الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 116 |  |  |
| | | from | 116 | | |
Abstract
This study was carried out in Ain Shams Center for Genetic Engineering and Biotechnology (ACGEB), Faculty of Agriculture, Ain Shams University, Egypt, during the period from 2020 to 2022 to investigate the molecular genetic studies of the effects of carbon nanotubes on the growth of cucumber plants (Cucmis satives) through two indvidual experiments, .i.e., in vitro and field experiments.
The field experiment was conducted when the seeds were first soaked for 5 minutes in different concentrations of multiwall carbon nanotubes; MWCNT solutions with different concentrations were prepared (1.0 mg/l, 2.0 mg/l and 4.0 mg/l), the MWCNT was previously dissolved in royal water by dissolving 0.1g MWCNT in 100 ml of royal water which was prepared by adding concentrated hydrochloric acid (37%) to concentrated nitric acid (98%). Control plant seeds ware soaked in solution with the same concentrations of royal water without adding MWCNT. Seeds were planted in plant pots at the beginning of May in the soil mixture (1:1:1 sand: peatmoss: pearlite). Morphological characteristics were observed and recorded after 46, 53, and 59 days and roots number and lengths observations were recorder after 87 days. Seed germination ratio, morphological characteristics (plant height, leaves number, root number and root length) and metabolic characteristics (Chlorophyll A, Chlorophyll B and carotin contents) were used as reference characteristics.
The in vitro experiment, 100 seed were first decontaminated by soaking in per-acetic acid 3% for 20 minutes, then planted in previously thermally sterilized hormone – free M.S - Murashinge and Skoog agar medium. After 21 days, stem nots were collected and were divided into 7 groups, each group contained 10 stem nots, in step 2, five of them were transferred to the agar media (the same composition) with 5 different concentrations of MWCNT (0.5 mg/l, 1.0 mg/l, 1.5 mg/l, 2.0 mg/l and 2.5 mg/l) in parallel with two other sets, as control, one of them was planted in medium provided with 0.5 ml/l of royal water without MWCNT and the other set were planted in medium without either MWCNT or royal water, the MWCNTs were previously dissolved in aqua regia; each amount of MWCNT was added to 0.5 ml of aqua regia at each treatment. The same four morphological characteristics; previously mentioned, were observed on the seedlings with the same method. Determination of chlorophyll A, B and total chlorophyll (A + B) as well as carotenoids content were determined in the samples taken randomly from the fourth true upper leaf after 21 days. In order to study the protein profile, isozyme profile and changes in gene expression, samples were first collected from the shoot parts of the plant (leaves) and different treatment preparations were undergone to the collected parts according to each methodology.
The obtained results could be summarized as follows:
5.1. In Vitro propagation
5.1.1. Multiplication Stage
• Several MWCNT concentrations were applied, and this boosted seedling performance and early growth. In response to the MWCNT treatments, it was discovered that shoot parameters such as plant length and leaf number had changed.
• Compared to the untreated control, the addition of various dosages of CNT to the culture media significantly increased the quantity and length of the roots, especially the MWCNT 1.5 mg/l treatment followed by MWCNT 1.5 mg/l treatment for roots number
5.1.2. Photosynthetic pigments
• The CNT2.5 and CNT2.0 treatments caused a high increase in chlorophyll-A chlorophyll-B and total chlorophyll contents, whereas the other CNT treatments did not find to make a significant change in this trait relative to the control.
• The increases in carotenoid contents were also observed in the 2.5mg CNT/l supplemented seedlings by approximately two folds relative to the untreated control.
5.1.3. Total protein
• The SDS-PAGE of total protein showed change in intensity of protein bands comparing to control, increase of most protein bands visibly starting from the concentration 0.1 mg CNT/l moreover, the heaviest protein bands were between 25 KD and 35 KD and they were at the concentrations 1 mg/l, 1.5 mg/l and 2 mg/l showing that they are the most effective concentrations for inducing these change in proteins.
• Comparing to control, new band at 20 KD emerged with all concentrations of CNT starting from 0.5 mg/l to 2.5 mg/l proving the ability of MWCNT to induce expression of proteins which were not expressed usually.
• The SDS-PAGE of total protein showed also that, the treatment with aqua regia alone decreases the total protein expression comparing to all treatments even with the control.
5.1.4. Peroxidases isozyme
• Peroxidase 2 was almost stable in all samples except for the sample of aqua regia where it was less expressed than the other samples making the treatment with only aqua regia decreasing the expression of PO2. The heaviest expression of peroxidase was at the concentration 1 mg CNT/l.
• Peroxidase 3, 4 and 5 analysis showed contradicting and variable results. The heaviest expressions are at control, 0.5 mg/l, 1.5 mg/l and 2 mg/l and it decreases dramatically at 2.5mg/l. These results showed that MWCNT don’t have the same effect on all peroxidases but there is specific optimum concentration for each kind of peroxidase.
5.2. Field study
• Concerning the effect of multi-walled carbon nanotubes (MWCNT) on the seed germination, T3 (4 CNT) recorded the heighest values of seed germination.
• Shoots characteristics, including plant length and leaves No. were found to be altered in response to the MWCNT treatments.
• In comparison to the untreated control, the T3 (4 CNT) and T2 (2CNT) treatments significantly improved the plant length and leaves No.. Soaking cucumber seeds with different doses of MWCNT or royal water led to the significant increases in roots number and length compared to the untreated control, especially the T3 (4CNT) treatment followed by T2 (2CNT) treatment for roots number and root length.
5.2.2. Photosynthetic pigments
• The T3 (4CNT) treatments caused a high increase in Carotenoids contents, whereas these treatments did not find to make a significant (P ≤ 0.05) change in chlorophyll-A trait relative to the control.
• The increases in chlorophyll-B and total chlorophyll contents were also observed in the T2 (2CNT) relative to the untreated control.
5.3. Gene expression using RT-PCR:
• The PCR analysis showed that CNTs contribute to plant growth and development by influencing the expression of genes essential to cell division and growth, at T1 CNTs affected the gene expression of ACO1 and ACO2 genes significantly compared to controls groups as these genes are important regulators of many developmental and physiological processes such as seed dormancy, germination, vegetative growth, flowering, climacteric fruit ripening, and senescence.
• Also CNT affected the gene expression of PINI, RNAXL1 and YUC genes significantly compared to control groups; these genes play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic response, while Auxins control the growth of plants by promoting cell division and causing elongation in plant cells (the cells get longer).
Conclusion
In conclusion, our results-demonstrated that cucumber seedlings are affected by water soluble multi walled Carbon nanotubes as followed:
• The effect of MWCNTs on the plant is in a concentration-dependent manner.
• At middle concentrations, MWCNTs have beneficial physiological effects on cucumber in-vitro multiplication development.
• MWCNTs have great effect on gene expression of the genes that control growth and morphological traits of the plant.
• MWCNT, a low-cost nanomaterial, has the potential to significantly increase plant growth, performance, physiology, antioxidants, and secondary metabolism, all of which could be economically relevant for agriculture.