APOMIXIS IN AMELANCHIER LAEVIS,SHADBUSH (ROSACEAE,MALOIDEAE)

We studied ovule and megagametophyte development in tetraploid (n = 34) individuals of Amelanchier laevis in Maine. Nomarski differential interference contrast microscopy of cleared, whole ovules and conventional microscopy of sectioned, stained material show no clear evidence for the successful completion of meiosis. Instead, the megasporocyte or its derivatives degenerate and one to six nearby cells develop into aposporous initials. Usually more than one of these divide to form eight-nucleate, Polygonum-type megagametophytes. The egg apparently forms a proembryo parthenogenetically, but seed maturation requires pollination. This evidence for apospory and pseudogamy, the first to be reported in Amelanchier, conforms to the general pattern found in other apomictic genera of the Maloideae.     

Characteristics of ovary, ovule and mature megagametophyte in Rhododendron L. (Ericaceae) and their taxonomic significance

PALSER, B. F., PHILIPSON, W. R. & PHILIPSON, M. N., 1991. Characteristics of ovary, ovule and mature megagametophyte in Rhododendron L. (Ericaceae) and their taxonomic significance. The ovary, ovule and megagametophyte at the time the latter is mature are described for 177 species representing all subgenera, sections and most subsections recognized in Rhododendron. All three, but particularly the ovary, vary considerably. The ovary is compared among species as to size; shape of apex–tapered vs. depressed; relative dimensions; indumentum–five basic hair types; wall structure including crystal distribution and frequency, stomata, cuticular ornamentation; locule number, shape and size relative to radius; wall thickness relative to radius; placenta size, shape, depth of cleft, level of cleft junction, presence or not of a decurrent placental stalk ridge; number of ovules, their arrangement and orientation on placenta; and presence and distribution of internal stomata and hairs. The nectary which girdles the base of the ovary also varies in size, shape, indumentum, stomata and some internal features. The ovule is anatropous, unitegmic and tenuinucellate with the lateral and micropylar nucellus disappearing completely and the megagametophyte elongating into the micropyle in all species. Also common to almost all are an endothelium, hypostase, differentiated epidermis–most often tanniniferous, and starch in integument around egg apparatus and micropyle. There are differences in size, shape, proportion occupied by gametophyte and micropyle, thickness of integument, degree of differentiation of some features, amount and area of starch and occurrence of chalazal and micropylar tails or of incipient micropylar and/or chalazal appendages. The megagametophyte, which develops according to the Polygonum pattern, has two distinct portions, the chalazal bounded by the endothelium with small antipodal cells at its end and the usually broader micropylar part within the micropyle containing a rather large egg apparatus with distinctive synergids; starch is present in the central cell and the polar nuclei are most often fused. Differences occur in proportions of the parts to one another, amount of starch, etc. Sixty-three characters were entered onto a computer, clustered by two different techniques and dendrograms constructed. Personal analysis and both dendrograms show section Vireya to be characterized by a syndrome of distinctive features and clearly separated from the rest of the genus. Subgenus Hymenanthes also has its particular syndrome but is somewhat less distinct from the remainder of the genus. Even less distinct but still grouped together are species of section Choniasirum and of section Sciadorhodion. Many species of section Rhododendron and of subgenus Tsutsusi tend to cluster together but section Rhododendron and most sections of the azalea complex are more generalized and moderate in the ovary, ovule and megagametophyte characters and do not separate sharply from one another.     

