Improved development of microspore-derived embryo cultures of Brassica napus cv Topaz following changes in glutathione metabolism

Glutathione has been shown to play an important role during embryo development in both plant and animal systems. The effects of altered glutathione metabolism during microspore-derived embryos (MDEs) of Brassica napus were investigated following exogenous application of reduced glutathione (GSH), its oxidized form (GSSG) and buthionine sulfoximine (BSO), an inhibitor of glutathione de novo synthesis. Applications of BSO which lowered the cellular glutathione redox status, i.e. GSH/(GSH + GSSG), enhanced significantly the quality of the embryos and their ability to convert into viable plants. Histological analyses revealed that inclusions of BSO in the culture medium altered the pattern of storage product accumulation in the embryos and improved the architecture of the shoot apical meristems (SAMs). Compared with their control counterparts which showed severe signs of SAM deterioration, such as the formation of intercellular spaces and differentiation of the meristematic cells, BSO-treated embryos had well-organized SAMs. The improved SAM organization observed in the presence of BSO also correlated with the proper localization pattern of WUSCHEL , a SAM molecular marker gene which was miss-expressed in control embryos. The beneficial effects of BSO on embryo development and conversion were ascribed to the increasing levels of ABA. The concentration of this growth regulator in BSO-treated embryos was always higher than that of control embryos during the second half of the maturation period. Furthermore, many structural alterations induced by BSO could be reproduced in embryos cultured in the presence of ABA. Taken together, these results suggest that a lowering of the glutathione redox status during embryo development may represent a metabolic switch needed for increasing the endogenous levels of ABA, which is required for successful completion of the developmental program.     

Applications of DL-buthionine-[S,R]-sulfoximine deplete cellular glutathione and improve white spruce (Picea glauca) somatic embryo development

In white spruce (Picea glauca), an improvement of somatic embryo yield and quality can be achieved by applications of dl-buthionine-[S,R]-sulfoximine (BSO), which inhibits the biosynthesis of reduced glutathione (GSH), thereby switching the total glutathione pool towards its oxidized form (GSSG). Applications of BSO almost tripled the embryogenic output of two cell lines by increasing the number of embryos produced by 100 mg⁻¹ tissue from 65 to 154 in the (E)WS1 line and from 59 to 130 in the (E)WS2 line. This increase in embryo number was ascribed to a higher production of morphologically normal embryos with four or more cotyledons (group A embryos), at the expense of group B embryos, characterized by fewer cotyledons. The quality of the embryos produced, estimated by their post-embryonic performance, was also different between treatments. In both cell lines applications of BSO in the maturation medium increased the conversion frequency, i.e. root and shoot emergence, of group A embryos while it enhanced root emergence in group B embryos. Compared to their control counterparts, BSO-treated embryos had normal shoot apical meristems as in their zygotic counterparts. Such meristems were characterized by large apical cells and vacuolated sub-apical cells. They also lacked intercellular spaces, which were present in the apical poles of control embryos where they contributed to cell–cell separation and meristem degradation. Furthermore, storage product accumulation was also improved in the presence of BSO, with protein bodies prevailing over starch. These data show that an oxidized glutathione environment is beneficial for spruce embryo production in vitro.     

Development of white spruce somatic embryos: II. Continual shoot meristem development during germination

Summary This study compares the development of shoot apical meristems of white spruce somatic and zygotic embryos during germination. In mature somatic embryos, the functional part of the shoot apical meristem was bi-layered. After partial drying, a normal shoot meristem was formed from these two cell layers during germination. Other cells within the meristem were vacuolated and separated by intercellular air spaces. In the absence of the partial drying treatment, somatic embryos enlarged in size primarily due to vacuolation of cells and the formation of large intercellular air spaces. A majority of these somatic embryos failed to form a functional shoot apical meristem. Compared with somatic embryos, the shoot apical meristem of a mature zygotic embryo was well organized with a densely cytoplasmic apical layer. The cells within the meristem were tightly packed. Judging from the cell profiles during germination, all cells within the meristem of the zygotic embryo took part in the formation of the vegetative shoot apical meristem.     

