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The success of fertilization depends on an array of complex physical and physiological interactions between the pollen and the pistil. To better understand the fertilization process in Malagasy baobabs, pistil structure and pollen tube growth were investigated in three species of Adansonia: A. grandidieri (section Brevitubae), A. madagascariensis and A. rubrostipa (Longitubae section). The pistil consists of an ovary with 6–9 locules, a very long hollow type style and six to nine lobed wet, papillate stigma. The pollen tubes grow in intercellular secretions in the stigma and join the stylar canal to reach the locule of the ovary and then the micropyle of the ovules. The size of the central stylar canal shrinks as it approaches the ovary. This restriction leads to gametophytic competition by reducing the number of pollen tubes that reach the ovules. The pollen tubes reach the ovules four to six days after pollination. The pollen tubes of the two Longitubae species grow faster than those of A. grandidieri. This information on the progamic phase of baobabs is essential to understand the mode of reproduction of baobabs.

Vascular tissues are main routes of resource transport, which are crucial for the growth of fleshy fruit. Very few quantitative data of the total and active areas of xylem and phloem are available for herbaceous plants and their variabilities are unknown. In this study, histological approach and process-based modeling of tomato fruit growth were combined to evaluate the potential contribution of the pedicel anatomy to fruit mass variations. Eleven genotypes were described and the impact of water deficit was studied depending on stress intensity and stage of application. In parallel, MRI experiments allowed to better understand the source of variability in xylem flow along the main stem. Our results suggested that the genetic and water deficit-induced variations in the areas of vascular tissues in the pedicel partly contributed to fruit mass variability. Flow-MRI appeared to be a complementary non-destructive method to phenotype vascular tissues. Whereas the flow velocity in active xylem vessels was rather stable along the main stem, the decrease in the number of active vessels strongly reduces the effective flow from the bottom to the top of the plant. Fruitful interactions between modeling, histology and flow-MRI are promising and worth exploring, to predict water fluxes within plant architecture.

The occurrence of somaclonal variants (ca 5%) among populations of somatic embryo-derived oil palms (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 Sssl-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.

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.

La conservación in vitro de Dioscorea alata L. clon Caraqueño es fundamental para garantizar la propagación y distribución de material de plantación sano a los productores, y disponer de un banco in vitro de un clon de gran valor agronómico y comercial en la región oriental de Cuba. Con el fin de evaluar las modificaciones anatómicas que se producen en plantas de ñame en tres condiciones de cultivo in vitro: plantas conservadas por métodos de mínimo crecimiento, plantas regeneradas y plantas en fase de multiplicación en el medio MS 75 %, se realizó un análisis de la anatomía foliar y caulinar a partir de cortes transversales de la lámina foliar y del tallo, y cortes longitudinales y transversales de microtubérculos formados durante el proceso de conservación. Las hojas de las plantas conservadas mostraron menor espesor del mesófilo y la epidermis y el área de los haces conductores del tallo también fue menor, debido al proceso de stress durante la conservación in vitro. Sin embargo, durante la recuperación del material conservado a través de la regeneración y la multiplicación in vitro se restablecieron de manera normal estos parámetros. También se evidenció que los microtubérculos formados en la conservación in vitro, poseen parénquima amilífero con abundantes gránulos de almidón, capa delgada de parénquima cortical, y haces conductores poco desarrollados, todo lo cual indica la presencia de actividad meristemática.Palabras clave: caracterización anatómica, conservación in vitro, microtubérculos, ñame, segmentos nodales The in vitro conservation of Dioscorea alata L. clone Caraqueño is fundamental to guarantee the propagation and distribution of healthy plantation material to the farmers and the establishment of one in vitro bank of this clone of great agronomic and commercial value in the Oriental Region of Cuba. With the purpose of evaluating the anatomical modifications that take place in yam plants under three in vitro culture conditions: conserved plants by slow growth, regenerated plants and in plants multiplication phase in MS 75% medium, was carried out an analysis of the foliar and caulinar anatomy from transversal cuts of the foliar sheet and of the stem, and longitudinal and transversal cuts of microtubers formed during the conservation process. Smaller thickness of the mesophyll and of the epidermis in the leaves of the conserved plants were showed and the conductive sheaves area of the stem were also smaller, due to the stress process during the in vitro conservation. However during the recovery of the conserved material through the regeneration and the in vitro multiplication were reestablished to their normal state these parameters. It was also evidenced that the microtubers formed in the in vitro conservation, have reserve parenchyma with abundant starch granules, thin cortical parenchyma and conductive sheaves little developed were determined. All this characteristics indicated the presence of meristematic activity.Key words: Anatomical characterization; In vitro conservation; In vitro tubers; Nodal cutting; Yam

Background Drought 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. Results Pyrosequencing 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. Conclusions The 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. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0777-5) contains supplementary material, which is available to authorized users.

