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Öğe Abiotic stress of ZnO-PEG, ZnO-PVP, CuO-PEG and CuO-PVP nanoparticles enhance growth, sweetener compounds and antioxidant activities in shoots of Stevia rebaudiana Bertoni(Institution of Engineering and Technology, 2017) Javed, Rabia; Zia, Muhammad; Yücesan, Buhara; Gürel, EkremNanoparticles are known to play remarkable role as abiotic stress elicitors in plants. This study reports the comparative analysis of effects produced by capped [zinc oxide (ZnO)-polyethylene glycol (PEG), ZnO-polyvinyl pyrrolidone (PVP), copper oxide (CuO)-PEG, CuO-PVP] and uncapped (ZnO and CuO) nanoparticles on the medicinal plant, Stevia rebaudiana raised in vitro for the production of commercially important sweetener compounds. In context of shoot organogenesis, ZnO-PEG, ZnO-PVP, CuO-PEG, CuO-PVP were employed to the growth medium that resulted in increased growth parameters, and larger content of steviol glycosides as compared to the shoots raised in medium containing ZnO and CuO, revealed by high-performance liquid chromatography. In the meanwhile, non-enzymatic antioxidant activities including total phenolic content, total flavonoid content, total antioxidant capacity, total reducing power and 2,2-diphenyl-1-picryl hydrazyl-free radical scavenging activity were calculated and showed comparatively greater amounts in shoots grown in medium containing capped ZnO or CuO nanoparticles. Furthermore, the ZnO and its derivatives revealed to be more reactive at 1 mg/l of concentration. Whereas, the CuO and its derivatives produced greater response on Stevia at 10 mg/l concentration of nanoparticles. This study paves the way for more such studies encompassing capped and uncapped nanoparticles and their ultimate effect on in-vitro grown plant tissues for the production of active metabolites on industrial scale. © The Institution of Engineering and Technology 2017.Öğe Adventitious shoot regeneration from stem internode explants of Verbena Officinalis L., a medicinal plant(2010) Türker, Arzu Uçar; Yücesan, Buhara; Gürel, EkremVerbena officinalis L. (vervain) is a medicinal plant that shows several biological activities, including antimicrobial, antiinflammatory, analgesic, neuroprotective, hypnotic/sedative, gastroprotective, hepatoprotective, anticancer, cicatrizant, and antioxidant. This report describes an efficient plant regeneration system for V. officinalis via adventitious shoot development from stem internode and petiole explants. Stem internode explants were more effective than petiole explants in terms of both the number of shoots per explant and the percentages of explants forming shoots. Increasing benzyladenine (BA) concentrations steadily increased shoot formation up to 13.32 μM, but 22.22 μM BA caused a sharp decrease. The highest number of shoots (17.1 shoots per explant at 100% frequency) was obtained when 13.32 μM BA was combined with 5.71 μM indole-3-acetic acid (IAA). Rooting of the regenerated shoots was readily achieved when multiple shoots were singled out and cultured on medium containing varying concentrations of different auxins [IAA, indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid (2,4-D), or naphthalene acetic acid (NAA)]. IBA was more effective in terms of the number of roots developed, producing a mean of 7.1 roots per shoot at 4.92 μM, whereas IAA was more effective for the frequency of shoots developing roots: 100% of the shoots rooted at 5.71 μM IAA. More than 90% of the regenerants survived through the hardening off process, when they were transferred to foam cups and kept under growth room conditions for 2 weeks before finally being moved to room conditions at low humidity. Approximately 3 months after the transfer to room conditions, the flowering of the regenerated plants could be observed.Öğe Alkaloid production from cell suspension cultures obtained from osmotic-stressed callus lines of Catharanthus roseus(2003) Akçam-Oluk, Esin; Demiray, Hatice; Gürel, EkremCatharanthus roseus callus cultures established were first exposed to osmotic stress using polyethylene glycol (9.0%, w/v)(MW 3350) in solid MS medium supplemented with 20 g/l sucrose, 2 mg/l ?