Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells

M. Domoki, J. Györgyey, J. Bíró, T. P. Pasternak, A. Zvara, S. Bottka, L. Puskás, D. Dudits, A. Fehér

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Abstract

Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

Original languageEnglish
Pages (from-to)543-551
Number of pages9
JournalBBA - Gene Structure and Expression
Volume1759
Issue number11-12
DOIs
Publication statusPublished - Nov 2006

Fingerprint

2,4-Dichlorophenoxyacetic Acid
Medicago sativa
Protoplasts
Mental Competency
Genes
Embryonic Development
Gene Expression
Gene expression
Embryonic Structures
Cell Division
Proteins
Cells
Monomeric GTP-Binding Proteins
Nucleosomes
Ribosomal Proteins
Transferases
Cell culture
Functional groups
Culture Media
Real-Time Polymerase Chain Reaction

Keywords

  • 2,4-dichlorophenoxyacetic acid
  • cDNA subtraction
  • Gene expression
  • Medicago sativa L.
  • Somatic embryogenesis

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology
  • Biophysics

Cite this

@article{fd293e1ccd464d558fda698da43c3123,
title = "Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells",
abstract = "Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.",
keywords = "2,4-dichlorophenoxyacetic acid, cDNA subtraction, Gene expression, Medicago sativa L., Somatic embryogenesis",
author = "M. Domoki and J. Gy{\"o}rgyey and J. B{\'i}r{\'o} and Pasternak, {T. P.} and A. Zvara and S. Bottka and L. Pusk{\'a}s and D. Dudits and A. Feh{\'e}r",
year = "2006",
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language = "English",
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pages = "543--551",
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TY - JOUR

T1 - Identification and characterization of genes associated with the induction of embryogenic competence in leaf-protoplast-derived alfalfa cells

AU - Domoki, M.

AU - Györgyey, J.

AU - Bíró, J.

AU - Pasternak, T. P.

AU - Zvara, A.

AU - Bottka, S.

AU - Puskás, L.

AU - Dudits, D.

AU - Fehér, A.

PY - 2006/11

Y1 - 2006/11

N2 - Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

AB - Alfalfa leaf protoplast-derived cells can develop into somatic embryos depending on the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D) in the initial culture medium. In order to reveal gene expression changes during the establishment of embryogenic competence, we compared the cell types developed in the presence of 1 and 10 μM 2,4-D, respectively, at the time of their first cell divisions (fourth day of culture) using a PCR-based cDNA subtraction approach. Although the subtraction efficiency was relatively low, applying an additional differential screening step allowed the identification of 38 10 μM 2,4-D up-regulated transcripts. The corresponding genes/proteins were annotated and representatives of various functional groups were selected for more detailed gene expression analysis. Real-time quantitative PCR (RT-QPCR) analysis was used to determine relative expression of the selected genes in 2,4-D-treated leaves as well as during the whole process of somatic embryogenesis. Gene expression patterns confirmed 2,4-D inducibility for all but one of the 11 investigated genes as well as for the positive control leafy cotyledon1 (MsLEC1) gene. The characterized genes exhibited differential expression patterns during the early induction phase and the late embryo differentiation phase of somatic embryogenesis. Genes coding for a GST-transferase, a PR10 pathogenesis-related protein, a cell division-related ribosomal (S3a) protein, an ARF-type small GTPase and the nucleosome assembly factor family SET protein exhibited higher relative expression not only during the induction of somatic embryogenesis but at the time of somatic embryo differentiation as well. This may indicate that the expression of these genes is associated with developmental transitions (differentiation as well as de-differentiation) during the process of somatic embryogenesis.

KW - 2,4-dichlorophenoxyacetic acid

KW - cDNA subtraction

KW - Gene expression

KW - Medicago sativa L.

KW - Somatic embryogenesis

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U2 - 10.1016/j.bbaexp.2006.11.005

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M3 - Article

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EP - 551

JO - Biochimica et Biophysica Acta - Gene Structure and Expression

JF - Biochimica et Biophysica Acta - Gene Structure and Expression

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