International audience; Knowledge of the cell separation processes underlying organ abscission is limited mainly to the eudicot model systems tomato and Arabidopsis, while less is known about the mechanisms in crop species in general, and monocots in particular. Here we describe a simple phenotypic screen for oil palm plant lines with delayed or non-abscising fruit. The phenotypic test was used on the two species of oil palm including Elaeis guineensis and E. oleifera, in addition to their interspecific hybrids, and hybrids backcrossed to E. guineensis. A large variation in abscission phenotypes was observed. Histological analysis revealed similarities and differences in the abscission zone (AZ) of E. guineensis and E. oleifera, and dominant traits were observed in the AZ of the interspecific crosses. The expression of abscission marker genes for a polygalacturonase (EgPG4) and an ethylene synthesis ACO synthase (EgACO) were monitored during abscission of a randomly selected set of plants and in a non-abscising individual identified by the phenotypic test.

A simple and efficient cryopreservation protocol for coconut zygotic embryos has been developed. Embryos were inoculated in Petri dishes on medium containing 3.2 M glucose, which were placed in hermetically closed containers containing 80 or 160 g silica gel for 48 or 24 h. Moisture content of embryos at the end of this treatment varied between 0.25 and 0.65 g g(-1) DW, depending on the accession. Embryos were then transferred for cryopreservation by rapid immersion of cryotubes in liquid nitrogen. After rapid re-warming, embryos were transferred to culture medium containing Eeuwens mineral elements for germination. This protocol was applied to ten accessions representative of coconut genetic diversity, with germination percentages of cryopreserved embryos between 13.7% and 74.7%.

UMR AGAP - équipe PHIV - Plateforme d'histocytologie et d'imagerie cellulaire végétale; Root and stem rot (RSR) disease caused by Fusariurn oxysporurn f. sp. radicis-vanillae (Forv) is the most damaging disease of vanilla (Vanilla planifolia and V. x tahitensis, Orchidaceae). Breeding programs aimed at developing resistant vanilla varieties are hampered by the scarcity of sources of resistance to RSR and insufficient knowledge about the histopathology of Forv. In this work we have (i) identified new genetic resources resistant to RSR including V planifolia inbreds and vanilla relatives, (ii) thoroughly described the colonization pattern of Forv into selected vanilla accessions, confirming its necrotic non-vascular behavior in roots, and (iii) evidenced the key role played by hypodermis, and particularly lignin deposition onto hypodermal cell walls, for resistance to Forv in two highly resistant vanilla accessions. Two hundred and fifty-four vanilla accessions were evaluated in the field under natural conditions of infection and in controlled conditions using in vitro plants root-dip inoculated by the highly pathogenic isolate Fo072. For the 26 accessions evaluated in both conditions, a high correlation was observed between field evaluation and in vitro assay. The root infection process and plant response of one susceptible and two resistant accessions challenged with Fo072 were studied using wide field and multiphoton microscopy. In susceptible V planifolia, hyphae penetrated directly into the rhizodermis in the hairy root region then invaded the cortex through the passage cells where it induced plasmolysis, but never reached the vascular region. In the case of the resistant accessions, the penetration was stopped at the hypodermal layer. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to Forv. The thickness of lignin constitutively deposited onto outer cell walls of hypodermis was highly correlated with the level of resistance for 21 accessions tested. The accumulation of p-coumaric and sinapic acids, two phenolic precursors of lignin, was observed in the resistant plants inoculated with Fo072, but not in the susceptible one. Altogether, our analyses enlightened the mechanisms at work in RSR resistant genotypes and should enhance the development of novel breeding strategies aimed at improving the genetic control of RSR of vanilla.

Histocytological characteristics of Eucalyptus urophylla 9 Eucalyptus grandis shoot apical meristems (SAMs) were described, comparing five outdoor and in vitro sources of akin genotypes differing in their physiological age. The size and the number of cells of the five zones identified within each SAM, i.e. the two tunica layers (L1 and L2), the central mother cells (CMC), the peripheral zone (PZ) and the combination of these four zones (4CZ) varied according to physiological age and plastochron phase. These five zones were significantly larger with higher numbers of cells for SAMs from mature and juvenile trees than for those from physiologically rejuvenated, in vitro mature and in vitro juvenile plants. However, these origin-related differences were not significant for SAMs in their early plastochron phase, to become obvious in a more advanced plastochron stage. Individual cell and nuclear measurements confirmed the rationale of distinguishing within SAM zones, characterized by specific cell and nuclear sizes liable to vary according to physiological age. The various histocytological investigations carried out established that SAM cell characteristics appeared to be the more reliable indicators of phase change. This was particularly true for the nucleoplasmic ratio and for more qualitative differences observed also at the nuclear level. SAM nuclei of the two in vitro origins were more evenly stained by naphtol blue-black, uniformly light for the juvenile source, whereas the mature source showed also darker nuclei. In contrast, SAM nuclei from outdoor origins had more chromocenters, darker and diffusely spread for the mature source than for the rejuvenated and the juvenile origins, where they were more peripherally distributed and where the nucleoli appeared more clearly. These results were discussed with respect to physiological ageing and in vitro culture influence, and suggest a determining influence of SAM cell nuclei on phase change phenomenon of arborescent species.

