Theses supervised by Prof. Dr. Ekrem Gürel
12 theses · Bolu Abant İzzet Baysal University
In vitro effect of nanoparticles on sugar beet (Beta vulgaris) and its secondary metabolites (betalain and betaine) production
Sugar beet (Beta vulgaris L.) is considered the most desirable and productive economic and food crop. Many studies have been based on the development of this crop. With the widespread applications of nanoparticles in the field of plants that have received great interest, due to their amazing results and promising capabilities that are considered so far only the "tip of the iceberg", this research was conducted to investigate the effect of two nanoparticles on the sugar beet plants in several respects; seed germination, morphology, chlorophyll A and B content, and carotenoids, as well as two of the most important sugar beet metabolites, betaine and betalain. TiO2 NPs and ZnO NPs were applied in four different concentrations separately (0, 5, 15, and 25 mg/ml) on two genotypes of sugar beet plants, monogerm (SG833) and multigerm (SG2020), by adding the NPs to the MS medium. Using a UV-spectrophotometer, chlorophyll, carotenoids, betaine, and betalain were estimated. Plants exposed to NPs did not have a significant difference in terms of seed germination, chlorophyll contents, or carotenoids compared to the control. In comparison to the control group, it showed a statistically significant difference and improvement for betaine at low concentrations of NPs and gradually decreased with increasing concentrations in both genotypes. In contrast to the multigerm genotype (SG2020), which showed a statistically significant increase in the content of betaxanthin at a concentration of 15 mg/ml (p<0.01) and 25 mg/ml (p<0.05) of ZnO NPs compared to the control. Regarding betacyanin, only the plants exposed to ZnO NPs at the highest concentration, which is 25 mg/mL, showed a statistically significant increase (p< 0.05), compared to the control plants. It is indicated that the response of plants during exposure to NPs depends on several factors, including the type of nanoparticles, their concentration, and the exposure time; moreover, it varies between plant species and even between genotypes within the same plant species.
Determining agro-morpohological and molecular characteristics of Bolu province wheat landrace and bred wheats and modeling their spatial distribution by geographic information systems and remote sensing
Landraces are more important than ever today due to both breeding studies and people's tendency towards healthier and higher quality products. It is necessary to know and understand genetic and agro-morphological structures as well as geographical distribution of landraces for serious management of breeding and production studies. In this study, the genetic, agro-morphological characteristics and geographical distributions of local populations of Triticum monococcum L. ssp. monococcum (IZA), Triticum aestivum L. cv. Çalıbasan and Triticum aestivum L. cv. Sarı Serez in Bolu province were researched. Genetic polymorphisms between populations and variants of these two species were investigated with 5 SCoT markers. Thousand kernels weight (TKW), flag leaf area (FLL), flag leaf length (FLL), flag leaf width (FLW), spike length (SL), plant height (PH), upper node length (UNL), number of kernels per spike (KPS), spikes fertile per square meter (SFPS), and yield (YLD) were investigated as agro-morphological variables. Moreover, topographic (elevation, slope, aspect), climate (precipitation, maximum-minimum-mean temperatures and bioclimate), soil (bulk density, coarse fragment, sand, silt, clay, cation exchange capacity (CEC), nitrojen, organic carbon, pH, great soil groups and land use capability) and CORINE (land use/cover) complementary database (raster maps) of Bolu province were created and relationships between agro-morphological and these environmental variables were investigated by correlation analysis. All in all, in IZA wheat TKW-SL, TKW-FLL, TKW-elevation, FLL-elevation, SL-elevation, YLD-precipitation (in April) shows significant (p<0.01) and high correlations at values of 0.692, 0.644, 0.793, 0.773, 0.702, 0.554, respectively. According to the results of start codon targeted (SCoT) analysis markers, primers amplified 48 polymorphic fragments with an average of 9.60. The average polymorphism information content (PIC) value of SCoT markers was 0.08. As a result of dendrogram analysis, T. aestivum and T. monococcum ssp. monococcum divided into two main clusters with 62% genetic similarity. Using yield and the environmental variables strongly effective on yield, a yield model of IZA wheat that shows potential growing areas was developed by multiple regression and mapped by GIS at the end of this study.
