الفهرس 
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Abstract
The Madagascar periwinkle Catharanthus roseus (L.) G. Don (Apocynaceae) produces a wide range of monoterpenoid indole alkaloids (MIAs). Some of these secondary metabolites possess therapeutical value. Monomeric MIAs, ajmalicine and serpentine are used in the treatment of hypertension, while the dimeric MIAs, vincristine and vinblastine, are powerful anti-cancer drugs in widespread use in cancer chemotherapy. Cell cultures of C. roseus have long been considered to be sources of medicinally important MIAs, but have suffered -#102;-#114;-#111;-#109; their characteristically low productivity. The optimization of medium composition, including the supply of biosynthetic precursors, and genetic engineering are among the various strategies that have been followed to increase alkaloid production both in vitro and in vivo. br establishment of tissue culture system is required prior to further exploration of the biosynthetic capabilities of various cell cultures. The results of this part were as follows” br 1- the red purple variety contains the highest amount of vincristine and vinblastine, therefore, this variety was chosen. br 2- Seeds of Egyptian Catharanthus roseus (L.) G. Don. were surface sterilized under aseptic conditions of laminar flow hood, using 12 % H2O2 for 5 min. Then they were aseptically germinated on half-strength of solid basal MS medium. After 4-6 weeks of old seedling, leaves were excised and used as a source of plant materials and calli production. br 3- three explant types were used: hypocotyl (undeveloped lower stem), cotyledons, and mature leaves. Transversal leave sections proved to be the best explant resulted in production of healthy callus with good size, shape and color. br 4- Cultures were incubated in 16 light and 8 dark at 22-25°C induction. All culture media used in this study were adjusted to pH= 5.6-5.8 before solidification with 0.8% agar. br 5- Four plant hormones were used in this study, two auxins (2,4 D and NAA) and two cytokinins (kinetin and BA). Combinations of these plant hormones were tested for their effects on callus growth. qualitative and quantitative differences were observed. br 6- The best callus was obtained when the plant growth regulators were used in the recombination of 1 mg/L 2.4D + 0.1 mg/L Kin after four weeks. It was also observed that replacing 2.4D with NAA resulted in the same result, but the callus takes 6 weeks to reach the desired size and shape. br Among a number of other components in the medium nutritional factors and phytohormones such as auxins and kinetins have shown the most remarkable effects on growth and productivity of plant metabolites. br In this study, three different sucrose concentrations (40, 50, and 60 g/L) and three concentrations of benzyl adenine (0.1, 0.2 and 0.4mg/L) in addition to two concentrations of jasmonic acid (10 µM and 100 µM) were studied to determine their influence on growth and alkaloid formation in Catharanthus roseus callus cultures. . The results of this part were as follows. br 1- Treatment with 60 g/L sucrose resulted in the highest increase in the callus weight comparing to the other sucrose treatments. Among the three benzyl adenine treatments the highest increase in the callus weight was obtained after the treatment of 0.4mg/L. On the other hand, planting the callus on medium containing 100 µM of jasmonic acid resulted in the highest increase in the callus weight. br 2- Molecular genetic techniques were used for tracing gene expression of three candidate genes proved to be effective in the biosynthesis pathways of the above mentioned alkaloids, tryptophan decarboxylase (tdc), strictosidine synthase (str1) and cyp72A1. Molecular analysis indicated that the two genes tdc and cyp72A1were upregulated in the treatments of 60 g/L of sucrose, 0.4mg/L of benzyl adenine as well as the treatment of 10 µM jasmonic acid while str1 gene was downregulated in the samethree treatments. br HPLC analysis br A stock standard solution of vinblastine sulphate was prepared with dissolving 5mg in 5ml of methanol; volume of 50μL was injected into the HPLC column. The mobile phase consisted of gradient increasing of acetonitrile in water, marinating the 0.1% of triethylamine concentration constant through the whole experiment, with a flow rate of 1.0mL/min. br br 1. The HPLC chromatogram obtained for the standard vinblastine sulphate shows one major peak appears at 26.242 and two minor peaks at 15.052, 29.143 minutes. The peak appearing at 26.242 min represents the major peak and considered as the vinblastine sulphate. br 2. The HPLC chromatograms of samples derived -#102;-#114;-#111;-#109; sucrose treatments (40, 50, and 60 g/L) of C. roseus calli, demonstrated four main peaks at ~28.6, 30.3, 31.7, and 32.8 retention times comparing to only one peak at 16.6 in control sample. br 3. Regarding benzyl adenine treatments, the extracted samples -#102;-#114;-#111;-#109; the first two treatments (0.1mg/L and 0.2mg/L) demonstrated four main peaks at ~27.4, 30.66, 31.35, and 32.5 retention times but they totally absent in both 0.4mg/L treatment and the control sample. br 4. In jasmonic acid treatment, the HPLC chromatograms of 10µM and 100µM samples demonstrated four main peaks at ~ 28.1, 31.7, 32.4, and 33 retention times but they totally absent in control sample. br 5. Unfortunately, all the samples didn’t show vinblastine sulphate peak, this may attributed to the low amount of starting materials, or the low quantity of the same compound in the different tissue culture samples. The most promising point in this study, the existence of different alkaloid compounds in the extracts which need extensive chemical studies to know the type of compounds and their biological activities as anticancer agents. br Molecular analysis br In the last part of this study, real-time quantitative RT-PCR using SYBRGreen I assay was used to analyze the changes in expression of a three of Catharanthus roseus genes (Str1, tdc and cyp72A1) in response to different media additives (different concentration of sucrose, benzyl adenine and Jasmonic Acid) br 1. Primers designed for three of the target genes (Str1, tdc and cyp72A1) and the one endogenous reference gene (CrActin) produced single band of expected size. br 2. Two endogenous reference genes were evaluated. The calculated ΔCT values indicated little variation in the expression of endogenous reference gene CrActin between the treated and untreated samples, so CrActin was chosen as the endogenous reference. br 3. Of the three putative genes analyzed, cyp72A1 showed maximum folding of expression (4.2) between treated and untreated callus under BA2 treatment. This manifested the influence of BA in up-regulating this gene br 4. str1 gene under Ja2 treatment showed minimum folding (0.3) between treated and untreated callus. br 5. The remaining genes represented comparable expression in all treatments. Str1 gene was up-regulated in all treatments except 4% sucrose treatment (0.7) and as mentioned before Ja2 treatment (0.3), and about tdc gene, it was up-regulated in all treatments except 4% sucrose (0.9), while cyp72A1 gene was up-regulated in all treatments. br 6. This study has shown that differential gene expression can be detected unequivocally by real-time PCR with SYBR Green I assay. It also demonstrated the sensitivity of the assay and its ability to detect subtle changes in gene expression.