Microsurgery reveals regional capabilities for pattern reestablishment in somatic carrot embryos

The extent to which regions of a somatic embryo were committed to a particular developmental fate was explored by surgically removing portions of somatic embryos and observing patterns of regeneration. Through a variety of excisions that resulted in tissue slices ranging from less than 10% to nearly 90% of the original embryo mass, we observed only a few cases where such isolates completely abandoned preexisting patterns of organized growth. Instead, most subcultured portions of the embryonic axis restored all, or part of, a missing complement of the organism. At the shoot apex, a single lost cotyledon was replaced by new cotyledonary structures, although these usually occurred as multiple pairs of cotyledons. If both cotyledons were removed, secondary axes, each with its own cotyledons, typically formed at the embryo midlength. When embryos were divided into shoot and root pieces, the shoot pole usually regenerated a new root, while the original root and rapidly elongated and matured days earlier than uncut controls. Surprisingly, cotyledon regeneration from excised root sections occurred at much greater frequency when the root piece comprised only 10-25% of the embryo mass; larger portions of the root pole rarely produced recognizable shoot structures. These studies indicate that several discrete regions of the embryo are committed to specific types of patterned growth, and that continuity between certain of these regions is required for the maintenance of axial polarity.     

The role of polarity in the development of the hydrozoan planula larva

Summary These experiments were done in order to define the role that polarity plays during embryogenesis in hydrozoans.Parts of hydrozoan embryos isolated at different developmental stages from early cleavage to postgastrula will regulate to form normal planulae. During this process, the original anterior-posterior axis of the part is conserved. In normal embryos the posterior pole of the anterior-posterior axis is congruent with the site where the polar bodies are given off and with the site where the first cleavage is initiated. By centrifuging fertilized eggs, it is possible to create embryos in which the first cleavage initiation site does not correspond to the site where the polar bodies are given off. In these embryos the posterior pole of the anterior-posterior axis corresponds to the first cleavage initiation site. When parts of these embryos are isolated at different stages they also regulate to form normal planulae. The axial properties of these planulae are determined by the site of first cleavage initiation.The interactions between regions of the embryo with different axial properties were studied by grafting together parts in such a way as to create embryos with abnormal axial arrangements. Following gastrulation interactions take place between the grafted parts leading to the formation of normal planulae with a new set of axial properties.Blastula stage embryos can be dissociated into single cells and the cells can be reaggregated. These reaggregates form normal planulae. Polarity can be entrained in the reaggregates by grafting a small piece of tissue from any part of an intact blastula to the reaggregate. These cells organize the formation of an axis of symmetry with an appropriate orientation with respect to the graft.     

In vitro regeneration of commercial cultivars of subterranean clover

Regeneration of subterranean clover (Trifolium subterraneum L.) was achieved by both shoot organogenesis and somatic embryogenesis. Shoots derived via organogenesis were initiated from the hypocotyls of mature imbibed seed. The hypocotyl, including the emerging radicle, was sliced longitudinally into two halves and cultured on shoot induction medium. After 30 days, adventitious shoots were formed from the hypocotyl region while the radicle showed no development. Shoots were then subcultured onto shoot multiplication medium and finally onto a root initiation medium. Histological studies revealed that shoots arose de novo and did not originate from pre-existing meristems. In the second regeneration protocol, shoot apical meristems from young seedlings were induced to form callus. Following four to six weeks culture in the dark, somatic embryos appeared spontaneously on the calli. A majority of embryos had a well-defined root pole, two cotyledonary lobes, and were capable of germination, albeit at a low frequency. Regenerated plants obtained from both protocols appeared phenotypically normal.     

Transformation of shoots into roots in Arabidopsis embryos mutant at the TOPLESS locus

We describe a novel phenotype in Arabidopsis embryos homozygous for the temperature-sensitive topless-1 mutation. This mutation causes the transformation of the shoot pole into a root. Developing topless embryos fail to express markers for the shoot apical meristem (SHOOT MERISTEMLESS and UNUSUAL FLORAL ORGANS) and the hypocotyl (KNAT1). By contrast, the pattern of expression of root markers is either duplicated (LENNY, J1092) or expanded (SCARECROW). Shifts of developing topless embryos between permissive and restrictive temperatures show that apical fates (cotyledons plus shoot apical meristem) can be transformed to basal fates (root) as late as transition stage. As the apical pole of transition stage embryos shows both morphological and molecular characteristics of shoot development, this demonstrates that the topless 1 mutation is capable of causing structures specified as shoot to be respecified as root. Finally, our experiments fail to show a clear link between auxin signal transduction and topless-1 mutant activity: the development of the apical root in topless mutant individuals is not dependent on the activity of the predicted auxin response factor MONOPTEROS nor is the expression of DR5, a proposed 'auxin maximum reporter', expanded in the apical domain of topless embryos.     

