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debal deb-GM_Mustard-environmental risk assessment-dmh-11-Jul14-GEAC.pdf

Debal Deb
Debal Deb

The document summarizes potential environmental risks from the commercialization of DMH-11, a transgenic mustard hybrid in India. It notes that the male sterility trait introduced through the barnase gene is capable of spreading to non-GM mustard populations through outcrossing. Modeling shows the frequency of the barnase gene will increase over generations, spreading male sterility and potentially causing crop losses. Concerns are also raised about a lack of study on the impacts of herbicide use and potential for herbicide tolerant superweeds. Methodological flaws are identified in field trials conducted to assess the crop.

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Environmental Risks of DMH-11 & Unscientific Biosafety Trials Dr. Debal Deb Centre for Interdisciplinary Studies http://cintdis.org (Member, Agro-Biodiversity Expert Committee, National Biodiversity Authority)
Presentation will cover 1. Population Genetics of MS (Male Sterility) Trait 2. Spread of HT (Herbicide Tolerance) Trait 3. Breakdown of MS 4. Methodological Flaws in Trials 5. False Assumption about Cross Pollination & Crossability between Indian Mustard and Relatives
1 The Risk of barnase Male Sterility Transmission to Non-GM Mustard Populations
The Transmissibility of Transgenic Male Sterility Trait 1. The barnase transgene-induced MS is capable of transmission to non-GM plants. 2. Gametes from GM MS plants will contain barnase gene. 3. After outcrossing, the recipient non-GM plant will bear seeds with hemizygous Barnase/B combinations. B is Barnase; B is absence of Barnase 硫 is Barstar; 硫 is absence of Barstar
VarunaBn 3.6 EH-2 modbs 2.99 B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 DMH-11 DMH-11 B / B 硫 / 硫 B /B 硫 / 硫 B / B 硫 / 硫 B / B 硫/硫 MS B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 MS B/B 硫 / 硫 B/B 硫 / 硫 B/ B 硫/硫 2 2 4 2 2 DMH-11 DMH-11 18.76 % will be MS in F1 25% will be MS in F2
Offspring Genotype Phenotype Genotype Frequency Prob. F1 BB 硫硫 p1 2 1/16 BB 硫硫 2 p1p2 1/8 BB 硫硫 Male Sterile p2 2 1/16 BB 硫硫 2 p1p3 1/8 BB 硫硫 2 ( p1p4 + p2p3 ) 1/4 BB 硫硫 Male Sterile 2 p2p4 1/8 BB 硫硫 p3 2 1/8 BB 硫硫 2 p3p4 1/16 BB 硫硫 p4 2 1/16 Trait Haplotype Frequency GM B 硫 p1 B 硫 p2 B 硫 p3 Non- GM B 硫 p4 Total Male Sterile % = 18.76
Genotype B B B B B B Frequency p2 2 p (1 p) (1 p)2 Fitness w11 w12 w22 Regardless of the Presence of barstar (硫) Gene, the Frequency of barnase (B) Gene will Continue to Increase Every Generation. We May Assume, Realistically, w11> w12 > w22 because Farmers will preferentially Grow Plants containing barnase gene If Not for Herbicide Tolerance. After Selfing in the DMH-11 Population, the B allele Frequency in the Next Generation will be p' = [p2 w11 + p (1 p) w12] / [p2 w11 + p (1 p) w12 + p2 w22] Thus, the Difference in Allele Frequency between p and p is: p = [p2 (1 p) (w11 w12) + p (1 p)2 (w12 w22) / H where H = p2 w11 + 2 p (1 p) w12 + (1 p)2 w22 Barnase Gene Frequency : Will It Increase Over Generations?
Selection and Fitness Advantage in GM Mustard Farmers Adoption of Glufosinate Herbicides, will Entail Fitness Disadvantage for All Bar Gene-Deficient Genotypes (incl. Non-GM Genotypes), and Advantage for All barnase-Bar-barstar Containing Plants. In Absence of Herbicides, Fitness of the GM Mustard is Likely to be Greater than Non-GM Mustard, Primarily Due to promised/ hyped Yield Benefit. Even a minor advantage weightage will lead to a monotonic increase in the barnase gene in the population.
Estimation of Frequency of barnase Allele, with 3 Sets of Simulated Fitness Values: W11 w12 w22 Ser. 1 1 1 0.8 Ser. 2 1 1 0.5 Ser. 3 1 0.6 0.5 The DU Reports Assertion that The frequency of the "new allele" will remain same generation after generation is WRONG.
