RESUMO
BACKGROUND: The application of ribo nucleic acids for molecular studies requires high integrity and quality of extracted total RNA samples. In addition, the need to transfer RNA samples at room temperature without special treatments such as ice and liquid nitrogen storage according to international transport laws highlights the importance of low cost alternative methods such as RNA air-drying, lyophilisation and transportable agents. In this study, the quality and quantity of air-dried RNA samples from leaf, petiole and bark tissues of different olive genotypes using several RNA extraction methods were compared with lyophilized ground leaves and RNAlater-stored tissue samples before precipitation. The quality of RNA and prepared libraries were checked by several techniques including agarose and polyacrylamide gel electrophoresis, Agilent quality control, RT-PCR amplification of housekeeping and viral genes and high throughput sequencing. RESULTS: Although RNA value varied amongst cultivars, RNA extraction with TRIzol™ Reagent in fresh extractions and samples stored in RNAlater before RNA extraction resulted in 455.26 ng/µL and 63.46 ng/µL (mean value of cultivars) as the highest RNA concentration averages, respectively. RNA samples extracted by TRIzol™ Reagents and stored for a short term at - 80 °C before air-drying showed the third highest concentration (44.87 ng/µL). The synthesized cDNAs quality for PCR amplification of housekeeping genes (Rbc 1 and Nad 5) and partial genomes of Arabis mosaic virus and Cucumber mosaic virus showed satisfactory results in RNA samples extracted by TRIzol™ Reagents despite its variation amongst cultivars. CONCLUSIONS: Considering the difficulties in the extraction of high quality and quantity RNA in olive for molecular analyses, this study demonstrated that RNA extraction method based on TRIzol™ Reagent can be considered for virobiome studies of both fresh and air-dried samples.
RESUMO
BACKGROUND: In vitro culture of olive, as an economically valuable tree, has fundamentally a genotype-dependant low micropropagation rate which needs to be improved in already established and newly released cultivars. Various plant tissue culture media, planting systems and growth factors were evaluated in two promissing Iranian olive cultivars 'Amin' and 'Meshkat' and the commercial Spanish cultivar 'Arbequina'. RESULTS: The results showed that cultivars have their specific optimal media, i.e. 'Amin' in the MS with 4 mg/L zeatin, 'Arbequina' in the OM with 1 mg/L zeatin, and 'Meshkat' in the OM and MS with 2 mg/L zeatin, which produced significantly a higher number of axillary shoots than other media. The results also indicated a significant improvement in the growth indices of 'Amin' (number of axillary shoots) when cultured using periodical mini bioreactor (PMB) in the VS medium. In comparison with VS, OM did not reveal any significant differences when both culturing systems (PMB and semi-solid media (SSM)) were used. Regarding the effect of carbon source and light intensity, mannitol and 2000 cd sr m-2 greatly enhanced 'Arbequina' growth indices (main shoot length and growth quality). The results of genetic stability of callus induced shoots (CIS) and meristem induced shoots (MIS) revealed that 2C DNA value assessed by partec flow cytometery (FCM) had 0.01, 0.03 and 0.08 pg discrepencies in 'Amin', 'Arbequina' and 'Meshkat', repectively. The Amplified Fragment Length Polymorphism (AFLP) results also indicated that the cultivars were classified regardless of the micropropagation origin (CIS or MIS), except for 'Arbequina'. The AFLP findings showed that 'Arbequina' had the highest dispersal (7-38%) in CIS and MIS, while the Iranian cultivar of 'Meshkat' (5-9%) had the highest stability. CONCLUSIONS: This study indicated the importance of in vitro growth parameters for improving the micropropagation indices of olive cultivars. It showed that optimized protocols (OM, PMB, zeatin, mannitol and 2000 cd sr m-2) co-produced larger calli resulting in indirect organogenesis. Based on FCM and AFLP analysis, it can be concluded that true-to-typeness of micropropagated olive was cultivar-dependent.
RESUMO
Recent studies suggest that dietary virgin olive oil (VOO) reduces hypoxia-reoxygenation injury in rat brain slices. We sought to extend these observations in an in vivo study of rat cerebral ischemia-reperfusion injury. Four groups, each consisting of 18 Wistar rats, were studied. One group (control) received saline, while three treatment groups received oral VOO (0.25, 0.5, and 0.75 mL/kg/day, respectively). After 30 days, blood lipid profiles were determined, before a 60-min period of middle cerebral artery occlusion (MCAO). After 24-h reperfusion, neurological deficit scores, infarct volume, brain edema, and blood brain barrier permeability were each assessed in subgroups of six animals drawn from each main group. VOO reduced the LDL/HDL ratio in doses of 0.25, 0.5, and 0.75 mL/kg/day in comparison to the control group (p < 0.05), and offered cerebroprotection from ischemia-reperfusion. For controls vs. doses of 0.25 vs. 0.5 vs. 0.75 mL/kg/day, attenuated corrected infarct volumes were 207.82 +/- 34.29 vs. 206.41 +/- 26.23 vs. 124.21 +/- 14.73 vs. 108.46 +/- 31.63 mm3; brain water content of the infarcted hemisphere was 82 +/- 0.25 vs. 81.5 +/- 0.56 vs. 80.5 +/- 0.22 vs. 80.5 +/- 0.34%; and blood brain barrier permeability of the infarcted hemisphere was 11.31 +/- 2.67 vs. 9.21 +/- 2.28 vs. 5.83 +/- 1.6 vs. 4.43 +/- 0.93 micro-g/g tissue (p < 0.05 for measures in doses 0.5 and 0.75 mL/kg/day vs. controls). Oral administration of VOO reduces infarct volume, brain edema, blood brain barrier permeability, and improves neurologic deficit scores after transient MCAO in rats.
