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APO2001

A sexy apomixer in como

^a University of Zürich, CH-8008 Zürich, Switzerland
^b CINVESTAV-Plant Biotechnology Unit 36500 Irapuato, Mexico
^c University of Zürich, CH-8008 Zürich, Switzerland

Long before promiscuity was discovered to bear significant risk, many flowering plants had partially abandoned the pleasures of their sexual life to evolve one of the most intriguing reproductive alternatives found in nature. Apomixis is an asexual method of reproduction through seed that circumvents meiosis and fertilization to culminate in the autonomous development of an embryo. Thus, unlike sexual reproduction, which yields genetically diverse progeny, apomixis produces clonal offspring. The production of clonal, genetically identical, seed bears great potential for applications in plant breeding and seed production (Hanna and Bashaw, 1987; Savidan, 1992; Koltunow et al., 1995). Apomixis has evolved several times independently from sexual ancestors and can be viewed as a modification of the sexual reproductive program. In angiosperms, sexual reproduction entails complex interactions between a variety of tissues. Female reproductive development occurs in a specialized organ, the ovule, where usually a single cell becomes committed to the reproductive pathway (the megaspore mother cell, or MMC). After meiosis, a single reduced product, the functional megaspore, divides mitotically to form the mature female gametophyte or embryo sac. The usually seven-celled embryo sac contains the egg cell and the binucleate central cell, both of which get fertilized. In the male reproductive organs, meiosis produces a tetrad of reduced spores, all of which divide mitotically to form the male gametophytes (pollen). The male gametophyte consists of two sperm cells, which are contained in a large vegetative cell that delivers the sperm cells to the female gametophyte. During double fertilization, one sperm fuses with the egg to form the zygote and the second sperm fuses with the central cell to form the endosperm.

In apomictic plants, this sexual developmental program is bypassed or deregulated at various steps (Koltunow, 1993; Grossniklaus, 2001): (1) meiosis is altered or absent to produce an unreduced female gametophyte with the full complement of maternal chromosomes (apomeiosis); (2) fertilization is avoided, producing an autonomous embryo (parthenogenesis); and (3) endosperm development is initiated autonomously or sexually; in the latter case, embryo sac development or fertilization is often modified to adjust to a different genomic context (Savidan, 2000; Grossniklaus et al., 2001). In contrast to the modified female reproductive program, pollen formation usually is unaffected in apomicts.

During the last two decades, the introduction of apomixis into sexual crops has been perceived as one of the most promising challenges faced by agricultural biotechnology. Apomixis could allow the fixation of any genotype, however complex, including that of high yielding F1 hybrids. The enormous potential of this trait was realized as early as the 1930s by Navashin and Karpenko (cited by Asker, 1971). Simplified apomixis breeding programs would allow an immediate fixation of any genotype and the production of self-perpetuating improved hybrids and could promise social and economic benefits that would challenge those of the Green Revolution (Vielle-Calzada et al., 1996a; Grossniklaus et al., 1998a).

The 2nd International Conference on Apomixis took place in Como, Italy, from April 24 to 28, 2001. The scientific program was organized by Lucia Colombo, Thomas Dresselhaus, Yves Savidan, and Rod Scott and was sponsored by the European Union, the Food and Agriculture Organization of the United Nations, the Institut de Recherche pour de Développement (IRD), the Italian National Research Center, and many private sponsors. Following on the success of the 1st International Apomixis Meeting held at College Station, Texas, in 1995, the Como conference attracted 170 participants from 27 countries. All meeting abstracts of invited speakers and poster sessions are available at http://www.apomixis.de.

Leo Beukeboom (University of Leiden, The Netherlands) gave the opening lecture on "Origin and Genetics of Parthenogenesis in Animals." Asexual reproduction is a widespread phenomenon across the animal and plant kingdoms. There are differences in both the terminology and the mechanisms of asexual reproduction in plants and animals. Parthenogenesis (virgin birth), first observed by Bonnett in 1745, is the development of an egg without fertilization. Asexual reproduction in animals can occur either by mitotic parthenogenesis (apomixis) or by meiotic parthenogenesis (automixis). In contrast to the phenomenon in plants, apomixis in animals is rarely facultative, and most forms of automixis occur exclusively in animals. A wide range of cytological mechanisms underlie mitotic or meiotic parthenogenesis in animals. Beukeboom presented examples of asexual reproduction in freshwater flatworms (Polycelis nigra), in which B chromosomes may be associated with parthenogenetic lineages. Although much is known of the molecular mechanisms of fertilization in animals, remarkably little is known about the mechanisms of parthenogenesis.

MECHANISMS AND EVOLUTION OF APOMIXIS

Early studies demonstrated that apomixis is controlled genetically. Typically, a single dominant mendelian trait is associated with apomixis, although more complex modes of inheritance have been reported (see accompanying Insight article). Cytological descriptions emphasized distinctions between different apomictic mechanisms and have led to a simplified classification that is based on the origin and the location of cells initiating apomictic development (reviewed in Koltunow, 1993). In diplospory, the MMC undergoes an aberrant meiosis or divides mitotically, producing unreduced spores that eventually form the embryo sac containing an egg cell with the full genetic complement of the mother. In apospory, a cell in the ovule other than the MMC produces the unreduced embryo sac. Anna Koltunow (Commonwealth Scientific and Industrial Research Organization, Adelaide, Australia) described the enormous variability existing in apomictic processes. In Hieracium, apomictically derived embryo sacs usually are produced through apospory. However, several loci modify the timing of apomictic initiation, the frequency at which apomictic embryo sacs are formed, and the mode of progression of apomictic development (Koltunow et al., 1998, 2000; Bicknell et al., 2000). These findings indicate that the major locus associated with apomixis might create a competence for a variety of reproductive developmental processes in the ovule. Koltunow hypothesized that sporophytic cells in the ovule play an active role in modulating and controlling reproductive development. Transformation-induced alterations in ovule development that result in significant changes of the mode of apomixis in Hieracium add support to this hypothesis.

For Yves Savidan (IRD, Montpellier, France), close examination of developmental processes in the ovule indicates that apomictic embryo sacs develop more precociously than their sexually derived counterparts. However, embryo sac development is always normal, no matter how strongly meiotic processes are disturbed. This suggests a crucial role for the timing of developmental events during reproduction and implies that all types of gametophytic apomixis may relate to the ectopic expression of the same master regulatory gene(s). In this view, distinct mechanisms would result solely from differences in the timing of apomictic induction within the ovule. Daniel Grimanelli (IRD and Centro Internacional de Mejoramiento de Maíz y Trigó [CIMMYT], México) has cytologically analyzed the apomictic mechanism of Tripsacum dactyloides, focusing on chromosome and chromatin dynamics and the characteristics of the cytoskeleton during apomeiosis. A wide phenotypic variability occurs during apomictic initiation, even within a single genotype. Many of the developmental abnormalities characteristic of apomeiotic processes in Tripsacum have striking similarities to defects found in meiotic mutants of maize. Although these meiotic mutants have a strong impact on fertility, similar abnormalities do not compromise apomictic seed formation. This suggests that developmental events occurring after apomictic initiation can compensate for, and rescue, meiotic defects.

