Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes

Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported.</p> <p>Results</p> <p>Seven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (<it>NADHD</it>), actin (<it>ACT</it>), β-tubulin (<it>TUB</it>), glyceraldehyde-3-phosphate-dehydrogenase (<it>GADPH</it>), histone H3 (<it>H3</it>), elongation factor 1-alpha (<it>EF</it>) and 18S rRNA (<it>rRNA</it>) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. <it>ACT</it>, <it>EF</it>, and <it>rRNA </it>were the most stable genes as identified by the three different analysis methods. In addition, the use of <it>ACT, EF </it>and <it>GAPDH </it>as reference genes was illustrated by analysing 1-<it>FEHII </it>(<it>FEHII</it>) expression in chicory root and leaf tissues. These analyses revealed the biological variation in <it>FEHII </it>transcript expression among the tissues studied, and between individual plants.</p> <p>Conclusions</p> <p>geNorm, NormFinder, and BestKeeper analyses indicated that <it>ACT</it>, <it>EF </it>and <it>rRNA </it>had the highest expression stability across leaf and root tissues, while <it>GAPDH </it>and <it>NADHD </it>showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as <it>ACT </it>and <it>EF </it>allows accurate normalisation of gene expression in chicory leaf and root tissues.</p

An innovative integrated approach based on DNA walking to identify unauthorized GMOs

&lt;p&gt;In the next coming years, the frequency of unauthorized genetically modified organisms (GMOs) being present in the European food/feed chain will increase significantly. Rice already constitutes a challenge for laboratories developing methods to detect unauthorized GMOs. Indeed, in 2012, several genetic modified rices were detected in products imported from Asia, mainly from China. Therefore, we have developed a strategy to identify unauthorized GMOs containing a pCAMBIA family vector, frequently present in transgenic plants. The presented integrated approach is performed in two main successive steps on Bt rice grains. First, the potential presence of unauthorized GMOs is assessed by the qPCR SYBR&amp;reg;Green technology targeting the terminator 35S (t35S) pCAMBIA element, which allows discriminating pCAMBIA family vectors. Second, its presence is confirmed via the characterization of the junction between the transgenic cassette and the rice genome. To this end, a DNA walking strategy is applied using a first reverse primer followed by two semi-nested PCR rounds using primers that are each time nested to the previous reverse primer. The sensitivity of the method was assessed. This innovative approach allows to rapidly identifying the transgene flanking region and presents the advantage to be easily implementable in GMO routine analysis by the enforcement laboratories.&lt;/p&gt;</p

Differential expression of fructan 1-exohydrolase genes involved in inulin biodegradation in chicory (Cichorium intybus) cultivars

Fructan 1-exohydrolase (1-FEH; FEH) enzymes are involved in inulin degradation in the roots of chicory. rehgiH FEH expression in cold temperatures can decrease the quality and the quantity of the inulin. This is the case at the end of the growing season and during cold storage. Little is known at molecular level whether the expression levels of fructan 1-exohydrolase genes vary among chicory cultivars, especially during cold storage of roots. Real-time RT-PCR is the most sensitive method for the detection of low abundance mRNAs. Quantitative real time PCR (RT-qPCR) analysis of the FEH mRNAs (including FEHI and FEHII) in cold stored roots of three chicory cultivars was studied. RT-qPCR results showed variable levels of FEHII and FEHI expression in different hgitonehis of the chicory cultivars studied. Moreover, longer cold storage of roots significantly induced expression levels of FEHII in some chicory phenotypes. These results could be used in breeding programmes to increase inulin content in root chicory by selecting genotypes based on their FEH expression profiles

Integrated DNA walking system to characterize a broad spectrum of GMOs in food/feed matrices

Background: In order to provide a system fully integrated with qPCR screening, usually used in GMO routine analysis, as well as being able to detect, characterize and identify a broad spectrum of GMOs in food/feed matrices, two bidirectional DNA walking methods targeting p35S or tNOS, the most common transgenic elements found in GM crops, were developed. These newly developed DNA walking methods are completing the previously implemented DNA walking method targeting the t35S pCAMBIA element. Results: First, the newly developed DNA walking methods, anchored on the sequences used for the p35S or tNOS qPCR screening, were tested on Bt rice that contains these two transgenic elements. Second, the methods were assessed on a maize sample containing a low amount of the GM MON863 event, representing a more complex matrix in terms of genome size and sensitivity. Finally, to illustrate its applicability in GMO routine analysis by enforcement laboratories, the entire workflow of the integrated strategy, including qPCR screening to detect the potential presence of GMOs and the subsequent DNA walking methods to characterize and identify the detected GMOs, was applied on a GeMMA Scheme Proficiency Test matrix. Via the characterization of the transgene flanking region between the transgenic cassette and the plant genome as well as of a part of the transgenic cassette, the presence of GMOs was properly confirmed or infirmed in all tested samples. Conclusion: Due to their simple procedure and their short time-frame to get results, the developed DNA walking methods proposed here can be easily implemented in GMO routine analysis by the enforcement laboratories. In providing crucial information about the transgene flanking regions and/or the transgenic cassettes, this DNA walking strategy is a key molecular tool to prove the presence of GMOs in any given food/feed matrix

