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     

Multinucleate storage cells in Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) and the effect of seed parasitism by the chalcid Megastigmus spermotrophus Wachtl

Megagametophytes of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) accumulated storage products following fertilization. As megagametophytes matured, the number of nuclei per cell rose, resulting in syncytial storage cells. Studies carried out on trees in France and Canada confirmed that such previously unreported, free nuclear cells were a normal part of late megagametophyte development. Unfertilized megagametophytes showed that some binucleate cells before degeneration resulted in empty seed. Insect parasitism prevented megagametophyte abortion in unfertilized ovules. Oviposition by a torymid chalcid wasp (Megastigmus spermotrophus Wachtl) early in megagametophyte development resulted in normal megagametophyte development. Around the time of plant egg maturation, binucleate and trinucleate cells were observed. As megagametophytes matured, multinucleate mature storage cells rich in proteins, lipids and starch were formed. The insect was able to induce identical nuclear behaviour in infested, unfertilized megagametophytes, as that of uninfested, fertilized megagametophytes.     

FINE STRUCTURAL DEVELOPMENT OF THE MEGAGAMETOPHYTE OF ZEA MAYS FOLLOWING FERTILIZATION

The changes in cytoplasmic fine structure during the interval between pollination and zygotic division in the megagametophyte of Zea mays are reported. The rate of membrane synthesis appears to be low in the mature unfertilized megagametophyte. This megagametophyte is suggested to be in a state of relative metabolic inhibition. The rate of membrane synthesis is high in the zygote and free nuclear endosperm. After fertilization the swelling of the mitochondria in all cytoplasms is interpreted to reflect a presumed increase in metabolic rate relative to growth. In the zygote and endosperm, endoplasmic reticulum (ER) production is associated with the nuclear envelope. In all of the material the ribosomes not associated with the ER are helical polysomes which increase in length after fertilization. On the basis of permanganate staining, the ribonucleoprotein of the endosperm is suggested to be different from that of the other cytoplasms. The specific plastid form of each cell type does not change during the first few hours after fertilization. Dictyosomes are most numerous and active in the antipodals. They are least numerous in the egg and zygote. The smallest dictyosome vesicles are in the synergids. Dictyosome number and vesicle size increase in the egg and central cell after fertilization. Similarities among the megagametophytes of several angiosperms are enumerated.     

Megagametophytes of Douglas fir (Pseudotsuga menziesii) and hybrid larch (Larix × eurolepis) in culture: Multiplication of neck cells and the formation of binucleate cells

Summary To study the effect of culturing on megagametophytes of Douglas fir (Pseudotsuga menziesii) and hybrid larch (Larix × eurolepis), cones were collected at the time of fertilization and the megagametophytes were removed, then placed on medium. We used a modified Murashige and Skoog medium supplemented with 5% lactose and 10% polyethylene glycol 4000. A variety of cell types proliferated including prothallial, neck, and jacket cells. Some of these multiplying cells showed a binucleate condition. The prothallial cells of the apex divided and expanded. The neck cells formed clusters composed of more cells than normally found in situ; though otherwise they showed ultrastructural similarity to neck cells in situ. These neck cells had large numbers of active Golgi complexes, numerous large and small vacuoles, coated vesicles, smooth vesicles, a well-developed endoplasmic reticulum, and thickened cell walls. These are the first reports of neck cell multiplication and induction of a binucleate state for gymnosperm megagametophyte cells in vitro.     

