الفهرس 
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Abstract
he present thesis is concerned with studying the impact of Titanium dioxide nanoparticles on Spearmint plant physiological parameters (photosynthetic pigments, minerals uptake, essential oil quantity, and composition), besides evaluating their effectiveness at the molecular level by applying the RAPD technique for all treatments that are investigated.
The utilized TiO2NPs were synthesized from two sources with different particle sizes: 1- chemical source (15.3 nm) (purchased from the Nanotechnology Research Center, Faculty of Engineering, Minufiya University).
2- Biological source (47.5 nm) (prepared in the laboratory, by exerting many pre-trials to select the most effective method for preparing the biologically synthesized TiO2 NPs). Nanoparticle size was measured by the XRD (X-Ray Diffraction) technique at the Metallurgical Development Research Center, Al Tbin, Cairo.
The experiments were established on Mentha spicata L. (spearmint) leaves under physical conditions at the greenhouse as mentioned above by applying six treatments of foliar spraying Titanium dioxide nanoparticles: 25 mg L-1 Chemically synthesized nano- Titanium dioxide (CNT), 25 mg L-1 Chemically synthesized nano- Titanium dioxide with excess Nitrogen (CNTN), 50 mg L-1 Biologically synthesized nano- Titanium dioxide (BNT), 50 mg L-1 Biologically synthesized nano- Titanium dioxide with Nitrogen (BNTN), Positive control (PC), and Negative control (NC).
The obtained results can be summarized as follows:
I. Characterization of TiO2 NPs by XRD technique
Several trials were carried out to select the most effective nitrogen source to be used in media preparation to obtain the most appropriate amount, effective size, and suitable biological anatase Titanium dioxide nanoparticles (B-TiO2NPs). As a result, the TiO2 nanoparticles were analyzed using the XRD technique, revealing particle sizes of 43.9 nm, 60.7 nm, 69.5 nm, and 70.2 nm in different media.
The best medium for making biological Titanium dioxide nanoparticles was found to be a mixture of peptone, sucrose, and yeast extract in ratio 2:2:1.
Thus, the TiO2 NPs utilized in the main experiments of this study possess particle sizes of 15.3 nm for chemically synthesized anatase Titanium dioxide nanoparticles and 47.5 nm for biologically synthesized anatase Titanium dioxide nanoparticles, as determined via XRD analysis.
II. Estimation of photosynthetic pigments (chl. a, chl. b, and carotenoids) in spearmint leaves in pre-trial experiment
Three different concentrations (25 mg L-1, 50 mg L-1, and 100 mg L-1) of chemically and biologically synthesized Titanium dioxide nanoparticles were carried out as a pilot experiment to select the most significant concentrations to be applied for all experiments. The content of pigments (chlorophyll a, Chlorophyll b, Total Chlorophyll, and Total Carotenoids) was measured. Results showed that spearmint plants that were treated with 25 mg L-1 chemically synthesized Titanium dioxide nanoparticles recorded the highest content of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, while the highest effective concentration of Chlorophyll a and b for plants that were treated with biologically synthesized Titanium dioxide nanoparticles was 50 mg L-1.
III. Estimation of photosynthetic pigments (chl. a, chl. b, and carotenoids) in spearmint leaves upon the application of chosen concentrations of chemical and biological synthesized TiO2 NPs (the main experiment)
According to the results, the application of 50 mg L-1 biologically synthesized Titanium dioxide nanoparticles resulted in the highest chlorophyll a. content. In contrast, Positive control plants exhibited the highest chlorophyll b content.
However, the foliar application of Titanium dioxide nanoparticles on spearmint leaves significantly improved chlorophyll b contents. All treatments of Titanium dioxide nanoparticles improved the total chlorophyll contents, whereas the highest value was recorded in 50 mg L-1 biologically synthesized Titanium dioxide nanoparticles. Additionally, the highest content of total carotenoids was recorded in plants that were treated with 25 mg L-1 chemically synthesized Titanium dioxide nanoparticles.