Key divisions in the early Arabidopsis embryo require POL and PLL1 phosphatases to establish the root stem cell organizer and vascular axis

Arabidopsis development proceeds from three stem cell populations located at the shoot, flower, and root meristems. The relationship between the highly related shoot and flower stem cells and the very divergent root stem cells has been unclear. We show that the related phosphatases POL and PLL1 are required for all three stem cell populations. pol pll1 mutant embryos lack key asymmetric divisions that give rise to the root stem cell organizer and the central vascular axis. Instead, these cells divide in a superficially symmetric fashion in pol pll1 embryos, leading to a loss of embryonic and postembryonic root stem cells and vascular specification. We present data that show that POL/PLL1 drive root stem cell specification by promoting expression of the WUS homolog WOX5. We propose that POL and PLL1 are required for the proper divisions of shoot, flower, and root stem cell organizers, WUS/WOX5 gene expression, and stem cell maintenance.     

Histological comparison of single somatic embryos of maize from suspension culture with somatic embryos attached to callus cells

An embryogenic suspension culture of Zea mays, genotype 4C1, was obtained from friable callus that was cultured on solid medium and had been obtained from zygotic embryos. The suspension contained non-dividing elongated cells, clusters of dividing isodiametric cells, and globular, ovoid, and polar stages of somatic embryos. The single somatic embryos were blocked in shoot meristem formation: when transferred to regeneration medium they developed a root and, at the shoot side, a green cap with meristematic cells, but a scutellum and leaf primordia were not formed. In medium containing 2,4-dichlorophenoxy acetic acid, somatic embryos formed embryogenic callus aggregates, consisting of globular stage somatic embryos attached to each other via undifferentiated callus cells. These somatic embryos developed into mature embryos with the zygotic histological characteristics, such as scutellum and leaf primordia, in maturation medium, and then regenerated into plants in regeneration medium. By omitting the maturation phase, regeneration occurred via organogenesis. Polyembryos, i. e. embryos attached to each other without callus tissue in between, behaved as single somatic embryos. It is concluded that the attached callus tissue provides a factor that stimulates scutellum and leaf primordia formation.Abbreviations CMM callus maintenance medium - 2,4D 2,4-dichlorophenoxy acetic acid - PCV packed cell volume - MS Murashige and Skoog medium     

The regeneration potential of wheat shoot meristems in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Tissue cultures capable of plant regeneration were successfully initiated from extremely immature shoot meristems of 21 randomly selected genotypes of wheat on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). By means of scanning electron microscopy it was demonstrated that cultures consisted of teratomatous primordia, which were kept in a proliferating budding state by the 2,4-D. These are characteristic of cereal tissue cultures. Release of the primordia and outgrowth of normal shoots and roots occurred when the cultures were no longer exposed to 2,4-D. Shoot primordia which were clearly identifiable were always associated with root primordia in a quasi-bipolar fashion. Sometimes regions assumed the shape of zygotic embryos, but the transition from apparently normal embryos with scutellum to abnormal configurations with shoot and root regions was gradual. The differences between genotypes in shoot regeneration potential was minimal compared to cultures derived from explants which were taken from regions temporally and spatially more distant from the shoot apex. It is concluded that the ability to give rise to cultures capable of shoot regeneration was lost within a fraction of a millimeter distance from the apical meristem in many genotypes. The proliferating tissues were subcultured at regular intervals over a period of one year and the regeneration potential was monitored. Areas capable of shoot regeneration tended to deteriorate more or less rapidly and were overgrown by root-type tissue in a number of genotypes. The results are discussed in the context of the frequently observed, but largely unexplained, variability in the regeneration potential of cereal tissue cultures.     

Promoter trap markers differentiate structural and positional components of polar development in Arabidopsis.

To investigate mechanisms involved in establishing polar organization in Arabidopsis embryos and seedlings, we used promoter trapping to identify molecular markers (beta-glucuronidase fusion genes) expressed in spatially restricted patterns along the apical-basal axis. Three markers were identified that are expressed, respectively, in the embryonic and seedling root tip (POLARIS), cotyledons and shoot and root apices (EXORDIUM), and root cap (COLUMELLA). Each marker was crossed into the mutants hydra and emb30, which are defective in embryonic and seedling morphogenesis. All three markers were expressed in hydra mutants in patterns similar to those observed in phenotypically wild-type embryos and seedlings. In emb30 mutants, the EXORDIUM marker was expressed in cotyledons but not in the expected position of shoot and root meristems, and the marker COLUMELLA was not expressed at all, which is consistent with the view that the emb30 mutant, but not hydra, lacks shoot and root meristems. However, POLARIS was expressed in the basal part of hydra embryos lacking an embryonic root and in the basal parts of both hydra and emb30 seedlings. Expression of POLARIS is inducible by exogenous auxin and suppressed by cytokinin but is unaffected by inhibitors of polar auxin transport or cell division. We conclude that POLARIS differentiates positional aspects of polar development from structural aspects.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of chestnut (<Emphasis Type="Italic">Castanea sativa</Emphasis> Mill.) cotyleodon explants: responses to growth regulators and developmental aspects

