Mechanisms Of Somatic Embryogenesis Research Articles

Transcriptomes analysis reveals novel insight into the molecular mechanisms of somatic embryogenesis in Hevea brasiliensis

BackgroundSomatic embryogenesis (SE) is a promising technology for plant vegetative propagation, which has an important role in tree breeding. Though rubber tree (Hevea brasiliensis Muell. Arg.) SE has been founded, few late SE-related genes have been identified and the molecular regulation mechanisms of late SE are still not well understood.ResultsIn this study, the transcriptomes of embryogenic callus (EC), primary embryo (PE), cotyledonary embryo (CE), abnormal embryo (AE), mature cotyledonary embryo (MCE) and withered abnormal embryo (WAE) were analyzed. A total of 887,852,416 clean reads were generated, 85.92% of them were mapped to the rubber tree genome. The de novo assembly generated 36,937 unigenes. The differentially expressed genes (DEGs) were identified in the pairwise comparisons of CE vs. AE and MCE vs. WAE, respectively. The specific common DEGs were mainly involved in the phytohormones signaling pathway, biosynthesis of phenylpropanoid and starch and sucrose metabolism. Among them, hormone signal transduction related genes were significantly enriched, especially the auxin signaling factors (AUX-like1, GH3.1, SAUR32-like, IAA9-like, IAA14-like, IAA27-like, IAA28-like and ARF5-like). The transcription factors including WRKY40, WRKY70, MYBS3-like, MYB1R1-like, AIL6 and bHLH93-like were characterized as molecular markers for rubber tree late SE. CML13, CML36, CAM-7, SERK1 and LEAD-29-like were also related to rubber tree late SE. In addition, histone modification had crucial roles during rubber tree late SE.ConclusionsThis study provides important information to elucidate the molecular regulation during rubber tree late SE.

Transcriptome analysis identifies genes involved in the somatic embryogenesis of Eucalyptus

BackgroundEucalyptus, a highly diverse genus of the Myrtaceae family, is the most widely planted hardwood in the world due to its increasing importance for fiber and energy. Somatic embryogenesis (SE) is one large-scale method to provide commercial use of the vegetative propagation of Eucalyptus and dedifferentiation is a key step for plant cells to become meristematic. However, little is known about the molecular changes during the Eucalyptus SE.ResultsWe compared the transcriptome profiles of the differentiated and dedifferentiated tissues of two Eucalyptus species – E. camaldulensis (high embryogenetic potential) and E. grandis x urophylla (low embryogenetic potential). Initially, we identified 18,777 to 20,240 genes in all samples. Compared to the differentiated tissues, we identified 9229 and 8989 differentially expressed genes (DEGs) in the dedifferentiated tissues of E. camaldulensis and E. grandis x urophylla, respectively, and 2687 up-regulated and 2581 down-regulated genes shared. Next, we identified 2003 up-regulated and 1958 down-regulated genes only in E. camaldulensis, including 6 somatic embryogenesis receptor kinase, 17 ethylene, 12 auxin, 83 ribosomal protein, 28 zinc finger protein, 10 heat shock protein, 9 histone, 122 cell wall related and 98 transcription factor genes. Genes from other families like ABA, arabinogalactan protein and late embryogenesis abundant protein were also found to be specifically dysregulated in the dedifferentiation process of E. camaldulensis. Further, we identified 48,447 variants (SNPs and small indels) specific to E. camaldulensis, including 13,434 exonic variants from 4723 genes (e.g., annexin, GN, ARF and AP2-like ethylene-responsive transcription factor). qRT-PCR was used to confirm the gene expression patterns in both E. camaldulensis and E. grandis x urophylla.ConclusionsThis is the first time to study the somatic embryogenesis of Eucalyptus using transcriptome sequencing. It will improve our understanding of the molecular mechanisms of somatic embryogenesis and dedifferentiation in Eucalyptus. Our results provide a valuable resource for future studies in the field of Eucalyptus and will benefit the Eucalyptus breeding program.

Evidence of the Regulatory Roles of Candidate miRNAs During Somatic Embryogenesis in Lilium davidii var. unicolor

microRNAs (miRNAs) play an essential role in the regulation of large gene networks involved in plant somatic embryogenesis (SEG), but the regulatory mechanism of SEG mediated by miRNAs remains unclear. We used small RNA (sRNA) and transcriptome sequencing methods to identify and profile conserved miRNAs and target genes in five classic embryogenic cultures of Lilium davidii var. unicolor. A total of 35,450 unigenes and 1,727,322 sRNA sequences were obtained from the high-throughput sequencing data. After sequence alignment, 314 known miRNAs belonging to more than 47 miRNA families, 44 novel miRNAs and 32 miRNA*s were identified in L. davidii var. unicolor. miR159 was the largest miRNA family and exhibited the highest expression, and 711 target genes were predicted as the corresponding targets for 273 known miRNAs. In addition, we identified miRNAs that are differentially expressed during SEG and explored the expression profiles of 12 miRNAs and 8 targets. The corresponding relationships between the lda-miR167-WRKY/BGAL, lda-miR319-PCF, lda-miR395-FtsZ and lda-miR408/lda-miR397-LAC pairs were ultimately validated via 5′ rapid amplification of cDNA ends (5′ RACE). Furthermore, we summarized candidate miRNA sequence information, monitored the dynamic expression profiles of miRNAs and tested the interactions between miRNAs and their targets. The data revealed the complex regulatory roles of miRNAs during SEG in L. davidii var. unicolor, and these findings contribute to the miRNA functional annotation of Liliaceae and help elucidate the miRNA-mediated regulatory network during plant SEG.

