jenny p. c. chong; o. w. liew deputy principal (academic)’s office technology centre for life...
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Jenny P. C. Chong; O. W. LiewDeputy Principal (Academic)’s OfficeTechnology Centre for Life Sciences Singapore PolytechnicSingapore 139651Email : [email protected]; [email protected]
B. Q. Li ; A. K. Asundi School of Mechanical & Aerospace EngineeringNanyang Technological UniversitySingapore 639798Email : [email protected]; [email protected]
Development of Transgenic
“Indicator Plants” for Fibre
Optic Detection of Drought
Stress Conditions
INTRODUCTION
•Regimented management•Open field application
: non-precise: not “needs-based”
Traditional farming
Negative ecological consequences•Soil physio-chemical properties•Biodiversity•Groundwater quality•Environment
Unsustainable agricultural practices•Imbalanced resource distribution affecting crop yield, quality and profit.
Turnkey Technologies
Maximum Crop Production and Quality
Environmentally FriendlyProfitable
PRECISION AGRICULTURE-Precise and Site-specific resource management
Agricultural Science + Engineering
Modern IT (track crop status)
Link with resource management (feedback online control system)
Targeted resource utilisation
‘Needs-Based’ Application of Water
Transgenic ‘Indicator plants’
Prototype Fibre Optic
Spectroscopic Analyser
Online irrigation system
Assessment of Abiotic Stress in Plants
Biochemical Approach
Biophysical Approach
•Destructive
•Costly
•Laborious
•Time consuming
•Laboratory-based
•Non-invasive
•Cost-effective
•Relatively easy & fast
•Instantaneous, Real-time
•Field scale, Portable
TRANSGENIC ‘INDICATOR PLANT’ Drought-responsive
PromoterSensitive to Osmotic Changes
Reporter gene leads to green fluorescence emission in response
to drought stressEGFP Drought-responsive
Promoter
Plant DNA
EGFP Drought-responsive Promoter
Drought Responsive Promoter - Inducible
Fluorescent Protein gene -Stress reporter
Regenerate genetically-
modified ‘INDICATOR
PLANT’
Agrobacterium-mediated gene transfer
Cloning
ANALYSIS OF RESPONSE TO DEHYDRATION
Agrobacterium-mediated gene
transfer
Drought-responsive
PromoterEGFP
Plant Vector
3’Ter
Molecular Characterization
Dehydration Stress
Characterisation of Inducible Gene Expression
Transcriptional Translational
Fluorescence Stereoscope
Fibre optic SpectroscopyCorrelation
Correlation
REGENERATED TRANSGENIC
INDICATOR PLANTS
Verification of transgene insertion
Crown Gall: Nature’s Example
of Cross-Border Gene Transfer
Agrobacterium is a naturally occurring soil bacterium that transfers genes carried in its T-
DNA into plant cells following bacterial infection. The subsequent expression of these
genes results in distinctive tumour morphologies, the so-called crown gall tumours. This natural process has been
adapted by scientists and developed into a powerful tool to manipulate the genetic
constitution of plants.
Agrobacterium is a naturally occurring soil bacterium that transfers genes carried in its T-
DNA into plant cells following bacterial infection. The subsequent expression of these
genes results in distinctive tumour morphologies, the so-called crown gall tumours. This natural process has been
adapted by scientists and developed into a powerful tool to manipulate the genetic
constitution of plants.
ENHANCED GREEN FLUORESCENT PROTEIN
A red-shifted variant of GFP isolated from the jellyfish, Aequorea victoria.
Low molecular weight light emitting protein.
Requires only O2 and excitation by blue light (488nm) to emit visible green fluorescence (509nm).
Fluorescence intensity is 35 x higher than wild-type GFP. Relatively resistant to photobleaching.
PROPERTIES
Drought stress inducible
promoter drives EGFP production
in regenerated ‘Indicator plant’
The plant emits green fluorescence when illuminated by blue light.
Drought-responsive Promoter
EGFPLight source
Probe
Laptop computer
Emission spectrum
Spectrometer
BIODETECTION APPROACH
Pattern of dehydration-induced EGFP Expression
Non-uniform spatial distribution of fluorescence
Randomly localised in various plant parts
Leaves
Vascular tissues of stems
Optical Detection of EGFP
Gradual increase in emission intensity over 0,1, 2 and 24h dehydration stress
Baseline levels throughout dehydration stress
Drought-Stressed Transgenic Petunia Drought-Stressed Wild-type Petunia
Autofluorescence Typical Excitation Typical Emission
source wavelength (nm) wavelength (nm)
NADH and NADPH 360 – 390 440 – 470
Flavins 380 – 490 520 – 560
Lignins 488 530
Chlorophyll 488 685 (740)
Knight & Billinton (2001) Biophotonics International
SOURCES OF AUTOFLUORESCENCE
EGFP excitation max
Future Work Plant Host:
Overcome endogenous background autofluorescence
Fluorescent Reporter:
Study the utility of other fluorescent protein reporters with more suitable properties than EGFP
Fibre Optic Spectroscopic Analyzer:
Increase detection sensitivity to address weak inducible expression.
Acknowledgement
• This work was supported by the Singapore Polytechnic and Singapore Totalisator Board under Model Project No. 11-27801-36-M087
• We are grateful for the diligent assistance of our project students
- Lim Lishi
- Janice Chan QiaoHui
- Geraldine Tan Shu Hui