Non-embryogenic Cells Research Articles

H3K9 methylation patterns during somatic embryogenic competence expression in tamarillo (Solanum betaceum Cav.)

The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.

Global transcriptome profiling reveals differential regulatory, metabolic and hormonal networks during somatic embryogenesis in Coffea arabica

BackgroundSomatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial and error approach. We report the first global scale transcriptome profiling performed at all developmental stages of SE in coffee to unravel the mechanisms that regulate cell fate and totipotency.ResultsRNA-seq of 48 samples (12 developmental stages × 4 biological replicates) generated 90 million high quality reads per sample, approximately 74% of which were uniquely mapped to the Arabica genome. First, the statistical analysis of transcript data clearly grouped SE developmental stages into seven important phases (Leaf, Dedifferentiation, Primary callus, Embryogenic callus, Embryogenic cell clusters, Redifferentiation and Embryo) enabling the identification of six key developmental phase switches, which are strategic for the overall biological efficiency of embryo regeneration. Differential gene expression and functional analysis showed that genes encoding transcription factors, stress-related genes, metabolism-related genes and hormone signaling-related genes were significantly enriched. Second, the standard environmental drivers used to control SE, i.e. light, growth regulators and cell density, were clearly perceived at the molecular level at different developmental stages. Third, expression profiles of auxin-related genes, transcription factor-related genes and secondary metabolism-related genes were analyzed during SE. Gene co-expression networks were also inferred. Auxin-related genes were upregulated during dedifferentiation and redifferentiation while transcription factor-related genes were switched on from the embryogenic callus and onward. Secondary metabolism-related genes were switched off during dedifferentiation and switched back on at the onset of redifferentiation. Secondary metabolites and endogenous IAA content were tightly linked with their respective gene expression. Lastly, comparing Arabica embryogenic and non-embryogenic cell transcriptomes enabled the identification of biological processes involved in the acquisition of embryogenic capacity.ConclusionsThe present analysis showed that transcript fingerprints are discriminating signatures of cell fate and are under the direct influence of environmental drivers. A total of 23 molecular candidates were successfully identified overall the 12 developmental stages and can be tested in many plant species to optimize SE protocols in a rational way.

Grape ASR Regulates Glucose Transport, Metabolism and Signaling.

To decipher the mediator role of the grape Abscisic acid, Stress, Ripening (ASR) protein, VvMSA, in the pathways of glucose signaling through the regulation of its target, the promoter of hexose transporter VvHT1, we overexpressed and repressed VvMSA in embryogenic and non-embryogenic grapevine cells. The embryogenic cells with organized cell proliferation were chosen as an appropriate model for high sensitivity to the glucose signal, due to their very low intracellular glucose content and low glycolysis flux. In contrast, the non-embryogenic cells displaying anarchic cell proliferation, supported by high glycolysis flux and a partial switch to fermentation, appeared particularly sensitive to inhibitors of glucose metabolism. By using different glucose analogs to discriminate between distinct pathways of glucose signal transduction, we revealed VvMSA positioning as a transcriptional regulator of the glucose transporter gene VvHT1 in glycolysis-dependent glucose signaling. The effects of both the overexpression and repression of VvMSA on glucose transport and metabolism via glycolysis were analyzed, and the results demonstrated its role as a mediator in the interplay of glucose metabolism, transport and signaling. The overexpression of VvMSA in the Arabidopsis mutant abi8 provided evidence for its partial functional complementation by improving glucose absorption activity.

Symplasmic isolation marks cell fate changes during somatic embryogenesis.

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.

Histocytological Study of Somatic Embryogenesis in the Tree Cinnamomum camphora L. (Lauraceae)