The Monotonic Increase in barnase Transgene Frequency in Mustard Population: What Does it Imply? Male Sterile Trait will Spread in the Population through Outcrossing with Non-GM Mustard Sub- Populations With an Increasing Frequency of the Gene (due to Greater Farm Level Selection Advantage), More and More Proportions of the Non-GM Mustard Crop Population will Become MS, Leading to Crop Loss.
Even If DMH-11 Seeds are Replaced Every Year, the Out-crossing of the First DMH-11 plants with NON-GM Mustard in & around the DMH-11 Field will Spread the barnase, Bar and barstar Genes Every Subsequent Generation. After the First Year of Out-Crossing with Non-GM Mustard, the F1 Progeny will contain 25% MS.
B / B 硫 / 硫 B / B 硫/ 硫 B / B 硫 / 硫 B/ B 硫 / 硫 B / B 硫/ 硫 B/ B 硫 / 硫 MS DMH-11 Plant Non-GM Plant DMH-11 25% MS F1 Outcrossing with Non-GM Mustard will Produce 25% MS Offspring Application of Herbicides will Eliminate 25% of the F1 Population. In the Absence of Herbicide Spray, Panmyxis in F1 will Produce Hybrids with 14.29% MS Phenotype in F2 WIldtype
F1 Gamete Weightage Gametes from genotype [B B 硫 硫] : B 硫 1 B 硫 1 B硫 1 B 硫 1 Gametes from genotype (MS) [B B 硫 硫] : B 硫 2 1 (No pollen) B 硫 2 1 (No pollen) Gametes from genotype [B B 硫 硫] : B 硫 2 B硫 2 Gametes from genotype [B B 硫硫]: B 硫 4 B 硫 : B 硫 : B硫 : B 硫 1 2 3 8 F2 Generation (after Outcrossing with non-GM Crop B 硫) [BB 硫硫] [BB 硫硫] [BB硫硫] [BB 硫硫] 1 2 3 8
F2 PROGENY FROM F1 (DMH-11 X Non-GM) PROGENY B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 Gametes from F1 Progeny: B B B B 硫 硫 硫 硫 1 : 2 : 3 : 8 1 2 3 8 F2 GENOTYPES MS 1/7 = 14.286% B / B 硫/ 硫 This MS plant Will NOT produce Pollen B 硫 B 硫 X
Yield Losses for Farmers will Ensue from: Increasing Male Sterility, Leading to Proportionate Loss of Seeds When F2 Seeds are Used, Loss of Hybrid Vigour Reducing Crop Yield In the DMH-11 Crop, Glufosinate Spray will Cause 6.25% yield loss in F1 progeny. In Case of DMH-11 Outcrossing with A Non-GM Mustard, Glufosinate Application will Cause 25% Loss in F1 and 57.14% Loss in the F2 progeny
Precision? Predictability? Summary Report (p. 9) states Variable Expression of bar gene - from location to location - from plant to plant in the same line - from leaf to leaf in the Same Plant If this Unpredictability and Uncertainty Prevails Even in the Plants HOMOZYGOUS for bar gene, * What is the Level of Certainty of Expression of the Other Transgenes? * How Truthful is the Promise to Farmers of the Crops Advantage?
2 Herbicide Use: Is There Scope for Complacency?
Assumption: Farmers Dont Use Herbicides in Mustard Cultivation False Assumption in the Case of (Eastern) Indian Farmers. Herbicides used at Harvest extensively not to eradicate weeds. HAVE REGULATORS STUDIED: Herbicide Tolerant Superweeds? Health Impacts? Impact of Bar gene Flow to Non-GM Mustard and Related Species (esp. B. rapa and B. napus)? The Glufosinate Herbicide Employed in Developing the New Mustard Hybrid is a Trojan Horse for Marketing and Cultivation of the Herbicide as Well as the Hybrid Seeds.
3 Male Sterility Breakdown in Varuna barnase
The Central Compliance Committee field visits also note this down at various trial locations Non-standardisation of test protocol an issue as seen in reports Lies in summing up : No sterility breakdown observed in extensive backcrosses and numerous field observations; Pollination behaviour of all the lines remained true to their expected characteristics. How much of the Yield of Varuna-Bn line is due to Male Sterility Breakdown and how much due to Cross Pollination?