Development and validation of a standardized double-blind, placebo-controlled food challenge matrix for raw hazelnuts

Background: Double-blind, placebo-controlled food challenge (DBPCFC) is considered the gold standard for food allergy diagnosis. However, this test is rarely performed routinely in clinical practice because of various practical issues, e.g. the lack of a standardized matrix preparation. The aim of this study was to develop and validate a convenient DBPCFC matrix, that can easily be implemented in daily clinical practice. The focus of this study was the blinding of hazelnuts, whereby the hazelnuts retained as much as possible their allergenicity and could be mixed homogenously in low-doses to the matrices. Methods: A basophil-activation test (BAT), microbial tests and an LC-MS/MS test were performed to assess respectively the allergenicity of the used hazelnuts, the microbial stability of the novel developed matrices and the homogeneity of the hazelnuts in the matrices. A sensory test was conducted to validate the blinding of the hazelnuts in the matrices. A pilot DBPCFC study included eight patients as proof of concept. Results: The BAT-test gave the first insights concerning the retained allergenicity of the hazelnuts. The microbial safety could be assured after 12 months of storage. Sufficient masking was assessed by several sensory tests. Homogeneous hazelnut distribution could be achieved for the different hazelnut concentrations. The DBPCFC's results showed diverse allergic responders (from no reactions to distinct objective symptoms). Conclusion: A novel stable and validated DBPCFC matrix using raw hazelnuts has been developed that allows easy preparation in a standardized way for convenient use in daily clinical practice

Regeneration ability and genetic transformation of root type chicory (Cichorium intybus var. sativum)

To develop an efficient protocol for shoot regeneration of root chicory (Cichorium intybus var. sativum), some factors, including different concentrations of plant growth regulators in Murashige and Skoog (MS) medium, type of explants and genotypes were evaluated. Initiation of callusing were best achieved in MS medium supplemented with 1-naphthaleneacetic acid (NAA) (0.1 mg l-1) plus 6-Benzylaminopurine (6-BAP) (1 mg l-1), indole-3-acetic acid (IAA) (0.01 mg l-1) plus 6-BAP (1.0 mg l-1), and IAA (0.5 mg l-1) plus (0.5 mg l-1) 6-BAP combinations on leaf and cotyledon explants. Explant-derived calli were able to produce multiple adventitious shoots in MS medium containing IAA (0.5 mg l-1) plus 6-BAP (0.5 mg l-1). MS medium containing indole-3-butylric acid IBA (1 mgl-1) efficiently induced rooting on elongated shoots. Various responses to the number of generated shoots were observed when regeneration abilities of different chicory cultivars were examined. Among root and “Witloof” cultivars, ‘Melci’ and ‘Hera’ belong to the root cultivars and exhibited higher shoot regeneration ability. Using the optimized regeneration method, genetic transformation of ‘Melci’ with Agrobacterium tumefaciens strain C58C1 RifR (pGV2260) (pTJK136) was successfully carried out. Histochemical GUS assay, polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis of putative transformed plants confirmed successful integration of the T-DNA into the chicory genome. Expression of the neomycine phosphotransferase (NPTII) in the regenerated plants was also shown by well-developed roots on root inducing medium containing 100 mg l-1 kanamycin. This simple, efficient and reproducible protocol could be useful for inducing somaclonal variation and genetic modification of root chicory cultivars to broaden genetic variation and transferring of important genes.Keywords: Chicory, regeneration, transformation, 1-Naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), 6-Benzylaminopurine (6-BAP

Current and new approaches in GMO detection: challenges and solutions

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market as well as to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owed to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g. Loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g. PCR capillary gel electrophoresis, microarray and Luminex®), more accurate quantification of GM targets (e.g. digital PCR) or characterization of partially known (e.g. DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g. NGS) GMO. The benefits and drawbacks of these methods are discussed in this review

Development of an LC-MS/MS method for the detection of traces of peanut allergens in chili pepper

The recent detection of nuts (including peanut) in spices across the globe has led to enormous recalls of several spices and food products in the last two years. The lack of validated detection methods specific for spices makes it difficult to assess allergen presence at trace levels. Because of the urgent need for confirmation of possible peanut presence in chili peppers, an LC-MS/MS method was optimized and developed for this particular food matrix. Although several studies optimized LC-MS detection strategies specific for peanuts, the presence of complex components in the spices (e.g., phenolic components) makes method optimization and validation necessarily. Focus was laid on validation of the method with real incurred chili peppers (whereby a known amount of peanut is added) at low concentrations, to deal with possible matrix interferences. LC-MS/MS proves to be a good alternative to the currently most applied methods (ELISA and RT-PCR) and can be used as a complementary method of analysis when results are unclear. Peanut marker peptides were selected based on their abundancy in digested incurred chili peppers. The limit of detection was determined to be 24 ppm (mg peanut/kg), a level whereby the risk for potential allergic reactions is zero, considering the typical portion size of spices. The chili pepper powder under investigation proved to contain low levels of peanuts after LC-MS/MS, ELISA, and RT-PCR testing