Effects of selenium on sulfate transporter genes in rice and the functional characterization of the OsSULTR1;1 gene in selenium homeostasis in arabidopsis
Sulfate transporters in rice are reported to be involved in abiotic and heavy metal stresses. However, the regulation of these transporters and their functional characterization in relation to selenate treatment remain elusive. The focus of this research was to analyze the expression profiles of rice sulfate transporters at vegetative and reproductive stages. The transporters exhibited diverse expression patterns in response to Se treatment and displayed tissue specificity, suggesting their varied functional roles. OsSULTR1;1 was selected as a candidate gene due to its expression under low sulfur (S) conditions (15 µM) and its high root tissue specificity, both of which were further enhanced by Se treatment. Subcellular localization revealed that the OsSULTR1;1 protein localizes to the plasma membrane, implicating its role in root uptake of Se. To further characterize this gene, it was genetically transformed into Arabidopsis thaliana. Overexpression (OX) of the gene in Arabidopsis resulted in enhanced growth, development, and uptake of mineral nutrients compared to wild-type (WT) plants, suggesting a role in efficient nutrient acquisition. OX plants subjected to time- and concentration-dependent Se treatment accumulated significant levels of Se in their tissues. Se was found to be toxic for both OX and WT plants at higher concentrations (10, 25, and 50 µM), suggesting its beneficial effects at low concentrations. The significantly enhanced Se levels in seed and shoot tissues of OX plants compared to WT plants indicate the functional role of OsSULTR1;1 in Se uptake from the rhizosphere. A rhizosphere acidification assay revealed that OX Arabidopsis plants exude more efficiently, causing a significant change in rhizosphere pH compared to WT plants. This suggests a secondary active transport mechanism. OX plants exhibited increased tolerance to abiotic stresses such as heat, cadmium, lead, and aluminum. Taken together, these findings suggest that OsSULTR1;1 is a potential candidate gene for Se accumulation for biofortification purposes. It may also play a role in plant tolerance to climate change and environmental stresses, potentially contributing to sustainable agricultural productivity under global warming conditions.
Comparison of different seed priming technologies for seed germination and plant growth in sugar beet (Beta vulgaris L.)
Environmental stress is a significant threat to crop production and quality. The effect of ecological changes, shortage of natural resources, more demand for food, and increased crop production, more attention has turned to improving seed quality and crop plant production through implementing efficient strategies. Seed priming, an innovative technology, has emerged as a pivotal strategy in this pursuit. This research was carried out to determine the effects of hydro-priming and nano-priming on seed germination, α-amylase activity, chlorophyll and MDA content in the sugar beet genotype SG188 (developed by Songül GÜREL). Seeds were soaked in water and in solutions containing different concentrations (5,10, and 15 ppm) of TiO2 NPs for 3,6,9 hours and experiments were repeated three times. As the first step, the seed was primed and stored at 4˚C for 10 and 45 days as a post-priming application. Before sowing seeds, after 10 days of priming, α-amylase activity, chlorophyll and MDA content of seeds were measured in primed seeds and the control. Then, growth and development parameters were checked after 10 days of germination. It showed that the germination rate in the primed-seed group was relatively higher than in the control. Next, Image J software showed that seeds soaked with nanoparticles 5,10,15 ppm for 3 hours had a positive impact and increased root length compared to the control. In the next step, after 45 days of priming, seedlings were analyzed similarly to seedlings after 10 days post-priming in terms of morphology, physiology, and biochemistry by ANOVA. In addition, a comparison of post-priming treatments showed that 45 days post-priming led to a boost of α-amylase enzyme activity compared to 10 days post-priming.
Analyses of promoters in Arabidopsis thaliana of 3 beta HSD and 5 beta POR genes isolated from digitalis species
Digitalis ferruginea, known as foxglove, is famous for the production of secondary metabolites; i.e., cardiac glycosides. The progesterone 5β-reductase (5βPOR1 and 5βPOR2) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are important key step genes in the pathway of biosynthesis of cardenolide in Digitalis species. For the first time, promoters of 3β-HSD, 5βPOR1 and 5β POR2 were isolated from genomic DNA of Digitalis ferruginea and sequence analysis was performed. Sequence information of the promoters revealed that these promoters have abundant cis-elements related to light responses. 5βPOR1promoter specific cis-elements are circadian and heat shock. 5βPOR2promoter specific regulatory elements are defense, stress and methyl jasmonate. Wound response element was found specifically in the 3β-HSDpromoter. Functional characterization of the 3β-HSDpromoter::GUS in Arabidopsis plants transformed by floral dip transformation showed activity in cotyledons, newly emerged leaves, vascular tissues of shoot and root, trichomes, shoot apical meristem (SAM) and hydathodes during plant growth and developmental stages. Under the treatments of 3% sucrose or 2% mannitol intensively enhanced GUS activity in the seedlings. In the presence of NaCl (150 mM) promoter expression activity was downregulated. 5βPOR1promoter::GUS activity was the highest in 5 and 10 days old seedlings in all tissues except root apical meristem, then decreased in 15 days old seedlings. 5βPOR2promoter::GUS activity was confined to vascular tissues in cotyledons in 5 and 15 days old seedlings. In 10 days old seedlings, promoter activity was intensively found in cotyledons, vascular tissues of cotyledons, hypocotyl and root adjacent to hypocotyl. Promoter activity was intensive in hypocotyl of 15 days old seedlings. The promoter sequences and functional characterization in Arabidopsis, revealed that the genes are light regulated and the 3β-HSD have role in plant growth, development and abiotic stress tolerance. Further, the genes 5βPOR1 and 5βPOR2 also have crucial role in normal plant growth during developmental stages.
