Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10⁻⁴M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. Received: 22 April 2000 / Accepted: 8 June 2000     

Developmental pattern formation of somatic embryos induced in cell suspension cultures of cowpea [Vigna unguiculata (L.) Walp]

The sequence of events in the functional body pattern formation during the somatic embryo development in cowpea suspensions is described under three heads. Early stages of somatic embryogenesis were characterized by both periclinal and anticlinal cell divisions. Differentiation of the protoderm cell layer by periclinal divisions marked the commencement of somatic embryogenesis. The most critical events appear to be the formation of apical meristems, establishment of apical-basal patterns of symmetry, and cellular organization in oblong-stage somatic embryo for the transition to torpedo and cotyledonary-stage somatic embryos. Two different stages of mature embryos showing distinct morphology, classified based on the number of cotyledons and their ability to convert into plantlets, were visualized. Repeated mitotic divisions of the sub-epidermal cell layers marked the induction of proembryogenic mass (PEM) in the embryogenic calli. The first division plane was periclinally-oriented, the second anticlinally-oriented, and the subsequent division planes appeared in any direction, leading to clusters of proembryogenic clumps. Differentiation of the protoderm layer marks the beginning of the structural differentiation in globular stage. Incipient procambium formation is the first sign of somatic embryo transition. Axial elongation of inner isodiametric cells of the globular somatic embryo followed by the change in the growth axis of the procambium is an important event in oblong-stage somatic embryo. Vacuolation in the ground meristem of torpedo-stage embryo begins the process of histodifferentiation. Three major embryonic tissue systems; shoot apical meristem, root apical meristem, and the differentiation of procambial strands, are visible in torpedo-stage somatic embryo. Monocotyledonary-stage somatic embryo induced both the shoot apical meristem and two leaf primordia compared to the ansiocotyledonary somatic embryo.     

Study of aberrant morphologies and lack of conversion of somatic embryos of chickpea (Cicer arietinum L.)

Summary Somatic embryos which originated from mature embryo axes of the chickpea (Cicer arietinum L.) showed varied morphologies. Embryos were classified based on shape of the embryo and number of cotyledons. “Normal” (zygotic-like) embryos were bipolar structures with two cotyledons and a well-developed shoot and root apical meristem, whereas “aberrant” embryos were horn-shaped, had single and multiple cotyledons, and were fasciated. Histological examination revealed the absence of a shoot apical meristem in horn-shaped embryos. Fasciated embryos showed diaxial fusion of two embryos. Secondary embryogenesis was also observed, in which the embryos emerged from the hypocotyl and cotyledonary region of the primary somatic embryo. This report documents the absence of an apical meristem as a vital factor in the lack of conversion of aberrant somatic embryos.     

Formation, Maintenance and Function of the Shoot Apical Meristem in Rice

In higher plants, the process of embryogenesis establishes the plant body plan (body axes). On the basis of positional information specified by the body axes, the shoot apical meristem (SAM) and root apical meristem (RAM) differentiate at fixed positions early in embryogenesis. After germination, SAM and RAM are responsible for the development of the above-ground and below-ground parts, respectively, of the plant. Because of the importance of SAM function in plant development, the mechanisms of SAM formation during embryogenesis and of SAM maintenance and function in post-embryonic development are priority questions in plant developmental biology. Recent advances in molecular and genetic analysis of morphogenetic mutations in Arabidopsis have revealed several components required for SAM formation, maintenance and function. Although these processes are fundamental to the life cycle of every plant, conservation of the components does not explain the diversity of plant morphologies. Rice is used as a model plant of the grass family and of monocots because of the progress in research infrastructure, especially the collection of unique mutations and genome information. In comparison with the dicot Arabidopsis, rice has many unique organs or processes of development. This review summarizes what is known of the processes of SAM formation, maintenance and function in rice.     

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.     

Studies on the behavior of organelles and their nucleoids in the root apical meristem of Arabidopsis thaliana (L.) Col.

