MECHANISMS OF APOMIXIS IN ELYMUS RECTISETUS FROM EASTERN AUSTRALIA AND NEW ZEALAND

Megasporogenesis was examined in cleared ovaries of 23 accessions of hexaploid Elymus from southeastern Queensland, northeastern New South Wales, the Australian Capital Territory, and the South Island of New Zealand. Apomixis was confined to the 17 accessions that morphologically corresponded to E. rectisetus (Nees in Lehm.) Löve et Connor. Female meiotic development followed the Polygonum type. Apomeiotic development was delayed relative to meiotic development because of a lengthy period of MMC vacuolation and nuclear stretching that occurred in place of meiosis I. Amitosis was evident in up to possibly five percent of the MMC's during nuclear stretching. A subsequent mitotic division facultatively functioned as meiosis II or the first embryo-sac mitosis to yield a 2n megaspore dyad, a hemidyad with an incomplete crosswall, or a directly binucleate embryo sac. Nuclear stretching generally resumed in the chalazal daughter nucleus from the apomeiotic division, but was not seen later in embryo sac development. When a dyad formed, its chalazal member would enlarge and develop into the embryo sac. The organized embryo sac was of the conventional eight-nucleate, seven-celled structure prior to antipodal proliferation, regardless of meiotic or apomeiotic origin. Microsporocyte meiosis was normal in both sexuals and apomicts. Deposition of a slightly birefringent substance, possibly callose, was deficient around megasporocytes, megaspores, and microsporocytes in the apomicts.     

Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a β-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution. Received: 18 April 2000 / Accepted: 10 July 2000     

Detection of the apomictic mode of reproduction in maize-Tripsacum hybrids using maize RFLP markers

Polyploid plants in the genus Tripsacum, a wild relative of maize, reproduce through gametophytic apomixis of the diplosporous type, an asexual mode of reproduction through seed. Moving gene(s) responsible for the apomictic trait into crop plants would open new areas in plant breeding and agriculture. Efforts to transfer apomixis from Tripsacum into maize at CIMMYT resulted in numerou intergeneric F₁ hybrids obtained from various Tripsacum species. A bulk-segregant analysis was carried out to identify molecular markers linked to diplospory in T. dactyloides. This was possible because of numerous genome similarities among related species in the Andropogoneae. On the basis of maize RFLP probes, three restriction fragments co-segregating with diplospory were identified in one maize-Tripsacum dactyloides F₁ population that segregated 1∶1 for the mode of reproduction. The markers were also found to be linked in the maize RFLP map, on the distal end of the long arm of chromosome 6. These results support a simple inheritance of diplospory in Tripsacum. Manipulation of the mode of reproduction in maize-Tripsacum backcross generations, and implications for the transfer of apomixis into maize, are discussed.     

Heterochronic features of the female germline among several sexual diploid <Emphasis Type="Italic">Tripsacum</Emphasis> L. (Andropogoneae,Poaceae)

One element of gametophytic apomixis is unreduced embryo sac (ES) formation, which often occurs precociously displacing or replacing meiosis and causing apospory or diplospory, respectively. This study evaluated a premise that apomixis may evolve in hybridogenous plants that contain duplicate sets of allelically divergent ovule development heterochrony genes. The duplicate sets of genes would belong to duplicate genomic regions that are recombinationally isolated from each other (no gene flow) by allopolyploidy or paleopolyploidy, and this isolation would genetically stabilize apomixis. For apomixis to evolve, the ancestral donors of the duplicate regions must have differed from each other in timing of megasporogenesis, ES formation and embryony such that epigenetic misexpressions, or competitions in expression, of the duplicate heterochrony genes in hybridogenous derivatives would cause apomixis. Herein, we report substantial heterochrony in onset timing of germline stages among several sexual diploid Tripsacum genotypes, which may have been progenitors of apomictic polyploid Tripsacum. Tripsacum floridanum and Tripsacum zopilotense genotypes entered meiosis early. The former advanced rapidly through ES formation, but the latter entered a lengthy lag phase prior to ES formation. In two Tripsacum dactyloides var. dactyloides genotypes, meiosis occurred late and was followed by a distinct lag phase prior to ES formation. Likewise, the T. dactyloides var. meridonale genotype entered meiosis late, but the lag phase was brief. These differences appear to reflect allelic diversity at loci responsible for onset timing of different germline development stages within and across species and possibly across the recombinationally isolated duplicate chromosome regions in the Tripsacum paleopolyploid haplome (x = 18). Unique combinations of divergent alleles in hybridogenous plants coupled with polyploidy induced gene misexpressions may be required for apomixis to evolve. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Dosage effects in the endosperm of diplosporous apomictic Tripsacum (Poaceae)