Summary Adventious root and shoot formation was obtained from cotyledon fragments of chestnut (Castanea sativa Mill.) and these processes followed two phases. In a first stage after detachment of the embryonic axis, the cotyledon fragments in culture formed a cotyledon petiole, which elongated for about 6d. Thereafter, root primordia arose at the tip of the cotyledon petioles, followed by normal root development. In some cases, the cotyledon, petioles showed adventitious shoot regeneration from a nodular structure previously formed at the end of the petioles. The presence or absence of growth regulators did not significantly influence root regeneration, whereas cytokinins stimulated shoot formtion. The processes of root and shoot differentiation were studied also at the histological level. Observation with a light microscope showed that the developing root apical meristems were connected with a vascular bundle of the cotyledon petiole. Similarly, shoot bud meristem connections were observed with vascular tissue inside the nodular structure.     

Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)

Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube.     

SOME ASPECTS OF MORPHOGENESIS IN CUSCUTA GRONOVII

More than 100 primary root segments from dormant seeds, 100 radicular segments from immature embryos, 450 whole immature embryos, and a number of whole seedlings of C. gronovii were studied under in vitro cultivation in the presence of such growth-regulating factors as: coconut milk, casein hydrolysate, auxins, kinetin, adenine, gibberellic acid, and additional B vitamins as supplements to White's medium. In normal embryogeny early swelling of the basal pole of the embryo and profuse production of epidermal hairs upon germination are superficial characteristics of Cuscuta which resemble seedling root growth in other plants. At no time is there evidence of a primary-root meristem, nor at any time do cotyledons develop. It was not possible to induce root formation experimentally from any of the starting embryonic materials, nor from stems which were cultured to maturity culminating in the production of flowers after 6 months of in vitro cultivation, nor from callus derived from the radicular pole of the embryo. The undifferentiated radicular pole frequently gave rise to rapidly growing callus from which new shoots were formed spontaneously, although some immature embryos completed normal embryogeny. Embryo size and subsequent type of development were not strictly correlated but there was a tendency toward the following patterns. Embryos less than 0.4 mm in length lost their original form, callused evenly throughout and became masked by development of buds over the entire surface. Embryos 0.4–1.2 mm were most apt to retain their original form; callus developed at the radicular pole but not at the shoot pole. Embryos 1.5 mm and larger frequently gave rise to radicular callus but also grew normal shoots. Embryo inoculants which had begun to coil were most apt to complete normal embryogenesis. No correlation could be drawn between the addition of specific growth regulators or combinations of regulators and specific patterns of development. It is suggested that in the evolution of Cuscuta from autotrophic ancestors, the loss of both cotyledons and roots was a single embryonic event and so complete that even under the influence of a highly modified biochemical environment it would be impossible to derive roots from dodder tissue. The usual interpretation of the dodder haustorium as a highly modified adventitious root in all probability is not valid. Is is further suggested, however, that this loss of root and cotyledon is not a necessary consequence of the evolution of the parasitic mode of nutrition, nor of the gross adult growth form.     

Sugarcane somatic embryogenesis: a scanning electron microscopy study

Spindles of CUBA 87-51 sugarcane were cultured in Murashige and Skoog (MS) basal medium and supplemented with different nutrients. Embryogenic and non-embryogenic callus obtained were comparatively studied by scanning electron microscopy (SEM). Samples of embryogenic callus cultured in regeneration medium (MS without 2.4 dichlorophenoxyacetic acid) were taken at different times for analyzing the sequential process. Distinctive features of two types of callus are shown by SEM: cells organized in embryos are noted in embryogenic callus; while elongated, disorganized cells can be seen in non-embryogenic callus. The characteristics of the embryos during plant regeneration are described. Sugarcane embryoid stages are: globular, globular with lateral notch and scutellum. In this process also appear shoot meristems, leaf and root primordia and finally, true leaves and roots. It is concluded that callus plant regeneration from young leaf segments of sugarcane mainly occur via somatic embryogenesis.     

