SEED MORPHOLOGY OF MONOTROPA UNIFLORA L. (ERICACEAE)

Mature seeds of Monotropa uniflora L., an achlorophyllous mycotrophic perennial, underwent imbibition and were processed for study using modern histological and histochemical techniques. The seeds ranged from 0.6-0.8 mm in length and 0.12-0.15 mm in width and exhibited integumentary winglike structures at either end. As in other members of the Ericaceae, seeds are unitegmic, tenuinucellate, albuminous, and form both micropylar and chalazal endosperm haustoria. A two-celled reduced embryo was observed in all seeds, except one where the embryo was three-celled. Protein granules in the thick walled endosperm were found to contain an aniline blue-fluorescent material that may be a calloselike carbohydrate. The limited amount of seed food reserves and the retardation of embryo differentiation may reflect specialized germination requirements.     

ADVENTIVE EMBRYOGENESIS IN CITRUS AND ITS RELATION TO POLLINATION AND FERTILIZATION

Cytological and histological studies of seeds from three facultative apomictic Citrus cultivars show that adventive embryos develop, as a rule, from the first few cell layers of the nucellus adjacent to the embryo sac in the micropylar half and occasionally from the chalazal end. The adventive embryos initiated in nucellar tissue away from the embryo sac and most of those initiated from the chalazal end of the nucellus do not develop beyond the one-celled stage. When two or more embryos are developing in the same seed, the successful development of a given embryo depends on its location in relation to access to nutrients from the endosperm. The presence of a zygote and triploid endosperm in seeds with adventive embryos, the abortion of seed when endosperm degenerates, and the lack of seed set without pollination indicate that pollination and fertilization are essential for in vivo adventive embryogenesis.     

Cell-to-cell movement of green fluorescent protein reveals post-phloem transport in the outer integument and identifies symplastic domains in Arabidopsis seeds and embryos

Developing Arabidopsis (Arabidopsis thaliana) seeds and embryos represent a complex set of cell layers and tissues that mediate the transport and partitioning of carbohydrates, amino acids, hormones, and signaling molecules from the terminal end of the funicular phloem to and between these seed tissues and eventually to the growing embryo. This article provides a detailed analysis of the symplastic domains and the cell-to-cell connectivity from the end of the funiculus to the embryo, and within the embryo during its maturation. The cell-to-cell movement of the green fluorescent protein or of mobile and nonmobile green fluorescent protein fusions was monitored in seeds and embryos of plants expressing the corresponding cDNAs under the control of various promoters (SUC2, SUC3, TT12, and GL2) shown to be active in defined seed or embryo cell layers (SUC3, TT12, and GL2) or only outside the developing Arabidopsis seed (AtSUC2). Cell-to-cell movement was also analyzed with the low-molecular-weight fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonate. The analyses presented identify a phloem-unloading domain at the end of the funicular phloem, characterize the entire outer integument as a symplastic extension of the phloem, and describe the inner integument and the globular stage embryo plus the suspensor as symplastic domains. The results also show that, at the time of hypophysis specification, the symplastic connectivity between suspensor and embryo is reduced or interrupted and that the embryo develops from a single symplast (globular and heart stage) to a mature embryo with new symplastic domains.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.     

小叶兜兰的果实生长和雌配子体发育

为了解濒危兰科植物小叶兜兰(Paphiopedilum barbigerum Tang et Wang)胚珠和雌配子体的发育过程,采用常规石蜡切片技术对其果实的生长动态进行了研究。结果表明,授粉后60~75 d的蒴果内种子数量迅速增加,到授粉后120 d时种子充满整个蒴果。授粉后40 d的胎座上分化形成多数由1层表皮细胞包被1列细胞的胚珠原基;授粉后60 d时位于胎座指状结构末端处紧靠表皮细胞下方的孢原细胞分化为大孢子母细胞。之后,大孢子母细胞经过减数分裂和有丝分裂最终形成成熟胚囊;授粉后135 d胚囊发育成熟,附着在胎座上的种子个体分化明显。小叶兜兰胚囊的发育类型为双孢子葱型,胚珠为倒生胚珠,薄珠心,单珠被,成熟胚囊为8核。这为小叶兜兰的生殖生物学及繁殖体系的建立提供理论依据。     

Hormonal Complex and Ultrastructure of Maturing Aesculus hippocastanum Seeds

The content and temporal changes in the endogenous IAA, cytokinins, gibberellin-like compounds (GLC), and ABA were determined during horse chestnut (Aesculus hippocastanum L.) seed development (the stages of embryo axis development, its active growth, and storage compound deposition). The active growth of the embryo was characterized by the highest amounts of free phytohormones. Later, by the end of seed maturation, we observed the accumulation of the bound forms of IAA and ABA and a trend to a decrease in the content of free IAA, zeatin, and GLC (butanol fraction). The electron-microscopic examination of the embryo from the mature seed demonstrated that some structural components of the cytoplasm were similar in the cells of embryo axes and cotyledons. During the entire period of maturation, the embryo cells preserved native vacuoles and protein bodies were not formed. Thus, the structure of cotyledonary and axial cells and the distribution of free and bound phytohormones in the horse-chestnut seeds are similar to those in maturing seeds characterized by exogenous dormancy.     

