Regulation Of Somatic Embryogenesis Research Articles

Genetic and Molecular Control of Somatic Embryogenesis.

Somatic embryogenesis is a method of asexual reproduction that can occur naturally in various plant species and is widely used for clonal propagation, transformation and regeneration of different crops. Somatic embryogenesis shares some developmental and physiological similarities with zygotic embryogenesis as it involves common actors of hormonal, transcriptional, developmental and epigenetic controls. Here, we provide an overview of the main signaling pathways involved in the induction and regulation of somatic embryogenesis with a focus on the master regulators of seed development, LEAFY COTYLEDON 1 and 2, ABSCISIC ACID INSENSITIVE 3 and FUSCA 3 transcription factors whose precise role during both zygotic and somatic embryogenesis remains to be fully elucidated.

Secretome-Derived Cultured Cell System: Overview Towards Extracellular Protein Characterization and Biotechnological Applications

Secretome released by plant cells into the extracellular space, play crucial roles during development, embryonic potential acquisition, nutrient backing and stress acclimation. The dynamic nature of the extracellular proteome presents the challenge of identifying an array of extracellular proteins involved in the regulation of somatic embryogenesis in embryogenic suspension cultures. Extracellular proteins produced by cell cultures are perceived here as a central node of overlapping regulator factor network of totipotent somatic embryo developmental process. This paper reviews in a morphogenetic aspect the biological processes associated with extracellular protein-derived plant cultured cells and explores their prospective biotechnological applications in laboratories and biofactories retated to cell signaling and metabolism, developmental process, and biotic / abiotic stress tolerance. The role of extracellular proteins in acquisition and maintenance of embryonic potential and their relevance are especially emphasized.

TaTCP-1, a Novel Regeneration-Related Gene Involved in the Molecular Regulation of Somatic Embryogenesis in Wheat (Triticum aestivum L.).

The lower regeneration rate of wheat calli is the main factor restricting the development of transgenic wheat plants. Therefore, improving the regeneration rate of wheat callus is a precondition for developing genetic engineering-based wheat breeding approaches. In the present study, we explored the molecular mechanism of wheat regeneration and aimed to establish an efficient system for transgenic wheat. We isolated and identified a regeneration-related gene, TaTCP-1 (KC808517), from wheat cultivar Lunxuan 987. Sequence analysis revealed that the ORF of TaTCP-1 was 1623bp long encoding 540 amino acids. The TaTCP-1 gene was expressed in various wheat tissues. Further, the level of TaTCP-1 expression was higher in calli and increased gradually with increasing callus induction time, reaching a peak on the 11th day after induction. Moreover, the expression level of TaTCP-1 was higher in embryogenic calli than in non-embryonic calli. The TaTCP-1 protein was localized to the nucleus, cytoplasm, and cell membrane. The callus regeneration rate of wheat plants transformed with TaTCP-1-RNAi reduced by 85.09%. In contrast, it increased by 14.43% in plants overexpressing TaTCP-1. In conclusion, our results showed that TaTCP-1 played a vital role in promoting wheat regeneration, and regulated the somatic embryogenesis of wheat. These results may have implications in the genetic engineering of wheat for improved wheat production.

Expression of somatic embryogenesis-related genes in sugarcane (Saccharum officinarum L.)

The low frequency of plantlet regenerates from the somatic embryogenesis (SE) callus in sugarcane becomes a problem to produce its seed. Plant growth regulators were able to increase the regeneration frequency of SE to normal plantlets, such as 2,4-dichlorophenoxyacetic acid (2,4-D) as promoting callus induction. Since molecular mechanisms involved SE in sugarcane have not been reported, expression of Baby Boom (BBM) and Leafy Cotyledon (LEC) genes related to SE had investigated. The effect of difference concentration of 2,4-D on callus induction and expression of somatic embryogenesis-related genes (BBM and LEC) in sugarcane were important information for the increasing quantity and quality of seed production. The percentage of callus formation and embryogenic callus in the Murashige and Skoog’s (MS) medium contained 4 mg L−1 2,4-D after 6 week cultivation were 76% and 86%, respectively. The MS + 4 mg L−1 2,4-D medium was recommended for the large-scale embryogenic callus production from sugarcane explant. The high-level expression of BBM and LEC was shown in the embryogenic callus, which suggested that the expressions of both genes were believed to play on the somatic embryogenesis regulation in sugarcane.