A COMPARATIVE STUDY OF OVULE AND MEGAGAMETOPHYTE DEVELOPMENT IN FIELD-GROWN AND GREENHOUSE-GROWN PLANTS OF GLYCINE MAX AND PHASEOLUS AUREUS (PAPILIONACEAE)

A study of ovule and megagametophyte development in field- and greenhouse-grown plants of Glycine max (L.) Merrill and Phaseolus aureus Roxb. reveals several consistent features for both species. These features include: a multiple archesporium, enlargement of a primary sporogenous cell directly into a megasporocyte, production of unequal dyad cells, a functional chalazal megaspore, Polygonum-type development, and a hypostase. A filiform apparatus was not observed in either species. Several marked differences in development also occur. Phaseolus usually produces one sporogenous cell per ovule; Glycine produces 2–3 sporogenous cells per ovule. Meiosis II is synchronous in Phaseolus but nonsynchronous in Glycine. Linear tetrads are produced in Phaseolus, whereas linear and T-shaped tetrads are found in Glycine. Starch grains accumulate in the mature megagametophyte of Glycine but are absent at that stage in Phaseolus. The usefulness of the modified clearing fluid, benzyl benzoate-4½, for the study of ovule and megagametophyte development in Glycine max and Phaseolus aureus is here demonstrated. In addition, the study indicates for both species that megagametophyte development in plants grown under field conditions is markedly similar to development in plants grown in the more uniform conditions of the greenhouse. Accordingly, these findings suggest generally that embryological data collected from plants grown under greenhouse conditions will reflect those from plants found in nature.     

THE USE OF A NEW CLEARING TECHNIQUE FOR THE STUDY OF EARLY OVULE DEVELOPMENT,MEGASPOROGENESIS, AND MEGAGAMETOGENESIS IN FIVE SPECIES OF CORNUS L.

The application of a new clearing technique proved successful for the study of ovule development, megasporogenesis, and megagametogenesis in Cornus asperifolia Michaux., C. stricta Lam., C. amomum Miller, C. florida L., and C. alternifolia L. These were the first studies of embryological features in C. asperifolia and C. stricta. Consistent features for both species include: slightly crassinucellate nucelli, megaspore mother cells, and equal dyads with the upper member obliquely aligned to the parietal cell. Alignment of nucelli of the 4-nucleate megagametophyte is not the same for both species. More than one mature 8-nucleate megagametophyte per nucellus was recorded for C. asperifolia. Embryological features not revealed in previous investigations of the other three species include: equal dyad cells for C. alternifolia and C. amomum, although disintegration of defunct megaspores is variable for both species; vacuole formation at the micropylar end at the 2-nucleate stage of C. amomum; a 4-nucleate stage in C. amomum and C. alternifolia with different alignments of nuclei; synergids that disintegrate rapidly in both species although antipodal shapes vary for each; and fusion of polar nuclei at varying locations within different species. A prominent hypostase is formed in all five species. Because of embryological features, it is recommended that C. forida be separated from the genus Cornus.     

DEVELOPMENT OF THE OVULE AND MEGAGAMETOPHYTE IN SAXIFRAGA HIERACIFOLIA

Wiggins , Ira L. (Stanford U., Stanford, Calif.) Development of the ovule and megagametophyte in Saxifraga hieracifolia. Amer. Jour. Bot. 46(10): 692–697. Illus. 1059.—Buds of Saxifraga hieracifolia collected in the vicinity of Point Barrow, Alaska, fixed, sectioned, and stained by standard methods, revealed that the archesporial cell in the ovule of this species is hypodermal and gives rise to the megaspore mother cell and a small number of parietal cells. Occasionally 2 megaspore mother cells occur within an ovule. Meiosis in the megaspore mother cell produces a linear tetrad of megaspores, the chalazal one of which normally gives rise to a monosporic, Polygonum-type megagametophyte. The polar nuclei fuse near the chalazal end of the megagametophyte and the antipodal cells disintegrate prior to fertilization. A distinct filiform apparatus and a marked lateral “spur” develop on each synergid. Vacuolation in the egg cell and in the synergids follows the usual pattern. Only a single integument surrounds the nucellus.     

