Utilizing Science & Technology and Innovation for Development A novel approach for assessing the impacts of metal oxide nano-particles on the physiological, biochemical, and ultrastructural characteristics in tobacco plant and the systemic movement of plant RNA viruses. Marriott Hotel- Amman, August 13th, 2015

Project Team Principle Investigator: Dr. Rami Alkhatib/Jordan University of Science and Technology/Biotechnology and Genetic Engineering Department /Jordan Co-Investigator: Dr. Fares Howari/ Director Office of Sustainable Eco Innovation, Zayed University/ UAE Co-Investigator: Dr. M-Ali AL-Omari (AL-Akhras)/JUST/ Jordan Co-Investigator: Dr. Borhan Albiss/ JUST/ Jordan Co-Investigator: Dr. Laith AL-Eitan/ JUST/ Biotechnology and Genetic Engineering Department /Jordan Co-Investigator: Dr. Nour Abdo/ Public Health/ Jordan University of Science and Technology/Jordan

Brief Description Our knowledge to explore the physiological and metabolic impact of metal oxide nano-particles on plant is poorly understood. The perception about the systemic movement of plant RNA viruses and the factors involved in this process is not fully elucidated. Agricultural damages caused by pathogenic attack (insect, viral, and fungal diseases) are enormous, and because of the environmental concerns, the communities avoid the total dependence on chemical applications.

Justifications  Using microscopy techniques will enable us to localize where metal oxide nano- particles are accumulating in the plant organs. the first pioneer study  To the best of our knowledge, this will be the first pioneer study to examine the effect of metal oxide nano-particles on the systemic movement of plant RNA viruses and the factors involved in this process.  To date, the physiological and biochemical effects of different concentrations of metal oxide nano-particles in plants are still not fully understood.

Objectives To determine the biochemical (carbohydrate content, protein content, Proline concentration) effects of metal oxide nano-particles. To elucidate the effects of of metal oxide nano-particles on the nano-machine systemic movement of certain plant RNA viruses (Turnip vein-clearing virus (TVCV), Tobacco musaic virus (TMV), and Tobacco etch virus (TEV) using microscopy and molecular techniques. To examine the physiological (shoot height, root length, root and shoot dry mass, photosynthetic rate, stomatal conductance, transpiration rate, and chlorophyll content); and ultrastructural (chloroplast morphology, thickness of cell wall, and localization of metal oxide nano-particles in the plant different tissues).

Scope of work/Duration Estimated Budget three directions/ phases This Research proposal has three directions/ phases: Duration: 36 months (each scope: 12 months) 117,000 JD Estimated Budget : 117,000 JD Phase I Conducting a physiological and biochemical experiments as well as analyzing all physiological and biochemical parameters to examine the effects of metal oxide-nano particles. Phase II To localize where metal oxide-nano particles are accumulating in the plant tissues using SEM and TEM. Phase IV Test the effects of metal oxide nano-particles on the nano-machine systemic movement of certain plant RNA viruses (Turnip vein-clearing virus (TVCV), Tobacco musaic virus (TMV), and Tobacco etch virus (TEV) using microscopy and molecular techniques.

Methodology of Implementation Tobacco (Nicotiana tabacum var. Turkish) will be grown hydroponically in a growth chamber at a photosynthetic photon flux density (PPFD) of 250–300 µmol m -2 s -1 under controlled temperature (23–30 ºC), and humidity (60 ± 5%). Different concentrations of Metal oxide nano-particles will be added to the Hoagland solution. Metal oxide nano-particles with diameters of approximately 9 nm and18 nm will be prepared according to the chemical co-precipitation method. Prepare metal oxide nano-particles Grow Tobacco hydroponically Treat Tobacco with different concentrations at different conditions

Ultrastructural parameters will be examined using both TEM and SEM devices. Viral presence or absence will be detected using immunolabeling techniques in microscopy. Methodology of Implementation Physiological and biochemical parameters will be measured using different techniques and kits (i.e. carbohydrate conc., protein conc., and proline conc.) Test and measure Physiological and biochemical parameters Examine Ultrastructural parameters using both TEM and SEM devices. Use immunolabeling techniques in microscopy to investigate Viral presence or absence

Expected Output The ultrastructural study will allow us to localize where these particles accumulate and show their role in the plant growth. Knowing the plant factors that may be responsible for the systemic movement inhibition, if any, will allow us to genetically engineer them and produce plants resistance to viral infection. Elucidate the physiological and biochemical effects of metal oxide nano-particles in the plants will shed more light about how these particles enhance or inhibit the plant growth.

Impact Producing plants more resistance to biotic and abiotic stress which minimize our dependence on chemical pesticides. Preventing plant viral infections especially in crops which will reduce the cost and loss of these crops. Enhancing seed germination and plant production especially for concentrations improving the physiological and biochemical factors in the plant.

Sustainability Identifying and characterizing the plant factors responsible for the virus movement inhibition and genetically engineered them to produce crops with highly virus resistance. We plan to secure fund for this part from Genetic Engineering Companies. Applying the genetically engineered product to improve agriculture in general. Based on the biochemical, physiological parameters, and the toxicity of metal oxide nanoparticles, other plants species (mainly crops) will be used in the future.

Action Plan Project Direction A: Physiological and biochemical analyses Stage 1: Measuring all physiological parameters (shoot height, root length, dry mass for shoot and root, photosynthetic parameters, and stomatal conductance) Stage 2: Measuring all the biochemical parameters (carbohydrate conc., protein conc., proline conc., and GABA conc.) Stage 3: Statistical analyses for all parameters measured. Stage 4: Finalizing the data and writing a manuscript. This project will be conducted over a period of 36 months after funding the proposal.

Project Direction B: Metal oxide nano-particles localization in plant tissues Stage 1: Conducting a microscopy study for localizing metal oxide nano-particles accumulation in both root and shoot Stage 2: Image processing and preparing them for publication. : Stage 3: Finalizing the data and writing a manuscript. Action Plan In the first year after the proposal acceptance and receiving the funds and Reagents, Kits, consumables

Project Directions C: Plant-virus interaction : Stage 1: Plant inoculation with RNA viruses, then apply metal oxide nano-particles for 14 days. Stage 2: Examine the presence or the absence of the virus in the upper leaves using TEM. Stage 3: Writing manuscript for publication. Action Plan