Jenny P. C. Chong; O. W. Liew Deputy Principal (Academic)’s Office Technology Centre for Life Sciences Singapore Polytechnic Singapore B. Q. Li ; A. K. Asundi School of Mechanical & Aerospace Engineering Nanyang Technological University Singapore

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 Promoter Sensitive to Osmotic Changes Reporter gene leads to green fluorescence emission in response to drought stress EGFP 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 Promoter EGFP Plant Vector 3’Ter Molecular Characterization Dehydration Stress Characterisation of Inducible Gene Expression TranscriptionalTranslational Fluorescence Stereoscope Fibre optic Spectroscopy 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.

ENHANCED GREEN FLUORESCENT PROTEIN A red-shifted variant of GFP isolated from the jellyfish, Aequorea victoria. Low molecular weight light emitting protein. Requires only O 2 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 EGFP Light 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 PetuniaDrought-Stressed Wild-type Petunia

Autofluorescence Typical Excitation Typical Emission source wavelength (nm) wavelength (nm) NADH and NADPH 360 – – 470 Flavins 380 – – 560 Lignins Chlorophyll (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 M087 We are grateful for the diligent assistance of our project students - Lim Lishi - Janice Chan QiaoHui - Geraldine Tan Shu Hui