COMPARATIVE MORPHOLOGY OF THE CARPEL IN THE ROSACEAE. V. POMOIDEAE: AMELANCHIER,ARONIA, MALACOMELES,MALUS, PERAPHYLLUM,PYRUS, SORBUS

The carpels of 2 groups of pomoid genera, Amelanchier, Malacomeles, Peraphyllum and Aronia, Malus, Pyrus, and Sorbus, were analyzed morphologically. Open sutures are associated with a lesser extent of tegumentary fusion and ovular bundle–wing bundle fusion than are closed sutures. However, in the genera as a whole (and particularly in Aronia and Sorbus), the extent of sutural closure is inversely related with the amount of intercarpellary adhesion and with the fusion of carpels to the floral cup. In the Amelanchier group and in Malus and Pyrus, ovular- and wing-bundle fusion is directly related with intercarpellary adhesion. Malus and Pyrus have closer structural resemblances with one another than they have with Aronia and Sorbus.     

The megagametophyte in Anarthria (Anarthriaceae,Poales) and its implications for the phylogeny of the Poales

The development and structure of the megagametophyte of Anarthria (Anarthriaceae), Aphclia, and Centrolepis (Centrolepidaceae) are described. Anarthriaceae has tenuinucellate ovules and the Polygonum type of megagametophyte development, both characters typical of the Poales. However, it lacks the anticlinally elongated nucellar epidermis and numerous large starch bodies observed in the megagametophyte of Centrolepidaceae, both characters also present in Restionaceae. This relatively generalized megagametophyte structure is consistent with data from the chloroplast genome, which suggest that Anarthriaceae are not as closely related to Restionaceae as previously assumed. New data from the megagametophyte are analyzed cladistically together with other available information on the poalean families. The results show that there are two possible positions for Anarthriaceae: either as sister to Poaceae. Joinvilleaceae, Restionaceae, Ecdeiocoleaceae, and Restionaceae, or as sister to only the latter three families. The new data also allow a critical reevaluation of the phylogenetic position of Centrolepidaceae, which is either basal to the poalean clade (based on microgametophyte data), or embedded in the Restionaceae (based on anther structure and megagametophyte data).     

Comparisons of within-population genetic variation in sexual and agamospermousAmelanchier (Rosaceae) using RAPD markers

We compared genetic variation of sexualAmelanchier bartramiana and facultatively agamospermous (asexually seed-producing)A. laevis at one site where the two species are sympatric. We analyzed 77 random amplified polymorphic DNA (RAPD) markers in 29A. bartramiana individuals and 76 RAPD markers in 31A. laevis individuals. The two species do not differ significantly in mean genetic variation. However, 22.4% of genetic similarity values betweenA. laevis individuals exceed the highest value ofA. bartramiana and may represent the effect of agamospermy. Variation withinA. laevis may be the result of sexuality, hybridization, polyploidy, and other factors.     

Genome size and environmental factors in the genus Pinus

Nuclear 1C DNA content in haploid megagametophyte tissue of 18 North American and one exotic Pinus species was determined using scanning microspectrophotometry. The nuclear DNA content in root meristematic cells of Zea mays L. ssp. mays, inbred line Va35 (4C = 10.31 pg) was used as a standard. DNA content measured by microspectrophotometry was verified using laser flow cytometry with two additional standards, Hordeum vulgare cv. Sultan (2C = 11.12 pg) and P. eldarica (2C = 47.30 pg). DNA values obtained by both methods were significantly correlated (r = 0.987). The 1C nuclear DNA content ranged from 21 pg to 31 pg. The ratio of DNA content in embryo tissue of P. eldarica to that in megagametophyte tissue was 1.72 by scanning microspectrophotometry and 1.74 by laser flow cytometry. To date, this is the most comprehensive data set available for North American Pinus species. Relationships between genome size of 18 North American Pinus species and climatic factors and indices of growth were investigated using regression and correlation analyses. Positive correlations were observed between nuclear DNA content and growth indices, minimum seed-bearing age, and seed dimensions. Strong negative correlations were observed between nuclear DNA content and two climatic factors, the lowest mean annual and monthly precipitation (excluding January) and the highest mean monthly spring air temperature. These correlations suggest that the large genome size and its variation in Pinus are adapted responses to the habitats of these species.     