Despite Reporting No Male Sterility Breakdown, Field Trials in Ludhiana shows Male Sterility Breaking Down to the Extent of 7.43% in MS Plants in Varunabarnase. Calculation: In 10 Sample Plants, 548 Pods, with 12 seeds/Pod in Fertile Plants = total 6576 Seeds 176 Pods, with 3 seeds/Pod in MS Plants = total 528 Seeds Thus, Proportion of MS breakdown, as indicated by Seeds produced in MS Plants = 100 [528/ (528+6576)] = 7.43%
4 Unreliable Methodology and Spurious Data
The GM Crop Developers State with Reference to Pollen Flow That: 1. The Maximum Distance of Bee Pollination is 20 m. Absurd. Bee Pollination Distance Can Exceed 6 km. [see Pasquet et al. 2008. PNAS 105: 13456-61] Specifically for Mustard, gene flow was detected at the 800 m limit of the experiment. [Beckle et al. 2003. Ecol. Appl. 13: 1276-1294 ] 2. "Farmers who want to keep their own [sic] seed are advised to collect seed from the centre of the field." It's Irrational to Assume that Bees will Visit only the Peripheral Plants, and Plants at the Centre of the Field Only will Self-Pollinate!
Test for Aggressiveness and Weediness Record of Plant Height Leaf Traits Floral Traits are Irrelevant and Misleading. WHERE ARE DATA OF POSSIBLE COMPETITIVE ADVANTAGE OF GM MUSTARD - With Herbicide Exposure? - When Outcrossed with Non-GM Cultivars? VOLUNTEER BEHAVIOUR OVER A FEW SEASONS NOT STUDIED
Pod Shattering This trait not studied in any quantitative fashion A very important trait for farmers More importantly, this trait is related to biosafety risks (weediness, contamination of non-GM)
Counts of Beneficial Insects Predators: Coccinelid Beetles, Lacewings, Syrphid Larvae Pollinators: Honeybees No Other Taxa are Considered in the Tests. Mustard fields are habitats for Odonates Ants Gryllids Spiders WHY NO EFFORT TO TEST EFFECTS OF GMO ON THESE ARTHROPODS?
Periodic Observation Method: Unreliable, with No Complementary Method For Aphids - Suction Sampler; Drop Cloth For Beetle Adults - Drop Cloth, Vacuum/ Suction, Sweep net, Pitfall trap, Videography For Beetle Larvae Drop Cloth, Sweep Net, Pitfall trap For Honeybees (which species? Only one? Really?) - Sweep Net; Soap bowl; Videography For Lacewings - Sweep Net, Videography Pedigo & Buntin 1993. Handbook of sampling methods for arthropods in agriculture; Woltz & Landis 2014. J. Appl. Entomol. 138: 475484
Counts of Insect Pests: An Ecologically Unfeasible Scenario Trial Location Mustard Aphid Painted Bug Leaf Miner Cabbage Butterfly Mustard Sawfly Termites BRL-I, 1st Year (2010-11) Kumher Nil Nil Nil Nil Nil Alwar Nil Nil Nil Nil SG nagar Nil Nil Nil Nil Nil BRL-II, 2nd Year (2011-12) Kumher Nil Nil Nil Nil Nil Alwar Nil Nil Nil Nil Nil SG nagar Nil Nil Nil Nil Nil Nil BRL-III, 3rd Year (2014-15) Delhi Nil Nil Nil Nil Nil Bhatinda Nil Nil Nil Nil Nil Nil Ludhiana Nil Nil Nil Nil Nil Nil Ref : 1) BRL Trial Reports
STATISTICS IN THE TRIAL: INACCURATE & DECEITFUL Predators Varuna-Barnase EH-2 Dmh-11 Coccinelids (Grubs + Beetles) 60/70/80/90/100 /110/120 DAS 8.75 6.50 10.25 Chrysoperla Larvae Nil Nil Nil Honey bee visits per 5 minutes 13.1 15.56 19.44 Honey bee visits per 10 minutes 26.94 31.94 36.63 Questions Mean per Replicates or per Day of test? ditto ditto
A Lesson in Averaging (using Excel) NOT CLEAR WHAT WAS CALCULATED HERE TWO OF THE ABOVE ARE INVALID, IF USED
Doctored Report: INSECT DATA (Rabi 2011) Mustard Aphid All Other Prey COCCINELID ABUNDANCE Tables 8 A & 10 A 4.9 Nil Nil Tables 8 B & 10 B 3.26 Nil 10.25 Tables 8 C & 10 C Nil Nil Nil A Global Ecological Rule * Low or No Predator in Presence of Prey is Possible. * When Prey Abundance is Low, 3 Times Higher Predator Abundance is Ecologically Unfeasible. - The Trial Report Boldly Defies This Rule !
