Induction of autonomous endosperm in Lupinus luteus,Helleborus niger and Melandrium album by in vitro culture of unpollinated ovaries

Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules.     

Megagametogenesis in Halophila johnsonii, a threatened seagrass with no known seeds,and the seed-producing Halophila decipiens (Hydrocharitaceae)

Megagametogenesis, the development of a megaspore into an embryo sac, has been identified in the seagrass Halophila johnsonii, a threatened species with no known sexual reproduction or seeds. Megagametogenesis in H. johnsonii was compared with megagametophyte development in Halophila decipiens, a related species known to readily produce viable seeds. In both species, ovules were structurally similar, megaspore mother cells were seen in premeiotic ovules, and linear tetrads and megagametophytes with two to eight nuclei were present in postmeiotic ovules. However, H. decipiens postmeiotic ovules had a chalazal pouch that was absent in the postmeiotic ovules of H. johnsonii. Late-stage H. decipiens ovules also contained embryos, indicating that they had been fertilized, whereas all late-stage H. johnsonii ovules were degrading and showed no signs of fertilization. These observations suggest that meiosis does occur in H. johnsonii megasporocytes, leading to the formation of viable megagametophytes and egg cells that could be fertilized if pollination occurred. Thus, the lack of seed set is due to a lack of pollination rather than any loss of capacity to produce seeds in this species.     

Degree of pectin methyl esterification in endosperm cell walls is involved in embryo bending in Arabidopsis thaliana

The endosperm is a transitory structure involved in proper embryo elongation. The cell walls of mature seed endosperm are generally composed of a uniform distribution of cellulose, unesterified homogalacturonans, and arabinans. Recent studies suggest that changes in cell wall properties during endosperm development could be related to embryo growth. The degree of methyl esterification of homogalacturonans may be involved in this endosperm tissue remodelling. The relevance of the degree of homogalacturonan methyl esterification during seed development was determined by immunohistochemical analyses using a panel of probes with specificity for homogalaturonans with different degrees of methyl esterification. Low-esterified and un-esterified homogalacturonans were abundant in endosperm cells during embryo bending and were also detected in mature embryos. BIDXII (BDX) could be involved in seed development, because bdx-1 mutants had misshapen embryos. The methyl esterification pattern described for WT seeds was different during bdx-1 seed development; un-esterified homogalacturonans were scarcely present in the cell walls of endosperm in bending embryos and mature seeds. Our results suggested that the degree of methyl esterification of homogalacturonans in the endosperm cell wall may be involved in proper embryo development.     

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     

Downregulation of egg cell-secreted EC1 is accompanied with delayed gamete fusion and polytubey

One major player known to be essential for successful gamete interactions during double fertilization in Arabidopsis thaliana is the recently identified family of egg cell-secreted EC1 proteins. Both gamete fusion events are affected in EC1-deficient female gametophytes. Here, we show that the number of ovules with unfused sperm cells is considerably higher than the number of undeveloped seeds in the same ec1-RNAi knockdown lines. We found that some sperm cells are able to fuse with the female gametes even 2 to 3 days after pollination, as reflected by delayed embryo and endosperm development, and by polytubey. We propose that the egg cell secretes EC1 proteins upon sperm arrival to promote rapid sperm activation, thereby accelerating gamete fusion and preventing polytubey.     

The progamic phase, embryo and endosperm development in an intraspecific Tulipa gesneriana L. cross and in the incongruent interspecific cross T. gesneriana × T. agenensis DC.