Occurrence of unique three-celled megagametophyte and single fertilization in an aquatic angiosperm-Dalzellia zeylanica (Podostemaceae-Tristichoideae)

Angiosperms are characterized by the occurrence of double fertilization. However, Podostemaceae is considered an exception with the presence of only single fertilization (syngamy) though two male gametes are formed conventionally. To determine the cause for the failure of double fertilization in Dalzellia zeylanica (Gardn.) Wight, we closely tracked the movement of the male gametes from the time of pollen tube initiation to the time of entry into the megagametophyte to affect fertilization. We report for the first time, the presence of a novel type of three-nucleate/three-celled mature megagametophyte consisting of two synergids and an egg cell in D. zeylanica. Therefore, of the two male gametes formed in this plant, one fuses with the egg cell resulting in syngamy, whereas the other male gamete eventually degenerates due to the absence of its partner i.e. single polar nucleus of the central cell that degenerates prior to the entry of the pollen tube into the synergid. The present work not only highlights the highly reduced nature of megagametophyte but also the occurrence of single fertilization resulting in sperm selection in D. zeylanica.     

Einfluß von Textur,Gehalt an organischer Substanz,Dichte und Luftgchalt des Bodens auf die Vermehrung von Heterodera schachtii Schmidt, 1871 an Zuckerrüben

Defense reactions of embryo and megagametophyte (endosperm) of European black pine (Pinus nigra Arn.) were studied by in vitro technique. As a tester, basidiomycete Phaeolus schweinitzii (Fr.) Pat. was used. Both, defense reaction of embryo and very strong defense reaction of megagametophyte were found. Some substances which may be involved in defense reactions are discussed.     

Diploidization in megagametophyte-derived cultures of the gymnosperm Larix decidua

Tested haploid embryogenic lines (n=12) of Larix dedicua Mill, initiated from megagametophyte tissue were maintained on half-strength LM medium without growth regulators. The cultures were analyzed for ploidy level after 1–9 years. All lines tested were found to have doubled (2n=24) their chromosome number at the end of the experiment, though there were a few lines that still gave occasional haploid counts. Flow cytometric data of embryogenic tissue confirmed these results. Protoplasts were stained in ethidium bromide, and cultured human leucocytes and chicken erythrocytes were used as internal standards. Haploid megagametophytes from immature seeds of L. decidua and known diploid culture lines of a related hybrid (L. x eurolepis) were also analyzed by flow cytometry. Haploid reference material had 12.3–13.6 pg DNA per cell, whereas formerly haploid callus lines had an average of 25.0 pg DNA per cell. The one exception was a known, genetically unstable line of L. decidua (34.8 pg DNA per cell). The diploid cell line of L. x eurolepis had 27.6 pg DNA per cell. The results show that spontaneous diploidization of megagametophyte lines is relatively rapid and that both haploid and dihaploid lines are embryogenic in larch.     

Evolution of microsporangium numbers in Microseris (Asteraceae: Lactuceae)

The genus Microseris contains species with disporangiate stamens and species with tetrasporangiate stamens. We determined the number of microsporangia per stamen in serial sections of heads from all 13 species of Microseris, its close relative Uropappus lindleyi, and the two allopolyploid species of Slebbinsoseris. Four Microseris species, three diploid and one tetraploid, have two microsporangia per stamen; all other species investigated have four. The most parsimonious assumption is that the disporangiate condition is derived and arose once in the evolution of Microseris. The inheritance of the number of microsporangia per stamen in crosses between M. bigelovii (disporangiate) and M. douglasii (tetrasporangiate) was determined. Segregation of microsporangium number per stamen in F2s derived from these crosses is quantitative rather than Mendelian. The average number of microsporangia per stamen in the F2 plants ranges from 2.0 to 4.0. There is a predominance of tetrasporangiate stamens in the F1 and in most F2 plants. The observed pattern of inheritance suggests a major gene with dominance and quantitative modifiers.     