FORMATION OF 2N MEGAGAMETOPHYTES IN DIPLOID TUBER-BEARING SOLANUMS

Development of megaspores and megagametophytes was analyzed for several diploid potato clones (Solanum spp.) that exhibit either high (HI) or low (LO) seed set when crossed as female with the tetraploid cultivated potato S. tuberosum Group Tuberosa. The objectives were to determine the relationship between ploidy and diam of nuclei and nucleoli, and to determine the mechanism(s) and frequencies of 2n megagametophyte formation. Sizes of nuclei and nucleoli were found to depend on ploidy. For HI clones, the distributions of sizes indicated that doubling occurred during meiosis, and that 30 to 50% of the megaspores and megagametophytes were 2n rather than haploid. Omission of the second meiotic division led to formation of second division restitution (SDR) 2n megagametophytes. Only one HI clone had abnormal meiosis I, in addition to omission of meiosis II in some meiocytes; this clone seemed to produce not only 1n and 2n, but also 4n megagametophytes. The results indicated that high crossability of the HI clones as female with tetraploids largely was due to formation of SDR 2n megagametophytes, a finding strongly supporting the hypothesis that sexual polyploidization is the driving force behind polyploidization of Solanums. The results contribute to increasing evidence that meiotic mutants and abnormalities play an important role in angiosperm evolution.     

中华水韭雌配子体及胚胎发育的初步研究

中华水韭（Isoetes sinensis Palmer）属水韭科（Isoetaceae）草本拟蕨类,是长江中下游特有的古老物种,对环境污染敏感,为国家一级重点保护的极度濒危的野生植物。本研究通过人工培养,按不同发育阶段,用常规石蜡切片法连续观察了雌配子体及胚胎发育的全过程。结果显示：大孢子培养15～20 d萌发,雌配子体为球状、属壁内发育型,每个雌配子体着生8～20个颈卵器。成熟的颈卵器发育有多种可能,但只有颈沟细胞和腹沟细胞都退化,卵才有受精的机会。合子首先进行横向分裂,培养约50 d发育成数十个细胞的球形胚胎,约80 d雌配子体的细胞萎缩,第一叶及其叶舌、第一根结构更明显,出现第二叶。文中还初步探讨了中华水韭濒危的生殖生物学机制。     

A NEW CLEARING-SQUASH TECHNIQUE FOR THE STUDY OF OVULE DEVELOPMENT IN ANGIOSPERMS

The simple, efficient method described here for the study of ovule and megagametophyte development in angiosperms provides for the extension of investigation beyond the limits imposed by the traditional but arduous section technique. Excised pistils previously fixed in FPA₅₀ and stored in 70 % ethanol are placed in a clearing fluid composed of lactic acid (85 %), chloral hydrate, phenol, clove oil, and xylene (2:2:2:2:1, by weight). After 24 hr, ovules dissected from the ovularies are transferred with some of the fluid to a slide, covered so that the cover glass is supported laterally by two permanently affixed covers, and examined with phase contrast optics. The unique action of the clearing fluid permits the study of cellular structure with the phase oil objective focused at any focal plane within the ovule. Downward focusing thus reveals a series of optical sections in the sagittal, frontal, or transverse plane depending on the orientation of the ovule. Orientation can be altered by a slight shifting of the cover glass on the lateral support mounts. The ovules become quite fragile in the clearing fluid. Pressure applied to the cover glass gradually breaks the ovule apart without disrupting the structural integrity of individual cells. This squash procedure provides for extending observations to cytological features of megasporocytes, megaspores, and megagametophytes previously identified in intact ovules. The new method is applied here to the study of ovule development in two unrelated species, Cassia abbreviata Oliver var. granitica Bak. f. (Leguminosae) and Ludwigia uruguayensis (Camb.) Hara. (Onagraceae). For best results, the ovules of Ludwigia must be pretreated in lactic acid (85 %) for 24 hr prior to application of the clearing fluid. Other methods for pretreatment likely will be required as the technique is applied to a wider range of flowering plant species.     

Protein Bodies in the Pine Megagametophyte in vitro Culture: Ultrastructural, Histochemical and Electrophoretic Observations

The megagametophytes of the European black pine (Pinus nigra Arn.) were cultured on modified MS medium. After 10 d, protein bodies showed well-marked degradation on freeze-etched replicas and in preparations observed by scanning electron microscopy. After 20 d of cultivation, the megagametophyte cells were completely empty. Proteins secreted into the agar medium were determined by electrophoresis and 15 different proteins, in the range of 6.5 to 71 kDa, were identified.     

