PROJECT PROFILE Title : “Referral Centre for Virus Testing of Tissue Culture Raised Plants” Duration : Five years Date of Start : 28th September 2006 Date of Termination : 27th 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 Major Objectives RESEARCH APPROACH 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 SERVICE APPROACH SERVICE APPROACH Prepared write-up on Standard Operating Procedures (SOPs) for virus testing of tissue culture raised plants to be followed by Accrediated Test Laboratories (ATLs) SERVICE APPROACH Imparted training to personnel from Accredited Test Laboratories and other Institutions Personnel(s) Shivkant Shukla ATL s BCIL Date 12.12.06 – 16.12.06 R.P.Pant NRC for Orchids, Sikkim 30.10.07 – 08.11.07 Raj Verma 12.11.07 – 17.11.07 Rahul Mishra S.R. Kalpana IARI Regional station, Pune Plant Quarantine Station, Chennai ATLs Technical Personnels (12 participants) CPRI, SBI, IIHR, IISR, GKVK, IHBT, NRC Banana 28.04.08 – 03.05.08 28.01.08 – 01.02.08 ATLs Technical undergoing training SERVICE APPROACH Supplied Diagnostic Reagent to various laboratories for virus detection Polyclonal antiserum Laboratories Papaya ringspot virus Indian Institute of Horticultural Research, Bangalore IARI Regional Station, Pune Citrus tristeza virus Indian Institute of Horticultural Research Bangalore IARI Regional Station, Kalimpong Service Approach Testing of plant samples for freedom from viruses Import Materials National Plant Quarantine Station, Rangpuri, New Delhi M/S Team Seeds, New Delhi China Cucumber, Pepper M/S Gautam Trading Co, Delhi Pakisthan Lotus M/S Nunhems India (P) Ltd, Hyderabad Malaysia Egg Plant, Papper M/S Sungro Seeds Ltd, Delhi Mauritius Cabbage, Carrot, Ridge Gourd Nath Biogene Ltd. Aurangabad Bangladesh Maize Hybrid Export Materials National Plant Quarantine Station, Rangpuri, New Delhi 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 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/- RESEARCH & DEVELOPMENT APPROACH Developed polyclonal antiserum to coat protein of PRSV using recombinant technology M Amplification RT-PCR PRSV infected leaf by 1 M 1 2 3 4 Cloning in pGEMT Easy ~850 bp Vector ~850 bp Amplification of coat protein gene, Lane 1: helathy; Lane 2: infected sample Mobilization of coat protein gene into expression vector (pMAL-C2X) M 1 Production of polyclonal antiserum 2 2 3 4 5 6 Purification of expressed coat protein ~850 bp 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 sample A405 nm (1 h) Papaya sample Indian isolates Andhra Pradesh Hyderabad A405 nm (1 h) Uttar Pradesh 0.13 Jharkhand Ranchi 98 0.10 Ranchi 07 0.61 Karnataka Bangalore 0.40 Dharwad 1 0.33 Dharwad 2 1.20 Madhya Pradesh Gorakhpur 0.10 Lucknow 0.42 Tamil Nadu Coimbatore 1 0.59 Coimbatore 2 0.38 Coimbatore 3 0.25 Trichi 0.20 West Bengal 0.52 Indore 1.80 Sikkim 0.25 Raipur 1 0.73 Delhi 0.05 Raipur 2 0.28 Gujarat 0.28 Bangladesh 0.29 Healthy control 0.00 Maharastra Solapur 1.75 Pune 1 1.83 Pune 2 0.57 RESEARCH & DEVELOPMENT APPROACH Developed polyclonal antiserum to coat protein of CTV using recombinant technology M 1 2 3 4 M CTV infected material Amplification RT-PCR ~700 bp by 1 2 3 4 5 6 7 8 9 Cloning in pGEMT Easy ~700 bp Mobilization of coat protein gene into expression vector (pMAL-C2X) M M S IS FT W F4 F5 F8 F11MBP S IS FT W F4 F5 F8 F11MBP M ~66 Kd ~66 Kd 50 Kd 50 Kd 1 2 3 4 5 6 7 Purification of expressed coat protein ~700 bp 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 Developed repository of diagnostic reagents for virus diagnosis Virus Antiserum ALFALFA MOSAIC VIRUS AMV APPLE STEM GROOVING VIRUS ASGV ARABIS MOSAIC VIRUS ArMV CITRUS TRISTEZA VIRUS CTV CUCUMBER MOSAIC VIRUS CMV CYMBIDIUM MOSAIC VIRUS CyMV GRAPEVINE LEAF ROLL VIRUS GaLRV Virus Primers CITRUS TRISTEZA VIRUS CTV CUCUMBER MOSAIC VIRUS CMV CYMBIDIUM MOSAIC VIRUS CyMV IRIS YELLOW SPOT VIRUS IYSV ODONTOGLOSSUM RINGSPOT VIRUS ORSV GRAPEVINE VIRUS 1-3 IRIS YELLOW SPOT VIRUS IYSV ODONTOGLOSSUM RINGSPOT VIRUS ORSV PEPPER MILD MOTTLE VIRUS PMMoV POTATO LEAFROLL VIRUS PLRV TOBACCO RATTLE VIRUS TBRV TOMATO RINGSPOT VIRUS ToRSV Acknowledgements • Department of Biotechnology DBT, New Delhi • Indian Agricultural Research Institute (IARI) , New Delhi • Research Associates- Sudeep Bag and Surekha Agarwal