Conifer Embryos Research Articles

Deep learning for automated segmentation and counting of hypocotyl and cotyledon regions in mature Pinus radiata D. Don. somatic embryo images.

In commercial forestry and large-scale plant propagation, the utilization of artificial intelligence techniques for automated somatic embryo analysis has emerged as a highly valuable tool. Notably, image segmentation plays a key role in the automated assessment of mature somatic embryos. However, to date, the application of Convolutional Neural Networks (CNNs) for segmentation of mature somatic embryos remains unexplored. In this study, we present a novel application of CNNs for delineating mature somatic conifer embryos from background and residual proliferating embryogenic tissue and differentiating various morphological regions within the embryos. A semantic segmentation CNN was trained to assign pixels to cotyledon, hypocotyl, and background regions, while an instance segmentation network was trained to detect individual cotyledons for automated counting. The main dataset comprised 275 high-resolution microscopic images of mature Pinus radiata somatic embryos, with 42 images reserved for testing and validation sets. The evaluation of different segmentation methods revealed that semantic segmentation achieved the highest performance averaged across classes, achieving F1 scores of 0.929 and 0.932, with IoU scores of 0.867 and 0.872 for the cotyledon and hypocotyl regions respectively. The instance segmentation approach demonstrated proficiency in accurate detection and counting of the number of cotyledons, as indicated by a mean squared error (MSE) of 0.79 and mean absolute error (MAE) of 0.60. The findings highlight the efficacy of neural network-based methods in accurately segmenting somatic embryos and delineating individual morphological parts, providing additional information compared to previous segmentation techniques. This opens avenues for further analysis, including quantification of morphological characteristics in each region, enabling the identification of features of desirable embryos in large-scale production systems. These advancements contribute to the improvement of automated somatic embryogenesis systems, facilitating efficient and reliable plant propagation for commercial forestry applications.

Size regulation of the lateral organ initiation zone and its role in determining cotyledon number in conifers.

Unlike monocots and dicots, many conifers, particularly Pinaceae, form three or more cotyledons. These are arranged in a whorl, or ring, at a particular distance from the embryo tip, with cotyledons evenly spaced within the ring. The number of cotyledons, nc, varies substantially within species, both in clonal cultures and in seed embryos. nc variability reflects embryo size variability, with larger diameter embryos having higher nc. Correcting for growth during embryo development, we extract values for the whorl radius at each nc. This radius, corresponding to the spatial pattern of cotyledon differentiation factors, varies over three-fold for the naturally observed range of nc. The current work focuses on factors in the patterning mechanism that could produce such a broad variability in whorl radius. Molecularly, work in Arabidopsis has shown that the initiation zone for leaf primordia occurs at a minimum between inhibitor zones of HD-ZIP III at the shoot apical meristem (SAM) tip and KANADI (KAN) encircling this farther from the tip. PIN1-auxin dynamics within this uninhibited ring form auxin maxima, specifying primordia initiation sites. A similar mechanism is indicated in conifer embryos by effects on cotyledon formation with overexpression of HD-ZIP III inhibitors and by interference with PIN1-auxin patterning. We develop a mathematical model for HD-ZIP III/KAN spatial localization and use this to characterize the molecular regulation that could generate (a) the three-fold whorl radius variation (and associated nc variability) observed in conifer cotyledon development, and (b) the HD-ZIP III and KAN shifts induced experimentally in conifer embryos and in Arabidopsis. This quantitative framework indicates the sensitivity of mechanism components for positioning lateral organs closer to or farther from the tip. Positional shifting is most readily driven by changes to the extent of upstream (meristematic) patterning and changes in HD-ZIP III/KAN mutual inhibition, and less efficiently driven by changes in upstream dosage or the activation of HD-ZIP III. Sharper expression boundaries can also be more resistant to shifting than shallower expression boundaries. The strong variability seen in conifer nc (commonly from 2 to 10) may reflect a freer variation in regulatory interactions, whereas monocot (nc = 1) and dicot (nc = 2) development may require tighter control of such variation. These results provide direction for future quantitative experiments on the positional control of lateral organ initiation, and consequently on plant phyllotaxy and architecture.