Histocytological studies were conducted on primary, secondary, and malformed embryos produced during somatic embryogenesis of Cinnamomum camphora L. to better understand its development. Exploring its callus types and structures provided a theoretical basis for clarifying the mechanism of somatic embryogenesis, which may shed light on the mechanism of zygotic embryogenesis. We used immature zygotic embryos as explants to induce somatic embryos, forming many embryogenic calli that differentiated into mature somatic embryos. Our results showed that somatic embryogenesis of C. camphora was similar to that of zygotic embryos. We have been dedifferentiated four types of callus. Compared with non-embryogenic cells, embryogenic cells had a closer arrangement, larger nucleus, thicker cytoplasm, more starch granules and easier to stain into black. Somatic embryogenesis had two pathways: direct (predominate) and indirect (rare). Embryogenic cells of C. camphora could have either an internal or external origin, the latter being primary, for which occurrence sites include epidermis and near-epidermis (little internally). Mostly arising from single cells, C. camphora follows two developmental pathways: single-cell equal as opposed to unequal, wherein both divide to form multi-cell proembryos. However, multicellular origins can occasionally occur and feature physiological isolation during somatic embryo development. This development has four embryo stages: globular, heart-shaped, torpedo, and cotyledon, with procambium cells apparent in globular embryos and late cotyledons forming “Y-shaped” vascular bundles. Secondary embryos were present in all stages, directly occurring on primary embryo’s germ and radicle end surfaces. We conclude that secondary and primary embryos of C. camphora undergo similar developmental processes. At the same time, conjoined cotyledon embryos and morphological abnormal embryos were found, with an internal origin more likely to generate abnormal embryos.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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NEP-TC a rRNA Methyltransferase Involved on Somatic Embryogenesis of Tamarillo (Solanum betaceum Cav.).

Somatic embryogenesis (SE) is an important biotechnological tool for large-scale clonal propagation and for embryogenesis research. Moreover, genetic transformation and cryopreservation procedures in many species rely on efficient SE protocols. We have been studying different aspects related to SE induction and somatic embryo development in tamarillo (Solanum betaceum Cav.), a small tree from the Solanaceae family. Previous proteomic analyses identified a protein (NEP-TC, 26.5 kDa) consistently present in non-embryogenic calluses of tamarillo, but absent in the embryogenic ones. In this work, the role of NEP-TC during SE was assessed by gene expression analysis and immunolocalization. The results obtained demonstrated that NEP-TC is a putative member of the SpoU rRNA methylase family. This protein, present in the cytoplasm and nucleus, is expressed in non-embryogenic cells and not expressed in embryogenic cells. Slightly enhanced SE induction levels in tamarillo plants with NEP-TC down-regulated levels also supports the role of this protein on SE induction. Heterologous expression was used to confirm NEP-TC rRNA methyltransferase activity, with enhanced activity levels when rRNA was used as a substrate. These data relate a putative member of the SpoU methylase family with plant morphogenesis, in particular with SE induction.

Identification and characterization of cell cultures with various embryogenic/regenerative potential in cotton based on morphological, cytochemical, and cytogenetical assessment

Somatic embryogenesis (SE) plays a vital role in genetic transformation and massive propagation of important agronomical and economical crops. Here, we conducted a systematic assessment of the morphological, cytochemical, and cytogenetical characteristics of six culture strains with various embryogenic/regenerative potential during SE process in cotton. Results indicated that the six cell culture strains had stable ploidy levels, and did not reveal any relationship between the cytogenetic state and their morphogenetic potential. Moreover, the six culture strains were compared via double staining with Evans blue and Acetocarmine to efficiently distinguish embryogenic and non-embryogenic cells and determine the embryogenic nature of the calli. In addition, the kind of auxins added in medium affected not only growth property, color, size of cell clumps but also ploidy level and regeneration ability. By combining analysis of morphological, cytochemical, and cytogenetical characteristics of the cell cultures, we are able to obtain and maintain homogeneous cell population with high morphogenic and regeneration ability and establish efficient somatic embryogenesis and regeneration system from short-term cell cultures in upland cotton, which highlight the application of biotechnological approaches in crop breeding, and above all, to better understand totipotency of cells in higher plants.

Somatic embryogenesis induction of litchi (Litchi chinensis Sonn.) from leaves of mature trees

