Suspensor Region Research Articles

Using Giant Scarlet Runner Bean (Phaseolus coccineus) Embryos to Dissect the Early Events in Plant Embryogenesis.

The giant embryo of the scarlet runner bean (Phaseolus coccineus) has been used historically to investigate the molecular and developmental processes that control the early events of plant embryo development. In more recent years, our laboratory has been using scarlet runner bean embryos to uncover the genes and regulatory events that control embryo proper and suspensor region differentiation shortly after fertilization. In this chapter we describe methods that we have developed to isolate scarlet runner bean embryos at the globular stage of development, and capture embryo proper and suspensor regions by either hand dissection or laser capture microdissection (LCM) for use in downstream genomic analysis. These methods are also applicable for use in investigating the early events of common bean (Phaseolus vulgaris) embryo development, a close relative of scarlet runner bean, which also has a giant embryo in addition to a sequenced genome.

Erratum to: Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.

Using giant scarlet runner bean embryos to uncover regulatory networks controlling suspensor gene activity.

One of the major unsolved issues in plant development is understanding the regulatory networks that control the differential gene activity that is required for the specification and development of the two major embryonic regions, the embryo proper and suspensor. Historically, the giant embryo of scarlet runner bean (SRB), Phaseolus coccineus, has been used as a model system to investigate the physiological events that occur early in embryogenesis—focusing on the question of what role the suspensor region plays. A major feature distinguishing SRB embryos from those of other plants is a highly enlarged suspensor containing at least 200 cells that synthesize growth regulators required for subsequent embryonic development. Recent studies have exploited the giant size of the SRB embryo to micro-dissect the embryo proper and suspensor regions in order to use genomics-based approaches to identify regulatory genes that may be involved in controlling suspensor and embryo proper differentiation, as well as the cellular processes that may be unique to each embryonic region. Here we review the current genomics resources that make SRB embryos a compelling model system for studying the early events required to program embryo development.

Direct somatic embryogenesis and plantlet regeneration in oil palm

Direct embryogenesis without an intervening callus phase from cotyledonary nodes of germinated immature zygotic embryos of hybrids viz. DG1 and DG21 of oil palm (Elaeis guineensis) is reported here. Direct embryogenesis was achieved when the cotyledonary nodes of germinated immature zygotic embryos were cultured in dark for 8 weeks on Eeuwens media (Y3) supplemented with 40 μM 2,4-Dichlorophenoxyacetic acid (2,4-D), 40 μM α-Naphthaleneacetic acid (NAA), 10 μM 2,4,5-Trichorophenoxyacetic acid (2,4,5-T), 10 μM Thiadiazuron (TDZ), 10 μM 6-Benzyladenine (BA). The globular embryos with clear suspensor region appeared directly on the explants and multiplied. On subculture to fresh media, the other stages such as torpedo and heart shaped embryos were seen. On transfer to light in Y3 media containing BA (2 μM) and ABA (1 μM) they matured into complete plantlets. In 2% of the cultures secondary embryogenesis also was seen. Along with several other advantages of direct somatic embryogenesis this protocol opens up the prospect of genetic transformation in this important commercial crop.

A promoter from the loblolly pine PtNIP1;1 gene directs expression in an early-embryogenesis and suspensor-specific fashion.

The PtNIP1;1 gene encodes an aquaglyceroporin that is expressed early in embryogenesis and appears to be expressed preferentially in the suspensor [V.T. Ciavatta et al. (2001) Plant Physiol 127:211-224]. An 899-bp fragment 5' to the PtNIP1;1 open reading frame (NIP(-899)) was cloned from loblolly pine (Pinus taeda L.) genomic DNA and fused to the beta-glucuronidase (GUS) reporter gene. The resulting plasmid, pNIP-GUS, was transformed into Norway spruce (Picea abies L.) embryogenic cultures by co-bombarding with a plasmid containing a bar gene construct as a selectable marker. The identity of lines selected on medium containing the herbicide Basta and showing beta-glucuronidase activity was confirmed by polymerase chain reaction as harboring GUS. Histochemical GUS assays of these lines revealed GUS activity in all cells of proembryogenic masses. During early embryogeny, GUS staining was intense in the suspensor region but not detectable in embryonal masses. GUS staining was absent by mid-embryogeny. By contrast, a control transgenic line, transformed with EuCAD-GUS, expressed GUS throughout embryo development. These results suggest that NIP(-899) contains elements that drive early embryogenesis-specific expression and suspensor-specific expression. This is the first example of a suspensor-specific promoter in conifers.