 The development of the embryo and endosperm has been investigated in an intraspecific Tulipa gesneriana cross and in the incongruent cross T. gesneriana ×T. agenensis at intervals of 10 days, from 12 to 82 days after pollination (DAP). In both tulip crosses, the zygote gives rise to an apparently undifferentiated cell mass, the proembryonal cell mass, on which a suspensor then develops. Subsequently, a globular embryo is formed on top of the suspensor. This embryo finally elongates, giving rise to a spindle-shaped embryo. The cellular endosperm fills the whole embryo sac in mature seeds, except for a region immediately around the embryo. In both crosses, aberrant developments were found. In the intraspecific T. gesneriana cross, the pollen tubes did not open in a number of ovules. In other ovules, the pollen tubes seemed to have opened, but an embryo or endosperm was not found or only endosperm was observed. In the cross T. gesneriana ×T. agenensis, fewer pollen tubes entered the ovules than in the intraspecific T. gesneriana cross. The ovules with embryo and endosperm formation of the incongruent interspecific cross showed, in general, retarded development in comparison with the intraspecific T. gesneriana cross. The first globular embryos and spindle-shaped embryos were found at the later fixation dates and the relatively lower number of spindle-shaped embryos at 82 DAP had a shorter average length. The number of ovules with deformations in embryo and/or endosperm development was also higher in the cross T. gesneriana × T. agenensis in comparison with the intraspecific T. gesneriana cross. Between 87% and 100% of the ovules with embryo and endosperm development showed normal development in the intraspecific T. gesneriana cross, while in the incongruent interspecific cross, from 22 DAP, between 17% and 56% of the ovules showed normal development. Of those ovules with aberrations in embryo and/or endosperm formation, about 80% had a deformed endosperm, of which more than 50% also contained a deformed embryo. Embryos of the incongruent cross might be saved by the application of embryo rescue techniques. Received: 10 December 1996 / Revision accepted: 23 April 1997     

Unexpected DNA content in the endosperm of Cupressus dupreziana A. Camus seeds and its implications in the reproductive process

 Nuclear DNA content of embryo and endosperm from mature and immature Cupressus dupreziana A. Camus seeds was estimated using laser flow cytometry. Relative DNA content of endosperm nuclei corresponded to four ploidy levels: 2C, 4C, 6C and 8C. The embryo nuclei invariably exhibited a diploid pattern. In all endosperm tissue analyzed no haploid nucleus was found. This is problematic since, in gymnosperms, endosperm and female gametes originate from one functional haploid megaspore produced by meiosis. The possible origin and derivation of C. dupreziana endosperm are discussed in light of previous results concerning the two other Mediterranean cypresses, C. sempervirens and C. atlantica. Received: 15 January 1998 / Revision accepted: 27 March 1998     

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     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

POLLINATION,FERTILIZATION AND OVULE ABORTION IN OXALIS MAGNIFICA

Pollination, fertilization and ovule abortion were studied in Oxalis magnifica (Rose) Knuth, a strongly self-incompatible herb that regularly matures only a fraction of its ovules. Examination of cleared ovules indicated that among 9 individuals the average number of ovules fertilized ranged from 48–92%. The remaining ovules either failed to produce female gametophytes, or more commonly contained unfertilized female gametophytes, despite large numbers of compatible pollen grains that were placed on stigmas. Abortion of fertlized ovules could be detected first by the flattened and enlarged appearance of the endosperm nuclei, followed by visible deterioration of the embryo. Among individuals the rate of embryo abortion varied from 3.4–47.9%. At lower levels of pollination an almost one-to-one relationship existed between the number of pollen grains placed on stigmas and the number of seeds matured in the capsule. No threshold number of pollen grains necessary for successful pollen tube growth and fertilization could be demonstrated. Reduction in seed number through embryo abortion provides an opportunity for selection among developing seeds. The potential for this form of selection varies widely among individuals of Oxalis magnifica, which showed a 14-fold variation in the average percentage of aborted ovules.     

Apomixis in <Emphasis Type="Italic">Eulaliopsis binata</Emphasis>: characterization of reproductive mode and endosperm development

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds with essentially clonal embryos, stimulating wide interest from biologists and plant breeders for its ability to fix heterosis. Eulaliopsis binata (Poaceae), is identified here as a new apomictic species. Embryological investigation indicates that the developmental pattern of embryo sac formation in E. binata represents gametophytic apospory, the embryo originating from an unreduced cell, without fertilization and the mode of endosperm development was autonomous. Sexual embryo sacs were found with a frequency of 1–4% depending on the biotype. The DNA content of nuclei (C-value) in mature seeds was screened by flow cytometry (FCSS) and demonstrated that the endosperm was derived autonomously without fertilization and the three biotypes of E. binata showed varying degrees of apomixis. The Wide-leaf type showed obligate apomixis whereas the Slender-leaf and the Red-haulm type displayed facultative apomixis. In addition, adventitious embryos were observed on the wall of ovary, integument and nucellus cells, indicating that E. binata produces embryos via a mixture of apospory and adventitious embryony.     

Embryological Studies of Codonopsis pilosula Nannf.