Gynogenesis induction and doubled haploid plant production in sugar beet (Beta vulgaris)
The first chapter contains a brief introduction and a general literature review. Sugar was first extracted from sugar beet in 1747, with an approximate rate of 1.6% extractable sugar. During more than two centuries, researchers' attempts increased the sugar amount up to 20%. Majority of those successes were because of conventional breeding methods. In the early 19th century, a spontaneously induced haploid plant was discovered. The doubled haploid technique provides researchers with a complete homozygous plant, which is of high value for breeders, and let them shorten breeding duration for biennial plants from about ten years to two years. However, for sugar beet, from 1945 doubled haploid sugar beet technique was employed towards breeding sugar beets with other beneficial traits other than sugar concentration, i.e. resistance or tolerance to biotic or abiotic stresses. Several doubled haploid methods were examined, categorized in vivo and in vitro methods. Androgenesis, the most favorable methods mostly resulted in callus or diploid plants instead of haploid or doubled haploid plants. Except for gynogenesis, none of the applied methods was promising. Therefore, the only option was gynogenesis to be employed in sugar beet doubled haploid breeding programs. Sugar beet is an inter-breeding allogamous plant. Thus, there is a considerable variation among different varieties, cultivars, and genotypes. As a result, the available methods reported in the scientific literature sometimes cannot be successfully implemented for other genotypes. Sugar beet is an economically important crop for the countries in the temperate region of the northern hemisphere, including Turkey, which is a sugar beet seed importer country from western countries, e.g. Germany, the Netherland, Denmark, or Sweden. To produce sugar beet sustainably in Turkey, the country needs to breed and produce the required seeds and locally adaptable genotypes. Therefore, this project was launched to be a bridge between two parts of a long-term breeding program, started from more than a decade ago and is continued. The aims of these experiments reported in four chapters in the present thesis were haploid sugar beet induction through in vitro culture of unfertilized ovules (Chapter II); ameliorating hyperhydricity of the gynogenic explants (Chapter III); Doubled haploid sugar beet induction (Chapter IV); Increasing the propagation rate of the doubled haploid explants (Chapter V). The summaries of the chapters are provided in the following paragraphs. The second chapter describes a protocol studying the effects of an interaction between cold pretreatment of six genotypes of sugar beet inflorescences at 4 °C for one week or more and 6-benzylaminopurine (BAP) concentrations (1 or 2 mg L‒1) to increase the response rate of haploid embryo induction.. Ovules were removed from the unfertilized flowers and cultured on in vitro media. The interaction of three variables was examined, i.e. genotype, cold pretreatment, and hormonal treatment. In comparison with freshly cultured ovules, cold pretreatment for one week almost doubled the mean of haploid plantlet induction rate. Supplementing BAP at 2 mg L‒1 nearly doubled the induction rate of the cultured ovules, followed by 1 mg L‒1 BAP in comparison with the hormone-free medium. Interaction of 2 mg L‒1 BAP with one-week cold pretreatment induced the highest gynogenesis rate, but the hormonal treatment resulted in hyperhydricity. There was a considerable variation among the genotypes in their responses to the treatments. Genotype and cold pretreatment also showed a significant interaction. Cold pretreatment for longer than one week, i.e. 2–5 weeks resulted in similar or lower amounts of gynogenesis in comparison with the control (freshly cultured ovules). Ovules of one of the genotypes (SG3) treated with one-week cold pretreatment and 1 mg L−1 BAP produced the highest percentage of regenerants. BAP at 1 or 2 mg L−1 increased the gynogenesis rates 1.7 and 2 fold, respectively. Cold pretreatment increased the haploid embryo induction from a mean of 6.49% to 11.3% after one-week cold pretreatment. The third chapter describes a study involving the effects of media with different concentrations of BAP and/or kinetin (Kin) as hormonal treatments, sucrose, and a solidifying agent (Phytagel) on haploid sugar beet explants' proliferation and hyperhydricity. After inducing haploid embryos and initial propagation of the explants, they were treated with ten different concentrations of the above-mentioned chemicals over six weeks. After applying