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Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies

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Abstract

Key message

Suitable internal control genes to normalize qPCR data from different stages of embryo development and germination were identified in two representative conifer species.

Abstract

Clonal propagation by somatic embryogenesis has a great application potentiality in conifers. Quantitative PCR (qPCR) is widely used for gene expression analysis during somatic embryogenesis and embryo germination. No single reference gene is universal, so a systematic characterization of endogenous genes for concrete conditions is fundamental for accuracy. We identified suitable internal control genes to normalize qPCR data obtained at different steps of somatic embryogenesis (embryonal mass proliferation, embryo maturation and germination) in two representative conifer species, Pinus pinaster and Picea abies. Candidate genes included endogenous genes commonly used in conifers, genes previously tested in model plants, and genes with a lower variation of the expression along embryo development according to genome-wide transcript profiling studies. Three different algorithms were used to evaluate expression stability. The geometric average of the expression values of elongation factor-1α, α-tubulin and histone 3 in P. pinaster, and elongation factor-1α, α-tubulin, adenosine kinase and CAC in P. abies were adequate for expression studies throughout somatic embryogenesis. However, improved accuracy was achieved when using other gene combinations in experiments with samples at a single developmental stage. The importance of studies selecting reference genes to use in different tissues or developmental stages within one or close species, and the instability of commonly used reference genes, is highlighted.

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Abbreviations

SE:

Somatic embryogenesis

qPCR:

Real-time quantitative PCR

C t :

Cycle threshold

ACT:

Actin

ATUB:

α-Tubulin

EF1:

Elongation factor-1α

AK:

Adenosine kinase

UBI:

Ubiquitin

HISTO3:

Histone 3

SAND:

SAND protein family

CAC:

Clathrin adaptor complex subunit

HEATS:

Heat shock protein

REDUC:

Ether reductase protein

V n :

Pairwise variation

L1L :

Leafy cotyledon 1-like

PpRab1 :

Pinus pinaster Rab GTPase

CTAB:

Hexadecyltrimethylammonium bromide

PVPP:

Polyvinylpyrrolidone

DEPC:

Diethylpyrocarbonate

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Acknowledgments

This work has been supported by Fundação para a Ciência e Tecnologia (FCT, Portugal) through projects PTDC/AGR-GPL/102877/2008, P-KBBE/AGR-GPL/0001/2009 and grant SFRH/BD/32037/2006 (MS). The authors acknowledge Prof. Sara von Arnold and Dr. David Clapham (SLU, Uppsala, Sweden) for providing the Picea embryogenic cell line used in these experiments.

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  1. Instituto de Biologia Experimental e Tecnológica (iBET), Apartado 12, 2780-901, Oeiras, Portugal

    José J. de Vega-Bartol, Raquen Raissa Santos, Marta Simões & Célia M. Miguel

  2. Instituto de Tecnologia Química e Biológica-Universidade Nova de Lisboa (ITQB-UNL), Av. da República, Qta. Do Marquês, 2780-157, Oeiras, Portugal

    José J. de Vega-Bartol, Raquen Raissa Santos, Marta Simões & Célia M. Miguel

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  1. José J. de Vega-Bartol
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  2. Raquen Raissa Santos
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  3. Marta Simões
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  4. Célia M. Miguel
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Correspondence to Célia M. Miguel.

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Communicated by J. Bennett.

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de Vega-Bartol, J.J., Santos, R.R., Simões, M. et al. Normalizing gene expression by quantitative PCR during somatic embryogenesis in two representative conifer species: Pinus pinaster and Picea abies . Plant Cell Rep 32, 715–729 (2013). https://doi.org/10.1007/s00299-013-1407-4

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