Dataset Information

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

ABSTRACT: BACKGROUND: Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in E. globulus microcuttings. RESULTS: By the use of two distinct algorithms, geNorm and NormFinder, we have assessed gene expression stability of eleven candidate reference genes in E. globulus: 18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI and 33380. The candidate reference genes were evaluated in microccuttings rooted in vitro, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: IDH and SAND for geNorm, and H2B and TUA for NormFinder. Both programs identified UBI and 18S as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the ARGONAUTE1 gene was evaluated in relation to the most stable candidate genes indicated by each algorithm. CONCLUSION: Our study showed that expression stability varied between putative reference genes tested in E. globulus. Based on the AGO1 relative expression profile obtained using the genes suggested by the algorithms, H2B and TUA were considered as the most suitable reference genes for expression studies in E. globulus adventitious rooting. UBI and 18S were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.

SUBMITTER: de Almeida MR

PROVIDER: S-EPMC2955024 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

de Almeida Márcia R MR Ruedell Carolina M CM Ricachenevsky Felipe K FK Sperotto Raul A RA Pasquali Giancarlo G Fett-Neto Arthur G AG

BMC molecular biology 20100920

<h4>Background</h4>Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this techniq ...[more]

PMID: 20854682

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Project description:Background: Air pollution is one of the biggest problems in the world, and it is generated by industrial production, vehicular flow and use of fossil fuels, leaving aside other important emission sources such as vegetation. The aim of this research is to quantify the emissions of natural volatile organic compounds produced by the forest species: Eucalyptus globulus L., Pinus radiata and Alnus acuminata in Riobamba, Ecuador. Methods: Identification of plant coverings in the years 2014 and 2017was performed using geographic information systems tools, complemented with the application of the Guenther model for the calculation of monoterpenes and other organic volatile compounds; thus, to analyze the relationship between meteorological variables and concentrations of volatile organic compounds and nitrogen dioxide per species. Results: Mathematical calculation of emissions in Riobamba showed that Eucalyptus globulus L. registered higher emissions in the years 2014-2017, followed by Pinus radiata and Alnus acuminata. These emissions are due to the vegetation cover covering each species. The analysis of volatile organic compounds in forest plantations in air is directly related to the emissions represented in the environment and correlated with the meteorological variables of temperature, global solar radiation and wind velocity. The proposed method manages to estimate concentrations of monoterpenes and volatile organic compounds for the two examined seasons, presenting the influence of the species introduced in this study such as Eucalyptus globulus L. and Pinus radiata, with a reduction in their emissions (less area found in the year 2017, with respect to 2014). However, the emission of Alnus acuminata can be quantified only in 2017, since in 2014 no records of this species were found. Conclusions: Volatile organic compound concentrations in the air are directly related to the emissions represented spatially and correlated with the meteorological variables of temperature, global solar radiation and wind velocity.

Project description:Adventitious root (AR) development takes place in an intricate cellular environment. Reactive oxygen species (ROS) and antioxidant defenses, triggered by wounding in cuttings, can modulate this process. A comparative assessment of biochemical and anatomical parameters at critical rooting stages in hard- (Eucalyptus globulus Labill.) and easy- (Eucalyptus grandis W.Hill ex Maiden) to-root species was carried out. Microcuttings from seedlings were inoculated in auxin-free AR induction medium and, after 96 h, transferred to AR formation medium for a period of 24 h. Samples were collected upon excision (Texc) and at the 5th day post excision (Tform). Delayed xylem development, with less lignification, was recorded in E. globulus, when compared to E. grandis, suggesting lower activity of the cambium layer, an important site for AR development. Superoxide was more densely present around the vascular cylinder at both sampled times, and in greater quantity in E. globulus than E. grandis, declining with time in the former. Hydrogen peroxide was localized primarily along cell walls, more intensely in the primary xylem and phloem, and increased significantly at Tform in E. globulus. Ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT) activities were generally higher in E. grandis and varied as a function of time in E. globulus. Soluble guaiacol peroxidase (GPRX) activity increased from Texc to Tform in both species, whereas cell wall-bound GPRX activity increased with time in E. grandis, surpassing E. globulus. Flavonoid content increased with time in E. grandis and was higher than E. globulus at Tform. Principal component analysis showed that species- and time-derived differences contributed to almost 80% of the variance. Overall, data indicate that E. grandis shows higher cambium activity and tighter modulation of redox conditions than E. globulus. These features may influence ROS-based signaling and phytohormone homeostasis of cuttings, thereby impacting on AR development. Besides being players in the realm of AR developmental differences, the specific features herein identified could become potential tools for early clone selection and AR modulation aiming at improved clonal propagation of this forest crop.

Dataset Information

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

Publications

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill.

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