Autonomous endosperm induction by in vitro culture of unfertilized ovules of Viola odorata L.

Autonomous endosperm was found in unfertilized ovules of V. odorata L. cultured on MS medium supplemented with 2,4-D as a sole growth regulator or on media with 2,4-D and BAP or kinetin. Frequency of endosperm induction was approximately 9% in ovules analyzed. The induction rate depended mainly on genotype of the donor plant, and to lesser degrees, on floral stage, flower series and medium type. Multinuclear endosperms consisting of 10–37 nuclei were found in ovules after as few as 4 days of culture. In some ovules at this stage, the egg cell and two polar nuclei were present. The process of endosperm degeneration began after 3 weeks of culture. In some ovules, degenerating autonomous endosperm was observed up to the 7th week. Parthenogenetic development of egg cells or apogamy did not accompany autonomous endosperm, supporting the hypothesis of independent pathways for embryo and endosperm development. Received: 1 December 1998 / Revision accepted: 6 April 1999     

COTTON EMBRYOGENESIS: THE EARLY DEVELOPMENT OF THE FREE NUCLEAR ENDOSPERM

An electron microscope study was made of the central cell and the development of the free nuclear endosperm surrounding the zygote and synergids during the first three days after pollination. The cytoplasm of the central cell, concentrated around the partially-fused polar nuclei, contains many ribosomes, mitochondria and large, dense, starch-containing plastids, some dictyosomes and lipid bodies, and long, single cisternae of rough endoplasmic reticulum (RER) that frequently terminate in whorls. Dense, core-containing microbodies are closely associated with the RER. After fertilization the cytoplasm of the 2-and 4-nucleate endosperm shows an increase in number of dictyosomes, and in amount of RER which becomes stacked in arrays of parallel cisternae. Cup-shaped plastids are associated with many long, helical polysomes. Perinuclear aggregates of dense, granular material also appear after fertilization. Granular aggregates and helical polysomes disappear after the first few divisions of the primary endosperm nucleus. During the second and third days of development there is an increase in dictyosome number and RER proliferation, and endosperm nuclei become deeply lobed. Concurrently, there is a sharp decline in the starch and lipid reserves of the central cell and elaborate transfer walls are formed at the micropylar end of the embryo sac and on the outer surface of the degenerating synergid. The transfer walls contain groups of small, membrane-bound vesicles, and are associated with large numbers of mitochondria and with the smooth endoplasmic reticulum.     

In vitro culture promotes partial autonomous endosperm development in unfertilized ovules of wild-type Arabidopsis thaliana var. Columbia

Partial endosperm development without paternal genome involvement was induced in unpollinated ovaries of wild-type Arabidopsis thaliana cultured in vitro. Unpollinated pistils were cultured on hormone-free Murashige and Skoog (MS) medium with addition of 6% sucrose and supplemented with: benzylaminopurine (BAP; 2 mg l^–1) combined with naphthylacetic acid (NAA; 0.1 mg l^–1), 2,4-dichlorophenoxyacetic acid (2,4-D; explants exposed to 1-h auxin shock 20 or 40 mg l^–1, and transferred to hormone-free MS medium). Initiation of autonomous endosperm (AE) development was induced on all media used in 54 ovules from 39 cultured ovaries (26%), with an average frequency of 1.4 ovules/ovary. The highest frequency of partial endosperm formation occurred on media combining the two growth regulators BAP and NAA (59% of ovaries had ovules with AE), although endosperm development was also induced on hormone-free medium (in 20.5% of ovaries). The number of AE nuclei ranged from 2 to ~50, depending on the day of culture and medium; neither cellularization nor differentiation on specific regions typical for endosperm of wild-type Arabidopsis, were noted. Fertilization independent endosperm most probably originated from the secondary nucleus, but involvement of the polar nuclei could not be excluded, as indicated by nuclear size and structure. In vitro conditions did not influence egg cell proliferation. Gynogenic embryos were observed neither in the ovules with autonomous endosperm nuclei nor in ovules without endosperm induction.     