Whereas in Tripsacum the MMC proceeds directly to divide mitotically, in dandelions (Taraxacum sp) the MMC enters prophase of meiosis I without subsequent chromosome pairing. This attempted first division results in a restitution nucleus enclosing a complete set of chromosomes that undergoes the second meiotic division (diplospory). Hans de Jong (Wageningen University, The Netherlands) presented a detailed genetic analysis of apomixis based on interploidy crosses between sexual diploids and apomictic triploids of Taraxacum. The occurrence of recombinants in which the initiation of apomixis and the autonomous development of embryo and endosperm were uncoupled indicates that apomixis is controlled by at least three loci. A dominant trait linked to a microsatellite marker and located on one of the nucleolar organizer chromosomes appears to control diplospory. Interestingly, this marker is absent in sexual diploid progeny, suggesting that haploid pollen grains cannot transmit this trait. Emidio Albertini (University of Perugia, Italy) confirmed that apospory in Poa pratensis can be uncoupled from the mechanisms controlling the parthenogenetic development of embryos. Although parthenogenesis is seemingly contingent on apomeiosis, the reverse is not the case. The variability of the degree of parthenogenesis suggests that it is likely not controlled by a discrete locus and may be under complex control.

Apomixis is reported in more than 400 species belonging to 40 families. However, it is particularly prevalent within the Asteraceae, Poaceae, and Rosaceae. Focusing on the distribution and evolutionary history of apomixis, John Carman (Apomyx, Inc., Logan, UT) emphasized that only 127 of more than 14,000 genera of flowering plants contain apomictic species, most of which have appeared during or after the Pleistocene. Carman suggests that apomicts may have arisen by wide hybridization of ancestral sexual parents having distinct phenotypic traits related to reproduction (Carman, 2001). To test this hybridization-derived floral asynchrony hypothesis, Carman's group has documented variation of reproductive traits among sexual relatives of well-known apomicts (Tripsacum and Antennaria), finding that they are heterozygous and polygenic. Many forms of reproductive variation, including apomixis, may have arisen after hybridization of sexual ancestors with divergent reproductive traits. In many agamic complexes which are composed of individuals of varying ploidy levels, diploid genotypes usually are sexual and polyploid genotypes usually are apomictic. Tim Sharbel (Max Planck Institute for Chemical Ecology, Jena, Germany) studied the evolution of apomixis and polyploidy in the Arabis holboellii agamic complex. Using chloroplast haplotypes identified in diploid, aneuploid, and triploid individuals, he concluded that polyploidy arose repeatedly and independently within this complex (Sharbel and Mitchell-Olds, 2001). The variation in reproductive mode and population structure suggests that apomixis might have a single evolutionary origin, followed by multiple instances of phenotypic expression of this trait.

BREEDING APOMIXIS INTO SEXUAL CROPS

Among the grasses, apomixis occurs in several economically important forage genera (e.g., Pennisetum, Brachiaria, Paspalum, and Poa). At least three groups have attempted the introgression of apomixis into sexual crops via wide hybridization using backcrossing (BC) strategies combined with embryological or cytogenetic studies. For close to 20 years, Wayne Hanna (United States Department of Agriculture–Agricultural Research Service, Tifton, GA) and his colleagues have attempted the transfer of apomixis from Pennisetum squamulatum to pearl millet (P. glaucum) (Hanna et al., 1998). By screening large populations to identify partially male fertile apomictic plants, backcrossing has progressed to the BC₇ generation. Apomictic tetraploid BC₇ plants have 28 or 29 chromosomes and closely resemble pearl millet. However, they form very little viable seed, a problem possibly related to the dosage sensitivity of endosperm development (Morgan et al., 1998). Seed sterility may be overcome by exploiting the genetic diversity found within the tetraploid germplasm pool.

Following the pioneering work of D.F. Petrov, who realized the potential of introgressing apomixis into maize by wide hybridization more than 50 years ago, Victor Sokolov's group (Institute of Cytology and Genetics, Novosibirsk, Russia) attempted to transfer apomixis from T. dactyloides to maize using tetraploid female parents. F1 hybrids having 20 chromosomes from maize and 36 from Tripsacum were apomictic but male sterile. Recurrent backcrossing to male diploid or tetraploid maize resulted in apomictic genotypes invariably containing the same nine Tripsacum chromosomes. The absence of any additional Tripsacum chromosomes resulted in the loss of apomixis, suggesting polygenic control. Moreover, apomeiosis and parthenogenesis segregate and are controlled by different loci (Sokolov and Khatypova, 2001). Inspired by the work conducted in Russia, Yves Savidan launched an initiative to transfer apomixis from Tripsacum to maize in the early 1990s. Large population screening, flow cytometry, and genomic in situ hybridization were used to obtain BC₃ plants that have 20 chromosomes from maize and 18 from Tripsacum. Because little sexuality was present in BC₃ plants, the acquisition of subsequent BC populations was difficult (Savidan, 2000). A plant with a chromosome responsible for apomixis has yet to be found among the BC₄ generation. Olivier Leblanc (IRD-CIMMYT, Mexico) indicated that the maize genome severely alters the expression of apomixis in members of these BC populations. Specific attributes necessary for the proper expression of apomixis in maize could be related to gene dosage effects of specific modifiers or to parent-of-origin–dependent expression (i.e., genomic imprinting) of key regulatory genes that control embryo sac development and/or early seed formation.

ISOLATION OF GENES CONTROLLING APOMIXIS

Basic knowledge of the genetic and molecular regulation of female reproductive development in apomictic species remains poor. Although the in-heritance of the trait has been studied in several species, efforts to isolate the genes that control apomixis are at an early stage. The development of the genetic and molecular tools necessary to establish an apomictic model system is well under way (Bicknell, 1994, 2001), and the first mutants altered in apomictic developmental pathways have been isolated. Ross Bicknell and his team (Institute for Food and Crop Research, Lincoln, New Zealand) have implemented -ray and insertional mutagenesis strategies in Hieracium spp. At least two mutants have been isolated that have lost the ability to form apomictic seed but still reproduce sexually, indicating that apomixis and sexuality can be uncoupled in Hieracium. In both mutants, the differentiation of aposporous initials early during ovule development is affected. Further characterization promises insights into the molecular nature of the genes involved in apomictic initiation.

Peggy Ozias-Akins and co-workers (University of Georgia, Tifton) have mapped apomixis to a single locus in both Pennisetum ciliare and Pennisetum squamulatum (Ozias-Akins et al., 1998; Roche et al., 1999), two species related to pearl millet. Detailed analysis of P. squamulatum revealed no recombination between 12 markers and the apomixis locus. Interestingly, no sequences that cross-hybridize to four of these markers are present in sexual individuals of a segregating population, suggesting that the region is hemizygous or highly divergent in the apomicts. On the basis of marker conservation between the two species, bacterial artificial chromosome clones linked to apomixis were identified, revealing a series of duplications that might explain the absence of meiotic recombination in this chromosomal region. Informative bacterial artificial chromosome clones have been physically mapped using fluorescence in situ hybridization. Fulvio Pupilli's group (Consiglio Nazionale delle Richerche, Perugia, Italy) has mapped the apomixis locus in Paspalum simplex using selected rice probes as restriction fragment length polymorphism anchor markers and amplified fragment length polymorphism (AFLP) markers. They confirmed that apomixis segregates as a single dominant trait and identified five rice markers tightly linked to apomixis (Pupilli et al., 2001). As in Pennisetum, the genomic region linked to apomixis shows no signs of meiotic recombination. The authors concluded that the apomixis locus is contained in a chromosomal area that is syntenic to a 15-centimorgan region on rice chromosome 12.