Induction and development somatic embryos of litchi (Litchi chinensis Sonn.) from leaflet explants obtained from mature litchi trees were investigated. Somatic embryogenesis was significantly influenced by the cultivar, plant growth regulators, position of leaflet and orientation of leaf explants. Adaxial-face up orientation of leaf explants significantly enhanced embryogenesis in comparison with adaxial-face down orientation. Maximum number of somatic embryos were obtained using median position from basal leaflet with adaxial-face up orientation cultured on Murashige and Skoog (MS) medium supplemented with 1.0 mg/l 2, 4 dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l 6-benzylaminopurine (BA) (37.67). Two types of salts media (MS and Gamborg’s B5) as well as different sucrose concentrations were examined for development and maturation of somatic embryos. MS salts medium with 45g/l sucrose was effective on size of mature somatic embryos (8.00 > 0.5 cm SE and 6.33 < 0.5 cm SE) compared with B5 salts medium (4.33 > 0.5 cm SE and 4.66 < 0.5 cm SE) on the same concentration of sucrose. Development of somatic embryos occurred on the media without growth regulators, heart and torpedo stage somatic embryos were obtained. Somatic embryos enlarged more and turned to milky opaque in color and finally reached to mature milky cotyledonary stage. Scanning electron microscopy revealed different morphological development of somatic embryos. In addition, Anatomical observation showed differences in the embryogenic and non-embryogenic cells. Furthermore, anatomical observation for development stages of somatic embryos.

Identification and expression analysis of genes involved in somatic embryogenesis of banana

Somatic embryogenesis (SE) is a process in which somatic cells go through morphological and biochemical changes to form somatic embryos. These embryos are capable of regenerating into new plants. The present study aims to understand the molecular basis of these changes during the different stages of SE in banana cv. Grand Naine. 16 SE related genes belonging to 8 gene families i.e. PINHEADED (PIN), YUCCA (YUC), PICKLE (PKL), TOPOISOMERASE (TOP), HISTINE KINASES (CRE), SOMATIC CELL RECEPTOR KINASE (SERK), GLOBULIN (GLB), and KNOTTED (KNAT) were identified from the banana genome database. In-silico analysis of all 16 genes was performed to find out their structural features, phylogenetic relationship, subcellular, and chromosomal localization. Phylogenetic analysis of these genes with their homologs in other plant species (gymnosperm, monocot, and dicot) confirmed their close evolutionary association with monocot. We studied the differential expression pattern of all 16 genes for the exposure of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in callus, embryogenic cell suspension (ECS), and non-embryogenic cell suspension (NECS) cultures of cv. Grand Naine. The higher expression of MaPIN1 in embryogenic callus and ECS suggests its possible role in SE of banana. The prominent expression of MaCRE2 and MaCRE3 found in NECS revealed that these genes might be involved in other cell functions except embryogenesis. The present study reported that MaPIN1 could be a potential marker for the embryogenicity in banana.

Comparative metabolomics and glycolysis enzyme profiling of embryogenic and nonembryogenic grape cells.

A novel biological model was created for the comparison of grapevine embryogenic cells (EC) and nonembryogenic cells (NEC) sharing a common genetic background but distinct phenotypes, when cultured on their respective most appropriate media. Cytological characterization, 1H‐NMR analysis of intracellular metabolites, and glycolytic enzyme activities provided evidence for the marked metabolic differences between EC and NEC. The EC were characterized by a moderate and organized cell proliferation, coupled with a low flux through glycolysis, high capacity of phosphoenolpyruvate carboxylase and glucokinase, and high oxygen consumption. The NEC displayed strong anarchic growth, and their high rate of glycolysis due to the low energetic efficiency of the fermentative metabolism is confirmed by increased enolase capacity and low oxygen consumption.

Profiling the onset of somatic embryogenesis in Arabidopsis

BackgroundTotipotency is the ability of a cell to regenerate a whole organism. Plant somatic embryogenesis (SE) is a remarkable example of totipotency because somatic cells reverse differentiation, respond to an appropriate stimulus and initiate embryo development. Although SE is an ideal system to investigate de-differentiation and differentiation, we still lack a deep molecular understanding of the phenomenon due to experimental restraints.ResultsWe applied the INTACT method to specifically isolate the nuclei of those cells undergoing SE among the majority of non-embryogenic cells that make up a callus. We compared the transcriptome of embryogenic cells to the one of proliferating callus cells. Our analyses revealed that embryogenic cells are transcriptionally rather than metabolically active. Embryogenic cells shut off biochemical pathways involved in carbohydrate and lipid metabolism and activate the transcriptional machinery. Furthermore, we show how early in SE, ground tissue and leaf primordia specification are switched on before the specification of a shoot apical meristem.ConclusionsThis is the first attempt to specifically profile embryogenic cells among the different cell types that constitute plant in vitro tissue cultures. Our comparative analyses provide insights in the gene networks regulating SE and open new research avenues in the field of plant regeneration.

