Alain Rival

Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis

Elaeis guineensis Jacq.) currently hampers the scaling-up of clonal plant production. In order to investigate the relationship between the “mantled” somaclonal variant and possible alterations in genomic DNA methylation rate, two complementary approaches have been used. HPLC quantification of relative amounts of 5-methyl-deoxycytidine has shown that global methylation in leaf DNA of abnormal regenerants is 0.5–2.5% lower than in their normal counterparts (20.8% vs 22%, respectively). When comparing nodular compact calli and fast growing calli, yielding respectively 5% and 100% of “mantled” plantlets, this decrease was up to 4.5% (from 23.2 to 18.7%). An alternative method, the SssI-methylase accepting assay, based on the enzymatic saturation of CG sites with methyl groups, gave convergent results. This work demonstrates that a correlation exists between DNA hypomethylation and the “mantled” somaclonal variation in oil palm.

Epigenetic imbalance and the floral developmental abnormality of the in vitro-regenerated oil palm Elaeis guineensis

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.

Potential use of Cu2+, K+ and Na+ for the destruction of Caulerpa taxifolia: differential effects on photosynthetic parameters.

Chemical techniques were investigated in order to eradicate Caulerpa taxifolia, a green alga spreading at a remarkable rate in the Mediterranean Sea. The action of copper, potassium and sodium ions on survival rates and photosynthetic parameters was compared, in order to optimise the conditions of further in situ treatments. The lethal doses were determined and the impact of the studied cations on photosynthesis and respiration rates and PSII photochemistry was analysed from measurements of net oxygen exchanges and chlorophyll fluorescence. The Cu2+ concentrations required to obtain 100% mortality were 15 × 102 to 104 times lower than those of K+ and Na+. Respiration was slightly affected whatever the salt concentration,while photosynthesis could be totally inhibited depending on the applied treatment. Changes in the structure of the Ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO, EC: 4.1.1.39) were also detected when C. taxifolia under went cation treatments (10 mg L-1 Cu2+, 1h; 20 gL-1 K+, 3 h; 20 g L-1 Na+, 1 h). Given the high concentration and long incubation periods required with K+ and Na+ ions, these cations are not suitable to be used in situ. Our results make possible the utilisation of copper cations following technical approaches such asion-exchange textile covers, which allows a controlled release of cupric ions without dissemination in the marine environment.

Cold hardening and sucrose treatment improve cryopreservation of date palm meristems

Date palm (Phoenix dactylifera L.) cv. Khenizi caulogenic meristems were initiated from achlorophyllous leaves excised from in vitro shoot cultures and then proliferated on a specific culture medium supplemented with 70 g dm−3 sucrose. Regeneration rates obtained when using standard vitrification, droplet-vitrification, and encapsulation-vitrification protocols reached 26.7, 60.0, and 40.0 %, respectively. Only explants smaller than 3 mm in diameter were found to survive cryogenic treatments. Sucrose preculture, cold hardening and loading solution pretreatments showed significant effects on regeneration rates. Moreover, our results indicate that both sucrose preculture and cold acclimation of explants increased proline content. Cryopreservation of date palm tissue with high proliferation capacity can directly benefit large scale micropropagation projects.

Carbon metabolism in #in vitro# cultures of date palm: the role of carboxylases (PEPC and RubisCO)

While describing major trends of carbon metabolism during the initiation and expression of somatic embryogenesis in date palm (Phoenix dactylifera L., cv. Deglet Nour), we have investigated the role of two carboxylases, namely PEPC (Phosphoenolpyruvate carboxylase, EC 4.1.1.31) and RubisCO (Ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39), in embryogenic and non-embryogenic cultures. The detection of PEPC activity on polyacrylamide native gels after electrophoresis revealed the presence of 3 active isoforms in crude extracts from the embryogenic (E) callus strain, whereas only a single band was present in the non-embryogenic (NE) one. The level of PEPC specific capacity was of the same order (3.9 ± 1.2 μmol CO2 h−1 mg−1 TSP) in both types of cultures. Further changes in carboxylase (PEPC and RubisCO) activities during the growth and development of somatic embryo–derived plantlets were also analysed. The PEPC/RubisCO ratio was found to progressively decrease (from 17.7 to 0.2) throughout the in vitro development of plantlets, due to a substantial depletion of PEPC activity, which decreased from 5.3 to 1.2 μmol CO2 h−1 mg−1 TSP. Concomitantly, RubisCO assumed greater importance (from 0.3 to 5.3 μmol CO2 h−1 mg−1TSP) and became the main route for inorganic carbon fixation. Western blot analysis using polyclonal antibodies raised against PEPC and RubisCO purified from tobacco leaves confirmed this trend in terms of relative enzyme abundance.

