The CDKA gene is linked to the cellular control. This gene was isolated from coconut palm (Cocos nucifera L) and a detailed expression analysis was done during somatic embryogenesis. Analysis of the deduced amino acid sequence showed the most important residues to be conserved. The highest homology was with Picea abies (96% similarity). Expression of the putative CnCDKA gene steadily increased during embryogenic callus formation phase when embryogenic competence is attained. In situ hybridization specified the localization of the transcripts as being mainly in a few cell layers within the meristematic centres in embryogenic calli at 90 day cultures. Analysis of CnCDKA expression at different somatic embryo formation stages showed that the expression was decreased progressively with the lowest expression in germinated somatic embryos. (Résumé d'auteur

The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 muM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem.

In our coconut laboratory micropropagation has been the subject of research for nearly three decades, as this plant species is highly recalcitrant for in vitro regeneration and so far only achieved through somatic embryogenesis as the sole path for coconut regeneration. Of all the explants tested, plumules have proved to be the most responsive and the process efficiency has been improved by indirect embryogenesis and thereafter secondary embryogenesis and callus multiplication, this strategy is currently applied in floral explants. Two different approaches have been used to find ways to have a more efficient protocol. The first one, a direct and practical method, included plant hormones and activated charcoal. On the other hand, the indirect approach consisted in basic studies on: morphohistological development, biochemical and physiological aspects such as uptake of exogenous auxin, levels of endogenous auxin; shoot apical meristem formation and maintenance (KNOX gene family); the occurrence and expression of genes related to the cell cycle control (Cyclin-Dependent Kinase), and somatic embryogenesis (Somatic Embryogenesis-Related Kinase); and the establishment of a transformation protocol. A better understanding of the somatic embryogenesis of coconut was achieved by these approaches. This way, in the short term there is no doubt that we will have mass propagation options based not only in plumule explants but also on rachillae, unfertilized ovary, and leaf explants.

Previous reports have demonstrated that gibberellins promote seed germination, including in vitro germination of zygotic embryos of coconut. Genes that control the cell cycle seem to play important roles during germination. In this study, complete full-length sequences of two E2F-like genes, CnE2F1 and CnE2F2, were obtained. Deduced amino acid sequences of both genes showed conserved domains characteristic of E2F genes. Both genes showed high homology with Arabidopsis thaliana E2Fa and E2Fb, which are transcriptional activators. The expression profiles of CnE2F genes were evaluated in in vitro zygotic embryos with or without gibberellic acid. In control embryos, expression of CnE2F1 showed a peak at day 2, and, gradually this dropped to similar levels as those at day 0. Gibberellic acid (GA3) treatment promoted expression at day 2, higher than that of the control. For CnE2F2, peak expression was detected at day 8 of culture. However, with GA treatment, CnE2F2 expression profile increased by day 2. The expression profile of a cyclin-dependent kinase (CnCDKA), another gene related to the cell cycle control, was also studied. In control embryos, expression of this gene showed two peaks at days 2 and 8, compared with day 0. A different profile was shown with GA treatment. In general, major changes in expression profiles were observed from days 2-8 when morphological changes in zygotic embryos were not yet observed.