Transcription Factors: Their Role in the Regulation of Somatic Embryogenesis in Theobroma cacao L. and Other Species.

Transcription factors are proteins that help with the control and regulation in the transcription of the DNA to mRNA by binding to special DNA sequences. With the aim to understand more about gene transcription regulation in Theobroma cacao L., this review outlines the principal transcription factors that were reported in other plants especially Arabidopsis thaliana and attempts at looking for the homologies with transcription factors in T. cacao. The information cited in this work is about the initiation, development, and maturation of the cacao somatic embryos and other crops. It is important to underline that there are very few publications in T. cacao discussing transcription factors that control the somatic embryogenesis process, but there is some information about transcription factors in other crops that we have used as a guide to try to understand this process.

Genome-wide identification, classification and analysis of HD-ZIP gene family in citrus, and its potential roles in somatic embryogenesis regulation

The homeodomain–leucine zipper (HD-Zip) transcription factors, which belong to a class of Homeobox proteins, has been reported to be involved in different biological processes of plants, including growth and development, photomorphogenesis, flowering, fruit ripening and adaptation responses to environmental stresses. In this study, 27 HD-Zip genes (CsHBs) were identified in Citrus. Based on the phylogenetic analysis and characteristics of individual gene or protein, the HD-Zip gene family in Citrus can be classified into 4 subfamilies, i.e. HD-Zip I, HD-Zip II, HD-Zip III, and HD-Zip IV containing 16, 2, 4, and 5 members respectively. The digital expression patterns of 27 HD-Zip genes were analyzed in the callus, flower, leaf and fruit of Citrus sinensis. The qRT-PCR and RT-PCR analyses of six selected HD-Zip genes were performed in six citrus cultivars with different embryogenic competence and in the embryo induction stages, which revealed that these genes were differentially expressed and might be involved in citrus somatic embryogenesis (SE). The results exhibited that the expression of CsHB1 was up-regulated in somatic embryo induction process, and its expression was higher in citrus cultivars with high embryogenic capacity than in cultivars recalcitrant to form somatic embryos. Moreover, a microsatellite site of three nucleotide repeats was found in CsHB1 gene among eighteen citrus genotypes, indicating the possible association of CsHB1 gene to the capacity of callus induction.

Regulation of somatic embryogenesis in Panax ginseng C. A. Meyer cell cultures by PgCDPK2DS1

We isolated the full-length cDNA of PgCDPK2DS1 gene, the expression of which was significantly increased at early stages of embryo development in cell cultures of ginseng P. ginseng 2c3. Interest in this gene also was supported by its nonstandard structure: the amino acid sequence of the PgCDPK2DS1 gene contained only the N-terminal domain and 80% of the kinase domain. Overexpression of the PgCDPK2DS1 gene in nonembryonic calli 1c resulted in the appearance of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture 1c-2d. Also, expression of the plant embryogenesis marker genes WUS and SERK significantly increased in cell culture 1c-2d. The observed embryo-like structures were at early stages of embryo development; attempts to obtain an adult plant from these embryo-like structures were unsuccessful. Overexpression of PgCDPK2DS1 gene in the embryonic cell culture PG resulted in a decrease of embryonic structures in the PgCDPK2DS1-transgenic ginseng cell culture PG-2d. Moreover, expression of the plant embryogenesis marker genes WUS and SERK and expression of the endogenous PgCDPK2DS1 significantly decreased in the cell culture PG-2d. Thus, for the first time it was shown that the PgCDPK2DS1 gene is involved in the regulation of somatic embryogenesis in P. ginseng cell cultures.

Temporal regulation of somatic embryogenesis in guava (Psidium guajava L.)