Honey Bee Visitation Counts : What Exactly was Counted? A) Foraging Rate The No. of Flowers Probed by an Individual Bee in X minutes e.g. Honeybees: 9.2 Flowers/min; Beetles: 2.2/min (Couvillon et al. 2015 Psyche, Article ID 134630) B) Visitation Counts No. of Bees Observed per Flower in X minutes e.g. A. dorsata: 7/ Fl/ 5 min; A. florea: 1/ Fl/ 5 min. (Balachandran et al. 2014 Curr. Sci.106: 1379-86) C) Visitation-Abundance No. of Bees in a Patch of Flowers in X minutes e.g. 7.53 Bees/ 10 Flowers/ 5 min. (Sivaram et al. 2013 Braz. Arch. Biol. Tech. 56: 365-70) The Trial Reports: Incompetent Methodology: Untrustworthy.
Statistical Fallacy, Again! Bee Visitation in 10 min is Not Double that in 5 min in the Field!! ( Unless The Bees are Tutored) Predators Varuna-Barnase EH-2 DMH-11 Honey bee visits per 5 minutes 13.1 15.56 19.44 Honey bee visits per 10 minutes 26.94 31.94 36.63 At this rate, Honey Bee Visits to DMH-11 would Exceed 2332 if Observed for 10 hr !
Crossability Studies The Report Falsely Claims: No Reports are Available on Naturally Occurring Inter-specific Crosses among Cultivated and Wild Species of Brassica Cenospecies in India. There is a Legion of Studies Reporting Natural Crossability between B. juncea and B. napus, (Oilseed Rape) and B. rapa (Yellow Sarson). Bing et al. 1996; Gupta 1997; Choudhary & Joshi 1999; Ghosh Dastidar & Varma 1999; Choudhary et al. 2002; Gupta et al. 2006 The Frequency of Hybridization between B. napus oleifera and B. juncea in Nature is 5.91%. Salisbury 2006
CURRENT TESTING & DATA RELATED TO DMH-11 TRANSGENIC MUSTARD COMPLETELY UNRELIABLE AND EVEN DOCTORED FOR ANY DECISION- MAKING MANY RISKS REMAIN UNASSESSED
References Beckle, HL, S.I. Warwick, H Nair, G. Seguin-Swartz 2003. Gene flow in commercial fields of herbicide-resistant canola (Brasscia napus). Ecol. Appl. 13: 1276-94 Bing, D.j., R K Downey and G F W Rakow 1996. Hybridization among Brasscia napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapois arvensis under open pollinated conditions in the field. Plant Breeding 115: 470-73. Canadian Food Inspection Agency . Biology of Brassica juncea (Canola/ Mustard). Biology Document BIO2007-01: A companion document to the Directive 94-08 (Dir94-08) Assessment Criteria for Determining Environmental safety of Plants and Novel Traits. http://inspection.gc.ca Chaudhary, B R and P. Joshi ,1999. Interspecific hybridization in Brassica. Proc. 10th Intl. Rapeseed Congr, Australia. Contribution No. 510. Chaudhary, B R, P. Joshi and S R Rao 2002. Cytogenetics of Brassica juncea X Brassica rapa hybrids and patters of variation in the hybrid derivatives. Plant Br. 121: 292-96. Ghosh Dastidar, N and N S Varma 1999. A study on intercrossing between transgenic B. juncea and other related species. Proc. 10th Intl. Rapeseed Congr, Australia. Contribution No 244. Gupta, S.K. 1997. Production of interspecific and inergeneric hybrids in Brassica and Raphanus. Cruciferae Newslett. Eucarpia 19: 21-22. Gupta, K, D. Prem, N I Nashaat and A Agnihotri 2006. Response of interspecific Brassica juncea/ B. rapa hybrids and their advanced progenies to Albugo candida (white blister). Plant Pathol. 55: 679-89. Pasquet, R S, A Peltier, M B Hufford, E Oudin, J Saulnier, L. Paul, J T Knudsen, H R Herren and P Gepts 2008. Long-distance pollen flow assessment through evaluation of pollinator foraging range suggests transgene escape distances. PNAS 105: 13456-61. Rakow, G. and D L Woods 1984. Outcrossing in rape and mustard under Saskatchewan prairie conditions. Can. J. Pl. Sci. 67: 147-51. Salisbury, P. 2006. Biology of Brassica juncea and potential gene flow from B. juncea to other Brassicaceae species in Australia. Faculty of Land and Food Resources, University of Melbourne. Miscellaneous Report PAS 2006/1.

