Loss of DNA methylation affects somatic embryogenesis in Medicago truncatula

To investigate the involvement of methylation of DNA in somatic embryogenesis we initiated a comparative study using Medicago truncatula lines that have different capacities to produce somatic embryos. Treatment with the demethylating drug 5-azacytidine caused a loss of regeneration capacity in the embryogenic line by arresting the production of somatic embryos. Analysis with methylation-sensitive enzymes showed disruption of somatic embryogenesis competence to be correlated with rDNA demethylation. Our data suggest production of somatic embryos depends on a certain level of DNA methylation.     

Epigenetics of long-term somatic embryogenesis in <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.: DNA methylation and recovery of embryogenic potential

In Theobroma cacao L., declined embryogenic potential was observed in regenerated somatic embryos from long-term secondary somatic embryogenesis (SE). In order to explore the relationship between DNA methylation and the long-term secondary SE, the embryogenic potential and global DNA methylation levels in young (12 months-old), aged (36 months-old) and extra somatic embryogenesis (39 months-old) subjected to different 5-Azacytidine (5-azaC) treatments were comparatively assessed. Global DNA methylation levels increased in aged somatic embryos with long-term in vitro culture, but 5-azaC-supplemented treatments resulted in unaltered levels. In addition, DNA methylation pattern during SE was not affected by 5-azaC. DNA methylation increased during SE expression. Interestingly, the extra SE induction showed that aged somatic embryos can recovery the embryogenic potential in treatment supplemented with 5-azaC at specific concentration. The outcome of this study suggested that the long-term SE in cacao induced the decline on embryogenic potential, which can be reversible trough 5-azaC supplementation. Besides, increased DNA methylation levels might be a response to the stress conditions that plant cells were exposed to during SE.     

Cloning mature holm oak trees by somatic embryogenesis

Key message

Integuments from holm oak developing ovules were suitable initial explants to obtain embryogenic lines from which plants could be regenerated.

Abstract

The implementation of multivarietal forestry as part of breeding strategies is expected to provide more productive forest plantations. To achieve this, a reliable and effective method for mass production of clonal plants is needed. Somatic embryogenesis is considered the enabling technology for implementing multivarietal forestry. The holm oak (Quercus ilex L.) is a Mediterranean evergreen tree of economic interests because of the acorn production for animal feed and edible fungi mycorrhization. The aim of this work was to obtain clonal plants by inducing somatic embryogenesis in tissues of female flowers from mature trees. The influence of the developmental stage of the explant, the genotype and medium composition, and the effect of arabinogalactan proteins on the induction frequency, were assessed. Somatic embryogenesis induction (frequency ranging from 1.2 to 3.2 %) was restricted to ovules excised at an advanced stage of development, when they were at least 3–4 mm wide and the rest of the ovules within the ovary had aborted. Somatic embryos arose from the integuments of those fertilized ovules. Embryogenic response was obtained on media with and without plant growth regulators. All the genotypes that were cultured on medium containing “as reported by Schenk and Hildebrandt (Can J Bot 50:199–204, 1972)” SH macronutrients could be captured. Treatments including Larix arabinogalactan proteins did not improve induction, while those from Acacia inhibited the embryogenic response. Several embryogenic lines were multiplied by repetitive embryogenesis on medium lacking plant growth regulators. Mature somatic embryos of three genotypes were germinated at frequencies ranging from 41 to 58 %, and converted into plants at frequencies from 11 to 30 %, depending on the genotype.     

Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor

Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot.     

The Arabidopsis Somatic Embryogenesis Receptor Kinase 1 Gene Is Expressed in Developing Ovules and Embryos and Enhances Embryogenic Competence in Culture

We report here the isolation of the Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (AtSERK1) gene and we demonstrate its role during establishment of somatic embryogenesis in culture. The AtSERK1 gene is highly expressed during embryogenic cell formation in culture and during early embryogenesis. The AtSERK1 gene is first expressed in planta during megasporogenesis in the nucellus [corrected] of developing ovules, in the functional megaspore, and in all cells of the embryo sac up to fertilization. After fertilization, AtSERK1 expression is seen in all cells of the developing embryo until the heart stage. After this stage, AtSERK1 expression is no longer detectable in the embryo or in any part of the developing seed. Low expression is detected in adult vascular tissue. Ectopic expression of the full-length AtSERK1 cDNA under the control of the cauliflower mosaic virus 35S promoter did not result in any altered plant phenotype. However, seedlings that overexpressed the AtSERK1 mRNA exhibited a 3- to 4-fold increase in efficiency for initiation of somatic embryogenesis. Thus, an increased AtSERK1 level is sufficient to confer embryogenic competence in culture.     