Developmental changes in carboxylase activities in in vitro cultured coconut zygotic embryos : comparison with corresponding activities in seedlings

Phosphoenolpyruvate Carboxylase (PEPC; EC: 4.1.1.31) and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (RubisCO; EC: 4.1.1.39) enzyme specific activities were measured during the in vitro development of coconut (Cocos nucifera L.) zygotic mature embryos into plantlets and compared with those of palms produced by conventional seed germination. At the time of initiation of germination, high PEPC and low RubisCO activities were measured in both cultured and conventionally germinated embryos, thus indicating an anaplerotic CO2 fixation. During both in vitro and in planta development, RubisCO progressively took over and became the main route for inorganic carbon fixation. The in vitro-grown coconut plantlets showed a faster decrease in their PEPC:RubisCO ratio than the seedlings, suggesting that an earlier transition from a heterotrophic to an autotrophic mode of carbon fixation takes place in the in vitro-derived material. Just before acclimatization, the RubisCO activity in in vitro-derived plantlets (2.83 µmol CO2h−1mg−1TSP) was lower than that in seedlings (6.98 µmol CO2h−1mg−1TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material

Assessment of genetic diversity among accessions of two traditional leafy vegetables ( Acmella uliginosa (L.) and Justicia tenella (Nees) T.) consumed in Benin using amplified fragment length polymorphism (AFLP) markers

Amplified fragment length polymorphism (AFLP) markers were used to evaluate the genetic diversity and explore the genetic relationship among accessions of Acmella uliginosa and Justicia tenella , two leaf vegetables collected from different areas in the northwest and northeast parts of Benin (West Africa). The total number of exploitable amplicons revealed with genomic DNA from A. uliginosa was 224 with an average of 50.5% polymorphic amplicons. Using DNA from J. tenella , we obtained 34% of polymorphic amplicons from a total of 418. The coefficient of dissimilarity varied from 0.01 to 0.67 and from 0.17 to 0.62 for Acmella and Justicia , respectively. Low genetic diversity was observed among Acmella accessions although three distinct clusters could be differentiated. Contrarily, a great genetic diversity was observed among J. tenella accessions. In addition to this, most of the clusters were heterogeneous and showed the relationship between accessions collected from northeast and northwest. Our results confirm the robustness of AFLP techniques for genetic diversity studies and they provide the first set of molecular data for these two species. Key words: Amplified fragment length polymorphism (AFLP), genetic diversity, leafy vegetable, Benin.

Effect of medium sucrose on the photosynthetic capacity of coconut vitroplants formed from zygotic embryos

The coconut palm is an important source of oil and other products in tropical countries. Coconut populations in the Caribbean area, including Florida (USA), Mexico, Belize and Honduras have being lowered by a severe disease called lethal yellowing (LY) that has killed millions of plants (Oropeza and Zizumbo, 1997). In order to control this disease it is necessary to implement plant-breeding programs to increase both LY resistance and yields. For a successful breeding program it is important to have safe and efficient ways to exchange plant material from country to country. It is clear that the in vitro culture of zygotic embryos meets the need for phytosanitary requirements for plant material transport from country to country. However, the acclimatisation of plants derived from tissue cultured zygotic embryos is not effective enough and shows poor survival rates (Ashburner et al., 1994). It is important to study the physiology of these vitroplants to determine if the problem is related to problems in controlling water loss when vitroplants are transferred from the in vitro container to the field or to problems with the development of photosynthetic ability.

Genetic characterization of two traditional leafy vegetables (Sesamum radiatum Thonn. ex Hornem and Ceratotheca sesamoides Endl.) of Benin, using flow cytometry and amplified fragment length polymorphism (AFLP) markers

Amplified fragment length polymorphism (AFLP) markers and flow cytometry were applied for the genetic characterization of wild and cultivated accessions of Sesamum radiatum and Cerathoteca sesamoides ; two neglected and underutilized species of traditional leafy vegetable consumed in Benin. The average 2C nuclear DNA content per nucleus was found to be 1.99 ± 0.06 and 1.05 ± 0.06 pg for S. radiatum and C. sesamoides , respectively which correspond to estimated genome size of 1946,22 Mpb for S. radiatum and 1026,9 Mpb for C. sesamoides. No variation in DNA content could be detected within accessions from each analysed species. Also, no relation was found between nuclear DNA content, ecogeographical origin and the status (cultivated or wild) of the analyzed accessions. AFLP markers revealed low diversity within the accessions analyzed. Results from the study contributed to a better characterization of S. radiatum and C. sesamoides accessions and will help in defining both genetic resources conservation and breeding strategies. Key words : Amplified fragment length polymorphism (AFLP) markers, Ceratotheca sesamoides, flow cytometry, genetic diversity, ploidy, Sesamum radiatum, leafy vegetables.

Applying Epigenetics in Plant Breeding: Balancing Genome Stability and Phenotypic Plasticity

The correct implementation of epigenetic mechanisms is often a prerequisite for the timely regulation of genome expression and structure and ultimately for the development of higher plants. Developmental regulation is thus playing a paramount role in the elaboration of yields in agricultural crops. However, numerous studies have shown that this tight control includes a certain degree of freedom as epigenetic regulations can be loosened in the course of the reproductive development, after hybridization or as part of the response to environmental constraints – both in vitro and in vivo – whereas genome stability is globally maintained. As a result, several modified epigenetic marks and associated altered gene or transposable element expression can eventually give rise to qualitative or quantitative phenotypic changes on the long term. The present chapter is intended to present the main concepts governing epigenetic regulation of gene expression in higher plants and to review its potential applications for the selection of heritable phenotypes. It illustrates how epigenetic variations can be smartly used in breeding schemes and which questions remain to be addressed in order to make such integration successful. The Next Generation Sequencing revolution has also impacted our approach of plant epigenetics as more genomes, epigenomes and transcriptomes and are made available for crop plants and the simplistic Arabidopsis model is being questioned.