Developmental pathways of somatic embryogenesis

Somatic embryogenesis is defined as a process in which a bipolar structure, resembling a zygotic embryo, develops from a non-zygotic cell without vascular connection with the original tissue. Somatic embryos are used for studying regulation of embryo development, but also as a tool for large scale vegetative propagation. Somatic embryogenesis is a multi-step regeneration process starting with formation of proembryogenic masses, followed by somatic embryo formation, maturation, desiccation and plant regeneration. Although great progress has been made in improving the protocols used, it has been revealed that some treatments, coinciding with increased yield of somatic embryos, can cause adverse effects on the embryo quality, thereby impairing germination and ex vitro growth of somatic embryo plants. Accordingly, ex vitro growth of somatic embryo plants is under a cumulative influence of the treatments provided during the in vitro phase. In order to efficiently regulate the formation of plants via somatic embryogenesis it is important to understand how somatic embryos develop and how the development is influenced by different physical and chemical treatments. Such knowledge can be gained through the construction of fate maps representing an adequate number of morphological and molecular markers, specifying critical developmental stages. Based on this fate map, it is possible to make a model of the process. The mechanisms that control cell differentiation during somatic embryogenesis are far from clear. However, secreted, soluble signal molecules play an important role. It has long been observed that conditioned medium from embryogenic cultures can promote embryogenesis. Active components in the conditioned medium include endochitinases, arabinogalactan proteins and lipochitooligosaccharides.     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统.整个体细胞胚胎发生过程主要包括3个步骤:胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换.过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理.然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量.此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要.因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义.     

松柏类植物体细胞胚胎发生的研究进展

松柏类植物的体细胞胚胎发生既是繁育的一种手段,又是研究胚胎发育过程中结构、生理和分子事件的一种重要的模式系统。整个体细胞胚胎发生过程主要包括3个步骤：胚性组织的诱导和增殖、体细胞胚的成熟以及体细胞胚的萌发和转换。过去为了提高胚胎发育过程所做的努力主要都集中在胚的成熟阶段,这是因为一直认为能否成功再生的关键在于胚发育成熟阶段的处理。然而,在过去几年里,结合生理生化以及分子生物学的研究发现,胚胎发生的早期阶段对于完成整个发育过程也是至关重要的,早期阶段培养条件的优化可以显著提高培养过程中体细胞胚的数量和质量。此外,萌发过程培养条件的调节对于提高成熟体细胞胚的萌发率和转换率也很重要。因此,这些新的研究成果对于改善松柏类植物体细胞胚胎发生中的胚的诱导率和转换率低的现象具有重要的意义。     

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.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Efficiency,genotypic variability,and cellular origin of primary and secondary somatic embryogenesis of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.

Summary The development of efficient tissue culture systems for cacao holds the potential to contribute to the improvement of this tropical erop by providing a rapid and efficient vegetative propagation system for multiplication of elite genotypes. It may also find application in facilitation of germplasm movement across quarantine borders, enhancement of germplasm conservation via cryo-preservation, and development of genetic transformation systems. Somatic embryogenesis using floral tissue explants was previously the only tissue culture procedure for regeneration of cacao. We report the development of a secondary embryogenesis system utilizing primary somatic embryo cotyledon explants, which results in up to a 30-fold increase in somatic embryo production compared to primary somatic embryogenesis. The influence of genotype on the efficiency of the system was evaluated. To understand the cellular origins and developmental pathways operative in this system, we investigated the morphological changes occurring over time using light and scanning electron microscopy. While primary embryos arise from clusters of cells forming embryonic nodules, secondary embryos arise predominantly from the division of single cells, in a pathway reminiscent of zygotic embryogenesis. These results have important significance to the application of tissue culture to cacao improvement programs.     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis

The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E) and non-embryogenic (NE) callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L^-1) of activated charcoal (AC). Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L^-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days) in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project), including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.     

秦艽(Gentiana macrophylla Pall.)离体培养再生过程中体细胞胚的发生(简报)

本文以秦艽叶片和茎段作为外植体,通过离体培养对秦艽植株再生途径进行研究。愈伤组织在添加2mg/L 2,4-D和0.5mg/L BA的MS培养基上诱导,两周内可出现愈伤组织。愈伤组织在相同激素配比并附加500mg/L LH的MS培养基上继代。愈伤组织的分化在添加有0.1mg/L 2,4-D和0.5mg/L BA的MB培养基上进行。通过显微观测,疑似体细胞胚可以在叶片和茎段的愈伤组织上产生。形态学和组织学的分析进一步证实了秦艽离体再生过程中体细胞胚发生的现象。体细胞胚和合子胚一样,也经历球形、心形、鱼雷和子叶胚等发育时期。相对独立的结构说明秦艽的体细胞胚可能是单细胞来源。体细胞胚在愈伤组织的表面和内部都有出现。在本实验中,体细胞胚发生途径是在秦艽愈伤组织形成后观察到的唯一再生途径。     

Molecular characterization of a Cyrtochilum loxense Somatic Embryogenesis Receptor-like Kinase (SERK) gene expressed during somatic embryogenesis