the treatments, the mean of proliferation and the mean of hyperhydricity of the explants were compared. It was observed that Kin with a reasonable amount of proliferation and minimum rate of hyperhydricity performed better than BAP in different concentrations and combinations. Highest proliferation with the least hyperhydricity was obtained with 0.2 mg L−1 Kin, 10 g L−1 sucrose, and 6.5 mg L−1 Phytagel. The variables were negatively correlated (τb = −.648, n = 36, p < .001). The fourth chapter describes a detailed study of a highly efficient protocol to multiply the number of haploid plants in sugar beet and subsequent chromosome doubling. In this chapter, the interactions between cold pretreatment, seven genotypes of sugar beet, and Kin to improve haploid embryo induction were studied. In addition, the effects of the color of ovules, flower bud position, and comma‑form ovule on haploid embryo induction were investigated. Cold pretreatment for one-week, Kin supplementation, and genotype were influential in stimulating the ovules. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. Two-way and three-way interactions of the variables were also statistically significant. Kin at 0.05 or 0.5 significantly induced more gynogenic embryo induction in comparison with the control. The difference in gynogenic embryo induction between the most and the least responsive genotypes was about 4-fold. The effect of interaction between cold pretreatment and Kin was most prominent when 0.05 mg L−1 was used. However, for freshly cultured ovules, Kin was not statistically significant. The hormonal treatments' effects on the genotypes were different, e.g. a genotype (SG5) was highly benefitted from 0.5 mg L−1 Kin and reached as much as 24% of induction, whereas another genotype (SG4) was not responsive to any of the hormonal treatments. When the effect of ovule position on inflorescence was studied, it was observed that the ovules removed from the flowers grew on the lower part of the inflorescence were more responsive and produced more gynogenic embryos than the ones removed from the upper part of the inflorescence. The ovules turned brown after one month produced higher percentages of the gynogenic embryo as compared with the ovules remained white. Colchicine at 5 g L–l for 5 min was used to double the chromosome number of the haploid plantlets because the treatment over 3 or 7 min resulted in lower amounts of doubled haploid plants. However, the genotype responses to the doubling treatments were not significantly different. In the fifth chapter, with an aim of increasing the number of doubled haploid explants, the effects of five levels of proline (0.0, 0.1, 0.2, 0.3 or 0.4 mM) on the explants' proliferation, propagation, and shoot length were compared. The amino acid was supplemented to the most productive medium that is described in chapter III which contained 0.2 mg L−1 Kin, 10 g L−1 sucrose, and 6.5 mg L−1 Phytagel (i.e., treatment HT9). Proline at 0.3 mM induced the highest amount of proliferation and propagation, while proline-free medium resulted in the lowest amount of proliferation, and induced one of the lowest amounts of propagation. Proline at 0.3 mM induced the shortest shoots, whereas 0.1 mM proline induced the longest shoots. The proliferated explants were suitable for propagation (τb = 0.822, SE = 0.027, n = 75, p < 0.001). However, both proliferation and propagation showed negative correlation (τb = –0.565 and –0.601, SE = 0.061 and 0.054, respectively, n = 75, p < 0.001). For the first time, our results show beneficial effects of proline on in vitro proliferation and propagation of sugar beet. The six chapter covers the main conclusions. In summary, it can be noted that haploid plantlet induction rate can be improved by cold pretreatment of inflorescences for one week at 4 °C. Moreover, BAP supplementation may induce more gynogenesis. However, the higher level of BAP may lead to higher abnormal development of emerged structures, e.g. hyperhydricity and necrosis. The technique appears highly genotype-dependent. However, Kin seemed a better alternative than BAP in inducing non-hyperhydric plantlets. Ovule color and the position of the flower bud on the inflorescence showed influential in gynogenesis, which was statistically significant. Proline at 0.4 mM might be deleterious to in vitro growth of sugar beet. Proline at 0.3 mM induced more proliferation. Although proline at 0.1 mM was less favorable, it yielded better proliferation and propagation rates in comparison with the proline-free medium. The longest shoots were produced by 0.1 mM proline, while the shortest ones grew on the medium with 0.3 mM proline.
