Transcript Slide 1

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