The effect of the suspensor and gibberellic acid on Phaseolus vulgaris embryo protein synthesis

The role of the suspensor in the early development of the dicot embryo has been described as merely an anchor or, conversely, as the major route of nutrients into the embryo. In order to further elucidate the role of the suspensor we have examined protein synthesis in early 0.2-mm and late heart stage 0.5-mm Phaseolus vulgaris (var. Taylor's Horticultural) embryos in tissue culture. Protein synthesis was examined in embryos and suspensors. Our results showed that in 0.2-mm embryos virtually all protein synthesis was dependent on an attached suspensor. Maximum protein synthesis in 0.5-mm embryos was observed when embryos were cultured attached to the suspensor. The levels were moderately decreased when the embryo was cultured detached from or without the suspensor. Gibberellic acid at 10(-6) to 10(-7) M elicited the same protein diversity and greater [35S]methionine incorporation than did the attached suspensor in 0.2-mm embryos. Embryos of 0.5 mm did not appear to be differentially responsive to various gibberellin concentrations.     

Suspensor,gibberellin and in vitro development of Phaseolus coccineus embryos

Summary Embryos of Phaseolus coccineus in different stages of development (from 0.5 to 5 mm in length) were grown in vitro. Both intact embryos (with suspensor) and embryos deprived of suspensor were studied. It was found that removal of the suspensor has no effect on the development of embryos which have reached a length of 5 mm. With younger embryos, removal of the suspensor reduces embryo development, the negative effect being the greater the younger the embryo. It was shown that gibberellic acid (GA₃) concentrations of 10^-8 to 10^-6M can replace the suspensor in heart-shaped and early cotyledonary embryos (0.5 to 1.5 mm in length), whereas they reduce the development of suspensor-deprived embryos of later stages (embryos 2 to 3 mm in length) as compared with intact embryos of similar size grown on hormone-free medium. GA₃ concentrations of 10^-5 and 10^-4M are generally inhibitory and may stimulate callus formation in some embryos. The present data and those of Alpi et al. (1975) concur in ascribing a major role to gibberellins in characterizing the physiological function of the suspensor in early embryogenesis in Phaseolus coccineus.Abbreviation GA gibberellic aid     

The osmotic property and fluorescent tracer movement of developing orchid embryos of Phaius tankervilliae (Aiton) Bl

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.     

In vitro Culture of Immature Embryos of Cytisus laburnum

Immature embryos of Cytisus laburnum L. were cultivated in vitro and four culture media, different techniques of substrate preparation, sucrose concentration and the effect of suspensor removal were tested. The best results were obtained with N6 medium supplemented with 2 mg dm⁻³ glycine and set up using a double-layer culture system, in which the top layer had a higher osmotic potential than the bottom one. These conditions allowed normal embryogenic development in up to 45 % of early globular embryos, that were able to develop until a complete maturity. Osmotic potential and mineral nutrients of the medium demonstrated to be crucial for the successful culture and their effects were dependent on embryo age at the time of excision. The presence of an intact suspensor showed to be beneficial only for early globular embryos while older developmental stage embryos were not significantly affected. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metabolic regulation in C4 photosynthesis: Phosphoenol pyruvate carboxylase and 3C intermediates of the photosynthetic carbon reduction cycle

Summary Gibberellins and auxins were extracted from embryos and suspensors of Phaseolus coccineus L. at two stages of development: A) heart-shaped embryo and B) cotyledonary embryo with suspensor in the initial stage of degeneration. The time interval between the two stages was 5–6 days.In both embryos and suspensors, gibberellin (GA)-like activity was found in three fractions: F-1 (ethyl acetate fraction at pH 8.0), F-2 (free GAs) and F-3 (bound GAs). At stage A, the total GA activity in the suspensor was about 30 times greater than in the embryo and the bound GAs contributed by about 90% to the total GA content. A dramatic decrease in level of bound GA-like substances was found in suspensors at stage B, when the level of total GAs in the embryo had increased to 10 times that at stage A. This might suggest a transport of GAs from the suspensor to the embryo. In both embryo and suspensor, qualitative changes in GAs with shift in activity of the fractions tested occurred at the two developmental stages.The methanolic extracts of stage A suspensors showed two inhibitors, one much more active than the other, and two large peaks of growth promoting activity at Rf 0.4–0.7; in stage A embryos, the general activity of the extracts was lower and the promoting effect was spread over Rf 0.3–0.9.The present results seem to support the view that the suspensor plays a role in embryogenesis by acting as a site of synthesis of growth regulators needed by the embryo.Abbreviations F-1 ethyl acetate fraction at pH 8.0 - F-2 free gibberellins - F-3 bound gibberellins - GA gibberellic acid - Stage A heart-shaped embryo - stage B cotyledonary embryo with suspensor in the initial stage of degeneration     

