Effect of sucrose concentrations on Stevia rebaudiana Bertoni tissue culture and gene expression.

Stevia rebaudiana (Bert.) Bertoni is known as sweet plant which it contains a high level of steviol glycosides in the leaves.  This plant has been used from centuries ago as a sweetener for tea. One of the most important steviol glycosides is stevioside that is attractive for diabetic persons. Tissue culture is the only rapid process for the mass propagation of stevia. One of the most important factors in the medium is sucrose that is a necessary for plant growth. In the present study, we use nodal segments of the stem as explants in mediums with different sucrose concentration (50 mM, 100mM and 150mM). Several morphological traits were measured in a 28 day period. Results analysis showed a significant variation between treatments. The highest growth rate, rooting and leaf production was obtained in medium with 100mM sucrose. The correlation between measured traits was significant at the 0.01 level. To investigation of UGT74G1, UGT76G1, UGT85C2 and KS genes expression that are involved in the synthesis of SGs, RT- PCR was done with the housekeeping gene of as internal control. There were significant differences between all media. The results showed thatsucrose 100 mM containing media was more desirable than others for expression of UGT76G1 and UGT85C2 genes. Whereas, the best medium for expression of UGT74G1 was sucrose 150 mM and sucrose 50 mM for KS gene. Totally, it seems that sucrose at a concentration of 100 mMprovides the best condition for stevia growth and steviol glycosides production.

Effect of salinity on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

Stevia rebaudiana Bertoni is a famous medicinal plant for its low calorific value compounds which are named steviol glycosides (SGs) and they are 150-300 times sweeter than sugar. Among various SGs, stevioside and rebaudioside A considered to be the main sweetening compounds.  Soil salinity is one of the most essential stress in the world. Salinity affects the survival and yield of crops. In current study the effects of salinity and osmotic stress caused by different concentration of NaCl (0, 20, 40, 60 and 80 mM) on morphological traits, genes expressionand amount of both stevioside and rebaudioside Aunder in vitro conditions has been investigated. The morphological traits such as bud numbers, root numbers, shoot length (after 15 and 30 days) were evaluated. With increasing salinity, the values of all studied morphological traits decreased. To investigation of UGT74G1 and UGT76G1 genes expression that are involved in the synthesis of SGs, RT-PCR was done and there were significant differences between all media. The highest expression of both genes was observed in plantlets grown on MS media (with NaCl-free). Also, the lowest amounts of gene expression of the both genes were seen in MS+ 60 mM NaCl. Based on HPLC results, the highest amount of both stevioside and rebaudioside A were observed in plantlets grown in MS media (with NaCl-free). Finally, it can be concluded that stevia can survive under salt stress, but it has the best performance in the lower salinity.

Effect of KH2PO4 on gene expression, morphological and biochemical characteristics of stevia rebaudiana Bertoni under in vitro conditions.

Stevia rebaudiana is one of the most important biologically sourced and low-calorie sweeteners Bertoni that has a lot of steviol glycosides. Tissue culture is the best for propagation of stevia and micro nutrients can affect both morphological traits and steviol glycosides production. Therefore, the effect of different concentrations of KH2PO4on stevia growth factors and gene expression had been studied by tissue culture methods, RT-PCR and HPLC. According the results, bud numbers had increased significantly in MS + 0.034 mMKH2PO4 media and the highest measured length was seen in plants grown under MS + 0.034 mM KH2PO4 treatment. Also, the highest growth rate (1.396 mm/d) was observed in MS + 0.034 mMKH2PO4.The best concentration of KH2PO4 for expression of UGT74G1 was 0.00425mMand the best one for UGT76G1 expression was 0.017mM. Interestingly, the best media for both stevioside and rebaudioside A accumulation was 0.017mM KH2PO4containing media. There was positive correlation between the best media for gene expression and the best one for steviol glycosides production.

Comparison of two DNA extraction protocols from leave samples of Cotinus coggygria, Citrus sinensis and Genus juglans.

High quality DNA is essential for molecular research. Secondary metabolites can affect the quantity and quality DNA. In current research two DNA isolation methods including CTAB and Delaporta (protocols 1 & 2 respectively) were applied in three leave samples from Cotinus coggygria, Citrus sinensis and Genus juglans that their leaves are rich of secondary metabolites. We successfully isolated DNA from C. coggygria, C. sinensis and Genus Juglans using the two protocols described above. Good quality DNA was isolated from C. coggygria, C. sinensis and Genus Juglans using protocol 1, while protocol 2 failed to produce usable DNA from these sources. The highest amount of DNA (1.3-1.6) was obtained from them using protocol 1. As we discovered, procedure 1 may work better for plants with secondary metabolites.

Septal cholinergic deafferentation of the dentate gyrus results in a loss of a subset of neuropeptide Y somata and an increase in synaptic area on remaining neuropeptide Y dendrites.

Removal of cholinergic septal inputs using the immunotoxin 192 IgG-saporin reduces the number of interneurons containing neuropeptide Y (NPY)-immunoreactivity in the rat dentate gyrus by approximately 30% [Milner et al., J. Comp. Neurol. 386 (1997) 48-59]. The goal of the present study was to determine if NPY-containing neurons that survive deafferentation have any distinguishing morphological and/or microenvironmental features. For this, 2 or 24 weeks after intracerebroventricular injections of 192 IgG-saporin, NPY-immunolabeled neurons in the hilus of the dentate gyrus were examined by electron microscopy. Neither the size nor morphological traits of NPY-labeled perikaryal or dendritic profiles from lesioned compared to control rats at either time-point differed significantly. However, at both time-points, NPY-containing somatal profiles from immunolesioned rats compared to controls had a reduced percentage of their plasmalemmal surface apposed to unmyelinated axon profiles and an increased percentage of their surface occupied by astrocytic profiles. At the 24 week time-point, these differences were statistically significant. The primary contributing factor for these changes was the absence of a subgroup of NPY-labeled somatal profiles in lesioned rats compared to controls which was: (a) distinguished by frequent appositions of unmyelinated axons (from 15 to 35%) to the plasmalemmal surface; and (b) located primarily in the central hilar region. Unlike NPY-containing somata, changes associated with NPY-labeled dendritic profiles were exclusively related to associated presynaptic profiles at the 24 week time-point. In lesioned rats compared to controls at this time-point, NPY-containing dendritic profiles had a concurrent increase in the percentage of the plasmalemmal surface occupied by active zones and the size of terminals contacting them. The present results combined with those of our earlier study suggest that septal cholinergic deafferentation results in: (a) the loss of a distinct subpopulation of hippocampal NPY-containing neurons; and (b) an increase in total active zone area suggesting a strengthening of synaptic connections to the surviving population of NPY-containing neurons in the long term.