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debal deb-GM_Mustard-environmental risk assessment-dmh-11-Jul14-GEAC.pdf

  • 1. Environmental Risks of DMH-11 & Unscientific Biosafety Trials Dr. Debal Deb Centre for Interdisciplinary Studies http://cintdis.org (Member, Agro-Biodiversity Expert Committee, National Biodiversity Authority)
  • 2. Presentation will cover 1. Population Genetics of MS (Male Sterility) Trait 2. Spread of HT (Herbicide Tolerance) Trait 3. Breakdown of MS 4. Methodological Flaws in Trials 5. False Assumption about Cross Pollination & Crossability between Indian Mustard and Relatives
  • 3. 1 The Risk of barnase Male Sterility Transmission to Non-GM Mustard Populations
  • 4. The Transmissibility of Transgenic Male Sterility Trait 1. The barnase transgene-induced MS is capable of transmission to non-GM plants. 2. Gametes from GM MS plants will contain barnase gene. 3. After outcrossing, the recipient non-GM plant will bear seeds with hemizygous Barnase/B combinations. B is Barnase; B is absence of Barnase 硫 is Barstar; 硫 is absence of Barstar
  • 5. VarunaBn 3.6 EH-2 modbs 2.99 B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 DMH-11 DMH-11 B / B 硫 / 硫 B /B 硫 / 硫 B / B 硫 / 硫 B / B 硫/硫 MS B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 MS B/B 硫 / 硫 B/B 硫 / 硫 B/ B 硫/硫 2 2 4 2 2 DMH-11 DMH-11 18.76 % will be MS in F1 25% will be MS in F2
  • 6. Offspring Genotype Phenotype Genotype Frequency Prob. F1 BB 硫硫 p1 2 1/16 BB 硫硫 2 p1p2 1/8 BB 硫硫 Male Sterile p2 2 1/16 BB 硫硫 2 p1p3 1/8 BB 硫硫 2 ( p1p4 + p2p3 ) 1/4 BB 硫硫 Male Sterile 2 p2p4 1/8 BB 硫硫 p3 2 1/8 BB 硫硫 2 p3p4 1/16 BB 硫硫 p4 2 1/16 Trait Haplotype Frequency GM B 硫 p1 B 硫 p2 B 硫 p3 Non- GM B 硫 p4 Total Male Sterile % = 18.76
  • 7. Genotype B B B B B B Frequency p2 2 p (1 p) (1 p)2 Fitness w11 w12 w22 Regardless of the Presence of barstar (硫) Gene, the Frequency of barnase (B) Gene will Continue to Increase Every Generation. We May Assume, Realistically, w11> w12 > w22 because Farmers will preferentially Grow Plants containing barnase gene If Not for Herbicide Tolerance. After Selfing in the DMH-11 Population, the B allele Frequency in the Next Generation will be p' = [p2 w11 + p (1 p) w12] / [p2 w11 + p (1 p) w12 + p2 w22] Thus, the Difference in Allele Frequency between p and p is: p = [p2 (1 p) (w11 w12) + p (1 p)2 (w12 w22) / H where H = p2 w11 + 2 p (1 p) w12 + (1 p)2 w22 Barnase Gene Frequency : Will It Increase Over Generations?
  • 8. Selection and Fitness Advantage in GM Mustard Farmers Adoption of Glufosinate Herbicides, will Entail Fitness Disadvantage for All Bar Gene-Deficient Genotypes (incl. Non-GM Genotypes), and Advantage for All barnase-Bar-barstar Containing Plants. In Absence of Herbicides, Fitness of the GM Mustard is Likely to be Greater than Non-GM Mustard, Primarily Due to promised/ hyped Yield Benefit. Even a minor advantage weightage will lead to a monotonic increase in the barnase gene in the population.
  • 9. Estimation of Frequency of barnase Allele, with 3 Sets of Simulated Fitness Values: W11 w12 w22 Ser. 1 1 1 0.8 Ser. 2 1 1 0.5 Ser. 3 1 0.6 0.5 The DU Reports Assertion that The frequency of the "new allele" will remain same generation after generation is WRONG.
  • 10. The Monotonic Increase in barnase Transgene Frequency in Mustard Population: What Does it Imply? Male Sterile Trait will Spread in the Population through Outcrossing with Non-GM Mustard Sub- Populations With an Increasing Frequency of the Gene (due to Greater Farm Level Selection Advantage), More and More Proportions of the Non-GM Mustard Crop Population will Become MS, Leading to Crop Loss.