Genes specific to apomictic development may be identified by comparing gene expression in sexual and apomictic ovules among closely related genotypes of the same species (Vielle- Calzada et al., 1996b). Sexual and apomictic genotypes have been characterized in the genus Brachiaria. Julio Rodrigues from Vera Carneiros's group (Embrapa, Brasilia, Brazil) reported on a differential display strategy to isolate differentially expressed transcripts during specific stages of ovary development in Brachiaria brizantha. Several dozen fragments were specific to either sexuals or apomicts and are being analyzed further. Similarly, Gianni Barcaccia (University of Padova, Italy) is investigating gene expression during flowering of mutants of alfalfa that fre-quently form unreduced 2n egg cells. A collection of 40 polymorphic cDNA-AFLP clones was isolated by differential display.

In a special lecture on "Approaches to Capturing Wild Apomictic Genes Combining Genetics and Genomics," Michael Freeling (University of California, Berkeley) presented a strategy to identify regulatory regions. Nick Kaplinski, a graduate student with Freeling, developed a sliding window–type algorithm that identifies regions of conserved noncoding sequences both within and between species. Such an inverse genetics approach can be used to identify regions that are under functional selective pressure.

OVULE AND FEMALE GAMETOPHYTE DEVELOPMENT

The initiation of apomictic development occurs at early stages of ovule development, during the differentiation of meiotic or apomeiotic products. What leads to the meiotic or apomeiotic commitment of cells within the ovule? Do megaspores sense their position and communicate with other sporophytic cells? It is becoming apparent that meiosis and female gametophyte development are controlled by regulatory genes that may be deregulated in space and time in apomicts (Koltunow, 1993; Grossniklaus, 2001). In fact, many events relevant to apomixis, such as the initiation of embryo sac development, autonomous activation of the egg cell, and modified fertilization mechanisms, are functions of the developing female gametophyte. Yet, we know little about the genetic control of these events, even in sexual model species. A better understanding of the molecular mechanisms underlying embryo sac development and its interactions with sporophytic cells in the ovule could enhance our understanding of apomixis dramatically.

Ueli Grossniklaus and his team (University of Zürich, Switzerland) use Arabidopsis as a model system to identify genes involved in megasporogenesis and embryo sac development. Using an enhancer detection strategy (Sundaresan et al., 1995), they identified mutants affected in female gametophyte development and double fertilization. Some of them may be relevant to the developmental alterations that ensure the normal endosperm formation observed in many apomicts. For example, a new signaling pathway between male and female gametophytes was identified by the feronia mutant: a wild-type pollen tube was unable to release its sperm cells into a mutant feronia embryo sac, suggesting an active role of the female gametophyte in this process. Grossniklaus and colleagues also identified genes expressed in specific cell types of the ovule and embryo sac, many of which encode regulatory proteins. Promoters controlling expression in the ovule at the site of apomictic initiation, the MMC, or the egg could be used to induce elements of apomixis by expressing candidate genes in specific cell types.

In Arabidopsis, female meiosis results in three cells that die, whereas a single megaspore produces the embryo sac. Wei-Cai Yang (Institute of Molecular Agrobiology, Singapore) used transposon mutagenesis (Sundaresan et al., 1995) to investigate cell fate determination among the megaspores. He identified a tagged mutant with embryo sacs consisting of large multinucleated cells. These cells may be derived from several surviving megaspores, indicating a defect in cell specification, or may result from aberrant cellularization events in the embryo sac. The isolation of regulatory genes implicated in cell fate determination may be related directly to the developmental alternatives associated with apomictic initiation. In that regard, the role of transcription factors implicated in the regulation of ovule development in sexual species is particularly relevant. The work of Rebecca Favaro and colleagues in Lucia Colombos's group (University of Milan, Italy) demonstrated that the expression of a MADS box gene (AGL11) during ovule and seed formation in Arabidopsis is controlled by an intron that is crucial for determining the temporal and spatial context in which AGL11 acts.

CELL CYCLE AND MEIOSIS

Critical aspects of cell cycle regulation and meiosis differ between apomicts and sexuals. Unlike sexually reproducing plants, apomicts do not undergo recombination during meiosis I, and they produce unreduced female gametes. Greater understanding of the (mis)regulation of the cell cycle and meiosis will facilitate the development of systems to induce parthogenesis and apomeiosis.

To maintain genetic stability and fertility, polyploid crops (e.g., wheat) must both pair and segregate the closely related chromosomes correctly during meiosis. In polyploids, homologous chromosomes must accurately distinguish each other from homeologous chromosomes. Peter Shaw (John Innes Centre, Norwich, UK) described how the Ph1 locus controls the specificity of somatic and meiotic chromosome association in wheat. The Ph1 locus restricts chromosome pairing and recombination to true homologs. Thus, in hexaploid wheat with Ph1 deletions, pairing and recombination can occur between homeologs and homologs. In the absence of Ph1, nonhomologously associated centromeres fail to separate at the beginning of meiosis. Shaw discussed how the Ph1 locus promotes the specificity rather than the induction of centromere association (Martinez-Perez et al., 2001).

Screens for male sterility in Arabidopsis by Hong Ma's group (Pennsylvania State University, University Park) identified genes regulating chromosome segregation during male meiosis. Two male sterile mutants (ask1 and sds) were shown to have abnormal chromosome segregation during meiosis I. Premature homolog dissociation occurs in the sds mutant near the end of prophase I, whereas the homologs seem to remain attached at anaphase I in the ask1 mutant. The ASK1 gene is most similar to yeast SKP1, which is a subunit of the SKp1-Cullin-1-F-box (SCF) ubiquitin ligase complex. Although the yeast and human SKP1 genes regulate the mitotic cell cycle, it was not known until recently that these proteins also could be required for meiosis (Yang et al., 1999). Molecular determination of the developmental mechanisms underlying male and female meiosis is under way by Tom Gerats' team (University of Gent, Belgium), which is undertaking cDNA-AFLP transcript profiling in Petunia. Transcript profiling techniques were applied to male meiosis, focusing on the identification of genes involved in synapsis and recombination. Reproducible cDNA-AFLP patterns were obtained using RNA from only three developing anthers. In a pilot study, 100 (5%) differentially expressed transcripts were identified, including previously characterized meiotic genes (e.g., DMC1-like protein).

Fifty years ago, Böcher reported unreduced pollen development in the apomict A. holboellii (Böcher, 1951). Alexei Kravtchenko (Russian Academy of Sciences, Novosibirsk, Russia) conducted a cytological reexamination of unreduced pollen formation in diploid and triploid A. holboellii accessions. The first meiotic division was equational, and omission of the second division produced unreduced microspores. For both triploid and diploid plants, pollen viability was highly variable. Yet, some triploids exhibited greater than 90% pollen fertility, an advantage for apomixis research on A. holboellii.

Although many meiotic mutants have been described in plants, only six genes involved in meiosis have been cloned. Christine Horlow (Institut National de la Recherche Agronomique, Versailles, France) showed that the Arabidopsis SWITCH1 (SWI1) protein is required for both sister chromatid cohesion and bivalent formation (Mercier et al., 2001). Horlow has identified a second swi1 allele (swi1-2) that affects both male and female meiosis, whereas swi1-1 was reported to be female specific (Montamayor et al., 2000). The cytological behavior of swi1-2 during metaphase in male meiocytes is intriguing: instead of the normal 5 bivalents, 20 chromatids are observed that segregate aberrantly.