Establishment and biochemical characterization of tamarillo (Solanum betaceum Cav.) embryogenic cell suspension cultures

Somatic embryogenesis (SE) is an important biotechnological tool with great potential for large-scale cloning. In Solanum betaceum Cav. (tamarillo), embryogenic (EC) and non-embryogenic (non-EC) cells can be obtained from the same explant on auxin-containing medium, making this system ideal for the evaluation of biochemical changes occurring during embryogenic induction. Liquid cultures offer additional possibilities for the analysis of factors controlling SE induction, and the main objectives here were the establishment of cell suspensions and the characterization of the extracellular protein profiles in EC and non-EC cultures. Growth kinetics of liquid cultures, starting with different amounts of EC or non-EC callus or with different weight per volume ratios, were analyzed. Embryogenic suspension cultures were efficiently established starting with 40 mg of cells in 20 mL of liquid medium. Mass spectrometry and fluorometric techniques were employed to identify extracellular proteins, their hydrolytic activity, and the main classes of proteases secreted into the media of EC or non-EC cultures. Extracellular protein profiles revealed quantitative and qualitative differences between EC and non-EC suspension cultures, mainly for several hydrolytic enzymes, such as glucanases and xylanases. Proteolytic activity analysis found serine proteases, aspartic proteases, and metalloproteases in EC cultures, whereas serine proteases were dominant in non-EC lines. For the first time, a protocol for the growth of tamarillo EC and non-EC suspensions was achieved. Moreover, the comparison of protein profiles between EC and non-EC lines showed pronounced differences in the proteolytic and glycolytic enzymes secreted.

Genome-wide analysis of transcription factors during somatic embryogenesis in banana (Musa spp.) cv. Grand Naine.

Transcription factors BABY BOOM (BBM), WUSCHEL (WUS), BSD, LEAFY COTYLEDON (LEC), LEAFY COTYLEDON LIKE (LIL), VIVIPAROUS1 (VP1), CUP SHAPED COTYLEDONS (CUC), BOLITA (BOL), and AGAMOUS LIKE (AGL) play a crucial role in somatic embryogenesis. In this study, we identified eighteen genes of these nine transcription factors families from the banana genome database. All genes were analyzed for their structural features, subcellular, and chromosomal localization. Protein sequence analysis indicated the presence of characteristic conserved domains in these transcription factors. Phylogenetic analysis revealed close evolutionary relationship among most transcription factors of various monocots. The expression patterns of eighteen genes in embryogenic callus containing somatic embryos (precisely isolated by Laser Capture Microdissection), non-embryogenic callus, and cell suspension cultures of banana cultivar Grand Naine were analyzed. The application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in the callus induction medium enhanced the expression of MaBBM1, MaBBM2, MaWUS2, and MaVP1 in the embryogenic callus. It suggested 2, 4-D acts as an inducer for the expression of these genes. The higher expression of MaBBM2 and MaWUS2 in embryogenic cell suspension (ECS) as compared to non-embryogenic cells suspension (NECS), suggested that these genes may play a crucial role in banana somatic embryogenesis. MaVP1 showed higher expression in both ECS and NECS, whereas MaLEC2 expression was significantly higher in NECS. It suggests that MaLEC2 has a role in the development of non-embryogenic cells. We postulate that MaBBM2 and MaWUS2 can be served as promising molecular markers for the embryogencity in banana.

Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba

Somatic embryogenesis is considered a convenient tool for investigating the regulating mechanisms of embryo formation; it is also a feasible system for in vitro regeneration procedures, with many advantages in woody species. Nevertheless, trees have shown recalcitrance to somatic embryogenesis, and its efficiency remains very low in many cases. Consequently, despite the clear potential of somatic embryogenesis in tree breeding programs, its application is limited since factors responsible for embryogenesis initiation have not yet been completely elucidated.In the present work, we investigated key cellular factors involved in the change of developmental program during leaf somatic embryogenesis initiation of white oak (Quercus alba), aiming to identify early markers of the process. The results revealed that pectin esterification, auxin accumulation and DNA demethylation were induced during embryogenesis initiation and differentially found in embryogenic cells, while they were not present in leaf cells before induction or in non-embryogenic cells after embryogenesis initiation. These three factors constitute early markers of leaf embryogenesis and represent processes that could be interconnected and involved in the regulation of cell reprogramming and embryogenesis initiation.These findings provide new insights into the mechanisms underlying plant cell reprogramming, totipotency and embryogenic competence acquisition, especially in tree species for which information is scarce, thus opening up the possibility of efficient manipulation of somatic embryogenesis induction.

