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.     

Somatic embryogenesis in conifers—A case study of induction and development of somatic embryos in Picea abies

Vegetatively propagated material offers many advantages over seed material in forest tree breeding research and in reforestation programmes. Evidence is accumulating to suggest that using somatic embryos in forestry is a viable option. However, before somatic embryos can be used optimally in forestry, basic research aimed at increasing the number of responsive genotypes as well as the age of the primary explant is needed. This in turn requires the establishment of a basic understanding of the physiological and molecular processes that underlie the development of somatic embryos. The functions of genes and their developmental and tissue specific regulation are studied using transient and stable transformation techniques.The process of somatic embryogenesis can be divided into different steps: (1) initiation of somatic embryos from the primary explant, (2) proliferation of somatic embryos, (3) maturation of somatic embryos and (4) plant regeneration. Cortical cells in the primary explant are stimulated to go through repeated divisions so that dense nodules are formed from which somatic embryos differentiate. The first formed somatic embryos continue to proliferate and give rise to embryogenic cell lines. Embryogenic cell lines of Picea abies can be divided into two main groups A and B, based on morphology, growth pattern and secretion of proteins. Our results suggest that extracellular proteins play a crucial role in embryogenesis of Picea abies. Somatic embryos from group A can be stimulated to go through a maturation process when treated with abscisic acid. Mature somatic embryos can develop into plants.Abbreviations ABA abscisic acid - BA N⁶-benzyladenine - 2,4-D dichlorophenoxy acetic acid     

Differential gene expression during somatic embryogenesis in the maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) inbred line H99

Somatic embryogenesis is a complex developmental process that offers great potential for plant propagation. Although many studies have shown that the generation of embryonic cells from somatic cells is accompanied by the synthesis of RNA and DNA and by elevated enzymatic activity, the mechanism of the onset of somatic embryogenesis is not well understood. cDNA-amplified fragment length polymorphism analysis was used to evaluate the gene expression pattern in embryogenic and non-embryogenic of the inbred maize line H99 during the process of embryogenesis. We identified a total of 101 candidate genes associated with the formation of maize embryonic calli. Based on the sequence analysis, these genes included 53 functionally-annotated TDFs involved in such processes as energy production and conversion, cell division and signal transduction, suggesting that somatic embryogenesis undergoes a complex process. Two full-length cDNA sequences, encoding KHCP (kinesin heavy chain like protein) and TypA (tyrosine phosphorylation protein A), and partial sequences, encoding ARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor) homologs, were isolated from embryonic calli of maize and named ZmKHCP, ZmTypA and ZmARF-GEP, respectively. Finally, the real-time qRT-PCR results showed that the expression levels of the three genes were significantly higher in the embryonic calli than the non-embryonic calli. Thus, this study provides important clues to understanding the induction of somatic embryogenesis in maize. The candidate genes associated with the formation of embryonic calli may offer additional insights into the mechanism of somatic embryogenesis, and further research on the three candidate genes may determine their role in increasing the rate of induction of embryonic calli, which may aid in the development of cultivars through transgenic breeding.     

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes     

The histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.)

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the somatic embryogenesis (SE) mechanisms. In the present study, the histodifferentiation events involved during the acquisition and development of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) was investigated. Zygotic embryos were inoculated on SE induction medium, and at 14 days the first divisions of the procambial and perivascular cells were observed. This region progressed to the formation of meristematic masses at 21 days, indicating their procambial and perivascular origin. Primary calli emerged at 45 days of culture, followed by progression to embryogenic calli at 90 days. The formation of proembryos (PE) from the meristematic cells occurred at 135 days of cultivation. The PE were isolated from the tissue of origin by the slight thickening of the cell wall, indicating their unicellular origin. When transferred to the maturation phase, differentiation of the somatic embryos at different developmental stages (globular and torpedo) was observed. The differentiated somatic embryos presented protoderm, procambial strands and plumules. Afterwards, they were transferred to culture medium without growth regulators in which conversion of the somatic embryos from torpedo stage into plants was observed. These results enable a greater understanding of the SE process and plantlet formation in E. guineensis.     

Somatic embryogenesis from pea embryos and shoot apices

Conditions were defined for plant regeneration via somatic embryogenesis in pea, using explants from immature zygotic embryos or from shoot apices. For the induction of somatic embryos, an auxin (picloram or 2,4-dichlorophenoxyacetic acid) was required. Embryogenic callus originated from embryonic axis tissue of immature embryos and from the axillary-bud region and the plumula of shoot apices. A clear effect of embryo size on somatic embryogenesis was shown. There were differences in frequency of somatic embryogenesis among the five genotypes used in the study. Additions of BA to auxin-containing medium reduced embryo production. Histological examinations confirmed the embryogenic nature of the immature embryo cultures and revealed that somatic embryos originated from the meristematic areas near the callus surface.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Morpho-histological study of direct somatic embryogenesis in endangered species Frittilaria meleagris

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm⁻³ 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.     