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.     

The effects of reduced and oxidized glutathione on white spruce somatic embryogenesis

Summary The glutathione-glutathione disulfide redox pair was utilized to improve white spurce somatic embryo development. Mature cotyledonary-stage somatic embryos were divided into two groups (A and B) based on morphological normality and the ability of the mature somatic embryos to convert into plantlets. Group A embryos had four or more cotyledons and converted readily upon germination after a partial drying treatment. Group B embryos had three or fewer cotyledons with a low conversion frequency. The addition of reduced glutathione (GSH) at a concentration of 0.1 mM resulted in an increase in embryo production (total population) with a mean total number of 64 embryos per 100 mg embryogenic tissue as well as an increase in post-embryonic root growth. However, at a higher concentration (1 mM), GSH inhibited embryo formation. The manipulation of the tissue culture environment via the inclusion of glutathione disulfide (GSSG), at concentrations of 0.1 and 1.0 mM, enhanced the development of better-quality embryos. This quality was best exemplified when embryos forming four or more cotyledons increased by at least twofold to 73.9% when treated with 1.0 mM GSSG, compared to 38% in control. Furthermore, this improved quality was reflected by an increased conversion frequency. A 20% increase in the ability of the somatic embryo to produce both root and shoot structures during post-embryonic development was noted when embryos were matured on maturation medium supplemented with 1.0 mM GSSG over the control.     

Induction of Zygotic Polyembryos in Wheat: Influence of Auxin Polar Transport

The effects of two auxin polar transport inhibitors, N-1-naphthylphthalamic acid (NPA) and 3,3[prime],4[prime],5,7-pentahydroxyflavone (quercetin), on attaining bilateral symmetry from radial symmetry during early wheat embryogenesis were investigated by using an in vitro culture system. Although NPA and quercetin belong to two different classes of auxin transport inhibitors, the phytotropins and the flavonoids, respectively, they induced the same specific abnormal phenotypes during embryo development. These abnormal embryos differentiated multiple meristems (i.e., multiple shoot and root meristems) and multiple organs (i.e., multiple coleoptiles and scutella). Multiple shoot apical meristem phenotypes were characterized by partly multiplied embryonic axes and supernumerary scutella. The differentiation of multiple primary roots in addition to multiple shoot meristems and multiple scutella led to the formation of polyembryos. The occurrence of multiple shoot meristem phenotypes depended on the concentration of the inhibitor and the developmental stage of the isolated embryo. Embryos treated with NPA or quercetin developed multiple radicle phenotypes less frequently than they developed multiple shoot meristem phenotypes. Our observations suggest that the root meristem differentiates later than the shoot meristem. Our data support the hypothesis that polar transport of auxin has a determining influence on the differentiation of the embryonic axis and the scutellum.     

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.     

In situ localization of storage protein mRNAs in developing meristems of Brassica napus embryos

Probes derived from cDNA clones of napin and cruciferin, the major storage proteins of Brassica napus, and in situ hybridization techniques were used to examine changes in the spatial and temporal distribution of storage protein messages during the course of embryogeny, with a special emphasis on the developing apical meristems. Napin mRNAs begin to accumulate in the cortex of the axis during late heart stage, in the outer faces of the cotyledons during torpedo stage and in the inner faces of the cotyledons during cotyledon stage. Cruciferin mRNAs accumulate in a similar pattern but approximately 5 days later. Cells in the apical regions where root and shoot meristems develop do not accumulate storage protein messages during early stages of embryogeny. In the upper axis, the boundary between these apical cells and immediately adjacent cells that accumulate napin and cruciferin mRNAs is particularly distinct. Our analysis indicates that this boundary is not related to differences in tissue or cell type, but appears instead to be coincident with the site of a particular set of early cell divisions. A major change in the mRNA accumulation patterns occurs halfway through embryogeny, as the embryos enter maturation stage and start drying down. Final maturation of the shoot apical meristem is associated with the development of leaf primordia and the accumulation of napin mRNAs in the meristem, associated leaf primordia and vascular tissue. Cruciferin mRNAs accumulate only in certain zones of the shoot apical meristem and on the flanks of leaf primordia. Neither type of mRNA accumulates in the root apical meristem at any stage.     