Cell cycle control is a complex process that is mediated largely by protein kinases, which activate proteins with specific functions during the cell cycle. Danny Geelen (University of Gent, Belgium) reviewed current research on cell cycle progression that is under way in Dirk Inzé's laboratory (Joubes et al., 2000; Stals et al., 2000; de Veylder et al., 2001). Transgenic studies in which the function of key cell cycle regulators is disrupted demonstrated that the cell cycle is integrated with plant development. The Inzé group has conducted an AFLP-based screening for cell cycle genes in tobacco BY-2 cells using an aphidicoline blocker. Approximately 500 cell cycle–modulated expressed sequence tags (ESTs) have been identified, and 50% of the clones isolated exhibited no significant homology with known proteins. Jim Murray (University of Cambridge, UK) focused on the roles of D-type cyclins (cycD) in modulating cell division rates that affect growth and development (Meijer and Murray, 2000). The CycD genes play an important role in deciding whether a cell enters a division cycle. Cytokinin activates cell division through the induction of CycD3 at the G1-S cell cycle transition (Riou-Khamlichi et al., 1999). Overexpression of CycD2 reduces the length of the G1 phase, causing faster cell cycling and accelerated plant development (Cockcroft et al., 2000). The constitutive overexpression of the CycD2 cyclin in the shootmeristemless Arabidopsis mutant led to a restoration of vegetative growth and long-lived plants.

FERTILIZATION AND PARTHENOGENESIS

Parthenogenesis is a critical element of apomixis whereby an (unreduced) egg cell initiates embryogenesis without fertilization. The molecular mechanisms that trigger parthenogenesis in apomictic plants remain largely unknown. Helmut Bäumlein (Institut für Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany) discussed embryological and molecular studies on apomeiosis in Poa pratensis and parthenogenesis in wheat that his group conducted in close cooperation with Fritz Matzk. Genetic and ploidy analysis (Matzk et al., 2000) of crosses between obligate sexual and apomictic Poa lines demonstrated that apomixis was dominant and that apomeiosis and parthenogenesis are not linked. A molecular approach based on SMART subtractive suppression hybridization identified cDNAs from (apo)meiotic stages specific to obligate sexual and apomictic lines. The "Salmon system" of wheat produces a high fraction of parthenogenetic offspring, and isogenic sexual lines are available (Matzk, 1996). Bäumlein and co-workers isolated candidate egg cell–specific genes from an egg cell cDNA library of these sexual and parthenogenetic lines.

In apomicts, embryogenesis occurs completely without the contribution of the paternal genome. Thomas Dresselhaus (University of Hamburg, Germany) presented data suggesting that maize differs from Arabidopsis with regard to the time of activation of the paternal genome during early embryogenesis (Vielle-Calzada et al., 2000). In maize, the switch from maternal to zygotic control of gene expression appears to occur 18 to 24 hr after fertilization. Using reproductive cells isolated in vitro, Dresselhaus and co-workers developed a procedure that allows for the isolation of mRNAs present in individual cell types of the female gametophyte or the zygote. This approach led to the identification of differentially expressed genes (Cordts et al., 2001) and is being implemented in Tripsacum, allowing comparative studies of gene expression between sexual and apomictic cells.

Jean-Emmanuel Faure (Ecole Normale Superieure, Lyon, France) presented a cytological study of Arabidopsis fertilization using confocal laser scanning microscopy (Christensen et al., 1997). Characterization of the time course of fertilization indicates that (1) synergids degenerate at 7 hr after pollination (HAP), (2) there is a rapid change in egg cell polarity, (3) karyogamy occurs 8 to 9 HAP, and (4) the first division of the primary endosperm nucleus occurs 9 to 12 HAP. Faure's group identified mutants that affect fertilization among 4000 -ray–mutagenized M1 plants. They confirmed phenotypes for a collection of 200 families containing putative early fertilization mutants. Maura Cardarelli (University La Sapienza, Rome, Italy) discussed the effects of the Agrobacterium rhizogenes rolB gene on gamete development and embryogenesis. rolB expression driven by AtDMC1 caused sterility that was likely attributable to a delay in anther dehiscence, whereas rolB expression under the control of the FBP7 promoter caused floral defects and a slight delay in anther dehiscence.

EMBRYOGENESIS

Despite the existence of large collections of mutants that affect plant embryogenesis, the molecular basis underlying the developmental steps leading to egg cell activation and early embryo development remains poorly understood. Bob Goldberg (The Seed Institute and University of California, Los Angeles) discussed a genomics approach toward the understanding of plant embryogenesis. The giant embryos of scarlet runner bean allowed the microdissection and isolation of cDNAs from either the embryo proper or the suspensor. EST sequencing was used to identify differentially expressed genes in the embryo proper (2863 ESTs, 50% unique) and the suspensor (3138 ESTs, 59% unique). Approximately 10 to 12% of the ESTs exhibited no homology with genes currently in databases. On the basis of these expression data, the suspensor appears to be the major source of gibberellic acid in the developing embryo. The Seed Institute also uses Affymetrix chips for transcript profiling in Arabidopsis wild-type and mutant seed. The profiles show highly complex changes during the early steps of seed development.

Sacco de Vries (Wageningen University, The Netherlands) discussed the role of the Arabidopsis somatic embryogenesis receptor–like kinase (AtSERK1) in ovule and embryo development. SERK encodes a leucine-rich repeat transmembrane receptor kinase (Schmidt et al., 1997) that is similar to animal receptors. The use of fluorescence spectral imaging microscopy to study physical interactions in plant cells showed that AtSERK1 can oligomerize in vivo (Shah et al., 2001) and interact directly with the kinase-associated protein phosphatase (KAPP) phosphatase. Plants overexpressing AtSERK1 have an increased potential for somatic embryogenesis in culture. To investigate the potential to induce embryogenesis in planta, the de Vries team developed a multiplex marker system for testing AtSERK1–overexpressing F2 plants. Fixed heterozygosity is indicative of apomixis. Indeed, lines expressing AtSERK1 in the ovule produced progeny without a recombination event among 14 markers tested, whereas control crosses did not yield such progeny among more than 1000 F2 plants. This could be attributable to apomictic embryo initiation or suppressed recombination in these plants. Ed Schmidt (Genetwister Technologies, Wageningen, The Netherlands) focused on the family of transmembrane Receptor Kinases–like SERK (RKS) genes. There are 16 RKS members in Arabidopsis, which can be grouped into three classes. Genetwister Technologies characterizes the developmental function of the RKS family using transgenic plants, which either overexpress or cosuppress the different RKS genes.

Kim Boutilier's presentation (Plant Research International, Wageningen, The Netherlands) focused on the engineering of adventitious embryony. Using subtractive hybridization, Boutilier isolated an embryo-expressed gene called BABY BOOM (BBM) from microspore embryo cultures of Brassica napus. BBM encodes an AP2 domain transcription factor. When expressed constitutively, it induces the spontaneous formation of somatic embryos in young seedlings. Because the BBM overexpression phenotype is restricted to seedlings, Boutilier uses tissue-specific promoters with the aim of inducing somatic embryos in ovules. Although L1 expression of BBM extends the tissue range and penetrance of somatic embryo production, it is still restricted to seedlings. BBM-expressing lines show a cytokinin overproduction phenotype, and mutants such as amp1 (Chin-Atkins et al., 1996) and other backgrounds affecting cytokinin levels enhance the BBM somatic embryo phenotype.