Isolation and identification of substances inducing formation of tracheary elements in cultured carrot-root slices

Data are presented on the cytokinin status of seeds and seed components, at different stages of development in Phaseolus coccineus L., as determined with the soybean callus growth bioassay: A change in cytokinin types according to developmental stage occurred: from biologically very active less polar types (zeatin=Z) at early stages to more polar types (zeatin glucoside=Z₉G and zeatin riboside=Zr), with relatively low biological activity, at intermediate and late stages of seed development: When cytokinins were analyzed separately in embryos (embryo proper) and suspensors at two embryonic stages: heart-shaped (A) and middle cotyledonary embryos (stage B) respectively, it was found that: i) at stage A, the suspensor showed cytokinin activity at the level of Z, 2iPA (2-isopentenyladenosine) and Zr, whereas more polar cytokinins (Z₉G, Zr) were present in the embryo; ii) at stage B, when the embryo seems to become autonomous for cytokinin supply, there was a relative abundance of active cytokinins (Z, 2iPA) in the embryo to which Z₉G activity in the suspensor corresponded. It is concluded that the suspensor plays an essential role in embryogenesis by acting as a hormone source to the early embryo.Abbreviations GA gibberellic acid - 2iPA 2-isopentenyladenosine - Stage A heart-shaped embryo - siage B middle cotyledonary embryo - Z zeatin - Z₉G zeatin glucoside - Zr Zeatin riboside     

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. Received: 30 June 1999 / Revision received: 15 November 1999 / Accepted: 3 December 1999     

Macromolecular synthesis during plant embryogeny: rates of RNA synthesis in Phaseolus coccineus embryos and suspensors

A comparative study of RNA metabolism as an indicator of major changes of tissue organization, cell number, and physiology in the two developmentally and cytologically distinct parts of the bean embryo, the organogenetic part and the suspensor, was carried out. The metabolism of RNA was determined separately for these two parts of embryos removed aseptically from seeds at different times during embryogeny and incubated in culture medium containing ³H-adenosine. Equilibration of ATP in the nucleotide pool, ATP pool size and specific activity, total RNA content, rate of RNA synthesis in culture, rate of RNA synthesis and specific activity during embryogeny, and total protein content were determined. Synthetic activity of the suspensor was highest early in development and then declined, whereas synthetic activity of the organogenetic part increased throughout development. These changes may reflect developmental and functional differences in the two parts of the embryo.     

Frequenza di cellule con « DNA puffs » nel sospensore di Phaseolus coccineus in momenti diversi dello sviluppo embrionale

Abstract

Morphological aspects of Phaseolus coccineus suspensor cells at different periods of embryo development. — Embryo suspensor cells of Phaseolus coccineus have been analysed at different periods of seed and embryo development in field-grown plants and the frequency of cells involved in « DNA puffs » formation has been determined. The collected data show that: a) the relation between seed development and embryo development is not constant at different times in the season (from July to November); b) the frequency of cells showing « DNA puffs » is influenced by the above ratio. This fact seems to indicate that embryos of comparable developmental stages (as estimated by cotyledon length) show different metabolic patterns in relation to seed development and enviromental factors.     

Identification of a factor that complementarily inhibits somatic embryogenesis with vanillyl benzyl ether in Japanese larch

In Japanese larch (Larix leptolepis Gordon), a well-developed suspensor forms during somatic embryogenesis. The suspensor is the essential tissue for development of the embryo proper. In high-cell-density culture, the embryogenic cells proliferate, but no somatic embryos form because suspensor development is suppressed. Previously, we identified vanillyl benzyl ether (VBE) as a novel factor suppressing suspensor development from the high-cell-density conditioned medium (HCM), but the inhibitory effect of VBE was weaker than that of HCM added. Therefore, this study attempted to identify another inhibitory factor in the culture medium. Induction of somatic embryos was performed in a medium containing both VBE and a fraction of each chromatogram extracted from the culture medium. Results of the bioassay showed that a fraction had strong inhibitory activity with VBE, but weak activity without it. By physicochemical analyses of the fraction, 4-[(phenylmethoxy)methyl]phenol was identified as an inhibitory factor of larch somatic embryogenesis.     