Induction of somatic embryogenesis in Pinus roxburghii Sarg.

Embryogenic calli were initiated from embryonic explants of Pinus roxburghii using female gametophytes containing immature pre-cotyledonary embryos. Zygotic embryos were collected at different developmental stages and cultured on various media. Initiation of embryogenic calli was achieved in pre-cotyledonary zygotic embryos of a 0.1-mm to 1.2-mm embryonal head on Douglus fir cotyledon revised medium (DCR) medium supplemented with 2,4-D or NAA and BA. Embryogenic callus development was initiated from the suspensor region of immature embryos. The method of immature embryo culture was significant as rapid embryogenic callus development occurred in megagametophytes where the suspensor was stretched onto the medium from the cut micropylar end. Sixty embryogenic lines were established from 2500 explants cultured during one season. A pro-embryo with six to eight meristematic cells and suspensor of six to ten long, vacuolated cells dominated the early phase of the callus development. Cleavage polyembryony occurred in proliferating callus, constituting a method of multiplication of these somatic embryos. Somatic embryos developed to stage-I and stage-II embryos on DCR medium supplemented with 5 μM 2,4-D or 10 μM NAA.

Somatic Embryogenesis in Cephalotaxus harringtonia Embryo-Megagametophyte Co-culture

Somatic embryogenesis was initiated from Cephalotaxus harringtonia (Forbes) K. Koch embryo culture. Explants consisted of embryo and megagametophyte halves both cut longitudinally. They were removed aseptically from mature seeds and grown together on a solid Murashige and Skoog modified medium supplemented with 5 mg ▪ l −1 2,4-dichlorophenoxyacetic acid. Embryogenic cultures started from callus after three or more months on the primary medium. The embryogenic callus originated from the suspensor region of the embryo. All chromosome counts made in the cells of the embryonic structures demonstrated a diploid stage, which suggest that they originated from zygotic embryo tissue. The early stages of somatic embryogenic development were achieved, i.e., formation of small clusters consisting of an embryonal region made up of isodiametric meristematic cells. A more advanced stage was reached in some cultures in which the distal embryonal end of the embryo appeared smooth and opaque. The ultrastructural characteristics of the embryos, the two types of embryo cells, embryonal and suspensor cells, as well as their contents were similar to those already reported in the case of somatic embryogenesis of other conifers.

Two alternative pathways of somatic embryo origin from polyembryonic mature stored seeds of Pinus koraiensis Sieb et Zucc.

Individual mature stored seeds of Pinus koraiensis sometimes contain several viable zygotic embryos originated through the processes of simple and cleavage polyembryony. To induce the embryonic process, isolated zygotic embryos were cultured on five different media all supplemented with 10 μM 2,4-dichlorophenoxyacetic acid and 5 μM 6-benzyladenine. Two alternative pathways of somatic embryo origin were revealed. The first pathway was associated with the production of a friable, translucent callus in the hypocotyls–cotyledon region of the dominant zygotic embryo. The second pathway was related to the proliferation of a translucent, moist, and mucilaginous tissue (termed embryonal–suspensor mass) in the suspensor region of the dominant zygotic embryo. Both types of tissues contained early somatic embryos. Regression analysis has shown a strong negative correlation between the frequencies of formation of embryogenic callus and embryonal–suspensor mass both at 3 and 8 weeks of culture (r = − 0.85; p = 0.07 and r = −0.71; p = 0.17, respectively). Key words: Pinus koraiensis; polyembryonal seeds; somatic embryogenesis; embryogénie callus; embryonal–suspensor mass.

