Obtaining sexual genotypes for breeding in buffel grass

Buffel grass (Cenchrus ciliaris L. syn. Pennisetum ciliare (L.) Link) is a species that is highly tolerant to drought and is used primarily as forage in drier regions throughout the subtropics and tropics. It reproduces mainly by apomixis and the acquisition of obligate sexual genotypes or facultative apomicts with high levels of sexuality is required for performing crosses and plant improvement. The aim of this study was to obtain sexual genotypes from controlled crosses using obligate apomictic cultivars and a sexual line. Twelve putative hybrid F1 plants were selected morphologically and two of them were identified as sexual genotypes by PCR using specific primers for reproductive mechanism. Cytoembryological analysis showed 65.5 and 71.3% meiotic embryo sacs in these plants and their hybrid nature was corroborated by AFLP. Both highly sexual genotypes could be used as female parents in crosses for obtaining improved cultivars of buffel grass.     

Apomixis and sexuality in diploid and tetraploid accessions of Brachiaria decumbens

 Meiotic and aposporous embryo sacs and the initial steps of parthenogenetic embryogenesis and endosperm formation were investigated in diploid and tetraploid accessions of Brachiaria decumbens in two environments, differing mainly in day length: early summer and late autumn. Both diploid and tetraploid accessions were facultative apomicts. Di(ha)ploids showed a much lower level of apomixis (10% to15%) than tetraploids (80% to 95%). No obligate sexual diploids were found; thus, their occurrence in natural populations is obscure. It is suggested that reproduction in B. decumbens, as in other agamic complexes of the Paniceae tribe, in general, approximates a diploid-tetraploid-(di)haploid reproductive cycle which does not involve triploids. The dihaploids were fertile and survived in nature. Development of the reproductive structures depended on the environment. In autumn, in contrast to early summer, many meiotic and aposporous embryo sacs degenerated during development, leading to a significant reduction in the proportion of parthenogenetic embryos. Whether this effect can be attributed to day length or simply to age remains to be investigated. The ratio of aposporous to sexual embryo sacs was relatively stable over the two seasons. Received: 15 April 1998 / Revision accepted: 13 October 1998     

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.     

Allelic ratios and sterility in the agamic complex of the Maximae (Panicoideae): evolutionary role of the residual sexuality

The agamic complex of the tribe Maximae is constituted by two pools characterized by their own breeding system (sexuality on the one hand, facultative apomixis on the other hand). Facultative apomicts shows two types of embryo-sacs (aposporic or meiotic). Presence of aposporic embryo-sacs is genetically controlled by the dominant allele A (all apomictic plants are Aaaa). Meiotic embryo-sacs, constituting residual sexuality, can allow crosses between apomicts and theoretically the generation of other genotypes than Aaaa. Additionally, some cases of sterile apomicts appears in a breeding system known to bypass it. This led us to study these two paradoxes. In fact, the sterility would exist when the ratio A/a is greater than 0.25. This would prevent the total overrunning of an apomictic population by AAAA genotypes. The role of residual sexuality is then to allow generation of true sexuals (aaaa) from apomicts (Aaaa).     

A modified method of flow cytometric seed screen simplifies the quantification of progeny classes with different ploidy levels

Flow cytometric analysis of ten bulked seeds is proposed to quantify particular embryo ploidy classes in Hieracium. The method is recommended 1) for the detection and quantification of residual sexuality in facultative apomicts, which can generate progeny from heteroploid crosses, 2) for the quantitative screening of pollen donors with different ploidy levels, based on the fertilization success of the maternal plant, and 3) for the screening of parents producing a high proportion of polyhaploids.     

Effect of pollination timing on the rate of apomictic reproduction revealed by RAPD markers in paspalum notatum