Somatic embryogenesis is crucial for the propagation of endangered Ecuadorian orchid species, among them Cyrtochilum loxense, in view of the fact that their number in nature or in collections is quite reduced. One of the genes expressed during somatic and zygotic embryogenesis is Somatic Embryogenesis Receptor-like Kinase (SERK). Despite the development of somatic embryogenesis protocols for orchids, no SERK genes have been isolated from this family. This is the first report on the isolation of a full-length orchid SERK sequence, namely that of Cyrtochilum loxense (ClSERK). The identity of ClSERK was inferred by the presence of all domains typical of SERK proteins: a signal peptide, a leucine zipper domain, five LRRs, a serine proline-rich domain, a transmembrane domain, a kinase domain, and the C-terminal region. We have observed that the ClSERK gene is highly expressed in embryogenic calluses generated from protocorms at the time of appearance of embryonic morphological features. At later stages when embryos become well visible on calluses, ClSERK gene expression decreases. Compared to early stages of embryo formation on calluses, the expression detected in leaf tissue is far lower, thus suggesting a role of this gene during development.     

Expression of the QrCPE gene is associated with the induction and development of oak somatic embryos

Somatic embryogenesis is a powerful tool for plant regeneration and also provides a suitable material for investigating the molecular events that control the induction and development of somatic embryos. This study focuses on expression analysis of the QrCPE gene (which encodes a glycine-rich protein) during the initiation of oak somatic embryos from leaf explants and also during the histodifferentiation of somatic embryos. Northern blot and in situ hybridization were used to determine the specific localisation of QrCPE mRNA. The results showed that the QrCPE gene is developmentally regulated during the histodifferentiation of somatic embryos and that its expression is tissue- and genotype-dependent. QrCPE was strongly expressed in embryogenic cell aggregates and in embryogenic nodular structures originated in leaf explants as well as in the protodermis of somatic embryos from which new embryos are generated by secondary embryogenesis. This suggests a role for the gene during the induction of somatic embryos and in the maintenance of embryogenic competence. The QrCPE gene was highly expressed in actively dividing cells during embryo development, suggesting that it participates in embryo histodifferentiation. The localised expression in the root cap initial cells of cotyledonary somatic embryos and in the root cap of somatic seedlings also suggests that the gene may be involved in the fate of root cap cells.     

Involvement of gibberellin and cytokinin in the formation of embryogenic cell clumps in carrot (Daucus carota)

Somatic embryogenesis of carrots is a typical example of the totipotency of plant cells. However, little is known about the process of change from somatic cells to embryogenic cells. To test the involvement of plant hormones in the acquisition process of embryogenic potency, we investigated the effects of plant growth regulators and their inhibitors on auxin-induced direct somatic embryogenesis of carrots. Gibberellin (GA) inhibited the early stage of embryogenic cell differentiation/development to the globular stage and uniconazole, an inhibitor of GA synthesis, promoted the secondary embryogenesis from the primary embryo. Purine riboside, an anticytokinin, inhibited direct somatic embryogenesis, and this effect was nullified by the application of cytokinin (CK). These results show that GA and CK regulate the early stage of auxin-induced somatic embryogenesis in carrots.     

Detection of a <Emphasis Type="Italic">SERK</Emphasis>-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.     

Enhanced production of recombinant human gastric lipase in turnip hairy roots

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.     

黄山栾树体细胞胚的发生和组织学观察

黄山栾树无菌苗的节间和叶柄离体培养后,其体细胞胚发生的结果表明：节间愈伤组织可诱导产生体细胞胚,而叶柄愈伤组织则生根：节间愈伤组织诱导培养基为MS＋3．0mg．L～2,4．D＋0．5～3．0mg．L-1NAA;节间胚性愈伤组织诱导培养基为MS＋2．0nag．L-2,4-D;胚性愈伤组织转移到无植物生长调节剂的MS培养基上可发育成正常植株。组织学观察表明,体细胞胚在胚性愈伤组织中有的发生于愈伤组织表层细胞,有的发生在愈伤组织内部。黄山栾树体细胞胚的形成经历球形胚、心形胚、鱼雷胚和子叶胚几个阶段,这与合子胚的发育途径相似。     

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

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

Auxin-induced WUS expression is essential for embryonic stem cell renewal during somatic embryogenesis in Arabidopsis

Somatic embryogenesis requires auxin and establishment of the shoot apical meristem (SAM). WUSCHEL ( WUS ) is critical for stem cell fate determination in the SAM of higher plants. However, regulation of WUS expression by auxin during somatic embryogenesis is poorly understood. Here, we show that expression of several regulatory genes important in zygotic embryogenesis were up-regulated during somatic embryogenesis of Arabidopsis. Interestingly, WUS expression was induced within the embryonic callus at a time when somatic embryos could not be identified morphologically or molecularly. Correct WUS expression, regulated by a defined critical level of exogenous auxin, is essential for somatic embryo induction. Furthermore, it was found that auxin gradients were established in specific regions that could then give rise to somatic embryos. The establishment of auxin gradients was correlated with the induced WUS expression. Moreover, the auxin gradients appear to activate PIN1 polar localization within the embryonic callus. Polarized PIN1 is probably responsible for the observed polar auxin transport and auxin accumulation in the SAM and somatic embryo. Suppression of WUS and PIN1 indicated that both genes are necessary for embryo induction through their regulation of downstream gene expression. Our results reveal that establishment of auxin gradients and PIN1-mediated polar auxin transport are essential for WUS induction and somatic embryogenesis. This study sheds new light on how auxin regulates stem cell formation during somatic embryogenesis.