Mini review: Application of the somatic embryogenesis technique in conifer species

The somatic embryogenesis (SE) process is better suited to large-scale production and automation than other clonal propagation methods such as the rooting of cuttings. SE is becoming a key technique to promote the asexual industrialization of conifers. Furthermore, somatic embryos are an ideal material to study the molecular mechanism of conifer embryo development, as the processes of somatic and zygotic embryo development are very similar. This brief review introduces the culturing techniques of the SE process in conifers and outlines the progress and deficiencies in conifer SE research. Emphasis is placed on the patterning formation of conifer somatic embryos.

Two-stage patterning dynamics in conifer cotyledon whorl morphogenesis.

Conifer embryos, unlike those of monocots or dicots, have variable numbers of cotyledons, even within the same species. Cotyledons form in a single whorl on a dome-shaped embryo. The closely spaced cotyledons are not found outside this ring, indicating a radial control on where they can form. Polar transport of the hormone auxin affects outgrowth of distinct cotyledons, but not the radial aspect of the whorl or the within-whorl spacing between cotyledons. A quantitative model of plant growth regulator patterning is needed to understand the dynamics of this complex morphogenetic process. A two-stage reaction-diffusion model is developed for the spatial patterning of growth regulators on the embryo surface, with a radial pattern (P1) constraining the shorter-wavelength cotyledon pattern (P2) to a whorl. These patterns drive three-dimensional (3-D) morphogenesis by catalysing local surface growth. Growth driven by P2 generates single whorls across the experimentally observed range of two to 11 cotyledons, as well as the circularly symmetric response to auxin transport interference. These computations are the first corroboration of earlier theoretical proposals for hierarchical control of whorl formation. The model generates the linear relationship between cotyledon number and embryo diameter observed experimentally. This accounts for normal integer cotyledon number selection, as well as the less common cotyledon fusings and splittings observed experimentally. Flattening of the embryo during development may affect the upward outgrowth angle of the cotyledons. Cotyledon morphogenesis is more complex geometrically in conifers than in angiosperms, involving 2-D patterning which deforms a surface in three dimensions. This work develops a quantitative framework for understanding the growth and patterning dynamics involved in conifer cotyledon development, and applies more generally to the morphogenesis of whorls with many primordia.

Effect of light conditions on anatomical and biochemical aspects of somatic and zygotic embryos of hybrid larch (Larix × marschlinsii).

In conifers, mature somatic embryos and zygotic embryos appear to resemble one another physiologically and morphologically. However, phenotypes of cloned conifer embryos can be strongly influenced by a number of invitro factors and in some instances clonal variation can exceed that found in nature. This study examines whether zygotic embryos that develop within light-opaque cones differ from somatic embryos developing in dark/light conditions invitro. Embryogenesis in larch is well understood both in situ and invitro and thus provides a suitable system for addressing this question. Features of somatic and zygotic embryos of hybrid larch, Larix × marschlinsii, were quantified, including cotyledon numbers, protein concentration and phenol chemistry. Somatic embryos were placed either in light or darkness for the entire maturation period. Embryos at different developmental stages were embedded and sectioned for histological analysis. Light, and to a lesser degree abscisic acid (ABA), influenced accumulation of protein and phenolic compounds in somatic and zygotic embryos. Dark-grown mature somatic embryos had more protein (91·77 ± 11·26 µg protein mg(-1) f.wt) than either dark-grown zygotic embryos (62·40 ± 5·58) or light-grown somatic embryos (58·15 ± 10·02). Zygotic embryos never accumulated phenolic compounds at any stage, whereas somatic embryos stored phenolic compounds in the embryonal root caps and suspensors. Light induced the production of quercetrin (261·13 ± 9·2 µg g(-1) d.wt) in somatic embryos. Mature zygotic embryos that were removed from seeds and placed on medium in light rapidly accumulated phenolics in the embryonal root cap and hypocotyl. Delaying germination with ABA delayed phenolic compound accumulation, restricting it to the embryonal root cap. In larch embryos, light has a negative effect on protein accumulation, but a positive effect on phenol accumulation. Light did not affect morphogenesis, e.g. cotyledon number. Somatic embryos produced different amounts of phenolics, such as quercetrin, depending on light conditions. The greatest difference was seen in the embryonal root cap in all embryo types and conditions.