Investigation of physiological roles of strigolactones in sugar beet (Beta vulgaris L.) under in vitro tissue culture and ex vitro abiotic stress conditions
Strigolactones (SLs), as carotenoid-derived compounds and recently introduced plant hormones, have wide-ranging biological roles ranged from both shoot and root architecture, plant communication in the rhizosphere, stimulation of germination in root parasitic plants, such as Striga, Orobanche and Phelipanche species, seed germination, responses to environmental stresses, modulators of root and shoot development in response to nutrient-deficient conditions, regulation of plant defense, and stimulation of secondary growth. Since this class of plant hormones has various biological roles in the growth and development, the recognition of responsible genes, which have not been recognized yet in sugar beet (Beta vulgaris), will provide a valuable knowledge in order to select better and productive genotypes, or to be manipulated in vitro, then to breed this highly important and industrial crop species. On the other hand, environmental stresses like salinity and drought lead to a reduction in the productivity of plants due to their adverse effects on plant growth. Different phytohormones are involved in stress responses; however, the role of strigolactones (SL) in this important respect has not been elucidated yet. In addition to the above-mentioned importance, deciphering SLs roles in in vitro culture of sugar beet may contribute to a greater success in its tissue culture methods and optimization of the certain stages. To know more, it is encouraged to read the Introduction section of the thesis (Chapter I). Therefore, this thesis has been defined to evaluate the roles of MAX1 gene in biosynthesis or signaling of SLs and hopefully to suggest/introduce the responsible gene for the first time in sugar beet (Chapter II). It will include estimates how SLs could contribute to alleviating the adversely affected plant conditions under abiotic stresses, which are one of the undeniable problems for plant production throughout the world (Chapter III). In addition, one part of the thesis research is to understand the SLs effects on tissue culture of sugar beet from germination to root growth (Chapter IV and V). The next purpose is investigating the relationship between SLs and auxin as a hormonal interaction (Chapter VI). In addition, it is hoped that such findings will contribute to agricultural research activities aiming at the development of plant varieties with high abiotic stress tolerance. Chapter II describes an experiment for the evaluation of a putative ortholog of MAX1 gene in sugar beet. The effects of strigolactone hormones (rac-GR24, (±)-strigol and (±)-5-deoxystrigol) and one SL inhibitor (TIS108) on the expression level of Beta vulgaris subsp. vulgaris the gene encoding Cytochrome P450 711A1 were studied. A few sets of different primer pairs for this gene and a reference gene (β-actin) were designed to estimate the expression level of the gene of interest quantitatively. Before starting the main experiments, a few sets of pre-experiments were devised to check the feasibility and reliability of the main experiment. The pre-experiments were done for checking the spraying conditions, and examining the quality and quantity of the molecular results. Since the results of the pre-experiments were highly promising, the main experiments were done. To evaluate the effects of exogenously applied SLs on the expression of the gene of interest, sugar beet cv. Serenada seeds were sown in pots. They were sprayed with four levels (0, 2.5, 5, and 7.5 µM) of the chemicals (rac-GR24, strigol, 5-deoxystrigol, and TIS108), once every two days for seven times, each time with 5 or 10 ml of an aqueous solution of the chemicals. After applying the treatments, leaf samples of the plants were collected, their whole RNA was extracted, cDNAs were synthesized, and the changes in the expression levels of the gene were investigated based on a quantitative PCR (qPCR) method using BioRad CFX connect Real-Time PCR instrument. The results obtained from the qPCR analysis indicated that exogenous application of the SL hormones decreased the expression of the gene. On the other hand, application of the SL inhibitor increased the expression of the gene of interest. Generally, the decrease and increase in the expression of the gene were respectively inversely and directly proportional to the concentrations of the applied chemicals. Chapter III describes an experiment designed to evaluate the effects of exogenous application of SLs on salinity- and drought-stress exposed sugar beet plants growing in pots. After analyzing the properties of soil, a pre-experiment was carried out to assess the response of the sugar beet plants to different levels of salinity and drought stresses. The salinity pre-experiment was performed by irrigating the potted plants with 50 ml of an aqueous solution of NaCl containing 150 mM, 200 mM, or 250 mM over ten