-naphtalene acetic acid (NAA) and 3 mg/l 6-benzylamino purine (BAP). Cell suspensions were then initiated from stressed (S) and non-stressed (NS) calli in liquid LS medium. When LS medium was employed, only ajmalicine was detected at 1.308 ?g/ml and 0.405 ?g/ml concentrations for the S and NS cell lines, respectively. None of the other alkaloids normally found in the intact plant, such as vinblastine, vincristine and vindoline was detected at the end of experiments carried out with both media. From these results it can be concluded that, application of the osmotic stress to Catharanthus roseus cell suspension cultures increases ajmalicine production capacity, although it does not trigger the production of other alkaloids involved.Öğe Applications of carbon nanomaterials in the plant system: A perspective view on the pros and cons(Elsevier, 2019) Verma, Sandeep Kumar; Das, Ashok Kumar; Gantait, Saikat; Kumar, Vinay; Gürel, EkremWith the remarkable development in the field of nanotechnology, carbon-based nanomaterials (CNMs) have been widely used for numerous applications in different areas of the plant system. The current understanding about the CNMs' accumulation, translocation, plant growth responses, and stress modulations in the plant system is far from complete. There have been relentless efforts by the researchers worldwide in order to acquire newer insights into the plant-CNMs interactions and the consequences. The present review intends to update the reader with the status of the impacts of the different CNMs on plant growth. Research reports from the plant biotechnologists have documented mixed effects (which are dependent on CNMs' concentration) of the CNMs' exposure on plants ranging from enhanced crop yield to acute cytotoxicity. The growth and yield pattern vary from species to species and are dependent on the dosage of the CNMs applied. Studies found an increase in vegetative growth and yield of fruit/seed at lower concentration of CNMs, but a decrease in these observables were also noted when higher concentrations of CNMs were used. In general, at lower concentrations, CNMs were found to be effective in enhancing (water uptake, water transport, seed germination, nitrogenase, photosystemand antioxidant activities), activating (water channels proteins) and promoting (nutrition absorption); all these change when concentrations are raised. All these aspects have been reviewed thoroughly in this article, with a focus on the recent updates on the role of the CNMs in augmenting or retarding plant growth. Sections have been devoted to the various features of the CNMs and their roles in inducing plant growth, phytotoxic responses of the plants and overall crop improvement. Concluding remarks have been added to propose future directions of research on the CNMs-plant interactions and also to sound a warning on the use of CNMs in agriculture. (C) 2019 Elsevier B.V. All rights reserved.Öğe Bioremediation applications with fungi(Springer International Publishing Ag, 2018) Sağlam, Necdet; Yeşilada, Özfer; Sağlam, Semran; Apohan, Elif; Sam, Mesut; İlk, Sedef; Emul, Ezgi; Gürel, EkremIndustrial wastewaters such as olive oil mill wastewater (OOMW) and alcohol factory wastewater (vinasse) with high polluting characteristics affect the ecosystem seriously. The environmental impact of these wastewaters is rather severe due to their organic matter content and dark color. Therefore, it is illegal to dispose these wastewaters directly into the environment. In addition of wastewater treatments, decolorization of Kraft mills is one of the serious problems in pulp and paper industries because of their high color contents. The discharges of Kraft mills present a threat for environment and especially surface waters. These colored effluents contain serious amounts of chlorinated and oxidized lignin compounds. Remediation of this kind of wastewaters by conventional treatment methods has difficult and challenging processes. As a solution, the fungi have been chosen for bioremediation of wastewaters as efficient biological systems as they are able to remove the color and threatful organic content. Mycoremediation is an effective and ecofriendly method for the bioremediation of this type of wastewaters. This method has several advantages over chemical or physical remediation. Besides, fungal