Transposon tagging of the Defective embryo and meristems gene of tomato.

The shoot and root apical meristems (SAMs and RAMs, respectively) of higher plants are mechanistically and structurally similar. This has led previously to the suggestion that the SAM and RAM represent modifications of a fundamentally homologous plan of organization. Despite recent interest in plant development, especially in the areas of meristem regulation, genes specifically required for the function of both the SAM and RAM have not yet been identified. Here, we report on a novel gene, Defective embryo and meristems (Dem), of tomato. This gene is required for the correct organization of shoot apical tissues of developing embryos, SAM development, and correct cell division patterns and meristem maintenance in roots. Dem was cloned using transposon tagging and shown to encode a novel protein of 72 kD with significant homology to YNV2, a protein of unknown function of Saccharomyces cerevisiae. Dem is expressed in root and shoot meristems and organ primordia but not in callus. The expression pattern of Dem mRNA in combination with the dem mutant phenotype suggests that Dem plays an important role within apical meristems.     

Failure to establish a functional shoot meristem may be a cause of conversion failure in somatic embryos of Daucus carota (Apiaceae)

A carrot cell culture line was shown to be highly embryogenic, but plantlet recovery (conversion) was low (about 14%). The majority of somatic embryos that did not convert showed pronounced vacuolation in the apical notch, leading to their inability to form primary leaves and thereby convert. Comparisons of developing meristems in the shoot apical notch of converting somatic and zygotic embryos revealed similarities in cytoplasmic density and meristem organization between the two populations. Abscisic acid (ABA) was shown to significantly increase conversion in somatic embryos of globular, torpedo, and preplantlet stages (62%, 62.5% and 40%, respectively). Somatic embryos that were treated with 50 μM ABA showed retention of the highly cytoplasmic cells in the apical notch. Histological analysis showed a resemblance between shoot apices of converting somatic embryos and ABA-treated somatic embryos. ABA may be an induction agent for meristematic organization, or perhaps may cause cells of the apical notch to extend competence for determination as meristematic cells.     

Role of 2,4-dichlorophenoxyacetic acid (2,4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2,4-D

The relationship between cell expansion and cell cycling during somatic embryogenesis was studied in cultured bent-cotyledon-stage zygotic embryos of a transgenic stock of Arabidopsis thaliana harboring a cyclin 1 At:β-glucuronidase (GUS) reporter gene construct. In embryos cultured in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D), following a brief period of growth by cell expansion, divisions were initiated in the procambial cells facing the adaxial side at the base of the cotyledons. Cell division activity later spread to almost the entire length of the cotyledons to form a callus on which globular and heart-shaped embryos appeared in about 10 d after culture. Anatomical and morphogenetic changes observed in cultured embryos were correlated with patterns of cell cycling by histochemical detection of GUS-expressing cells. Although early-stage somatic embryos did not develop further during their continued growth in the auxin-containing medium, maturation of embryos ensued upon their transfer to an auxin-free medium. In a small number of cultured zygotic embryos the shoot apical meristem was found to differentiate a leaf, a green tubular structure, or a somatic embryo. Contrary to the results from previous investigations, which have assigned a major role for the shoot apical meristem and cells in the axils of cotyledons in the development of somatic embryos on cultured zygotic embryos of A. thaliana, the present work shows that somatic embryos originate almost exclusively on the callus formed on the cotyledons. Other observations such as the induction of somatic embryos on cultured cotyledons and the inability of the embryo axis (consisting of the root, hypocotyl, and shoot apical meristem without the cotyledons) to form somatic embryos, reaffirm the important role of the cotyledons in somatic embryogenesis in this plant.     

