Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Genetic Modifications of Chlorogenic Acid Content in Eggplant (solanum Melongena L.) by Agrobacterium-Mediated Transformation Technique(01. Izmir Institute of Technology, 2023) Tanyolu, Tanya Nil; Frary, AnneEggplant is a nutritious vegetable that is low in lipids and high in fiber, vitamins, and minerals. Eggplant is a good source of phenolic compounds, particularly chlorogenic acid (CGA), which is associated with several health benefits, such as reducing the risk of diabetes and cardiovascular diseases. Among CGA-rich fruits, eggplant has a geographically wide distribution while coffee is limited to tropical regions. For that reason, eggplant can be used as alternative to coffee for CGA production. The goal of this research was to develop transgenic eggplant that has increased CGA content using the Agrobacterium-mediated transformation technique. The over-expression of the Myb1 gene, which is related to CGA synthesis and responsible for the production of a transcription factor that activates the phenylpropanoid pathway in eggplant, was used. The Myb1 gene was isolated, amplified, and inserted into the PRI910 vector system before being transformed into Agrobacterium tumefaciens. After plant transformation, seven regenerated plants were verified as transgenic by PCR analysis. Young leaves from transgenic eggplants were used for mRNA expression analysis and metabolite analyses. A two-fold increase in Myb1 gene expression levels for three of the transformed plants was observed and five plants showed a significant increase in 3-CGA content. Expression of Myb1 correlated with CGA content measured by HPLC analysis. Specifically, the two-fold increases in Myb1 expression of three plants corresponded with two-fold increases in their leaf 3-CGA content. In the future, these genetically modified plants could serve a crucial role in better understanding the pathway of CGA production in eggplants, and may also prove useful in generating valuable CGAs for both economic and medicinal purposes. Subsequent research can be directed towards determining the CGA content via measurement of 5-CGA and examining CGA content in different parts of the fruit such as flesh and peel. Study of the next generation (T1 plants) will also be critical for confirming the stability of the transformation.Master Thesis Genetic Modification of Glycoalkaloid Content in Eggplant by Agrobacterium-Mediated Transformation Technique(Izmir Institute of Technology, 2022) Gültekin, Helin; Frary, AnneEggplant is one of the first plantsthat was cultivated in the Old World. It is economically and agriculturally important for many countries such as Turkey. In addition, eggplant has been used medicinally since ancient times. The medicinal effects of eggplant come from its secondary metabolites. One of the most useful secondary metabolites is steroidal glycoalkaloids (SGA). SGAs have been used in cancer treatment research. The biosynthesis of SGAs in plants is controlled by enzymes and transcription factors. GAME 9 is a transcription factor that has been investigated in tomato and potato and has been shown to be an important element in the production pathway. In this research, a copy of eggplant’s own GAME 9 transcriptional factor was transformed to the eggplant genome with Agrobacterium-mediated transformation. To this end, the GAME 9 gene was amplified from the eggplant genome and inserted into the pSoup/pGreen0029 vector system. This vector system was inserted into Agrobacterium. Eggplant seedling explants were incubated with transgenic Agrobacterium. The transformation of regenerated plants was confirmed with PCR and efficiency was found to be 1.3%. RNA expression levels were checked by RT-QPCR for 12 confirmed transgenic plants. On the metabolite level, the leaves of transgenic plants were assayed for SGA content using Dragendorff’s reagent method. The transgenic plants’ mRNA and metabolite levels showed plant to plant variation due to the random nature of transgene insertion. Of the 12 transgenic eggplant, nine plants had significantly increased production of both mRNA (at least 2-fold difference) and metabolite levels. In addition, two plants did not show any difference in mRNA level but their SGA amount was significantly increased. The remaining plant had a 0.16-fold decrease in mRNA level but a significantly greater level of SGA than control plants. In the future, these transgenic plants can help illuminate the SGA production pathway of eggplant and be used for the production of economically and medicinally valuable SGAs.Master Thesis Assessment of Liposomal Formulations and Biological Activities of Eggplant Glycoalkaloids(Izmir Institute of Technology, 