Gabriella Consonni (Universita degli Studi di Milano, Italy) presented a characterization of the maize mutant fused leaves (fdl). The fdl mutant causes organ fusion and affects both embryo organization and seedling growth: epidermal cells of the coleoptile and first leaf, and the first and second leaves are joined by a single cell wall.

ENDOSPERM DEVELOPMENT

For agricultural applications, it is essential that endosperm development in engineered apomicts is normal. Most apomictic seed formation requires fertilization of the central cell (pseudogamy). This poses a problem for the transfer of apomixis to sexual crops because maize and likely most cereals require a ratio of maternal to paternal genomes of 2m:1p for normal endosperm development (Lin, 1984). Fertilization of an unreduced central cell with a reduced sperm will yield a 4m:1p ratio, which is expected to cause seed abortion. In contrast, natural apomicts are either insensitive to unbalanced endosperm or circumvent the problem by altering embryo sac development or the mechanism of fertilization (Grossniklaus et al., 2001).

Abed Chaudhury (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia) provided an overview of ongoing research on the fis (fertilization-independent seed) class of Arabidopsis mutants. The FIS class includes MEDEA (MEA), FIS2, and FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) (Grossniklaus et al., 1998b; Luo et al., 1999; Ohad et al., 1999). Mutations in these genes lead to maternal effect seed abortion, and mutant central cells are capable of endosperm development in the absence of fertilization, a component of apomixis (reviewed in Grossniklaus et al., 2001). Chaudhury's group made promoter::GUS fusions for all three FIS class genes, showing that the expression of MEA and FIS2 is similar but differs significantly from the FIE pattern (Luo et al., 2000). Only maternally inherited copies are expressed early in seed development, suggesting regulation by genomic imprinting. Seed abortion can be prevented if a demethylated paternal genome is introduced. This effect is independent of FIS gene activity (Luo et al., 2000). Interestingly, a demethylated paternal genome appears to have an effect on gene expression from the maternal genome.

Robert Fischer (The Seed Institute and University of California, Berkeley) focused on the suppression of endosperm development by the FIS genes. FIE and MEA encode WD40 and suvar3-9, enhancer-of-zeste, Trithorax (SET) domain proteins of the Polycomb group. FIE and MEA proteins interact directly, like their mammalian and Drosophila homologs, which form a multiprotein complex (Luo et al., 2000; Spillane et al., 2000; Yadegari et al., 2000). Fischer reviewed data showing silencing of the paternal mea locus in the endosperm (Kinoshita et al., 1999; Vielle-Calzada et al., 1999). He discussed how these findings fit with the parental conflict theory for the evolution of genomic imprinting (Haig and Westoby, 1991) and with the observation that many apomicts require fertilization of the central cell for endosperm formation.

Problems associated with the engineering of autonomous endosperm in apomicts also were reviewed by Rod Scott (University of Bath, UK). Endosperm abortion may be caused by the nonequivalence of paternal and maternal genomes at imprinted loci. Autonomous endosperm development observed in the Arabidopsis fie mutant can be enhanced by combining it with hypomethylation (Vinkenoog et al., 2000). On the basis of the parental conflict theory (Haig and Westoby, 1991), Scott proposes that endosperm size in plants is an assay for gametic "gender" as exemplified by interploidy crosses (Scott et al., 1998). In Arabidopsis, modifications of gametic gender may be achieved through changes in the methylation profile, which either "maternalize" or "paternalize" the gametic genome, or as a result of mutants such as fie, which paternalize maternal gametes (Adams et al., 2000; Vinkenoog et al., 2000).

Fred Berger (Institut National de la Recherche Agronomique, Lyon, France) discussed the recent work of Boisnard-Lorig et al. (2001), in which a HISTONE::YFP fusion protein was used to demonstrate that syncytial endosperm is divided into three distinct mitotic domains. Enhancer detection screens (Haseloff, 1999) were used to isolate GFP-based endosperm markers, such as KS117, which is expressed initially in the entire endosperm but then is restricted to the chalazal region. Using this marker, it was found that in fis class mutants endosperm polarization is disturbed (Sorensen et al., 2001). The KS117 line has been used in a -ray mutagenesis screen for mutants that alter the expression of KS117. Among 4000 M1 plants, 60 lines with disturbed KS117 GFP expression were identified.

José Gutierrez (University of Oxford, UK) presented the isolation of candidate imprinted genes that are expressed differentially in maize endosperm depending on parental origin. Allelic display polymerase chain reaction was used to screen for imprinted maize genes in reciprocal crosses between different inbred lines of maize. Loci that exhibit either maternal-specific or paternal-specific expression were identified at early endosperm stages (10 days after pollination; 15 candidates) and at later endosperm stages (30 days after pollination; 31 candidates). The group of Angelo Viotti (Consiglio Nazionale delle Richerche, Milan, Italy) investigates epigenetic phenomena in maize endosperm. Some alleles of the -zein and -tubulin genes derived from specific inbred lines are subject to genomic imprinting (Lund et al., 1995a, 1995b). Two of the six -tubulin genes displayed a correlation between DNA demethylation at the locus and increased RNA accumulation in the endosperm. -zein and -tubulin genes are hypomethylated only when transmitted through the female. Viotti's results confirm that the imprinting status of certain zein and tubulin alleles in maize endosperm can be influenced by interactions of parental factors and are cross and genotype dependent (Ciceri et al., 2000).

Hilde-Gunn Opsahl-Ferstad (Agricultural University of Norway, Aas) focused on the defective kernel1 (dek1) and crinkly4 (cr4) genes that regulate cell identity in the cereal endosperm (Becraft et al., 2001; Olsen, 2001). The Olsen group is studying several of the Pioneer Hi-Bred "Trait Utility System for Maize" Mutator-induced mutants to isolate genes involved in the control of aleurone cell identity. Promoter studies of the LIPID TRANSFER PROTEIN1 (LTP1) and LTP2 upstream sequences have shown LTP2 to direct expression in cereals similar to the patterns described in dicots, whereas the LTP1 directs a different expression pattern.

ECONOMIC AND ECOLOGICAL IMPLICATIONS OF APOMIXIS

The long gestation period for the development of apomixis technology (Savidan, 2000) has allowed reflection on both economic (Jefferson, 1994; Bicknell and Bicknell, 1999) and ecological issues (van Dijk and van Damme, 2000) regarding the deployment of apomictic crops in agriculture. Apomictic varieties are being developed for a few (mainly forage grass) species. Jorge Gonzalez (Universidad Autónoma Agraria Antonio Narro, Saltillo, México) presented such a breeding program for new disease-tolerant buffelgrass (Penn. ciliare) cultivars. Contiguous planting of a single cultivar of this obligate apomict over large geographic areas in the United States and Mexico since 1949 led to genetic vulnerability to blight disease. To develop new disease-resistant cultivars, Gonzalez and colleagues crossed sexual lines with the apomictic clone Zaragoza-115. Progeny testing and multilocation evaluation trials over several years led to the release of the hybrid AN-17-PS for commercial seed production last year.