  • 11. Even If DMH-11 Seeds are Replaced Every Year, the Out-crossing of the First DMH-11 plants with NON-GM Mustard in & around the DMH-11 Field will Spread the barnase, Bar and barstar Genes Every Subsequent Generation. After the First Year of Out-Crossing with Non-GM Mustard, the F1 Progeny will contain 25% MS.
  • 12. B / B 硫 / 硫 B / B 硫/ 硫 B / B 硫 / 硫 B/ B 硫 / 硫 B / B 硫/ 硫 B/ B 硫 / 硫 MS DMH-11 Plant Non-GM Plant DMH-11 25% MS F1 Outcrossing with Non-GM Mustard will Produce 25% MS Offspring Application of Herbicides will Eliminate 25% of the F1 Population. In the Absence of Herbicide Spray, Panmyxis in F1 will Produce Hybrids with 14.29% MS Phenotype in F2 WIldtype
  • 13. F1 Gamete Weightage Gametes from genotype [B B 硫 硫] : B 硫 1 B 硫 1 B硫 1 B 硫 1 Gametes from genotype (MS) [B B 硫 硫] : B 硫 2 1 (No pollen) B 硫 2 1 (No pollen) Gametes from genotype [B B 硫 硫] : B 硫 2 B硫 2 Gametes from genotype [B B 硫硫]: B 硫 4 B 硫 : B 硫 : B硫 : B 硫 1 2 3 8 F2 Generation (after Outcrossing with non-GM Crop B 硫) [BB 硫硫] [BB 硫硫] [BB硫硫] [BB 硫硫] 1 2 3 8
  • 14. F2 PROGENY FROM F1 (DMH-11 X Non-GM) PROGENY B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 B / B 硫 / 硫 Gametes from F1 Progeny: B B B B 硫 硫 硫 硫 1 : 2 : 3 : 8 1 2 3 8 F2 GENOTYPES MS 1/7 = 14.286% B / B 硫/ 硫 This MS plant Will NOT produce Pollen B 硫 B 硫 X
  • 15. Yield Losses for Farmers will Ensue from: Increasing Male Sterility, Leading to Proportionate Loss of Seeds When F2 Seeds are Used, Loss of Hybrid Vigour Reducing Crop Yield In the DMH-11 Crop, Glufosinate Spray will Cause 6.25% yield loss in F1 progeny. In Case of DMH-11 Outcrossing with A Non-GM Mustard, Glufosinate Application will Cause 25% Loss in F1 and 57.14% Loss in the F2 progeny
  • 16. Precision? Predictability? Summary Report (p. 9) states Variable Expression of bar gene - from location to location - from plant to plant in the same line - from leaf to leaf in the Same Plant If this Unpredictability and Uncertainty Prevails Even in the Plants HOMOZYGOUS for bar gene, * What is the Level of Certainty of Expression of the Other Transgenes? * How Truthful is the Promise to Farmers of the Crops Advantage?
  • 17. 2 Herbicide Use: Is There Scope for Complacency?
  • 18. Assumption: Farmers Dont Use Herbicides in Mustard Cultivation False Assumption in the Case of (Eastern) Indian Farmers. Herbicides used at Harvest extensively not to eradicate weeds. HAVE REGULATORS STUDIED: Herbicide Tolerant Superweeds? Health Impacts? Impact of Bar gene Flow to Non-GM Mustard and Related Species (esp. B. rapa and B. napus)? The Glufosinate Herbicide Employed in Developing the New Mustard Hybrid is a Trojan Horse for Marketing and Cultivation of the Herbicide as Well as the Hybrid Seeds.
  • 20. The Central Compliance Committee field visits also note this down at various trial locations Non-standardisation of test protocol an issue as seen in reports Lies in summing up : No sterility breakdown observed in extensive backcrosses and numerous field observations; Pollination behaviour of all the lines remained true to their expected characteristics. How much of the Yield of Varuna-Bn line is due to Male Sterility Breakdown and how much due to Cross Pollination?