enzymes have a great potential for detoxifying and screening most of the environmental pollutants. In the last decade, fungal enzymes have a new application area on sensor technology. Biosensors are able to utilize fungal enzymes including an electrode that may be used for the detection of pollutants such as phenolic compounds. The objective of this chapter is to summarize recent developments in mycoremediation of wastewaters, especially OOMW and vinasse. Here, the role of biotechnologically important fungi such as yeast, molds, and white rot fungi in the mycoremediation of wastewaters is reviewed. The various mycoremediation methods for effective bioremediation and recent developments for fungal enzymes are also discussed.Öğe Biotechnology applications for sugar beet(Taylor & Francis Inc., 2008) Gürel, Ekrem; Gürel, Songül; Lemaux, Peggy G.Sugar beet (Beta vulgaris L.) is an important industrial crop, being one of only two plant sources from which sucrose (i.e., sugar) can be economically produced. Despite its relatively short period of cultivation (ca. 200 years), its yield and quality parameters have been significantly improved by conventional breeding methods. However, during the last two decades or so, advanced in vitro culture and genetic transformation technologies have been incorporated with classical breeding programs, the main aim being the production of herbicide-and salt-tolerant, disease- and pest-resistant cultivars. Among the many applications of in vitro culture techniques, sugar beet has benefited the most from haploid plant production, protoplast culture, and somaclonal variation and in vitro cell selection. Several genetic transformation technologies have been developed, such as Agrobacterium-meditated, PEG-mediated, particle bombardment, electroporation, sonication and somatic hybridization, the first two being the most successful. Development of herbicide- and salt-tolerant, virus-, pest/nematode-, fungus/Cercospora- and insect-resistant sugar beet has been demonstrated. However, only herbicide-tolerant varieties have been approved for commercialization but not yet available in the marketplace; rhizomania-resistant varieties are being evaluated in field trials. Transgenic plants that convert sucrose into fructan, a polymer of fructose, were also developed. Initial attempts to increase sucrose yields produced promising results, but it still requires additional work. Despite marked progress in improving regeneration and transformation of sugar beet, genotype dependence and low regeneration and transformation frequencies are still serious restrictions for routine application of in vitro culture and, more importantly, transformation technologies. Selected food safety and environmental impact, as well as regulatory and public acceptance issues relating to transgenic sugar beet are also discussed.Öğe Bolu yöresine ait bazı bitkilerin doku kültürü yöntemiyle çoğaltılması(2006) Türker, Uçar Arzu; Gürel, Ekrem; Türker, Hakan; Yücesan, Bahtiyar Buhara; Cansever, Esra-Öğe Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey(Elsevier France-Editions Scientifiques Medicales Elsevier, 2013) Şahin, Günce; Verma, Sandeep Kumar; Gürel, EkremElimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 mu g ml(-1) thidiazuron (TDZ) and 0.25 mu g ml(-1) indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 mu g g(-1)) > D. trojana (1385.75 mu g g(-1)) > D. cariensis (1038.65 mu g g(-1)) > D. davisiana (899.86 mu g g(-1)) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides. 2013 Elsevier Masson SAS. All rights reserved.Öğe Callus and root development from leaf explants of sugar beet (Beta vulgaris L.): Variabilit? at variety, plant and organ level(1997) Gürel, EkremIn this study , the nature of variability observed in previous experiments, when leaf explants of sugar beet (Beta vulgaris L.) were cultured in vitro, was investigated at different levels of classification (i.e., variety, plant and organ). Large differences in rooting capacity were observed when 10 commercial varieties were compared. Significant inter-plant and inter-leaf (intra-plant) variability was found to exist within a variety but explants taken from different positions within the leaf behaved similarly. A two-fold variation in polyphenol-oxidase activity was found within a population of the variety Primo, but no relationship existed between enzyme activity and either root formation or the blackening of the explant tissue when compared by culturing explants taken from the same leaves used for enzyme assays, although a weak negative correlation between the degree of blackening and rooting capacity was observed.