DNA methylation was analyzed in Acacia mangium Willd. buds collected from in vitro and field growing shoots from the same clone and its juvenile offspring. Shoot tips from in vitro microshoots showed higher levels of DNA methylation for juvenile (23.4%) than for mature (21.8%) plant material, irrespective of their morphology. Likewise, under the same in vitro conditions, the apical buds collected from microshoots exhibiting the juvenile morphology displayed higher DNA methylation rates (23.2%) than those characterized by the mature phyllode morphological type (22.1%), regardless of the age of the plant material source. In outdoor conditions, apical buds collected from mature tree growing shoots were significantly less methylated (20.9%) than the resting axillary buds located just underneath (25.2%) and than the apical buds from the in vitro microshoots of the same clone and mature-like phyllode morphology (22.6%). Overall, the DNA methylation levels found for our Acacia mangium plant materials were consistent with those reported for other Angiosperms, but showed, similarly to other recent findings in different species, that DNA methylation in plants does not always increase with ageing. This was discussed in relation to other phase-related traits and more particularly foliage characteristics, which are conspicuous markers of ageing in this heteroblastic species.

The current article presents the investigations into the effect of the laurina mutation on the functioning and size of the shoot apical meristem (SAM) in Coffea arabica. This monolocus and Mendelian mutation is known to have pleiotropic effects on tree shape and dwarfism. A comparison between the wild type C. arabica var. Bourbon and its natural dwarf mutant C. arabica var. laurina, also called Bourbon pointu, was carried out leading to three main results: (1) the effects appeared immediately after the emergence of the buttress but did not affect the dome-shaped SAM (size and shape); (2) the effects were located at the peripheral zone and maintained subsequently within the leaf primordia; (3) the effects consisted of reduction in both the size of primordia and the height of incipient internode, consequently resulting in dwarfism of mature leaves and internodes. By contrast, the laurina mutation had no effect on the relationship between the phyllochron and the plastochron, the decussate and opposite phyllotaxis, and the relative timing of SAM functioning within the plastochron.

Plant roots have a large range of functions, including acquisition of water and nutrients, as well as structural support. Dissecting the genetic and molecular mechanisms controlling rice root development is critical for the development of new rice ideotypes that are better adapted to adverse conditions and for the production of sustainably achieved rice yield potential. Most knowledge regarding the gene networks involved in root development has been accumulated in the model dicotyledon plant species Arabidopsis thaliana. Rice, the model monocotyledon species, presents several singularities compared to A. thaliana, including a root architecture characterized by a fibrous root system comprising five types of embryonic and postembryonic roots. The anatomy and morphology of the rice root system, which is typical for a cereal, differs from that of A. thaliana, for instance, by the presence of a lysigenous cortex and additional cell layers compared to the dicotyledon model. Moreover, the structure and functions of the root apical meristem (RAM) of rice are distinct from those of A. thaliana. Recently, several rice root mutants have been identified via forward or reverse genetics, and these will aid in forming hypothesis to characterize either the divergence or conservation of genetic pathways relative to A. thaliana. Furthermore, these mutants will help to identify key genes in rice roots that may be missing in A. thaliana. This review summarizes both classical and recent data concerning the molecular genetics of rice root development, including root anatomy and morphology, RAM structure, RAM patterning, and root mutants.

The effect of growth regulators on induction of androgenesis in coconut was investigated using seven different growth regulators at various concentrations and combinations. Three auxins (1-naphthalene acetic acid-NAA, indoleacetic acid-IAA, picloram) and three cytokinins (2-isopentyl adenine-2-iP, kinetin, zeatin) were tested either alone or in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), using modified Eeuwens Y3 liquid medium as the basal medium. Among the tested auxins, 100 [mu]M NAA in combination with 100 [mu]M 2,4-D enhanced the production of calli/embryos (123) whereas IAA and picloram showed negative and detrimental effects, respectively, for androgenesis induction over 100 lM 2,4- D alone. Kinetin and 2-iP enhanced the production of calli/ embryos when 100 [mu]M 2,4-D was present in the culture medium. Both cytokinins at 10 [mu]M yielded the highest frequencies of embryos (113 and 93, respectively) whereas zeatin (1 or 2.5 [mu]M) had no impact on microspore embryogenesis. When calli/embryos (produced from different treatments in different experiments) were subcultured in somatic embryo induction medium (Y3 medium containing 66 [mu]M 2,4-D), followed by maturation medium (Y3 medium without growth regulators) and germination medium (Y3 medium containing 5 [mu]M-6-benzyladenine- BA and 0.35 [mu]M gibberellic acid-GA3), plantlets were regenerated at low frequencies (in most treatments ranging from 0% to 7%).

Anther culture was used to obtain dihaploid (DH) coconut plants and their ploidy level was determined by flow cytometric analysis. Simple sequence repeat (SSR) marker analysis was conducted to identify the homozygous diploid individuals. Ploidy analysis showed that 50% of the tested plantlets were haploid and 50% were diploid. Polymorphic fragments of the mother palm and their segregation patterns in anther-derived plantlets were used to determine the origin of the diploid plantlets. Using a diagnostic SSR marker (CNZ43), all the diploid plantlets tested were identified as being derived from microspores (i.e. were homozygous) and were thus candidates for use in coconut breeding programs.