  • 21. Despite Reporting No Male Sterility Breakdown, Field Trials in Ludhiana shows Male Sterility Breaking Down to the Extent of 7.43% in MS Plants in Varunabarnase. Calculation: In 10 Sample Plants, 548 Pods, with 12 seeds/Pod in Fertile Plants = total 6576 Seeds 176 Pods, with 3 seeds/Pod in MS Plants = total 528 Seeds Thus, Proportion of MS breakdown, as indicated by Seeds produced in MS Plants = 100 [528/ (528+6576)] = 7.43%
  • 23. The GM Crop Developers State with Reference to Pollen Flow That: 1. The Maximum Distance of Bee Pollination is 20 m. Absurd. Bee Pollination Distance Can Exceed 6 km. [see Pasquet et al. 2008. PNAS 105: 13456-61] Specifically for Mustard, gene flow was detected at the 800 m limit of the experiment. [Beckle et al. 2003. Ecol. Appl. 13: 1276-1294 ] 2. "Farmers who want to keep their own [sic] seed are advised to collect seed from the centre of the field." It's Irrational to Assume that Bees will Visit only the Peripheral Plants, and Plants at the Centre of the Field Only will Self-Pollinate!
  • 24. Test for Aggressiveness and Weediness Record of Plant Height Leaf Traits Floral Traits are Irrelevant and Misleading. WHERE ARE DATA OF POSSIBLE COMPETITIVE ADVANTAGE OF GM MUSTARD - With Herbicide Exposure? - When Outcrossed with Non-GM Cultivars? VOLUNTEER BEHAVIOUR OVER A FEW SEASONS NOT STUDIED
  • 25. Pod Shattering This trait not studied in any quantitative fashion A very important trait for farmers More importantly, this trait is related to biosafety risks (weediness, contamination of non-GM)
  • 26. Counts of Beneficial Insects Predators: Coccinelid Beetles, Lacewings, Syrphid Larvae Pollinators: Honeybees No Other Taxa are Considered in the Tests. Mustard fields are habitats for Odonates Ants Gryllids Spiders WHY NO EFFORT TO TEST EFFECTS OF GMO ON THESE ARTHROPODS?
  • 27. Periodic Observation Method: Unreliable, with No Complementary Method For Aphids - Suction Sampler; Drop Cloth For Beetle Adults - Drop Cloth, Vacuum/ Suction, Sweep net, Pitfall trap, Videography For Beetle Larvae Drop Cloth, Sweep Net, Pitfall trap For Honeybees (which species? Only one? Really?) - Sweep Net; Soap bowl; Videography For Lacewings - Sweep Net, Videography Pedigo & Buntin 1993. Handbook of sampling methods for arthropods in agriculture; Woltz & Landis 2014. J. Appl. Entomol. 138: 475484
  • 28. Counts of Insect Pests: An Ecologically Unfeasible Scenario Trial Location Mustard Aphid Painted Bug Leaf Miner Cabbage Butterfly Mustard Sawfly Termites BRL-I, 1st Year (2010-11) Kumher Nil Nil Nil Nil Nil Alwar Nil Nil Nil Nil SG nagar Nil Nil Nil Nil Nil BRL-II, 2nd Year (2011-12) Kumher Nil Nil Nil Nil Nil Alwar Nil Nil Nil Nil Nil SG nagar Nil Nil Nil Nil Nil Nil BRL-III, 3rd Year (2014-15) Delhi Nil Nil Nil Nil Nil Bhatinda Nil Nil Nil Nil Nil Nil Ludhiana Nil Nil Nil Nil Nil Nil Ref : 1) BRL Trial Reports
  • 29. STATISTICS IN THE TRIAL: INACCURATE & DECEITFUL Predators Varuna-Barnase EH-2 Dmh-11 Coccinelids (Grubs + Beetles) 60/70/80/90/100 /110/120 DAS 8.75 6.50 10.25 Chrysoperla Larvae Nil Nil Nil Honey bee visits per 5 minutes 13.1 15.56 19.44 Honey bee visits per 10 minutes 26.94 31.94 36.63 Questions Mean per Replicates or per Day of test? ditto ditto
  • 30. A Lesson in Averaging (using Excel) NOT CLEAR WHAT WAS CALCULATED HERE TWO OF THE ABOVE ARE INVALID, IF USED
  • 31. Doctored Report: INSECT DATA (Rabi 2011) Mustard Aphid All Other Prey COCCINELID ABUNDANCE Tables 8 A & 10 A 4.9 Nil Nil Tables 8 B & 10 B 3.26 Nil 10.25 Tables 8 C & 10 C Nil Nil Nil A Global Ecological Rule * Low or No Predator in Presence of Prey is Possible. * When Prey Abundance is Low, 3 Times Higher Predator Abundance is Ecologically Unfeasible. - The Trial Report Boldly Defies This Rule !