 Imprinting in the endosperm of angiosperms, a phenomena by which expression of alleles differs depending on whether they originate from the male or female parent, has been shown to explain most failure of interploidy or interspecific crosses in plants. Because of imprinting, seeds develop normally only if a specific dosage is represented in the endosperm, with the relative contributions of genomes in the ratio of two maternal doses to one paternal dose (2m:1p). In Tripsacum, a wild relative of maize, all polyploids reproduce through the diplosporous type of apomixis. Diplospory results from meiotic failure in megasporocytes that develop into eight-nucleate unreduced female gametophytes. The male gametophytes remain unaffected. Flow cytometry was used to determine ploidy levels in the endosperm of both apomictic and sexual Tripsacum accessions. In both cases, fertilization appeared to involve only one sperm nucleus. Therefore, endosperm of apomictic Tripsacum develops normally even though the ratio of genomic contributions deviates from the normal 2m:1p ratio. Ratios of 2:1, 4:1, 4:2, 8:1 and 8:2 were observed, depending on both the ploidy level of the parents and the mode of reproduction. Thus, specific dosage effects are seemingly not required for endosperm development in Tripsacum. These findings suggest that evolution of diplosporous apomixis might have been restricted to species with few or no imprinting requirements, and the findings have strong implications regarding the transfer of apomixis to sexually reproducing crops. Received: 17 February 1997 / Revision accepted: 7 July 1997     

New facts about callose events in the young ovules of some sexual and apomictic species of the Asteraceae family

Callose (β-1,3-glucan) is one of the cell wall polymers that plays an important role in many biological processes in plants, including reproductive development. In angiosperms, timely deposition and degradation of callose during sporogenesis accompanies the transition of cells from somatic to generative identity. However, knowledge on the regulation of callose biosynthesis at specific sites of the megasporocyte wall remains limited and the data on its distribution are not conclusive. Establishing the callose deposition pattern in a large number of species can contribute to full understanding of its function in reproductive development. Previous studies focused on callose events in sexual species and only a few concerned apomicts. The main goal of our research was to establish and compare the pattern of callose deposition during early sexual and diplosporous processes in the ovules of some Hieracium, Pilosella and Taraxacum (Asteraceae) species; aniline blue staining technique was used for this purpose. Our findings indicate that callose deposition accompanies both meiotic and diplosporous development of the megaspore mother cell. This suggests that it has similar regulatory functions in intercellular communication regardless of the mode of reproduction. Interestingly, callose deposition followed a different pattern in the studied sexual and diplosporous species compared to most angiosperms as it usually began at the micropylar pole of the megasporocyte. Here, it was only in sexually reproducing H. transylvanicum that callose first appeared at the chalazal pole of the megasporocyte. The present paper additionally discusses the occurrence of aposporous initial cells with callose-rich walls in the ovules of diploid species.

     

An <Emphasis Type="Italic">Hieracium</Emphasis> mutant, <Emphasis Type="Italic">loss of</Emphasis><Emphasis Type="Italic">apomeiosis 1</Emphasis> (<Emphasis Type="Italic">loa1</Emphasis>) is defective in the initiation of apomixis