Öğe Callus and root development from leaf explants of sugar beet (Beta vulgaris L.): Variability at variety, plant and organ level(1997) Gürel, EkremIn this study , the nature of variability observed in previous experiments, when leaf explants of sugar beet (Beta vulgaris L.) were cultured in vitro, was investigated at different levels of classification (i.e., variety, plant and organ). Large differences in rooting capacity were observed when 10 commercial varieties were compared. Significant inter-plant and inter-leaf (intra-plant) variability was found to exist within a variety but explants taken from different positions within the leaf behaved similarly. A two-fold variation in polyphenol-oxidase activity was found within a population of the variety Primo, but no relationship existed between enzyme activity and either root formation or the blackening of the explant tissue when compared by culturing explants taken from the same leaves used for enzyme assays, although a weak negative correlation between the degree of blackening and rooting capacity was observed.Öğe Cardenolide estimation in callus-mediated regenerants of Digitalis lamarckii Ivanina (dwarf foxglove)(Springer, 2014) Yücesan, Buhara; Müller-Uri, Frieder; Kreis, Wolfgang; Gürel, EkremDigitalis cardenolides can regulate heart rhythms and are effective agents in cancer chemotherapy, in particular, for treating prostate and breast cancer. In this study, an optimized and efficient plant tissue culture protocol was established using callus cultures of Digitalis lamarckii Ivanina, commonly known as dwarf foxglove. Lamina explants developed callus when cultured on Linsmaier and Skoog (LS) medium containing different concentrations of 6-benzyladenine (BA; 4.4, 13.3, or 22.2 mu M) and alpha-naphthalene acetic acid (NAA; 2.7, 5.4, or 10.8 mu M). The highest incidence of callus formation (100%) was achieved on LS medium containing 13.3 mu M BA and 10.8 mu M NAA. Indirect shoot regeneration was achieved when the callus explants were cultured on LS medium supplemented with varying concentrations of BA (0.4, 1.1, or 2.2 mu M) and/or gibberellic acid (0.7 or 1.4 mu M) for 8 wk. Following the rooting of shoots on LS medium supplemented with either indole-3-acetic acid (ranging from 1.4 to 5.7 mu M) or NAA (1.3 to 5.2 mu M), lamina and petiole tissues of the 4-mo-old regenerated plants were compared for their cardenolide contents. Lamina extracts showed nearly three times higher cardenolide accumulation than petiole extracts. Of the cardenolides analyzed by reverse-phase high-performance liquid chromatography, neo-odorobioside G and glucogitoroside were abundant in lamina extracts (170.3 and 143.9 mg/kg dry weight, respectively). The regeneration protocol described in this study can be used for the in vitro production of certain cardenolides from D. lamarckii.Öğe Cardiotonic glycoside determination in in vitro and ex vitro samples of Digitalis lamarckii Ivan, an endemic species to Anatolia(Georg Thieme Verlag Kg, 2010) Yücesan, Buhara; Gürel, EkremDigitalis lamarckii Ivan is an endemic species to Turkey. Digitalis species are medicinally and economically important as they contain cardiac glycosides that strengthen diffusion and regulate the heart rhythm [1]. Moreover, preliminary studies have revealed that digoxin and digitoxin are also effective agents in several cancer treatments [2]. In this study cardenolide patterns in in vitro and ex vitro (from natural populations) samples of D. lamarckii were studied. In in vitro samples, digoxin was predominantly found in different tissues (lamina, petiole or whole shoot) of 12 or 18-week old regenerants. Digoxin content was, in general, lower in the 18-week old regenerants than 12-week old ones (Fig.1).