ICAR-National Bureau of Plant Genetic Resources(Indian Council of Agricultural Research) New Delhi 110012

www.nbpgr.ernet.in

DNA-based GM Detection

DNA based detection has become a challenge with the increase in number and complexity of GM events commercialised. NBPGR is the nodal agency for issuance of import permit and quarantine processing along with molecular testing of transgenics for research purposes as per the Plant Quarantine(Regulation of Import into India) Order 2003 after technical clearance for import by Review Committee on Genetic Manipulation.

Molecular testing of imported transgenics

Imported transgenics are being tested for specific transgenic elements and also to ensure the absence of embryogenesis deactivator gene (Genetic UseRestriction Technology) using Polymerase Chain Reaction (PCR) based assays.245 imports of 15 GM crops have been tested so far.

Trend of Import of Transgenics

GM-free Conservation of Ex-situ Collections

GM Detection Research Facility (GDRF) at ICAR-NBPGR has been designated as National Referral Laboratory to detect the presence or absence of LMOs and GMOs under sub- section (1) of Section 4 of the Seeds Act, 1966 Notification (DAC&FW, MoA&FW, Govt. of India) dated 15 November 2017 in the Gazette of India.

The facility is accredited as per international standards ISO:IEC17025:2005 by the National Accreditation Board for Testing and Calibration Laboratories (NABL), a constituent Board of Quality Council of India.

The National Genebank at ICAR NBPGR, houses the second largest ex-situ collections in the world,conserving >0.43 million accessions of 1,760 crop species. DNA-based diagnostics have been employed to monitor adventitious presence of transgenes in conserved ex-situ collections of cotton, brinjal, maize, okra and Indian mustard.None of these accessions showed presence of transgenes.

Crop wise import

Year of Import

Loop-mediated Isothermal Amplification (LAMP) :

For screening of GM crops with the following targets:Promoters: (i) Cauliflower mosaic virus 35S promoter (ii) Figwort mosaic virus promoterMarker Genes: (i) aadA, (ii) nptII, (iii) uidATransgenes: (i) cry1Ac, (ii) cry2Ab2, (iii) cp4-epsps

For Event-specific:Cotton events: (i) MON531 (Bollgard®I), (ii) MON15985 (Bollgard®II) Maize events: (i) Bt11, (ii) GA21, (iii) MON810, (iv) MON89034, (v) NK603, (vi) TC1507

GMO Matrix for 141 Indian GM events of 21 crops*106 genetic elements were targeted

Real-time PCR-based Multi-target SystemTaqMan® Real-time PCR based multi-target system comprising the pre-spotted lyophilized primers and probes developed for six crops* for simultaneous detection of 47 targets including 21 event-specific sequences, five construct regions, 15 transgenic elements and six taxon- specific targets*Cotton, eggplant, maize, potato, rice, soybean

1. DNA extraction 2. Add Taqman Universal PCR Master mix onto the prespotted plate and load the plate

3. Real time PCR amplification

4. Data Analysis

1-4 hrs 15-30 mins 1.5 hrs

Visual & Real-time LAMP based technologies

Cost-effective GM Detection Technologies / Strategies

Visual and real-time LAMP assays developed for rapid and on-site GM detection, in combination with fast and efficient DNA extraction method.

Ten most frequently present targets: [P-35S], [T-nos], [Os-Msca1], [cry1Ab], [cry1Ac], [cry1c], [cry2Ab], [GA20 oxidase1], [nptII], [bar] identified to screen these events.The matrix would facilitate efficient, rapid and cost-effective screening. *Brinjal, cabbage, cauliflower, chickpea, corn, cotton, groundnut, mustard, okra, papaya, potato, rapeseed, rice, rubber, sorghum, soybean, sugarcane, tobacco, tomato, watermelon and wheat.

Gel Profile of Hexaplex PCR

Gel Profile of Triplex PCRGel Profile of Decaplex PCR

Detection and Quantification of GM Crops/Events Qualitative and quantitative PCR and Real-time PCR assays developed for detection of more than 40 GM events of 12 crops (brinjal, cabbage, cauliflower, cotton, maize, mustard, okra, potato, rice, soybean, tomato and wheat).

Event-specific real-time PCR assay for Bt brinjal event EE-1

LOD: 0.01%

Limit of Detection (LOD) : 0.1-0.01%

Detection of Bt Brinjal Event EE1

Technologies for GM Detection, Identification and Quantification

Screening of GM CropsFor checking the GM status of a sample irrespective of crop and GM trait:- Hexaplex PCR assay targeting six marker genes: aadA, bar, hpt, nptII, pat and uidA - Octaplex PCR assay targeting seven screening elements: P-35S, P-nos, T-35S, aadA, nptII, pat, uidA, and Sad1 endogenous gene

Detection and Quantification of Bt Cotton Events- Decaplex and triplex PCR assays, targeting specific transgenes, constructs, events and endogenous Sad1 gene, for detection of five commercialized Bt cotton events, viz., MON531 (Bollgard I®), MON15985 (Bollgard II®), Event1, GFM-cry1A and MLS-9124.- Transgene- and construct-specific real-time PCR assays for quantification of Bt cotton events.