UMR DAP, équipe AFEF, Correspondance: costes@supagro.inra.fr Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Genetic control of plant size and shape is a promising perspective, particularly in fruit trees, in order to select desirable genotypes. A recent study on architectural traits in an apple progeny showed that internode length was a highly heritable character. However, few studies have been devoted to internode cellular patterning in dicotyledonous stems, and the interplay between the two elementary cell processes that contribute to their length, i.e. cell division and elongation, is not fully understood. The present study aimed at unravelling their contributions in the genetic variation of internode length in a selection of F(1) and parent genotypes of apple tree, by exploring the number of cells and cell shape within mature internodes belonging to the main axes. The results highlighted that both the variables were homogeneous in samples collected either along a sagital line or along the pith width, and suggest that cell lengthening was homogeneous during internode development. They allowed the total number of cells to be estimated on the internode scale and opened up new perspectives for simplifying tissue sampling procedures for further investigations. Differences in internode length were observed between the genotypes, in particular between the parents, and partly resulted from a compensation between cell number and cell length. However, genetic variations in internode length primarily involved the number of cells, while cell length was more secondary. These results argue for an interplay between cellular and organismal control of internode shape that may involve the rib meristem.

Immature seeds isolated from pods of carob (Ceratonia siliqua L.) were used as a source of explants for callogenesis and somatic embryogenesis. Suitable developmental Stages of immature seed explants, and in vitro culture conditions for the production of translucent callus were identified. The addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to the culture medium was found to be necessary for callogenesis. Both the developmental Stage of the explant and the 2,4-D concentration had significant effects on the percentage of callus production. A high proportion (67.0 ± 1.0%) of immature seed at Stage 2 gave rise to callus when cultured on a medium containing 9.0 µM 2,4-D, which was significantly higher than all the other treatments tested. Embryogenic calli consisted of a white-translucent mass proliferating all around the explants and gave rise to somatic embryos. On day-45 of culture, transverse sections of callus showed de-differentiated cells located on the surface, which gave rise to pro-embryos. On day-70, early somatic embryos were observed as globular white structures, and others were at the heart-shaped stage. On day-90, complete somatic embryos were initiated which had attained the cotyledonary stage, characterised by a defined meristematic area and separation of a protoderm and pro-vascular bundles. (Resume d'auteur)

Little is known of the mechanisms that induce the dedifferentiation of a single somatic cell into a totipotent embryogenic cell that can either be regenerated or develop into an embryo and subsequently an entire plant. In this Opinion article, we examine the cellular, physiological and molecular similarities and differences between different plant stem cell types. We propose to extend the plant stem cell concept to include single embryogenic cells as a totipotent stem cell based on their capacity to regenerate or develop into an embryo under certain conditions. Our survey suggests that differences in chromatin structure might ensure that meristem-localized stem cells have supervised freedom and are pluripotent, and that embryogenic stem cells are unsupervised, autonomous and, hence, freely totipotent.

Key message Hypocotyl semi-dwarfism in BP was only due to less cell number. Daylight was required to inhibit cell division in the mutant. ABA, IAA and cytokinins would be involved. Abstract Coffea arabica 'Laurina' is a natural mutant of Coffea arabica 'Bourbon' (B) and is known under the trade name 'Bourbon Pointu' (BP). Under daylight, the laurina mutation leads to pleiotropic effects, including semi-dwarf hypocotyls. At the opposite, semi-dwarfism of BP seedlings disappeared under darkness conditions. The first step was to describe the morphological impact of the mutation in seedlings growing under daylight by comparison with seedlings growing under darkness. As the hypocotyl length was mainly affected, the second step was to investigate histological modifications in the organ comparing B and BP seedlings growing under daylight. Result of this investigation indicated that the mutation does not impact on cell length. Moreover, cytometry analyses showed absence of endoreduplication. Actually, the mutation influenced the cell number and this effect appeared before the 40th day after sowing. The length difference of hypocotyls between B and BP was due to lower cell number in BP, indicating possible involvement of phytohormones. Investigations showed the decrease of cytokinin and auxin levels in BP compared to B, while the cytokinin/auxin ratio remained constant in both varieties. By contrast, abscisic acid content increased in BP. Concurrently these results indicate the lowering of cell division, due to the mutation.

When fused to " Pr AlSAP " promoter, transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Pr AlSAP can be used for engineering abiotic stress tolerance. We previously showed that ectopic expression of a stress-associated protein gene from Aeluropus littoralis (AlSAP) enhances tolerance to multiple abiotic stresses in tobacco, wheat and rice. The ortholog of AlSAP in rice is OsSAP9. Here, we demonstrate that AlSAP transcripts accumulate in Aeleuropus in response to multiple abiotic stresses and at a higher level in roots, while those of OsSAP9 are preferentially induced by cold and heat treatments and accumulate preferentially in leaves of rice. In silico analysis of the AlSAP promoter "Pr AlSAP " predicted several cis-acting elements responsible for gene regulation by dehydration, salt, heat, ABA, SA, wounding and tissue-specific expression. The Pr AlSAP promoter was fused to the gusA gene and used to produce transgenic rice plants. Transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Indeed, accumulation of gusA transcripts was higher in roots than in leaves and induced by salt, drought, cold and heat treatments. GUS activity was confirmed in roots, coleoptiles, leaves and glumes, but absent in the root cell elongation zone and in dry seeds. A wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Altogether, these results indicate that the regulatory regions of two ortholog genes "AlSAP" and "OsSAP9" have diverged in the specificity of the signals promoting their induction, but that the trans-acting elements allowing the correct spatiotemporal regulation and stress induction of Pr AlSAP exist in rice. Therefore, the AlSAP promoter appears to be an interesting candidate for engineering abiotic stress tolerance in cereals.