Expression of a fungal ferulic acid esterase in suspension cultures of tall fescue (Festuca arundinacea) decreases cell wall feruloylation and increases rates of cell wall digestion

In the cell walls of grasses ferulic acid is esterified to arabinosyl residues in arabinoxylans that can then undergo oxidative coupling reactions to form ferulate dehydrodimers, trimers and oligomers which function to cross-link cell-wall polysaccharides, limiting cell wall degradability. Fungal ferulic acid esterase can release both esterified monomeric and dimeric ferulic acids from these cell wall arabinoxylans making the cell wall more susceptible to further enzymatic attack and increasing cell wall degradability. Non-embryogenic cell suspension cultures of Festuca arundinacea expressing a Aspergillus niger ferulic acid esterase (faeA) targeted to either the apoplast, or endoplasmic reticulum under the control of a constitutive actin promoter, or to the vacuole under the control of a soybean heat shock promoter, were established and FAE activity determined in the cells and medium during a growth cycle. Analysis of the ester-linked ferulates of the cell walls showed that all three transformed cell lines had both reduced ferulate levels and increased levels of xylanase mediated release of wall phenolics on autodigestion as well as increased rates of cell wall digestion in a simulated rumen environment, when compared to control non-transformed cells.

ARS-Media for Excel: A Spreadsheet Tool for Calculating Media Recipes Based on Ion-Specific Constraints.

ARS-Media for Excel is an ion solution calculator that uses “Microsoft Excel” to generate recipes of salts for complex ion mixtures specified by the user. Generating salt combinations (recipes) that result in pre-specified target ion values is a linear programming problem. Excel’s Solver add-on solves the linear programming equation to generate a recipe. Calculating a mixture of salts to generate exact solutions of complex ionic mixtures is required for at least 2 types of problems– 1) formulating relevant ecological/biological ionic solutions such as those from a specific lake, soil, cell, tissue, or organ and, 2) designing ion confounding-free experiments to determine ion-specific effects where ions are treated as statistical factors. Using ARS-Media for Excel to solve these two problems is illustrated by 1) exactly reconstructing a soil solution representative of a loamy agricultural soil and, 2) constructing an ion-based experiment to determine the effects of substituting Na+ for K+ on the growth of a Valencia sweet orange nonembryogenic cell line.

MRI-Based Visualization of the Relaxation Times of Early Somatic Embryos

Abstract The large set of scientific activities supported by MRI includes, among others, the research of water and mineral compounds transported within a plant, the investigation of cellular processes, and the examination of the growth and development of plants. MRI is a method of major importance for the measurement of early somatic embryos (ESE) during cultivation, and in this respect it offers several significant benefits discussed within this paper. We present the following procedures: non-destructive measurement of the volume and content of water during cultivation; exact three-dimensional differentiation between the ESEs and the medium; investigation of the influence of ions and the change of relaxation times during cultivation; and multiparametric segmentation of MR images to differentiate between embryogenic and non-embryogenic cells. An interesting technique consists in two-parameter imaging of the relaxation times of the callus; this method is characterized by tissue changes during cultivation at a microscopic level, which can be monitored non-destructively.

A reliable protocol for the stable transformation of non-embryogenic cells cultures of grapevine (Vitis vinifera L.) and Taxus x media

