1. PROJECT PROFILE Title : “Referral Centre for Virus Testing of Tissue Culture Raised Plants” Duration : Five years Date of Start : 28 th September 2006 Date of Termination : 27 th September 2011 Project Cost (Rs.) : 75.05 Lakhs Project Personnels Principal Investigator: R.K. Jain Co-Investigators : Padma Ramachandran/ V.K. Baranwal / Bikash Mandal Research Associates : Sudeep Bag / Surekha Agarwal

2. Major Objectives  Confirmation of results on diagnosis of diagnostic test centers, whenever required  Development of diagnostic probes/kits for viruses  Development, standardization and validation of virus test protocols  Training of personnel of diagnostic centers  Maintenance of reference virus cultures and diagnostic reagents RESEARCH APPROACH SERVICE APPROACH

3. Prepared write-up on Standard Operating Procedures (SOPs) for virus testing of tissue culture raised plants to be followed by Accrediated Test Laboratories (ATLs)

4. Imparted training to personnel from Accredited Test Laboratories and other Institutions SERVICE APPROACH Personnel(s)ATL sDate Shivkant ShuklaBCIL12.12.06 – 16.12.06 R.P.PantNRC for Orchids, Sikkim30.10.07 – 08.11.07 Raj VermaIARI Regional station, Pune 12.11.07 – 17.11.07 Rahul Mishra S.R. Kalpana Plant Quarantine Station, Chennai 28.01.08 – 01.02.08 ATLs Technical Personnels (12 participants) CPRI, SBI, IIHR, IISR, GKVK, IHBT, NRC Banana 28.04.08 – 03.05.08 ATLs Technical undergoing training

5. SERVICE APPROACH Supplied Diagnostic Reagent to various laboratories for virus detection Polyclonal antiserumLaboratories Papaya ringspot virusIndian Institute of Horticultural Research, Bangalore IARI Regional Station, Pune Citrus tristeza virusIndian Institute of Horticultural Research Bangalore IARI Regional Station, Kalimpong

6. Service Approach Testing of plant samples for freedom from viruses Import Materials National Plant Quarantine Station, Rangpuri, New Delhi M/S Team Seeds, New DelhiChinaCucumber, Pepper M/S Gautam Trading Co, DelhiPakisthanLotus Export Materials National Plant Quarantine Station, Rangpuri, New Delhi M/S Nunhems India (P) Ltd, Hyderabad MalaysiaEgg Plant, Papper M/S Sungro Seeds Ltd, DelhiMauritiusCabbage, Carrot, Ridge Gourd Nath Biogene Ltd. AurangabadBangladeshMaize Hybrid Status Not Known ---Chambal Agritech Ltd. Chandigarh---Potato ----M/S Sheel Biotech Ltd., New Delhi----Banana ----Merino Industries Ltd. Hapur----Potato -----Jain Irrigation System Ltd., Jalgaon-----Banana

7. SERVICE APPROACH Resource generation through testing of samples for freedom from viruses and other pathogens Tissue culture industries National Plant Quarantine Station Rs. 1,40,000/-

8. PRSV infected leaf Developed polyclonal antiserum to coat protein of PRSV using recombinant technology Amplification by RT-PCR Amplification of coat protein gene, Lane 1: helathy; Lane 2: infected sample ~850 bp Cloning in pGEMT Easy Vector M 1 2 3 4 ~850 bp M 1 2 M 1 2 3 4 5 6 ~850 bp Mobilization of coat protein gene into expression vector (pMAL-C2X) Purification of expressed coat protein Production of polyclonal antiserum RESEARCH & DEVELOPMENT APPROACH

9. Detection of Papaya ring spot virus (PRSV) in infected papaya and other hosts samples collected from different geographical locations by direct antigen coated enzyme linked immunosorbent assay (DAC-ELISA) Papaya sampleA 405 nm (1 h) Indian isolates Andhra Pradesh Hyderabad0.13 Jharkhand Ranchi 980.10 Ranchi 070.61 Karnataka Bangalore0.40 Dharwad 10.33 Dharwad 21.20 Madhya Pradesh Indore1.80 Raipur 10.73 Raipur 20.28 Maharastra Solapur1.75 Pune 11.83 Pune 20.57 Papaya sampleA 405 nm (1 h) Uttar Pradesh Gorakhpur0.10 Lucknow0.42 Tamil Nadu Coimbatore 10.59 Coimbatore 20.38 Coimbatore 30.25 Trichi0.20 West Bengal0.52 Sikkim0.25 Delhi0.05 Gujarat0.28 Bangladesh0.29 Healthy control0.00

10. Developed polyclonal antiserum to coat protein of CTV using recombinant technology RESEARCH & DEVELOPMENT APPROACH Amplification by RT-PCR CTV infected material M 1 2 3 4 ~700 bp Cloning in pGEMT Easy Mobilization of coat protein gene into expression vector (pMAL-C2X) ~700 bp M 1 2 3 4 5 6 7 8 9 ~700 bp M 1 2 3 4 5 6 7 Purification of expressed coat protein ~66 Kd 50 Kd ~66 Kd 50 Kd M S IS FT W F4 F5 F8 F11MBP M : Marker; S: Soluble fraction; IS: Insoluble fraction; FT: Flowthrough; W: Wash; F4-11: eluted protein fraction Validation of Antiserum at IIHR, Bangalore and IARI, Regional Station, Kalimpong

11. Developed repository of diagnostic reagents for virus diagnosis Virus Antiserum ALFALFA MOSAIC VIRUSAMV APPLE STEM GROOVING VIRUSASGV ARABIS MOSAIC VIRUSArMV CITRUS TRISTEZA VIRUSCTV CUCUMBER MOSAIC VIRUSCMV CYMBIDIUM MOSAIC VIRUSCyMV GRAPEVINE LEAF ROLL VIRUSGaLRV GRAPEVINE VIRUS 1-3 IRIS YELLOW SPOT VIRUSIYSV ODONTOGLOSSUM RINGSPOT VIRUSORSV PEPPER MILD MOTTLE VIRUSPMMoV POTATO LEAFROLL VIRUSPLRV TOBACCO RATTLE VIRUSTBRV TOMATO RINGSPOT VIRUSToRSV Virus Primers CITRUS TRISTEZA VIRUSCTV CUCUMBER MOSAIC VIRUSCMV CYMBIDIUM MOSAIC VIRUSCyMV IRIS YELLOW SPOT VIRUSIYSV ODONTOGLOSSUM RINGSPOT VIRUSORSV

12. Acknowledgements Department of Biotechnology DBT, New Delhi Indian Agricultural Research Institute (IARI), New Delhi Research Associates- Sudeep Bag and Surekha Agarwal