Öğe Clonal propagation and synthetic seed production from nodal segments of Cape Gooseberry (Physalis Peruviana L.), a tropical fruit plant(2015) Yücesan, Bahtiyar Buhara; Mohammed, Aliyu; Arslan, Merve; Gürel, EkremPhysalis peruviana L. contains polyphenols and carotenoids with antiinflammatory and antioxidant activities used against diabetes. To establish an efficient regeneration system using nodal segments excised from 4-week-old germinated seedlings, direct plant regeneration, without additional rooting stage, was achieved on LS medium containing 0.5 mg/L 6-benzylaminopurine (BAP), kinetin (KIN), thidiazuron (TDZ), or gibberellic acid (GA3), alone or in combination with 0.25 mg/L indole-3-acetic acid (IAA) or indole-3- butyric acid (IBA), after 2 weeks of incubation. The highest mean numbers of shoots and well-developed roots were obtained on LS medium containing solely 0.5 mg/L TDZ, producing 5.3 shoots and 3.3 roots per explant after 2 weeks of incubation. Direct shoot and root formation were also recorded on LS medium containing no plant growth regulators. Due to the high regeneration capacity of nodal segments, synthetic seed production was also investigated using the sodium alginate (NaAlg) encapsulation technique. Four different matrix compositions, including NaAlg with or without LS medium containing 3% (w/v) sucrose alone or in combination with 0.5 mg/L abscisic acid (ABA) as a growth retardant were tested for the regrowth performance of synthetic seeds after storage at 4 °C up to 70 days. The highest regrowth (100%) was observed at 28 days of storage for all matrix compositions. All plantlets were acclimatized to the soil and then progressively transferred to the field. The fruits were harvested after 5 months. This study might provide a new insight through protocol development for micropropagation and synthetic seed production of many solanaceous species with economical relevance.Öğe Common mullein (Verbascum thapsus L.): recent advances in research(Wiley, 2005) Türker, Arzu Uçar; Gürel, EkremCommon mullein (Verbascum thapsus L.) is a medicinal plant readily found in roadsides, meadows and pasture lands and has been used to treat pulmonary problems, inflammatory diseases, asthma, spasmodic coughs, diarrhoea and migraine headaches. Although it has been used medicinally since ancient times, the popularity of common mullein has been increasing commercially for the past few years. Today, the dried leaves and flowers, swallow capsules, alcohol extracts and the flower oil of this plant can easily be found in health stores in the United States. The use of common mullein extracts in folk medicine begun recently to be supported by an increasing number of research studies. This paper thoroughly reviewes all the scientific research related to Verbascum thapsus L. including plant tissue cultures and the biological properties of this plant.Öğe CRISPR/Cas9-mediated immunity in plants against pathogens(Caister Academic Press, 2017) Sameeullah, Muhammad; Khan, Faheem Ahmed; Özer, Göksel; Aslam, Noreen; Gürel, Ekrem; Karadeniz, TuranGlobal crop production is highly threatened due to pathogen invasion. The huge quantity of pesticides application, although harmful to the environment and human health, is carried out to prevent the crop losses worldwide, every year. Therefore, understanding the molecular mechanisms of pathogenicity and plant resistance against pathogens is important. The resistance against pathogens is regulated by three important phytohormones, viz. salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Here we review the possible role of CRISPR technology to understand the plant pathogenicity by mutating genes responsible for pathogen invasion or up-regulating the phytohormones genes or resistant genes. Thus hormone biosynthesis genes, receptor and feeding genes of pathogens could be important targets for modifications using CRISPR/Cas9 following multiplexing tool box strategy in order to edit multiple genes simultaneously to produce super plants. Here we put forward our idea that the genes would be either mutated in case of plant receptor protein targets of pathogens or up-regulation of resistant genes or hormone biosynthesis genes will be better choice for resistance against pathogens.