Journal Article

Ectopic expression of <i>LEAFY COTYLEDON1-LIKE</i> gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of <i>Helianthus annuus</i> × <i>H. tuberosus</i>

A. Chiappetta, M. Fambrini, M. Petrarulo, F. Rapparini, V. Michelotti, L. Bruno, M. Greco, R. Baraldi, M. Salvini, C. Pugliesi and M. B. Bitonti

in Annals of Botany

Published on behalf of The Annals of Botany Company

Volume 103, issue 5, pages 735-747
Published in print March 2009 | ISSN: 0305-7364
Published online January 2009 | e-ISSN: 1095-8290 | DOI:
Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus × H. tuberosus

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  • Ecology and Conservation
  • Evolutionary Biology
  • Plant Sciences and Forestry


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Background and Aims

The clone EMB-2 of the interspecific hybrid Helianthus annuus × H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence.


Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography–mass spectrometry–selected ion monitoring method and an immuno-cytochemical approach.

Key Results

Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency.


In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Keywords: Helianthus annuus × H. tuberosus; auxin; epiphylly; LEAFY COTYLEDON1-LIKE gene; somatic embryogenesis

Journal Article.  7082 words.  Illustrated.

Subjects: Ecology and Conservation ; Evolutionary Biology ; Plant Sciences and Forestry

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