Reaction-Diffusion Patterns in Plant Tip Morphogenesis: Bifurcations on Spherical Caps

We study a chemical reaction-diffusion model (the Brusselator) for pattern formation on developing plant tips. A family of spherical cap domains is used to represent tip flattening during development. Applied to conifer embryos, we model the chemical prepatterning underlying cotyledon ("seed leaf") formation, and demonstrate the dependence of patterns on tip flatness, radius, and precursor concentrations. Parameters for the Brusselator in spherical cap domains can be chosen to give supercritical pitchfork bifurcations of patterned solutions of the nonlinear reaction-diffusion system that correspond to the cotyledon patterns that appear on the flattening tips of conifer embryos.

Somatic embryogenesis for mass propagation of elite Spruce families: effect of storage time on somatic embryogenesis initiation

Background Somatic embryogenesis (SE) is the only clonal propagation method that has potential for large scale production of elite conifer plants from the breeding programs. Methods that support bioreactor-based methods for SE propagation are developed [1,2]. Samples of somatic embryos can be stored indefinitely under liquid nitrogen for future plant production. Somatic embryo cultures are also studied as model systems for conifer embryo development to address fundamental research questions, or used as material for genetic transformation to study gene function in conifers. In addition to utilizing SE for masspropagation of known elite clones previously tested in field tests, the SE technology offers an opportunity to directly capture and increase the value of small samples of elite seeds from the breeding programs. Furthermore, by direct masspropagation of families through SE, the value of the elite seed is increased; however without the cost of clonal testing. This is arguably an alternative approach to the traditional approach of only utilizing clonal field-tested material for SE masspropagation [3].The aim of this project was to investigate the effect from seed storage time on the rate of somatic embryo initiation for the purpose of optimizing the use for SE over time of small valuable seed samples. This was done by isolating ZE from seeds of Norway spruce that had been stored for various times, and were collected from different parts of Sweden. Material and methods Plant material Nineteen batches of Norway spruce (Picea abies) seeds from commercial seed orchards in southern, middle and northern parts of Sweden were provided by the forest companies supporting the project.

Organization, anatomy, and fungal endophytes of a Triassic conifer embryo

Much is known about the Paleozoic conifers and the conifers assignable to extant families that appear in the Jurassic; however, relatively little is known about the transitional conifers of the early Mesozoic, especially the Voltziales. To better understand the evolution of this group, we aim to increase knowledge of voltzialean anatomy and morphology. A fossil embryo of the group is described in this study. • Several permineralized seeds, one containing a well-preserved embryo, were collected from the early Middle Triassic Fremouw Peak locality in the Central Transantarctic Mountains. Samples were prepared using the standard acetate peel technique and studied in transmitted light. • The embryo belongs to Parasciadopitys aequata, a member of the Voltziales. The embryo and megagametophyte tissues are exquisitely preserved. The embryo is colonized by two distinct fungi. Sporocarps of the fungi are found in the megagametophyte and in the space between the megagametophyte and nucellus. The additions of embryo and megagametophyte characters to the description of P. aequata have made it one of the most completely known fossil taxa reproductively. • Preservation of fossil embryos, although rare, can expand the array of characters available in tracing the evolutionary history of plants. The embryo of P. aequata shares similarities with embryos of other extinct and extant conifer families. The association of the embryo with Combresomyces cornifer and Mycocarpon asterineum increases our understanding of the roles of microorganisms in Triassic ecosystems.

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.

