Expression analysis of somatic embryogenesis-related SERK, LEC1, VP1 and NiR ortologues in rye (Secale cereale L.)

The genetic basis of the regeneration process in cultured immature embryos of rye (Secale cereale L.) was analyzed. The experiments were designed to reveal differences between the in vitro culture responses of two inbred lines: L318 (a high regeneration ability) and L9 (a low potential for regeneration). The rye ortologues of plant genes previously recognized as crucial for somatic embryogenesis and morphogenesis in vitro were identified. Using oligonucleotide primers designed to conserved regions of the genes Somatic Embryogenesis Receptor-like Kinase (SERK), Leafy Cotyledon 1 (LEC1), Viviparous 1 (VP1) and NiR (encoding ferredoxin-nitrite reductase), it was possible to amplify specific homologous sequences from rye RNA by RT-PCR. The transcript levels of these genes were then measured during the in vitro culture of zygotic embryos, and the sites of expression localized. The expression profiles of these genes indicate that their function is likely to be correlated with the in vitro response of rye. In line L9, increased expression of the rye SERK ortologue was observed at most stages during the culture of immature embryos. The suppression of ScSERK expression appears to start after the induction of somatic embryogenesis and lasts up to plant regeneration. The rye ortologues of the LEC1 and VP1 genes may function in a complimentary manner and have a negative effect on the production of the embryogenic callus. The expression of the rye NiR ortologue during in vitro culture reveals its importance in the process of plant regeneration

Evaluation of locked nucleic acid and TaqMan probes for specific detection of cashew nut in processed food by real time PCR

Cashew (Anacardium occidentale) nut can trigger serious reactions in allergic patients, including anaphylaxis and death. Labelling the presence of cashew nuts in food products is mandatory and consequently, sensitive and specific analytical methods must be developed. In this study, Ana o allergen coding sequences have been sequenced in several cashew varieties. Two hydrolysis probes, locked nucleic acid (LNA) and TaqMan, have been designed and their efficiency, sensitivity, limit of detection and specificity for Ana o 1 coding-sequence detection have been compared. Reliable Real Time PCR assays to detect and quantify up to 10 ppm of cashew nuts in complex mixtures have been developed. Moreover, the influence of boiling and autoclave treatment on cashew nut detectability has been analysed by qPCR, showing both probes similar performance. This analytical method was able to detect up to 1000 ppm with good functionality in autoclave treated samples. Boiling did not affect cashew nut detectability. Both hydrolysis probes are suitable for Ana o 1 coding sequence detection. Applicability of the assay has been studied by analysing several food products, and comparing the results with those of a commercial ELISA kit