Influence of growth regulators on callogenesis and somatic embryo development in date palm (Phoenix dactylifera L.) Sahelian cultivars.

This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2 mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5 mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.

In vitro conservation of oil palm somatic embryos for 20 years on a hormone-free culture medium: characteristics of the embryogenic cultures, derived plantlets and adult palms.

This study was conducted over a period of 20 years, to assess the problems involved in developing subcultures over a very long period, of oil palm (Elaeis guineensis Jacq.) somatic embryos which were maintained in vitro on a Murashige and Skoog mineral-based culture medium, without growth regulators. Analysis of the proliferation rate of the embryogenic cultures, along with the survivability of the regenerated plantlets after their transfer into soil and of the flowering of the derived adult palms has been conducted for cultures maintained in vitro during 1 to 20 years. From the ninth year of maintenance, the tissue quality of the somatic embryos gradually began to decline. However, after more than 20 years, 30% of the 20 clones tested still continued to proliferate satisfactorily on the same maintenance medium, keeping their multiplication potential intact. Even though a depressive effect of the age of the lines has been observed on the survival capacity of plants under natural conditions, it is noteworthy that among the clones originating from 20-year-old cultures only eight of them (40%) have exhibited the "mantled" floral abnormality. Different hypotheses concerning the origin of the disruptions observed on the in vitro cultures, plantlets and adult palms that occur over a very long period of in vitro conservation are discussed.

Contrasting globulin and cysteine proteinase gene expression patterns reveal fundamental developmental differences between zygotic and somatic embryos of oil palm.

Oil palm (Elaeis guineensis Jacq.) somatic embryos differ from zygotic embryos in that they accumulate only small amounts of storage proteins. We compared the balance between deposition and degradation of storage proteins during zygotic or somatic embryogenesis and germinative growth in the two types of embryos. During mid to late zygotic embryogenesis, storage proteins accumulated and globulin 7S (GLO7A) gene transcripts were detected, whereas neither protease activity nor cysteine proteinase (CPR) gene transcripts were detected. Globulin degradation occurred after 8 days of in vitro germination in zygotic embryos and was accompanied by a decrease in GLO7A transcripts. Transcripts of three cysteine proteinase genes of the papain family were detected as early as Day 2 of in vitro germination. Several proteolytically active protein bands were identified by zymography, and CPR-like proteins were detected with an antibody raised against the Vicia sativa L. cysteine proteinase CPR1. Protease activities and CPR-like proteins were observed from Day 8 onward when globulin degradation occurred. During somatic embryogenesis and subsequent germinative growth, only small amounts of storage proteins accumulated, even though GLO7A transcripts were detected. Two of the three cysteine proteinase genes were expressed throughout both somatic embryogenesis and germinative growth. Protease activities and CPR-like protein species were detected in somatic embryos at several developmental stages. In contrast to zygotic embryogenesis, the accumulation of globulins and their subsequent mobilization appear to be concomitant processes during somatic embryogenesis, which could explain the low accumulation of storage proteins in somatic embryos.

Determination of flower structure in Elaeis guineensis: do palms use the same homeotic genes as other species?

AIMS: In this article a review is made of data recently obtained on the structural diversity and possible functions of MADS box genes in the determination of flower structure in the African oil palm (Elaeis guineensis). MADS box genes play a dominant role in the ABC model established to explain how floral organ identity is determined in model dicotyledon species such as Arabidopsis thaliana and Antirrhinum majus. In the monocotyledons, although there appears to be a broad general conservation of ABC gene functions, the model itself needs to be adapted in some cases, notably for certain species which produce flowers with sepals and petals of similar appearance. For the moment, ABC genes remain unstudied in a number of key monocot clades, so only a partial picture is available for the Liliopsida as a whole. The aim of this article is to summarize data recently obtained for the African oil palm Elaeis guineensis, a member of the family Arecaceae (Arecales), and to discuss their significance with respect to knowledge gained from other Angiosperm groups, particularly within the monocotyledons. SCOPE: The essential details of reproductive development in oil palm are discussed and an overview is provided of the structural and functional characterization of MADS box genes likely to play a homeotic role in flower development in this species. CONCLUSIONS: The structural and functional data provide evidence for a general conservation of the generic 'ABC' model in oil palm, rather than the 'modified ABC model' proposed for some other monocot species which produce homochlamydeous flowers (i.e. with morphologically similar organs in both perianth whorls), such as members of the Liliales. Our oil palm data therefore follow a similar pattern to those obtained for other Commelinid species in the orders Commelinales and Poales. The significance of these findings is discussed.

