Marc Lartaud

Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique

SummaryA culture procedure using temporary immersion in a liquid medium was tested for somatic embryogenesis of Hevea brasiliensis (Müll. Arg.). Embryogenic callus was placed under regeneration conditions, either on a gelled medium (Phytagel, Sigma, St. Louis, MO) or in a container designed for temporary immersion. The latter technique has some advantages over the use of a gelled medium during both the early steps of somatic embryogenesis, i.e., embryo development, and later on, i.e., during maturation, desiccation and germination. Somatic embryo production in a liquid medium was three to four times greater than on a semi-solid medium: 400 embryos/g fresh weight under the best embryogenesis induction conditions. Somatic embryogenesis had to be initiated on a gelled medium before the embryogenic callus was transferred to temporary immersion, and the amounts of 3,4- dichlorophenoxyacetic acid and N6-benzyladenine had to be reduced. Temporary immersion resulted in substantially more consistent, synchronized somatic embryo development, reducing the number of abnormal embryos by half and stimulating germination. All of the late events could be carried out in the temporary immersion container. Effective drying conditions were achieved after 12 wk without immersion and without selection of the embryos. Temporary immersion during germination greatly stimulated root development (+60%) and epicotyl emergency (+35%), combined with increased synchronization and a substantially reduced workload.

Development of coffee somatic and zygotic embryos to plants differs in the morphological, histochemical and hydration aspects.

In Coffea arabica L., the development of direct sowing of somatic embryos (SE) in planting substrate, with subsequent nursery production of plants, has promoted the industrialization of somatic embryogenesis. However, plant conversion rates are still low and require improvements to enhance the cost-effectiveness of commercial micropropagation. With the aim of improving plant regeneration from SE, we studied the morphological and histological criteria and water characteristics during germination and plant conversion of zygotic embryos (ZE) and SE. At the cotyledonary stage, SE produced in a 1 l RITA(®) temporary immersion bioreactor (area 55.8 cm(2)) were morphologically similar in size (2-3 mm) but abnormal as compared with mature ZE. Protein and starch reserve levels were extremely low throughout germination and conversion to plantlets, while the water status remained steady [water content (WC) from 76 to 87%, Ψ from -0.37 to -0.47 MPa, pressure potential from 0.69 to 0.24 MPa]. In ZE, spectacular hydration occurred during the first 3 weeks (WC from 37 to 75%; Ψ from -6.24 to -1.0 MPa). Cotyledons remained undifferentiated for 10 weeks after sowing. Conversely, after only 3 weeks under germination conditions in a RITA(®) bioreactor, spongy and palisade parenchyma and stomata formed in SE cotyledons. The ZE plant conversion was faster than that of SE (14 vs. 22 weeks) and more efficient (rates 96 vs. 55%), with much more substantial hypocotyl and cotyledon development. The use of a new 5 l MATIS(®) bioreactor (area 355 cm(2)), designed especially to favor embryo dispersion and light transmittance to SE, markedly improved the embryo-to-plantlet conversion rate (91%). These results highlight the morphological heterogeneity and lack of protein reserves in SE at the beginning of the germination phase and marked differences in water characteristics. However, they also reveal high phenotypic plasticity, leading to a highly efficient plantlet conversion rate due to better embryo dispersion and light transmittance in more horizontal bioreactors.

Reactive Oxygen Species and Cellular Interactions Between Mycosphaerella fijiensis and Banana

Globally, the banana plant (Musa spp) is the fourth most important crop after rice, wheat and corn (based on production in tons). It is cultivated in more than 100 tropical and subtropical countries, mainly by small producers and is a fundamental food source for millions of people. Black leaf streak disease (BLSD), caused by Mycosphaerella fijiensis Morelet (sexual phase) or Paracercospora fijiensis (Morelet) Deighton (asexual phase), is the main disease affecting the world’s banana culture. This disease has a wide geographical distribution accounting for losses exceeding 50% of global banana production. We conducted a comparative histocytological study on the kinetics of the infection process using three banana genotypes with phenotypes that differ in resistance to BLSD: Grand Naine (Susceptible), Pisang Madu (Partially Resistant) and Calcutta 4 (Resistant). Experiments were conducted under controlled conditions with the objective of characterizing the cellular interaction processes between M. fijiensis and Musa acuminata. Conidia germination occurred 24 hours after inoculation. Germination rates were high (97%) and there were no significant differences between the three genotypes (P > 0.147). The Peroxidase enzyme and H2O2 were associated with a hypersensitivity-like reaction in the resistant genotype Calcutta 4, indicating a possible role of the enzyme or its product as defense mechanisms against M. fijiensis in banana plants.

Variations of DNA methylation in Eucalyptus urophylla×Eucalyptus grandis shoot tips and apical meristems of different physiological ages.

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei.

Identification of candidate genes for drought tolerance in coffee by high-throughput sequencing in the shoot apex of different Coffea arabica cultivars

BackgroundDrought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes.ResultsPyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi.ConclusionsThe full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.

Uvitex2B: a rapid and efficient stain for detection of arbuscular mycorrhizal fungi within plant roots

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and β-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.

Characteristics of Acacia mangium shoot apical meristems in natural and in vitro conditions in relation to heteroblasty

Morphological and histocytological characteristics of Acacia mangium shoot apical meristems (SAMs) were assessed in natural and in vitro conditions in relation to heteroblasty. In the natural environment, SAMs with a mature-phyllode morphology were much bigger, contained more cells with larger vacuolated area, or vacuome, and lower nucleoplasmic ratios than those from the juvenile type (Juv). In these latter, nuclei appeared more voluminous, evenly and lightly stained, with clearly distinguishable nucleolei and less abundant chromocenters. In vitro, where reversions from mature to juvenile morphological traits do occur unpredictably, heteroblasty was less obvious in the SAM characteristics examined. In vitro SAMs corresponding to the juvenile and mature types showed similarities with outdoor Juv SAMs, but could be distinguished from these latter by a much larger vacuome that might be induced by the culture conditions. These findings encourage pursuing the investigations at the chromatin and nucleolus level in SAM zones where heteroblasty-related differences have been detected.