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
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 centersMaintenance of reference virus cultures and diagnostic reagents
RESEARCH APPROACH
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)
Imparted training to personnel from Accredited Test Laboratories and other Institutions
SERVICE APPROACH
Personnel(s) ATL s Date Shivkant Shukla BCIL 12.12.06 – 16.12.06
R.P.Pant NRC for Orchids, Sikkim 30.10.07 – 08.11.07
Raj Verma IARI Regional station, Pune
12.11.07 – 17.11.07
Rahul MishraS.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
SERVICE APPROACH
Supplied Diagnostic Reagent to various laboratories for virus detection
Polyclonal antiserum Laboratories
Papaya ringspot virus Indian Institute of Horticultural Research, BangaloreIARI Regional Station, Pune
Citrus tristeza virus Indian Institute of Horticultural Research BangaloreIARI 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
Export Materials
National Plant Quarantine Station, Rangpuri, New Delhi
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
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/-
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
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)
Indian isolates
Andhra Pradesh
Hyderabad 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
Indore 1.80
Raipur 1 0.73
Raipur 2 0.28
Maharastra
Solapur 1.75
Pune 1 1.83
Pune 2 0.57
Papaya sample A405 nm (1 h)
Uttar 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
Sikkim 0.25
Delhi 0.05
Gujarat 0.28
Bangladesh 0.29
Healthy control 0.00
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 F11MBPM 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
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
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
Virus Primers
CITRUS TRISTEZA VIRUS CTV
CUCUMBER MOSAIC VIRUS CMV
CYMBIDIUM MOSAIC VIRUS CyMV
IRIS YELLOW SPOT VIRUS IYSV
ODONTOGLOSSUM RINGSPOT VIRUS ORSV
Acknowledgements
• Department of Biotechnology DBT, New Delhi
• Indian Agricultural Research Institute (IARI) , New Delhi
• Research Associates- Sudeep Bag and Surekha Agarwal