Functional characterization of MADS box genes involved in the determination of oil palm flower structure.

In order to study the molecular regulation of flower development in the monoecious species oil palm (Elaeis guineensis), cDNAs of 12 MADS box genes from this plant belonging to seven distinct subfamilies were previously isolated and characterized. Here studies carried out on five of these genes, each likely to be involved in floral morphogenesis: EgSQUA1 (SQUAMOSA subfamily); EgAGL2-1 (AGL2 subfamily); EgGLO2 (GLOBOSA subfamily); EgDEF1 (DEFICIENS subfamily); and EgAG2 (AGAMOUS subfamily), are described. In order to determine where and when in the plant these genes are likely to function, their spatial and temporal patterns of expression were studied during the development of male and female inflorescences, either of normal phenotype or displaying a homeotic flowering abnormality known as mantled. In parallel, the phenotypic effects of ectopically expressing these genes in transgenic Arabidopsis thaliana plants were analysed. The data suggest a broad conservation of floral homeotic gene functions between oil palm and previously described model species, although a few minor variations in the zones of activity of certain genes cannot be excluded. The data also indicate distinct molecular identities for the morphologically similar floral organs of whorls 1 and 2. They also reveal reduced expression of putative B, C/D, and E class genes in mantled flowers, which undergo a homeotic transformation comparable to B class mutants of model species.

Somaclonal variation in micropropagated oil palm. Characterization of two novel genes with enhanced expression in epigenetically abnormal cell lines and in response to auxin.

In vitro micropropagation based on somatic embryogenesis provides an efficient means to multiply selected genotypes of oil palm (Elaeis guineensis Jacq.). Despite its considerable potential, somatic embryogenesis can yield plants bearing a homeotic flowering abnormality known as mantled. Because the mantled abnormality is epigenetic, it cannot be detected with conventional structural molecular markers. Thus, to develop a means of discriminating among callus cultures carrying or lacking the mantled abnormality, we used a gene expression approach. We describe two novel oil palm genes, EgM39A and EgIAA1, both of which display increased transcript accumulation in epigenetically abnormal calli. EgIAA1 codes for an oil palm relative of the Arabidopsis thaliana (L.) Heynh. AXR3/IAA17 protein involved in early auxin response and EgM39A codes for a protein of unknown function sharing sequence similarities with asparagine synthetases. In addition to their enhanced expression in somaclonal variant callus lines, both genes displayed increased transcript accumulation in response to auxin treatment. Normal seed-derived zygotic embryos germinated in the presence of auxin accumulated increased amounts of EgIAA1 transcripts after a few hours of treatment, suggesting a role in auxin response similar to that demonstrated for IAA genes in other species. The EgM39A gene also displayed enhanced transcript accumulation in auxin-treated zygotic embryos. Although only a small increase was seen after 24 h, greater changes were observed after 15 days. Both genes show potential as early markers of clonal conformity and may help to elucidate the nature of the epigenetic changes causing the mantled abnormality.

MADS box genes in oil palm (Elaeis guineensis): patterns in the evolution of the SQUAMOSA, DEFICIENS, GLOBOSA, AGAMOUS, and SEPALLATA subfamilies.

MADS box genes code for a large family of transcription factors which regulate development in higher plants, notably flower formation. We describe here a study of members of the MADS box gene family in oil palm (Elaeis guineensis Jacq.), a representative of the family Arecaceae and order Arecales, a key group of monocotyledons which has been unreported in previous phylogenetic reconstructions of the different recognized clades of MADS box genes. In this study, 13 oil palm MADS box genes were identified and characterized. They were found to belong to five different subfamilies, namely, the previously defined SQUAMOSA, AGAMOUS, AGAMOUS-like2, DEFICIENS, and GLOBOSA groups. Genes belonging to each of these groups play a critical role in the determination of flower structure as defined by the ABCDE model. The in planta expression profiles of the oil palm MADS box genes were studied by RT-PCR and phylogenetic sequence diversity within individual subfamilies was investigated by comparing their deduced protein sequences with those of other angiosperms. Most of the oil palm sequences studied were observed to group with distinct supported clades within their subfamily. Some unexpected groupings were observed between monocot sequences (including oil palm ones) of non-Poaceae origin, probably illustrating the importance of obtaining adequate taxon representation in monocot molecular phylogenies.

