Genetic diversity and reproductive biology in ecotypes of the facultative apomict Hypericum perforatum L

Apomixis is a mode of asexual reproduction through seed. Progeny produced by apomixis are clonal replicas of a mother plant. The essential feature of apomixis is that embryo sacs and embryos are produced in ovules without meiotic reduction or egg cell fertilisation. Thus, apomixis fixes successful gene combinations and propagates high fitness genotypes across generations. A more profound knowledge of the mechanisms that regulate reproductive events in plants would contribute fundamentally to understanding the evolution and genetic control of apomixis. Molecular markers were used to determine levels of genetic variation within and relationship among ecotypes of the facultative apomict Hypericum perforatum L. (2n = 4x = 32). All ecotypes were polyclonal, being not dominated by a single genotype, and characterised by different levels of differentiation among multilocus genotypes. Flow cytometric analysis of seeds indicated that all ecotypes were facultatively apomictic, with varying degrees of apomixis and sexuality. Seeds set by haploid parthenogenesis and/or by fertilisation of aposporic egg cells were detected in most populations. The occurrence of both dihaploids and hexaploids indicates that apospory and parthenogenesis may be developmentally uncoupled and supports two distinct genetic factors controlling apospory and parthenogenesis in this species. Cyto-embryological analysis showed that meiotic and aposporic processes do initiate within the same ovule: the aposporic initial often appeared evident at the time of megaspore mother cell differentiation. Our observations suggest that the egg cell exists in an active metabolic state before pollination, and that its parthenogenetic activation leading to embryo formation may occur before fertilisation and endosperm initiation.     

植物无融合生殖相关基因研究进展

无融合生殖是指不经过精卵融合即可形成胚从而进行种子繁殖后代的一种特殊的无性生殖方式, 无融合生殖胚的形成没有父本的参与, 其后代是母本基因型的完整克隆, 因此是植物杂种优势固定与利用的一种最理想的途径, 具有巨大的潜在利用价值, 被誉为“无性革命”。按其胚体发生的途径, 无融合生殖可分为二倍体孢子生殖、无配子生殖和不定胚生殖三种类型。本文介绍了植物胚发育、胚乳发育、减数分裂等涉及无融合生殖过程的相关基因的研究进展, 同时介绍了可能与植物无融合生殖途径调控相关的几个基因片段的研究情况。     

Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy

Apomixis is facultative in characterized members of the genus Hieracium. The three components that comprise the apomictic mechanism include apospory followed by autonomous embryo and endosperm formation. The time of aposporous embryo sac initiation and mode of embryo sac formation are different in Hieracium piloselloides (D3) and Hieracium aurantiacum (A3.4). Genetic studies have shown that a single dominant locus encodes all three components of apomixis in both species (Bicknell et al. 2000). We histologically examined a range of related, genetically characterized apomictic Hieracium plants derived from D3 and A3.4 to assess conservation of the apomictic mechanism in different genetic backgrounds. The plants varied in ploidy, and also in the amount of DNA introduced from sexual Hieracium pilosella (P4). An apomictic hybrid from a cross between the two apomicts was also examined. The developmental processes observed in the parental apomicts were not conserved in the examined plants and alterations occurred in the components of apomixis. One plant also exhibited adventitious embryony. The results show that other genetic factors can modify apomixis with respect to time of initiation, spatial location, and mode of developmental progression. Both the apomictic locus and the modifiers are essential for efficient penetrance of the trait in Hieracium. Some of the findings in Hieracium correspond with observations in Ranunculus and this is discussed in terms of models for apomictic development and the control of apomixis in crops. Received: 21 June 1999 / Revision accepted: 17 November 1999     

Self-fertility and polyembryony in South American yellow trumpet trees (Handroanthus chrysotrichus and H. ochraceus, Bignoniaceae): a histological study of postpollination events

Handroanthus chrysotrichus shows pollination-dependent self-fertility, polyploidy, and adventitious polyembryony, and it is closely related to H. ochraceus, for which apparently conflicting reports of self-incompatibility and apomixis have been published. The present study aims to investigate the polyembryony in these species by means of histological analysis of ovule/seed development in unpollinated, selfed, and crossed pistils/fruits (in H. chrysotrichus only) as well as seed germination experiments. Experimental pollinations were carried out to evaluate breeding systems in the studied populations, and the results indicated self-fertility in both species. Adventitious embryo precursor cells (AEPs) were formed in the ovules of unpollinated, selfed, and crossed pistils. However, unfertilized ovules never develop into seeds, and fertilization/endosperm initiation clearly stimulates the formation of AEPs in pollinated pistils. The inability of AEP-bearing unfertilized ovules to initiate endospermogenesis clearly shows that fertilization is needed for adventitious embryo development. Consequently, formation of AEPs is required but is not sufficient for apomictic reproduction in H. chrysotrichus. Analysis of the positions of multiple embryos in the endosperm indicated that fertilized ovules are able to develop into seeds even in the absence of a zygotic embryo. The development of AEPs in ovules of H. chrysotrichus foregoes the stage in which activation of selfed pistil rejection takes place in H. impetiginosus, a species with late-acting self-incompatibility. Our study supports the hypothesis that the self-fertility in H. chrysotrichus (and perhaps also in H. ochraceus) resulted from the emergence of pseudogamous apomixis, favored by the physiological peculiarities of the late-acting self-incompatibility and possibly related to polyploidy.     

