Strigolaktonlarin şeker pancarinda (Beta vulgaris L.) fizyolojik etkilerinin in vitro doku kültürü ve ex vitro abiyotik stres koşullari altinda i̇ncelenemsi

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.