Acquisition of embryogenic competence during somatic embryogenesis

This review focuses on investigation in acquisition of embryogenic competence during somatic embryogenesis in the last five decades. In tissue culture, differentiated somatic cells acquire embryogenic competence and proliferate as embryogenic cells during the induction phase. These embryogenic cells are important because they differentiate to form somatic embryos at a later time. Various molecular and structural markers for detecting embryogenic cells or enhancing embryogenic competence are summarized and implications of the findings are discussed.     

Inheritance of the ability for regeneration via somatic embryogenesis in Cyclamen persicum

Objective of the studies was to analyse the mode of inheritance of the regeneration ability of Cyclamen persicum via somatic embryogenesis. Three genotypes inbred to I differing concerning their in vitro response were selfand cross-pollinated to produce I , F and F progenies. These progenies were tested in vitro by culturing unpollinated ovules on callus induction medium containing 9.05 mM 2,4-D (2,4-dichlorophenoxyacetic acid) and 3.94     

Genetic improvement of the somatic embryogenesis and regeneration in soybean and transformation of the improved breeding lines

Somatic embryos of soybean [Glycine max (L.) Merrill] have been used to generate transgenic plants by particle bombardment. The induction and proliferation of somatic embryos from immature cotyledons are dependent on the genotype of the cultivar. Whereas somatic embryogenesis and plant regeneration are inefficient in most cultivars, they are efficient in the cultivar Jack. We previously established a breeding line, QF2, by the integration of null mutations of each subunit of the major seed storage proteins glycinin and β-conglycinin, but the embryogenic response of this line is insufficient to allow efficient transformation. We have now backcrossed QF2 to cultivar Jack in order to combine the null traits with competence for somatic embryogenesis. The backcrossed breeding lines selected on the basis of the absence of the major storage proteins exhibited an improved capacity for the induction and proliferation of somatic embryos compared with that of QF2. The induced somatic embryogenic tissue of these breeding lines was successfully used for the production of transgenic plants by particle bombardment. These results also indicate that somatic embryogenesis in soybean is genetically controlled and inherited in a manner independent of the null traits of the major seed storage proteins.     

Evidence for in vitro induced mutation which improves somatic embryogenesis in Asparagus officinalis L.

Summary Somatic embryogenesis from different genotypes of Asparagus officinalis L. could be obtained by in vitro culture of shoot apices. Apices were first cultured on an auxin-rich inducing medium and then transferred onto a hormone-free development medium. All genotypes tested in this way produced a few somatic embryos. In some experiments, during the development phase, a new kind of friable highly embryogenic tissue appeared in a random manner. These tissues could be continuously subcultured on a hormone-free medium and were named embryogenic lines. Five of these embryogenic lines regenerated plants from somatic embryos. These regenerated plants exhibited an increased embryogenic response compared to the parent plants; e.g. apex culture produced somatic embryos without any auxin treatments. For one of the embryogenic lines, a genetic analysis showed that the improved embryogenic response of regenerated plants was controlled by a mendelian dominant monogenic mutation.Abbreviations LSEA low somatic embryogenesis ability - HSEA high somatic embryogenesis ability - NAA 1-naphthaleneacetic acid     

New insights into plant somatic embryogenesis: an epigenetic view

Somatic embryogenesis plays a significant role in plant regeneration and requires complex cellular, molecular, and biochemical processes for embryo initiation and development associated with plant epigenetics. Epigenetic regulation encompasses many sensitive events and plays a vital role in gene expression through DNA methylation, chromatin remodelling, and small RNAs. Recently, regulation of epigenetic mechanisms has been recognized as the most promising occurrences during somatic embryogenesis in plants. A few reports demonstrated that the level of DNA methylation can alter in embryogenic cells under in vitro environments. Changes or modification in DNA methylation patterns is linked with regulatory mechanisms of various candidate marker genes, involved in the initiation and development of somatic embryogenesis in plants. This review summarizes the current scenario of the role of epigenetic mechanisms as candidate markers during somatic embryogenesis. It also delivers a comprehensive and systematic analysis of more recent discoveries on expression of embryogenic-regulating genes during somatic embryogenesis, epigenetic variation. Biotechnological applications of epigenetics as well as new opportunities or future perspectives in the development of somatic embryogenesis studies are covered. Further research on such strategies may serve as exciting interaction models of epigenetic regulation in plant embryogenesis and designing novel approaches for plant productivity and crop improvement at molecular levels.     