HISTOLOGICAL ANALYSIS OF INDIRECT SOMATIC EMBRYOGENESIS INDUCED FROM ROOT EXPLATS OF OIL PALM (Elaeis guineensis Jacq)

ABSTRACT Oil palm is economically important as a crop with high oil production. Indirect somatic embryogenesis in oil palm requires a long time for callus induction and plant formation, and it is important to study the embryogenic potential of calli and the mechanisms of somatic embryogenesis. The aim of this study was to test different growth regulators and spermine in induction of embryogenic calli in root explants of oil palm (Elaeis guineensis Jacq). Apex root explants of approximately 0.5 cm were isolated from plants cultivated in vitro and inoculated in Y3 culture medium in the following treatments: A - without growth regulators; B - 1 mg.L-1 picloram (4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid); C - 1 mg.L-1 picloram and 2 mg.L-1 2ip (2-isopentenyladenine); D - 2 mg.L-1 2ip; E - 1 mg.L-1 picloram and 2 mg.L-1 BAP (6-benzylaminopurine); F - 2 mg.L-1 BAP; and G - 14.5 mg.L-1 spermine. After six months of culturing, the calli induced in the treatments were analyzed by light microscopy. The calli induced in the treatments with 1 mg.L-1picloram (B) and treatment with 14.5 mg.L-1spermine (G) exhibited embryogenic characteristics, small and isodiametric cells, forming agglomerates, besides a large amount of starch. Calli of the best treatment (Y3 com 1 mg.L-1 de picloram) were inoculated in Y3 culture medium without addition of growth regulators. After eight months, calli were once more analyzed under light microscopy. All the treatments showed callus formation, except for treatments D and A. Calli of treatment B exhibited cells with embryogenic characteristics that developed somatic embryos.

Expression profiling of AUXIN RESPONSE FACTOR genes during somatic embryogenesis induction in Arabidopsis

Key messageExtensive modulation of numerous ARF transcripts in the embryogenic culture of Arabidopsis indicates a substantial role of auxin signaling in the mechanism of somatic embryogenesis induction.Somatic embryogenesis (SE) is induced by auxin in plants and auxin signaling is considered to play a key role in the molecular mechanism that controls the embryogenic transition of plant somatic cells. Accordingly, the expression of AUXIN RESPONSE FACTOR (ARF) genes in embryogenic culture of Arabidopsis was analyzed. The study revealed that 14 of the 22 ARFs were transcribed during SE in Arabidopsis. RT-qPCR analysis indicated that the expression of six ARFs (ARF5, ARF6, ARF8, ARF10, ARF16, and ARF17) was significantly up-regulated, whereas five other genes (ARF1, ARF2, ARF3, ARF11, and ARF18) were substantially down-regulated in the SE-induced explants. The activity of ARFs during SE was also monitored with GFP reporter lines and the ARFs that were expressed in areas of the explants engaged in SE induction were detected. A functional test of ARFs transcribed during SE was performed and the embryogenic potential of the arf mutants and overexpressor lines was evaluated. ARFs with a significantly modulated expression during SE coupled with an impaired embryogenic response of the relevant mutant and/or overexpressor line, including ARF1, ARF2, ARF3, ARF5, ARF6, ARF8, and ARF11 were indicated as possibly being involved in SE induction. The study provides evidence that embryogenic induction strongly depends on ARFs, which are key regulators of the auxin signaling. Some clues on the possible functions of the candidate ARFs, especially ARF5, in the mechanism of embryogenic transition are discussed. The results provide guidelines for further research on the auxin-related functional genomics of SE and the developmental plasticity of somatic cells.

Differential gene expression during somatic embryogenesis in the maize (Zea mays 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.

Use of somatic embryogenesis as a vehicle for cotton transformation

Cotton has been aptly described as the prosperity plant owing to its unrivalled economic importance as a source of feedstock, food and oil, as well as raw material for diverse industrial applications, ranging from textile and footwear to automobiles, energy, medical and pharmaceutical. As such, over 180 million people of the world depend on its production for livelihood. However, cotton production is grossly hampered, and has long been peaked in many regions where it is being grown. Without prejudice to the genetic improvement already made by conventional breeding with respect to yield and quality over the years, genetic transformation is arguably the last recourse for further development of cotton, especially with respect to the prevailing production constraints of insect pests, weeds, environmental stresses and diseases. This review therefore focuses on the use of somatic embryogenesis as a vehicle for cotton genetic transformation. It indeed attempts to overview the challenges of cotton transformation with respect to narrow genetic base coupled with the recalcitrant nature of the crop species, as well as the research success achieved so far. It then discusses the underlying mechanisms of somatic embryogenesis as well as the current constraints and various strategies being used to overcome them; all with the aim of motivating interest groups to initiate research activities in cotton biotechnology and to strive for its optimization for further genetic improvement. Key words: Somatic embryos, genetic transformation, plant regeneration, genetic regulation, classical breeding.