International audience; We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.

The genus Adansonia (baobabs, Malvaceae) includes nine tropical species grouped in the Brevitubae, Longitubae and Adansonia sections. The ultrastructure of pollen from eight species of baobabs was studied using transmission electron microscopy. The pollen grains correspond to 3-colporate and 4-colporate types. The ectexine is formed by a perforate tectum with isolated spinules. An important distinguishing feature between the Brevitubae section and the Longitubae and Adansonia sections was observed in the infratectum. The apertural region was similar in all the species studied here. However, the most unusual structure was observed in the apertural margins where the ectexine consisted of a thick lamellate annulus and the endexine had a cracked appearance; the intine was composed of a well-developed oncus under the endopore with unusual fibrillar structures and the outer layer had a remarkable structure consisting of columns. Despite the few differences observed in the structure of the pollen wall within the genus, an original arrangement of the structure found in the aperture of the Adansonia pollen grains studied provides additional information about the new types of apertural structures. This type of sporoderm adds to our knowledge of the diversity of angiosperm pollen. Moreover, this apertural structure is probably an adaptation that occurs during the formation of the pollen tube and of harmomegathy.

Studies on the development of protocols for the clonal propagation, through somatic embryogenesis, of coconut have been reported for the past three decades, mostly using inflorescence explants, but with low reproducibility and efficiency. Recent improvements in these respects have been achieved using plumular explants. Here, we report a developmental study of embryogenesis in plumule explants using histological techniques in order to extend our understanding of this process. Coconut plumule explants consisted of the shoot meristem including leaf primordia. At day 15 of culture, the explants did not show any apparent growth; however, a transverse section showed noticeable growth of the plumular leaves forming a ring around the inner leaves and the shoot meristem, which did not show any apparent growth. At day 30, the shoot meristem started to grow and the plumular leaves continued growing., At day 45, the explants were still compact and white in color, but showed partial dedifferentiation and meristematic cell proliferation leading to the development of callus structures with a translucent appearance. After 60 d, these meristematic cells evolved into nodular structures. At day 75, the nodular structures became pearly globular structures on the surface of translucent structures, from which somatic embryos eventually formed and presented well-developed root and caulinar meristems. These results allow better insights and an integrated view into the somatic embryogenesis process in coconut plumule explants, which could be helpful for future studies that eventually could lead us to improved control of the process and greater efficiency of somatic embryo and plantlet formation.

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.

International audience; In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

The comparison between the cultivar Bourbon and its mutant, the Bourbon pointu, of Coffea arabica led to five novel findings on fruit development and three main impacts of the mutation. Coffea arabica ‘Laurina’ (Bourbon pointu) is a natural mutant of Coffea arabica ‘Bourbon’. Relative to the ‘Bourbon’ cultivar, it is characterized by internode dwarfism, a Christmas tree shape, and lower caffeine content. The effects of the laurina mutation on fructification over time, the fruit structure and seed characteristics were studied here. Fruits of ‘Bourbon’ and ‘Bourbon pointu’ were monitored. The trees were grown in the same plot and flowered on the same day. Harvesting was done every 2 weeks from the 6th to the 26th week after flowering. Histological observations were carried out using multiphoton and conventional microscopes. The measurements concerned the fruit, parchment and seed. Five novel findings on fructification development were obtained: (1) a sigmoid model and non-linear regression efficiently described the phenomenon; (2) a precise relationship was defined between the qualitative stages of fructification and quantitative observations, thus revealing key weeks in this process; (3) the parchment had a mesocarpic origin; (4) a meristematic zone was present close to the parchment; and (5) an endocarp with three cell layers was visible in young fruits. Three effects of the laurina mutation were highlighted: (1) fruit growth ended 1 week earlier in ‘Bourbon’, but without difference in fruit length. In contrast, fruits were wider on average in ‘Bourbon’; (2) the parchment of narrow seeds in ‘Bourbon pointu’ was thicker than in other ‘Bourbon pointu’ and ‘Bourbon’ seeds; and (3) the narrow seed frequency in ‘Bourbon pointu’ depended on environmental conditions.