Cytoembryological Studies on Polyembryonic Line SB-1 of Oryza sativa: Polyembryong and Its Origin

Anatomical observations, genetic and cytoembryological studies were conducted to determine the cytological factors in relation to the production of diploid polyembryony within one embryo sac occurring in polyembryonic line SB-1 of O. sativa L. The results showed that the diploid polyembryony in SB-1 was not originated from the fertilization of synergids, but presumably from the megagametophytes with supernumerary egg cells. Potentially polyembryonic megagametophytes at maturity contained either a conventional 3-celled egg apparatus (92.60% of megagametophytes) or the supernumerary eggs forming either a 4-celled (5.2 %) or 5-celled (2.2 %) egg apparatus. In megagametophyte with one additional supernumerary egg cells were almost situated side by side in adjacent to the synergids and recessed from the micropyle, whereas in megagametophyte with two additional supernumerary eggs, one egg cell occupied the conventional possition and the other two lay above the synergids. The arrangement of embryos in polyembryonic caryopsis at various developmental stages was concordant to that of egg cells in megagametophytes with supernumerary eggs. Karyotype analysis showed that most of the twins and triplets were of 2n--2n and 2n-2n-2n respectively. Polyembryony was observed in Fo and F1 caryopsis of cross SB-1 × Malaihong (Malaihong is of non-polyembryony) and in Fl caryopsis of its reciprocal but not in Fo caryopsis. This implies that polyembryony occurring in SB-1 is of a dominant character of embryo sac. Anatomical observations proved that each seedling from twins or triplets was completely independent with no vascular bundle connected with each other. The causes of polyembryony within one embryo sac, the entry of supernumerary sperms and the origin of supernumerary egg cells are discussed.     

EMBRYOGENY AND REPRODUCTIVE BIOLOGY OF BOTHRODENDROSTROBUS MUNDUS (LYCOPSIDA)

Permineralized lycopsid megagametophytes and embryos from Upper Carboniferous strata (Westphalian A) at Burnley, England have been found within isolated megaspores assignable to Setosisporites. The specimens illustrate for the first time the reproductive biology and embryogeny of a free-sporing, bisporangiate, Paleozoic lycopod, and permit reinterpretation of the megagametophyte of Bothrodendrostrobus. Megagametophyte development is entirely endosporal. Embryogeny is comparable to that of the extant genus Isoetes, and is fundamentally different from that of the fossil lycopsid, Lepidocarpon—Lepidophloios. This further illustrates the diversity among Paleozoic lycopods and helps to clarify relationships among both fossil and extant lycopsid taxa.     

Parasitism of seed of Douglas fir (<Emphasis Type="Italic">Pseudotsuga menziesii</Emphasis>) by the seed chalcid, <Emphasis Type="Italic">Megastigmus spermotrophus</Emphasis>, and its influence on seed hormone physiology

Parasitism of Douglas fir (Pseudotsuga menziesii) megagametophytes by the seed chalcid, Megastigmus spermotrophus Wachtl, occurs naturally after pollination but before fertilization. In the absence of fertilization, the presence of insect larvae within the megagametophyte prevents abortion and the storage tissue continues to develop as if the seed had been fertilized. We investigated the effect of parasitism on the metabolism of abscisic acid (ABA), auxins, cytokinins, and gibberellins during early development of Douglas fir seeds. Hormones and hormone metabolites of infested and uninfested megagametophytes with or without pollination were analyzed by HPLC–ESI/MS/MS. At 1 week after Megastigmus introduction, the insect’s presence stimulated ABA accumulation in unpollinated megagametophytes compared to unpollinated, unparasitized megagametophytes. In pollinated material, parasitism did not stimulate ABA accumulation compared to levels present in unparasitized megagametophytes. In all four treatments, the metabolism of ABA occurred primarily through conjugation to the ABA glucose ester (ABAGE), while the 7′-, 8′- and, 9′-hydroxylation pathways were only minor. ABAGE levels declined with time in all treatments and this occurred to a greater extent in pollinated, parasitized megagametophytes, suggesting that the insect’s presence induced the dramatic decrease in ABAGE. Although there were temporal variations in the auxin, cytokinin, and gibberellin profiles of parasitized megagametophytes, the profiles were generally similar to those of unparasitized megagametophytes. Our results suggest that failure of parasitized megagametophytes to abort may be due to the insect inducing similar hormone profiles to those present during normal development of Douglas fir seed.     

THE ONTOGENY OF CARBONIFEROUS ARTICULATES: THE APEX OF SPHENOPHYLLUM

Twig apices of Sphenophyllum lescurianum, S. constrictum, and two new Sphenophyllum taxa are described in transverse and longitudinal section from middle and upper Pennsylvanian age specimens. In all of the species the single apical cell has the shape of a tetrahedron, with a triangular upper surface and three internal cutting faces. Segment cells are produced from each of the cutting surfaces in a dextrorse or sinistrorse direction, depending upon the species. The central portion of each segment cell contributes to the initiation of the procambium, while the remaining outer portion undergoes a vertical and subsequent horizontal division to form segment cells. Segment cells are aligned in vertical tiers beneath the respective apical cell cutting faces, with the individual leaves positioned directly beneath a tier of segment cells. Leaf primordia are first observed as a series of surface undulations below the apex, with an intercalary meristem located directly beneath each primordium. The vegetative apical organization of Sphenophyllum is demonstrated to be very similar to the type of organization found at the stem tips of Catamites and Equisetum.     

Characterization of the developmental stages of cypress zygotic embryos by two-dimensional electrophoresis and by cytochemistry

Biochemical changes that characterize megagametophyte and zygotic embryo development in the conifer Cupressus sempervirens L. (Cupressaceae) were studied by complementary methods of cytochemistry and two-dimensional electrophoresis (2-DE). These analyses revealed that early in their development megagametophytes and embryos were characterized by the predominant elaboration of starch in association with a low protein content. As their development proceeded, starch content gradually decreased while protein body synthesis progressively intensified, both in the megagametophyte and the embryo. In parallel, an increase in protein level as well as an accumulation of specific polypeptides could be observed in the two tissues. During maturation, protein bodies accumulated to high levels both in megagametophyte and embryo cells, whereas starch could no longer be detected. Protein levels were high in mature seeds and reached 12% and 8% of the megagametophyte and embryo DW, respectively. Some sets of polypeptides accumulated more specifically at this time in both megagametophyte and embryo. Some of these began to first accumulate in the megagametophyte during embryo development before their concentration rose in the embryo at cotyledonary stage. Others displayed a more specific-embryo accumulation pattern.     

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.     

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.     

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 SEED OF SOLANUM PHUREJA

Dnyansagab , Vishnu R., and Delmer C. Cooper . (U. Wisconsin, Madison.) Development of the seed of Solanum phureja. Amer. Jour. Bot. 47(3) : 176—186. Illus. 1960.—Ontogeny of the seed of Solanum phureja Juz. et Buk. is described. The megagametophyte, during the course of its development, ruptures the nucellus and at maturity lies in direct contact with the inner layer (endothelium) of the single massive integument. The mature megagametophyte, a 7-celled structure, consists of a 3-celled egg apparatus, an endosperm mother cell with fused polar nuclei and 3 persistent antipodals. Both 2- and 3-celled mature pollen grains are formed within anthers of the same flower; hence this character cannot be considered of any taxonomic value. Double fertilization occurs between 24 and 72 hr. after pollination. A cellular endosperm is formed, the peripheral layer acting as an absorbing tissue during the early ontogeny of the seed. Later this layer becomes organized as an aleurone layer and thereafter the source of nutrients is via the basal portion of the endosperm immediately adjacent to the apical end of the vascular tissue of the developing seed. Embryo development follows the Nicotiana variation of the Solanad type. The mature testa is composed of an outer layer of thick-walled epidermal cells, an inner layer of thin-walled cells and an intervening mass of disorganized tissue. In those instances where the ovule or young seed aborts, the endothelial cells of the integument become hyperactive and proliferate to such an extent that the space formerly occupied by the gametophyte or the developing endosperm and embryo becomes completely filled with endothelial tissue.