Progeny tests employing molecular markers allow the identification of individuals originated by sexual means among the offspring of a facultative apomict. The objective of this work was to evaluate the effect of the pollination timing on the proportion of sexually formed individuals in progenies of a facultative apomictic Paspalum notatum genotype. Progeny families of approx. 30 plants each were generated at five different pollination times: 1-3 d pre-anthesis; at anthesis; and 2, 4 and 6 d post-anthesis. Cytoembryological analyses indicated that approx. 17% of the ovules carried a meiotic cytologically reduced embryo sac in florets formed simultaneously with those used for crosses. The parental plants and the five F1 families were analysed using RAPD molecular markers. Ninety-five oligonucleotides were assayed on the progenitors in order to search for male-specific bands. Eight primers presenting clear polymorphic bands were selected for use in the progeny tests. The proportion of sexually produced progeny reached 3.4% before anthesis and 20 % at anthesis, while pollination after anthesis generated only maternal plants. A second progeny of 97 plants obtained from pollination at anthesis produced 16 off-type plants (16.5%), of which only one was a B(III) hybrid (2n + n). Our results indicate that pollination at anthesis allows the greatest potential for sexuality to be expressed in this facultative apomictic genotype. When pollination is delayed as soon as 2 d after anthesis, only the aposporous sacs develop endosperm through pseudogamy to set seed.     

Genetic characterization of apospory in tetraploid Paspalum notatum based on the identification of linked molecular markers

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F₁ population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F₁ individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.     

Genetic fingerprinting for determining the mode of reproduction in Paspalum notatum, a subtropical apomictic forage grass

 Paspalum is an important genus of the family Gramineae that includes several valuable forage grasses. Many of the species are polyploid and either obligate or facultative apomicts. Cyto-embryological observations of several tetraploid genotypes of P. notatum were performed to determine their mode of reproduction. Afterwards, selfed progenies of the genotypes F131, Q3664 and Q4117 were analysed using RFLP and RAPD genetic fingerprints to identify maternal and non-maternal (aberrant) plants, and to establish the degree of apomictic reproduction. Five maize clones and six primers were used for detecting genetic deviations from the maternal profile. Maize clones umc379, umc384 and umc318 and primers OPG10 and OPI4 were the most informative for discriminating between maternal and aberrant individuals within the progenies of F131 and Q3664. The combined results of three RFLP clones or 4–6 RAPD primers were necessary to ascertain the mode of reproduction in plants F131 and Q3664. The results obtained with the RFLP and RAPD markers were in agreement with the cyto-embryological studies in ascertaining the mode and degree of apomictic reproduction. Plant F131 showed a completely sexual reproductive behaviour, Q3664 an elevated expression of sexuality, while Q4117 was highly apomictic. A fingerprint analysis of an outcrossing population, aimed at the identification of hybrid plants, was also performed. Maize clones um318 and umc379 and primers OPC2 and OPC9 were used. The presence of specific bands belonging to the male parent permitted a rapid and easy detection of hybrids. The methodology described here can be applied both for the characterisation of P. notatum populations and to identify hybrid progenies in Paspalum breeding programs. Received: 5 March 1997 / Accepted: 13 May 1997     

Apomixis versus sexuality in blackberries (Rubus subgen.Rubus,Rosaceae)

InRubus L. a connection seems to exist between the degree of meiotic disturbances on the one hand, and the production of unreduced embryo sacs, pollen fertility and relative seed set on the other hand. Severe meiotic disturbances commonly encountered in apomictic taxa decrease pollen fertility and thereby seed set since pollen is necessary for endosperm development. By contrast interspecific hybrids between apomictic taxa appear to be sexual and exhibit high pollen fertilities, probably due to an improved meiosis. Thus, apomixis leads to a decreased fertility inRubus, not the opposite, as often discussed.     

EMBRYO SAC DEVELOPMENT IN THE CV. KS MALE-STERILE,FEMALE-STERILE LINE OF SOYBEAN (GLYCINE MAX)

Light microscopic observations were made on 22 ovules from fertile plants and 108 ovules from sterile plants of the cv. KS synaptic mutant, a highly male-sterile, female-sterile line of soybean [Glycine max (L.) Merr.] (2n = 2x = 40). Ovules of fertile siblings contained normal embryo sacs and embryos. Ovules from sterile plants contained various irregularities. The most consistent abnormality was the failure of the embryo sac to attain normal size. Small megasporocytes of irregular shape were seen; only one megasporocyte of normal shape and size was noted. No linear tetrads were found. However, two ovules contained nonlinear triads. A range from zero to 28 cells and nuclei, of various sizes, were identifiable in small megagametophytes and embryo sacs. Degeneration of these nuclei and cells was noted as early as the four-nucleate gametophyte stage. Other ovules contained degenerated nucellar centers without embryo sacs. Two ovules appeared to be normal. Late postpollination stages were marked by shrunken nucellus and integuments. The presence of pollen tube traces, endosperm, and aborting embryos in ovules of hand-pollinated flowers from sterile plants suggested that no incompatibility was involved. Degeneration of the gametophyte and embryo sac contents at many developmental stages indicated a wide array of effects, possibly resulting from meiotic irregularities similar to those seen in microsporogenesis of this mutant.     