The behavior of cell nuclei, mitochondrial nucleoids (mt-nucleoids) and plastid nucleoids (ptnucleoids) was studied in the root apical meristem of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole for light-microscopic autoradiography and microphotometry. Synthesis of cell nuclear DNA and cell division were both active in the root apical meristem between 0 μm and 300 μm from the central cells. It is estimated that the cells generated in the lower part of the root apical meristem enter the elongation zone after at least four divisions. Throughout the entire meristematic zone, individual cells had mitochondria which contained 1–5 mt-nucleoids. The number of mitochondria increased gradually from 65 to 200 in the meristem of the central cylinder. Therefore, throughout the meristem, individual mitochondria divided either once or twice per mitotic cycle. By contrast, based on the incorporation of [³H]thymidine into organelle nucleoids, syntheses of mitochondrial DNA (mtDNA) and plastid DNA (ptDNA) occurred independently of the mitotic cycle and mainly in a restricted region (i.e., the lower part of the root apical meristem). Fluorimetry, using a videointensified microscope photon-counting system, revealed that the amount of mtDNA per mt-nucleoid in the cells in the lower part of the meristem, where mtDNA synthesis was active, corresponded to more than 1 Mbp. By contrast, in the meristematic cells just below the elongation zone of the root tip, the amount of mtDNA per mt-nucleoid fell to approximately 170 kbp. These findings strongly indicate that the amount of mtDNA per mitochondrion, which has been synthesized in the lower part of the meristem, is gradually reduced as a result of continual mitochondrial divisions during low levels of mtDNA synthesis. This phenomenon would explain why differentiated cells in the elongation zone have mitochondria that contain only extremely small amounts of mtDNA. This work was supported by a Grant-in Aid (T.K.) for Special Research on Priority Areas (Project No. 02242102, Cellular and Molecular Basis for Reproduction Processes in Plants) from the Ministry of Education, Science and Culture of Japan and by a Grant-in Aid (T.K.) for Original and Creative Research Project on Biotechnology from the Research Council, Ministry of Agriculture, Forestry and Fisheries of Japan.     

Plant meristems: <Emphasis Type="Italic">CLAVATA3/ESR</Emphasis>-related signaling in the shoot apical meristem and the root apical meristem

The plant meristems, shoot apical meristem (SAM) and root apical meristem (RAM), are unique structures made up of a self-renewing population of undifferentiated pluripotent stem cells. The SAM produces all aerial parts of postembryonic organs, and the RAM promotes the continuous growth of roots. Even though the structures of the SAM and RAM differ, the signaling components required for stem cell maintenance seem to be relatively conserved. Both meristems utilize cell-to-cell communication to maintain proper meristematic activities and meristem organization and to coordinate new organ formation. In SAM, an essential regulatory mechanism for meristem organization is a regulatory loop between WUSCHEL (WUS) and CLAVATA (CLV), which functions in a non-cell-autonomous manner. This intercellular signaling network coordinates the development of the organization center, organ boundaries and distant organs. The CLAVATA3/ESR (CLE)-related genes produce signal peptides, which act non-cell-autonomously in the meristem regulation in SAM. In RAM, it has been suggested that a similar mechanism can regulate meristem maintenance, but these functions are largely unknown. Here, we overview the WUS–CLV signaling network for stem cell maintenance in SAM and a related mechanism in RAM maintenance. We also discuss conservation of the regulatory system for stem cells in various plant species. S. Sawa is the recipient of the BSJ Award for Young Scientist, 2007.     