This paper reports the studies of megasporogenesis and microsporogenesis, development of female and male gametophytes, fertilization, and development of embryo and endosperm, The anther wall consists of four layers, i.e. epidermis, endothecium, middle layer and tapetum. Part of the tapetum cells originates from the primary parietal cells, and the other part comes from the basic tissue of the anther partition. Tapeta? cells are uninucleate or binucleate, and belong to the secretory type. Microsporocyte originates directly from the primary sporogenous cell, Cytokinesis is of the simultaneous type. Arrangement of microspores in tetrad is isobilateral. Mature pollen grain is of the 2-celled type. The ovary is tricarpellum, trilocular with many ovules. The ovule is mono-integinous, tenui-nucellar and anatropous. The embryo sac originates from the single-archesporial cell. The one chalazal megaspore in linear tetrad is the functional megaspore. The development of embryo sac is of the Polygonum type. Before fertilization, two polar nuclei fuse in to a secondary nucleus and the antipodal cells degenerate. Fertilization is porogamy, fusion of one sperm with secondary nucleus is faster than that of one sperm with egg nucleus. The development of endosperm is of the cellular type. The first three divisions of endosperm ceils are regular. Two endosperm cells near the ends of chalaza and the micropyle develop into haustorium without division. The haustoria gradually degenerate at the late stage of globular embryo. The mature seeds contain abundant endosperm. The development of embryo is of the Solanad type. The suspensor consists of 12–20 cells. The optimum development of the suspensor is at the early stage of the globular embryo. It begins to degenerate after late globular stage. The embryo develops from proembryo, heartshaped embryo, dicotyledenous- to mature embryo.     

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.     

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.     

Ricinosomes provide an early indicator of suspensor and endosperm cells destined to die during late seed development in quinoa (Chenopodium quinoa)

Background and Aims

In mature quinoa (Chenopodium quinoa) seeds, the lasting endosperm forms a micropylar cone covering the radicle. The suspensor cells lie within the centre of the cone. During the final stage of seed development, the cells of the lasting endosperm accumulate protein and lipids while the rest are crushed and disintegrated. Both the suspensor and endosperm die progressively from the innermost layers surrounding the embryo and extending towards the nucellar tissue. Ricinosomes are endoplasmic reticulum-derived organelles that accumulate both the pro-form and the mature form of cysteine endopeptidase (Cys-EP), first identified in castor bean (Ricinus communis) endosperm during germination. This study sought to identify associations between the presence of ricinosomes and programmed cell death (PCD) hallmarks in suspensor and endosperm cells predestined to die during quinoa seed development.

Methods

A structural study using light microscopy and transmission electron microscopy was performed. To detect the presence of Cys-EP, both western blot and in situ immunolocalization assays were carried out using anti-R. communis Cys-EP antibody. A TUNEL assay was used to determine DNA fragmentation.

Results and Conclusions

Except for the one or two cell layers that constitute the lasting endosperm in the mature seed, ricinosomes were found in suspensor and endosperm cells. These cells were also the site of morphological abnormalities, including misshapen and fragmented nuclei, vesiculation of the cytosol, vacuole collapse and cell wall disorganization. It is proposed that, in suspensor and endosperm cells, the early detection of Cys-EP in ricinosomes predicts the occurrence of PCD during late seed development.     

龙须草无融合生殖的胚胎学证据

采用石蜡切片技术对龙须草(Eulaliopsis binata(Rotz)C.E.Hubb)进行了系统的胚胎学研究,证明龙须草为禾本科植物中一种新的无融合生殖材料.龙须草无融合生殖方式为无孢子生殖,在胚珠发育早期,多个珠心细胞特化为无孢子生殖原始细胞,由原始细胞发育为单核胚囊,经两次有丝分裂形成4核胚囊,进一步分化形成两种类型的成熟胚囊:(1)具1个卵细胞,1个助细胞和2个极核,占观察总数的67.6%;(2)具1个卵细胞,2个助细胞和1个极核,占观察总数的32.4%.胚囊发育属大黍型.多个无孢子生殖原始细胞可以同时发育,最后形成2个或多个胚囊,其比例为17.7%.胚珠内没有有性胚囊的发育.胚的发生有两种类型:(1)早发生胚(74%),开花前1～2 d,极核未分裂前卵细胞分裂形成胚;(2)迟发生胚(26%),开花后2～3 d,极核分裂形成多个胚乳游离核后,卵细胞启动分裂形成胚.存在多胚现象,多胚来自不同胚囊内卵细胞的孤雌生殖,多胚发生率为13%.胚乳由极核不经受精自发分裂产生.     

