AOBPreview originally published online on January 16, 2009
Annals of Botany 2009 103(5):735-747; doi:10.1093/aob/mcn266
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Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus
1 Università della Calabria, Dipartimento di Ecologia, Via P. Bucci, I-87036 Arcavacata di Rende (CS), Italy
2 Università di Pisa, Dipartimento di Biologia delle Piante Agrarie – Sezione di Genetica, Via Matteotti 1B, I-56124 Pisa, Italy
3 Consiglio Nazionale delle Ricerche, Istituto di Biometeorologia IBIMET – Sezione di Bologna, Via Gobetti 101, I-40129 Bologna, Italy
4 Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56124 Pisa, Italy
* For correspondence. E-mail b.bitonti{at}unical.it
Received: 28 July 2008 Returned for revision: 1 October 2008 Accepted: 27 November 2008 Published electronically: 16 January 2009
Background and Aims: The clone EMB-2 of the interspecific hybrid Helianthus annuus x 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.
Methods: 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.
Conclusions: 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.
Key words: Helianthus annuus x H. tuberosus, auxin, epiphylly, LEAFY COTYLEDON1-LIKE gene, somatic embryogenesis
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