BACKGROUND: A cryopreservation protocol has been established for oil palm somatic embryos (SEs), the efficiency of which must be evaluated, both in terms of regeneration and of long-term storage capacity, before its large-scale routine use.OBJECTIVE:To test the survival and recovery of 29 clones of oil palm somatic embryos cryostored for 20 years.MATERIALS AND METHODS:Clumps of SEs were pregrown for 7 days on medium containing 0.75 M sucrose, dehydrated in air-tight containers containing silica gel to moisture contents between 19-35% fresh weight, and then immersed directly in liquid nitrogen and stored in cryotanks for 20 years.RESULTS:Survival of SEs cryopreserved and rewarmed immediately displayed an average value of 19.1% for the 29 clones tested while survival of SEs rewarmed after 20 years of cryostorage was significantly higher, with an average of 33.2% for the 28 surviving clones. Out of these 28 surviving clones, three were lost due to contamination or regrowth decline, six produced only shoots and the rest proliferated.CONCLUSION:It is possible to cryostore oil palm SEs for extended periods and to regenerate proliferating cultures and plantlets from the cryopreserved material. The cryopreservation protocol established can thus be efficiently used to store oil palm germplasm and to manage large-scale production in industrial laboratories.

La graine du palmier a huile (Elaeis guineensis Jacq.) est une graine albuminee constituee d'un embryon de tres petite taille encapsule dans un albumen riche en lipides et en polysaccharides parietaux. Il est accepte que son comportement en conservation soit de type intermediaire, bien que le niveau de tolerance a la dessiccation de cette graine reste mal connu. La seule technologie pour la conservation a long terme des graines de palmier est donc la cryoconservation. De plus, cette graine possede une dormance particuliere et le traitement utilise pour la lever (80 jours a 40°C) rend la mise au point de technologies de conservation tres laborieuse chez cette espece. Dans un premier temps, nous avons developpe une technique de germination qui permet d'obtenir des resultats sur la survie des graines en seulement trois semaines. Nous avons egalement optimise les conditions de culture in vitro des embryons pour determiner leur viabilite apres traitement. Ces premiers essais sont informatifs quant a la nature de la dormance chez le palmier a huile. Ensuite, afin de connaitre la plage hydrique potentiellement utilisable en cryoconservation, le niveau de tolerance a la dessiccation des graines entieres et des embryons a ete mesure. Au sein de cette plage hydrique, l'embryon supporte sans dommage une immersion dans l'azote liquide sans pre-refroidissement controle, ce qui nous a permis de definir un premier protocole utilisable en routine pour la sauvegarde a long-terme des ressources genetiques de cette espece. En revanche, l'albumen ne supporte pas une immersion directe dans l'azote liquide. Pour obtenir la survie de la graine entiere et eviter la culture in vitro des embryons, un essai combinant quatre HR (humidite relative) de deshydratation, quatre vitesses et deux temperatures de pre-refroidissement, et deux vitesses de rechauffement a ete mis en oeuvre. Les premiers resultats de cet essai seront presentes. (Resume d'auteur)

PREMISE OF THE STUDY: The genome size of a species (C-value) is associated with growth, development and adaptation to environmental changes. Angiosperm C-values range 1200-fold and frequently vary within species, although little is known about the impacts of domestication on genome size. Genome size variation among related species of palms is of evolutionary significance because changes characterize clades and may be associated with polyploidy, transposon amplifications, deletions, or rearrangements. Further knowledge of genome size will provide crucial information needed for planning of whole genome sequencing and accurate annotations. We studied the genome size of Cocos nucifera and its variation among cultivars, and compared it to values for related palms from the Attaleinae subtribe.METHODS: Flow cytometric analysis of isolated nuclei from young palm leaves was used to estimate genome sizes of 23 coconut cultivars (Talls, Dwarfs, and hybrids) worldwide and 17 Cocoseae species. Ancestral genome size was reconstructed on a maximum likelihood phylogeny of Attaleinae from seven WRKY loci.KEY RESULTS: The coconut genome is large-averaging 5.966 pg-and shows intraspecific variation associated with domestication. Variation among Tall coconuts was significantly greater than among Dwarfs. Attaleinae genomes showed moderate size variation across genera, except polyploids Jubaeopsis caffra, Voanioala gerardii, Beccariophoenix alfredii, and Allagoptera caudescens, which had larger genomes.CONCLUSIONS: Our results contribute to the understanding of the relationship between domestication and genome size in long-lived tree crops and provide a basis for whole-genome sequencing of the coconut and other domesticated plants. Polyploidy evolved independently in two clades within Attaleinae.

In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA®) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA® bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4–6.6 cm long compared to 3.2–3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5–20 nodal segments per RITA® bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0–17.6 μM), 0.44 μM induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA® bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.

The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities.