days. The drought pre-experiment was performed by withholding irrigation for four, six, or eight days. The observed results from the pre-experiments suggested that 250 mM NaCl and withholding irrigation for six days were the proper conditions for conducting the main experiments. For the main experiments, ten-day-old seedlings were sprayed with 10 µM of the hormones (rac-GR24, St, and dSt) once or twice per day over two weeks. After collecting shoot samples, the treatment effects on morphology, catalase (CAT) enzyme activity, chlorophyll (Chl) content, and malondialdehyde (MDA) content were compared with water-sprayed control plants. Observation of the plants after running the main experiments indicated that all the hormonal treatments increased the plants' tolerance to salinity and drought stresses. In a general point of view, the hormonal treatment effects on increasing the amounts of Chl a and the Chl total (Chl T) were significant, whereas their effects on Chl b were not significant. However, the hormonal treatment effects on CAT enzyme activity in the stress-subjected plants were not statistically significant. On the other hand, the hormonal treatments generally decreased the amount of MDA in the plants. Chapter IV describes a method proposed to examine the effects of the SL hormones on the germination of sugar beet seeds. The applied hormonal treatments were rac-GR24, St, dSt, and TIS108, mixed in a half-strength MS medium, and supplemented with 10 g L−1 sucrose. The concentrations of the hormonal treatments were 0 as control, 2.5, 5 and 7.5 µM. The experiment was done in in vitro conditions. Fourteen days after sowing the seeds in the hormone-containing media, the percentage of the germinated seeds were calculated. The results indicated that the effects of the hormonal treatments on seed germination of sugar beet were not statistically significant. Chapter V describes a method to study SLs effects on in vitro tissue culture of sugar beet. In this experiment, to germinate the seeds, a half-strength MS was used for the common medium preparation. After germinating seeds in in vitro medium, the explants were subcultured on a common medium containing full-strength MS medium supplemented with 30 g L−1 sucrose, plus 0 as control, 2.5, 5 and 7.5 µM rac-GR24. One month after culture initiation, shooting pattern and root growth were observed. Treating sugar beet explants with rac-GR24 significantly decreased the number of leaves, the total length of leaves, and the total area of leaves. Similarly, rac-GR24 addition to the in vitro medium decreased the length of root in sugar beet explants. The decrease in the measured parameters was statistically significant, and it was inversely proportional to the concentration of rac-GR24 in the medium. Chapter VI describes a protocol to study the interactions of SL with auxin in in vitro conditions. After germinating seeds in the medium, the explants were subcultured on a common medium containing a full-strength MS medium supplemented with 30 g L−1 sucrose, but with varied amounts of SL and auxin hormones (rac-GR24 and IAA), and SL and auxin inhibitors (TIS108 and TIBA). Totally seven different combinations of the chemicals were prepared. Four weeks after culture initiation, the effects of the interaction of different plant growth regulators on the number of leaves, the length of leaves, the area of leaves, the length and the number of roots in sugar beet explants were observed. The effects of the applied treatments were statistically significant. The highest number of leaves and the longest leaves were observed for control, TIS108, and TIS108+IAA treatments, whereas the lowest numbers were recorded for the treatments containing TIBA+GR24, TIBA+GR24+IAA, and TIBA+GR24+IAA+TIS108. The largest leaf area was recorded for control and TIS108 treatments, while the smallest areas were produced in the media containing TIBA+GR24, TIBA+GR24+IAA, and TIBA+GR24+IAA+TIS108. The sugar beet explants growing in control conditions produced the longest roots, whereas the shortest ones were recorded for the explants treated in TIBA+GR24, TIBA+GR24+IAA, and TIBA+GR24+IAA+TIS108 supplemented media. Control, TIS108, and TIS108+IAA treatments produced the highest number of roots, but the other treatments resulted in the lowest number of roots. Chapter VII includes the conclusions driven from the experiments described in this research thesis. It seems that the studied gene (encoding Cytochrome P450 711A1) in sugar beet (B. vulgaris) is most likely an ortholog of MAX1 gene in Arabidopsis. The gene is likely involved in the biosynthesis pathway of SLs in sugar beet. In addition, SLs are effective in modifying sugar beet shoot and root architectures in in vitro conditions. SLs seem to have an inhibitory effect on the growth of the in vitro sugar beet explants. It seems that SLs can alleviate the deleterious effects of salinity and drought conditions for sugar beet plants. Moreover, it suggests that SLs can act directly and independent of auxin.