  • 32. Honey Bee Visitation Counts : What Exactly was Counted? A) Foraging Rate The No. of Flowers Probed by an Individual Bee in X minutes e.g. Honeybees: 9.2 Flowers/min; Beetles: 2.2/min (Couvillon et al. 2015 Psyche, Article ID 134630) B) Visitation Counts No. of Bees Observed per Flower in X minutes e.g. A. dorsata: 7/ Fl/ 5 min; A. florea: 1/ Fl/ 5 min. (Balachandran et al. 2014 Curr. Sci.106: 1379-86) C) Visitation-Abundance No. of Bees in a Patch of Flowers in X minutes e.g. 7.53 Bees/ 10 Flowers/ 5 min. (Sivaram et al. 2013 Braz. Arch. Biol. Tech. 56: 365-70) The Trial Reports: Incompetent Methodology: Untrustworthy.
  • 33. Statistical Fallacy, Again! Bee Visitation in 10 min is Not Double that in 5 min in the Field!! ( Unless The Bees are Tutored) Predators Varuna-Barnase EH-2 DMH-11 Honey bee visits per 5 minutes 13.1 15.56 19.44 Honey bee visits per 10 minutes 26.94 31.94 36.63 At this rate, Honey Bee Visits to DMH-11 would Exceed 2332 if Observed for 10 hr !
  • 34. Crossability Studies The Report Falsely Claims: No Reports are Available on Naturally Occurring Inter-specific Crosses among Cultivated and Wild Species of Brassica Cenospecies in India. There is a Legion of Studies Reporting Natural Crossability between B. juncea and B. napus, (Oilseed Rape) and B. rapa (Yellow Sarson). Bing et al. 1996; Gupta 1997; Choudhary & Joshi 1999; Ghosh Dastidar & Varma 1999; Choudhary et al. 2002; Gupta et al. 2006 The Frequency of Hybridization between B. napus oleifera and B. juncea in Nature is 5.91%. Salisbury 2006
  • 35. CURRENT TESTING & DATA RELATED TO DMH-11 TRANSGENIC MUSTARD COMPLETELY UNRELIABLE AND EVEN DOCTORED FOR ANY DECISION- MAKING MANY RISKS REMAIN UNASSESSED
  • 36. References Beckle, HL, S.I. Warwick, H Nair, G. Seguin-Swartz 2003. Gene flow in commercial fields of herbicide-resistant canola (Brasscia napus). Ecol. Appl. 13: 1276-94 Bing, D.j., R K Downey and G F W Rakow 1996. Hybridization among Brasscia napus, B. rapa and B. juncea and their two weedy relatives B. nigra and Sinapois arvensis under open pollinated conditions in the field. Plant Breeding 115: 470-73. Canadian Food Inspection Agency . Biology of Brassica juncea (Canola/ Mustard). Biology Document BIO2007-01: A companion document to the Directive 94-08 (Dir94-08) Assessment Criteria for Determining Environmental safety of Plants and Novel Traits. http://inspection.gc.ca Chaudhary, B R and P. Joshi ,1999. Interspecific hybridization in Brassica. Proc. 10th Intl. Rapeseed Congr, Australia. Contribution No. 510. Chaudhary, B R, P. Joshi and S R Rao 2002. Cytogenetics of Brassica juncea X Brassica rapa hybrids and patters of variation in the hybrid derivatives. Plant Br. 121: 292-96. Ghosh Dastidar, N and N S Varma 1999. A study on intercrossing between transgenic B. juncea and other related species. Proc. 10th Intl. Rapeseed Congr, Australia. Contribution No 244. Gupta, S.K. 1997. Production of interspecific and inergeneric hybrids in Brassica and Raphanus. Cruciferae Newslett. Eucarpia 19: 21-22. Gupta, K, D. Prem, N I Nashaat and A Agnihotri 2006. Response of interspecific Brassica juncea/ B. rapa hybrids and their advanced progenies to Albugo candida (white blister). Plant Pathol. 55: 679-89. Pasquet, R S, A Peltier, M B Hufford, E Oudin, J Saulnier, L. Paul, J T Knudsen, H R Herren and P Gepts 2008. Long-distance pollen flow assessment through evaluation of pollinator foraging range suggests transgene escape distances. PNAS 105: 13456-61. Rakow, G. and D L Woods 1984. Outcrossing in rape and mustard under Saskatchewan prairie conditions. Can. J. Pl. Sci. 67: 147-51. Salisbury, P. 2006. Biology of Brassica juncea and potential gene flow from B. juncea to other Brassicaceae species in Australia. Faculty of Land and Food Resources, University of Melbourne. Miscellaneous Report PAS 2006/1.
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