MoU signed with DSS Imagetech Private Limited, New Delhi facilitated by Agrinnovate India Limited (19 August 2015) for five cost-efficient/ rapid GMO screening technologies:1. Hexaplex PCR targeting six marker genes (nptII, aadA, bar, hpt, pat and uidA) 2. Duplex TaqMan real-time PCR targeting P-35S and T-nos 3. Visual Loop-mediated Isothermal Amplification (LAMP)-based technology targeting eight transgenic elements (P-35S, T-nos, aadA, nptII, uidA, cry1Ac, cry2Ab and cp4-epsps) 4. Real-time LAMP-based technology targeting transgenic elements (P-35S, T-nos, aadA, nptII, uidA, cry1Ac, cry2Ab2 and cp4-epsps) 5. TaqMan real-time PCR-based multi-target system covering 47 targets for screening

Transfer of DNA-based GM Detection Technologies to Public / Private Sector:

Laboratories using GMO Screening and Quantification Kits• National Collateral Management Services Ltd. Hyderabad and Indore

• Intertek India Pvt. Ltd. Gurugram

• NAWaL Analytical Laboratories, Hosur

• Cremica Food Industries Limited, Ludhiana

• Envirocare Labs Private Limited, Thane

• Geo Chem Laboratories Private Limited, Thane

• Avon Food Lab Pvt. Ltd., New Delhi

• Testex Labs, Mumbai

• Amitex India Labs, Indore

• Avi Agro Limited, Indore

• Vippy Soya Limited, Dewas

MoUs signed for PCR-based diagnostics for screening and detection of GM events of ten GM crops, viz., cotton, brinjal, maize, soybean, rice, mustard, cauliflower, okra, tomato, potato, employing singleplex and multiplex PCR assays targeting specific transgenes /markers/promoters/terminators/endogenous genes, with:

- Punjab Biotechnology Incubator (Agri and Food Testing Laboratory), Mohali (4 July 2014)- Basmati Export Development Foundation, Meerut (15 July 2013)- Amar Immunodiagnostics Private Limited, Hyderabad (10 May 2010)

Consultative Workshop on Harmonization of LMO/GM Detection Activities in the Country

Organized at ICAR-NBPGR on 21 August 2017.The laboratory representatives had interface with more than 20 delegates including assessors and accreditation officers nominated by National Accreditation Board for Testing and Calibration Laboratories (NABL), Quality Council of India.

International Training Programme on “DNA based GMO detection for seed testing and certification” for the delegates of Nepal sponsored by International Food Policy Research Institute (IFPRI), New Delhi, at ICAR-NBPGR from 30th November to 12th December 2015.

Proficiency testing results (within the Z score limit)

Quality Assurance and Global Harmonization

Participation in 25 Proficiency testings as per ISO/IEC 17043: 2010 for testing theunknown levels of different GM events of maize, soybean, oilseed rape, rice using Real-time PCR assays, since 2010:

- Twelve proficiency testings organized by the European Union Reference Laboratory for GM Food and Feed, European Commission, Joint Research Centre (EC-JRC), Ispra, Italy

- Twelve proficiency testings of Grain Inspection, Packers and Stockyards Administration, United States, Department of Agriculture (GIPSA-USDA)

- One proficiency testing of Programas Interlaboratoriais e Materiais de Referência – PRI MAR Laboratório Nacional Agropecuário – LANAGRO/MG Ministério da Agricultura, Pecuária e Abastecimento, Brazil

Network of GMO/LMO Testing Laboratories in India has been established by the GDRF sponsored by MoEF&CC, Govt. of India. Developed and hosted the Webpage on ‘Network of GMO Testing Laboratories (NGTL) of India’ with information on 18 GM Testing laboratories in the country (4 Referral and 14 others):

(http://gmolabs.nbpgr.ernet.in:9090/)

Screenshot of Webpage ‘Network of GMO Testing Laboratories of India’

Inter Laboratory Comparison Programme- Organised an Inter Laboratory Comparison Programme in 2017 where 12 GM testing laboratories participated from all over India.- Participated in five inter-laboratory Comparison Programmes in India for PCR based GM detection in cotton, maize and soy during 2013-2017.

Dr. Gurinderjit Randhawa Principal Scientist & Officer-Incharge, Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012E-mail: gurinder.randhawa@icar.gov.in Phone: +91-9811974396

Dr. Kuldeep SinghDirectorICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012E-mail: director.nbpgr@icar.gov.inPhone: 011-25841129, 011-25843697

For more information please contact :

Dr. Bikram S. Gill, Distinguished Professor of Plant Pathology, Wheat Genetics Resource Center, Kansas State University

Nigerian delegation led by Lucy Ogbadu, Director General, National BiotechnologyDevelopment Agency

© ICAR-National Bureau of Plant Genetic ResourcesPublished by: The Director, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012

Acknowledgements

Special thanks for financial support to strengthen the Facility: Department of Biotechnology, Department of Science & Technology, Ministry of Science & Technology, Govt. of India, United Nations Environment Programme (UNEP) Global Environment Facility (GEF)-World Bank, National Agricultural Innovation Project (NAIP), ICAR, Ministry of Agriculture and Farmers’ Welfare, Department of Agriculture, Co-operation and Farmers' Welfare (DAC&FW), Ministry of Environment, Forest and Climate Change, Govt. of India.

GM Detection Research Facility offers

- Hands-on training for 5-21 days - Contract services for GM testing- Collaboration with public/private sector for GM detection work- Development of customized GM detection assays for new GM events

Some Distinguished Visitors

Visit of RCGM Sub-Committee

Dr. Gurinderjit Randhawa Principal Scientist & Officer-Incharge, Division of Genomic Resources, ICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012E-mail: gurinder.randhawa@icar.gov.in Phone: +91-9811974396

Dr. Kuldeep SinghDirectorICAR-National Bureau of Plant Genetic Resources, New Delhi 110 012E-mail: director.nbpgr@icar.gov.inPhone: 011-25841129, 011-25843697

For more information please contact :