SummarySomatic embryogenesis was induced in immature zygotic embryos of guava (Psidium guajava L.) at 10-weeks post anthesis using 2 – 38 d pulses and continuous treatments with different concentrations (0.001 – 3.0 mg l–1) of 2,4-dichlorophenoxyacetic acid (2,4-D). Subsequent development of somatic embryo was observed by sub-culturing onto plant growth regulator (PGR)-free, full-strength Murashige and Skoog (MS) agar medium containing 3% (w/v) sucrose. The maximum responses were shifted gradually from 0.01 mg l–1 2,4-D (continuous or 38 d treatment), to 0.05 mg l–1 2,4-D (28 – 18 d treatment), to 0.1 mg l–1 2,4-D (14 – 12 d treatment) and, finally, to 0.5 mg l–1 2,4-D (10 – 8 d treatment).The highest frequency of embryogenesis (68.8%) and embryogenic intensity (69.2 embryos per explant per culture), as well as moderate frequencies of convertible elongated and short torpedo-stage somatic embryos (15% and 42.9%, respectively), with the highest efficiency (27.5) achieved in the 8 d treatment compared to continuous treatment of zygotic embryo explants with 0.5 mg l–1 2,4-D in MS agar medium containing 3% (w/v) sucrose at both the induction and development phases. Cellular pools of total, free, conjugated, and bound forms of various polyamines (e.g., putrescine, spermidine, and spermine) showed slow increases in the presence of 2,4-D during the 8 d induction phase. This was followed by rapid increases immediately after sub-culturing explant tissue onto the PGR-free MS development medium. The concentrations of various polyamines declined gradually over time on MS development medium. The production, metabolism, and use of different forms of various polyamines at various times (d) after culture initiation correlated with their temporal regulation during the induction and development phases of somatic embryogenesis. Exogenous application of different polyamines during the induction phase resulted in slightly increased frequencies, intensities, and efficiencies of embryogenesis, but with no observable change in the frequencies of the various categories of somatic embryos. The present study indicated that the process of somatic embryogenesis in guava may be less sensitive to PGRs and was induced even in the continuous presence of 2,4-D. Furthermore, somatic embryogenesis in guava was temporally regulated as a function of the exogenous concentration of 2,4-D. In addition, the temporal regulation of somatic embryogenesis was also associated with high levels of production, metabolism, and use of cellular polyamines during the different phases. Hence, improvements in the process of somatic embryogenesis in guava could be regulated by timed applications of 2,4-D alone, or in combination with polyamines, thereby modulating endogenous levels of the latter.

Multifunctionality of the LEC1 transcription factor during plant development

LEC1 acts as a key regulator of embryogenesis in Arabidopsis thaliana, but is involved in a wide range of functions, all the way from embryo morphogenesis to seed maturation. New data show that LEC1, partially in conjunction with abscisic acid, affects auxin synthesis, and both brassinosteroid and light signaling. The phenotype of LEC1 overexpressors confirms LEC1's known participation in the regulation of somatic embryogenesis, but also indicates additional roles in embryonic and extra-embryonic cell elongation. Here we present an integrated model of LEC1 function and suggest potential directions to be taken in future research in this important area of plant science.

Control of somatic embryogenesis and embryo development by AP2 transcription factors

Members of the AP2 family of transcription factors, such as BABY BOOM (BBM), play important roles in cell proliferation and embryogenesis in Arabidopsis thaliana (AtBBM) and Brassica napus (BnBBM) but how this occurs is not understood. We have isolated three AP2 genes (GmBBM1, GmAIL5, GmPLT2) from somatic embryo cultures of soybean, Glycine max (L.) Merr, and discovered GmBBM1 to be homologous to AtBBM and BnBBM. GmAIL5 and GmPLT2 were homologous to Arabidopsis AINTEGUMENTA-like5 (AIL5) and PLETHORA2 (PLT2), respectively. Constitutive expression of GmBBM1 in Arabidopsis induced somatic embryos on vegetative organs and other pleiotropic effects on post-germinative vegetative organ development. Sequence comparisons of BBM orthologues revealed the presence of ten sequence motifs outside of the AP2 DNA-binding domains. One of the motifs, bbm-1, was specific to the BBM-like genes. Deletion and domain swap analyses revealed that bbm-1 was important for somatic embryogenesis and acted cooperatively with at least one other motif, euANT2, in the regulation of somatic embryogenesis and embryo development in transgenic Arabidopsis. The results provide new insights into the mechanisms by which BBM governs embryogenesis.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-010-9674-8) contains supplementary material, which is available to authorized users.