Asexual seed formation (apomixis) in Hieracium aurantiacum occurs by mitotic embryo sac formation without prior meiosis in ovules (apomeiosis), followed by fertilization-independent embryo and endosperm development. Sexual reproduction begins first in Hieracium ovules with megaspore mother cell (MMC) formation. Apomixis initiates with the enlargement of somatic cells, termed aposporous initial (AI) cells, near sexual cells. AI cells grow towards sexually programmed cells undergoing meiosis, which degrade as the dividing nuclei of AIs obscure and displace them. Following Agrobacterium-mediated transformation of an aneuploid Hieracium aurantiacum apomict, a somaclonal mutant designated “loss of apomeiosis 1” (loa1) was recovered, which had significantly lost the ability to form apomictic seed. Maternal apomictic progeny were rare and low levels of germinable seedlings were primarily derived from meiotically derived eggs. Cytological analysis revealed defects in AI formation and function in loa1. Somatic cells enlarged some distance away from sexual cells and unlike AI cells, these expanded away from sexual cells without nuclear division. Surprisingly, many accumulated callose in the walls, a marker associated with meiotically specified cells. These defective AI (DAI) cells only had partial sexual identity as they failed to express a marker reflecting entry to meiosis that was easily detected in MMCs and they ultimately degraded. DAI cell formation did not lead to a compensatory increase in functional sexual embryo sacs, as collapse of meiotic embryo sacs was prevalent in the aneuploid somaclonal mutant. Positional cues that are important for AI cell differentiation, growth and fate may have been disrupted in the loa1 mutant and this is discussed. The authors Takashi Okada, Andrew S. Catanach and Susan D. Johnson made equal contributions to the data.     

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.     

Activity and localisation of sucrose synthase and invertase in ovules of sexual and apomicticBrachiaria decumbens

Summary Brachiaria decumbens has sexual and apomictic reproduction. Apomixis is facultative and of the aposporic type. In early stages of ovule development, differences in the pattern of callose deposition between sexual and apomictic plants were observed which points to possible differences in carbohydrate metabolism. Therefore, a comparative study on carbohydrate metabolism between a sexual diploid ecotype and an apomictic tetraploidB. decumbens was made. A histochemical determination of two enzymes responsible for sucrose degradation, sucrose synthase and invertase, was performed for all stages of ovule development. In addition, the concentrations of sucrose, glucose, and fructose were measured for each stage of ovule development, both for sexual and apomictic plants. The enzymes were localised by immunohistochemistry with heterologous antibodies. A distinct difference between sexual and apomictic plants was observed in the localisation of sucrose synthase activity as well as in the amount of activity, especially in the early stages of ovular development. Invertase activity localisation was comparable between ovules of the sexual and apomictic plants, but its activity is clearly higher in ovules of sexual plants. The localisation of the enzymes coincided with the place of activity. For both sexual and apomictic plants the amount of sucrose in the ovaries increased with the stage of ovule development. Differences in the amount of sucrose between sexual and apomictic plants in ovaries with ovules in comparable stages of development were detected. A delay in the onset of carbohydrate metabolism during early stages of ovule development characterises the apomictic plant.Abbreviations MMC megaspore mother cell - MC meiocyte - MS megaspore - AI apospore initial - CO coenocyte - MES mature embryo sac - SuSy sucrose synthase - INV invertase - BMM buthylmethyl methacrylate - DTT dithiothreitol - DAPI 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline     

Description of a fertilization-independent obligate apomictic species: Corunastylis apostasioides Fitzg

The Australian midge orchid Corunastylis apostasioides of the tribe Diurideae has completely eliminated any male contribution in the process of seed formation, which occurs directly from the maternal tissue by a process termed apomixis. Here, we report C. apostasioides to be an obligate apomictic species devoid of any sexuality and compare its development to a close sexual relative C. fimbriata (R. Br.) D.L. Jones & M.A. Clem. Apomictic characteristics in C. apostasioides include production of seed in absence of fertilization, frequently closed flowers, production of immature pollen in non-dehiscent anthers, expansion of ovaries despite the lack of fertilization and the absence of a citronella scent that is found in C. fimbriata produced to attract pollinating vinegar flies (Jones 2006). The nature of apomixis in C. apostasioides was examined by ovule histology and amplified fragment length polymorphism (AFLP) in each case drawing comparison with sexual C. fimbriata. In C. apostasioides the central megaspore mother cell undergoes diplosporic apomixis, while additional embryos are derived from nucellar or integument initials formed by sporophytic apomixis. Typical of apomicts, C. apostasioides is polyploid compared to the sexual C. fimbriata. The divergences of C. apostasioides from sexuality to apomictic development are discussed.     