The polar auxin transport inhibitor NPA impairs embryo morphology and increases the expression of an auxin efflux facilitator protein PIN during Picea abies somatic embryo development

Auxin and polar auxin transport have been implicated in controlling embryo patterning and development in angiosperms but less is known from the gymnosperms. The aims of this study were to determine at what stages of conifer embryo development auxin and polar auxin transport are the most important for normal development and to analyze the changes in embryos after treatment with the polar auxin inhibitor N-1-naphthylphthalamic acid (NPA). For these studies, somatic embryos of Norway spruce (Picea abies L. Karst) were used. Growth on medium containing NPA leads to the formation of embryos with poor shoot apical meristem (SAM) and fused cotyledons, and to a pin-formed phenotype of the regenerated plantlets. The effect of NPA on embryo morphology was most severe if embryos were transferred to NPA-containing medium immediately before cotyledon initiation and SAM specification. Indole-3-acetic acid (IAA) was identified by immunolocalization in developing embryos. The highest staining intensity was seen in early staged embryos and then decreased as the embryos matured. No clear IAA-maxima was seen, although the apical parts of embryos, particularly the protoderm, and the suspensor cells appear to accumulate more IAA, as reflected by the staining pattern. The NPA treatment also caused expanded procambium and a broader root apical meristem in embryos, and a significant increase in the expression of a PIN1-like gene. Taken together, our results show that, for proper cotyledon initiation, correct auxin transport is needed only during a short period at the transition stage of embryo development, probably involving PIN efflux proteins and that a common mechanism is behind proper cotyledon formation within the species of angiosperms and conifers, despite their cotyledon number which normally differs.

WOX2 and polar auxin transport during spruce embryo pattern formation

The WOX family of transcription factors and polar auxin transport (PAT) are both essential for embryonic patterning and thus normal embryo development in angiosperms. Recent analysis by us of WOX-related genes in Picea and Pinus suggests that they play fundamental roles during embryo development also in conifers.1 It has been proposed that there is a connection between the spatial separation of WOX2 and WOX8, and PAT in the formation of the apical-basal axis in Arabidopsis embryos and that both are involved in the regulation of the auxin efflux carrier PIN1. Auxin also seems to play a crucial role in apical-basal axis formation in conifer embryos based on studies using the polar auxin inhibitor NPA. We recently analyzed the expression of a PIN1-like gene in NPA-treated and untreated precotyledonary somatic spruce embryos and could see a significant upregulation of the PIN1-like gene in the NPA-treated embryos.2 Here we show that PaWOX2 is also significantly upregulated in the same embryos. Taken together, this suggests that PAT is involved in regulating both PIN1 and WOX2 expression in conifers and strengthens the evidence for the proposed connection between WOX and PIN genes in seed plants.

Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine

Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.

Inhibited polar auxin transport results in aberrant embryo development in Norway spruce

Current hypotheses concerning the role of polar auxin transport in embryo development are entirely based on studies of angiosperms, while little is known about how auxin regulates pattern formation in gymnosperms. In this study, different developmental stages of somatic embryos of Norway spruce (Picea abies) were treated with the polar auxin transport inhibitor 1-N-naphtylphthalamic acid (NPA). Effects of the treatments on auxin content, embryo differentiation and programmed cell death (PCD) were analysed. During early embryo development, NPA-treatment led to increased indole-3-acetic acid (IAA) content, abnormal cell divisions and decreased PCD, resulting in aberrant development of embryonal tube cells and suspensors. Mature embryos that had been treated with NPA showed both apical and basal abnormalities. Typically the embryos had abnormal cotyledon formation and irregular cell divisions in the area of the root meristem. Our results show that polar auxin transport is essential for the correct patterning of both apical and basal parts of conifer embryos throughout the whole developmental process. Furthermore, the aberrant morhologies of NPA-treated spruce embryos are comparable with several auxin response and transport mutants in Arabidopsis. This suggests that the role of polar auxin transport is conserved between angiosperms and gymnosperms.