Genetic characterization and floral development of female sterile and stubby head, two aposporous mutants of pearl millet

 Apomixis has never been reported in natural populations of pearl millet [Pennisetum glaucum (L.) R.Br.], although many wild relatives of pearl millet are obligate or facultative aposporous apomicts. Four-nucleate aposporous embryo sacs are formed from somatic cells of the nucellus that do not undergo meiosis. Two mutants of pearl millet, female sterile (fs) and stubby head, have two developmental characteristics in common: a significant reduction in head length compared with the wild-type and the formation of aposporous embryo sacs. Reproductive development in fs and stubby head mutants was examined in depth because of the potential for illuminating basic cellular or developmental factors that may function to alter embryo sac development. Genetic analysis of stubby head showed that this phenotype is conferred by genes at two loci linked in coupling within 29 cM. Crosses between fs and stubby head mutants showed that, despite the similarities in phenotypes, the mutations are at different loci. The mutants differ from wild-type in their inflorescence structure from the time of initiation of spikelet primordia through terminal differentiation of the ovule. Both mutations could be categorized as meristic, since a change in inflorescence branch or organ number was common and gynoecium development varied. We speculate that heterochronic development of the floral meristem and organ initiation/specification programs may be the underlying mechanism for phenotypic changes in these mutants throughout the floral phase. Received: 25 October 1996 / Accepted: 13 March 1997     

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.     

APOMIXIS IN THE POA SECUNDA COMPLEX

Observation of cleared ovules of Poa secunda Presl showed that plants produce aposporous embryo sacs, and emasculation studies showed that they do not set seed without pollen. The ratio of sexual to asexual embryo sacs on a given plant does not correlate with percent stainable pollen, nor does pollen stainability correlate with meiotic regularity or with chromosome number. Percent apomictic ovules, percent pollen stainability, and regularity of meiosis all vary within a given population; they also vary if the same plant is moved from one site to another. Although there is morphological variation in the species, none of the reproductive features correlates with morphology. Pollen from any morph will stimulate seed set in any other morph, and hybrids have been produced in low frequency. Plants will also set seed using their own pollen. In its reproductive biology, P. secunda exhibits many similarities with P. pratensis L.     

Inorganic salts modify embryo sac development in sexual and aposporous Cenchrus ciliaris

Summary Sexual and aposporously apomictic plants of buffelgrass (Cenchrus ciliaris L.) form megaspore tetrads. In sexual plants the chalazal megaspore develops into a single Polygonum type embryo sac. In aposporous plants the megaspores degenerate, and one or more un-reduced nucellar cells form Panicum type embryo sacs. Apospory is conditioned by gene A; the dominant allele of gene B is epistatic to A and preserves sexual reproduction. We recently observed that heavy application of (NH₄)₂SO₄ to the soil induced multiple embryo sacs in a sexual line. Therefore we tested the effect of salt stress on embryo sac formation in sexual and aposporous genotypes. One molar solutions of CaCl₂, NaCl, (NH₄)₂SO₄, NH₄Cl, NaNO₃, or Na₂SO₄ were applied to the soil of greenhouse plants every day or two starting at the archespore stage. Some of the pistils in salt-treated plants of sexual genotypes AaBb, aaBb, and aabb showed features not seen in untreated controls: (1) multiple Polygonum type embryo sacs in 1%–7% of pistils depending upon the salt; (2) embryo sacs without antipodals (0%–7%); (3) embryo sacs protruding through the micropyle (1%–16%). Some pistils of salt-treated obligately aposporous lines, but not controls, developed Polygonum type embryo sacs (4%–13%) and protruding embryo sacs (0%–6%). There was no ion specificity for induction of abnormal features. We postulate that salt stress suppresses the developmental priority of nucellar embryo sacs over megaspores in aposporous lines and of the chalazal megaspore over other megaspores in all lines. This may permit megaspores of aposporous plants to form reduced Polygonum type gametophytes, and permit more than one megaspore to form reduced embryo sacs in all lines. Protrusion of sacs and failure of antipodal formation in reduced embryo sacs may be the consequence of uncoordinated expansion of the embryo sacs and surrounding tissue.Joint contribution of the Department of Biology, The Pennsylvania State University, and USDA-ARS, U.S. Regional Pasture Research Laboratory. Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA     