Determination of Rebaudioside-a content of in vitro propagated stevia (Stevia rebaudiana Bertoni)
Stevia rebaudiana Bertoni is a perennial shrub which produces zero-calorie diterpeneglycosides in leaves. This study involves two main parts; seed germination and best nutrient combinations for in vitro propagation. In most of our experiments, number of leaves and nodes, shoot and root length, and steviolglycoside content were studied. The seeds of stevia were germinated in vitro under different light densities ranging from 0.8 to 5 klux. Of all the light densities tested, 5.0 klux was found to be the most effective for seed germination. Another experimental results with HPLC analysis showed that the control treatment produced nearly 10 times higher Reb-A than Fe-deficient treated plants. These results suggest that Fe affects growth and Reb A content in stevia plant. According to the results, highest Reb-A was found in stock 5 solution of Murashige and Skoog (St5) deficient medium (2.72%) with the lowest stevioside content (0.86 %) as compared to the other St5 treatments. This is the first study to observe the effects of different concentration of some selected microlements on in vitro culture of stevia plants.
Tıbbı bir bitki olan yabani hindibanın (Cichorium intybus L.) in vitro çoğaltılması
An effcient protocol has been developed for the regeneration of plantlets of witloof chicory, a medicinal plant. After sterilization, chicory seeds were germinated on hormone-free MSMO basal medium (control medium). Leaf petiole and lamina explants were placed onto medium containing various concentrations of KIN and IAA alone or in combinations. Lamina explants cultured on medium containing 0.5 mg/l KIN + 0.3 mg/l IAA had the best response for the shoot formation (19.65 shoots per explant) after 3 weeks cultivation. For later experiments, lamina explants were used due to its higher rate of shoot regenaration capacity. In additon to KIN and IAA, different types of plant growth regulators (NAA, BAP and TDZ) were also tested for shoot formation. In this case, callus formation was remarkable on medium containing 0.3 mg/l NAA in combination with 0.5 mg/l BAP or KIN.Although TDZ at low concentrations in combination with 0.3 or 1.0 mg/l IAA had particular response for the shoot formation, the leaf morphology was abnormal when the well-developed chicory leaves were considered. For rooting stage, shoots were placed in auxin rich medium containing different types of auxins, (2,4-D, NAA, IAA and BAP). The best root formation per explant was found in medium containing 0.5 mg/l IAA (4.24 roots per explant). Moreover, mean values of root number, shoot and root lengths per explant were reduced at higher concentrations of IAA. For chicory, 2,4-D or NAA was not preferable for root formation, since, during root induction, 2,4-D or NAA even at low concentrations reduced both root and shoot development. The seedlings developed on medium containing IAA or IBA were placed in the pots and transferred to the growth chamber in order to be adapted to non-sterile conditions (acclimatization). Thus, in vitro propagation of witloof chicory was completed after 8- 10 weeks from initiation.
Anadolu endemik Digitalis L. türlerinin in vitro çoğaltımı ve kardiyak glikozitlerinin üretilmesi
Members of the genus Digitalis L. are medicinally and economically important plants as they contain cardiac glycosides that increase contractions of the heart and regulate heart rhythms. Several methods for high-frequency in vitro regeneration of endemic Digitalis species of Anatolia were established testing different types of basal medium and explants excised from in vitro germinated seedlings. Of those tested medium formulations, Linsmaier and Skoog (1965), supplemented with 0.5 mg/L thidiazuron (TDZ) and 0.25 mg/L indole-3-acetic acid (IAA), was selected mainly for the optimization of later tissue culture protocols of Digitalis davisina Heywood, D. cariensis Jaub. et Spach em. Werner and D. trojana Ivan. For D. lamarckii Ivan., benzyl-amino-purine (BA) and naphtylene-acetic-acid (NAA) were more productive in terms of shoot formation.As an explant source, flamingo-bill type (FBT) explants in general were more productive than other tested hypocotly, leaf or root explants. In addition to shoot regeneration, callus induction was employed in some species (D. cariensis and D. lamarckii) on LS or Murashige and Skoog medium (1962) containing various concentrations of BA and NAA. Callus cultures obtained from D. lamarckii only was able to produce multiple shoots. Regeneration frequency varied from species to species producing a range of 1 to 9 shoots per explant within 6 or 8 weeks of in vitro cultivation periods. Cardenolide profiles of leaf materials of endemic Anatolian Digitalis collected from different locations at various vegetation periods (budding, flowering or seed setting) and of tissue culture samples (leaf, complete shoots, etc.) produced in vitro were also investigated using two instrumental analysis; thin layer chromatography (TLC) and reverse phase high performance liquid chromatography (RP-HPLC). TLC analysis provides us new insights as a fingerprint pattern of the cardenolides depending on various biotic and/or abiotic factors (physiological ages, different locations or vegetation periods). HPLC analysis was substantiated in terms of quantitative analysis of cardenolides detected by TLC. In that, glucogitorosid content, a major cardenolide, found in our endemic species, was directly correlated with plant growth and development by means of exogenously added growth regulators or seasonal factors as well as the geography where they distributed.