Cell Differentiation and Morphogenesis Are Uncoupled in Arabidopsis raspberry Embryos.

We identified two Arabidopsis embryo mutants, designated as raspberry1 and raspberry2, by screening T-DNA-mutagenized Arabidopsis lines. Embryogenesis in these mutants is indistinguishable from that of wild-type plants until the late-globular stage, after which raspberry1 and raspberry2 embryos fail to undergo the transition to heart stage, remain globular shaped, and proliferate an enlarged suspensor region. raspberry1 and raspberry2 embryo-proper regions enlarge during embryogenesis, become highly vacuolate, and display prominent convex, or "raspberry-like" protuberances on their outer cell layers. In situ hybridization studies with several embryo cell-specific mRNA probes indicated that the raspberry1 and raspberry2 embryo-proper regions differentiate tissue layers in their correct spatial contexts and that the regulation of cell-specific genes within these layers is normal. Surprisingly, a similar spatial and temporal pattern of mRNA accumulation occurs within the enlarged suspensor region of raspberry1 and raspberry2 embryos, suggesting that a defect in embryo-proper morphogenesis can cause the suspensor to take on an embryo-proper-like state and differentiate a radial tissue-type axis. We conclude that cell differentiation can occur in the absence of both organ formation and morphogenesis during plant embryogenesis and that interactions occur between the embryo-proper and suspensor regions.

Cyto-histological Studies on Somatic Embryos of Coffee: Ultrastructural Aspects.

A histological and ultrastructural study of somatic embryogenesis in coffee embryos was made by light microscopy and transmission electron microscopy. A comparative study on somatic and zygotic embryos of coffee was also performed. The cells in the suspensor region of somatic embryos were filled with large-and small-sized granules containing polyphenol substances stainable in black. The granule size increased until the elongated-embryo stage and thereafter it decreased with embryo development. Such granules were very small in cotyledonary embryo cells. The continuous and discontinuous cuticular layers were detected on both the somatic, globular embryos and the zygotic proembryos by treatment of iodine/potassium iodide-sulfuric acid-silver proteinate. This preparation also permitted the identification of the deposition of fine silver particles inside the polyphenol granules. In addition the plastids were filled with several starch grains of large and small sizes. Similarly, cells of the zygotic proembryos and young embryos were also filled with polyphenol granules and plastids containing starch grains. It is interesting to note that mucilaginous materials cover the surface of both the embryogenic callus and the suspensor region of somatic embryos and are also present in the intercellular spaces of these tissues. These materials were also covering zygotic embryos situated in the embryo cavity of seeds. These observations led to the conclusion that the somatic and zygotic embryos have several similarities at the ultrastructural level.

Somatic Embryogenesis in Pinus patula

Summary Embryogenic callus was initiated from cultured Pinus patula Scheide. et Deppe. megagametophyte explants containing immature zygotic embryos. A average of 2.6 % of the gametophytes containing at least one embryo produced embryogenic tissue. Cones used for explant isolation were collected on a weekly basis during the summer months from December 1991 to February 1992. Embryogenic tissue was induced on both MSG and DCR (Douglas-fir Cotyledon Revised) basal media. The highest induction frequency was on a DCR1 medium supplemented with 0.5 mg L−1 BA and 3.0 mg L−12,4-D, using L-glutamine as the major nitrogen source. Histological examination showed that three distinct types of tissue were produced. A translucent to white mucilaginous tissue was observed proliferating at the micropylar end of the female gametophyte from the suspensor region of the embryo. This embryogenic tissue was induced in the latter half of the collection period, at a stage when cleavage polyembryony was manifest in the immature zygotic embryos. Both stage 1 (translucent suspensor) and stage 2 (opaque apex) somatic embryos were produced. Compact, crystalline non-embryogenic tissue developed from the surface of the female gametophyte throughout the collection period. The third type of tissue consisted of a mixture of embryogenic and non-embryogenic tissues, resulting in sphaeroblast production. This tissue type was usually limited to explants collected prior to the initiation of cleavage polyembryony. Anatomical studies showed the tissue types to be morphologically different, although ultrastructural differences were not as marked.

