الفهرس 
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Abstract
This study was conducted at Micropropagation Technology Laboratory (MPT) at the Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, Egypt and in Genetics Department of the Faculty of Agriculture - Ain Shams University - during the period from 2018-2023 on the potato cultivar Desiree.
The aim of the study was to obtain potato tubers of the cultivar Desiree with a low sweetening ratio when preserved and stored in refrigerators at a temperature of 4ºC.
The potato is a plant belonging to the Solanaceae family, and it is a seasonal tuber crop with very high economic and nutritional value, both in Egypt or in the globe. Therefore, farmers resort to storing tubers, whether in refrigerators or looms, to ensure their presence throughout the year.
Storing the tubers in refrigerators at a temperature of 4ºC leads to the phenomenon of sweetening, which occurs as a result of the decomposition of starch in the tubers into sugars, and this phenomenon leads to the production of acrylamide when these tubers are used in industry or home use, whether in the form of chips or fried potatoes.
Acrylamide is one of the substances that contribute to cancer and is called the specter of death, as it destroys the nervous system and negatively affects the fetus. Therefore, women are be advised during pregnancy to avoid eating fried and fast foods, and the frequent consumption of french fries, baked goods and fast foods exposes them to weak immunity and affects in a negative way on the liver, heart, kidneys and bones, so it is necessary to get rid of the sources of acrylamide.
Through the applications of agricultural genetic engineering and genomic modification, it is possible to obtain potato tubers when stored in refrigerators, producing lower levels of sugar through mutation. Using CRISPR technology in the starch phosphorylase gene, which is one of the most important genes in the vital pathway for starch analysis when stored. The CRISPR technology was used by designing sgRNA sequences and thus affects the biological pathway for the production of sugars in potato tubers and the production of tubers with low glycation.
During this study, genetic mutation of Desiree potato plants was carried out by transgenic using Agrobacterium strain LBA4404 containing the pCAS9-TPC plasmid that carries the sgRNA sequences to be transferred to the potato leaves, after the regeneration stages of leaves used in the experiment and reaching the full plant stage. Confirmation experiments were carried out both at the molecular level by polymerase chain reaction at the DNA level to ensure that plants containing genome editing or at the chemical level by measuring the sugar percentage in the tubers after storage, for a period of 90 days and 120 days or at the bioinformatics level by means of the Sanger sequencing.
The laboratory steps used to obtain genetically modified plants can be summarized as follows:
1- Design of sgRNA sequences and formation of annealed oligos was applied.
2- The sgRNA sequences were transferred into the pChimera cloning plasmid, then transferred into the pCAS9-TPC expression plasmid by digestion and ligation steps, and then transferred into the LBA 4404 Agrobacterium strain.
3- Performing the steps of genetic transfer of the leaves of the plants under study through transformation with Agrobacterium carrying sgRNA.
4- After transformation steps of the leaves, the leaves were incubated on a MS medium consisting of 5 mg 2-4, D/L for five days in the dark and then transferred to media containing 1 mg/L of indol acetic acid, Benzyl adenine and 10 mg/L of gibberellic acid for 72 days in illumination. After callus production and plant shoots, statistical analysis was performed to determine the percentage of callus and plant shoots production.
5- The resulting branches were tested by extracting the DNA of each branch and testing it using the polymerase chain method to identify the genome editing plants.
6- After obtaining the genome editing plants, they were planted in plastic greenhouses to obtain tubers, which can be carried out analytical experiments on them to calculate the proportion of sugars that produced in the tubers after storing them in the refrigerator at a temperature of 4°C for 90 and 120 days.
Through the previous steps, genetically modified tubers with low sugar levels were obtained.
The obtained results can be summarized as follows:
1- After three weeks of genetic transfer of Desiree cultivar by Agrobacterium strain LBA 4404 carrying sgRNA sequences on the pCAS9-TPC plasmid, the percentage of callus were 100%.
2- After 9-10 weeks, the result of regeneration and branches formation of the cultivar Desiree was 64%.
3- After 72 days of the experiment, the DNA was extracted from the plants and detecting genome editing plants by the polymerase chain reaction using the bar gene and the result was six plants containing the bar gene that showed a clear band at the expected size 400bp in plants numbers 2, 4, 11, 12, 24 and 26, respectively.
4- Sanger sequencing using primers sd1 and sd2 for the transformation plants, and three plants (DE 2, DE 7 and DE 27) were obtained containing genome editing in the sgRNA region and the pam site. The genome editing detection was deletion and substitution type using ICE software.
5- After 90 days and 120 days of storage, the ratio of monosaccharide sugars in the tubers obtained from the experiment was analyzed. The proportion of monosaccharide after 90 days in the plants of Desiree Control, Desiree 2 (DE 2), Desiree 7 (DE 7) and Desiree 27 (DE 27) were 54.466, 29.224, 35.078, and 35.862 mg, respectively, and after 120 days, the proportion of sugars were 105.249, 83.212, 56.674 and 34.109 mg, respectively.
6- from all the previous results, we conclude that Desiree 2 (DE 2) line was obtained with genome editing and containing a vector with bar gene, otherwise; Desiree 7 (DE 7) and Desiree 27 (DE 27) lines were obtained, that didn’t contain the bar gene, which indicate that the vector was eliminated inside the cells of plants after the occurrence of genome editing.
In the world, Potato tubers (Solanum tuberosum L.) are one of the most essential agro-economically food crop, so to ensure its presence throughout the year for food processors and to extend their shelf life; it should be stored in cold temperature. Despite the benefits of storage at low temperatures, it causes undesirable phenomenons, one of them is cold-induced sweetening, which reduces the quality and the commercial value of the potato tubers. Moreover, the potato tubers produce the acrylamide formation, which is regarded ″Probable human carcinogen″ when it used as chips or fried. CRISPR technology ″Clustered Regularly Interspaced Short Palindromic Repeats″ has been effectively applied in crop improvement more than a decade.
In the current study, the single guide RNA (sgRNA) sequence of starch phosphorylase L gene was cloned into pCAS9-TPC vector that transformed into LBA4404 Agrobacterium strain and used in leaves transformation of Desiree cultivar, after regeneration steps; the regenerated plants were used in screening of genome editing by Sanger sequencing and Inference of CRISPR editing (ICE) program.
The result of molecular detection and sequencing showed that the Desiree 2 (DE 2), Desiree 7 (DE 7) and Desiree 27 (DE 27) lines have mutations in sgRNA sequence that showed a significant reduction in monosaccharide’s concentration of tubers after cold storage for 90 and 120 days, the results were 29.224, 35.078 and 35.862 gm in DE 2, DE 7 and DE 27; respectively after 90 days and 83.212, 56.674 and 34.109 gm after 120 days.