Given current controversies regarding transgenic crop plants in agriculture, Peter van Dijk (Netherlands Institute of Ecology, Heteren) suggested that biosafety issues, which will undoubtedly arise (Ellstrand, 2001), should be considered well in advance of future apomixis technology deployment. He looked at the deployment of apomixis technology from an evolutionary and population genetics viewpoint (van Dijk and van Damme, 2000). Three hypothetical problems were identified with apomictic crops: (1) invasive weeds, (2) novel weeds, and (3) infectious apomixis, which might reduce genetic diversity by freezing the gene pool. Overall, van Dijk concluded that if pollen production is necessary in engineered apomictic crops, pollen flow of a dominant apomixis transgene should be prevented. The use of inducible or conditional systems to control apomixis could be a means to prevent such gene flow.

In recent years, there has been an increased industrial interest in the development of apomixis technology. Marc Albertsen (Pioneer Hi-Bred, IA) provided a well-balanced perspective on the factors that will determine how apomixis is deployed in commercial or subsistence farming. Albertsen raised the question of who will have access to proprietary apomixis technology (and the necessary supporting technologies) and under what terms. Both intellectual property management and freedom-to-operate issues will have a major bearing on which beneficiaries will have access to apomixis technology. From a strictly commercial perspective, approaches are needed that allow financial returns on technology investment and to ensure that "donated" technology is not used competitively against the original developers and their customers (G. Graaf, A. Bennett, B. Wright, and D. Zilberman, unpublished data; see http://www.cnr.berkeley.edu/csrd/technology/ipcmech/). From a less commercial perspective, there are humanitarian arguments for the provision of proprietary technologies to improve the well-being of poorer clients such as subsistence farmers in developing countries. Albertsen concluded that no single approach may encompass both requirements and that new strategies to deal with intellectual property rights and licensing practices will have to be developed.

Richard Jefferson (Center for the Application of Molecular Biology to International Agriculture, Canberra, Australia) continued the theme of how apomixis technology will be applied in agriculture by focusing on the challenge to deliver apomixis technology as a public good. This issue arose at a meeting funded by the Rockefeller Foundation in 1998 when the Bellagio Declaration was issued by many of the leading apomixis researchers (http://billie.btny.purdue.edu/apomixis). Jefferson focused on how current intellectual property management limits the application of enabling technologies such as apomixis to solely commercial objectives. He expressed concern that, unless the research community paid greater attention to the terms of research agreements and promoted nonexclusive licensing, a small number of multinational companies could exercise monopolistic control over apomixis tech-nology worldwide. The current exclusionary intellectual property management of both the private and public sector are nonsustainable business strategies. Many publicly funded bodies are adopting exclusive licensing models that are more suited to commercial than to public good objectives. Access to proprietary enabling technologies could be achieved by a consortium approach (enabling technology cooperative) that would develop proprietary technologies but adopt a broad nonexclusive licensing policy, allowing it to act as a clearinghouse for innovative proprietary technologies (G. Graaf, A. Bennett, B. Wright, and D. Zilberman, unpublished data; see http://www.cnr.berkeley.edu/csrd/technology/ipcmech/).

CONCLUSION

Research presented at the 2nd International Apomixis Conference coalesced around several themes, some of which are new and some of which have a long but unfinished history in apomixis research. A wide range of topics—the importance of existing apomictic mechanisms and their evolutionary implications, the isolation of genes controlling apomixis, the molecular and genetic basis of ovule and female gametophyte development, and the economic and ecological implications of apomixis—were discussed during plenary talks. It is increasingly apparent that sexuality and apomixis are interrelated and that they need to be investigated simultaneously to obtain a complete understanding of plant reproduction at the developmental, ecological, and evolutionary levels. Indeed, a number of changes were evident in the content and focus of apomixis research since the 1st International Apomixis Meeting. There is now a greater acceptance that genetic and molecular approaches to study sexuality can yield insights into the regulation and components of apomixis. This was particularly evident in the number of new groups entering this field. In addition to the impact of sexual model species such as Arabidopsis and maize, it is evident that a number of apomicts (Hieracium, Taraxacum, Tripsacum, Paspalum, and A. holboellii) are emerging as models that are amenable to molecular and genetic analyses. A number of new techniques (e.g., flow cytometry, transcript profiling, differential screening methods, and enhancer detection) will increase our knowledge of apomixis in the coming years and bring us a step closer to the engineering of apomixis in sexual crops.

Footnotes

¹ grossnik@botinst.unizh.ch

References

Adams, S., Vinkenoog, R., Spielman, M., Dickinson, H.G., and Scott, R.J. (2000). Parent-of-origin effects on seed development in Arabidopsis thaliana require DNA methylation. Development 127, 2493–2502.[Abstract]

Asker, S. (1971). Some viewpoints on Fragaria x Potentilla intergeneric hybridization. Hereditas 67, 181.

Becraft, P.W., Brown, R.C., Lemmon, B.E., Olsen, O.-A., and Opsahl-Ferstad, H.-G. (2001). Developmental biology of endo-sperm development. In Current Trends in the Embryology of Angiosperms, S.S. Bhojwani and S.S. Soh, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 353–374.

Bicknell, R.A. (1994). Hieracium: A model system for studying the molecular genetics of apomixis. Apomixis Newsletter 7, 8–10.

Bicknell, R.A. (2001). Model systems to study the genetics and developmental biology of apomixis. In The Flowering of Apomixis: From Mechanisms to Genetic Engineering, Y. Savidan, J.G. Carman, and T. Dresselhaus, eds (Mexico, D.F.: CIMMYT, IRD, European Commission DG VI), pp. 111–120.

Bicknell, R.A., and Bicknell, K.B. (1999). Who will benefit from apomixis? Biotechnology and Development Monitor 37, 17–20.

Bicknell, R., Borst, N.K., and Koltunow, A.M. (2000). Monogenic inheritance of apomixis in two Hieracium species with distinct developmental mechanisms. Heredity 84, 228–237.

Böcher, T.W. (1951). Cytological and embryological studies in the amphi-apomictic Arabis holboellii complex. Danske Videnskabernes Selskab Biologiske Skrifter 6, 2–58.

Boisnard-Lorig, C., Colon-Carmona, A., Bauch, M., Hodge, S., Doerner, P., Bancharel, E., Dumas, C., Haseloff, J., and Berger, F. (2001). Dynamic analyses of the expression of the histone::yfp fusion protein in Arabidopsis show that syncytial endosperm is divided in mitotic domains. Plant Cell 13, 495–509.[Abstract/Free Full Text]

Bonnet, C. (1745) Traite d'Insectologie ou Observations sur les Pucerons. (Paris: Durand).

Carman, J.G. (2001). The gene effect: Genome collision and apomixis. In The Flowering of Apomixis: From Mechanisms to Genetic Engineering, Y. Savidan, J.G. Carman, and T. Dresselhaus, eds (Mexico, D.F.: CIMMYT, IRD, European Commission DG VI), pp. 95–110.

