Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Cytological evidence of maternal meiotic errors in a line of chickens with a high incidence of triploidy.

Direct evidence of the nature of maternal meiotic errors in a selected line of chickens with a high incidence of triploidy was obtained by using cytologically marked paternal gametes derived from a closely related avian species. Matings were made by artificial insemination of female chickens of the selection line and a control line with semen from ring-necked male pheasants. A total of five triploid, one pentaploid, and 21 diploid hybrid embryos were karyotyped. Each triploid hybrid embryo contained one set of paternal pheasant chromosomes and two sets of maternal chicken chromosomes, providing irrefutable cytological evidence that the triploids were derived from diploid ova produced by females of the selection line. The pentaploid hybrid contained one set of paternal pheasant chromosomes and four sets of maternal chicken chromosomes, indicating that it had been derived from a tetraploid ovum. Females of the selection line are thought to have a genetically mediated susceptibility to nondisjunction which is responsible for the high incidence of meiotic errors. Evidence is provided that the non-disjunction occurs at both meiosis I and meiosis II.     

The level of chromosomal DNA methylation in mice in the early stages of embryogenesis studied by the action of restriction endonucleases on the chromosomes]

The degree of genome methylation was estimated on chromosomal slides of mouse zygotes, morulae and embryos of 10 day of gestation using in situ digestion with restriction endonucleases MspI and HpaII. The chromosome preparations of all the embryo stages were made by the same method. The degree of methylation was evaluated by the appearance of chromosomes after digestion and by staining with the Giemsa stain. At the zygote stage, both maternal and paternal genomes are more methylated than chromosomes of the next stages of development. The paternal genome is more methylated than maternal. The homologous chromosomes of morulae and 10 day embryos were identical and the pattern of G-banding was formed.     

Developing embryo and cyclic changes in the uterus of Peripatus (Macroperipatus) acacioi (Onychophora,Peripatidae)

Female reproductive tracts of the viviparous neo-tropical onychophoran Peripatus acacioi have been examined at different times throughout the year, and the altering relationship between the developing embryo and the uterus is described. Depending on her age and time of year, the female may have one or two generations of embryos within her uterus. The uterine wall consists of a thin outer epithelium and basal lamina, three layers of muscles, and a thick basal lamina beneath an inner epithelium lining the uterus lumen. These layers are consistent along the length of the uterus apart from the inner epithelial lining, which varies according to position in the uterus and the developmental stage of embryos contained in the uterus. Early embryos are positioned along the length of the uterus and therefore have space in which to grow. During cleavage and segment formation, each embryo is contained within a fluid-filled embryo cavity that increases in size as the embryo grows. Morulae and blastulae are separated by lengths of empty uterus in which the epithelial lining appears vacuolated. Until the process of segment formation is complete, the embryos are attached to a placenta by a stalk and remain in the same part of the upper region of the uterus. As these embryos grow, the lengths of vacuolated cell-lined uterus between them decrease. Each embryo cavity is surrounded by the epithelial sac, the maternal uterine epithelium, which becomes overlaid by a thin layer of cells, the embryo sac, which is believed to be of embryonic origin. The placenta is a syncytial modification of the epithelial sac located at the ovarian end of each embryo cavity covered by the embryo sac and is analogous to the mammalian noninvasive epitheliochorial placenta. Segment-forming embryos have their heads directed toward the ovary. As the embryo gets longer during segment formation, its posture changes from coiled to flexed. Once segment formation is complete, the embryo loses contact with its stalk, an embryonic cuticle forms, and the embryo turns around so that its head is directed toward the vagina. The embryo escapes from its embryo sac and moves to the lower part of the uterus. In the lower part of the uterus, the straightened fetuses are first unpigmented but subsequently become pigmented as the secondary papillae on the body surface form and an adult-type cuticle forms beneath the embryonic cuticle. While the embryos are contained within their embryo cavities, nutrients are supplied by the placenta. Throughout development the mouth is open and in the mature fetus the gut is lined by peritrophic membrane and material is present in the gut lumen. Trachea have been observed only in fetuses that were ready for birth. Insemination, cyclical changes in the uterine epithelium, and the nature of the cuticle shed at parturition are discussed. © 1995 Wiley-Liss, Inc.     

Allele‐specific expression of the MAOA gene and X chromosome inactivation in in vitro produced bovine embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre‐implantation bovine embryo development by characterizing the allele‐specific expression pattern of the X chromosome‐linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4‐, 8‐ to 16‐cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT‐PCR‐RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele‐specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4‐, 8‐ to 16‐cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele‐specific expression of an X‐linked gene that is subject to XCI in in vitro bovine embryos from the 4‐cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. 77: 615–621, 2010. © 2010 Wiley‐Liss, Inc.     