The cellular and molecular biology of conifer embryogenesis

Gymnosperms and angiosperms are thought to have evolved from a common ancestor c. 300 million yr ago. The manner in which gymnosperms and angiosperms form seeds has diverged and, although broad similarities are evident, the anatomy and cell and molecular biology of embryogenesis in gymnosperms, such as the coniferous trees pine, spruce and fir, differ significantly from those in the most widely studied model angiosperm Arabidopsis thaliana. Molecular analysis of signaling pathways and processes such as programmed cell death and embryo maturation indicates that many developmental pathways are conserved between angiosperms and gymnosperms. Recent genomics research reveals that almost 30% of mRNAs found in developing pine embryos are absent from other conifer expressed sequence tag (EST) collections. These data show that the conifer embryo differs markedly from other gymnosperm tissues studied to date in terms of the range of genes transcribed. Approximately 72% of conifer embryo-expressed genes are found in the Arabidopsis proteome and conifer embryos contain mRNAs of very similar sequence to key genes that regulate seed development in Arabidopsis. However, 1388 loblolly pine (Pinus taeda) embryo ESTs (11.4% of the collection) are novel and, to date, have been found in no other plant. The data imply that, in gymnosperm embryogenesis, differences in structure and development are achieved by subtle molecular interactions, control of spatial and temporal gene expression and the regulating agency of a few unique proteins.

Spatially quantitative control of the number of cotyledons in a clonal population of somatic embryos of hybrid larch Larix x leptoeuropaea.

Many conifer embryos, both in natural seeds and in clonal populations of somatic embryos, display variability in the number of cotyledons. In hybrid larch, Larix x leptoeuropaea (synonymous with L. x marschlinsii Coaz), such variability has previously been reported in somatic embryos, together with a decrease in the average cotyledon number when benzyladenine (BA) is applied exogenously. Described here is a spatially quantitative study with the aim of throwing some light on the way cotyledon number is determined, and hence the mechanism of cotyledon formation. Stock cultures of embryogenic tissue were maintained and later made embryogenically active by standard methods. Development through cotyledon formation was followed by optical microscopy with quantitative measurement of embryo diameter and number of cotyledons. SEMs of representative stages and cotyledon numbers were done for purposes of illustration in this account. Existing mathematics of waveforms on a disc were cast into a form suitable to compare with the quantitative data. The number of cotyledons is linearly related to the diameter of the apical surface of the embryo (which approximates a circular disc) at the time of first appearance of the cotyledon primordia. This linearity is a constant-spacing phenomenon between adjacent primordia. Addition of BA to the medium restricts the range of apical diameters without changing inter-cotyledon spacing. Slope/intercept ratio of the linear plot matches expectation for initiation of cotyledon pattern as a harmonic waveform on a circular disc. The entire pattern of cotyledon primordia arises as a single entity coordinated by a mechanism with wave-forming properties. This is explicable by diverse mechanisms, especially either mechanical buckling ('biophysical') or reaction-diffusion kinetics ('physicochemical').

Overdominant lethals as part of the conifer embryo lethal system

In pines, self-pollination rates can be as high as 34% yet only 5% of viable seed is a product of self-fertilization. This decline in selfed seed viability is the consequence of post-fertilization exclusion mechanisms operating via the embryo lethal system. Recent molecular marker dissection studies suggest that the embryo lethal system is composed of semilethal factors dispersed across the genome, but it is not clear whether overdominant lethal factors are rare or representative. The study objective was to determine if overdominance was rare for the embryo lethal system in conifers. Three cohorts of selfed offspring from a single Pinus taeda parent were genotyped for nuclear microsatellites. Maximum likelihood tests based on distorted segregation ratios for single markers and for interval mapping were used to infer the degree of dominance. Four hypotheses about overdominance lethal factors were tested: (1) overdominant lethal factors rarely occur within the embryo lethal system, (2) overdominant lethal factors are rarely detected because they are transient and display stage-specific expression, (3) overdominant lethal factors are rarely detected due to tight linkage with rare marker alleles and (4) dominance estimation is unbiased by gametic selection. Four out of the seven chromosomal segments were linked to an overdominant lethal factor. One of these four segments had symmetric overdominance, an effect which persisted from embryo maturity through germination. Four overdominant lethal factors were linked to common and rare marker alleles. Gametic selection was not a source of bias in dominance estimation. Overdominant or pseudo-overdominant lethal factors are a common component of the conifer embryo lethal system.