An imaging method using spectral analysis combined with advanced linear unmixing was used to allow histolocalization of natural autofluorescent compounds such as hydroxycinnamic acid (chlorogenic acid) and xanthone (mangiferin) in living cells and tissues (mature coffee leaves). The tested method included three complementary steps: 1/ visualization of natural autofluorescence and spectrum acquisition with a multiphoton microscope; 2/ identification of some compounds using previous information on the chemical composition of the tissue, obtained from litterature; and 3/ localization of candidate compounds by spectral imaging. The second part of the study consisted of describing the histochemical structure of leaves during their development. This revealed very fast histochemical differentiation of leaves during the first week after their emergence. Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared. This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization. This also highlighted the paternal CAN origin of the leaf structure in the allotetraploid species ARA. The limits and advantages of the method without staining are discussed relative to classical epifluorescence microscopy under UV light. This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

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−1 TSP) was lower than that in seedlings (6.98 µmol CO2h−1mg−1 TSP) of the same age. Nevertheless, after acclimatization, RubisCO activities were comparable in both in vitro and in planta germinated material

International audience; Deciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This "iron map" of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments.

In a previous work, we demonstrated that expression of TdPIP2;1 in Xenopus oocytes resulted in an increase in Pf compared to water injected oocytes. Phenotypic analyses of transgenic tobacco plants expressing TdPIP2;1 generated a tolerance phenotype towards drought and salinity stresses. To elucidate its stress tolerance mechanism at the transcriptional level, we isolated and characterized the promoter region of the TdPIP2;1 gene. A 1060-bp genomic fragment upstream of the TdPIP2;1 translated sequence has been isolated, cloned, and designated as the proTdPIP2;1 promoter. Sequence analysis of proTdPIP2;1 revealed the presence of cis regulatory elements which could be required for abiotic stress responsiveness, for tissue-specific and vascular expression. The proTdPIP2;1 promoter was fused to the β-glucuronidase (gusA) gene and the resulting construct was transferred into rice (cv. Nipponbare). Histochemical analysis of proTdPIP2;1::Gus in rice plants revealed that the GUS activity was observed in leaves, stems and roots of stably transformed rice T3 plants. Histological sections prepared revealed accumulation of GUS products in phloem, xylem and in some cells adjacent to xylem. The transcripts were up-regulated by dehydration. Transgenic rice plants overexpressing proTdPIP2;1 in fusion with TdPIP2;1, showed enhanced drought tolerance, while wild type plants were more sensitive and exhibited symptoms of wilting and chlorosis. These findings suggest that expression of the TdPIP2;1 gene regulated by its own promoter achieves enhanced drought tolerance in rice.

PDF version of the authors can be published in January 2013; International audience; 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.

Background Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process. Results The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700–5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation. Conclusions The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species.

International audience; Successful grafting in plants requires the development of a functional vascular system between the scion and the rootstock. Understanding the spatial organization of the graft interface is important to the evaluation of new rootstock genotypes and to the development of new grafting technologies. Until now the graft interface has only been studied using 2D classical histology and low resolution 3D magnetic resonance imaging. Here we investigate the ability of X-ray tomography to examine the graft interface of Vitis vinifera in high resolution and in 3D. Data were collected using a Skyscan 1076, scanning parameters, such as. X-ray energy, filter selection, pixel size and rotation angles, were optimized to study the particularities of the graft interface. The X-ray tomography technique was then used to evaluate graft quality. Two young vines were compared; one graft was classified as of 'good' quality, whereas the other was classified as of 'bad' quality. We were able to distinguish the "omega cut", the pith, the phloem and the xylem vessels in the images. The analysis shows several differences between the two vines. In the good graft, tissues appear well-connected in the wood and phloem, and had a regular structure; the wood appears homogenous with a lot of vessels that form a compact mass. By contrast, in the bad graft, the structures appear disorganized and not completely connected. Numerous new vessels, continuous between the scion and the rootstock, are visible in the "good graft" whereas only few ones are visible in the "bad one". It is the first time, to our knowledge, that 3D imaging of the graft interface and the vascular connections across it have been reported, opening new avenues for graft quality assessment in woody plants.

International audience; 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.

International audience; It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.

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.

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 beta-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.

Background and Aims Oil palm, an unbranched perennial monocotyledon, possesses a single shoot apical meristem (SAM), which is responsible for the initiation of the entire above-ground structure of the plant. To compare the palm SAM structure with those of other monocots and to study variations in its structure throughout the life of the plant, its organization was characterized from the embryonic stage to that of the reproductive plant. Methods SAM structure was studied by a combination of stained histological sections, light and confocal microscopy, and serial section-based three-dimensional reconstructions. Key Results The oil palm SAM is characterized by two developmental phases: a juvenile phase with a single tunica-corpus structure displaying a gradual increase in size; and a mature phase characterized by a stable size, a modified shape and an established histological zonation pattern. In mature plants, fluctuations in SAM shape and volume occur, mainly as a consequence of changes in the central zone, possibly in relation to leaf initiation. Conclusions Development of the oil palm SAM is characterized by a juvenile to mature phase transition accompanied by establishment of a zonal pattern and modified shape. SAM zonation is dynamic during the plastochron period and displays distinct features compared with other monocots. (Resume d'auteur)