2019) Tatlıdil, Engin; Frary, AnneBillions of dollars are spent every year in the world for cancer treatments and research. In recent years, bioactive compounds are being tested as promising therapeutics. Among these compounds, eggplant glycoalkaloids: solasonine and solamargine are known to be effective against skin cancer and diseases. However, these compounds are water insoluble. This reduces transdermal drug delivery and the efficacy of solasonine and solamargine. Nanocarriers are used for transdermal drug delivery of water insoluble molecules. In this study, liposomes were used as nanocarriers to increase drug delivery of solasonine and solamargine. In the first stage of the study, empty liposomes produced from four different lecithin types with hydration temperatures of 40°C, 45°C and 50°C were evaluated according to their physical, chemical stability and drug loading capacity criteria at three different storage temperatures (4°C, 25°C, 37°C). The liposome formulation which was most suitable for the continuation of the study was determined. In the second stage of the study, solasonine and solamargine loaded liposomes were produced according to the formulation determined in the first stage and these liposomes were evaluated according to their physical, chemical stability, zeta potentials and drug leakage rate criteria for 3 months and it was determined that the drug loaded formulation was stable during the monitoring process. Furthermore, the release profiles of the drugs in different release media were determined and also the efficacy of the free and encapsulated states of solasonine and solamargine were tested in HaCaT and SCC-25 cell lines and IC50 values were determined.Master Thesis Elecidation of Selenium Tolerance Mechanisms in Puccinellia Distans (jacq.) Parl, Using a Transcriptomic Approach(Izmir Institute of Technology, 2018) Kök, Aysu Başak; Frary, Anne; Eroğlu, SeçkinSelenium (Se) has a versatile chemistry. Therefore, it is present in a wide variety of materials on Earth. Although it is not an essential element for plants, Se provides several survival and ecological advantages. Puccinellia distans (P.distans) is weeping alkali grass which has a relatively fast growth rate and produces a large biomass. It can also hyperaccumulate extreme concentrations of boron and tolerate high levels of salinity. It is known that some plant species can tolerate high concentrations of soil salts and boron while hyperaccumulating Se in their leaves. Therefore, in this study, the Se accumulation and tolerance capacity of P. distans were investigated, and the plant’s Se tolerance mechanism was elucidated using a transcriptomic approach. The results of this study indicated that P. distans accumulated more than 100 mg Se kg-1 in its shoots. Therefore, P. distans was discovered to be a novel Se accumulator plant. Biochemical analyses and RNA sequencing results helped us to decipher the Se tolerance and accumulation mechanism of P. distans. Accordingly, the tolerance mechanism was found to be associated with the upregulation of genes involved in sulfate, selenocompound and antioxidant pathways. Here, we suggest that upregulation of Se assimilation and stress responses genes may have been due to induction of jasmonic acid signaling. In addition, we propose that the cell wall may play an important role in restriction of Se movement to the cytoplasm. Also, we put forward that sequestration of selenate inside the vacuole may be a way that Se accumulates in cells.Master Thesis Molecular Genetic Analyses in Turkish Plum (prunus Cerasifera)(Izmir Institute of Technology, 2018) Çakır, Gülsüm; Frary, AnnePlum is an economically important and popular fruit in Turkey, which ranks in sixth place in world plum production. This fruit is attractive to consumers with its beautiful scent and its delicious taste. In addition, it is an important plant species with its wealthy mineral, vitamin and phytochemical content. The genus Prunus is classified into three groups: the European, the Asian and the American plums. P. cerasifera, which was used in this study, belongs to the European group. P. cerasifera (2n=2x=16) is a Turkish plum drupe species. Since there are many subspecies, it can grow naturally in many parts of Turkey. The genetic diversity and population structure of P. cerasifera have not been studied using molecular techniques. Here, we studied the genetic diversity of 66 P. cerasifera accessions collected from Turkey at the molecular level using 47 sequence related amplified polymorphism (SRAP) primer combinations. The SRAP marker combinations showed reasonable polymorphism. A dendrogram was constructed using the Dice coefficient and unweighted neighbor joining algorithm. The dendrogram revealed