Regulation of Somatic Embryogenesis in Higher Plants

Somatic embryogenesis is the developmental process by which somatic cells undergo restructuring to generate embryogenic cells. These cells then go through a series of morphological and biochemical changes that result in the formation of a somatic or non-zygotic embryo capable of regenerating plants. Somatic embryogenesis represents a unique developmental pathway that includes a number of characteristic events: dedifferentiation of cells, activation of cell division, and reprogramming of their physiology, metabolism, and gene expression patterns. Valuable studies have elucidated the developmental processes pertaining to the control parameters of somatic embryogenesis. These studies have emphasized the transitional state from somatic to embryogenic cells, identified differentially expressed genes in nonembryogenic and embryogenic calli, isolated varieties of genes that are likely involved in the embryogenic pathway, compared specific gene products that accumulate during different stages of somatic embryogenesis, and discovered molecular or protein markers for somatic embryogenesis. An understanding of embryogenic pathway initiation, and origin of somatic embryos (unicellular or multicellular) is critical to scientific and biotechnological applications. Cell tracking has been successfully applied to determine the fate of embryogenic cells. Identification of hormone-inducible genes has yielded clues to the control of gene expression during embryogenic development. Characterization of genes taking part in signal transduction pathways, such as somatic embryogenesis receptor-like kinases, has generated great interest in the switching of several signal cascades during somatic embryogenesis, and has identified how genes encoding transcription factors such as BBM, LEC1, and LEC2 could be used to regulate somatic embryo development. Protein markers are useful probes for defining embryogenic potential and for marking different phases in plant development. This review provides a systematic and comprehensive analysis of current advances in the development of somatic embryogenesis, as well as the cellular and molecular mechanism of somatic embryogenesis in higher plants. The developmental pathway, differential gene expression, and extracellular protein markers during somatic embryogenesis are especially emphasized.

Proteomic Analysis of Somatic Embryogenesis in Cyclamen persicum Mill

Cyclamen persicum Mill. is a widely grown ornamental species that is clonally propagated by somatic embryogenesis. To better understand the biology of somatic embryo development in C. persicum, detailed proteomic (two-dimensional gel electrophoresis) and mass spectrometric analyses of somatic embryos at globular, torpedo, and germinating stages of development, along with nonembryogenic callus and zygotic embryos, were conducted. Of ~460 proteins resolved in two-dimensional gels, 35 proteins were differentially expressed and could be reproducibly displayed across an isoelectric focusing range of 5 to 8. Among those proteins, five were constitutively expressed, 13 were upregulated, nine were downregulated, and eight were deemed as novel proteins during the torpedo stage. A total of 35 protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and only four proteins were identified and these were available in public protein databases. The remaining protein spots were subsequently analyzed by MALDI-TOF-TOF-MS, and six proteins were then identified. These findings suggested that specific proteins are involved in the regulation of somatic embryogenesis.

Regulation of somatic embryogenesis by plant growth regulators in sugarcane

Effect of plant growth regulators on morphogenesis was studied using callus derived from inflorescence segments of sugarcane cv. Co-91010 and CoC-671 cultured on MS medium supplemented with 2,4-D, malt extract, L-glutamine, casein hydroly sate and coconut water (CW). Comparison was made of different media combinations of CW (5,10%), kinetin (lmg/l), zeatin (0.5mg/l) andTDZ (0.5mg/l) to study callus growth and regeneration. Results showed that CW (5%) + zeatin (0.5mg/l) was more effective over other growth regulators for callus induction while CW 5% was found effective for the regeneration of healthy plants in cv. Co-91010. Different levels of proline and thioproline were tested on the induction of embryogenie callus and subsequent plant regeneration in var. CoC-671. Proline at 2.61M showed vigorous growth of embryogenie callus compared to thioproline. Plants regenerated were hardened and grown in the green house to maturity.

Induction of secondary somatic embryogenesis in hybrid larch (Larix x leptoeuropaea) as related to ethylene

Induction of secondary somatic embryogenesis was studied with hybridlarch (Larix x leptoeuropaea)cotyledonary somatic embryos obtained after 3, 4, 5 and 6 weeks of culture on amaturation medium supplemented with abscisic acid. Almost all 3-week maturedcotyledonary somatic embryos can develop embryonal masses whereas only 78, 27and 12% of them are able to do so after 4, 5 and 6 weeks of maturation,respectively. During the first week of culture on the induction medium, somaticembryos with high embryogenic potential (i.e. 3-weekmatured) release little ethylene (less than 1.5 nL h−1g−1 FW), whereas those which have almost completelylosttheir ability to induce embryonal masses (i.e. 6-weekmatured) produce much more ethylene. Thereafter, ethylene production by bothtypes of embryos is very similar at around 5–6 nLh−1 g−1 FW. Enrichment of theatmosphere with ethylene, or addition of 2-chloroethylphosphonic acid(ethephon)or ACC in the induction medium strongly reduced the induction of secondarysomatic embryogenesis. Moreover, inhibitors of ethylene action(AgNO3and 2,5-norbornadiene) improved the development of embryonal masses fromsomaticembryos, particularly from the 6-week maturated ones. The results obtainedclearly suggest that ethylene is involved in the regulation of somaticembryogenesis in hybrid larch. The possible relationship between somaticembryogenic potential and ethylene biosynthesis by the explants or sensitivityof the latter to ethylene is discussed.