IV. Determination of mineral uptake in spearmint leaves by ICP-MS technique
The effects of TiO2NPs from different sources (Biological and Chemical) on macro, micronutrient, and heavy metals concentrations were investigated. The foliar spraying of TiO2 NPs, in general, showed a significant positive effect on the concentration of all Micronutrients (Cu, Fe, Mn, Zn, and B) except for the treatment of BNTN, however, the highest value was recorded in plants that were treated with 25 mg L-1 chemically synthesized Titanium dioxide nanoparticles. On the other hand, the concentration of macronutrients (N, P, K, Ca, and Mg) showed different responses to TiO2NPs at which the nitrogen content was increased in CNT, CNTN, and its highest value was estimated in PC. On the contrary, Phosphorous content was significantly decreased in all treatments compared with the negative control. Additionally, the highest contents of macronutrients (K, Ca, and Mg) were observed in plants that were treated with chemically synthesized Titanium dioxide nanoparticles at a concentration of 25 mg L-1. Similarly, heavy metals (Cr, Sr, Ag, In, Ba, Pb, Bi, and Ti) also displayed their highest concentrations under the same treatment. Conversely, plants treated with biologically synthesized Titanium dioxide nanoparticles at a concentration of 50 mg L-1 exhibited the highest value of Al.
V. Measuring the spearmint oil quantity
Hydro-distillation extraction of mint oil for all treatments was carried out. The highest volatile oils content (1.54 %) was present in plants that were treated with 50 mg L-1 biologically synthesized Titanium dioxide nanoparticles, while the lowest value (0.93 %) was estimated in 25 mg L-1 chemically synthesized Titanium dioxide nanoparticles.
VI. Identifying the spearmint oil composition
Gas chromatography-mass spectroscopy analysis was used to identify the spearmint oil composition which was affected by the foliar application of Titanium dioxide nanoparticles. Results indicated that there was an effective variation in mint oil composition within the different treatments of TiO2 NPs compared with the negative control, at which some compounds were increased or decreased from NC and in some cases, new compounds appeared or disappeared in others.
The CNT treatment yielded the highest variation in spearmint oil composition, leading to an increase or appearance of important pharmaceutical compounds including pinenes, limonene, epicubenol, eucalyptol, germacrene D, isoborneol, caryophyllene, and cis-calamenene. Additionally, the toxic isopulegone compound was no longer present. The positive effect of TiO2 NPs on oil composition was observed in all treatments as well, but with lower ratios than CNT-treated plants.
VII. Evaluating the treatments by Random amplified polymorphic DNA (RAPD) technique:
Random Amplified Polymorphic DNA (RAPD) markers were also used for the assessment of genetic variation between different treatments of Titanium dioxide nanoparticles on Mentha spicata L. in comparison with the control. A total of 10 arbitrary sequence primers were evaluated. All 10 primers used for the RAPD analysis showed consistent band patterns. In total, scorable bands were observed with the primers. The total number of amplicons produced per primer varied from 7 for OPA-09 and OPB-10 to as many as 13 bands for OPA-4. The average number of bands per primer was 9.9 Out of 99 bands, 31 were polymorphic (31.31%). The average number of polymorphic RAPD bands was 3.1 per primer. The highest similarity (94%) with negative control was recorded in spearmint leaf treated with Titanium dioxide nanoparticles that were chemical source-derived plus nitrogen, while spearmint leaf treated with Titanium dioxide nanoparticles that biological source-derived plus nitrogen was found to show the least similarity (88%) with negative control. In this concern, RAPD results have been suggested to be useful fast methods for comparing genetic changes and variation in plants.
Overall, the results of this investigation indicated that, the foliar application of TiO2 NPs on Mentha spicata L. has a significant positive effect on micronutrients, some macronutrients, and pigment contents, besides improving the spearmint oil quality by increasing the percentage of some effective compounds or/ and decreasing the percentage of toxic ones, the differences within treatments were also affirmed by the genetic markers of RAPD technique.