We analysed the GMO content of corn samples by polymerase chain reaction following the appropriate optimization of the reaction. The analysis included two main steps: extraction of DNA from the sample, and detection of the GMO content by polymerase chain reaction. The polymerase chain reaction is an in vitro method to multiply chromosomatic or...cloned DNA (cDNA) sequences through the enzymatic pathway. The reaction is sensitive enough to produce DNA in sufficient amount for the analysis from a single DNA. We identified the PCR products by agarose gel electrophoresis. When optimizing the reaction, the MgCl2 concentration, reaction time and temperature have to be taken into consideration. The temperature of the anellation has to be increased until the highest specificity and yield is reached. If the temperature of the anellation is too high, the primer is linked to non-specific sites as well; in the gel visualization, more lines can be seen at one sample. If the temperature of the anellation is too high, the primer is insufficiently linked or is not linked at all (too few lines in the gel visualization). After optimization, the GMO content in the unknown sample can be determined along with the appropriate positive and negative controls.
Under the GENOMNANOTECH Debrecen Regionális Egyetemi Tudásközpont (GND RET) research program in summer 2007 in UD Centre for Agricultural Sciences and Engineering Faculty of Agricultural Science Institute of Horticulture experiments were launched to transform tobacco plant by plastid transformation technics.
Our aim was to adopt first in
Under the „Molecular farming” research program (product vaccines and substances for medical use with gene manipulated plant) in 2007 in UD Centre for Agricultural Sciences and Engineering Faculty of Agricultural Science Institute of Horticulture Department of Plant Biotechnology experiments were launched to transform tobacco plant by PAF an...tifungal protein. Our aim was to learn the transformation technics. We chose the
Nicotiana tabacum and PAF as model systems.
Our work was to express several different paf constructions in plants with nuclear and plastid transformation too. After that we confirmed the presence of paf gene in the level of DNA and RNA.
Relative gene expression levels of transgene gshI (γ-glutamylcysteine synthetase cloned from E. coli) were analyzed by qRT-PCR in two transgenic poplar (Populus × canescens) clones (11ggs and 6lgl) and wild type (WT). An extremely high expression level of transgene gshI was observed in the 6lgl clone (13.5-fold) compared to 11ggs (1.0) sample...s, which level was doubled (1.8-fold) in the DHAC (5.6-dihydro-5'-azacytidine hydrochloride) treated (at 10-4 M for 7 days) 6lgl samples but not in the 11ggs clone (0.4-fold). Contrary to this result, relative copy number of transgene gshI in the 6lgl clone was found to be less 60% less (1.0) then in the 11ggs samples (1.6). Relative expression levels of proper poplar gene gsh1-poplar showed significantly higher responsiveness to DHAC treatment than transgene gshI with the highest expression level in the untransformed (WT) poplar
clone (19.7-fold) compared to transformed 6lgl (8.7-fold) and 11ggs (2.5-fold) clones. For internal controls constitutively expressed housekeeping genes a-tubulin were applied. For data analysis, the 2−ΔΔCt method was used. DHAC applied in long-term cultures (27 days) at low concentrations (10-8 – 10-6 M) showed morphogenetic activity by initiating de novo root development of leaf discs.