Intergeneric pollen – megagametophyte relationships of conifers in vitro

 Germinating pollen from larch (Larix occidentalis), Sitka spruce (Picea sitchensis) and white pine (Pinus monticola) were co-cultured with megagametophytes dissected from cones of other genera (Pseudotsuga menziesii, Larix×eurolepis and Pinus monticola). Pollen was presented to megagametophytes possessing archegonia which were either alive, degenerating or dead. In addition, pollen was presented to fertilized megagametophytes and to megagametophytes that had been cut in half. Megagametophyte penetration by pollen tubes and male gamete release into archegonia were verified by serial sections of glycomethacrylate-embedded specimens. Pollen tubes penetrated through any part of the apex of the megagametophyte. Division of the body cell into the two gametes was regularly observed. Delivery of gametes was confirmed between spruce and larch. Pollen tubes also penetrated fertilized megagametophytes, dead or degenerating archegonia as well as wounded and/or cut surfaces. This demonstrates the inability of the male gametophyte to optimize its mating efforts, since it is unable to differentiate between healthy and unhealthy archegonia. The megagametophyte cells are unable to optimize male selection. They may produce secretions of a generally attractive nature, as pollen is attracted to the apex of the megagametophyte, but archegonia themselves do not produce pollen-specific signals of either a promotive or inhibitory nature. These results open new avenues for the development of novel breeding strategies where natural breeding barriers may be bypassed. Received: 19 March 1998 / Accepted: 29 April 1998     

EXTRAFLORAL NECTARIES IN EBENACEAE: ANATOMY,MORPHOLOGY, AND DISTRIBUTION

Previous superficial reports, but only one anatomical study of one species, have supported the view that extrafloral nectaries (EFNs) of the unusual Benincasa type (i.e., with several layers of small nectariferous cells) of “Flachnektarien” (surface nectary) are common in Ebenaceae. An anatomical survey was made of 107 species (24% of the 450 species) of the four genera commonly accepted (Diospyros—73, Euclea—13, Maba— 12, Royena—9). There are 1–15 abaxial EFNs per leaf (two species each of Euclea and Royena had none). They were all of the Benincasa type, subtended by an irregularly 1–2-layered sheath of cells with extremely thick radial walls. The pattern of minor vein association allowed recognition of two subtypes: apovascular (two or more subglandular layers between vein and sheath) and paravascular (one subglandular separating layer). Apovascular EFNs are smaller but more numerous per leaf, paravascular EFNs are larger but fewer per leaf. The apovascular subtype is most common in North America and Asia; a mixture of both subtypes occurs elsewhere; Euclea has only the latter subtype. Both subtypes have the most extensive vascular association yet described for Flachnektarien. They appear to have become specialized early in the family's evolution, before extensive migration occurred.     

Western white pine (Pinus monticola Dougl.) reproduction: I. Gametophyte development

Male and female gametophyte development are described from light and transmission electron microscope preparations of ovules from first and second year Pinus monticola Dougl. seed cones. In the first year of development, pollen tubes penetrate about one-third the distance through the nucellus. The generative cell and tube nucleus move into the pollen tube. The megagametophyte undergoes early free nuclear division. First-year seed cones and pollen tubes become dormant in mid-July. In the second year, seed cones and pollen tubes resume development in April and the pollen tubes grow to the megagametophyte by mid-June. Early in June the generative cell undergoes mitosis, forming two equal-size sperm nuclei that remain within the generative cell cytoplasm. The generative cell has many extensions and abundant mitochondria and plastids. The megagametophyte resumes free nuclear division, then cell wall formation begins in early July. Cell wall formation and megagametophyte development follow the pattern found in other Pinaceae. Three to five archegonial initials form. The primary neck cell divides, forming one tier of neck cells. Jacket cells differentiate around each central cell. The central cell enlarges and becomes vacuolate; then vacuoles decrease in size and the cell divides, forming a small ventral canal cell and a large egg. Plastids in the central cell engulf large amounts of cytoplasm and enlarge. This process continues in the egg, and the peripheral cytoplasm of the egg becomes filled with transformed plastids. Mitochondria migrate around the nucleus, forming a perinuclear zone. The wide area of egg cytoplasm between these two zones has few organelles. A modified terminology for cells involved in microgametophyte development is recommended. Received: 9 December 1999 / Revision accepted: 30 April 2000     