Carbohydrate status during somatic embryo maturation in Norway spruce

Summary The development of Norway spruce (Picea abies (L.) Karst.) somatic embryos on a maturation medium was accompanied by changes in nonstructural carbohydrate status. During embryo maturation, the content of total soluble sugars in the embryonal suspensor mass decreased and the partitioning between sucrose and hexoses changed considerably in favor of sucrose. Developing somatic embryos were mainly responsible for these changes. Osmotic stress caused by the presence of 3.75% polyethylene glycol (PEG) in the maturation medium (decrease in osmotic potential by 52.5 kPa) resulted in dramatic changes in the content of endogenous saccharides. There was a lower total carbohydrate content in the embryonal suspensor mass grown on the medium containing PEG in comparison with the untreated control. Isolated embryos from later stages of embryo development contained mainly sucrose with a small amount (20%) of fructose and nearly no glucose. A further increase in PEG concentration in the medium (7.5%; decrease in osmotic potential by 112.5 kPa compared to the maturation medium) led to a large increase in the total endogenous sugar content. This increase in sugars was a result of the enhanced content of sucrose, fructose, and glucose. The increased glucose content was in contrast to embryos grown on the medium with lower or no PEG content.     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

Embryo abortion and histological features in the interspecific cross between <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L. and <Emphasis Type="Italic">P. coccineus</Emphasis> L

In this paper, the causes of early embryo abortion in the reciprocal crosses between Phaseolus vulgaris L. (a cultivar) and Phaseolus coccineus L. (a wild form) were studied. Methacrylate resin sections, 3–5 μm thick, of 3 to 14 day-old seeds were used to examine the embryo developmental stages and the state of seed tissue. It was observed that, embryos aborted at different developmental stages (globular to early cotyledon) depending on the maternal parent. The use of P. coccineus cytoplasm resulted in a higher number of abortion than in reciprocal crosses. Many of them took place between 5 and 6 days after pollination (DAP). Histological analyses permitted to observe that the embryo development was slower in the cross between P. coccineus and P. vulgaris, compared to parental seeds. It would be related to a deficient endosperm development in reciprocal crosses and, in some extent, hypertrophy of the suspensor might be the main cause of early embryo abortion. Then, it would be practical to overcome this incompatibility by rescuing the embryo at the globular stage of development.     

油菜裂外壁小孢子胚在分子水平上具有胚体/胚柄分化

早期合子胚取材困难, 难以开展相关研究。前人的工作表明, 油菜(Brassica napus)裂外壁小孢子胚胎发生系统能够较好地模拟合子胚的分化模式, 因而可替代早期合子胚胎作为研究材料。但目前尚缺乏该胚胎发生系统中胚胎具有胚体/胚柄分化的分子水平的证据。该文首次证明了油菜WOX家族基因能够用于标记胚体/胚柄的分化过程, 利用胚柄标记基因BnWOX8的表达模式, 从分子水平上证明了带胚柄的裂外壁小孢子胚的确存在胚体/胚柄的分化。研究结果为充分利用油菜裂外壁小孢子胚胎发生系统, 解决早期胚胎取材困难的问题奠定了坚实的基础。同时, 建立了活体激光切割分离特定细胞的技术, 结合用于少量细胞RNA提取的活体特异细胞RNA提取技术, 为鉴定少量特异分化细胞的基因表达模式提供了一个可行且明确的解决方案。     

Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death

Somatic embryogenesis is a useful tool to propagate conifers vegetatively. However, a major limitation in many pine species is the low quality of cotyledonary somatic embryos. The aim of this study has been to elucidate the developmental pathway of somatic embryos in Scots pine (Pinus sylvestris), to identify deviations from the normal pathway and to identify processes that might disturb normal development. Initially we compared the developmental pathway of somatic embryogenesis in representative cell lines yielding cotyledonary embryos with normal and abnormal morphology. Early embryos carrying suspensor cells in excess of the normal number (supernumerary) were more frequent in cell lines giving rise to abnormal cotyledonary embryos. In this study we show that the frequency of early somatic embryos with supernumerary suspensor cells increased after treatment with the auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Furthermore, the yield of developing embryos increased significantly after treatment with the antiauxin 2-(4-chlorophenoxy)-2-methylpropionic acid (PCIB), but the morphology of the embryos was not affected. The number of cells undergoing PCD was analyzed using a TUNEL-assay. The frequency of TUNEL-positive cells was high both in proliferating cultures and during differentiation of early somatic embryos. However, the pattern of TUNEL-positive cells was similar in normal somatic embryos and in embryos with supernumerary suspensor cells. Together our results suggest that the presence of supernumerary suspensor cells in early somatic embryos of Scots pine is caused by disturbed polar auxin transport and results in aberrant embryo development.     

An expanded role for the TWN1 gene in embryogenesis: defects in cotyledon pattern and morphology in the twn1 mutant of Arabidopsis (Brassicaceae)

The suspensor is a specialized basal structure that differentiates early in plant embryogenesis to support development of the embryo proper. Suspensor differentiation in Arabidopsis is maintained in part by the TWIN1 (TWN1) gene, which suppresses embryogenic development in suspensor cells: twn1 mutants produce supernumerary embryos via suspensor transformation. To better understand mechanisms of suspensor development and further investigate the function of TWN1, we have characterized late-embryo and post-embryonic development in the twn1 mutant, using seedling culture, microscopy, and genetics. We report here that the twn1 mutation disrupts cotyledon number, arrangement, and morphology and occasionally causes partial conversion of cotyledons into leaves. These defects are not a consequence of suspensor transformation. Thus, in addition to its basal role in suspensor differentiation, TWN1 influences apical pattern and morphology in the embryo proper. To determine whether other genes can similarly affect both suspensor and cotyledon development, we looked for twinning in Arabidopsis mutants previously identified by their abnormal cotyledon phenotypes. One such mutant, amp1, produced a low frequency of twin embryos by suspensor transformation. Our results suggest that mechanisms that maintain suspensor identity also function later in development to influence organ formation at the embryonic shoot apex. We propose that TWN1 functions in cell communication pathways that convey local positional information in both the apical and basal regions of the Arabidopsis embryo.     

Contrasting pattern of somatic and zygotic embryo development in alfalfa (Medicago sativa L.) as revealed by scanning electron microscopy

Scanning electron microscopy has been used to investigate the morphological changes occurring during the development of alfalfa somatic embryos. Embryos were initiated from callus, transferred to suspension culture and matured on solid agar medium. This developmental pattern was compared to that of zygotic embryos developing in ovulo. Somatic embryos begin as distinct pro-embryos within the callus tissue pieces placed in suspension culture. They become globular and heart-shaped while on solid agar medium and then undergo cotyledon elongation and maturation. Somatic embryos develop comparatively slower at early stages of development and faster at the later stages than zygotic embryos. They lack a well-defined suspensor and have a very rough, poorly-differentiated epidermis, the first layer of which is lost after pro-embryo formation. The cotyledons of somatic embryos are multiple and poorlydeveloped; there appears to be a correlation between the amount of surface roughness of the developing embryo and the extent to which polycotyledony occurs.     

In silico identification and characterization of putative differentially expressed genes involved in common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) seed development

Two genotypes of common bean (Phaseolus vulgaris L.) were studied to determine the structural cause of seed abortion in this species. In the non-abortive control (wild-type, cultivar BAT93), the histological analysis revealed a classical pattern of seed development and showed coordinated differentiation of the embryo proper, suspensor, endosperm tissue and seed coat. In contrast, the ethyl methanesulfonate (EMS) mutant (cultivar BAT93) showed disruption in the normal seed development leading to embryo abortion. Aborted embryos from these degenerate seeds showed abnormalities in suspensor and cotyledons at the globular, heart, torpedo and cotyledon stages. Exploring the feasibility of incorporating the available online bioinformatics databases, we identified 22 genes revealing high homology with genes involved in Arabidopsis thaliana embryo development and expressed in common bean immature seeds. The expression patterns of these genes were confirmed by RT–PCR. All genes were highly expressed in seed tissues. To study the expression profiles of isolated genes during Phaseolus embryogenesis, six selected genes were examined by quantitative RT–PCR analysis on the developing embryos of wild-type and EMS mutant plants. All selected genes were expressed differentially at different stages of embryo development. These results could help to improve understanding of the mechanism of common bean embryogenesis.