Regulation of somatic embryogenesis in celery cell suspensions

The regulation of somatic embryogenesis in celery (Apium graveolens L.) was studied to determine means of increasing its efficiency. Highly embryogenic cell lines were achieved by inducing cell cultures from in vitro plants which were previously regenerated from somatic embryos ('secondary' cell lines). The early detection of embryogenic potential of new cell lines was found to be regulated by 2,4-dichlorophenoxyacetic acid, mannitol and culture duration. Less frequent subculturing allowed embryogenic potential to be expressed earlier. Increased synchronization of celery somatic embryos was induced by two means: adding abscisic acid to the regeneration medium; and segregating the embryos by sieving them through serial metal mesh screens. When abscisic acid was removed from the growth medium, its effect became quickly transient. Embryos of 1400/~m in size provided the best growth rate and uniformity.

Regulation of somatic embryogenesis in celery cell suspensions

The regulation of somatic embryogenesis in celery (Apium graveolens L.) was studied to determine means of increasing its efficiency. Highly embryogenic cell lines were achieved by inducing cell cultures from in vitro plants which were previously regenerated from somatic embryos (‘secondary’ cell lines). The early detection of embryogenic potential of new cell lines was found to be regulated by 2,4-dichlorophenoxyacetic acid, mannitol and culture duration. Less frequent subculturing allowed embryogenic potential to be expressed earlier. Increased synchronization of celery somatic embryos was induced by two means: adding abscisic acid to the regeneration medium; and segregating the embryos by sieving them through serial metal mesh screens. When abscisic acid was removed from the growth medium, its effects became quickly transient. Embryos of 1400 µm in size provided the best growth rate and uniformity.

Role of ABA in the Regulation of Somatic Embryogenesis

Abstract Abscisic acid (ABA), a naturally occurring plant growth inhibitor, exerts a number of striking effects on the development of somatic embryos grown in suspension cultures. When added at moderate concentrations to populations of proembryos, ABA permits normal embryonic growth and maturation but inhibits diversions from this pathway. It prevents the initiation and growth of new embryonic centers and the malformation of cotyledons and shoots. In fully formed embryos, it inhibits their germination or conversion into plantlets. ABA also influences cellular differentiation, affecting both anatomy and biochemistry. Its role is to permit the normal progression of the embryonic programs. With ABA, somatic embryos closely resemble zygotic embryos in both structures and behavior. Practically, the manipulation of endogenous or exogenous ABA levels in populations of somatic embryos increases the frequency of embryos that reach maturity and facilitates their manipulation in large scale propagation and mechanized delivery.

Regulation of Somatic Embryogenesis and Organogenesis in Allium carinatum L.

Summary Callus cultures were derived from roots, shoots and aerial bulbils of Allium carinatum L. Regeneration was achieved either via somatic embryogenesis or organogenesis according to the type of explant. Shoot regeneration was also induced with cultures of isolated flower heads. In all instances the frequency of regeneration was high and regeneration took place for a period of more than 2 years on media with or without growth regulators.

Relationship between polyamine and ethylene biosynthesis in plants and its significance for morphogenesis in cell cultures.

The regulation of somatic embryogenesis in plant cell cultures is a topic of considerable interest, not only because of its potential applications for micropropagation and genetic engineering in plants but also as a prime example of the process of controlled development and differentiation. Therefore, it is not surprising that a vast amount of literature has accumulated discussing the morphogenetic and biochemical events underlying embryogénic development in cell cultures. The complexity of interaction of various chemical factors within the plant tissue and those that are externally applied, has been stressed in a number of reviews (Steward, 1976; Street, 1977; Ammirato, 1983, a, b, 1984; Sung et al., 1984; Raghavan, 1986).