Evolution of gametophytic apomixis in flowering plants: an alternative model from Maloid Rosaceae

Gametophytic apomixis, asexual reproduction involving megagametophytes, occurs in many flowering-plant families and as several variant mechanisms. Developmental destabilization of sexual reproduction as a result of hybridization and/or polyploidy appears to be a general trigger for its evolution, but the evidence is complicated by ploidy-level changes and hybridization occurring with facultative apomixis. The repeated origins of polyploid apomictic complexes in the palaeopolyploid Maloid Rosaceae suggest a new model of evolutionary transitions that may have wider applicability. Two conjectures are fundamental to this model: (1) that as previously suggested by Rutishauser, like many sexual flowering plants the polyploid apomicts require maternal–paternal balance in the second fertilization event that gives rise to the endosperm, and (2) that the observed variation in endosperm ploidy levels relates less to flexibility late in development than to the known variation in developmental origin of the megagametophyte between mechanisms loosely categorized as diplospory and apospory. The model suggests explanations for the relative frequencies of apospory and diplospory, and for the wide but incomplete associations of apospory with a pollination requirement (pseudogamy) and of diplospory with autonomous development of the endosperm. It is suggested that pollination from other taxa may provide some adaptive advantage to pseudogamous apospory. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

侧金盏胚和胚乳发育的细胞胚胎学研究

利用石蜡切片技术对毛茛科植物侧金盏胚及胚乳发育进行了研究,以明确其胚胎发育的特征,为毛茛科植物的系统研究提供资料。结果表明:(1)侧金盏胚的发育属于柳叶菜型,胚乳发育为核型;初生胚乳核的分裂早于合子的第一次分裂。(2)种子成熟时,种胚尚未分化完全,尚处于球形胚后期或心形胚早期阶段,整个胚发育大约需要50~60d。(3)侧金盏种子存在明显的形态生理休眠现象,经后熟作用逐渐完成种胚的分化与生长,形成子叶形胚;侧金盏种子在相同处理条件下胚分化和发育的速度存在差异。     

An interpretation of genomic balance in seed formation in interspecific hybrids of Solanum

Summary To investigate the mechanisms of seed failure in intraspecific and interspecific crosses of Solanum two diploid, S. commersonii and Group Phureja, and one tetraploid species, S. acaule, species were crossed and the seeds were analyzed for embryo and endosperm development. Many seeds of certain crosses observed seven days after pollinations were found to contain abnormal embryos and degenerating endosperms. In some cases seeds contained an embryo with no endosperm, or an endosperm with no embryo. Other interspecific crosses which were predicted to fail actually produced seeds with normally developed embryos and endosperms. To further characterize the intra- and interspecific embryos and endosperms the nuclear DNA was measured. There are several ways to explain the ploidy levels of embryos and endosperms among the crosses, the occurrence of unreduced gametes in some cases and genomic instability in other cases. The latter resulted in chromosome loss at meiotic and mitotic divisions. Genomic balance in interspecific seeds is critical to both embryo and endosperm development.Scientific Journal Series Article No. 14636 of the Minnesota Experiment Station     

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.     

Molecular characterization of the genomic region linked with apomixis in<Emphasis Type="Italic"> Pennisetum/Cenchrus</Emphasis>

Apomixis is defined as asexual reproduction through seeds, although this outcome can be achieved by multiple pathways. Since little is known about the molecular control of these pathways, how they might intersect is also a mystery. Two of these pathways in the grass family, diplospory and apospory, are receiving attention from molecular biologists. Apospory in Pennisetum/Cenchrus, two genera of panicoid grasses, results in the formation of four-nucleate embryo sacs that lack antipodals. Sexual reproduction frequently aborts so that the resulting seed is composed of (1) a parthenogenetically derived embryo that is genetically identical to the mother and (2) endosperm formed through pseudogamy. The transmission of apomixis is associated with the transfer of a linkage block on a single chromosome. This linkage block contains repetitive sequences as well as hemizygous, low-copy DNA sequences. Fluorescence in situ hybridization has demonstrated that these DNA regions occur on only a single chromosome, but not its homologs, in the polyploid apomicts studied. Features of the apomixis-associated region resemble those of other chromosomal segments isolated from recombination and replete with "selfish" DNAs.     