Coordination of apical and basal embryo development revealed by tissue-specific GNOM functions

Flowering-plant embryogenesis generates the basic body organization, including the apical and basal stem cell niches, i.e. shoot and root meristems, the major tissue layers and the cotyledon(s). gnom mutant embryos fail to initiate the root meristem at the early-globular stage and the cotyledon primordia at the late globular/transition stage. Tissue-specific GNOM expression in the gnom mutant embryo revealed that both apical and basal embryo organization depend on GNOM provascular expression and a functioning apical-basal auxin flux: GNOM provascular expression in gnom mutant background resulted in non-cell-autonomous reconstitution of apical and basal tissues which could be linked to changes in auxin responses in those tissues, stressing the importance of apical-basal auxin flow for overall embryo organization. Although reconstitution of apical-basal auxin flux in gnom results in the formation of single cotyledons (monocots), only additional GNOM epidermal expression is able to induce wild-type apical patterning. We conclude that provascular expression of GNOM is vital for both apical and basal tissue organization, and that epidermal GNOM expression is required for radial-to-bilateral symmetry transition of the embryo. We propose GNOM-dependent auxin sinks as a means to generate auxin gradients across tissues.     

Comparative anatomy of embryogenesis in three species of Podostemaceae and evolution of the loss of embryonic shoot and root meristems

During embryogenesis in angiosperms, the embryonic shoot and root meristems are created at opposite poles of the embryo, establishing a vertical body plan. However, the aquatic eudicot family Podostemaceae exhibits an unusual horizontal body plan, which is attributed to the loss of embryonic shoot and root meristems. To infer the embryogenetic changes responsible for the loss of these meristems, we examined the embryogenesis of three podostemads with different meristem characters, that is, Terniopsis brevis with distinct shoot and root meristems, Zeylanidium lichenoides with reduced shoot and no root meristems, and Hydrobryum japonicum with no shoot and no root meristems. In T. brevis, as in other eudicots, the putative organizing center (OC) and L1 layer (=the epidermal cell layer) arose to generate a distinct shoot meristem initial, and the hypophysis formed the putative quiescent center (QC) of a root meristem. Z. lichenoides had a morphologically unrecognizable shoot meristem, because a distinct L1 layer did not develop, whereas the putative OC precursor arose normally. In H. japonicum, the vertical divisions of the apical cells of eight-cell embryo prevented putative OC initiation. In Z. lichenoides and H. japonicum, the putative QC failed to initiate because the hypophysis repeated longitudinal divisions during early embryogenesis. Based on their phylogenetic relationships, we infer that the conventional embryonic shoot meristem was lost in Podostemaceae via two steps, that is, the loss of a distinct L1 layer and the loss of the OC, whereas the loss of the embryonic root meristem occurred once by misspecification of the hypophysis.     

Structural and component characterization of meristem cells in Araucaria angustifolia (Bert.) O. Kuntze zygotic embryo

Araucaria angustifolia, the Brazilian pine, is an endangered native conifer with economic and ecological importance. The female cone develops seeds containing the zygotic embryo, which, at cotyledonary stage, shows well-developed meristems. Little is known about the structure of gymnosperm meristems. In the present work, the composition and morphological organization of Araucaria angustifolia shoot and root apical meristems were studied during embryo development, using histochemical and microscope analyses. Histochemical evaluation revealed the presence of cellulose within the cell wall, cells with the presence of total proteins that react with Coomassie Brilliant Blue, starch grains, and large nuclei with evident nucleoli in the cytoplasm. Scanning electron microscopy showed apical meristem surface morphology, and both scanning and transmission microscopy revealed a thin and irregular cell wall with plasmodesmata and within the cells, mitochondria, many vacuoles, lipid bodies, Golgi bodies, and many amyloplasts with endoplasmic reticulum surrounding them and large nuclei. Similar to angiosperm cells, A. angustifolia meristem cells exhibit pluripotent characteristics, such as apparatus for intercellular communication and differentiation.     

