Alterations of the glutathione redox state improve apical meristem structure and somatic embryo quality in white spruce (Picea glauca)

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.     

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.     

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.     

Influence of auxin on the establishment of bilateral symmetry in monocots

To study the influence of auxin on the shift from radial to bilateral symmetry during monocot embryogenesis, the fate of young wheat (Triticum aestivum L.) zygotic embryos has been manipulated in vitro by adding auxins, an auxin transport inhibitor and an auxin antagonist to the culture medium. The two synthetic auxins used, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), induced identical phenotypes. In the most severe cases, the shift from radial to bilateral symmetry was blocked resulting in continuous uniform radial growth. The natural auxin indole-3-acetic acid (IAA) induced the same phenotype. The effect of 2,4,5-T and 2,4D depended on their concentrations and on the developmental stage of the isolated embryos. In the presence of 2,3,5-triiodobenzoic acid (TIBA), an auxin transport inhibitor, the overall embryo symmetry was abnormal. The relative position of the shoot apical meristem in comparison with the scutellum was anomalous. The quality of shoot apical meristem and the scutellum differentiation was altered compared with normal developed embryos. No root meristem was differentiated. The effect of TIBA depends on its concentration and on the developmental stage of the isolated embryos. By contrast, 2-(pchlorophenoxy)-2-methylpropionic acid (PCIB) which is described as an auxin antagonist, has no visible direct effect on the embryonic symmetry. These observations indicate that auxin influences the change from radial symmetry to embryonic polarity during monocot embryogenesis. A model of auxin action during early wheat embryo development is proposed.     

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.     

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.     

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.     

The rates of deceleration of nuclear and organellar DNA syntheses differ in the progenitor cells of the apical meristems during carrot somatic embryogenesis

 The synthesis of DNA in nuclei and organellar nucleoids at the various stages of somatic embryogenesis in carrot (Daucus carota L. cv. Kurodagosun) was analyzed using anti-5-bromo-2′-deoxyuridine (BrdU) immunofluorescence microscopy. The active syntheses of both nuclear and organellar DNA started in the cells forming the embryo proper 3 d after the initiation of embryogenesis, but not in cells forming suspensor-like cell aggregates. In the early globular embryo, active DNA syntheses were continuously observed in the whole embryo proper, except for the progenitor cells of the root apical meristem (RAM) and shoot apical meristem (SAM). These were recognized as slowly cycling cells with a non-BrdU-labelled nucleus and strongly BrdU-labelled organellar nucleoids. At the heart- and torpedo-shaped embryo stages, both nuclear and organellar DNA syntheses were inactive in the presumptive RAM and SAM. Thus, slowing down of organellar DNA synthesis is not coupled with, but is later than, that of nuclear DNA synthesis in the progenitor cells of the embryonic RAM and SAM. These findings clearly indicate that the timing of DNA synthesis is similar in the progenitor cells of both the RAM and SAM in the early stages of somatic embryogenesis. Received: 18 January 2000 / Accepted: 2 March 2000     

Vernalization in Lolium temulentum L.: Responses of In Vitro Cultures of Mature and Immature Embryos, Shoot Apices and Callus

Response of vernalization to low temperature (2C) was studiedin a winter-annual form of Lolium temulentum L., using imbibedseeds, excised mature and immature embryos, shoot apical meristemsand callus tissue. Excised embryos, as early as 5 days afteranthesis, and excised shoot apices could be vernalized as effectivelyas imbibed seeds. Cold treatment of developing embryos withinthe ear, however, appeared to have no vernalizing effect. Plantsderived from callus by somatic embryogenesis showed varyingdegrees of vernalization response. The vernalization response in L. temulentum, as in winter annualcereals, appears to be located in the shoot apical meristemand the vernalized condition can be transmitted to new axillarymeristems formed from it. Lolium temulentum L. darnel, vernalization, embryo culture, apical meristem culture, callus culture     

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.     

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.     

The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo

In Arabidopsis embryogenesis, the primary root meristem originates from descendants of both the apical and the basal daughter cell of the zygote. We have isolated a mutant of a new gene named BODENLOS (BDL) in which the primary root meristem is not formed whereas post-embryonic roots develop and bdl seedlings give rise to fertile adult plants. Some bdl seedlings lacked not only the root but also the hypocotyl, thus resembling monopteros (mp) seedlings. In addition, bdl seedlings were insensitive to the auxin analogue 2,4-D, as determined by comparison with auxin resistant1 (axr1) seedlings. bdl embryos deviated from normal development as early as the two-cell stage at which the apical daughter cell of the zygote had divided horizontally instead of vertically. Subsequently, the uppermost derivative of the basal daughter cell, which is normally destined to become the hypophysis, divided abnormally and failed to generate the quiescent centre of the root meristem and the central root cap. We also analysed double mutants. bdl mp embryos closely resembled the two single mutants, bdl and mp, at early stages, while bdl mp seedlings essentially consisted of hypocotyl but did form primary leaves. bdl axr1 embryos approached the mp phenotype at later stages, and bdl axr1 seedlings resembled mp seedlings. Our results suggest that BDL is involved in auxin-mediated processes of apical-basal patterning in the Arabidopsis embryo.     