Localization of organelle DNA synthesis within the root apical meristem of rice

DNA synthesis in cell nuclei and organelles in the root apicalmeristem of rice was analysed by anti-BrdU immunofluorescencemicroscopy to determine whether there is a specific order ofthese events in monocot roots. In the root meristem, organelleDNAs were synthesized in a specific region in the distal partof the root apical meristem, and were not synthesized in theroot meristem‘s proximal region or the elongation zone.In contrast, cell nuclear DNA was synthesized throughout theroot apical meristem, except in the quiescent centre. In theroot cap of rice, DNA synthesis in both cell nuclei and organellenucleoids was detected only in the two layers of cells at theproximal end, which is a striking characteristic of monocotyledonousplants. Moreover, to determine quantitatively the activity ofDNA synthesis in cell nuclei and organelle nucleoids in micro-scalesections of plant tissues, we developed novel techniques formicro-scale hybridization and immuno-detection analysis. Atthe distal end of the root apical meristem, DNA levels of plastidsand mitochondria were 4-fold and 5-fold greater than those inthe elongation zone, respectively. Intracellular organelle DNAlevels dropped rapidly as the distance from the root tip increased.The activity of organelle DNA synthesis in the distal end ofthe root apical meristem was about 10-fold greater than thatin the elongation zone. Our present results confirm that nuclearand organelle DNA synthesis are not synchronized, but the latteroccurs preferentially before multiple cell divisions. Key words: Organelle DNA synthesis, organelle nucleoids (organelle nuclei), root apical meristem, anti-bromo-deoxyuridine immunofluorescence microscopy, rice.     

POPCORN functions in the auxin pathway to regulate embryonic body plan and meristem organization in Arabidopsis

The shoot and root apical meristems (SAM and RAM) formed during embryogenesis are crucial for postembryonic plant development. We report the identification of POPCORN (PCN), a gene required for embryo development and meristem organization in Arabidopsis thaliana. Map-based cloning revealed that PCN encodes a WD-40 protein expressed both during embryo development and postembryonically in the SAM and RAM. The two pcn alleles identified in this study are temperature sensitive, showing defective embryo development when grown at 22°C that is rescued when grown at 29°C. In pcn mutants, meristem-specific expression of WUSCHEL (WUS), CLAVATA3, and WUSCHEL-RELATED HOMEOBOX5 is not maintained; SHOOTMERISTEMLESS, BODENLOS (BDL) and MONOPTEROS (MP) are misexpressed. Several findings link PCN to auxin signaling and meristem function: ectopic expression of DR5(rev):green fluorescent protein (GFP), pBDL:BDL-GFP, and pMP:MP-β-glucuronidase in the meristem; altered polarity and expression of pPIN1:PIN1-GFP in the apical domain of the developing embryo; and resistance to auxin in the pcn mutants. The bdl mutation rescued embryo lethality of pcn, suggesting that improper auxin response is involved in pcn defects. Furthermore, WUS, PINFORMED1, PINOID, and TOPLESS are dosage sensitive in pcn, suggesting functional interaction. Together, our results suggest that PCN functions in the auxin pathway, integrating auxin signaling in the organization and maintenance of the SAM and RAM.     

FASCIATA genes for chromatin assembly factor-1 in arabidopsis maintain the cellular organization of apical meristems

Postembryonic development of plants depends on the activity of apical meristems established during embryogenesis. The shoot apical meristem (SAM) and the root apical meristem (RAM) have similar but distinct cellular organization. Arabidopsis FASCIATA1 (FAS1) and FAS2 genes maintain the cellular and functional organization of both SAM and RAM, and FAS gene products are subunits of the Arabidopsis counterpart of chromatin assembly factor-1 (CAF-1). fas mutants are defective in maintenance of the expression states of WUSCHEL (WUS) in SAM and SCARECROW (SCR) in RAM. We suggest that CAF-1 plays a critical role in the organization of SAM and RAM during postembryonic development by facilitating stable maintenance of gene expression states.     

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.     

Organelle DNA Synthesis in the Quiescent centre of Arabidopsis thaliana (Col.)