Analysis of expressed sequence tags from oil palm (Elaeis guineensis).

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm.

Reproductive developmental complexity in the African oil palm (Elaeis guineensis, Arecaceae).

Species of the palm family (Arecaceae) are remarkably diverse in their inflorescence and floral morphologies, which make them a particularly interesting group for studies of reproductive development and its evolution. Using light and scanning electron microscopy, we describe inflorescence and flower development in the African oil palm Elaeis guineensis from the initiation of the inflorescence meristem to flower maturity. In mature palms, the inflorescence develops over 2-3 years and is characterized by individual stages within which differentiation may be either relatively slow, as in the case of early inflorescence meristem development, or rapid, as in the case of flower organogenesis. The female inflorescence bears floral triads composed of single pistillate flowers flanked by two abortive staminate flowers, whereas the male inflorescence contains single functional staminate flowers. This suggests a possible evolutionary movement from an ancestral hermaphrodite inflorescence form containing fully functional floral triads to the situation of temporal dioecy observed at present. Wild type flowers are compared to those bearing an epigenetic homeotic abnormality, known as mantled, involving an alteration of the identity of the organs in the fertile and sterile androecium.

Characterization of a defensin gene expressed in oil palm inflorescences: induction during tissue culture and possible association with epigenetic somaclonal variation events.

From differential display studies performed on oil palm (Elaeis guineensis Jacq.) tissue cultures bearing or lacking an epigenetic homeotic flowering abnormality, known as mantled, EGAD1, a gene coding for a putative plant defensin, has been identified and characterized. In whole plants, transcripts of the EGAD1 gene were detected only in inflorescences. The closest characterized relative of the oil palm EGAD1 gene is the Petunia PPT gene, which is expressed principally in the pistil of the flower. The 77 amino acid polypeptide encoded by the EGAD1 gene displays strong similarities with a number of plant defensin proteins, which are thought to play a protective role and which have been shown in some cases to possess antifungal properties. Oil palm tissue cultures exhibit a generally strong induction of accumulation of EGAD1 transcripts, which were detected to differing extents at all stages of the tissue culture regeneration process. The 5' flanking region of the EGAD1 gene was found to contain two different types of potential cis-acting DNA element previously identified in the promoters of plant defence-related genes, which may explain the observed expression in tissue cultures. At the callus stage of the in vitro regeneration procedure, a differential accumulation of EGAD1 transcripts was observed which correlated with the presence or absence of the mantled flowering abnormality. EGAD1 gene expression may therefore be a marker of epigenetic somaclonal variation events.

Regulation of 7S globulin gene expression in zygotic and somatic embryos of oil palm.

We describe here the characterization and expression analysis of the oil palm GLO7A gene encoding a 7S globulin protein. Previous work carried out in our laboratory showed that 7S globulins accumulate in the oil palm zygotic embryo mostly between the 14 and 17 weeks after pollination. To investigate further the regulation of 7S globulin gene expression in both zygotic and somatic embryos of oil palm, we isolated a cDNA clone, GLO7A, for use as a probe in northern hybridization studies. The nucleotide sequence of the GLO7A cDNA reveals that it encodes a polypeptide of 572 amino acids (66 kDa) sharing significant sequence similarities with various vicilin-like proteins of both dicotyledonous and monocotyledonous plants. Northern hybridization analysis shows that 7S globulin mRNA accumulation in zygotic embryos is temporally regulated with a profile essentially the same as that observed at the protein level. In somatic embryos, 7S globulin proteins were found to occur in amounts approximately 80 times lower than those in zygotic embryos. This lack of 7S globulin protein accumulation in somatic embryos is mirrored by a low accumulation of the GLO7A mRNA. The in vitro production of 7S globulins (and more generally salt-soluble proteins) is improved by the addition to the culture medium of arginine, sucrose and ABA, the effects of these 3 components being additive. To investigate further the action of the 3 molecules of interest, we performed parallel studies on mRNA and protein abundance. Our studies of transcript accumulation suggest that ABA and sucrose act directly on mRNA synthesis or stability; however, it appears that there are also translational or post-translational regulatory factors which act to limit protein accumulation in somatic embryos. The GLO7A gene promoter was cloned and sequenced to assess whether GLO7A gene expression might be modulated by cis-acting promoter elements related to those found in other plants. Two motifs resembling ABREs (ABA-responsive elements) and one motif resembling a seed-specific promoter element were identified within the 5' flanking sequence.