One of the major intent of metabolic engineering in cell culture systems is to increase yields of secondary metabolites. Efficient transformation methods are a priority to successfully apply metabolic engineering to cell cultures of plants that produce bioactive or therapeutic compounds, such as Vitis vinifera and Taxus x media. The aim of this study was to establish a reliable method to transform non-embryogenic cell cultures of these species. The V. vinifera cv. Gamay/cv. Monastrell cell lines and Taxus x media were used for Agrobacterium-mediated transformation using the Gateway-compatible Agrobacterium sp. binary vector system for fast reliable DNA cloning. The Taxus x media and Vitis cell lines were maintained in culture for more than 4 and 15 months, respectively, with no loss of reporter gene expression or antibiotic resistance. The introduced genes had no discernible effect on cell growth, or led to extracellular accumulation of phytoalexin trans-Resveratrol (t-R) in response to elicitation with methylated cyclodextrins (MBCD) and methyl jasmonate (MeJA) in the grapevine transgenic cell lines compared to the parental control. The method described herein provides an excellent tool to exploit exponentially growing genomic resources to enhance, optimize or diversify the production of bioactive compounds generated by grapevine and yew cell cultures, and offers a better understanding of many grapevine and yew biology areas.

Morphohistological and molecular profiles during the developmental stages of somatic embryogenesis of Musa acuminata cv. ‘Berangan’ (AAA)

The present study established a protocol for generating embryogenic cell suspension (ECS) cultures from an early stage embryogenic callus of Musa acuminata cv. ‘Berangan’. The necessary baseline data for morphohistological recognition and the selected gene expression for differentiating embryogenic and non-embryogenic cells were defined to enable early selection, as well as for quality assessment, which could aid in scaling up the production of these cells. In this study, male bud-derived callus on Murashige and Skoog (MS) medium containing 23 μM 2,4-dichlorophenoxyacetic acid (2,4-D) was transferred to M1 medium containing different concentrations of 2,4-D and picloram for somatic embryo initiation. ECSs established in liquid MS medium (M2a) containing 4.1 μM biotin, 10 mg/l ascorbic acid, 100 mg/l glutamine, 100 mg/l malt extract, 4.5 μM 2,4-D, 1.0 μM zeatin, and 20 g/l sucrose were used to study embryogenic development. The frequency of embryo development and maturation was influenced by the culture media. The highest frequency of embryo development (96.4 %) and maturation (66.4 %) was achieved on solid MS hormone-free medium. Almost 69 % of embryos were induced to form shoots on the MS medium containing 34 μM 6-benzylaminopurine (BAP). Histological study indicated that only embryos with distinct layer of protoderm having cells with prominent nucleus and dense cytoplasm were able to regenerate into plantlets. Endochitinase and phenylalanine ammonia-lyase (PAL) genes were expressed at all the developmental stages, and their expression level was tightly regulated during embryo development and reached its maximum in germinating embryos. Expression of these genes in embryogenic cells was higher than in non-embryogenic cells. The study findings suggested that these genes play an important role in the developmental stage and regeneration pathway of somatic embryos of banana.

Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L.

BackgroundIn Quercus suber, cork oak, a Mediterranean forest tree of economic and social interest, rapid production of isogenic lines and clonal propagation of elite genotypes have been achieved by developing in vitro embryogenesis from microspores and zygotic embryos respectively. Despite its high potential in tree breeding strategies, due to their recalcitrancy, the efficiency of embryogenesis in vitro systems in many woody species is still very low since factors responsible for embryogenesis initiation and embryo development are still largely unknown. The search for molecular and cellular markers during early stages of in vitro embryogenesis constitutes an important goal to distinguish, after induction, responsive from non-responsive cells, and to elucidate the mechanisms involved in embryogenesis initiation for their efficient manipulation. In this work, we have performed a comparative analysis of two embryogenesis pathways derived from microspores and immature zygotic embryos in cork oak in order to characterize early markers of reprogrammed cells in both pathways. Rearrangements of the cell structural organization, changes in epigenetic marks, cell wall polymers modifications and endogenous auxin changes were analyzed at early embryogenesis stages of the two in vitro systems by a multidisciplinary approach.ResultsResults showed that early embryo cells exhibited defined changes of cell components which were similar in both embryogenesis in vitro systems, cellular features that were not found in non-embryogenic cells. DNA methylation level and nuclear pattern, proportion of esterified pectins in cell walls, and endogenous auxin levels were different in embryo cells in comparison with microspores and immature zygotic embryo cells from which embryos originated, constituting early embryogenesis markers.ConclusionsThese findings suggest that DNA hypomethylation, cell wall remodeling by pectin esterification and auxin increase are involved in early in vitro embryogenesis in woody species, providing new evidences of the developmental pattern similarity between both embryogenesis pathways, from microspores and immature zygotic embryos, in woody species.