Öğe CuO nanoparticles significantly influence in vitro culture, steviol glycosides, and antioxidant activities of Stevia rebaudiana Bertoni(Springer Netherlands, 2017) Javed, Rabia; Mohamed, Aliyu; Yücesan, Bahtiyar; Gürel, Ekrem; Kausar, Rukhsana; Zia, MohammadCopper oxide (CuO) nanoparticles (NPs) synthesized through co-precipitation method were employed in MS media during in vitro culture of Stevia rebaudiana. Physiological characteristics, production of steviol glycosides, and antioxidative parameters were investigated in regenerated plants. CuO NPs had crystalline monoclinic cubic cuprous oxides with average size 47 nm. The NPs were applied at 0, 0.1, 1.0, 10, 100 and 1000 mg/L in MS media for direct organogenesis of S. rebaudiana from nodal segments. Shoot organogenesis was found highest (88.5%) at 10 mg/L CuO and average shoot length, mean number of shoot per explant, and fresh weight were also found significantly higher at the same concentration. High performance liquid chromatography (HPLC) illustrated significant rise of bioactive major steviol glycosides (rebaudioside A and stevioside) at 10 mg/L CuO NPs in MS media. The oxidative stress produced by CuO nanoparticles on S. rebaudiana was affirmed by antioxidant activities i.e. total antioxidant activity (TAC), total reducing power (TRP) and 2,2-diphenyl-1-picryl hydrazyl (DPPH)-free radical scavenging activity. The oxidative stress generated by NPs involved production of antioxidative molecules total phenolic content (TPC), total flavonoid content (TFC) depending on NPs concentration. The study concludes that copper oxide nanoparticles functions as a stimulator of bioactive components productions, and can be employed in in vitro batch cultures. © 2017, Springer Science+Business Media B.V.Öğe Development of an efficient plant regeneration system in sunflower (Helianthus Annuus L.)(1998) Gürel, Ekrem; Kazan, KemalWith the aim of developing an efficient plant regeneration system from cells or tissues of sunflower (Helianthus annuus L.)', we compared several regeneration protocols using different explant types and hormonal combinations. Somatic embryogenesis could be induced on cotyledon explants, especially from the basal (proximal) portion of the cotyledons, but genotypic vairation appeared to be the most critical factor for both somatic embryo and root production. Such a variation was more prominent when 10 different sunflower cultivars were compared for shoot production from shoot-tip explants. Further refinement of the culture conditions may be neceassary to improve the efficiency of somatic embryo production. Thin cell layers from hypocotyl segments were not found to be successful for plant regeneration.Öğe Differential effects of plant growth regulators on physiology, steviol glycosides content, and antioxidant capacity in micropropagated tissues of Stevia rebaudiana(De Gruyter Open Ltd, 2017) Rabia Javed; Yücesan, Bahtiyar Buhara; Zia M.; Gürel, EkremPlants naturally produce secondary metabolites on exposure to abiotic/biotic stress. Production of secondary metabolites, like phenols or flavonoids, is a defence mechanism against different stresses. This study explores the influence of plant growth regulators (PGRs) on the growth and secondary metabolites of in vitro grown tissues of zero calorie sweetener, Stevia rebaudiana. An efficient regeneration of this valuable plant under the influence of PGRs was conducted, and quantification of physiology parameters, steviol glycosides (SGs) and various antioxidant activities in callus, shoots and regenerated plants was performed. Significantly highest explant response was achieved by 2 mg/L of 6-benzyl aminopurine (BAP) and 0.5 mg/L of 2,4-dichlorophenoxy acetic acid (2,4-D) combination in case of callogenesis, while shoot and root organogenesis was best illustrated by control and 0.5 mg/L of indole acetic acid (IAA) treatment, respectively. Regarding SGs formation in leaves, significantly greater content (2.07% and 0.93%) was obtained by control in case of rebaudioside A (Reb A) and 2 mg/L of BAP in case of stevioside (ST). Leaves of rooted-shoots produced significantly highest Reb A (4.21%) and ST (2.22%) content in case of 1 mg/L of IAA and 0.25 mg/L of IAA inoculation in Murashige and Skoog growth medium, respectively. Similarly, differential effects of PGRs were elucidated in case of all in vitro grown tissues on antioxidant properties. The study concludes that secondary metabolites can be efficiently produced in in vitro culture so can be employed in bioreactors. © 2017 Institute of Molecular Biology, Slovak Academy of Sciences 2017.