Isolation and characterization of a diverse set of genes from carrot somatic embryos.

The early events in plant embryogenesis are critical for pattern formation, since it is during this process that the primary apical meristems and the embryo polarity axis are established. However, little is known about the molecular events that are unique to the early stages of embryogenesis. This study of gene expression during plant embryogenesis is focused on identifying molecular markers from carrot (Daucus carota) somatic embryos and characterizing the expression and regulation of these genes through embryo development. A cDNA library, prepared from polysomal mRNA of globular embryos, was screened using a subtracted probe; 49 clones were isolated and preliminarily characterized. Sequence analysis revealed a large set of genes, including many new genes, that are expressed in a variety of patterns during embryogenesis and may be regulated by different molecular mechanisms. To our knowledge, this group of clones represents the largest collection of embryo-enhanced genes isolated thus far, and demonstrates the utility of the subtracted-probe approach to the somatic embryo system. It is anticipated that many of these genes may serve as useful molecular markers for early embryo development.     

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.     

Glutathione redox regulation of in vitro embryogenesis

Production of embryos in culture via either somatic embryogenesis or androgenesis has long been used as a propagation tool and as a model system in the investigation of structural, physiological, and molecular events governing embryo development. Despite the similar external morphology to their zygotic counterparts, cultured embryos often fail to develop properly and convert into viable plants during post-embryonic growth. These deficiencies are the results of structural and physiological deviations ascribed to sub-optimal culture conditions. In an attempt to enhance embryo yield and quality we have conducted a series of investigations into the role of glutathione during embryogenesis. Changes in the glutathione redox state represent a key metabolic switch which triggers embryo growth. The imposition of a reduced environment during the early embryonic phases promotes cellular proliferation and increases the number of immature embryos, possibly by promoting the synthesis of nucleotides in support of energetic processes and mitotic activity. Continuation of embryo development is best achieved if the glutathione pool is experimentally switched towards an oxidized state; a condition which favors histodifferentiation and post-embryonic growth in both angiosperm and gymnosperms species. Among the structural events favored by the imposed oxidized environment is the proper formation of the shoot apical meristem (SAM), which acquires a “zygotic-like” appearance. The apical poles of treated embryos are well organized and display a proper expression and localization of meristem marker genes. These conditions are not met in control embryos which form abnormal SAMs characterized by the presence of intercellular spaces and differentiation of meristematic cells. Such meristems fail to reactivate at germination resulting in embryo abortion. Physiological and molecular studies have further demonstrated that the oxidized glutathione environment induces several responses, including changes in ascorbate metabolism, abscisic acid and ethylene synthesis, as well as alterations in storage product deposition patterns. This review attempts to relate these responses to the improved embryonic performance and proposes improved culture conditions to be applied for those cell lines and species recalcitrant to in vitro embryogenesis.     

细胞信号转导与植物体细胞胚发生

细胞信号转导主要是指胞间通讯的激素以及外界环境因子等作用于细胞表面（或胞内受体）后,如何跨膜传递形成胞内第二信使,以及其后的信息分子级联传递、诱导基因表达和引起生理反应的过程。植物体细胞胚发生的本质是基因的判别表达,因此细胞信号转导在此起关键作用。在植物组织培养过程中,外加的激素等因子通过细胞信号转导诱导基因差别表达,使体细胞转入胚胎发生过程,最终生长发育成完整植株。     

Analysis of expression profile of selected genes expressed during auxin-induced somatic embryogenesis in leaf base system of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and their possible interactions

Somatic embryogenesis is a notable illustration of plant totipotency and involves reprogramming of development in somatic cells toward the embryogenic pathway. Auxins are key components as their exogenous application recuperates the embryogenic potential of the mitotically quiescent somatic cells. In order to unravel the molecular basis of somatic embryogenesis, cDNA library was made from the regeneration proficient wheat leaf base segments treated with auxin. In total, 1440 clones were sequenced and among these 1,196 good quality sequences were assembled into 270 contigs and 425 were singletons. By reverse northern analysis, a total of 57 clones were found to be upregulated during somatic embryogenesis, 64 during 2,4-D treatment, and 170 were common to 2,4-D treatment and somatic embryogenesis. A substantial number of genes involved in hormone response, signal transduction cascades, defense, anti-oxidation, programmed cell death/senescence and cell division were identified and characterized partially. Analysis of data of select genes suggests that the induction phase of somatic embryogenesis is accompanied by the expression of genes that may also be involved in zygotic embryogenesis. The developmental reprogramming process may in fact involve multiple cellular pathways and unfolding of as yet unknown molecular events. Thus, an interaction network draft using bioinformatics and system biology strategy was constructed. The outcome of a systematic and comprehensive analysis of somatic embryogenesis associated interactome in a monocot leaf base system is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Invited review: Genetic regulation of somatic embryogenesis with particular reference to arabidopsis thaliana and Medicago truncatula