three groups and the similarity between accessions ranged from 0.04 to 0.66 with an average dissimilarity of 0.37. Population structure analysis identified three subpopulations (K=3). This is the first genetic diversity analysis of P. cerasifera using SRAP markers. Genetic diversity and population structure data can be useful for determining breeding strategies in P. cerasifera accessions.Master Thesis Characterization of World Spinach Genetic Collection by Using Molecular Markers(Izmir Institute of Technology, 2015) Göktay, Mehmet; Frary, AnneSpinach (Spinacia oleracea L.) belongs to the Amaranthaceae family and is a popular and nutritious vegetable. It is believed that this green leafy plant originated from Persia which is now modern Iran and neighboring countries. In this study we screened 176 spinach world collection germplasm accessions with 15 known SSR markers. The markers produced 58 bands with 57 identified as polymorphic. PIC values of the markers ranged between 0.01 and 0.44. Average PIC value was 0.28. Based on allele analysis with Darwin5 and STRUCTURE tools, 176 individual were clustered into three groups. The first cluster mostly consisted of accessions from Europe and USA and contained 69 samples. The second cluster mostly consisted of lines from Asia and neighboring countries and contained 89 samples. The third cluster did not represent any characteristic according to geographic region, thus it was called an intermixed cluster which contained 18 samples. The maximum genetic dissimilarity of spinach accessions was 0.551 and minimum was 0.019. Average genetic dissimilarity was 0.217. Moreover we sequenced S. oleracea L. cv. Universal nuclear genome via IIIumina MiSeq technology and genome assembly was performed to develop new spinach–specific SSR markers. As a result, 3853 SSRs were identified in the assembled genome and we successfully designed 3275 primer pairs for these identified SSR motifs. These newly developed SSR markers will be helpful to scientists who are interested in spinach genome diversity and breeding.Master Thesis Determination of Genetic Diversity and Population Structure in Faba Bean (vicia Faba L)(Izmir Institute of Technology, 2015) Göl, Şurhan; Frary, AnneFaba bean (Vicia faba L.) is an important legume species because of the high protein and starch content of its seeds. Broad bean can be grown in different climatic conditions and is an ideal rotation crop because of the symbiotic relationship between the plant and nitrogen fixing bacteria in its roots. Broad bean seeds are consumed as fresh vegetables in many countries throughout the world. However, the genetic diversity found in this germplasm has not yet been characterized and has not been systematically used in broad bean breeding programs. In this project, faba bean individuals obtained from International Center for Agricultural Research in the Dry Areas (ICARDA), Centre for Genetic Resources (CGN), Aegean Agricultural Research Institute (AARI), Nordic Gene Bank (NGB) and Australia (The University of Adelaide, Jeffrey Paull) were examined for their genetic diversity and population structure. For this purpose, 259 faba bean germplasm accessions were characterized using 32 SSR primers. A total of 302 polymorphic SSR fragments were analyzed. According the results, faba bean individuals were divided into two main clusters based on Neighbor-joining algorithm (r = 0.9062) with some clustering based on geographical origin as well as seed size. STRUCTURE 2.2.3 program was used to determine population structure. K was determined as 2 subpopulations. Cluster 1 had 87 individuals; cluster 2 had 162 individuals and 10 individuals were intermixed with results generally agreeing with the dendrogram analysis. A total of 45 well-characterized faba bean individuals were selected for the core collection to be used in breeding studies.Master Thesis Elucidation of Boron Hyperaccumulation and Tolerance Mechanisms in Puccinellia Distans (jacq.) Parl. Using Proteomics Approach(Izmir Institute of Technology, 2015) Bar, Cantuğ; Frary, AnneBoron can cause growth deficiency and yield loss in plants at both low and high concentrations. Turkey holds 72,5% of known world boron reserves, and boron toxicity causes major agricultural problems. In a search to identify resistant species, Puccinellia distans (weeping alkaligrass), growing near boron mining sites in Turkey, was found to tolerate toxic boron concentrations. This species has been studied at the level of gene transcription, however, investigation at the protein level has not yet been performed. In this work, the boron stress tolerance mechanism of P. distans was investigated with proteomic approaches. For this purpose, plants were divided into control