Digitalis ferruginea L. subsp. schischkinii (Ivan.) werner bitkisinin in vitro çoğaltımı ve fenolik analizi
An efficient in vitro regeneration protocol was developed from cotyledonary leaf and hypocotyl explants of Digitalis ferruginea L. subsp. schischkinii (Ivan.) Werner. After sterilization, seeds were put on germination media, then seedlings were used as explant source. Explants were cultured on MS medium with different concentrations and combinations of plant growth regulators (TDZ, BA, KIN, TDZ+IAA, BA+IAA, KIN+IAA, TDZ+IAA+GA3). The best shoot proliferation was obtained from hypocotyl explants cultured on medium containing 2.0 mg/l TDZ and 0.5 mg/l IAA, with a mean of 11.0 shoots per hypocotyl explant. Shoot formation was decreased to 6.5 shoots when the same concentration of TDZ and IAA was combined with 0.5 mg/l GA3. 1.0 mg/l BA combined with 0.5 mg/l IAA produced a mean of 6.16 shoots per hypocotyl explant. The combinations of KIN and IAA were employed and 2.0 mg/l KIN combined with 0.5 mg/l IAA produced a mean of 7.0 shoots per hypocotyl explant, which was the highest shoot number achieved.Consequently, hypocotyl explants were more effective for shoot production than cotyledonary leaf explants. Among all PGRs, the combination of TDZ and IAA was the most effective for shoot formation. Although not being as effective as the combinations with TDZ, different concentrations of KIN and BA also produced shoots, calli and roots. After shoot formation, healthy shoots were transferred to auxin-containing medium to induce roots. IAA was the most effective medium for the rooting of the regenerated shoots. Rooted shoots were transferred to pots containing soil and their acclimitization was achieved, 70% of plantlets being survived.In the last part of the study involving total phenolic analysis, rooted shoots were dried and analyzed in accordance with absorbance values by UV-spectrophotometer. The results revealed that the total phenolic contents were not significantly different among the samples of different treatments. While the highest amount of total phenolic content was 1149.7 ?g/g dw in samples regenerated on medium containing 0.2 mg/l TDZ combined with 1.0 mg/l IAA among TDZ and IAA combinations, for KIN and IAA combinations the highest total phenolic value was 1205.8 ?g/g dw in 0.5 mg/l KIN combined with 1 mg/l IAA.
Türkiye?ye endemik Digitalis L. türlerinin kallus kültürlerinde stress faktörleri ile kardenolitler de dahil belli metabolitler arasındaki ilişkinin araştırılması
The genus Digitalis L., whose Latin name is ?finger of a glove? referring to the shape of the flowers, is a member of the Plantaginaceae. Members of the genus Digitalis are medicinally and economically important plants which contain important cardioactive compounds used to treat heart problems. The plant is well-known in Turkey for its cardenolides (glycosides) content, which is used for myocardial infarction, edema, angina, cardiac dysfunction, hyertropy and arterial hypertension. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research is to obtain relationship between different stress treatments and content of cardiotonic glycosides (digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin) as secondary metabolites of commercial valuefor the pharmaceutical industry and to determine the antioxidant metabolites against stress conditions. The effects of different stress treatments (media strength,macronutrient deficiency, oxidative, chemical, temperature and UV) on cardiotonic glycoside accumulation in D. lamarckii Ivanina, D. trojana Ivanina, D. davisiana Heywood and D. cariensis Boiss. ex Jaub. et Spach were investigated. HPLC analysis revealed that all stress conditions were significantly effective at 5% significance level according to their control groups. The predominant cardiac glycoside was lanatoside C (Lan C) followed by digitoxin, digoxigenin, gitoxigenin and digoxin. No digoxin was detected in all treatments as well as in control groups. Moreover, the effects of these stress treatments on the total phenolics, proline content and antioxidant enzymes activities were investigated. The results revealed that all stress conditions significantly increased the total phenolic and proline content. It was also concluded that CAT and SOD activities were markedly stimulated under all stress conditions in all Digitalis species, except for high temperature treatment. In contrast, high temperature stress resulted in lower CAT and SOD activities. All these results indicated that different stress treatments have induced changes in the redox state of callus cells and suggest that this alteration stimulates cardenolide formation in Digitalis callus cultures.