RECIPROCAL DIFFERENCES IN INTRASPECIFIC CROSSES OF TOBACCO RESULT FROM EMBRYO DEATH

Tetraploid tobacco plants (Nicotiana tabacum) derived from cultured cell lines (TC) are partially cross-incompatible with their diploid progenitors (C). C × TC crosses (TC-derived tetraploids as the pollen parent) yield only 2% viable seed. The remaining seeds are normal size but lack an embryo (apoembryonic seeds). Apoembryonic seeds do not occur in the reciprocal TC × C crosses. Sections of ovules from C × TC crosses revealed that an embryo formed but that embryo growth slowed at 5 days postpollination and that by the 12th day following pollination, the embryo had disappeared although the ovule continued to develop. Endosperm degeneration occurred concurrently with embryo death. Culturing ovules from C × TC crosses has increased the yield of F₁ plants from 2% to as much as 25% indicating that embryo rescue is possible. Surviving F₁ plants from TC × C crosses have close to triploid chromosome numbers and are fertile.     

Changes in indole-3-acetic acid levels during tomato (Lycopersicon esculentum Mill.) seed development

Summary The changes in the level of indole-3-acetic acid (IAA) were investigated in seeds and fruit tissues-placenta and mesocarp-during tomato (Lycopersicon esculentum Mill.) zygotic embryogenesis, which was characterized through eight morphological embryo stages [from globular (stage 1) to mature embryo (stage 8)]. In whole seeds, IAA levels increased mainly at stage 3 (young torpedo) and at stage 5 (late torpedo stage). As the seed matured and dehydrated, IAA levels decreased and showed a new distribution pattern within seed structures, preferentially in endosperm tissue. IAA contents in fruit tissues were lower but followed the same pattern as those of seeds. These data support the hypothesis of IAA biosynthesis in seeds with a transient role of the endosperm at the end of embryo development and suggest a role of IAA in fruit and seed growth. Moreover a comparison of IAA and ABA changes suggests that IAA could be especially necessary for the beginning of embryo growth, whereas ABA could act mainly at the end of the growth phase.Abbreviations ABA abscisic acid - ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) - BHT butylhydroxytoluene - DW dry weight - ELISA enzyme linked immunosorbent assay - HPLC high performance liquid chromatography - IAA indole-3-acetic acid. PGRs: plant growth regulators     

The Development and Structure of the Late Embryo in Torreya grandis

The materials used in this investigation were collected during 1980–1983 from Zhuji county of Zhejiang province, China. Seed of Torrcya grandis is an important dry “fruit” and used for edible oil. It is endemic to China. The primordia of male strobili are differentiated before October in the first year, while those of female strobili occur later. The microspore mother cells and megaspore mother cells are found in March and April in the second year respectively. The fertilization takes place in August and the dormant embryo overwinters at the proembryo stage. Eventually the proembryo begins to differentiate and its development starts in July of the third year. Thus the interval from fertilization to latembryogeny of Torreya grandis lasts for about 11 months. When the seeds of Torreya grandis are shed 'in August the embryo within the seed is still immature. It requires a period of after-ripening. The experiments show that the embryo resumes to develop and differentiate during 1–3 months in stratification in moist sands. The development and structure of late embryo are characterized as follows: 1. The cotyledon of the mature embryo in Torreya grandis is of 15000 μm in length and 87% of the embryo. The hypocotyl is vary shert and only 13% of the embryo. This kind of structure of the embryo in Torreya is very rare among conifers and in some degree similar to that of Keteleeria. When seed is shed the meristem of cotyledon is just differentiated and only 100–200 μm in length at the end of July to the middle of August. As the seeds are stratified in moist sands for 1–3 months, the cotyledon increases about 100 times than in room temperature in Zhuji county. 2. There is a large secretory canal in either side between the procambium and the cortex of the mature embryo. The secretory canal consists of epithelial cells of 4–5 layers. It is very peculiar in conifers. 3. The shoot apex does not begin to differentiate, until the seed has been fallen from the tree. 4. The column of the root cap is rather short and consists of the cells of about 10 layers in height and 6 layers in width. 5. Proteins are only found in the focal zone of the free apex of the young embryo but without any starch grains. The starch is abundantly distributed in the opposite end from the root initials down to root cap and the entire transitional zone. It is interesting to note that neither proteins nor starch grains are found in the suspensor system. It is assumed that the protein may be the main form of storing material in the actively growing cells and tissues of embryo in Torreya grandis.     

ADVENTIVE EMBRYOGENESIS IN CITRUS (RUTACEAE) II. POSTFERTILIZATION DEVELOPMENT

Cytological and histological studies on postfertilization development of ovules were carried out in six facultatively apomictic Citrus cultivars. At the time of anthesis, adventive embryo initial cells (AEICs) were detected mainly in the cell layers of the nucellus around the chalazal half of the embryo sac. During the approximately 40 days rest period of the AEICs after fertilization, rapid cell division and enlargement in the endosperm and the chalazal half of the nucellus resulted in the split of AEICs into several separated areas forming the micropylar, lateral and chalazal islands surrounding the enlarging embryo sac. Both in diploid seeds with triploid endosperm and triploid seeds with pentaploid endosperm, the AEICs located in the micropylar half successfully developed into adventive embryos. In diploid seeds, almost all AEICs located in the chalazal half did not develop beyond the initial-celled stage, while in the triploid seeds, those located in the chalazal half occasionally developed into cotyledonary embryos. In seeds with aborted endosperm, the AEICs located in the chalazal half often developed into cotyledonary embryos. The chalazal expiants from normal seeds produced a large number of embryos in vitro. Four results can be summarized from these studies on adventive embryogenesis as follows: 1) All AEICs are initiated prior to anthesis. 2) Whether or not the AEICs successfully developed into adventive embryos is dependent upon their position in the seed. 3) The farther the AEICs are located from the micropylar end, the more adventive embryogenesis is suppressed by endosperm. 4) The degree of adventive embryogenesis in the chalazal half is affected by time and extent of malfunction of the endosperm. Under natural conditions, these regulatory systems of adventive embryogenesis contribute to high production of zygotic seedlings in apomictic Citrus species and cultivars.     

水稻PDER品系的特点和二倍体孢子生殖胚囊的形成和发育

对水稻（ＯｒｙｚａｓａｔｉｖａＬ．）早发生胚ＰＤＥＲ（ｐｒｅ－ｄｅｖｅｌｏｐｅｄｅｍｂｒｙｏｏｆｒｉｃｅ）品系的特点和细胞胚胎学研究表明,ＰＤＥＲ是二倍体植物２ｎ＝２４,约有５０％胚囊的卵细胞未经受精能自行发育形成胚,成熟种子的萌发和生长速度较常规正常水稻快。ＰＤＥＲ的大孢子母细胞经有丝分裂产生未减数的胚囊,即无融合生殖中的二倍体孢子生殖类型。在胚囊形成和发育过程中有如下几个特点：（１）孢原细胞至大孢子母细胞分裂前的过渡期持续时间较长,孢原细胞和大孢子母细胞的细胞质比周围的珠心细胞质稀淡。（２）大孢子母细胞经二次有丝分裂后形成直线排列的三个细胞（三分体）,珠孔端的两个解体,合点端的一个发育为功能细胞,有少数胚囊的三个细胞全部解体形成败育胚囊。（３）功能细胞经三次连续核分裂形成具八核七个细胞的成熟胚囊,它的结构与常规正常水稻基本相同,但助细胞呈长形而没有回抱着卵细胞。     

(1→4)-α-d-glucan phosphorylase [(1→4)-α-d-glucan:orthophosphate glucosyltransferase] isoenzymes from sweet corn seed embryo

The presence of multiple forms of phosphorylase [(1→4)-α-d-glucan:orthophosphate glucosyltransferase] in sugary maize seeds was demonstrated by polyacrylamide-gel-disc electrophoresis. The patterns of phosphorylase isoenzymes from immature and from germinating seeds were different. Most of the isoenzymes from embryo of germinated seeds precipitated at an ammonium sulfate concentration above 45% of saturation. The most cathodic band of the zymograms appeared on the third day of germination, then disappeared. This form of phosphorylase occurred only in the embryo of germinating seeds and it was absent both in the immature embryo and in the endosperm at any stage of development. The slow-moving embryo isoenzyme was purified through chromatography on DEAE-cellulose. Its kinetic properties and enlargement mechanism were studied.     

Changes in abscisic acid levels in embryo axes of Norway maple and sycamore seeds during maturation and dehydration

Changes in the abscisic acid (ABA) levels in embryo axes of seeds, belonging to the orthodox (Norway maple — Acer platanoides L.) and recalcitrant (sycamore — Acer pseudoplatanus L.) categories, were investigated throughout maturation using an ELISA (enzyme-linked immunosorbent assay) test. Concentration of ABA in embryo axes substantially differed depending on species and sampling date. ABA was always higher in Norway maple except at the end of seed maturation when ABA content was similar in both species. During maturation ABA decreased in both species but the decline was more marked in Norway maple than in sycamore (11 vs. 3 fold). These species also differed in the pattern of ABA changes, which in sycamore embryo axes was very regular, while in Norway maple a sharp decrease was recorded after acquisition by the seeds of tolerance to desiccation. Dehydration of embryo axes of Norway maple caused a further significant decrease of ABA level. In contrast, in dehydrated sycamore embryo axes ABA content did not decrease, but slightly increased. The role of ABA in desiccation tolerance and dormancy of Norway maple and sycamore seeds is discussed.     

Unfertilized ovules of Epilobium obcordatum (Onagraceae) continue to grow in developing fruits

To determine whether unfertilized ovules continue to grow when in an ovary containing fertilized ovules, we measured ovule lengths in developing fruits of Epilobium obcordatum that were harvested 4, 5, 8, and 10 d post pollination. We found that unfertilized ovules that were in the presence of fertilized ovules continued to grow and that there was a broad range of overlap in their sizes at all sampling times. This effect was found for two types of unfertilized ovules that occur throughout the length of the ovary: normal, unfertilized ovules, apparently bypassed by pollen tubes; and sterile ovules lacking an embryo sac. In addition, there is a position effect within developing fruits. Both fertilized and unfertilized ovules are larger at the stylar end. In six samples resulting from pollination with a single pollen tetrad, a total of 18 embryos were found, and the effect on unfertilized ovules, greatest at the stylar end, diminished with distance from the ovules with embryos. Our results are consistent with the interpretation that diffusible hormones produced by developing seeds cause nearby unfertilized ovules to grow. We conclude that caution is necessary when attempting to infer ovule fertilization histories from the appearances of ovules in developing and mature fruits. What are often inferred to be aborted seeds, in many cases, may not be seeds at all. They may be enlarged, unfertilized ovules.     

Endosperm-limited Brassicaceae seed germination: abscisic acid inhibits embryo-induced endosperm weakening of Lepidium sativum (cress) and endosperm rupture of cress and Arabidopsis thaliana

The endosperm is a barrier for radicle protrusion of many angiosperm seeds. Rupture of the testa (seed coat) and rupture of the endosperm are two sequential events during the germination of Lepidium sativum L. and Arabidopsis thaliana (L.) Heyhn. Abscisic acid (ABA) specifically inhibits the endosperm rupture of these two closely related Brassicaceae species. Lepidium seeds are large enough to allow the direct measurement of endosperm weakening by the puncture force method. We found that the endosperm weakens prior to endosperm rupture and that ABA delays the onset and decreases the rate of this weakening process in a dose-dependent manner. An early embryo signal is required and sufficient to induce endosperm weakening, which afterwards appears to be an organ-autonomous process. Gibberellins can replace this embryo signal; de novo gibberellin biosynthesis occurs in the endosperm and weakening is regulated by the gibberellin/ABA ratio. Our results suggest that the control of radicle protrusion during the germination of Brassicaceae seeds is mediated, at least in part, by endosperm weakening. We propose that Lepidium is an emerging Brassicaceae model system for endosperm weakening and that the complementary advantages of Lepidium and Arabidopsis can be used in parallel experiments to investigate the molecular mechanisms of endosperm weakening.     

Developmental anatomy and morphology of the ovule and seed of heliconia (heliconiaceae, zingiberales)

The developmental anatomy and morphology of the ovule and seed in several species of Heliconia were investigated as part of an embryological study of the Heliconiaceae and to provide a better understanding of their relationships with the other families of the Zingiberales. Heliconia species have an ovule primordium with an outer integument of both dermal and subdermal origin. The archesporial cell is divided into a megasporocyte and a single parietal cell, which in turn are divided only anticlinally to form a single parietal layer, disintegrating later during gametogenesis. The embryo sac was fully developed prior to anthesis. In the developing seed, the endosperm was nuclear, with wall formation in the globular stage; a nucellar pad was observed during embryo development, but later became compressed. The ripe fruit contained seeds enveloped by a lignified endocarp that formed the pyrenes, with each pyrene having an operculum at the basal end; the embryo was considered to be differentiated. Most of these characteristics are shared with other Zingiberales, although the derivation of the operculum from the funicle and the formation of the main mechanical layer by the endocarp are unique to the Heliconiaceae.     

四种石斛兰种胚发育进程研究

以玫瑰石斛、尖刀唇石斛、短棒石斛、兜唇石斛种子为材料,进行种胚非共生萌发研究,并对其种子形态和胚的发育进程进行了显微观察。结果表明:处于球形胚阶段的石斛兰种子,种胚吸胀后突破种皮,发育至吸收毛和芽生长点出现后,种胚形成原球体;种子萌芽后胚尚未成熟,只进入心形胚阶段。呈纺锤形种子的种皮两端形状不同,一端存在结点,呈弯曲状的尖形,另一端种皮呈收拢的圆口形。4种石斛兰种子,玫瑰石斛种子最长,为两端狭长的纺锤形;兜唇石斛种子最短,呈两端稍细的纺锤形。玫瑰石斛、短棒石斛、尖刀唇石斛种子胚培养需要5～10 d萌发;兜唇石斛种子和胚皆偏小,萌发需要30 d。石斛兰种胚和种皮吸水膨胀后,种胚向种皮的一端移动、脱出或种胚撕裂种皮中央后突破而出,形成裸胚。玫瑰石斛种子撕裂种皮后主要从种皮中央突破;短棒石斛、尖刀唇石斛、兜唇石斛部分种胚从种皮一端脱出,部分种胚则从中央撑破种皮脱出。充分膨胀、变绿后萌芽的裸胚,存在极性,顶部芽生长点萌动,下部出现成群散射状吸收毛。     

北柴胡胚和胚乳的发育及对其种子萌发的影响

利用常规石蜡制片技术对北柴胡胚和胚乳的发育及对其种子萌发的影响进行了观察。结果表明北柴胡胚的发育属于茄型,基细胞进行一次横分裂后不再分裂,因而胚柄不发达,且很早解体。胚乳的发育属于核型,初生胚孔核的分裂远远早于受精卵的分裂。对果实采收时期胚发育状况进行统计发现,在被测采收期果实中有20％的果实的胚处于球形胚阶段,70％处于心形胚,只有10％处于鱼雷胚,说明北柴胡种子采收时胚处于不同的发育阶段,存在形态后熟现象,这是北柴胡种子萌发难、萌发率低且出苗不整齐的主要因素。     

刺五加种子结构,后熟作用及其细胞化学研究

刺五加种子为扁肾形,种皮由一层细胞构成。种子脱落时,胚处于心形胚期,胚周围的胚乳细胞解体形成囊腔包囊胚,胚细胞原生质浓厚,胚乳细胞中贮存大量蛋白质和脂类,但两者均未见贮存多糖,有萌发潜能的种子只占全部种子的１２．８０％,种子经变温层积处理６个月即可完成后熟过程,其细胞化学特点是：处理１．５个月时胚细胞中开始积累多糖颗粒,至４个月时达最大量并一直保持至种子萌发。试验地种植条件下饱满种子经１８－２０个     

Imprinting of the MEDEA polycomb gene in the Arabidopsis endosperm.

In flowering plants, two cells are fertilized in the haploid female gametophyte. Egg and sperm nuclei fuse to form the embryo. A second sperm nucleus fuses with the central cell nucleus that replicates to generate the endosperm, which is a tissue that supports embryo development. MEDEA (MEA) encodes an Arabidopsis SET domain Polycomb protein. Inheritance of a maternal loss-of-function mea allele results in embryo abortion and prolonged endosperm production, irrespective of the genotype of the paternal allele. Thus, only the maternal wild-type MEA allele is required for proper embryo and endosperm development. To understand the molecular mechanism responsible for the parent-of-origin effects of mea mutations on seed development, we compared the expression of maternal and paternal MEA alleles in the progeny of crosses between two Arabidopsis ecotypes. Only the maternal MEA mRNA was detected in the endosperm from seeds at the torpedo stage and later. By contrast, expression of both maternal and paternal MEA alleles was observed in the embryo from seeds at the torpedo stage and later, in seedling, leaf, stem, and root. Thus, MEA is an imprinted gene that displays parent-of-origin-dependent monoallelic expression specifically in the endosperm. These results suggest that the embryo abortion observed in mutant mea seeds is due, at least in part, to a defect in endosperm function. Silencing of the paternal MEA allele in the endosperm and the phenotype of mutant mea seeds supports the parental conflict theory for the evolution of imprinting in plants and mammals.