Embryogenic and callus-specific proteins in somatic embryogenesis of the grass,Dactylis glomerata L.

Somatic embryogenesis occurs spontaneously in some monocotyledoneous callus and cell suspension cultures maintained in suitable culture conditions. Nevertherless, the processes involved in somatic embryo development, and factors inducing this differentiation, are poorly understood. In order to study the changes in protein composition accompanying embryogenesis in cell suspension cultures of Dactylis glomerata L., embryos of various sizes and “undifferentiated” callus cells were separated and their total cellular protein extracts analyzed by two-dimensional polyacrylamide gel electrophoresis. Several proteins could be identified that are specific for embryos or callus under various culture conditions. Three independent detection methods were employed: silver-staining of proteins, in vivo labeling of proteins with [³⁵S]methionine, and in vitro translation of poly(A)⁺ RNA. All culture conditions tested, including those that induce embryonic proteins in carrot, fail to induce embryonic proteins in D. glomerata callus cells.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Regeneration of zygotic-like microspore-derived embryos suggests an important role for the suspensor in early embryo patterning

The inaccessibility of the zygote and proembryos of angiospermswithin the surrounding maternal and filial tissues has hamperedstudies on early plant embryogenesis. Somatic and gametophyticembryo cultures are often used as alternative systems for molecularand biochemical studies on early embryogenesis, but are notwidely used in developmental studies due to differences in theearly cell division patterns with seed embryos. A new Brassicanapus microspore embryo culture system, wherein embryogenesishighly mimics zygotic embryo development, is reported here.In this new system, the donor microspore first divides transverselyto form a filamentous structure, from which the distal cellforms the embryo proper, while the lower part resembles thesuspensor. In conventional microspore embryogenesis, the microsporedivides randomly to form an embryonic mass that after a whileestablishes a protoderm and subsequently shows delayed histodifferentiation.In contrast, the embryo proper of filament-bearing microspore-derivedembryos undergoes the same ordered pattern of cell divisionand early histodifferentiation as in the zygotic embryo. Thisobservation suggests an important role for the suspensor inearly zygotic embryo patterning and histodifferentiation. Thisis the first in vitro system wherein single differentiated cellsin culture can efficiently regenerate embryos that are morphologicallycomparable to zygotic embryos. The system provides a powerfulin vitro tool for studying the diverse developmental processesthat take place during the early stages of plant embryogenesis. Key words: Brassica napus, microspore embryogenesis, pattern formation, polarity, suspensor, zygotic embryogenesis     

LEAFY COTYLEDON1 Is an Essential Regulator of Late Embryogenesis and Cotyledon Identity in Arabidopsis

LEAFY COTYLEDON1 (LEC1) is an embryo defective mutation that affects cotyledon identity in Arabidopsis. Mutant cotyledons possess trichomes that are normally a leaf trait in Arabidopsis, and the cellular organization of these organs is intermediate between that of cotyledons and leaves from wild-type plants. We present several lines of evidence that indicate that the control of late embryogenesis is compromised by the mutation. First, mutant embryos are desiccation intolerant, yet embryos can be rescued before they dry to yield homozygous recessive plants that produce defective embryos exclusively. Second, although many genes normally expressed during embryonic development are active in the mutant, at least one maturation phase-specific gene is not activated. Third, the shoot apical meristem is activated precociously in mutant embryos. Fourth, in mutant embryos, several genes characteristic of postgerminative development are expressed at levels typical of wild-type seedlings rather than embryos. We conclude that postgerminative development is initiated prematurely and that embryonic and postgerminative programs operate simultaneously in mutant embryos. The pleiotropic effects of the mutation indicate that the LEC1 gene plays a fundamental role in regulating late embryogenesis. The role of LEC1 and its relationship to other genes involved in controlling late embryonic development are discussed.     

Evidence for microspore embryogenesis in wheat anther culture

Summary In wheat, plants may be regenerated from microspores via direct embryogenesis or organogenesis or embryogenesis from callus. Light and scanning electron microscopy were used to carefully study morphogenesis of microspore-derived plants from anther culture on modified 85D12 starch medium and to determine whether the plants were formed via organogenesis or embryogenesis. Our results indicate that plants are formed via embryogenesis from microspores. Evidence for embryogenesis included the formation of the epidermis and a suspensorlike structure (21 days after culture), followed by initiation of an apical meristem, differentiation of the scutellum, and embryo elongation. At 28 days in culture, the embryo possessed a well-developed scutellum and axis with suspensor. Embryogenesis was further confirmed by coleoptile and radicle elongation during germination when the embryos were cultured on medium supplemented with kinetin with or without coconut water. In this system, an average 67 microspores per responsive anther began cell division but only 3.69 embryos were formed per responsive anther after 6 wk. Adventitious embryos could be induced if the embryos, once formed, remained on initiation medium for 10 wk instead of being transferred to regeneration medium. Developmental stages which may be amenable to changes that could enhance plant production were identified. The potential to use this information to enhance plant production is discussed.