Characterization of a dehydrin-like phosphoprotein (ECPP-44) relating to somatic embryogenesis in carrot

Somatic embryogenesis in carrot can be induced by the treatment of shoot apices with various kinds of stress chemicals. Using this system, we previously identified a phosphoprotein (ECPP-44) that appears to be involved in the induction of somatic embryogenesis. We have also isolated and characterized a cDNA encoding ECPP-44. In this study, to further characterize ECPP-44, we performed Western blot and immuno-precipitation analyses. Western blot analysis revealed that ECPP-44 was present in embryogenic cells, stress- and non-stress-treated tissues, and somatic embryos but was absent in non-embryogenic cells. Furthermore, ECPP-44 was found in some parts of the carrot plant, such as tap roots, leaves, and flowers (18–26 days after fertilization) but not in mature dry seeds. Interestingly, we could detect phosphorylated ECPP-44 in embryogenic cells and somatic embryos but not in non-embryogenic cells, tap roots, and non-stress-treated shoot apices by immunoprecipitation analysis, even though the protein existed. Our results suggest that ECPP-44 may perform some role in the induction or maintenance of embryogenic competence.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

Induction of somatic embryogenesis in woody plants

Somatic embryogenesis, the in vitro developmental program by which somatic cells are reprogrammed to undergo cellular and molecular changes that make them competent to produce somatic embryos, has been achieved with many woody plants. The program involves the stages of competence acquisition, induction and expression of the morphogenic pathway by the cultured cells and tissues. The ability to express the program in cultured cells/tissues is regulated by many factors, including genotype, explant type and age and culture conditions. In many woody plants, somatic embryogenesis was achieved with mature, immature explants or both. Juvenile tissues as immature and mature zygotic embryos are regarded best explants to establish embryogenic cultures in woody plants and potential to obtain the cultures decline with increasing maturity of the explant.     

Somatic Embryogenesis: Identified Factors that Lead to Embryogenic Repression. A Case of Species of the Same Genus

Somatic embryogenesis is a powerful biotechnological tool for the mass production of economically important cultivars. Due to the cellular totipotency of plants, somatic cells under appropriate conditions are able to develop a complete functional embryo. During the induction of somatic embryogenesis, there are different factors involved in the success or failure of the somatic embryogenesis response. Among these factors, the origin of the explant, the culture medium and the in vitro environmental conditions have been the most studied. However, the secretion of molecules into the media has not been fully addressed. We found that the somatic embryogenesis of Coffea canephora, a highly direct embryogenic species, is disrupted by the metabolites secreted from C. arabica, a poorly direct embryogenic species. These metabolites also affect DNA methylation. Our results show that the abundance of two major phenolic compounds, caffeine and chlorogenic acid, are responsible for inhibiting somatic embryogenesis in C. canephora.     

Somatic embryogenesis in oak (<Emphasis Type="Italic">Quercus</Emphasis> spp.)

Summary The present review summarizes the factors involved in controlling the process of oak somatic embryogenesis as a method for vegetative plant propagation and includes also data on artificial seed production, cryopreservation and transformation. One major limitation, the inability to initiate embryogenic cultures from mature trees, has been recently overcome. Leaves from selected cork oak trees with an age of 50 yr and more have been used to initiale somatic embryogenesis (SE) with a frequency of up to 20%. These findings offer encouraging prospects for cloning proven superior plant material and to integrate this propagation system into tree improvement programs. Once the process of SE has been initiated, the multiplication cycle proceeds via secondary embryogenesis, which can be maintained indefinitely. Problems are reported by the formation of anomalous embryos. The mutability of somatic embryogenic cell lines of various oak species has been monitored by flow cytometry and molecular markers. No somaclonal variation was detected applying random amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers, whereas DNA-content measurements via flow cytometry revealed tetraploidy in some cell lines after several years of continuous subculture. Maturation and low germination frequencies are the main bottlenecks for a broader use of this technique. Recently attention has been on embryo quality and parameters for conversion capacity such as high endogenous cytokinin level and low abscisic acid (ABA) level. Although oak is probably the species that is the most well-developed system for a broadleaved forest tree, data on growth performances of somatic embryo-derived plants are rare.     

Histology of somatic embryogenesis in <Emphasis Type="Italic">Coffea arabica</Emphasis> L.