Background Despite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented. Results The procedure was developed based on a range of developmental stages, from leaf initiation to senescence, of soil-grown Arabidopsis thaliana (L.) Heynh. Rigorous clearing of tissues, made possible by enhanced leaf permeability to clearing agents, allowed the optical sectioning of the entire leaf thickness by both confocal and multiphoton microscopy. The superior image quality, in resolution and contrast, obtained by the latter technique enabled the three-dimensional visualisation of leaf morphology at the individual cell level, cell segmentation and the construction of structural models. Image analysis macros were developed to measure leaf thickness and tissue proportions, as well as to determine for the epidermis and all layers of mesophyll tissue, cell density, volume, length and width. For mesophyll tissue, the proportion of intercellular spaces and the surface areas of cells were also estimated. The performance of the procedure was demonstrated for the expanding 6th leaf of the Arabidopsis rosette. Furthermore, it was proven to be effective for leaves of another dicotyledon, apple (Malus domestica Borkh.), which has a very different cellular organisation. Conclusions The pipeline for the three-dimensional imaging and analysis of plant leaves provides the means to include variables on internal tissues in leaf growth studies and the assessment of leaf phenotypes. It also allows the visualisation and quantification of alterations in leaf structure alongside changes in leaf functioning observed under environmental constraints. Data obtained using this procedure can further be integrated in leaf development and functioning models.

Anthers excised from male flowers of an adult coconut palm of Sri Lanka Tall cultivar were used to produce plants via androgenesis. Morphological aspects of the anther-derived structures and their plant regeneration pathways were studied. Under the culture conditions employed, both calli and embryo-mediated plant regeneration were observed. Two types of embryos with different germination patterns were identified-one with a germination point and the other type with a blunt haustorium without a prominent germination point which converted into germinated embryos. Three types of calli (embryogenic compact calli, embryogenic friable calli and non-embryogenic fast-growing calli) were also produced and the most commonly identified type was embryogenic compact calli that give rise to somatic embryos. Secondary somatic embryos were produced in both direct embryo and callus mediated embryogenesis. Normal plants with a single shoot were observed in low frequencies while weak plantlets with multiple shoot were observed in abundance. Under similar culture conditions, some plantlets showed vigorous growth whereas the majority had a slow growth rate. Keywords: Androgenesis, anther culture, callus, coconut ( Cocos nucifera L.), embryo, morphology doi :10.4038/jnsfsr.v38i1.1727 J.Natn.Sci.Foundation Sri Lanka 2010 38 (1): 69-74

Shoot apical meristem (SAM) domes derived from five different outdoor and in vitro sources of juvenile and mature Eucalyptus urophylla 9 Eucalyptus grandis akin genotypes were compared. Overall measurements of SAM dome height H and diameter D ranged from 2 to 35 lm and 20 to 80 lm, with significant differences according to the various physiological origins of plant material investigated. SAM domes from the mature trees ''Mat'' were taller than those from the rejuvenated ministock plants ''Rej''; from the in vitro microcuttings ''IVM'' of the same clone and also from the in vitro juvenile seedlings ''IVJ'', whereas outdoor seedlings ''Juv'' exhibited intermediate SAM dome height. SAM domes from the rejuvenated material ''Rej'', from the in vitro mature ''IVM'' and juvenile ''IVJ'' origins were also narrower than those from the outdoor seedlings ''Juv'' and to lesser extent than those from the mature trees ''Mat''. Overall, the mature source ''Mat'' displayed bigger and somehow sharper hemispherical domes than those from ''Rej'' and ''Juv'', physiologically more juvenile, or those from the in vitro origins ''IVM'' and ''IVJ'' which looked flatter and smaller. SAM dome height, diameter D and H/D values varied also significantly according to the plastochron. More specifically, H, D, and H/D SAM differences between the five origins were not significant during the early plastochron phase corresponding to leaf initiation, to become more salient as leaf structures started to elongate and to differentiate. This was particularly obvious for mature tree ''Mat'' SAM dome shapes which showed at this stage much higher H/D values than the other SAM sources. A shape index S used for characterizing more accurately dome shape confirmed these trends. These observations provide additional arguments to the view that juvenility in trees becomes more and more time- and shoot-tip restricted as ageing increases in the course of time during the ontogenetical process and could be ultimately confined to the most organogenic phase of SAM, from which shoot characteristics derive.

L'article original est publié par The American Society of Plant Biologists; International audience; Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB061311 (OsHKT2;1 mRNA), AJ491816 (OsHKT1;1 mRNA), and AJ491818 (OsHKT1;3 mRNA).; International audience; Plant growth under low K(+) availability or salt stress requires tight control of K(+) and Na(+) uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K(+) Transporters), permeable either to K(+) and Na(+) or to Na(+) only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na(+) transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na(+) only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na(+)-K(+) symport, Na(+) uniport, or inhibited states, depending on external Na(+) and K(+) concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K(+) and Na(+) accumulation in monocots.

