Jasmonic acid induces changes in growth and polypeptide composition of fern gametophytes

We studied morphology of gametophytes of the fern Platycerium bifurcation 20 to 45 days after spore sowing. In addition, we examined the effects of 0.01, 0.1, 1, 10 or 100 μM jasmonic acid (JA) on their growth and polypeptide composition. Gametophytes cultured for 20 days on modified Knop’s medium were oblong to round in outline. 3-5 rhizoids appeared mainly on the basal cell and 2-3 unicellular hairs formed on the margins. After 30 days in culture, slightly heart-shaped gametophytes produced numerous rhizoids and hairs. Gametophytes cultured for 45 days were cordate and the first antheridia were observed on the ventral side. Hairs, frequently branched, appeared on gametophyte surface. The effect of JA on the growth of gametophytes was age-dependent. After 20 and 30 days in culture, JA had no pronounced effect in comparison to the control (medium without JA), while after 40 days at concentrations exceeding 0.1 μM JA growth of gametophytes was inhibited. Analysis of soluble proteins from 2 month old JA-treated gametophytes revealed some alterations in polypeptide patterns when compared to the control. The most marked was an increase in a 92-93 kDa polypeptide band at 10 and 100 μM JA in the medium

Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms

<p>Abstract</p> <p>Background</p> <p>The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan^®and SYBR^®Green real-time PCR chemistries. In our study four alternative chemistries: Lux™, Plexor™, Cycling Probe Technology and LNA^®were extensively evaluated and compared using TaqMan^®chemistry as a reference system.</p> <p>Results</p> <p>Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics.</p> <p>Conclusion</p> <p>Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA^®technology is an alternative to TaqMan^®when designing assays for quantitative analysis. Because LNA^®probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan^®probe is difficult or even impossible due to sequence characteristics. Plexor™ on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.</p

Jasmonic acid induces changes in growth and polypeptide composition of fern gametophytes

We studied morphology of gametophytes of the fern Platycerium bifurcation 20 to 45 days after spore sowing. In addition, we examined the effects of 0.01, 0.1, 1, 10 or 100 μM jasmonic acid (JA) on their growth and polypeptide composition. Gametophytes cultured for 20 days on modified Knop’s medium were oblong to round in outline. 3-5 rhizoids appeared mainly on the basal cell and 2-3 unicellular hairs formed on the margins. After 30 days in culture, slightly heart-shaped gametophytes produced numerous rhizoids and hairs. Gametophytes cultured for 45 days were cordate and the first antheridia were observed on the ventral side. Hairs, frequently branched, appeared on gametophyte surface. The effect of JA on the growth of gametophytes was age-dependent. After 20 and 30 days in culture, JA had no pronounced effect in comparison to the control (medium without JA), while after 40 days at concentrations exceeding 0.1 μM JA growth of gametophytes was inhibited. Analysis of soluble proteins from 2 month old JA-treated gametophytes revealed some alterations in polypeptide patterns when compared to the control. The most marked was an increase in a 92-93 kDa polypeptide band at 10 and 100 μM JA in the medium

Piretro (Tanacetum cinerariifolium) del Nord Adriatico come fonte potenziale di insetticidi naturali

Pyrethrum Tanacetum cinerariifolium (Trevir.) Schulty-Bip., a species native to the Eastern Adriatic coastal mountains and islands, is a plant widely used in the production of natural insecticides, pyrethrins. The biosynthetic potential of Pyrethrum from two different locations in the Northern Adriatic for pyrethrin production was determined. In all the samples obtained, all 6 pyrethrins were detected, as measured by HPLC. The highest pyrethrin content was detected in the flower heads, which contained, on average, 1.2 % pyrethrins of dry weight. The pyrethrin content of flower heads from the Northern Adriatic populations is comparable with the content levels in conventional production of Pyrethrum, but not as high as the content levels for highly productive Pyrethrum clones from countries currently producing pyrethrins