Distinct nuclear organization, DNA methylation pattern and cytokinin distribution mark juvenile, juvenile-like and adult vegetative apical meristems in peach (Prunus persica (L.) Batsch)

Chromatin organization, nuclear DNA methylation and endogenous zeatin localization were investigated in shoot apical meristems (SAM) during juvenile and adult phases of peach (Prunus persica (L.) Batsch). The aim was to examine the extent to which these parameters could discriminate the juvenile and adult SAMs. Seedlings (juvenile, cannot flower), basal shoots (called juvenile-like, because they exhibit juvenile macroscopic traits) and apical shoots (competent to form flowers) of adult plants were chosen. Nuclear chromatin exhibited chromocentres that were peripherally distributed in SAMs of juvenile and juvenile-like shoots, but were diffusely spread in those of adult shoots. These patterns coincided with a peripheral labelling of DNA methylation in juvenile and juvenile-like meristem nuclei versus a diffuse labelling pattern in adult meristem nuclei. During vegetative growth (from March to June), the level of nuclear DNA methylation was higher in adult meristems than in juvenile and juvenile-like ones. The immunolocalization of zeatin in juvenile SAM was in the subapical region, but adult meristems exhibited a widespread localization or a signal confined within the boundaries of the central zone. The extent to which the acquisition of a strongly zonated pattern of these parameters as markers of floral competence in adult SAMs is discussed.     

The effect of osmoticum on ascorbate and glutathione metabolism during white spruce (Picea glauca) somatic embryo development.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.     

Formation and maintenance of the shoot apical meristem

Development in higher plants is characterized by the reiterative formation of lateral organs from the flanks of shoot apical meristems. Because organs are produced continuously throughout the life cycle, the shoot apical meristem must maintain a pluripotent stem cell population. These two tasks are accomplished within separate functional domains of the apical meristem. These functional domains develop gradually during embryogenesis. Subsequently, communication among cells within the shoot apical meristem and between the shoot apical meristem and the incipient lateral organs is needed to maintain the functional domains within the shoot apical meristem.     

Shoot meristem size is dependent on inbred background and presence of the maize homeobox gene, knotted1

The knotted1 (kn1) gene of maize is expressed in meristems and is absent from leaves, including the site of leaf initiation within the meristem. Recessive mutations of kn1 have been described that limit the capacity to make branches and result in extra carpels. Dominant mutations suggest that kn1 function plays a role in maintaining cells in an undifferentiated state. We took advantage of a Ds-induced dominant allele in order to screen for additional recessive alleles resulting from mobilization of the Ds element. Analysis of one such allele revealed a novel embryonic shoot phenotype in which the shoot initiated zero to few organs after the cotyledon was made, resulting in plants that arrested as seedlings. We refer to this phenotype as a limited shoot. The limited shoot phenotype reflected loss of kn1 function, but its penetrance was background dependent. We examined meristem size and found that plants lacking kn1 function had shorter meristems than non-mutant siblings. Furthermore, meristems of restrictive inbreds were significantly shorter than meristems of permissive inbreds, implying a correlation between meristem height and kn1 gene function in the embryo. Analysis of limited shoot plants during embryogenesis indicated a role for kn1 in shoot meristem maintenance. We discuss a model for kn1 in maintenance of the morphogenetic zone of the shoot apical meristem.     

Shoot organization genes regulate shoot apical meristem organization and the pattern of leaf primordium initiation in rice

The mechanism regulating the pattern of leaf initiation was analyzed by using shoot organization (sho) mutants derived from three loci (SHO1, SHO2, and SHO3). In the early vegetative phase, sho mutants show an increased rate of leaf production with random phyllotaxy. The resulting leaves are malformed, threadlike, or short and narrow. Their shoot apical meristems are relatively low and wide, that is, flat shaped, although their shape and size are highly variable among plants of the same genotype. Statistical analysis reveals that the shape of the shoot meristem rather than its size is closely correlated with the variations of plastochron and phyllotaxy. Rapid and random leaf production in sho mutants is correlated with the frequent and disorganized cell divisions in the shoot meristem and with a reduction of expression domain of a rice homeobox gene, OSH1. These changes in the organization and behavior of the shoot apical meristems suggest that sho mutants have fewer indeterminate cells and more determinate cells than wild type, with many cells acting as leaf founder cells. Thus, the SHO genes have an important role in maintaining the proper organization of the shoot apical meristem, which is essential for the normal initiation pattern of leaf primordia.