The behaviour of cell nuclei and organelle nucleoids (organellenuclei) was studied in the root apical meristem of 3-d-old seedlingsof Arabidopsis thaliana (Col.). Samples were embedded in Technovit7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole(DAPI) for observation of DNA. DNA synthesis in cell nucleiand organelle nucleoids was investigated using the incorporationof [³H] thymidine or 5-bromo-2'-deoxyuridine (BrdU). Incorporated[³H] thymidine and BrdU were detected by microautoradiographyor immunofiuorescence microscopy, respectively. Central cellsand cells just above the central cells of the quiescent centre(QC) showed an extremely low activity of DNA synthesis. However,DNA synthesis occurred in at least one organelle nucleoid ofall cells in the QC within 24 h. This suggests the cells inthe QC are quiescent with regard to nuclear DNA synthesis, butnot with regard to the organelle nucleoids. Key words: Arabidopsis thaliana, quiescent centre, root apical meristem, mitochondrial nucleoid (nuclei), plastid nucleoid (nuclei)     

Somatic embryogenesis in soybean via somatic embryo cycling

Summary The objectives of the present research were: a) to develop an efficient soybean embryogenic regeneration system characterized by a high frequency of explant response and a large number of somatic embryos per explant; b) to evaluate the factors affecting somatic embryogenesis via somatic embryo cycling; and c) to identify the origin of somatic embryos in the system. A highly improved and efficient system for soybean somatic embryogenesis was established using somatic embryo cotyledons and somatic embryo hypocotyl/radicle explants plated on α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented MS basal media. The system included somatic embryo cycling between liquid and solid medium and it consistently gave rise to a much higher frequency of explant response and a larger number of embryos per responding explant than those obtained from zygotic cotyledon explant tissues. Genotype, differences were observed for response in some of the treatments with cv “Fayette” being more responsive than “J103”. Histological studies revealed that somatic embryos induced in the somatic embryo cycling system originated almost exclusively from epidermal cells on both 2,4-D and NAA inductive media. The cells of the epidermis proliferated to produce somatic embryos directly without an intervening callus phase. A single-cell origin of somatic embryos was observed in cultures on a 40 mg/liter 2,4-D treatment. A large number of responding cells in the epidermis was also observed in the 10 mg/liter NAA treatment. The single-cell origin of somatic embryos from epidermal layers of the explant tissues should facilitate development of an efficient transformation system for soybean.     

Somatic embryo development in carrot is associated with an increase in levels of S-adenosylmethionine,S-adenosylhomocysteine and DNA methylation

Almost homogeneous populations representing different developmental stages of somatic embryos (globular, torpedo-shaped, plantlets) and vacuolated cells were obtained from a cell suspension culture of carrot. The concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and methylated DNA were determined in embryos at different developmental stages and were found to increase during somatic embryogenesis. The highest increase during embryogenesis was a 5-fold increase in the level of SAM. A considerable increase in the methylation index (SAM/SAH ratio) was also found. We propose that the levels of SAM and SAH may be involved in the control of somatic embryogenesis by affecting the level of DNA methylation, which in turn might cause differential changes in gene activation. An increase in the level of SAM may be a prerequisite for progression of embryogenesis and the development of complete embryos.     

Plant stem cells and their regulations in shoot apical meristems

Stem cells in plants, established during embryogenesis, are located in the centers of the shoot apical meristem (SAM) and the root apical meristem (RAM). Stem cells in SAM have a capacity to renew themselves and to produce new organs and tissues indefinitely. Although fully differentiated organs such as leaves do not contain stem cells, cells in such organs do have the capacity to re-establish new stem cells, especially under the induction of phytohormones in vitro. Cytokinin and auxin are critical in creating position signals in the SAM to maintain the stem cell organizing center and to position the new organ primordia, respectively. This review addresses the distinct features of plant stem cells and focuses on how stem cell renewal and differentiation are regulated in SAMs.     

Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants

 In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation. Received: 30 July 1999 / Accepted: 16 February 2000     

Establishment of the embryonic shoot apical meristem in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In higher plants, shoot organs such as leaves, branches, and flowers are generated from the shoot apical meristem (SAM), a small group of undifferentiated cells located at the tip of the shoot. The SAM maintains its pluripotency and simultaneously produces lateral organs at its periphery. The SAM arises during embryogenesis and its positioning requires axis-dependent embryo patterning and compartmentalization of the embryo apex. Here, we introduce major factors involved in these processes in Arabidopsis thaliana and discuss how the embryonic SAM is established.     

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.