Chin-Atkins, A.N., Hocart, C.H., Letham, D.S., Craig, S., Dennis, E.S., and Chaudhury, A.M. (1996). Increased en-dogenous cytokinins in the Arabidopsis amp-1 mutant corresponds with de-etiolation responses. Planta 198, 549–556.[Web of Science]

Christensen, C.A., King, E.J., Jordan, J.R., and Drews, G.N. (1997). Megagametogenesis in Arabidopsis wild type and the Gf mutant. Sex. Plant Reprod. 10, 49–64.[CrossRef]

Ciceri, P., Castelli, S., Lauria, M., Lazzari, B., Genga, A., Bernard, L., Sturaro, M., and Viotti, A. (2000). Specific combinations of zein genes and genetic backgrounds influence the transcription of the heavy-chain zein genes in maize opaque-2 endosperms. Plant Physiol. 124, 451–460.[Abstract/Free Full Text]

Cockcroft, C.E., den Boer, B.G., Healy, J.M., and Murray, J.A. (2000). Cyclin D control of growth rate in plants. Nature 405, 575–579.[CrossRef][Medline]

Cordts, S., Bantin, J., Wittich, P., Kranz, E., Lörz, H., and Dresselhaus, T. (2001). ZmES genes encode peptides with structural homology to defensins and are specifically expressed in the female gametophyte of maize. Plant J. 25, 103–114.[CrossRef][Web of Science][Medline]

de Veylder, L., Beemster, G.T., Beeckman, T., and Inzé, D. (2001). CKS1At overexpression in Arabidopsis thaliana inhibits growth by reducing meristem size and inhibiting cell-cycle progression. Plant J. 25, 617–626.[CrossRef][Web of Science][Medline]

Ellstrand, N.C. (2001). When transgenes wander, should we worry? Plant Physiol. 125, 1543–1545.[Free Full Text]

Grossniklaus, U. (2001). From sexuality to apomixis: Molecular and genetic ap-proaches. In The Flowering of Apomixis: From Mechanisms to Genetic Engineering, Y. Savidan, J.G. Carman, and T. Dresselhaus, eds (Mexico, D.F.: CIMMYT, IRD, European Commission DG VI), pp. 168–211.

Grossniklaus, U., van Lookeren Campagne, M.M., and Koltunow, A.M. (1998a). A bright future for apomixis. Trends Plant Sci. 3, 415–416.[CrossRef]

Grossniklaus, U., Vielle-Calzada, J.-P., Hoeppner, M., and Gagliano, W. (1998b). Maternal control of embryogenesis by MEDEA, a Polycomb-group gene in Arabidopsis. Science 280, 446–450.[Abstract/Free Full Text]

Grossniklaus, U., Spillane, C., Page, D.R., and Köhler, C. (2001). Genomic imprinting and seed development: Endosperm formation with and without sex. Curr. Opin. Plant Biol. 4, 21–27.[CrossRef][Web of Science][Medline]

Haig, D., and Westoby, M. (1991). Genomic imprinting in endosperm: Its effect on seed development in crosses between species, and between different ploidies of the same species, and its implications for the evolution of apomixis. Philos. Trans. R. Soc. Lond. Ser. B 333, 1–13.[CrossRef]

Hanna, W.W., and Bashaw, E.C. (1987). Apomixis: Its identification and use in plant breeding. Crop Sci. 27, 1136–1139.[Abstract/Free Full Text]

Hanna, W., Roche, D., and Ozias-Akins, P. (1998). Apomixis in crop improvement: Traditional and molecular approaches. In Advances in Hybrid Rice Technology. Proceedings of the 3rd International Symposium on Hybrid Rice 1996, S.S. Virmani, E.A. Siddiq, and K. Muralidharan, eds (Manila, Philippines: International Rice Research Institute Press), pp. 283–296.

Haseloff, J. (1999). GFP variants for multispectral imaging of living cells. Methods Cell Biol. 58, 139–151.[Web of Science][Medline]

Jefferson, R.A. (1994). Apomixis: A social revolution for agriculture? Biotechnology and Development Monitor 19, 14–16.

Joubes, J., Chevalier, C., Dudits, D., Heberle-Bors, E., Inzé, D., Umeda, M., and Renaudi, J.P. (2000). CDK-related protein kinases in plants. Plant Mol. Biol. 43, 607–620.[CrossRef][Web of Science][Medline]

Kinoshita, T., Yadegari, R., Harada, J.J., Goldberg, R.B., and Fischer, R.L. (1999). Imprinting of the MEDEA Polycomb gene in the Arabidopsis endosperm. Plant Cell 11, 1945–1952.[Abstract/Free Full Text]

Koltunow, A.M. (1993). Apomixis: Embryo sacs and embryos formed without meiosis or fertilization in ovules. Plant Cell 5, 1425–1437.[Free Full Text]

Koltunow, A.M., Bicknell, R.A., and Chaudhury, A.M. (1995). Apomixis: Molec-ular strategies for the generation of genetically identical seeds without fertilization. Plant Physiol. 108, 1345–1352.[CrossRef][Web of Science][Medline]

Koltunow, A.M., Johnson, S.D., and Bicknell, R.A. (1998). Sexual and apomictic development in Hieracium. Sex. Plant Reprod. 11, 213–230.[CrossRef]

Koltunow, A.M., Johnson, S.D., and Bicknell, R.A. (2000). Apomixis is not developmentally conserved in related, genetically characterized Hieracium plants of varying ploidy. Sex. Plant Reprod. 12, 253–266.[CrossRef]

Lin, B.-Y. (1984). Ploidy barrier to endosperm development in maize. Genetics 107, 103–115.[Abstract/Free Full Text]

Lund, G., Ciceri, P., and Viotti, A. (1995a). Maternal-specific demethylation and ex-pression of specific alleles of zein genes in the endosperm of Zea mays L. Plant J. 8, 571–581.[CrossRef][Web of Science][Medline]

Lund, G., Messing, J., and Viotti, A. (1995b). Endosperm-specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea mays L. Mol. Gen. Genet. 246, 716–722.[CrossRef][Medline]

Luo, M., Bilodeau, P., Koltunow, A.M., Dennis, E.S., Peacock, W.J., and Chaudhury, A.M. (1999). Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 96, 296–301.[Abstract/Free Full Text]

Luo, M., Bilodeau, P., Dennis, E.S., Peacock, W.J., and Chaudhury, A.M. (2000). Expression and parent-of-origin effects for FIS2, MEA, and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. USA 97, 10637–10642.[Abstract/Free Full Text]

Martinez-Perez, E., Shaw, P., and Moore, G. (2001). The Ph1 locus is needed to ensure specific somatic and meiotic centromere association. Nature 411, 204–206.[CrossRef][Medline]

Matzk, F. (1996). The "Salmon system" of wheat: A suitable model for apomixis research. Hereditas 125, 299–301.

Matzk, F., Meister, A., and Schubert, I. (2000). An efficient screen for reproductive pathways using mature seeds of monocots and dicots. Plant J. 21, 97–108.[CrossRef][Web of Science][Medline]

Meijer, M., and Murray, J.A.H. (2000). The role and regulation of D-type cyclins in the plant cell cycle. Plant Mol. Biol. 43, 621–633.[CrossRef][Web of Science][Medline]

Mercier, R., Vezon, D., Bullier, E., Motamayor, J.C., Sellier, A., Pelletier, G., and Horlow, C. (2001). The Arabidopsis SWI1 protein is required for both chromatid arm and centromere cohesion during meiosis. Genes Dev., in press.