Papaver somniferum regeneration by somatic embryogenesis and shoot organogenesis

Secondary somatic embryogenesis and shoot organogenesis from primary somatic embryos of Papaver somniferum L. are described. The embryos became malformed, the root meristem expressed dividing activity without position-dependent cell differentiation, causing abnormal development or arrested growth of primary somatic embryos. The adventitious shoots regenerated from embryo hypocotyl, but secondary somatic embryos had an epidermal origin close to the root meristem. The regeneration occurred without hormonal treatment, indicating endogenous nature of triggering signals. These signals are probably related to the integrity loss of morphogenetic steps during development of primary somatic embryos, which appeared to induce an activation of cells competent to regeneration. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

Patterns of junctional communication during development of the early amphibian embryo

Cell-cell communication through gap junctions was examined in Xenopus laevis embryos between the 16-cell and early blastula stages using Lucifer Yellow, Fluorescein, lead EDTA and dicyanoargentate as probes of junctional permeability. Injections were made into cells whose position was identified with respect to the primary cleavage axis and the grey crescent. FITC dextrans revealed cytoplasmic bridges between the injected cell and its sister only. In the animal pole at the 16-cell stage at the future dorsal side of the embryo, Lucifer Yellow was frequently and extensively transferred between cells through gap junctions. At the future ventral side gap junctional transfer of Lucifer Yellow was significantly less frequent and less extensive. The asymmetry of transfer between future dorsal and ventral sides of the animal pole was more marked at the 32-cell stage. In the vegetal pole also at the 32-cell stage, a dorsoventral difference in junctional permeability to Lucifer Yellow was observed. At the 64-cell stage the transfer of Lucifer Yellow was relatively frequent between cells lying in the same radial segment in the animal pole; transfer into cells outside each segment was infrequent, except at the grey crescent. At the 128-cell stage, Lucifer transfer between future dorsal or future ventral cells in the equatorial region was infrequent. A high incidence of transfer was restored at the future dorsal side at the 256-cell stage. At the 32-cell stage, fluorescein was infrequently transferred between animal pole cells although lead EDTA moved from cell to cell with high, comparable frequency in future dorsal and ventral regions. Dicyanoargentate always transferred extensively, both at the 32- and 64-cell stages. Treatment of embryos with methylamine raised intracellular pH by 0.15 units, increased the electrical conductance of the gap junction and produced a 10-fold increase in the frequency of Lucifer Yellow transfer through gap junctions in future ventral regions of the animal pole at the 32-cell stage.     

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.     

The potential for predation induced somatic embryogenesis in storage cotyledons

Using seven tropical rainforest species of north Queensland, Australia, we simulated partial predation and compared the development of intact embryos and embryos from which various proportions had been removed, including in some cases the original root–shoot axis. Embryos of all species contained storage reserve cotyledons and germinate in a hypogeal manner. Species mean embryo masses ranged from 0.4–48.1 g. Partial embryo predation treatments were: ½ embryo treatment; embryos separated into component cotyledons, and ¼ embryo treatment; cotyledons separated as above and subsequently cut in half. Germination was scored as production of roots and shoots, or roots or shoots only. Embryos from all species produced seedlings in all treatments, even after the removal of up to 75 percent of the cotyledonary reserve. Moreover, the proportion germinating were not different between intact embryos and i) separated cotyledons, ii) cotyledon halves that maintained or were adjacent to the embryonic axis (except for the largest seeded species), and iii) cotyledon halves that did not include the root–shoot axis (four species). Thus, production of roots and shoots, or roots or shoots only was largely independent of the presence of the embryonic root–shoot axis – implying that somatic cells in storage cotyledons are capable of differentiating into the full range of cell types typical of shoots and roots in the absence of the root–shoot axis. The generality of this response across all seven species suggests somatic embryogenesis in storage cotyledons may be a more widespread phenomenon in tropical floras than currently considered.     

Rescue of shoot apical meristems of chrysanthemum by culturing on root tips

A new method to regenerate plants from leaf primordia-free shoot apical meristem domes (LP-free SAMs) was developed by establishing the meristem dome on the cut surface of root tips. Ten days after culture, the viable rate of LP-free SAMs of chrysanthemum Piato attached to chrysanthemum root tips was >40%. Shoot regeneration was not observed from LP-free SAMs without the root tips. When LP-free SAMs of chrysanthemum were transferred to root tips of either petunia, cabbage, or carnation, the highest shoot regeneration rate was observed with cabbage root tips. Microscopic observation documented that the LP-free SAM temporarily adhered to the cut surface of the root tip of cabbage.Communicated by K.K. Kamo