Investigation on cell death in the megagametophyte of Araucaria bidwillii Hook. post-germinated seeds

The megagametophyte of the Araucaria bidwillii seed is a storage tissue that surrounds and feeds the embryo. When all its reserves are mobilized, the megagametophyte degenerates as a no longer needed tissue. In this work we present a biochemical and a cytological characterization of the megagametophyte cell death. The TUNEL assay showed progressive DNA fragmentation throughout the post-germinative stages, while DNA electrophoretic analysis highlighted a smear as the predominant pattern of DNA degradation and internucleosomal DNA cleavage only for a minority of cells at late post-germinative stages. Cytological investigations at these stages detected profound changes in the size and morphology of the megagametophyte nuclei. By using in vitro assays, we were able to show a substantial increase in proteolytic activities, including caspase-like protease activities during the megagametophyte degeneration. Among the caspase-like enzymes, caspase 6- and 1-like proteases appeared to be the most active in the megagametophyte with a preference for acidic pH. On the basis of our results, we propose that the major pathway of cell death in the Araucaria bidwillii megagametophyte is necrosis; however, we do not exclude that some cells undergo developmental programmed cell death.     

MORPHOLOGICAL STUDIES IN ALETRIS. I. DEVELOPMENT OF THE OVULE,MEGASPORES AND MEGAGAMETOPHYTE OF A. AUREA AND THEIR CONNECTION WITH THE SYSTEMATICS OF THE GENUS

Browne , Edward T., Jr . (U. of Kentucky, Lexington.) Morphological studies in Aletris. I. Development of the ovule, megaspores and megagametophyte of A. aurea and their connection with the systematics of the genus. Amer. Jour. Bot. 48(2): 143–147. Illus. 1961.—Development in a North American species of this variously classified genus has shown great similarity with the development in several genera of Hutchinson's Liliaceae-Narthecieae: Pleea, Tofieldia, Nanhecium and ∗∗∗Metaparthecium. The ovules are anatropous, bitegmic, crassinucellate and arranged in 4 rows in each locule of the tricarpellate pistil. There is a hypostase and an obturator. The primary archesporial cell is hypodermal. This undergoes a division to form a wall cell and the megaspore parent cell (MPC). The megaspores usually have a linear arrangement although occasionally a T-shaped tetrad may be formed. Most frequently the chalazal megaspore functions, but rarely the one adjacent to it may enlarge instead. Megagametophyte development is of the Polygonum type. A characteristic narrowed chalazal constriction is formed during the development of the megagametophyte. It is recommended on the basis of this information that Aletris be classified with the genera of the Liliaceae-Narthecieae.     

Genetic analysis of cinnamyl alcohol dehydrogenase in loblolly pine: single gene inheritance, molecular characterization and evolution

The gene encoding the monolignol biosynthetic enzyme cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) can be expressed in response to different developmental and environmental cues. Control of Cad gene expression could involve either differential regulation of more than one Cad gene or, alternatively combinatorial regulation of a single Cad gene. In loblolly pine (Pinus taeda L.), we found several electrophoretic variants (allozymes) of CAD and a high level of heterozygosity (h_e=0.46). Analysis of inheritance patterns of pine CAD allozymes gave segregation ratios that were consistent with Mendelian expectations for a single functional gene. The identity of the full-length Cad cDNA sequence was confirmed by alignment with peptide sequences obtained from purified active enzyme and by extensive similarity to Cad sequences from other species. Southern blot analysis of genomic DNA using the Cad cDNA as a hybridization probe gave simple patterns, consistent with our interpretation that pine Cad is a single-copy gene. Phylogenetic analysis and evolution rate estimates showed that Cad sequences are diverging less rapidly in the gymnosperms than in the angiosperms. The Cad mRNA was present in both lignifying tissues and a non lignifying tissue (the megagametophyte) of pine. The presence of a single gene suggests that different regulatory mechanisms for a single Cad gene, rather than differential regulation of several genes, can account for its expression in response to different cues.