Summary Investigations into the mechanisms of somatic embryogenesis (SE) have largely focused on the hormonal regulation of the process and a repertoire of strategies has been developed to regenerate many species via SE. However, the genes that regulate the induction and development of somatic embryos have not been defined. In the recent times, regeneration via overexpression of genes, such as WUSCHEL or LEAFY COTYLEDON, in Arabidopsis has started to provide a basis for understanding the genes involved in SE. This has gone hand in hand with the availability of genome sequence information and the availability of mutants in model plants such as Arabidopsis and Medicago. An improved understanding of zygotic embryogenesis and the maintenance and differentiation of stem cells in the shoot meristem also helps to provide novel insights into the mechanisms of SE. This review examines the current understanding of the genetic regulation of SE in the context of current molecular understanding of plant development.     

The embryogenic response of immature embryo cultures of durum wheat (Triticum durum Desf.): histology and improvement by AgNO3

Immature zygotic embryos of durum wheat cv Ardente were cultured vitro on 2,4-D to induce somatic embryogenesis. Five days after culture initiation, somatic proembryos were directly initiated from the scutellum of immature embryos. After 28 days, somatic embryos were fully developed with a scutellum-like structure. Histological observations between the first and the eighty day in culture showed a clear unicelllar origin for a few of these somatic embryos, whilst most of them originated from a meristematic multilayer. Furthermore, estimation of the mitotic index of outer epithelium, subepithelium and inner epithelium of the scutellum during the first week of culture, showed a strict epidermal origin of these early developed structures. The addition of 1 mg·L^–1 of AgNO₃ enhanced the induction of direct somatic embryogenesis (a more than 22 fold increase), affecting both the percentage of embryogenic explants and the number of somatic embryos per explant, suggesting the possible involvement of ethylene.     

Somatic Embryogenesis in Leguminous Plants

Abstract: This review examines recent advances in the induction and development of somatic embryos in leguminous plants. Emphasis has been given to identify the current trends and successful strategies for the establishment of somatic embryogenic systems, particularly in the economically important species. It appears that, in legumes, somatic embryogenesis can be realized relatively easily especially in young meristematic tissues such as immature embryos and developing leaves. In the majority of the species examined, chlorophenoxyacetic acids remained the most active inductive compounds; however, the new generation growth regulators such as thidiazuron are emerging as successful alternatives for high-frequency direct regeneration of somatic embryos, even from well differentiated explant tissues. Low-frequency embryo production, poor germination and conversion of somatic embryos into plantlets and somaclonal variation are the major impediments limiting the utility of somatic embryogenesis for biotechnological applications in legumes. These limitations, however, may be considerably reduced in the near future, as more newly developed growth regulators with specific morphogenic targets become available for experimentation. From the published data, it is apparent that more effort should be given to develop repetitive embryogenic systems with high frequency of germination and regeneration, since such systems will find immediate application in mass propagation and other crop improvement programmes. As our understanding of various morphogenic processes, including growth and differentiation of zygotic embryos, is fast expanding, it is conceivable that development of highly efficient somatic embryogenic systems with practical application can be anticipated, at least for the important leguminous crops, in the foreseeable future.     

The analysis of differential gene expression in early somatic embryogenesis on Lycium barbarum

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can induce somatic embryogenesis. Somatic embryogenesis of Lycium barbarum L. is controlled artificially by regulating 2,4-D concentration. The total RNA that was isolated from calluses, embryonic calluses and early somatic embryos was used for analyzing differential genes expression. We obtained three cDNAs from early somatic embryogenesis which were not found in calluses. The results indicate that these cDNAs were early embryogenesis-specific cDNAs and this gene expression was induced in cultured calluses after a transfer to an auxin- free medium. A cDNA library was constructed using poly(A)⁺-RNA derived from early somatic embryos of Lycium barbarism L. Two full-length cDNAs were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of the full-length cDNA only existed in embryogenic calluses and early somatic embryos of Lycium barbarum L. This revised version was published online in June 2006 with corrections to the Cover Date.