Production of micronucleoli and onset of cotton fiber growth

Experiments focused on the early development of fiber cells of cotton (Gossypium hirsutum L. cv. MD51 ne) ovules produced two novel findings: one biological, the other methodological. The first concerns a micronucleolus in the nucleus of fibers. This developmental marker appears at or a little before 4 days postanthesis (dpa) in about 10% of the fibers and increases thereafter to nearly 80% provided the fibers are growing on fertilized ovules. Micronucleoli are neither seen in nuclei of fibers at 0–2 dpa nor in nuclei of non-fiber cells. Consequently, it is postulated that they are the product of specific developmental genes associated with fiber growth. The second, methodological finding, involves a cytological means of directly counting the number of fibers produced on young ovules at 1–4 dpa. The method provides quantitative data unavailable in the past. We used this method to show that emasculation caused a temporary 24-h delay in the initiation of fibers, that 30% of the fibers are affected, and that at 3 dpa both fertilized and unfertilized ovules have about 14 500 fibers. These data indicate that the fibers on fertilized and unfertilized ovules represent the same cell populations, a finding heretofore unknown. Received: 24 October 1997 / Accepted: 5 January 1998     

竹节参雌配子体发育的研究

本文报道了竹节参(Panax japonicus C.A.Mey)雌配子体(胚囊)的发育过程。竹节参大孢子母细胞减数分裂产生线形排列的大孢子四分体。胚囊发育属蓼型,由合点端大孢子发育而成。游离核胚囊时期,胚囊珠孔端的细胞器种类和数量都较胚囊合点端多;胚囊合点端相邻的珠被细胞中有含淀粉粒的小质体,与胚囊珠孔端相邻的退化中的非功能大孢子中则有含淀粉粒的大质体和大类脂体。成熟胚囊中,反足细胞较早退化;极核融合成次生核;卵细胞高度液泡化,细胞器数量较少;助细胞则有丰富的细胞器和发达的丝状器。PAS反应表明,受精前的成熟胚囊中积累淀粉粒。次生核受精后,很快分裂产生胚乳游离核,到几十至数百个核时形成胚乳细胞。卵细胞受精后则要经过较长的休眠期。     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

Induced single fertilization in maize

 Bicellular pollen with one vegetative nucleus and one diploid arrested generative cell (”monospermic” pollen) was induced by trifluralin treatment of diploid maize plants at 7–9 days before flowering. The arrested generative cell (seemingly a diploid sperm cell) fused with the central cell of diploid plants and produced shriveled endosperm resembling that of a 2n×4n cross in maize. Dual pollination experiments with a purple embryo marker revealed single fertilization events in which the union of one sperm cell with the egg occurs but there is no union of a second sperm cell with the central cell. Singly fertilized ovules survived at least 4 days. Furthermore, many viable triploid plants were obtained. This technique therefore appears to have the potential for manipulating ploidy level in crops and may become useful in investigating fertilization mechanisms of angiosperms. Received: 1 October 1996 / Revision accepted: 8 January 1997     

ENZYMO-CYTOCHEMICAL OBSERVATIONS ON THE CORTICAL CHANGE IN THE EGGS OF CYPRINUS CARPIO AND CARASSIUS AURATUS

Acid phosphatase (AcPase) and cholinesterase (ChE) activities were examined by ultracytochemical techniques in the mature unfertilized and the fertilized eggs of Cyprinus carpio and Carassitus auratus , to reveal the differences among three kinds of structures, cortical alveoli, CA- and CB-granules, which discharge their contents on fertilization into the perivitelline space. Deposits of the reaction product for AcPase activity are localized on the plasmalemma of unfertilized eggs, in the cortical alveoli, cytoplasmic matrix, lamellae of the Golgi apparatus and sometimes in multivesicular bodies but not in CA- and CB-granules, mitochondria, rough endoplasmic reticulum or on the plasmalemma of fertilized eggs. Deposits of the reaction product for ChE activity are localized on the inner surface of the plasmalemma, in the cytoplasmic matrix, in mitochondria and on a small number of tubular or cisternal membranes of the endoplasmic reticulum in mature unfertilized eggs, and on the outer surface of the limiting membrane of CB-granules and on membranous structures (possibly Golgi lamellae) associated with their formation, in fertilized eggs. The deposits on the plasmalemma rapidly disappear almost completely, with discharge of the cortical alveoli soon after fertilization, but they are again seen on the inner surface of the plasmalemma when emiocytotic discharge of the CB-granules begins about 10 min after fertilization.     

大车前胚乳发育的超微结构研究

采用透射电镜技术对大车前(Plantago major L.)胚乳发育的超微结构进行了研究。结果表明:(1)大车前为细胞型胚乳;初生胚乳核经一次横分裂产生1个珠孔室细胞和1个合点室细胞;珠孔室两次纵向分裂一次横向分裂形成2层8个细胞,位于上层的4个细胞发育为4个珠孔吸器,位于下层的4个细胞发育为胚乳本体;合点室细胞进行一次核分裂,发育为两核的合点吸器。(2)珠孔吸器呈管状插入珠被组织,珠孔端细胞壁加厚呈现少量分支并具有壁内突,壁内突周围细胞质里分布着大量线粒体、粗面内质网、高尔基体、质体等,细胞核与核仁明显,细胞质浓厚,代谢活动旺盛;球胚期,珠孔吸器的体积呈现最大值,珠孔吸器周围的珠被组织均被水解,形成明显的空腔。珠孔吸器从珠被组织吸收并转运营养物质至胚乳本体,参与胚乳的构建与营养物质的贮藏。球胚后期,珠孔吸器逐渐退化。(3)4个胚乳本体原始细胞具旺盛的分生能力,经不断的平周与垂周分裂增加胚乳细胞数目,使胚乳本体呈现圆球体状,并将胚包围其中;珠孔吸器、合点吸器以及珠被绒毡层吸收转运的营养物质贮存在胚乳本体;球胚后期,随着胚柄的退化,胚体周围的胚乳细胞被水解,为发育的胚所利用。(4)合点吸器的2个细胞核与核仁巨大,线粒体、质体、高尔基体、内质网主要绕核分布,液泡化明显;胚体与胚乳本体的体积增大,逐渐将合点吸器向胚珠合点部位挤压,合点吸器周围的合点组织逐渐被水解,形成巨大空腔。合点吸器自珠心组织吸收并转运营养物质至胚乳本体,参与胚乳的结构构建与营养物质的贮藏。球胚后期,合点吸器逐渐失去功能,呈现退化状态。     

The ultrastructure of polymorphic pollen grains of Canna indica L.

Summary Our investigations on Canna indica L. indicate that the pollen of this species is polymorphic: there are two types of pollen — a larger type and a comparatively smaller type. Transmission electron microscopy (TEM) revealed the presence of small vacuoles containing tannic substances in the generative cell (GC) of the larger grains: the GC of the mature grain contained a higher quantity of tannins than the GC of the immature grain. Mitochondria, lipid bodies, rough endoplasmic reticulum (RER) and microtubular bundles were present in the cytoplasm of the GC. Numerous mitochondria, lipid bodies and plastids were also present in the vegetative cell (VC), with the mitochondria clustered around the vegetative nucleus. The plastids were observed to be associated with the RER cisterns. During the maturation process, the number of starch grains contained in the plastids decreased.     

Increased nuclear DNA content in developing cotton fiber cells

The nuclear DNA content of developing cotton fiber cells (Gossypium hirsutum, cv. MD51ne) increases ∼24% after 2 d postanthesis (dpa). The amount of nuclear DNA at 2 dpa is 5.4 ± 0.27 pg. At 3–4 dpa it increases to 6.7 ± 0.24 pg and by 5 dpa it is 6.8 ± 0.70 pg. These values were obtained by nuclear fluorescence after staining with Hoechst 33258. Human oral squamous cell nuclei were used as a DNA standard. Nuclear DNA content increases in fibers growing on either fertilized or unfertilized ovules. The increase also is detectable in Feulgen stained nuclei using two-wavelength cytospectrophotometry. All measurements were made on isolated fiber cell nuclei using a newly developed method tailored to cotton fiber cells. The results imply that during the early stages of development fiber cell nuclei either selectively amplify certain sequences or enter S-phase replicating a portion of their genome.     

Occurrence of Mitochondria in the Nuclei of Tobacco Sperm Cells

Tobacco sperm cells contain intact mitochondria within their nuclei with a frequency of 0.35 [plusmn] 0.13 per cell. These inclusions appear to originate from mitochondria found among chromatids in the highly elongated metaphase plate of the dividing generative cell. These organelles are apparently captured during the reconstitution of the nuclear envelope. Only sperm cells were observed to contain these nuclear mitochondria; generative cells, vegetative pollen cells, transmitting tissue cells, unfertilized egg cells, and central cells lacked them. Nuclear mitochondria were also seen in the nuclei of the egg and central cell after fusion with sperm nuclei, suggesting that nuclear mitochondria are transmitted into the zygote and primary endosperm cells during double fertilization. Organellar inclusions in the sperm nucleus provide a potential mechanism for transmitting organellar DNA into the next generation and could potentially facilitate the transfer of genetic material between the nucleus and other organelles.     

Ultrastructural investigations of Arbutus unedo-Laccaria amethystea mycorrhiza synthesized in vitro

Summary Anatomy and ultrastructure of the arbutoid mycorrhiza of Arbutus unedo-Laccaria amethystea from axenic culture are described. In comparison to non-inoculated roots, the rhizodermal cells of mycorrhizas are of greater volume, their nuclei are enlarged and show an irregular shape, plasmalemma and cytoplasm with mitochondria, plastids, endoplasmic reticulum and dictyosomes are increased. Several ontogenetical states are documented. The arbutoid mycorrhiza as a connecting link between ectomycorrhiza and ericoid mycorrhiza is discussed.     

Embryological study on gynogenesis in onion (Allium cepa L.)

Haploid induction in onion can, to date, be induced only via gynogenesis by culturing unfertilized flowers, ovaries or ovules. The process of haploid embryo induction has been macroscopically well studied, but only limited data exist from microscopic examination of ovule development status at the inoculation stage and of the origin of gynogenic embryos. Microscopic studies were carried out using individual donor plants with relatively high embryo induction frequencies (45.9 embryos formed per 100 flowers, on average, for 2 years). Ovaries from flower bud culture were fixed at 1 week intervals up to the 7th week of culture. These were compared with pollinated ovaries at 1 or 2 weeks after pollination. In total, 1428 unfertilized embryo sacs were examined. The results indicate that, at the time of inoculation, ovules within ovaries 2.0–3.0 mm in diameter contained two- or four-nucleate embryo sacs in the smallest ovaries to mature embryo sacs in the largest ovaries. It seems likely that the embryos are actually induced from ovaries cultured at the immature stage. After 1 or 2 weeks in culture, the egg apparatus primarily consisted of distinctly enlarged synergids and the egg cell, which was often detached from the micropylar pole. But free nuclear endosperm was also formed. From the 2nd to 7th week in culture, formation of haploid embryos (from globular to the almost mature cylindrical stage) was detected in 5.7% of the ovules. Their origin, for several reasons, was most likely the egg cell. In addition, ovules containing endosperm only (3.6%) and ovules containing the egg apparatus (0.5%) or both endosperm and embryo (0.4%) were detected. This observation is probably unique and has not yet been reported in other species studied. Received: February 2001 / Revision accepted: 20 April 2001     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

The Ultrastructural Study on the Early Embryonic Development of Radish

The ultrastructure of the mature embryo sac, the early stages of the embryo and endosperm development of common radish, Raphanur sativus was examined. The embryo sac consists of 7 cells with antipodal ceils disappeared when it matures. The egg cell is highly polarized. The wall surrounded the chalazal end of the egg cell is incomplete, showing a discontinuous structure of an electron dense material deposited intermittently in the space between the two plasma membranes of the egg cell and central cell. The synergid has filiform apparatus, rich in organelles and well developed ER. The two polar nuclei of the central cell are located near the egg apparatus because of the big vacuole, and the finger-like protrutions from the cell wall, as that in synergid, are found. The first division of the zygote occurs 4–5 days after pollination and the development of the embryo follows the Onagrad type, and the structure of the embryo cell is quite simple for containing small quantity of ER, plastids and other organelles. The primary endosperm nucleus deviates 2 days earlier than zygote. The endosperm is of nuclear-endosperm containing chloroplasts, well developed ER, and plentiful of mitochondria and golgi bodies and the nodule-like aggregation in both. the chalazal and micropylar ends of the embryo sac during the early development appeared, and cell wall starting at the micropylar end by freely-growing forms about 16 days after pollination.     

Quantitative analysis of intramembranous particles in the membranous system of rat liver cells at different stages of development and aging

The density of intramembranous protein particles was studied by freeze-fracture. Particle density on the fracture faces of the plasmalemma and the rough endoplasmic reticulum (RER), as well as the outer and inner membranes of the nucleus and the mitochondria in rat hepatocytes were quantified. Comparison among different age groups sampled days postcoitum (dpc), days postpartum (dpp), and months postpartum (mpp) shows age-related changes in particle density in each membrane system. With the exception of the RER, particle densities increased after the 16th dpc, reached a maximum at birth, and then decreased with increasing age. Simultaneously, the number nuclear pores shows a positive correlation with the particle density of the nuclear membranes. The particle density on the membranes of the RER shows a maximum on the 16th dpc, and on the 6th dpp. Thereafter, the density of the RER decreases slightly. In all membrane systems, the density of the particles on the external fracture faces is more variable than the density of the particles on the protoplasmic fracture faces.     

大葱卵器及受精后助细胞的超微结构

章丘大葱（Ａllium fistulosum L.cv.Zhangqiu)的卵器由１个卵细胞及２个助细胞组成,观察到不少卵器没有卵细胞,只有２个助细胞。卵细胞的核及大部分细胞质位于细胞的合点端,１个大液泡占据了细胞其他部位。卵细胞含有很多的核糖体及多聚核糖体、嵴明显的线粒体、粗面内质网、高尔基体具小泡,卵细胞似是一个活跃的细胞。细胞外被细胞壁,其合点端及侧方与助细胞共同壁不连续,助细胞有一较大的核,位于细胞膨大的部位,众多的小液泡遍布细胞中。核糖体及聚合核糖体、线粒体,粗面内质网及风心圆环状粗面内质丰富,高尔基体及小泡常见,反映了其活跃的代谢作用。助细胞合点端及侧方与卵细胞、中央细胞的共同壁不连续,与卵细胞共同壁含胞间连丝,壁不连续处,有不状多层膜结构伸入卵细胞质,显示助细胞可能对卵细胞提供营养,伟粉后,一个助细胞退化,宿存助细胞至随胚胚期尚存在,它经历了一个缓慢的退化过程,出现质壁分离,细胞质变稀,液泡扩大,细胞器逐渐减少,在椭形胚期,宿存助细胞核内的染色质及核仁消失,有细胞质侵入核内,因宿存助细胞壁变厚,细胞质出现现脂滴,宿存助细胞可能仍有合成功能,宿存助细胞壁出现若干无壁部位,细胞内的营养物质可能通过无壁部位向胚乳转运,供游离核胚乳及胚乳细胞化初期的发育。     

Sieve Element of Impatiens Sultanii: 2. Developmental Aspects

The sieve elements arise from vacuolate cells, and enlargementof one or more slime bodies increases the volume of cytoplasmrelative to that of the vacuole. The slime finally dispersesthroughout the region once occupied by the vacuole. A new term,mictoplasm, is proposed for the resulting mixture of non-membranouscytoplasmic material, including slime, with the contents ofthe vacuole. The nucleus disappears during development, butbefore losing its chromaticity, it apparently releases one ormore nucleoli into the cytoplasm. The extruded nucleoli areprominent during development but usually disappear with thetonoplast, nucleus, and dictyosomes as the cell matures. Atmaturity, small vesicles, plastids containing spherical starchgrains, and sparsely distributed mitochondria deficient in tubulesare attached to the plasmalemma. The sieve-plate connectingstrand develops in a pore site bearing a pair of callose plateletsand penetrated in the centre by a plasmodesms. The callose cylinderwhich surrounds the mature connecting strand is followed sothat the shape of a connecting strand in cross section is stellate.Mictoplasm and the plasmalemma are continuous from one cellto the next through the sieve-plate connecting strands.     

Development of fertilized ovules and their role in the growth of the pea pod

The histological development of fertilized ovules during fruit-set and development in pea ( Pisum sativum L. cv. Alaska) has been investigated. Killing the ovules on day 0 (anthesis) or day 1 prevented fruit-set and resulted in ovary degeneration. When the ovules were destroyed at later stages the ovaries developed, though the rate of growth of the pod was reduced significantly. Pollination in pea occurs normally the day before anthesis, and fertilization of the egg cell 32 to 48 h later. The first divisions of the zygote and endosperm nuclei started simultaneously (ca 48 h after pollination) but the endosperm developed more rapidly than the embryo; the embryo sac cavity was lined with free endosperm nuclei at the time of beginning suspensor elongation. Extracts of endosperm and ovule coats from ovules at day 7 after anthesis showed fruit-set activity in pea, the latter material having about 3 times more activity than the former per ovule basis. These results indicate that fertilization of the ovule is necessary for fruit-set in pea, and that compounds which induce fruit-set are probably synthesized in the ovules following fertilization.