Ultrastructural Studies on Rice Globular Embryos with Emphasis on Epidermis Initiation.

Cell ultrastructure was examined in rice embryos at the developmental stage between 42 and 72 HAA (hours after anthesis). Until 48 HAA, the cells gradually became cytoplasm-rich with a slight increase in the volume of the embryo exhibiting a globular shape. The globular embryo consisted of two parts : an embryo proper which was spherical in shape and a suspensor situated toward the micropylar end. From 48 to 66 HAA, cells of the embryo proper attained a very meristematic profile with vacuoles becoming small and few in number. On the other hand, in the suspensor region, cells and their vacuoles were larger than those of the embryo proper. Until 66 HAA, the absence of cuticular layer on the surface of globular embryo was ascertained since no materials reactive to the I2KI-H2SO4-AgP method were observed. At 72 HAA, however, a stable layer of cuticle began to appear on the surface of the embryo, extending from the ventral side and gradually covering the entire embryonal surface. Simultaneously, cell differentiation occurred in the internal tissues accompanied by ultrastructural changes. It was concluded from these results that the initiation of a cuticular layer is one of the first cytological events that lead rice embryos to organogenesis.

Somatic embryogenesis in European black pine (Pinus nigra Arn.)

Embryogenic callus was initiated from immature zygotic embryos of black pine on medium DCR supplemented with 2 mg 1-1 2,4-D and 0.5 mg 1-1 BAP. The diploid number of chromosomes confirmed the origin of callus from zygotic embryos. The callus was white, glossy, mucilaginous and contained somatic embryos consisted of an embryonic region with densely cytoplasmic cells and suspensor region with long vacuolated cells. Although somatic embryos with green cotyledons were recognisable after ABA treatment and subsequent transfer to growth-regulator free media whole plants have not yet been obtained.

An ultrastructural study on the early development of Zea mays somatic embryos

Friable embryogenic callus, obtained from immature embryos of Zea mays L., was cultured on N6 medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid, 6 mM proline, and 2% sucrose. Cultured tissue fragments containing several globular embryoids were excised and examined by light and electron microscopy to follow the early development of maize embryoids. The somatic embryos consist of an apical region and a suspensor region. Cells of the apical region are small, cytoplasm rich, and mitotically active. They contain much starch and numerous bundles of microtubules. Suspensor cells are larger and more vacuolated. A high metabolic activity in both cell types is indicated by the presence of many organelles, coated vesicles, and multivesicular bodies. Transition units appear to form intermediate stages between the embryogenic callus cells and the somatic embryo. A transition unit consists of a group of embryogenic cells and shows an apical and a basal region. The unit has many intercellular spaces, and within the cells areas with organelle-free cytosol are frequently observed. Key words: somatic embryogenesis, in vitro culture, ultrastructure, Zea mays L.

Initiation of embryogenic cultures and somatic embryo development in loblolly pine (Pinustaeda)

Immature zygotic embryo explants (isolated or with intact megagametophytes) from 10 loblolly pine (Pinustaeda L.) clones (7-34, 7-56, 11-9, 11-16, 11-25, 10-1003, 10-1007, 10-1011, 10-1018, and 10-1019) were surveyed for their potential to form embryogenic tissue from the suspensor region of zygotic embryos. After over 14 000 explants were cultured, embryogenic cultures were initiated from explants of 8 of the 10 clones; only explants from clones 11-25 and 10-1019 were not responsive. Embryogenic tissue was initiated from zygotic embryos with intact megagametophytes on MSG basal medium with no exogenous plant growth regulators or with 2–5 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0–1 mg/L N6-benzyladenine (BA). The highest initiation frequency (5%) was obtained from isolated zygotic embryos of clone 7-34 less than 0.5 mm in length just prior to cotyledon primordia development on DCR basal medium with 3 mg/L 2,4-D and 0.5 mg/L BA. Two types of embryogenic cultures were maintained on medium with 2,4-D and BA: (i) those that contained pre-embryonal masses of cells interspersed with unaggregated suspensorlike cells, but which rarely contained well-formed somatic embryos, and (ii) those that frequently contained well-formed somatic embryos. Somatic embryo development from both types of cultures progressed to a precotyledonary stage on medium with 2.6 mg/L abscisic acid.

