTLINE 10/04/2021 3 Department of Genetics and plant breeding

4. What is GMO??? 10/04/2021 4 Department of Genetics and plant breeding

5. 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 10/04/2021 Department of Genetics and plant breeding 5 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

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7. 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. 10/04/2021 Department of Genetics and plant breeding 7

8. 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 . 10/04/2021 Department of Genetics and plant breeding 8

9. 10/04/2021 9 Department of Genetics and plant breeding 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)

10. 10/04/2021 Department of Genetics and plant breeding 10 VERTICAL GENE TRANSFER HORIZONTAL GENE TRANSFER

11. 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” 10/04/2021 11 Department of Genetics and plant breeding

12. Causes of genetic pollution 10/04/2021 12 Department of Genetics and plant breeding

13. 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). 10/04/2021 13 Department of Genetics and plant breeding

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15. 10/04/2021 15 Department of Genetics and plant breeding (Rizwan et al ., 2019)

16. 10/04/2021 16 Department of Genetics and plant breeding (Rizwan et al ., 2019)

17. 10/04/2021 Department of Genetics and plant breeding 17 Impacts of genetic pollution Direct effects on non target organisms Genetically modified organisms might lead the non-GM organisms to extinction Impacts of transgenic crops on parasitoids 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

18. Direct effects on non-target organisms Transgenic pollen harms monarch larvae 10/04/2021 Department of Genetics and plant breeding 18 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)

19. Table 2. Impacts of transgenic crops on parasitoids 10/04/2021 Department of Genetics and plant breeding 19 (Gatehouse et al., 2011) Protein Transgenic plant Natural enemy Pest Effects on natural eneny Bt (cry 1AB) 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

20. Increased weediness 10/04/2021 Department of Genetics and plant breeding 20 Weediness There are apprehensions about GM crops becoming weeds Development of super weeds

21. s 10/04/2021 Department of Genetics and plant breeding 21

22. 10/04/2021 Department of Genetics and plant breeding 22 Yook et al . (2021)

23. 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 10/04/2021 Department of Genetics and plant breeding 23 Objectives

24. 10/04/2021 Department of Genetics and plant breeding 24 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)

25. 10/04/2021 Department of Genetics and plant breeding 25 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.

26. Phenotypic performances on reproductive traits and flowering phenology for parental soybeans and hybrids Parameter GR soybean Wild soybean F 1 hybrid F 2 hybrid First flowering 25 July 7 August 2 August 2 August End of f lowering 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 10/04/2021 Department of Genetics and plant breeding 26 Table 2 Yook et al. (2021)

27. 10/04/2021 Department of Genetics and plant breeding 27 Distance(m) 2013 2014 Tested seed no Survivors no. Hybrid s no. % Gene flow rate Tested seed no Survivors no. Hybrids no. % Gene flow rate - - - - 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

28. 10/04/2021 Department of Genetics and plant breeding 28 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

29. Stratagies to reduce genetic pollution Physical containment Biological containment 10/04/2021 29 Department of Genetics and plant breeding

30. Physical containment 10/04/2021 30 Department of Genetics and plant breeding 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)

31. 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 10/04/2021 Department of Genetics and plant breeding 31 31 (Daniel et al ., 2002)

32. Seed lethal system 10/04/2021 Department of Genetics and plant breeding 32 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)

33. Method Tight repression by R- locus Activity of seed specific promoter SL construct should be lethal to plants 10/04/2021 Department of Genetics and plant breeding 33

34. 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 10/04/2021 34 Department of Genetics and plant breeding 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

35. 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) 10/04/2021 35 Department of Genetics and plant breeding

36. 10/04/2021 Department of Genetics and plant breeding 36 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)

37. Materials and methods 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 10/04/2021 Department of Genetics and plant breeding 37 Yan et al . (2020)

38. 10/04/2021 Department of Genetics and plant breeding 38 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 .

39. 10/04/2021 Department of Genetics and plant breeding 39 Yan et al . (2020)

40. 10/04/2021 Department of Genetics and plant breeding 40 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

41. 10/04/2021 Department of Genetics and plant breeding 41 Pollen mediated gene flow under different intercropping

42. 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 Variety specific ( V- GURT) Trait specific (T- GURT 10/04/2021 42 Department of Genetics and plant breeding

43. 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. 10/04/2021 Department of Genetics and plant breeding 43

44. Components 4.Inducing substance ( Inducer) Mostly of chemical origin Biodegradable Nontoxic for the ecosystem Directly applicable in the field or in seeds 10/04/2021 Department of Genetics and plant breeding 44 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

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46. Site specific mutagenesis and Recombinase Zing finger nucleases TALENs CRISPER – Cas and EcoR 1 restriction endonucleases 10/04/2021 46 Department of Genetics and plant breeding Transgene mitigation

47. 10/04/2021 47 Department of Genetics and plant breeding Objectives To excise the transgene from the pollen using CinH R-S2 recombination system or a codon optimized serine resolvase CinH recombinase Materials and methods 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)

48. 10/04/2021 48 Department of Genetics and plant breeding Schematic illustration of CinH recombinase mediated transgene excision in pollen

49. 10/04/2021 Department of Genetics and plant breeding 49 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. 10/04/2021 Department of Genetics and plant breeding 51 Seggregation analysis of T1 progeny

52. 10/04/2021 Department of Genetics and plant breeding 52 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

53. 10/04/2021 Department of Genetics and plant breeding 53 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

54. 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 10/04/2021 54 Department of Genetics and plant breeding

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56. 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) 10/04/2021 56 Department of Genetics and plant breeding

57. Protocol for release of transgenic crops 10/04/2021 57 Department of Genetics and plant breeding

58. “BE A PART OF THE SOLUTION NOT PART OF POLLUTION ” 10/04/2021 Department of Genetics and plant breeding 58

59. 10/04/2021 Department of Genetics and plant breeding 59 Save PLAN(E)T Thank you!!!