Öğe Digitalis ferruginea L. türünün kardenolit sentez metabolizmasının moleküler yöntemlerle araştırılması ve kardenolit içeriğinin karşılaştırmalı analizi(2015) Gürel, Ekrem; Yücesan, Bahtiyar Buhara; Eker, Ismail; Ordu, Öznur Demir; Sammeullah, Muhammad; Mohammed, Aliyu; Ilal, Ayşe Tuğçe-Öğe Digitalis ferruginea subsp. ferruginea’ya ait 3?-HSD, P5?R1 ve P5?R2 Genlerinin Metabolik Mühendisliği: Transplastomik Nicotiana tabacum’da Metabolom ve Transgenik Arabidopsis thaliana’da Promotör Analizleri(2020) Gürel, Ekrem; Waheed, Mohammed Tahir; Yildirim, Muhammet Hakkı; Lössl, Andreas; Baloğlu, Mehmet CengizProje kapsamında tamamlandığımız çalışmalar, iki temel konu başlığı altında toplanabilir: (A) Kardiyak glikozit biyosentez genlerinin fonksiyonel analizleri; (1) kardiyak glikozitlerin sentezinden sorumlu üç anahtar genin (3?-HSD, P5?R1 ve P5?R2), Digitalis ferruginea L. subsp. ferruginea (yüksükotu) bitkisinden izole edilerek, tütün (Nicotiana tabacum) bitkisinin plastid genomuna aktarılması (transplastomik transformasyon), (2) kardiyak glikozitlerin miktarlarının üretilen bu transplastomik tütün bitkilerinde HPLC analizleri ile belirlenmesi, (3) NMR yardımıyla transplastomik tütün bitkilerinde metabolom analizlerinin yapılması, (4) farklı abiyotik stres koşulları altında transplastomik tütün bitkilerinin fizyolojik ve biyokimyasal tepkilerinin incelenmesi, (5) bu hedef genlere ait proteinlerin hücre içi lokalizasyonunun, yeşil floresan protein (GFP) geni yardımıyla, transgenik soğan zarı hücrelerinde gözlenmesi, ve (B) Kardiyak glikozit biyosentez genlerinin promotor analizleri; Agrobacterium tumefaciens aracılığı ile transforme edilen transgenik Arabidopsis bitkilerinde, farklı abiyotik stres uygulamalarının ardından, söz konusu bu hedef genlere ait promotorların izole edilmesi ve analizlerinin yapılması. Transplastomik tütün (Nicotiana tabacum) transformasyonu, hedef genlerin biyolistik yöntemle tütün plastomuna aktarılmıştır. Bunun için, standart moleküler klonlama yöntemleri kullanılarak, tekli veya ikili kardiyak glikozit biyosentez geni/genleri içeren plastomik vektörler hazırlanmıştır. Transplastomik transformasyonun teyidi ise, DNA ve protein düzeyinde, PCR çoğaltımı, Southern blot ve Western blot analizleri ile tamamlanmıştır. Genlerin hücre içi lokalizasyonları, GFP geninin biyolistik bombardıman yöntemiyle soğan zarı hücrelerine aktarılmasının ardından, konfokal mikroskop yardımıyla görüntülenmiştir. Transplastomik tütün bitkilerinin kardiyak glikozit tayinleri HPLC ile, metabolom analizleri ise NMR ile tamamlanmıştır. Ayrıca, farklı biyo/fizyo-kimyasal yöntemler takip edilerek, abiyotik stres faktörlerinin fizyolojik etkileri incelenmiştir. Promotorların fonksiyonel analizlerine yönelik olarak, GUS geni ile birleştirilmiş promotorları içerecek olan transgenik Arabidopsis thaliana bitkileri, Agrobacterium tumefaciens aracılığı ile ?floral dip? yönetmine göre elde edilmiş, ve farklı abiyotik stres koşulları ve bitki büyüme düzenleyicilerinin teşvik edici etkisi altında test edilmiştir. Fonksiyonel analizler ve stres tolerans testlerinin; sıcaklık, soğuk, yaralanma, tuz, ağır metal gibi farklı stres koşulları altında gen ifadesinin işleyişi ve bitki büyüme hormonlarının etkilerini ortaya koyacak yararlı bulgular üretmesi beklenmektedir. Ayrıca, bu bulguların, hem tıbbi ilaç geliştirme ve üretim araştırmalarına hem de abiyotik stres toleransı yüksek bitki çeşitleri geliştirmeye yönelik tarımsal araştırmalara katkı sağlayacağını ummaktayız. Diğer taraftan, transplastomik tütün bitkisinin yüksek miktarda kardenolit veya yeni (novel) metabolit/ler ürettiğinin teyit edilmesi ile, bu ürünlerin büyük ölçekli ticari üretimleri mümkün olabilecektir. Projenin bir diğer faydalı çıktısı da, transplastomik transformasyon teknolojisi, gen kaçışı riskini ortadan kaldırma potansiyeline sahip olduğu için, birçok yararlı uygulama alanı olan GDO teknolojisinin toplum nezdindeki var olan olumsuz algısının azaltılmasına katkı sağlayacaktır.