The objective of this study was to characterize the histodifferentiation of somatic embryogenesis obtained from leaf explants of C. arabica. Therefore, we histologically analyzed the respective stages of the process: leaf segments at 0, 4, 7, 15 and 30 days of cultivation, Type 1 primary calli (primary calli with embryogenic competence) and 2 (primary calli with no embryogenic competence), embryogenic calli, globular, torpedo and cotyledonary embryos, and mature zygotic embryos. Callus formation occurred after seven days of culture, with successive divisions of procambium cell. In this cultivation phase, it was found that Type 1 primary calli are basically formed by parenchymal cells with reduced intercellular spacing, whereas Type 2 primary calli are predominantly composed of parenchymal cells with ample intercellular spaces and embryogenic calli composed entirely of meristematic cells. After 330 days, it was evident from the differentiation of somatic embryogenesis that there was formation of globular somatic embryos, consisting of a characteristic protoderm surrounding the fundamental meristem. With the maturation of these propagules after 360 days, torpedo-stage somatic embryos arose, in which tissue polarization and early differentiation of procambial strands were verified. After 390 days, cotyledonary somatic embryos were obtained, where the onset of vessel elements differentiation was verified, a characteristic also observed in mature zygotic embryos. We concluded that somatic embryogenesis obtained from C. arabica leaves initiates from procambium cell divisions that, in the course of cultivation, produce mature somatic embryos suitable for regenerating whole plants.     

New Insights into Somatic Embryogenesis: LEAFY COTYLEDON1, BABY BOOM1 and WUSCHEL-RELATED HOMEOBOX4 Are Epigenetically Regulated in Coffea canephora

Plant cells have the capacity to generate a new plant without egg fertilization by a process known as somatic embryogenesis (SE), in which differentiated somatic cells can form somatic embryos able to generate a functional plant. Although there have been advances in understanding the genetic basis of SE, the epigenetic mechanism that regulates this process is still unknown. Here, we show that the embryogenic development of Coffea canephora proceeds through a crosstalk between DNA methylation and histone modifications during the earliest embryogenic stages of SE. We found that low levels of DNA methylation, histone H3 lysine 9 dimethylation (H3K9me2) and H3K27me3 change according to embryo development. Moreover, the expression of LEAFY COTYLEDON1 (LEC1) and BABY BOOM1 (BBM1) are only observed after SE induction, whereas WUSCHEL-RELATED HOMEOBOX4 (WOX4) decreases its expression during embryo maturation. Using a pharmacological approach, it was found that 5-Azacytidine strongly inhibits the embryogenic response by decreasing both DNA methylation and gene expression of LEC1 and BBM1. Therefore, in order to know whether these genes were epigenetically regulated, we used Chromatin Immunoprecipitation (ChIP) assays. It was found that WOX4 is regulated by the repressive mark H3K9me2, while LEC1 and BBM1 are epigenetically regulated by H3K27me3. We conclude that epigenetic regulation plays an important role during somatic embryogenic development, and a molecular mechanism for SE is proposed.     

Somatic embryogenesis in holm oak male catkins

Somatic embryogenesis (SE) of tree species is the most promising method for the implementation of multivarietal forestry and for biotechnological approaches. To date, however, the application of this technology to mature trees is restricted to a few species. This is the first report on the induction of SE from male catkins of 100-year-old holm oaks (Quercus ilex L.). Embryogenic competence was mainly dependent on genotype and restricted to the most advanced catkin developmental stage with distinguishable closed flowers along the axis. Following a three-stage treatment procedure, embryogenic response (frequencies up 3.3 %) was obtained in three [Remedio, Villar del Arzobispo (VA) and Hunde (HU)] out of the five genotypes evaluated. In the culture conditions tested, the preferred protocol to induce SE in holm oak catkins should include: induction on MS medium with 6-benzyladenine and naphthaleneacetic acid, subculture onto medium with a reduced concentration of both plant growth regulators and a final transference to medium without growth regulators. Under these conditions, cotyledonary-stage somatic embryos developed from brown calli with or without nodular structures. Secondary SE, favored by the addition of sorbitol to the manifestation medium, allowed the establishment of 14 embryogenic lines belonging to VA and HU genotypes. Histological observations of the proliferating cultures revealed the presence of globular, torpedo and cotyledonary somatic embryos. Somatic embryos were diploid as verified by flow cytometry analysis, suggesting that they originated from the perianthic tissue of the male flower.