Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools \u2018geNorm\u2019 and \u2018NormFinder\u2019. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). \u2018NormFinder\u2019 as well as \u2018geNorm\u2019 identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to \u2018NormFinder\u2019 COG complex component displayed the most stable expression whereas \u2018geNorm\u2019 indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum

Large impact of the apoplast on somatic embryogenesis in Cyclamen persicum offers possibilities for improved developmental control in vitro

<p>Abstract</p> <p>Background</p> <p>Clonal propagation is highly desired especially for valuable horticultural crops. The method with the potentially highest multiplication rate is regeneration via somatic embryogenesis. However, this mode of propagation is often hampered by the occurrence of developmental aberrations and non-embryogenic callus. Therefore, the developmental process of somatic embryogenesis was analysed in the ornamental crop <it>Cyclamen persicum </it>by expression profiling, comparing different developmental stages of embryogenic cell cultures, zygotic vs. somatic embryos and embryogenic vs. non-embryogenic cell cultures.</p> <p>Results</p> <p>The analysis was based on a cDNA microarray representing 1,216 transcripts and was exemplarily validated by realtime PCR. For this purpose relative transcript abundances of homologues of a putative receptor kinase, two different glutathione S-transferases (GST), a xyloglucan endotransglycosylase (XET) and a peroxidase (POX) were quantitatively measured by realtime PCR for three different comparisons. In total, 417 genes were found to be differentially expressed. Gene Ontology annotation revealed that transcripts coding for enzymes that are active in the extracellular compartment (apoplast) were significantly overrepresented in several comparisons. The expression profiling results are underpinned by thorough histological analyses of somatic and zygotic embryos.</p> <p>Conclusions</p> <p>The putative underlying physiological processes are discussed and hypotheses on improvement of the protocol for <it>in vitro </it>somatic embryogenesis in <it>Cyclamen persicum </it>are deduced. A set of physiological markers is proposed for efficient molecular control of the process of somatic embryogenesis in <it>C. persicum</it>. The general suitability of expression profiling for the development and improvement of micropropagation methods is discussed.</p

Selection of reference genes for normalization of quantitative real-time PCR in cell cultures of Cyclamen persicum

As a prerequisite for gene expression analyses in cell cultures of the ornamental crop Cyclamen persicum basic parameters for quantitative real-time polymerase chain reaction (qRT-PCR) have been established including the selection of reference genes using the software tools ‘geNorm’ and ‘NormFinder’. Five potential reference genes have been tested (elongation factor tu (Ef-Tu), putative ABC transporter ATPase, putative conserved oligomeric Golgi (COG) complex component, V-ATPase G subunit 1 and Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4)). ‘NormFinder’ as well as ‘geNorm’ identified Ef-Tu to be the least stable reference gene while the ranking of the most stable genes differed depending on the algorithm. According to ‘NormFinder’ COG complex component displayed the most stable expression whereas ‘geNorm’ indicated V-ATPase G subunit 1 and a putative ABC transporter ATPase to be the most reliable reference genes. Hence, we concluded to use a normalization factor calculated from the four reference genes V-ATPase G subunit 1, ABC transporter ATPase, Histone H3-K9 methyltransferase 4 (H3-K9-HMTase 4) and COG complex component for normalization of qRT-PCR in cell cultures of Cyclamen persicum

Gene expression of a putative glutathione S-transferase is responsive to abiotic stress in embryogenic cell cultures of Cyclamen persicum

Background: Cyclamen persicum is an economically important ornamental crop that is propagated exclusively through seeds as vegetative propagation using cuttings is not possible. Therefore a micropropagation method through somatic embryogenesis is of high interest; however the method suffers from low reliability concerning quality and quantity of the produced plantlets. A crucial step of the protocol is the removal of plant growth regulators (PGRs) that triggers embryo development. In order to get a better insight in this crucial step of the propagation process, a gene expression analysis has been set up using five different genes of glutathione S-transferases (GST) as these are known to be auxin responsive as well as stress reactive. Results: One out of the five genes of glutathione S-transferases (CpGST1) displayed a clear down-regulation 72 hrs after removal of PGRs compared to 4 hrs after, implying auxin responsiveness. However, a more detailed analysis including the time points 0, 4 and 72 hrs revealed an initial strong up-regulation after 4 hrs before it was down-regulated after 72 hrs. In comparison fold-changes of the additional four GST-genes were marginal. Comparing cultures on semisolid medium to that in suspension, transcript abundances of CpGST1 were clearly decreased in suspension culture. Conclusions: Against the initial hypothesis CpGST was not auxin responsive but stress reactive, probably especially indicating drought stress imposed on the cells upon transfer from submerged suspension culture to semisolid medium. Mechanical stress caused by shaking of suspensions cultures seemed to be less important