Montamayor, J.C., Vezon, D., Bajon, C., Sauvanet, A., Grandjean, O., Marchand, M., Bechtold, N., Pelletier, G., and Horlow, C. (2000). Switch1 (swi1), an Arabidopsis mutant affected in the female meiotic switch. Sex. Plant Reprod. 12, 209–218.[CrossRef]

Morgan, R.N., Ozias-Akins, P., and Hanna, W.W. (1998). Seed set in an apomictic BC3 pearl millet. Int. J. Plant Sci. 159, 89–97.[CrossRef]

Ohad, N., Yadegari, R., Margossian, L., Hannon, M., Michaeli, D., Harada, J.J., Goldberg, R.B., and Fischer, R.L. (1999). Mutations in FIE, a WD polycomb group gene, allow endosperm development without fertilization. Plant Cell 11, 407–416.[Abstract/Free Full Text]

Olsen, O.A. (2001). Endosperm development: Cellularization and cell fate specification. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 233–267.[CrossRef][Web of Science][Medline]

Ozias-Akins, P., Roche, D., and Hanna, W.W. (1998). Tight clustering and hemizygosity of apomixis-linked molecular markers in Pennisetum squamulatum implies genetic control of apospory by a divergent locus that may have no allelic form in sexual genotypes. Proc. Natl. Acad. Sci. USA 95, 5127–5132.[Abstract/Free Full Text]

Pupilli, F., Labombarda, P., Caceres, M.E., Quarin, Q.L., and Arcioni, S. (2001). The chromosome segment related to apomixis in Paspalum simplex is homoeologous to the telomeric region of the long arm of rice chromosome 12. Mol. Breed., in press.

Riou-Khamlichi, C., Huntley, R., Jacqmard, A., and Murray, J.A. (1999). Cytokinin activation of Arabidopsis cell division through a D-type cyclin. Science 283, 1541–1544.[Abstract/Free Full Text]

Riou-Khamlichi, C., Menges, M., Healy, J.M., and Murray, J.A. (2000). Sugar control of the plant cell cycle: Differential regulation of Arabidopsis D-type cyclin gene expression. Mol. Cell. Biol. 20, 4513-4521.[Abstract/Free Full Text]

Roche, D., Cong, P., Chen, Z., Hanna, W.W., Gustine, D.L., Sherwood, R.T., and Ozias-Akins, P. (1999). An apospory-specific genomic region is conserved between buffelgrass (Cenchrus ciliaris L.) and Pennisetum squamulatum Fresen. Plant J. 19, 203–208.[CrossRef][Web of Science][Medline]

Savidan, Y. (1992). Progress in research on apomixis and its transfer to major grain crops. In Reproductive Biology and Plant Breeding, Y. Dattée, C. Dumas, and A. Gallais, eds (Berlin: Springer-Verlag), pp. 269–279.

Savidan, Y. (2000). Apomixis: Genetics and breeding. Plant Breed. Rev. 18, 13–86.

Schmidt, E.D., Guzzo, F., Toonen, M.A., and de Vries, S.C. (1997). A leucine-rich repeat containing receptor-like kinase marks somatic plant cells competent to form embryos. Development 124, 2049–2062.[Abstract]

Scott, R.J., Spielman, M., Bailey, J., and Dickinson, H.G. (1998). Parent-of-origin effects on seed development in Arabidopsis thaliana. Development 125, 3329–3341.[Abstract]

Shah, K., Gadella, T.W.J., van Erp, H., Hecht, V., and de Vries, S.C. (2001). Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein. J. Mol. Biol. 309, 653–667.

Sharbel, T.F., and Mitchell-Olds, T. (2001). Recurrent polyploid origins and biogeographical variation in the Arabis holboellii complex (Brassicaceae). Heredity, in press.

Sokolov, V.A., and Khatypova, I.V. (2001). The development of apomictic maize: Update, problems and perspective. Genetika 32, in press.

Sorensen, M.B., Chaudhury, A.M., Robert, H., Bancharel, E., and Berger, F. (2001). Polycomb group genes control pattern formation in plant seed. Curr. Biol. 11, 277–281.[CrossRef][Web of Science][Medline]

Spillane, C., MacDougall, C., Stock, C., Köhler, C., Vielle-Calzada, J.-P., Nunes, S.M., Grossniklaus, U., and Goodrich, J. (2000). Interaction of the Arabidopsis Polycomb group proteins FERTILIZATION-INDEPENDENT ENDOSPERM and MEDEA mediates their common phenotypes. Curr. Biol. 10, 1535–1538.[CrossRef][Web of Science][Medline]

Stals, H., Casteels, P., van Montagu, M., and Inzé, D. (2000). Regulation of cyclin-dependent kinases in Arabidopsis thaliana. Plant Mol. Biol. 43, 583–593.[CrossRef][Web of Science][Medline]

Sundaresan, V., Springer, P.S., Volpe, T., Haward, S., Jones, J.D.G., Dean, C., Ma, H., and Martienssen, R.A. (1995). Patterns of gene action in plant development revealed by enhancer trap and gene trap transposable elements. Genes Dev. 9, 1797–1810.[Abstract/Free Full Text]

van Dijk, P., and van Damme, J. (2000). Apomixis technology and the paradox of sex. Trends Plant Sci. 5, 81–84.[CrossRef][Medline]

van Dijk, P., Tas, I.C.Q., Falque, M., and Bakx-Schoman, T. (1999). Crosses between sexual and apomictic dandelions (Taraxacum). II. The breakdown of apomixis. Heredity 83, 715–721.

Vielle-Calzada, J.P., Crane, C.F., and Stelly, D.M. (1996a). Apomixis: The asexual revolution. Science 274, 1322–1323.[Free Full Text]

Vielle-Calzada, J.-P., Nuccio, M.L., Budiman, M.A., Thomas, T.L., Burson, B.L., Hussey, M.A., and Wing, R.A. (1996b). Comparative gene expression in sexual and apomictic ovaries of Pennisetum ciliare (L.) Link. Plant Mol. Biol. 32, 1085–1092.[CrossRef][Web of Science][Medline]

Vielle-Calzada, J.-P., Thomas, J., Spillane, C., Coluccio, A., Hoeppner, M.A., and Grossniklaus, U. (1999). Maintenance of genomic imprinting at the Arabidopsis medea locus requires zygotic DDM1 activity. Genes Dev. 13, 2971–2982.[Abstract/Free Full Text]

Vielle-Calzada, J.-P., Baskar, R., and Grossniklaus, U. (2000). Delayed activation of the paternal genome during seed development. Nature 404, 91–94.[CrossRef][Medline]

Vinkenoog, R., Spielman, M., Adams, S., Fischer, R.L., Dickinson, H.G., and Scott, R.J. (2000). Hypomethylation promotes autonomous endosperm development and rescues postfertilization le-thality in fie mutants. Plant Cell 12, 2271–2282.[Abstract/Free Full Text]

Yadegari, R., Kinoshita, T., Lotan, O., Cohen, G., Katz, A., Choi, Y., Katz, A., Nakashima, K., Harada, J.J., Goldberg, R.B., Fischer, R.L., and Ohad, N. (2000). Mutations in the FIE and MEA genes that encode interacting polycomb proteins cause parent-of-origin effects on seed development by distinct mechanisms. Plant Cell 12, 2367–2382.[Abstract/Free Full Text]

Yang, M., Hu, Y., Lodhi, M., McCombie, W.R., and Ma, H. (1999). Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. Proc. Natl. Acad. Sci. USA 96, 11416–11421.[Abstract/Free Full Text]

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