Polycomb group gene function in sexual and asexual seed development in angiosperms

In sexually reproducing angiosperms, double fertilization initiates seed development, giving rise to two fertilization products, the embryo and the endosperm. In the endosperm, a terminal nutritive tissue that supports embryo growth, certain genes are expressed differentially depending on their parental origin, and this genomic imbalance is required for proper seed formation. This parent-of-origin effect on gene expression, called genomic imprinting, is controlled epigenetically through histone modifications and DNA methylation. In the sexual model plant Arabidopsis, the Polycomb group (PcG) genes of the plant Fertilization Independent Seed (FIS)-class control genomic imprinting by specifically silencing maternal or paternal target alleles through histone modifications. Mutations in FIS genes can lead to a bypass in the requirement of fertilization for the initiation of endosperm development and seed abortion. In this review, we discuss the role of the FIS complex in establishing and maintaining genomic imprinting, focusing on recent advances in elucidating the expression and function of FIS-related genes in maize, rice, and Hieracium, and particularly including apomictic Hieracium species that do not require paternal contribution and thus form seeds asexually. Surprisingly, not all FIS-mediated functions described in Arabidopsis are conserved. However, the function of some PcG components are required for viable seed formation in seeds formed via sexual and asexual processes (apomixis) in Hieracium, suggesting a conservation of the seed viability function in some eudicots.     

Problems of apomictic reproduction in the FamiliesCompositae andRosaceae

For many years several taxa belonging to the familiesCompositae andRosaceae have been subjected to an intensive research on apomixis. These two families, together with theGramineae have headed the list of the most thoroughly examined taxonomic groups in this field. At present, new methods are being elaborated and new problems solved within the following genera known to be apomictic, viz.Antennaria, Hieracium, Taraxacum, Alchemilla, Potentilla andRubus. Many general problems still require more attention in future studies. Seven of them are considered in the present article: 1. frequency of apomixis; 2. occurrence of apospory and diplospory within one taxon; 3. nonfunctional apospory; 4. facultativeness of autonomous apomixis; 5. origin of endosperm; 6. timing of embryo and endosperm development in apomicts; 7. instability of the endosperm type. BothCompositae andRosaceae produce suitable material for such research projects.     

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.     

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.     

Apomixis in hawkweed: Mendel's experimental nemesis

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.     

花椒和野花椒的无融合生殖

花椒与野花椒的胚囊发育类型属蓼型,成熟胚囊的卵器退化。花椒无雄花,不发生双受精,自发形成胚乳并产生珠心胚。野花椒虽有正常花粉,人工授粉后能萌发,但在花粉管长入胚囊之前卵器已解体,中央细胞中已形成胚乳游离核,因此也不发生双受精,由珠心细胞自发形成胚。这种现象是花椒和野花椒在长期进化过程中形成的一种十分特化的适应。     

APOMIXIS AND POLYEMBRYONY IN PACHIRA OLEAGINEA (BOMBACACEAE)

Baker , H. G. (U. California, Berkeley.) Apomixis and polyembryony in Pachira oleaginea (Bombacaceae) . Amer. Jour. Bot. 47(4): 296—302. Illus. 1960.–Trees identified as Pachira oleaginea Decne. are now widely cultivated in tropical and subtropical Africa. Specimens from Achimota, Ghana, are shown to contain 1—5 embryos in a single seed. The flowers, which open at night, are self-compatible but need to be pollinated for seed-setting to occur. Maturation of the fruit takes about 2 mo. but the seeds germinate soon after shedding. One embryo in each seed is usually much the largest and the only one to become established as a seedling. There is no relation between seed-size and numbers of embryos contained. Histological and cytogical studies show that adventitious embryony is responsible for the excess embryos following the formation of a normal sexually produced embryo (2n=72) and endosperm (3n=108). Adventitious embryos arise from single cells in the nucellus in the vicinity of the micropyle and appear to develop faster than the sexually produced embryo with consequent frequent apomictic reproduction. This is the first report of apomixis in the Malvales, which may have important practical consequences as P. oleaginea is a potential economic plant and may fruit only 3 yr. after sowing, rendering seed-propagation feasible.     

Light or high (30°C) temperature effects on l-[U-C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-¹⁴C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-¹⁴C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-¹⁴C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-¹⁴C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.     

黄花油点草胚胎发育研究

利用石蜡切片技术对百合科植物黄花油点草[Tricyrtis maculata(D.Don)Machride]双受精、胚及胚乳发育进行了研究,以明确其胚胎发育的特征,为百合科植物的系统研究提供生殖生物学资料。结果表明:(1)黄花油点草为珠孔受精;进入胚囊的2枚精子分别与卵细胞和中央细胞进行正常的双受精,其受精作用属有丝分裂前型。(2)受精后的初生胚乳核立即分裂,其发育方式为核型胚乳;早期的游离胚乳核沿胚囊的边缘分布,胚囊中央部位主要为胚乳细胞质,随着游离胚乳核数量的增加,胚乳核慢慢充满整个胚囊;当发育至球形胚早期阶段,在各胚乳核周围产生胚乳细胞壁,形成完整的胚乳细胞。(3)合子有较长的休眠时间,胚的发育方式为茄型;合子第一次有丝分裂为横裂,分裂后形成基细胞和顶细胞;基细胞经过3次横裂,形成一列胚柄细胞;顶细胞经过分裂形成胚体,依次形成球形胚、棒状胚和盾形胚。(4)种子成熟时胚无器官分化;成熟种子由种皮、胚和胚乳三部分组成。     

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.