Special symposium: In vitro plant recalcitrance transcript profiling: A tool to assess the development of conifer embryos

Embryogenesis is a phase of development that has been understudied at the molecular level, especially in conifers. Somatic embryogenesis, the asexual propagation of embryos, provides insights into development and has major applications in the forestry industry. Loblolly pine is the most important commercial species in the United States; however, it is recalcitrant to somatic embryogenesis methods. An appreciation of gene expression and the development of ‘expression markers’ would allow us to follow conifer embryogenesis more closely and to gain some insight into the metabolic states of zygotic and somatic embryos. We have employed the techniques of differential display to identify genes whose mRNA abundance changes over the course of development. We have isolated around 500 cDNAs and propose the establishment of a database of transcript levels in somatic and zygotic pine embryos over the course of development using cDNA arrays. Approximately one-third of our cDNAs have similarity to sequences in the Genbank. Our intention is to gain insight into cell physiology and biochemistry by identifying inducible transcripts. This information will form the basis of testable hypotheses regarding manipulation of embryo development in tissue culture. Experiments deriving from transcript profiling will provide insight into development. The expression markers' will allow a classification system more closely tied to metabolic state and the integration of these data into ongoing physiological research will lead to improved protocols for somatic embryogenesis.

Structures contributing to the completion of conifer seed germination

The variety of interpretations of the origin and role of the tissues surrounding the emerging radicle of conifer seeds prompted us to study changes during germination. The structures contributing to the opening of the seed coat and protrusion of the radicle in pine and spruce seeds were examined using light microscopy and field emission scanning electron microscopy. The opening of the seed coat was mainly mechanical and primarily affected by the enlargement of the imbibed endotesta cells lining the micropylar canal. The hypertrophied nucellar apex, swelling remnants and mucous substances in the micropyle further enhanced the testa splitting which was reinforced by the straightening of the folded nucellar cap. The expansion of the imbibed megagametophyte and the embryo were conducive to the seed coat opening only at a later stage. This was followed almost immediately by the appearance of the root cap showing an early geotropic response typical for taproots and assuring an immediate anchoring of the emerged embryo to the ground. The translucent tissue composed of elongating cell rows preceded and surrounded the protruding radicle and derived mainly from two origins: the micropylar end of the megagametophyte and the root cap. Observations about the origin of the sheath clarify the reasons for previous interpretations about the putative role of the massive embryo root cap in testa splitting and germination. The protective and adhesive function of the sheath is concluded to be essential to the conifer embryos, which present the epigeal type of germination.

The Culture of Young Conifer Embryos in Vitro

The Culture of Young Conifer Embryos in Vitro

Dissection, Staining, and Mounting of the Embryos of Conifers

A statement is given of the advantages of this special technic and its place in embryological investigations, including directions for selecting the proper stages in collecting conifer cones and ovules, their methods of dissection from living material and their preservation for later dissection. The choice of dissecting microscopes and dissecting instruments, as well as directions for staining embryos with phloxine which may be combined with slow dehydration in glycerin, or for staining with Delafield's or Heidenhain's hematoxylin which may be followed by the glycerin dehydration are described. Glycerin affords a convenient break for a temporary stopping place in this technic.Directions are given for transfer from glycerin thru 95% and absolute ethyl alcohol into other solvents such as diaphane solvent, essence of euparel or an easily prepared sandarac solvent. Other mounting media which have been used for conifer embryos are discussed—glycerin jelly, Venetian turpentine and Canada balsam—emphasizing the ...