While callogenesis in date palm can be initiated by culturing immature leaf segments on medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), it is difficult to obtain callus from certain genotypes. To better understand the mechanisms of callogenesis in date palm, we studied the developmental events underlying this process at the cellular level. The callogenic capacity of leaf segments depends on the state of cell differentiation. The most callogenic segments were within the leaf elongation zone, required polar auxin transport to initiate callogenesis and contained the highest quantities of free endogenous indole- 3-acetic acid. At the cellular level, callus induction involves two spatially and temporally separated events. The first event involves cells from the fascicular parenchyma in an equatorial plane perpendicular to the vascular axis that within 2 days of culture in the presence of 2,4-D, acquire structural and ultrastructural features typically observed in meristematic cells. The second event occurs 3 days later and is characterised by the modification of adjacent perivascular sheath cells. The latter cells become callogenic in that they reinitiated their cell cycles and undergo cell division leading to callus formation. These data provide evidence that callus initiation in leaf vascular tissue of date palm involves a sequential response of two distinct cell types to auxin and requires polar auxin transport.

The two varieties—Bourbon (B) and its natural mutant Bourbon pointu (BP)—of Arabica coffee (Coffea arabica L.) differ by an epigenetic, monolocus, and recessive laurina mutation that results in pleiotropic effects, such as tree dwarfism and tree-shape modification. The objective of the study was to search for the origin of the differences in size and shape of the tree both at the macroscopic (length and number of internodes, branching angle) and at the microscopic levels (size and number of cells within the internode pith). At the macroscopic level, the laurina mutation acted only by decreasing the internode size. Neither the angle of branching nor the number of internodes was influenced by the mutation. At the microscopic level, the mutation lowered mainly the number of cells present along the longitudinal axis of the internode, and, at a lesser extent, the cell height. Especially, the internode size decreasing explained both the tree dwarfism and the tree-shape modification. In fact, the laurina mutation strengthened the dwarfism of plagiotropic internodes when compared to that of the orthotropic ones, and such an impact was mainly due to a strong cell number decrease. To summarize, two major pleiotropic effects of the laurina mutation can be explained only by a modification of the trade-off between meresis and auxesis during the internode growth. This opens new perspectives for the characterization of the other effects at the hormonal level, and then, for the identification of the gene at the molecular level.

Coconut is a cross pollinating palm, propagated only by seeds. Tissue culture is the only vegetative propagation method available for coconut. Consistent callogenesis was obtained by culturing unfertilised ovaries at -4 stage in CRI 72 medium containing 100 mu M 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1% activated charcoal. Callusing was improved by application of 9 mu M thidiazuron (TDZ). Embryogenic calli were subcultured onto somatic embryogenesis induction medium containing 66 mu M 2,4-D. Stunted growth was observed in the somatic embryos after subculture onto CRI 72 medium containing abscisic acid (ABA). Maturation of somatic embryos could be achieved in Y-3 medium without growth regulators. Conversion of somatic embryos was induced by adding gibberellic acid (GA(3)) to conversion medium containing 5 mu M 6-benzyladenine (BA) while 2-isopentyl adenine (2iP) increased the frequency of plant regeneration. A total of 83 plantlets was produced from 32 cultured ovaries.

Bayoud, caused by #Fusarium oxysporum# f. sp.# albedinis# (Foa), is the most damaging disease of date palm in Morocco. In the present study we have investigated the effect of jasmonic acid (JA) on two defencerelated enzymes, namely peroxidases (POX) and polyphenoloxidases (PPO) in date palm seedlings root. Our data show that exogenous application of JA at a concentration of 50 [mu]M increased the activity of both enzymes. The increase of POX activity in the presence of JAwas much more important than that observed following infection with the pathogen. As compared to untreated plants, PPO activity was 2.2 and 1.3 times higher in BSTN and JHL cultivars respectively. In addition, PAGE analysis revealed increased band intensity of the major constitutive isoforms of POX and PPO in both JA-treated and Foa-treated seedlings. Close examination of symptomatic and asymptomatic plants showed that root tissues of symptomatic plants were massively colonized by Foa. Also, disease development in these plants appeared to involve a marked degradation of the host cell walls early during the process of pathogen invasion. In contrast, the presence of Foa in asymptomatic plants induced limited necrotic lesions (hypersensitive-reaction like lesions) that were probably involved in reducing the progression of the pathogen. Together, our findings indicate that JA is capable of enhancing date palm root resistance to infection by Foa via the activation of defence-related enzymes such as PPO and POX.

In date palm (Phoenix dactylifera L. cv. Ahmar, Arecaceae), as for many monocotyledons, callogenesis is a prerequisite for the initiation of somatic embryogenesis, and requires the presence of auxin in the medium. Immature leaf explants were cultivated in medium supplemented with either 1 or 54 μM 1-naphtaleneacetic acid in order to induce either rhizogenesis or callogenesis. Histological studies performed throughout the culture period established that precocious cell reactivation is similar in both morphogenetic pathways. Early cytological modifications are associated with cell reactivation and are observed in the pluripotent cells of perivascular sheaths. Divergence between the callogenesis and rhizogenesis pathways is observed later, during the subsequent determination and morphological differentiation phases. We established that in date palm, the rhizogenesis and callogenesis pathways are initiated from the same cell type, the ultimate developmental fate depending upon auxin concentration.