Callose pattern and polarization phenomena in the ovules in the F2-hybrids betweenOenothera hookeri andOe. suaveolens

The pattern of callose formation in meiotic cell walls and the order of megaspore degeneration and polarity during embryo sac development are investigated in F₂-plants ofOe. hookeri ×suaveolens and the reciprocal cross. All investigated characters are variable between the ovules in the same ovary. Plants differ in the frequency of the types of callose pattern and polarity of the embryo sacs. In segregating progenies different combinations of both characters are found. The genetic basis of the polarity phenomena during the embryo sac development is discussed. In our material no correlation can be seen between the callose pattern in the surrounding wall of the meiotic cell and the development of polarity in the later stages.     

Apomixis and sexuality in Paspalum simplex: characterization of the mode of reproduction in segregating progenies by different methods

Segregating progenies of crosses between sexual and apomictic genotypes of Paspalum simplex were analysed for the formation of meiotic versus aposporous embryo sacs, zygotic versus parthenogenetic embryos, and autonomous versus pseudogamous endosperms by using cytoembryological and flow cytometric analyses. Reduced and unreduced 8-nucleated embryo sacs were the final product of female gametophyte development in sexual and aposporous genotypes, respectively. An incomplete penetrance of parthenogenesis was detected in aposporous genotypes. The relative DNA content of endosperm nuclei revealed the normal 2:1 maternal to paternal ratio in sexuals and a 4:1 ratio in apomicts, indicating insensitivity of the apomictic genotypes to endosperm imprinting. Apospory, parthenogenesis and pseudogamy are located on a relatively large linkage group and are inherited together with previously developed molecular markers as a single genetic unit in segregating progenies.     

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

 Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus. Received: 20 January 1999 / Revision received: 12 July 1999 / Accepted: 17 August 1999     

Reproductive development in apomictic populations of <Emphasis Type="Italic">Arabis holboellii</Emphasis> (Brassicaceae)

Megasporogenesis, megagametogenesis and seed formation were analyzed cytologically in populations of Arabis holboellii originating from North America (Colorado) and Greenland. The Colorado population contained only triploid plants, while the Greenland population consisted of diploid and triploid plants. The penetrance of the apomictic trait was assessed at the level of embryo sac development. All populations showed facultative apomeiotic embryo sac development; however the penetrance of this trait differed between the populations. Apomeiotic and meiotic embryo sac development were characterized by diplosporous dyad formation (Taraxacum-type) and meiotic tetrad formation (Polygonum-type), respectively. Flow cytometric analyses of single mature seeds from all three populations suggest that only unreduced gametes participate in viable seed development. Pseudogamy was the predominant mode of endosperm formation; however, autonomous endosperm development was also observed. The fertilization of unreduced egg cells with unreduced pollen was observed at a low frequency in the Greenland populations. The mechanisms of apomictic reproduction in A. holboellii are discussed.     

Fertilization in maize indeterminate gametophyte1 mutant

Summary. Mature embryo sacs of the maize mutant indeterminate gametophyte1 displayed different cellular patterns compared to those of the wild type. About 40% of the ig1 embryo sacs contained three or more synergids and two or more egg cells at the micropylar end. During fertilization in embryo sacs with two synergids, both of them frequently degenerated and were penetrated by two pollen tubes. 75% of the embryo sacs containing three or more synergid cells were penetrated by two or more pollen tubes, although most of them had only one degenerated synergid. Multiple fusions between the sperm cells and eggs frequently occurred in the same embryo sac, which subsequently generated multiple embryos. There were two or more central cells in about 33% of ig1 embryo sacs. The largest central cell was usually adjacent to the egg apparatus and contained two unfused polar nuclei, while those extra central cells located at the chalazal end usually had a single nucleus. Fertilization occurred only between the male gamete and the largest binucleate central cell. The extra central cells eventually degenerated after fertilization.Present address: GI Basic Research Center, Mayo Clinic, Rochester, Minnesota, U.S.A.Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, Peoples Republic of China.