Because of their detrimental influence on plant genome structure and gene expression, Transposable Elements (TEs) are maintained in a transcriptionally inactive state through powerful and specific epigenetic mechanisms involving DNA methylation. However, these silencing processes can be alleviated under stress conditions, leading to the more or less transient reactivation of TEs and, in some cases, to the emergence of variant phenotypes. The mantled somaclonal variation resulting from the in vitro propagation of oil palm (Elaeis guineensis Jacq.) has been correlated with the occurrence of a genome-wide hypomethylation in both in vitro- cultivated and adult tissues, as well as with alterations in gene expression. The time-dependent increase of DNA methylation in long-term in vitro cultures retaining their embryogenic ability has also been demonstrated. In order to assess the contribution of TEs to a possible loss of genomic stability during long-term in vitro propagation of embryogenic suspensions, we evaluated the background genetic instability of the cultures using AFLP and used the retrotransposon-based S-SAP technique to detect evidence of retroelement mobility. Cultures derived from normal and mantled oil palms from two different genotypes were compared in the aim of identifying phenotype-associated TE insertions. (Texte integral)

Clonal regenerants of oil palm (Elaeis guineensis Jacq) can display a detrimental variant phenotype called mantled which affects the floral morphology of adult palms thus lowering oil yields in clonal offspring. To date, early detection markers of variant cell lines have failed to be identified. To circumvent this difficulty, we aimed to investigate the genetic stability of in vitro cultivated cells during proliferation in order to both evaluate the risk of somaclonal variation and to define new tissue culture strategies. Given the epigenetic nature of the mantled phenotype and the growing knowledge on the role of transposable element in the epigenetic control of gene expression, our present work focuses on the fate of retro-elements in oil palm cell suspensions propagated during one year. To address this question, we firstly undertook a partial sequencing of the oil palm genome in order to identify the diversity of transposable elements through bioinformatics analysis. In a second step, we used a S-SAP marker analysis for the assessment of dynamic changes in retrotransposons over the in vitro proliferation period and with respect to the mantled phenotype. A massive transcriptome analysis is currently undertaken on the same material which will provide features of retrotransposon activity during oil palm tissue-culture. Such a strategy will help us understanding the implication of transposable elements in genetic and epigenetic genome instability occurring during long term oil palm tissue-culture and furthermore to secure oil palm micropropagation process by developing a biomarker system. (Resume d'auteur)

BACKGROUND The large-scale clonal propagation of oil palm (Elaeis guineensis) is being stalled by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. SCOPE In the last 15 years, the prevailing point of view on the origin of the mantled floral phenotype has evolved from a random mutation event triggered by in vitro culture to a hormone-dependent dysfunction of gene regulation processes. In this review, we retrace the history of the research on the mantled variation in the light of the parallel advances made in the understanding of plant development regulation in model systems and more specifically in the role of epigenetic mechanisms. An overview of the current state of oil palm genomic and transcriptomic resources, which are key to any comparison with model organisms, is given. We show that, while displaying original characteristics, the mantled phenotype of oil palm is morphologically, and possibly molecularly, related to MADS-box genes mutants described in model plants. We also discuss the occurrence of comparable floral phenotypes in other palm species. CONCLUSIONS Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical palm. On the basis of recent results, we propose that future efforts should concentrate on the epigenetic regulation targeting MADS-box genes and transposable elements of oil palm, since both types of sequences are most likely to be involved in the mantled variant phenotype.

Epigenetics is the study of heritable changes in gene function that occur without a change in the DNA sequence. In recent years, this field has attracted much attention as more epigenetic controls of gene activities are being discovered. Such controls involve a complex interplay of DNA methylation, histone modifications, and RNA-mediated pathways from non-coding RNAs, notably silencing RNA (siRNA) and microRNA (miRNA). In plants, although epigenetic mechanisms help to protect cells from parasitic elements, this defence can complicate the genetic engineering process through transcriptional gene silencing. Furthermore, these phenomena have economic relevance, for example, in somaclonal variation: a genetic and phenotypic variation among clonally propagated plants from a single donor genotype. The loss of phenotypic fidelity is now a major impediment to the development of large scale propagation of plants through in vitro processes such as somatic embryogenesis. Examples of aberrant phenotypes in regenerated plants include abnormal leaf structures and variant floral morphology, both organs being of paramount importance for applications in horticulture and/or agriculture. Changes in DNA methylation have been hypothesised playing a key role in the mechanism underlying tissue-culture induced changes. Indeed, studies of both global methylation levels and the methylation of specific sites show that variation in DNA methylation occurs frequently during growth in tissue culture. In vitro plant regeneration, like somatic embryogenesis, bypasses the normal developmental process of fertilisation and plant development, thus potentially resulting in the instability of epigenetic patterns. The large-scale clonal propagation of oil palm (Elaeis guineensis Jacq.) is being hampered by the occurrence of the mantled somaclonal variation. Indeed, this abnormality which presents a homeotic-like conversion of male floral organs into carpelloid structures, hampers oil production since the supernumerary female organs are either sterile or produce fruits with poor oil yields. Beyond its primary interest in the search for discriminating markers against an economically crippling phenotype, the study of the mantled abnormality also provides a unique opportunity to investigate the regulation of reproductive development in a perennial tropical plant. The present interest on food and energy security as well as the concerns raised by the possibility of climate change further stress the need for a global comprehension of how crop plants react to their fluctuating environment and how their productivity can be affected. (Texte integral)