الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
 |  | from | 136 |  |  |
| | | from | 136 | | |
Abstract
SUMMARY
The main aim of the present study is to isolate the Mi gene from nematode-resistant tomato varieties and transfer it to cucumber varieties using chitosan molecules to form a complex of chitosan and a plasmid carrying the gene. This will activate the defense mechanism inside the cucumber plants after nematode infection. Accordingly, the gene was multiplied inside bacterial cells to produce a large quantity of the Mi gene loaded with chitosan particles to preserve it for a longer period. This will enhance plant resistance to nematodes and produce different cucumber varieties resistant to nematode infestation. Nematodes are one of the groups of the animal kingdom, and they are also called snake worms. Plant-parasitic nematodes cause major problems to major crops worldwide, including vegetables, fruits, and grain crops. Losses range from 8 to 20% of important crops worldwide.
Nematodes are small, translucent worms with bilateral symmetry, covered with a clear cuticle, parasitic, multicellular, live or parasitic, predatory,aquatic,terrestrial,entomopathogenic,ectoparasitic,endoparasitic, semi-parasitic, or sedentary organisms. The cuticle sheds when the nematode enters the successive larval stages. Plant-parasitic nematodes are small, measuring about 300-1000 microns, and some may reach 4 mm and 15-35 microns thick.All plant-parasitic nematodes belong to the Nematoda tribe; most of the plant parasitic genera belong to the order Tylenchida, and a few of them belong to the order Dorylaimida .
Tomato, Lycopersicum esculentum (syn. Solanum lycopersicum and Lycopersicon lycopersicum) is an herbaceous annual in the family Solanaceae and is grown for its edible fruit. The species originated in western South America, Mexico, and Central America.
The Cucumber (Cucumis sativa L) is one of the monoecious annual crops in the Cucurbitaceae family that has been cultivated by humans for over 3,000 years .Cucurbits form an important and big group of vegetable crops grown extensively in subtropical and tropics
countries.Cucumber ranks fourth after tomatoes, cabbage, and onion in Africa.
Although many methods of nematode control are used, the strategy of planting resistant varieties is considered one of the best alternative methods of nematode control. Traditional breeding methods have achieved clear successes in this field by producing varieties that are resistant or tolerant to nematode infection. However, the costs and length of time required for the breeding program for resistant varieties have led to the use of genetic engineering techniques to produce plants to which genes are transferred that are resistant to nematode infection.
Mi gene, one of the natural resistance genes (R genes for resistance to nematodes), was isolated from different tomato varieties (Solanum lycopersicum, Type D, Hybrid Ajyad 7, Hybrid Ajyad 19, Type A99) and transferred using chitosan particles to cucumber varieties (Hybrid F1, 20 rough and Sam 19) to activate the mechanical defense of cucumber plants after infection with root-knot nematodes, and the presence of the gene was confirmed through a PCR test.
Plasmid pEGAD was used to form a complex of chitosan/plasmid loaded with the Mi gene and propagated with DH5α bacteria. selection and screening of the genetically transformed cells were carried out using dishes containing LB medium to which the antibiotic ampicillin was added (for selection). Then, several white colonies and the plasmid containing it, followed by performing the Colony PCR test to ensure that cells containing the targeted expression plasmid were obtained.
Chitosan is a natural substance (dipolymerized polysaccharides) that is subject to some simple chemical reactions, including the disassembly of the sugar molecules in it by acetylcholine groups, which gives it advantages. It is similar to a magnet as it carries positive charges and works to attract any negative charges based on the degree of viscosity and the amount of charge.
The biological ability of chitosan molecules depends on the variation in the molecular weight of the chitosan and the degree of deacetylation to transform it into nanometer particles. Accordingly, the molecular weight of the chitosan used was estimated using the viscometry method, and the degree of deacetylation was determined using the titration method, and the optimal concentration obtained was 0.08%. After testing different concentrations of chitosan and their effect and degree of association with CS-DNA by performing electrophoresis technology and determining the best binding ratio between CS-DNA and chitosan (1:1).
The plants were injected with CS/pDNA molecules into the stems of the targeted cucumber varieties and left for 3 weeks to spread within the plants. The extent of the effect of the Mi gene on the cucumber varieties was observed and compared to the varieties to which the gene was not transferred.
Morphological symptoms were observed on different cucumber varieties carrying the Mi gene, which include increased leaf greenness because the Mi gene is expressed in the leaves, no yellowing of the plants, no deformities in the stems, no spots on the leaves, and plants grow normally while the plants infected with nematodes that do not carry the Mi gene showed yellowing of the plants, wilting of the plant, the appearance of some spots on the leaves, stunting of the plant, abnormal growth of the plant, and deformities in the stem.
Gene Expression is a powerful tool for studying gene expression and gene function, with many potential applications in the fields of molecular biology, genetics, medicine, and agriculture. Accordingly, samples were drawn, and total RNA was extracted for all cucumber varieties to which the gene was transferred. qPCR was also performed on the RNA molecules after they were converted by reverse transcriptase into cDNA, with the aim of measuring the gene expression of the Mi gene.
from the previous results, it can be concluded that it is possible to use nanosized particles of chitosan to form a complex with CS/pDNA molecules. This is used to stimulate defense mechanisms within the plant, with the purpose of increasing resistance and reducing the damage caused by Root-Knot Nematodes.