EMBRYO GROWTH AND SEED SIZE IN RAPHANUS SATIVUS: MATERNAL AND PATERNAL EFFECTS IN VIVO AND IN VITRO

Theories on the evolution of the angiosperm seed disagree as to the effects of different plant tissues on embryo growth. To examine the relative contributions of maternal and paternal genes on embryo growth, we conducted controlled crosses in the greenhouse with wild radish plants (Raphanus sativus), looked for maternal, paternal, and interaction effects on embryo development, and compared the performance of embryos within fruits and in embryo culture. Maternal plant identity affected fruit set, seeds per fruit, embryo developmental stage, and mean seed weight. In embryo culture, maternal effects were found for cotyledon size and embryo weight. Paternal effects were fewer or smaller in magnitude than maternal effects. The identity of the pollen donor affected embryo developmental stage and mean seed weight. In culture, paternal effects were detected for cotyledon size and embryo weight. Our results demonstrate that both maternal and paternal elements affect embryo growth. The fact that maternal effects are greater than paternal effects on embryo development in culture may result from cytoplasmic elements or maternal nuclear genes. Embryo performance in vivo compared to that in vitro varied among maternal plants. The interaction between an embryo and its endosperm and maternal tissues may be either positive or negative, depending upon the maternal plant and the embryo's developmental stage.     

A mechanism of cohesin‐dependent loop extrusion organizes zygotic genome architecture

Fertilization triggers assembly of higher‐order chromatin structure from a condensed maternal and a naïve paternal genome to generate a totipotent embryo. Chromatin loops and domains have been detected in mouse zygotes by single‐nucleus Hi‐C (snHi‐C), but not bulk Hi‐C. It is therefore unclear when and how embryonic chromatin conformations are assembled. Here, we investigated whether a mechanism of cohesin‐dependent loop extrusion generates higher‐order chromatin structures within the one‐cell embryo. Using snHi‐C of mouse knockout embryos, we demonstrate that the zygotic genome folds into loops and domains that critically depend on Scc1‐cohesin and that are regulated in size and linear density by Wapl. Remarkably, we discovered distinct effects on maternal and paternal chromatin loop sizes, likely reflecting differences in loop extrusion dynamics and epigenetic reprogramming. Dynamic polymer models of chromosomes reproduce changes in snHi‐C, suggesting a mechanism where cohesin locally compacts chromatin by active loop extrusion, whose processivity is controlled by Wapl. Our simulations and experimental data provide evidence that cohesin‐dependent loop extrusion organizes mammalian genomes over multiple scales from the one‐cell embryo onward.     

Paternal chromosome incorporation into the zygote nucleus is controlled by maternal haploid in Drosophila

maternal haploid (mh) is a strict maternal effect mutation that causes the production of haploid gynogenetic embryos (eggs are fertilized but only maternal chromosomes participate in development). We conducted a cytological analysis of fertilization and early development in mh eggs to elucidate the mechanism of paternal chromosome elimination. In mh eggs, as in wild-type eggs, male and female pronuclei migrate and appose, the first mitotic spindle forms, and both parental sets of chromosomes congress on the metaphase plate. In contrast to control eggs, mh paternal sister chromatids fail to separate in anaphase of the first division. As a consequence the paternal chromatin stretches and forms a bridge in telophase. During the first three embryonic divisions, damaged paternal chromosomes are progressively eliminated from the spindles that organize around maternal chromosomes. A majority of mh embryos do not survive the deleterious presence of aneuploid nuclei and rapidly arrest their development. The rest of mh embryos develop as haploid gynogenetic embryos and die before hatching. The mh phenotype is highly reminiscent of the early developmental defects observed in eggs fertilized by ms(3)K81 mutant males and in eggs produced in incompatible crosses of Drosophila harboring the endosymbiont bacteria Wolbachia.     

The paternal sex ratio chromosome in the parasitic wasp Trichogramma kaykai condenses the paternal chromosomes into a dense chromatin mass.

A recently discovered B chromosome in the parasitoid wasp Trichogramma kaykai was found to be transmitted through males only. Shortly after fertilization, this chromosome eliminates the paternal chromosome set leaving the maternal chromosomes and itself intact. Consequently, the sex ratio in these wasps is changed in favour of males by modifying fertilized diploid eggs into male haploid offspring. In this study, we show that in fertilized eggs at the first mitosis the paternal sex ratio (PSR) chromosome condenses the paternal chromosomes into a so-called paternal chromatin mass (PCM). During this process, the PSR chromosome is morphologically unaffected and is incorporated into the nucleus containing the maternal chromosomes. In the first five mitotic divisions, 67% of the PCMs are associated with one of the nuclei in the embryo. Furthermore, in embryos with an unassociated PCM, all nuclei are at the same mitotic stage, whereas 68% of the PCM-associated nuclei are at a different mitotic phase than the other nuclei in the embryo. Our observations reveal an obvious similarity of the mode of action of the PSR chromosome in T. kaykai with that of the PSR-induced paternal genome loss in the unrelated wasp Nasonia vitripennis.     

父系HBeAg 阳性流产胚胎绒毛中HBV-DNA 的表达

目的:探讨在父系HBeAg阳性的流产胚胎中,乙型肝炎病毒在绒毛中的表达。方法:募集仅父系感染乙型肝炎病毒组合,即母HBsAg(-)且父HBsAg(+)流产胚胎。按以下组合将入选对象分为4组:组1为父HBeAg(+)母HBsAb(+);组2为父HBeAg(+)母HBsAb(-);组3为父HBeAg(-)母HBsAb(+);组4为父HBeAg(-)母HBsAb(-),采用酶联免疫吸附实验(ELISA)对胎儿父、母亲血清进行乙肝抗原、抗体检测,并使用荧光定量PCR法对胚胎绒毛进行HBV DNA检测。结果:父系感染乙型肝炎病毒的142例胚胎中,仅在父系HBeAg阳性组别(1、2组)84例胚胎中发现3例绒毛HBV-DNA升高,阳性率为3.57%。其中父HBeAg(+)母HBsAb(-)组合中2例,父HBeAg(+)母HBsAb(+)组合中1例。父系HBeAg均阳性,母系HBsAb阳性与阴性组间子代绒毛HBV-DNA升高率差异无显著性(P>0.05)。结论:HBeAg阳性父亲可能更容易导致乙肝父婴垂直传播。     

多胚水稻品系APⅣ不同类型胶囊的受精及其胚胎形成

通过GMA半薄切片技术对APⅣ不同类型水稻(Oryza sativa L.)胚囊的受精及其胚胎发育的研究表明,APⅣ中5-2-l型胚囊的3个卵细胞在少数情况下都可受精并发育形成3个胚;但多数情况只有 1个或2个卵细胞受精发育成1个胚或2个胚。6-2-0型和5-3-0型胚囊多个卵受精频率都很低。由此证明APIV多胚是来自如5-2-1型胚囊的多卵卵器胚囊多个卵细胞都受精的结果,其中3胚来自3个卵细胞受精发育,2胚来自2个卵细胞受精发育。双套结构胚囊受精最为复杂,多数情况是受精不正常,只有少数子房大、小胚囊中的卵细胞都能正常受精。大胚囊中的卵细胞受精发育可能是形成所谓中位胚(远离珠孔端胚)的主要原因。     

Paternal cyclophosphamide exposure induces the formation of functional micronuclei during the first zygotic division

Paternal exposures to cancer chemotherapeutics or environmental chemicals may have adverse effects on progeny outcome that are manifested in the preimplantation embryo. The objectives of this study were to determine the impact of paternal exposure to cyclophosphamide, an anticancer alkylating agent, on the formation, chromatin origin and function of micronuclei in cleavage stage rat embryos. Male Sprague-Dawley rats were gavaged with saline or cyclophosphamide (6 mg/kg/day) for 4 weeks and mated to naturally cycling females to collect pronuclear zygotes and 2 to 8 cell embryos. Micronuclear chromatin structure was characterized using confocal microscopy to detect immunoreactivities for H3K9me3, a marker for maternal chromatin, and lamin B, a nuclear membrane marker. DNA synthesis was monitored using EdU (5-ethynyl-2'-deoxyuridine) incorporation. Fertilization by cyclophosphamide-exposed spermatozoa led to a dramatic elevation in micronuclei in cleavage stage embryos (control embryos: 1% to 5%; embryos sired by treated males: 70%). The formation of micronuclei occurred during the first zygotic division and was associated with a subsequent developmental delay. The absence of H3K9me3 indicated that these micronuclei were of paternal origin. The micronuclei had incomplete peri-nuclear and peri-nucleolar lamin B1 membrane formation but incorporated EdU into DNA to the same extent as the main nucleus. The formation of micronuclei in response to the presence of a damaged paternal genome may play a role in increasing the rate of embryo loss that is associated with the use of assisted reproductive technologies, parenthood among cancer survivors, and paternal aging.     

WHOLE-EMBRYO CULTURE AND THE STUDY OF MAMMALIAN EMBRYOS DURING ORGANOGENESIS

1. Simple and reliable methods are now available for growing rat and mouse embryos in culture at all stages of organogenesis. Primitive-streak embryos can be maintained for up to 5 days in culture while they develop to early foetal stages. Older embryos are maintained for progressively shorter periods and the most advanced stage that can be supported is equivalent to the rat foetus of 15 days' gestation. 2. The rates of protein synthesis and differentiation of the younger embryos in vitro are similar, and of head-fold embryos identical, to those in vivo. After the formation of the limb buds growth is slower, with protein synthesis more retarded than differentiation, resulting in embryos or foetuses that are well formed but smaller than in vivo. This slowing of growth of the older embryos in culture is probably caused by the lack of a functional allantoic placenta. 3. The embryos of some other species, including the guinea-pig, hamster, rabbit and opossum have also been maintained in culture during organogenesis but the results are not yet as good as those for rats and mice. 4. Maximum growth of rat embryos explanted with the visceral yolk sac intact is obtained in undiluted homologous serum, though adequate growth for many studies can be maintained in mixtures of serum with chemically defined tissue-culture media. The best results are obtained in serum prepared from blood centrifuged before clotting has occurred (I.C. serum) and heat-inactivated. The importance of a high concentration of serum in the culture medium may be related to the mechanisms for uptake, transport and digestion of macromolecules by the rodent yolk sac. 5. There is no convincing evidence for a changing rate of oxygen consumption during organogenesis but there is strong evidence for changes in energy metabolism. At the beginning of organogenesis, the embryo shows a high rate of anaerobic glycolysis and of pentose-shunt activity. During the following days these decline while activity of the Krebs' cycle and electron-transport system increases. Anoxia, or exposure of the embryo to carbon monoxide, increases glycolysis and reduces growth rate. 6. The earliest stages of the formation of the heart and blood circulation can be closely observed in culture. The heart rate of the 111/2-day rat embryo is about 160 beats per minute at 38°C, and falls by about 7% per degree for lower temperatures. Several drugs that are cardioactive in the adult also affect the frequency of the heartbeat in the embryo, and the pattern of response suggests that the adrenergic receptors in the embryo develop before the cholinergic receptors. Experiments in which embryo and yolk sac were cultured separately, as well as together, have indicated that haemopoiesis can occur in the embryo only after a migration of stem cells from the yolk sac. 7. Microsurgery has been successfully applied to embryos in culture in studies on morphogenetic movements, heart development, axial rotation, limb-bud regeneration and placenta formation. Biochemical studies of normal morphogenesis have been few, but one has shown a high rate of hyaluronate synthesis by the embryo which may be related to the maintenance and expansion of extracellular spaces and the formation of the neural folds. 8. Embryos are particularly sensitive to teratogenic agents during organogenesis. Teratogens that have been studied on whole embryos in culture include trypan blue, antisera, hyperthermia, anaesthetics, and abnormal concentrations of vitamins, oxygen and glucose. Many of the malformations induced have been similar to those obtained after administration of the same agents in vivo and have demonstrated a direct teratogenic effect on the embryo independent of the maternal metabolism. It is suggested that culture methods may provide a valuable additional screening procedure for new drugs and other potentially embryopathic agents.     

The mechanism of the mixed inheritance of chloroplast genes in Pelargonium

Summary The distributions are given of gene frequencies among embryos after G X W and W X G plastid crosses within and between eight Pelargonium cultivars and some of their inbred or hybrid derivatives.Two distinct segregation patterns are recognized. Homozygous type I female parents (Pr₁Pr₁) have a high frequency of progeny with only maternal alleles, are intermediate for biparental and low for paternal offspring. Heterozygous type II female plants (Pr₁Pr₂) have an equally high frequency of maternal and paternal offspring and a generally low biparental frequency. These correspond to L-shaped and U-shaped gene frequency distributions respectively in which the only modes are at 0 per cent (maternal embryos) and 100 per cent (paternal embryos), with no mode corresponding to the population mean and no sign of a Gaussian distribution.The extremely variable plastid gene frequencies are strongly influenced by the maternal nuclear genotype and by the plastid genotype in which the wild-type allele is always more successful than the mutant in strict comparisons.The relative frequencies of maternal and paternal zygotes, and the mean gene frequency among all the zygotes in a cross, are explicable in terms of the input frequencies of genes from the two parents, their degree of mixing, and by some form of selective replication of plastids. This selection is controlled by nuclear and plastid genotypes which may act in the same direction, to increase the frequency of either the maternal or the paternal alleles, or in opposition. But selection alone is inadequate to explain the shapes of the gene frequency distributions. Instead, a model is proposed in which the segregation or replication of plastids appears to have a strong random element, which results in random drift of gene frequencies within a heteroplasmic zygote or embryo.     

Preimplantation genetic diagnosis: State of the ART 2011

For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1–2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally ‘best’ embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates.     

Cytogenetic studies following high dosage paternal irradiation in the mealy bug,Planococcus citri

Summary In the mealy bug, Planococcus citri, following high dosage paternal irradiation (60,000–120,000 rep), the survivors are mostly female (about 30–40% of the unirradiated control value) whereas very few males survive (about 5% of control value). After lower doses of paternal irradiation (P. I.), however, few or no females survive while the normal number of males (never less than the control value) survive.The females developing after high dosage P. I. are gynogenetic and are triploid or diploid or 3N/2N or 2N/N mosaics (Chandra 1963).The cytology of X₁ embryos following 90,000 rep is described in this report, in comparison with data from embryos following lower doses (8,000 r) of P. I. and unirradiated controls, to illustrate the chromosomal mechanisms leading to the production of gynogenetic females and the probable reasons for lethality of X₁ males after heavy P. I.It has been shown that triploid females stem from a fusion nucleus of the first and second polar bodies. This triploid polar nucleus, which normally participates in the formation of a polyploid sector in the young embryo, undertakes a successful embryogeny in many embryos when the zygote nucleus is unable to do so because of the heavily damaged paternal complement of chromosomes. Since the chromosomes are characterized by holokinetic activity, the irradiated paternal set manages to divide with the maternal complement but did not always segregate as successfully. Restitution divisions of the zygotic nuclei result in haploid, hyperhaploid, diploid and polyploid nuclei. Most of the diploid gynogenetic females probably originate from diploid nuclei of zygotic origin although it is possible that a few diploid females and the 2N/N mosaic females develop from polar bodies.From a dissertation submitted to the University of California, in partial satisfaction of the requirements for the degree of Doctor of Philosophy.Supported in part by a National Science Foundation Grant (No. G-9772) to Professor Spencer W. Brown.N. I. H. Predoctoral Trainee in Genetics 1961–1962.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Determination by GISH and FISH of hybrid status in Lilium

In the genus Lilium, plants obtained from crosses, especially between distant relatives, are not always hybrids because embryos can develop as a result of apomixis. These plants constitute genetic material of the maternal parent only. In this study, verification of hybrid status of plants which have been obtained from the crosses 'Marco Polo'xLilium henryi and 'Expression'xL. henryi was performed through the use of cytological and molecular cytogenetic methods. According to cytological analyses, all genotypes tested had 2n = 2x = 24 chromosomes. Genomic in situ hybridisation (GISH) was used for hybrid verification. In hybrid plants, this method distinguished all paternal and maternal chromosomes at the stage of somatic metaphase and prophase. For GISH, paternal genomic DNA was used as a probe and maternal DNAs were used as blocks. Fluorescence in situ hybridisation (FISH) with 5S rDNA and 25S rDNA probes was used as the second method of hybrid verification. Selected chromosome markers based on genome-specific localisation of rDNA loci were used for analysis of the F1 hybrids obtained from the crosses 'Marco Polo'xL. henryi and 'Expression'xL. henryi. The presence of marker chromosomes characteristic for each of the paternal genotypes was a confirmation that the plants obtained were hybrids.     

Fertilization of Different Types of Embryo Sacs and Its Embryo Formation in Polyembryonic Rice Strain APⅣ

The ferflization and its embryo fonnation of different types of embryo sacs were studied by using the technology of GMA half-section in the APIV strain of polyembryonic rice ( Oryza sativa L. ). In rare cases,all three egg cells in the embryo sac of 5-2-1 type could fertilize and develop into three embryos. But in most cases only one or two egg cells fertilized and developed into one or two embryos in the respective type of embryo sac. The frequency of poly-egg fertilization in total all was very low in the embryo sac of 6-2-0 type and 5-3-0 type. These results indicated that the polyembryos in APIV originated mainly from overall fertilization and develotment of the embryo sac with poly-egg apparatus. This was observed,for example, in 5-2-1 type in which three embryos were fertilized and developed from three egg cells and two embryos from two egg cells. The fertlization process of double set of embryo sac was most complicated, all often abnormal. Only in few ovaries the egg cells in both large and small embeyo sac could fertilize simultaneously. The fertilization and development of egg cells in the large embryo sac might be the main cause of the formation of the so called "mid-seated embryo" (the embryo far from the micropyle end).