Studies on the early development of zygotic and synergid embryo and endosperm in polyembryonic rice ApIII

A morphological, cytological and embryological investigation was conducted on the early development of embryo and endosperm in polyembryonic rice (Oryza sativa L.) ApIII. We found that the percentage of single-, twin- and triple-seedlings from mature caryopses was 82.4–85.6%, 11.3–14.6% and 2.0–3.8%, respectively. From twin-seedlings one of the plants and in the triple-seedlings usually two seedlings were very weak having a mortality of about 50–60% and over 95%, respectively, and most of the dead seedlings were haploid. In addition, among 674 young caryopses the percentage of single-, double- and triple-embryo sacs was 77.60%, 18.10% and 4.30%, respectively. The development of both embryo and endosperm was observed in the embryo sacs of 653 young caryopses, while embryo development occurred in absence of endosperm in the embryo sacs of 21 ones. Furthermore, after pollen tube penetration, frequently one, rarely two, additional embryos arise via apogamy from the typical specialized synergid of normal egg apparatus with a collapsed cap-neck region that had a damaged filiform apparatus at the micropylar end. Finally, the formation of a strikingly degenerated endosperm cell region is a specialized functional structure that serves for the active transport of metabolites from endosperm to the developing embryo. Taken together, these results suggest that synergid apogamy seems to be the unique reason of polyembryony, poor growth and high mortality of many seedlings may be due to haploidy, and high genetic stability of polyembryony occurs in rice ApIII.     

Seed development in Malpighiaceae species with an emphasis on the relationships between nutritive tissues

Malpighiaceae ovules have a well-developed nucellus; previous observations indicate that during seed development, the endosperm does not proliferate, thus, remaining scarce. This study aimed at identifying the nutritive tissues during seed development in Malpighiaceae, focusing especially on the endosperm. We analysed the seed development of Janusia mediterranea, J. occhionii, Mascagnia cordifolia, and Tetrapterys chamaecerasifolia, which were collected and processed by traditional methods for light microscopy. Ovules are subcampylotropous, crassinucellate and unitegmic in Janusia and bitegmic in M. cordifolia and T. chamaecerasifolia. The nucellus is well developed and protrudes through the micropyle, touching the funicular obturator. During development, a pachychalaza is formed, and the integuments coalesce in bitegmic species. Through a series of nucellar cell divisions, the perisperm is formed. In Janusia species, the endosperm is not produced. In M. cordifolia and T. chamaecerasifolia, the endosperm is nuclear, but it is scarce and ephemeral. The mature seed is exalbuminous, and the perisperm is consumed, and thus, the mature embryo is total. The absence of endosperm in Janusia is newly observed for the family and indicates functional transfer for the abundant perisperm.     

Floral biology and ovule and seed ontogeny of Nymphaea thermarum,a water lily at the brink of extinction with potential as a model system for basal angiosperms

Background and Aims Nymphaea thermarum is a member of the Nymphaeales, of one of the most ancient lineages of flowering plants. This species was only recently described and then declared extinct in the wild, so little is known about its reproductive biology. In general, the complete ontogeny of ovules and seeds is not well documented among species of Nymphaea and has never been studied in the subgenus Brachyceras, the clade to which N. thermarum belongs.Methods Flowers and fruits were processed for brightfield, epifluorescence and confocal microscopy. Flower morphology, with emphasis on the timing of male and female functions, was correlated with key developmental stages of the ovule and the female gametophyte. Development of the seed tissues and dynamics of polysaccharide reserves in the endosperm, perisperm and embryo were examined.Key Results Pollen release in N. thermarum starts before the flower opens. Cell walls of the micropylar nucellus show layering of callose and cellulose in a manner reminiscent of transfer cell wall patterning. Endosperm development is ab initio cellular, with micropylar and chalazal domains that embark on distinct developmental trajectories. The surrounding maternal perisperm occupies the majority of seed volume and accumulates starch centrifugally. In mature seeds, a minute but fully developed embryo is surrounded by a single, persistent layer of endosperm.Conclusions Early male and female function indicate that N. thermarum is predisposed towards self-pollination, a phenomenon that is likely to have evolved multiple times within Nymphaea. While formation of distinct micropylar and chalazal developmental domains in the endosperm, along with a copious perisperm, characterize the seeds of most members of the Nymphaeales, seed ontogenies vary between and among the constituent families. Floral biology, life history traits and small genome size make N. thermarum uniquely promising as an early-diverging angiosperm model system for genetic and molecular studies.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.