Plant Molecular Biology Reporter

, Volume 20, Issue 1, pp 19–26 | Cite as

Single-embryo RT-PCR assay to study gene expression dynamics during embryogenesis inArabidopsis thaliana



We have developed a single-embryo RT-PCR protocol for studying gene expression during plant embryogenesis. Four genes,glyceraldhyde-3-phosphate dehydrogenase (GAPC), shoot-meristemless (STM), monopteros (MP), andshaggy-like kinase etha (ASKη), fromArabidopsis thaliana were used to test the sensitivity and reliability of this method by analyzing the differential signal intensities of their RT-PCR products. The method could detect genes expressed during embryogenesis at a single-embryo level and, therefore, can be used to identify phenotypes. When in vitro, embryogenesis also is used to control the time course of zygote development exactly. The single-embryo RT-PCR protocol becomes a powerful method to survey the dynamics of specific gene expression.

Key words

Arabidopsis thaliana embryogenesis gene expression RT-PCR single embryo 



shaggy-like kinase etha


days after pollination




glyceraldhyde-3-phosphate dehydrogenase




reverse transcriptase-polymerase chain reaction


ribonucleoside vanadyl complexes




  1. Adjaye J, Bolton V, and Monk M (1999) Development expression of specific genes detected in high-quality cDNA library from single human preimplantation embryos. Gene 237: 373–83.PubMedCrossRefGoogle Scholar
  2. Dornelas MC and Kreis M (1997) Three New cDNAs Related to SGG/GSK-3 (SHAGGY/Glycogen Synthase Kinase-3) fromArabidopsis thaliana. Plant Physiol 113: 306–306.Google Scholar
  3. Faure JE, Digonnet C, and Dumas C (1994) An in vitro system for adhesion and fusion of maize gametes. Science 263: 1598–160.PubMedCrossRefGoogle Scholar
  4. Hardtke CS and Berleth T (1998) The Arabidopsis gene MONOPTEROS encodes a transcription factor mediating embryo axis formation and vascular development. EMBO J 17(5): 1405–1411.PubMedCrossRefGoogle Scholar
  5. Jacqueline KP and Lipski J (2000) Single-cell RT-PCR as a tool to study gene expression in central and peripheral autonomic neurons. Autonomic Neuroscience: Basic and Clinical 86: 1–12.CrossRefGoogle Scholar
  6. Johansen FF, Lambolez B, Audinat E, Bochet P, and Rossier J (1995) Single cell RT-PCR proceeds without the risk of genomic DNA amplification. Neurochem Int 26: 239–243.PubMedCrossRefGoogle Scholar
  7. Karrer EE, Lincoln JE, Hogenhout S, Bennett AB, Bostock RM, MarTineau B, Lucas WJ, Gilchrist DG, and Alexander D (1995) In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA library. Proc Natl Acad Sci 92: 3814–3818.PubMedCrossRefGoogle Scholar
  8. Kawasaki E, Hattori N, Miyamoto E, Yamashita T, and Inagaki C (1999) Single-cell RT-PCR demonstrates expression of voltage-dependent chloride channels (CIC-1, CIC-2 and CIC-3) in outer hair cells of rat cochlea. Brain Research 838: 166–170.PubMedCrossRefGoogle Scholar
  9. Kranz E, Bautor J, and Lorz H (1991) In vitro fertilization with isolated, single gametes by electrofusion. Sex Plant Reprod 4: 12–16.Google Scholar
  10. Long JA and Barton MK (1996) A member of the KNOTTED class of homeodomain proteins encoded by the STM gene ofArabidopsis. Nature 379(6560): 66–69.PubMedCrossRefGoogle Scholar
  11. Preuss D (1999) Chromatin silencing andArabidopsis development: a role for Polycomb protein. Plant Cell 11: 765–767.PubMedGoogle Scholar
  12. Richert J, Kranz E, Lörz H, and Dresselhaus T (1996) A transcriptase-polymerase chain reaction assay for gene expression studies at the single cell level. Plant Science 114: 93–99.CrossRefGoogle Scholar
  13. Shih MC and Goodman HM (1991) Cloning and chromosomal mapping of Nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenase fromArabidopsis thaliana. Gene 104: 133–138.PubMedCrossRefGoogle Scholar
  14. Sun M-X, Moscatelli A, Yang H-Y, and Cresti M (2000) In vitro double fertilization inNicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy. Sex Plant Reprod 12: 267–275.CrossRefGoogle Scholar

Copyright information

© International Society for Plant Molecular Biology 2002

Authors and Affiliations

  1. 1.Key Laboratory of MOE for Plant Developmental BiologyWuhan UniversityWuhanPR. China

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