Characterization and expression pattern analysis of DcNAC gene in somatic embryos of Dendrobium candidum Wall Ex Lindl.

The plant-specific “no apical meristem” genes are transcription factors that play diverse roles in plant development and stress responses. However, whether the gene family is also involved in somatic embryogenesis remains unknown, and no NAC family genes have been identified from orchid species. Here, we cloned and characterized a new member of NAC family from somatic embryos of Dendrobium candidum. Phylogenetic analysis indicated that DcNAC fell into the NAM subgroup based on similarities in protein sequence. Expression pattern analysis indicated that DcNAC exhibited a specific expression pattern in tissues examined, high levels of DcNAC mRNA was detected in protocorm-like bodies, and predominantly in the shoot apical meristem region as detected by in situ hybrization. Subcellular localization analysis showed that DcNAC protein is restricted in the nucleus through transient expression in Nicotiana benthamiana. Thus, DcNAC is the first NAC family gene isolated from orchid species, and our results provide the foundation for future investigations on the molecular mechanisms of somatic embryogenesis and for genetic improvement of Dendrobium candidum.

Ontogeny of embryogenic callus in Medicago truncatula: the fate of the pluripotent and totipotent stem cells.

Understanding the fate and dynamics of cells during callus formation is essential to understanding totipotency and the mechanisms of somatic embryogenesis. Here, the fate of leaf explant cells during the development of embryogenic callus was investigated in the model legume Medicago truncatula. Callus development was examined from cultured leaf explants of the highly regenerable genotype Jemalong 2HA (2HA) and from mesophyll protoplasts of 2HA and wild-type Jemalong. Callus development was studied by histology, manipulation of the culture system, detection of early production of reactive oxygen species and visualization of SERK1 (SOMATIC EMBRYO RECEPTOR KINASE1) gene expression. Callus formation in leaf explants initiates at the cut surface and within veins of the explant. The ontogeny of callus development is dominated by the division and differentiation of cells derived from pluripotent procambial cells and from dedifferentiated mesophyll cells. Procambium-derived cells differentiated into vascular tissue and rarely formed somatic embryos, whereas dedifferentiated mesophyll cells were competent to form somatic embryos. Interestingly, explants incubated adaxial-side down had substantially less cell proliferation associated with veins yet produced similar numbers of somatic embryos to explants incubated abaxial-side down. Somatic embryos mostly formed on the explant surface originally in contact with the medium, while in protoplast microcalli, somatic embryos only fully developed once at the surface of the callus. Mesophyll protoplasts of 2HA formed embryogenic callus while Jemalong mesophyll protoplasts produced callus rich in vasculature. The ontogeny of embryogenic callus in M. truncatula relates to explant orientation and is driven by the dynamics of pluripotent procambial cells, which proliferate and differentiate into vasculature. The ontogeny is also related to de-differentiated mesophyll cells that acquire totipotency and form the majority of embryos. This contrasts with other species where totipotent embryo-forming initials mostly originate from procambial cells.

Comparative embryology and mammalian cloning

A hypothesis has been advanced that logically combines "contradictory" facts concerning the early mammalian development and shows a natural relationship between the embryos developing from a fertilized ovum and from cells of the inner cell mass of blastocyst. When studying the theoretical questions of cloning, it is necessary to take into consideration the peculiarities of prenatal mammalian ontogenesis, which make themselves evident upon comparison with other animals. The absence of yolk in the mammalian ovum defines sharp differences in the early development between mammals and other Amniota. The whole asynchronic cleavage results in the formation of the morula followed by the blastocyst, which hatches from zona pellucida and is implanted into the uterus tissue. This fact allows us to consider the blastocyst as a mammalian larva, which is fed thanks to maternal organism. It is known that, in the body of a larva (blastocyst), a new embryo develops from some somatic cells. This process is known as a polyembryony, which is typical for the development of some parasitic insects. The polyembryony in turn is a variant of somatic embryogenesis, which is a form of asexual reproduction. Thus, two different embryos, "conceptus" and "embryo proper", have different origin: the first forms by the sexual way and the second, by the asexual. The investigation of the mechanisms of somatic embryogenesis in mammals will help us to find conditions necessary for the full reprograming of donor somatic nuclei and provide the successful development of reconstructed embryos.

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

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.

Mechanisms of somatic embryogenesis in cell cultures: Physiology, biochemistry, and molecular biology

Mechanisms of somatic embryogenesis in cell cultures: Physiology, biochemistry, and molecular biology

Synchronization of Somatic Embryogenesis in a Carrot Cell Suspension Culture

Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. "Kurodagosun") suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis.