Early Embryogeny Research Articles

Erratum to: In vitro clonal propagation and stable cryopreservation system for Platycladus orientalis via somatic embryogenesis

Platycladus orientalis is a widespread conifer, which is native in eastern Asia, and has recently attracted much attention due to its ornamental value for landscape and gardens. However, native P. orientalis populations have been in decline over the past century. Here, we established an in vitro propagation and cryopreservation system for P. orientalis via somatic embryogenesis (SE). Whole megagametophytes with four development stages (Early embryogeny: E1 and late embryogeny: L1, L2, and L3) of zygotic embryos from immature P. orientalis cones were used as initial explants and cultured on three different basal media such as initiation medium (IM), Litvay (LV), and Schenk and Hildebrandt (SH). Both the developmental stage of zygotic embryos and kind of basal medium had a significant effect on embryogenesis induction with IM (P < 0.001, respectively). The highest frequency of embryogenic callus induction was obtained in megagametophytes with zygotic embryos at L2 stage, which ranged as high as 30%. The maturation medium containing IM basal salts, vitamins and amino acids, 15 g l−1 abscisic acid (ABA), 50 g l−1 maltose, and 100 g l−1 polyethylene glycol 4000 (PEG) was found to be the suitable medium for production of somatic embryos. The frequency of somatic embryo formation from both non-cryopreserved and cryopreserved cell lines was also tested. There were no statistical differences on the production of somatic embryos between non-cryopreserved and cryopreserved cells (P = 0.523). Genetic fidelity of the plantlets regenerated from non-cryopreserved and cryopreserved embryogenic cell lines was assessed by both random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analysis. There was no genetic instability in the regenerated plantlets from cryopreserved embryogenic cell lines. Both the SE protocol and cryopreservation protocols described here have the potential to contribute the conservation and clonal propagation of P. orientalis germplasm.

Expression of catalase and retinoblastoma-related protein genes associates with cell death processes in Scots pine zygotic embryogenesis.

BackgroundThe cell cycle and cellular oxidative stress responses are tightly controlled for proper growth and development of Scots pine (Pinus sylvestris L.) seed. Programmed cell death (PCD) is an integral part of the embryogenesis during which megagametophyte cells in the embryo surrounding region (ESR) and cells in the nucellar layers face death. In the present study, we show both the tissue and developmental stage specific expression of the genes encoding the autophagy related ATG5, catalase (CAT), and retinoblastoma related protein (RBR) as well as the connection between the gene expressions and cell death programs.ResultsWe found strong CAT expression in the cells of the developing embryo throughout the embryogenesis as well as in the cells of the megagametophyte and the nucellar layers at the early embryogeny. The CAT expression was found to overlap with both the ATG5 expression and hydrogen peroxide localization. At the late embryogeny, CAT expression diminished in the dying cells of the nucellar layers as well as in megagametophyte cells, showing the first signs of incipient cell death. Accumulation of starch and minor RBR expression were characteristic of megagametophyte cells in the ESR, whereas strong RBR expression was found in the cells of the nucellar layers at the late embryogeny.ConclusionsOur results suggest that ATG5, CAT, and RBR are involved in the Scots pine embryogenesis and cell death processes. CAT seems to protect cells against hydrogen peroxide accumulation and oxidative stress related cell death especially during active metabolism. The opposite expression of RBR in the ESR and nucellar layers alongside morphological characteristics emphasizes the different type of the cell death processes in these tissues. Furthermore, the changes in ATG5 and RBR expressions specifically in the megagametophyte cells dying by necrotic cell death suggest the genetic regulation of developmental necrosis in Scots pine embryogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0462-0) contains supplementary material, which is available to authorized users.

The embryo sac ofVanilla imperialis(Orchidaceae) is six-nucleate, and double fertilization and formation of endosperm are not observed

Double fertilization and subsequent endosperm formation are two of the most important synapomorphies of the angiosperms. Endosperm is generally lacking in Orchidaceae, but Swamy reported the formation of up to ten endosperm nuclei in Vanilla planifolia in 1947. The observation was documented only by line drawings and has not been confirmed; however, assumptions about endosperm formation occurring in Orchidaceae are primarily founded on Swamy's study. The current study provides the first detailed description of embryo sac formation and early embryogeny in Vanilla since Swamy, and is the first using modern imaging techniques. Flowers of Vanilla imperialis were artificially pollinated at 1-week intervals and the resulting fruits were fixed, embedded and sectioned for light microscopy of embryo sac formation, fertilization and early development of the embryo. Three-dimensional reconstructions of embryo sacs were obtained using confocal laser scanning microscopy on fixed ovules. The mature embryo sac contains only six nuclei as a result of the arrested development of the chalazal nuclei prior to the formation of three antipodals and a polar nucleus. Fertilization was observed 7 weeks after pollination, but double fertilization and the formation of endosperm did not occur. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177, 202–213.

Transcriptional changes of antioxidant responses, hormone signalling and developmental processes evoked by the Brassica napus SHOOTMERISTEMLESS during in vitro embryogenesis

Previous work showed that alterations in Brassica napus (Bn) SHOOTMERISTEMLESS (BnSTM) expression levels influence microspore-derived embryogenesis in B. napus. While over-expression of BnSTM increased microspore-derived embryo (MDE) yield and quality, down-regulation of BnSTM repressed embryo formation [16]. Transcriptional analyses were conducted to investigate the molecular mechanisms underpinning these responses. The induction of BnSTM resulted in a heavy transcriptional activation of genes involved in antioxidant responses, hormone signalling and developmental processes. Several antioxidant enzymes, including catalases, superoxide dismutases, and components of the Halliwell-Asada cycle were induced in embryos ectopically expressing BnSTM and contributed to the removal of reactive oxygen species (ROS). These changes were accompanied by elevated levels of ascorbate and glutathione, which have been shown to promote embryonic growth and development. Induction or repression of BnSTM altered the early cytokinin response, whereas late responses, modulated by Type-A Arabidopsis response regulators (ARRs), were induced in MDEs over-expressing BnSTM. Major differences between transgenic MDEs were also observed in the expression pattern of several auxin transporters and key developmental factors required for normal embryogenesis. While some of these factors, BABYBOOM1 (BBM1) and SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK), play a key role during early embryogeny, others, CYP78A5, LEAFY COTYLEDON1 and 2 (LEC1 and LEC2), as well as WOX2 and 9, are required for proper embryo development. Collectively these results demonstrate the involvement of BnSTM in novel developmental processes which can be utilized to enhance invitro embryogenesis.

Expression of a gymnosperm PIN homologous gene correlates with auxin immunolocalization pattern at cotyledon formation and in demarcation of the procambium during Picea abies somatic embryo development and in seedling tissues

In seed plants, the body organization is established during embryogenesis and is uniform across gymnosperms and angiosperms, despite differences during early embryogeny. Evidence from angiosperms implicates the plant hormone auxin and its polar transport, mainly established by the PIN family of auxin efflux transporters, in the patterning of embryos. Here, PaPIN1 from Norway spruce (Picea abies [L.] Karst.), a gene widely expressed in conifer tissues and organs, was characterized and its expression and localization patterns were determined with reverse transcription polymerase chain reaction and in situ hybridization during somatic embryo development and in seedlings. PaPIN1 shares the predicted structure of other PIN proteins, but its central hydrophilic loop is longer than most PINs. In phylogenetic analyses, PaPIN1 clusters with Arabidopsis thaliana (L.) Heynh. PIN3, PIN4 and PIN7, but its expression pattern also suggests similarity to PIN1. The PaPIN1 expression signal was high in the protoderm of pre-cotyledonary embryos, but not if embryos were pre-treated with the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). This, together with a high auxin immunolocalization signal in this cell layer, suggests a role of PaPIN1 during cotyledon formation. At later stages, high PaPIN1 expression was observed in differentiating procambium, running from the tip of incipient cotyledons down through the embryo axis and to the root apical meristem (RAM), although the mode of RAM specification in conifer embryos differs from that of most angiosperms. Also, the PaPIN1 in situ signal was high in seedling root tips including root cap columella cells. The results thus suggest that PaPIN1 provides an ancient function associated with auxin transport and embryo pattern formation prior to the separation of angiosperms and gymnosperms, in spite of some morphological differences.

Polar auxin transport controls suspensor fate

Polar auxin transport is critical for normal embryo development in angiosperms. It has been proposed that auxin accumulates dynamically at specific positions, which in early Arabidopsis embryos correlates with developmental decisions such as specification of the apical cell lineage, specification of the hypophysis, and differentiation of the two cotyledons. In conifers, pattern formation during embryo development is different, and includes a free nuclear stage, nondividing suspensor cells, presence of tube cells, lack of hypophysis, and formation of a crown of cotyledons surrounding the shoot apical meristem. We have recently shown that polar auxin transport is important for normal embryo development also in conifers. Here we suggest a model where auxin is transported from the suspensor cells to the embryonal mass during early embryogeny in conifers. This transport is essential for the developmental decisions of the tube cells and the suspensor, and affects both the amount of programmed cell death and the embryo patterning.

High-efficiency somatic embryogenesis and morphohistology and histochemistry of somatic embryo development in Larix leptolepis Gordon

A high-efficiency somatic embryogenesis protocol of Japanese larch (Larix leptolepis Gordon) has been established in our investigation. Calli were induced from immature zygotic embryos of female cones of L. leptolepis and then subcultured regularly on to a modified Gupta and Durzan (DCR) basal medium for 5 years. Embryogenic tissues showed distinct morphological changes during somatic embryo development when they were transferred to a maturation medium supplemented with abscisic acid (ABA) compared with the morphology in a medium lacking ABA. Histological observations indicated that polyembryony was a characteristic feature during early embryogeny and somatic embryos at later stages showed normal histodifferentiation. In addition, histochemical analysis revealed that abundant starch granules and proteins accumulated in mature embryos, indicating that they played important roles in the development and regeneration of normal plantlets from somatic embryos on hormone-free germination media

Re-thinking the embryo lethal system within the Pinaceae

Outcrossing, wind-pollinated members of the Pinaceae have high self-pollination rates yet produce few selfed seedlings. How are selfs selectively eliminated? Barriers to selfing have long been considered to be (i) avoidance of self-pollen capture, (ii) competition via simple polyembryony, and (iii) lower viability of selfed embryos. Here, reviews — and some revisions — are offered in addition to future research needs. First, avoiding self-pollen capture is largely ineffectual. Second, simple polyembrony is also a weak or nonexistent barrier against selfed embryos. Third, selfed embryos die at all stages, but a stage-specific death peak occurs during early embryogeny in some taxa and this death peak, if prevalent across a wider range of taxa, deserves a closer look. The death peak does not preclude the prevailing genetic model for embryo viability loci, but could indicate that other genetic mechanisms are operative. Molecular dissection shows that zygotic lethals are distributed across different chromosomal segments. To date, these are mostly semi-lethal rather than lethal, and they range from partially dominant to overdominant. A literature survey suggests that only 5 of the 10 genera within the Pinaceae ( Abies , Larix , Picea , Pinus , and Pseudotsuga ) have been well characterized with respect to selfing. Extreme inbreeding depression during embryo development may not be a shared feature among conifers as a group. A critical research question is whether dual death patterns are present in other genera, and if they are, alternative genetic models to account for the death peak. Addressing these questions has broad relevance to conservation, domestication, and management of closed populations, not just conifers or gymnosperms.

Propagation of Norway spruce via somatic embryogenesis

Somatic embryogenesis combined with cryopreservation is an attractive method to propagate Norway spruce (Picea abies) vegetatively both as a tool in the breeding programme and for large-scale clonal propagation of elite material. Somatic embryos are also a valuable tool for studying regulation of embryo development. Embryogenic cell lines of Norway spruce are established from zygotic embryos. The cell lines proliferate as proembryogenic masses (PEMs). Somatic embryos develop from PEMs. PEM-to-somatic embryo transition is a key developmental switch that determines the yield and quality of mature somatic embryos. Withdrawal of plant growth regulators stimulates PEM-to-somatic embryo transition accompanied by programmed cell death (PCD) in PEMs. This PCD is mediated by a marked decrease in extracellular pH. If the acidification is abolished by buffering the culture medium, PEM-to-somatic embryo transition together with PCD is inhibited. Cell death, induced by withdrawal of PGRs, can be suppressed by extra supply of lipo-chitooligosaccharides (LCOs). Extracellular chitinases are probably involved in production and degradation of LCOs. During early embryogeny, the embryos form an embryonal mass surrounded by a surface layer. The formation of a surface layer is accompanied by a switch in the expression pattern of an Ltp-like gene (Pa18) and a homeobox gene (PaHB1), from ubiquitous expression in PEMs to surface layer-specific in somatic embryos. Ectopic expression of Pa18 and PaHB1 leads to an early developmental block. Transgenic embryos and plants of Norway spruce are routinely produced by using a biolistic approach. The transgenic material is used for studying the importance of specific genes for regulating plant development, but transgenic plants can also be used for identification of candidate genes for use in the breeding programme.

Expression of the viviparous 1 (Pavp1) and p34cdc2 protein kinase (cdc2Pa) genes during somatic embryogenesis in Norway spruce (Picea abies [L.] Karst).

Detailed expression analysis of the Norway spruce (Picea abies [L.] Karst) Viviparous 1 (Pavp1) and p34cdc2 (cdc2Pa) genes was carried out during somatic embryogenesis. Pavp1, a gene associated with embryo development, was expressed in proliferating embryogenic suspension cultures in the absence of exogenous ABA. When somatic embryo formation was promoting by blocking proliferation, Pavp1 expression was reduced. During maturation, exogenous ABA induced increased Pavp1 expression, which peaked at the early cotyledonary stage of somatic embryogenesis. Following partial desiccation of mature somatic embryos at high relative humidity, Pavp1 expression persisted under germination conditions. Pavp1 expression was also detected in non-dormant immature male strobili and dormant terminal buds. These data confirm the functional conservation of Pavp1 during the evolution of seed plants and extend its function beyond the embryo. Cdc2Pa, a gene associated with the cell cycle, was up-regulated when the proliferation of embryogenic cells was blocked. Expression was again up-regulated in early embryogeny and again during germination. The implications of this up-regulation of cdc2Pa are discussed.

Re-organisation of the cytoskeleton during developmental programmed cell death in Picea abies embryos.

Cell and tissue patterning in plant embryo development is well documented. Moreover, it has recently been shown that successful embryogenesis is reliant on programmed cell death (PCD). The cytoskeleton governs cell morphogenesis. However, surprisingly little is known about the role of the cytoskeleton in plant embryogenesis and associated PCD. We have used the gymnosperm, Picea abies, somatic embryogenesis model system to address this question. Formation of the apical-basal embryonic pattern in P. abies proceeds through the establishment of three major cell types: the meristematic cells of the embryonal mass on one pole and the terminally differentiated suspensor cells on the other, separated by the embryonal tube cells. The organisation of microtubules and F-actin changes successively from the embryonal mass towards the distal end of the embryo suspensor. The microtubule arrays appear normal in the embryonal mass cells, but the microtubule network is partially disorganised in the embryonal tube cells and the microtubules disrupted in the suspensor cells. In the same embryos, the microtubule-associated protein, MAP-65, is bound only to organised microtubules. In contrast, in a developmentally arrested cell line, which is incapable of normal embryonic pattern formation, MAP-65 does not bind the cortical microtubules and we suggest that this is a criterion for proembryogenic masses (PEMs) to passage into early embryogeny. In embryos, the organisation of F-actin gradually changes from a fine network in the embryonal mass cells to thick cables in the suspensor cells in which the microtubule network is completely degraded. F-actin de-polymerisation drugs abolish normal embryonic pattern formation and associated PCD in the suspensor, strongly suggesting that the actin network is vital in this PCD pathway.

Up, down and up again is a signature global gene expression pattern at the beginning of gymnosperm embryogenesis

Somatic embryogenesis of a gymnosperm, Picea abies, represents a sequence of specifically regulated developmental stages including proembryogenic mass (PEM), PEM-to-embryo transition, and early and late embryogeny. Here, we report cDNA array analysis of expression patterns of 373 genes in the beginning of P. abies embryo development. The analysis revealed a group of 107 genes (29% of arrayed cDNAs) which were upregulated upon PEM-to-embryo transition, then downregulated during early embryogeny and finally upregulated again at the beginning of late embryogeny. This major gene expression pattern was abrogated in a developmentally arrested cell line that is unable to pass through the PEM-to-embryo transition. Thirty-five genes (9.4% of arrayed cDNAs) were found to be differentially expressed during normal embryonic pattern formation. Among them, 22 genes (5.9% of arrayed cDNAs) were directly associated with embryo pattern formation and can be considered as marker genes for early stages of P. abies embryogenesis. The majority of the marker genes encode for proteins involved in translation and posttranslational modification. Among them, 18 genes displayed the major expression pattern.

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.

Two waves of programmed cell death occur during formation and development of somatic embryos in the gymnosperm, Norway spruce.

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.

Laticifers: An historical perspective

This review describes the development of the laticifer concept, with emphasis upon the nonarticulated type, from early observations of plant exudates and “juices” to the presentation of laticifers by Esau (1953). Classical writers and herbalists described practical applications of these substances. With the advent of the microscope early investigators believed that these substances occurred in structures present in most, if not all, plants and, wrongly, equated these structures to the circulatory system in animals. Introduction of the term, latex, into botany derived from its early use as a term for a blood component by physicians, and not for analogy to milk. However, the origin of the terms, laticifer and laticiferous, remains uncertain. Initial studies of laticifers were marked by the controversy of whether they represented intercellular spaces or elongated cells. Confirmation of their cellular character led to the designation of nonarticulated and articulated laticifers. Nonarticulated laticifers were shown to arise during early embryogeny in some plants. The ontogenetic origin of the articulated laticifer was unclear to early workers, but new laticifers were detected to be formed by cambium activity. Nonarticulated laticifers were described to develop by intrusive growth whereby tips of the cell penetrated between adjacent cells. The coenocytic condition of the nonarticulated laticifer resulted from nuclear divisions along the cell positioned in the growth region of the shoot and the subsequent distribution of the daughter nuclei along the length of the cell.

Laboratory studies on the life span, growth, fecundity and embryonic development ofDaphnia cephalata King (Crustacea: Daphniidae)

Studies onDaphnia cephalata, relating to its longevity, instar duration, growth and fecundity have been made. Fifteen of the 22 individuals reared became primiparous at the fourth instar, the remaining at fifth. During growth, the total length, carapace length and carapace height increased by 2·95, 2·20 and 1·79 mm respectively and the growth curves for the 3 morphometric dimensions are of the same shape. About 200 eggs were produced during the life span of 52 days when the animals were reared at 27±2°C. The early embryogeny resembles other tropical daphnids but for differences in the developmental period in the brood chamber.

Lyonization and the lines of Blaschko.

The lines of Blaschko represent a nonrandom developmental pattern of the skin fundamentally differing from the system of dermatomes. Many nevoid skin lesions display an arrangement following these lines. This is a review of case reports providing photographically documented evidence that the lines of Blaschko become manifest in the heterozygous state of various X-linked gene defects such as incontinentia pigmenti, focal dermal hypoplasia, X-linked dominant chondrodysplasia punctata, X-linked hypohidrotic ectodermal dysplasia, and Menkes syndrome. Hence, a causal relationship between lyonization and the lines of Blaschko seems quite obvious. Although it should be borne in mind that other genetic mechanisms such as somatic mutations or chimerism may give rise to the same linear pattern, the datable embryologic event of X-inactivation seems most suitable to explain the origin and nature of the lines of Blaschko. Apparently, in women affected with X-linked skin disorders the lines of Blaschko visualize the clonal proliferation of two functionally different populations of cells during early embryogenesis of the skin. The typical dorsal V-shape and the abdominal S-figure of these lines may result from an interference of the transversal coherent proliferation with the longitudinal growth and flexion of the embryo. In contrast to Blaschko's original assumption, it is now clear that these lines are independent from the metameric structure of the human body. Obviously, they represent a marker of the normal development of human skin. Therefore, a thorough study of the distribution pattern of X-linked skin disorders in women may give us a better insight into the early embryogeny of the human integument.

Hybridization and sex pheromone responses in two closely related mealybug species (Homoptera: Pseudococcidae)

Abstract. Reciprocal crosses were made between the closely related species Planococcus citri and Planococcus ficus by using mating inducers. The pheromone responses of the hybrid offspring were bioassayed. The sex ratio of the offspring showed a striking shift in favour of females in comparison with homospecific matings mainly because of high mortality of male nymphs during the first instar. Hybrid males gave positive reactions only to pheromone extracts from the maternal species and showed no response to extracts from the paternal species. This result may be explained by the lecanoid chromosome system known for mealybug males, in which the paternal set of chromosomes becomes heterochro‐matic (condensed and genetically inactive) starting from early embryogeny. Further evidence is, however, presented that (at least in certain tissues) the paternal set reverts to the euchromatic state and to genetical activity.

Developmental regulation of cloned superabundant embryo mRNAs in soybean

We have investigated the developmental expression and reiteration frequency of genes encoding four soybean embryo mRNAs. A cDNA library of mid-maturation-stage mRNA sequences was constructed by cloning into pBR322. Plasmids representing distinct superabundant mRNA species (>10,000 molecules per cell) were selected from the library, and four clones were chosen for further investigation. Hybridization experiments with mRNA blots showed that three of the clones hybridized to a single mRNA, each of which had a unique size. One clone, however, was complementary to two mRNAs having distinct molecular weights. The prevalence of each mRNA was quantitated at distinct developmental periods. The results showed that the mRNAs accumulated during early embryogeny and then decayed prior to seed dehydration. In addition, the cloned transcripts were either undetectable, or present at very low levels (<1 molecule per 20 cells) in leaf cells, suggesting that their structural genes are regulated primarily at the transcriptional level. The reiteration frequency of genes encoding all four mRNAs was measured by hybridizing labeled, single-stranded plasmid DNA probes with an excess of embryo and/or leaf DNAs. The findings demonstrated that each message is encoded by a small multigene family containing ⪕5 members. No detectable differences in hybridization rates were observed with leaf and embryo DNAs, indicating an absence of selective gene amplification during embryogenesis.

Embryogeny ofPhaseolus coccineus: Growth and microanatomy

During early embryogeny, the development of the suspensor is rapid both in terms of size and fresh weight; structural differentiation can be observed as early as the proembryo stage with the formation of wall ingrowths. Ingrowths first appear in the outer wall of the suspensor cells adjacent to the integumentary tapetum, soon ingrowths begin to form in the inner suspensor cells as well. A basal-terminal gradation in nuclear size exists, with the largest nuclei in the basal suspensor cells. Cytologically, the suspensor cells appear to be very active, especially when the embryo reaches heart stage. Initially, the development of the embryo proper lags behind the suspensor, but its size and fresh weight increase rapidly as development proceeds. The volume of the liquid endosperm rises most rapidly during the late heart stage; and it is absorbed soon after. A cellular endospermic sheath surrounds the embryo, separating it from the liquid endosperm. Structural differentiation also occurs in the cellular endosperm cells with the formation of wall ingrowths in those cells that abut directly onto the integumentary tapetum. Both the suspensor and the cellular endosperm appear to remain active through the maturation of the seed. Storage bodies are formed in the cotyledons as well as in the embryonic axis. In the suspensor and the cellular endosperm, starch grains and lipid bodies can be found at the maturation stage.