and stress groups with optimal and toxic boron concentrations, respectively. Boron accumulation was observed in leaf tissues at the end of growing stages. Leaf and root tissues were checked for tissue boron concentrations at the end of their growth period with ICP-MS. Both leaves and roots were found to accumulate around 6 g B/kg dry tissue. Total proteins were extracted from leaf tissues of both groups and analyzed in LC-MS instrument to determine the differences between protein profiles. One hundred and nine significantly differentially expressed proteins were identified in this work. Three upregulated proteins were selected as candidate proteins for boron hyperaccumulation tolerance. This work proved successful in identification of some of the proteins responsible for boron hyperaccumulation in P.distans and paving the way for future studies to use P. distans for phytoremediation in areas with excess boron.Master Thesis Determination of Genetic Diversity and Population Structure in Eggplant(Izmir Institute of Technology, 2015) Altaye, Tesfa; Frary, AnneEggplant (Solanum melongena), an economically important crop in the genus Solanum, is known by different names in different countries. The diversity of some eggplant germplasm has studied using morphological, biochemical and molecular techniques by different scientists. Here, we studied the genetic diversity of 79 eggplant accessions collected from 28 countries in a molecular level and the efficiency of Sequence related amplified polymorphism also evaluated in comparison with other studies. The genetic diversity of 73 S. melongena accessions and six outgroups were assessed using ten sequence related amplified polymorphisms (SRAP) marker combinations. All the primer tested showed polymorphism and the average alleles registered per locus was 4.7. The primers included in the study showed moderate polymorphic information content. Genetic similarity was analyzed using Dice coefficient and the relation tree constructed using unweighted neighbor joining. The dendrogram revealed five groups and the similarity of the dendrogram ranged from 0.18 to 1 with mean value of 0.59. All the outgroups were distantly related to S. melongena accessions except the wild S. linnaeanum and S. incanum group C ,which showed strong similarity with the cultivated s. Melongena.The result reported in Dendrogram, PCoA and STRUCTURE analysis showed consistency. The genetic similarities registered in our accessions were not correlated with geographical diversity. Lack of exact provenance and accessions naming is one of the challenge beside uncoordinated work done to characterize eggplant accessions. Therefore, collecting worldwide accessions from different eggplant germplasm center and screening by power core may clarify the ongoing confusions of eggplant classification.Master Thesis Determination of Genetic Diversity and Antioxidant Content of the National Melon (cucumis Melo) Collection(Izmir Institute of Technology, 2008) Şığva, Hasan Özgür; Frary, Anne; Frary, AnneIn this study, characterization with 19 morphological criteria and two types of PCRbased molecular marker systems, Simple Sequence Repeats (SSRs, microsatellites) and Amplified Fragment Length Polymorphisims (AFLPs), were used to characterize genetic variability among 238 national melon (Cucumis melo L.) accessions and 12 different members of the Cucurbitaceae family.According to morphological analysis, all accessions were vigourous. The accessions showed a great variety of fruit shape, 53 (38.4%) accessions were globular (round), 51 (37.0%) were eliptical, 15 (10.9%) were ovate, 7 (5.1%) oblate, 2 (1.4%) elongate and one accession was flattened. Another agronomically important criterion was predominant fruit skin colour. A total of 55 (39.9%) accessions had light yellow predominant skin colour, 34 (24.6%) were pale green, 29 (21.0%) were orange, 9 (6.5%) were cream, 6 (4.4%) were green, 4 (2.9%) were blackish-green and 1 (0.7%) was dark green.In accordance with genetic characterization, a total of 345 polymorphic AFLP fragments (products of 10 AFLP primer combinations) and 93 SSR fragments (products of 12 SSR markers) were detected and used to calculate genetic distance using DICE matrix and UPGMA (Unweighted Pair Group Method) arithmetical averages. The average polymorphic AFLP fragments per combination was 34.5 and SSR fragments per marker was 7.75. The phylogenetic tree showed that groups were clearly separated by both marker systems. This study allowed the identification of the relationship between national melon accessions based on genetic similarity or differences.Forty three melon accessions were also analyzed for total water soluble antioxidant and total phenolic compound activities.