Diploid and haploid embryogenesis in Larixleptolepis, L. decidua, and their reciprocal hybrids

Diploid and haploid embryogenesis was induced in two Larix species (L. decidua and L. leptolepis) and their reciprocal hybrids. Diploid embryogenic tissue was initiated in immature zygotic embryos isolated with the micropylar half of the megagametophyte left attached. These were placed either on modified LM or MSG medium supplemented with the growth regulators 2,4-D and 6-benzyladenine. MSG medium was solidified with either gellan gum or agar. There was no appreciable difference in response between the two. Haploid embryogenesis was induced in isolated megagametophytes placed on modified LM medium supplemented with 2,4-D and 6-benzyladenine. Diploid embryogenic tissue was subcultured on medium with growth regulators, but haploid embryogenic tissue grew well on medium without growth regulators. There were few morphological differences between the diploid and haploid embryogenic tissue. In all species and hybrids, haploid cultures contained more coenocytic long cells. Binucleate cells were most common, but tetranucleate and octanucleate cells were also present. Haploid cultures showed poorer organization than the diploid ones, with only a few cultures having well-developed embryoids. Haploid tissue originated from expanded cells of the megagametophyte. Diploid tissue originated from the suspensor region of the zygotic embryo; it proliferated from isolated clusters of meristematic cells in early embryoids. Diploid and haploid cultures differed not only from the outset, but also in the mature embryoids they produced.

Organogenesis and embryogenesis in mature zygotic embryos of Picea sitchensis.

Differentiation of adventitious buds and somatic embryos from mature zygotic embryos of Picea sitchensis (Bong.) Carr. is described. Adventitious buds were formed on embryos pulse-treated with 250 microM benzyladenine for 2 h and cultured on medium lacking growth regulators. Buds were initiated at different frequencies and sites depending on when the BA-pulsed embryos were transferred to fresh culture medium. Embryogenic callus was formed when the zygotic embryos were cultured on medium containing 10 microM 2,4-dichlorophenoxyacetic acid and 5 microM benzyladenine. Although 50% of the embryos gave rise to embryogenic callus, only 20% of the callus continued to proliferate after subculture. Proliferation of new somatic embryos occurred from both the embryonic region and the suspensor region on previously formed somatic embryos. The pattern for development of adventitious buds and somatic embryos in Picea sitchensis is compared to that in Picea abies under similar culture conditions.

Somatic embryogenesis in Picea glauca (white spruce) and Picea mariana (black spruce)

Embryogenic callus was initiated from immature embryos of Picea glauca (white spruce) and Picea mariana (black spruce) cultured on defined media supplemented with 2,4-dichlorophenoxyacetic acid (1 × 10−5 M), N6-benzyladenine (5 × 10−6 M), and 1% sucrose. Seeds from cones stored at 4 °C for up to 3 months yielded embryogenic callus. Much higher frequencies of embryogenic callus were obtained from white spruce than from black spruce. Embryogenic callus contained loosely organized cells and somatic embryos of various sizes. The embryos consisted of a cluster of tiny dividing cells (embryonic region) with attached large vacuolated cells (suspensor region). Upon subculture of embrogenic callus to media either lacking growth regulators or with reduced concentrations (5 × 10−7 M, 2,4-dichlorophenoxyacetic acid and 5 × 10−6 M N6-benzyladenine) somatic embryos could be stimulated to develop into plantlets.