Diplospory and Parthenogenesis in Sexual x Agamospermous (Apomictic ) Erigeron (Asteraceae) Hybrids

Segregation for asexual seed production was evaluated for 130 experimental F1 hybrids resulting from a cross between diploid (2n=18) sexual Erigeron strigosus and triploid (2n=27) agamospermous Erigeron annuus. Paternity of hybrids was documented using 13 RAPD markers. The distribution of F1 chromosome numbers is bimodal, centering on diploid and triploid ploidal levels but with underrepresentation of diploids. Diplosporous versus meiotic megagametophyte development was ascertained microscopically for >/=100 ovules per plant. Diplospory ranges from 0% to 100% among all progeny but is uniformly low (0%-3%) for 17 diploid hybrids. The inheritance of diplospory in Erigeron appears to be best explained by a one-locus-two-allele polysomic model with selection against gametes homozygous for diplospory. Parthenogenesis, estimated via seed counts, ranges from 0% to 60% and apparently is contingent upon diplospory, as seed production was absent or very low in predominantly meiotic hybrids. However, the absence of parthenogenesis in many highly diplosporous hybrids indicates that these two aspects of agamospermous development are not strictly associated. The segregation of both diplospory and parthenogenesis in this population will permit further genetic dissection of these traits with molecular marker-based analyses.     

Embryological and genetic evidence of amphimixis and apomixis in <Emphasis Type="Italic">Boehmeria tricuspis</Emphasis>

Apomixis is a widespread alternative mode of sexual reproduction resulting in offspring that are genetically identical to the maternal plant. Boehmeria tricuspis (Hance) Makino is a perennial, wind-pollinated, herbaceous plant in the nettle family Urticaceae. The diploid B. tricuspis is monoecious but the triploid B. tricuspis is gynoecious, bearing female inflorescences only. Apomixis in B. tricuspis was first reported 50 years ago, but the mode of apomixis in the species has not been described yet. Here, we provide embryological observations of the embryo sac formation proving that triploid B. tricuspis reproduced apomictically following the Antennaria type of diplospory, and that the diploid individuals were the sexual genotype with the classical Polygonum-type maturation pattern of embryo sac development. A subsequent flow cytometry seed screen (FCSS) showed that the triploids were obligate apomicts with autonomous endosperm development, and the diploids reproduced sexually. In addition, a progeny test by molecular marker assays further demonstrated the above results.     

Synthesis and Deposition of Callose in Anthers and Ovules of Meiotic Mutants of Maize (Zea mays)

The pattern of callose deposition was studied in anthers and ovules of three meiotic mutants of Zea mays L. The synthesis of the callose wall in sporogenous cells was related to their transfer to meiotic division.     

Apomixis via recombination of genome regions for apomeiosis (diplospory) and parthenogenesis in Erigeron (daisy fleabane, Asteraceae)

Apomixis in daisy fleabanes (Erigeron annuus and E. strigosus) is controlled by two genetically unlinked loci that regulate, independently, the formation of unreduced female gametophytes (apomeiosis, diplospory) and autonomous seed formation (parthenogenesis). In this work, fully apomictic F2s were regenerated by crossing F1s bearing, separately, these two functional regions. Two triploid (3x = 2n = 27) highly diplosporous F1s served as seed parents to an aneuploid (2x + 1 = 2n = 19) meiotic pollen donor bearing four AFLP markers linked to parthenogenetic seed formation but producing only abortive embryos and endosperm. Of 408 hybrids, 21 (5.1%) produced seed. Nine of these putative apomicts were tetraploids (4x), likely combining an unreduced egg from the diplosporous seed parent and a haploid gamete from the pollen parent (3x + x). The other 12 hybrid apomicts were pentaploid, interpreted as arising from the fusion of an unreduced diplosporous egg with an unreduced sperm cell (3x + 2x). Analysis indicated that all but three of the 21 synthetic apomicts recombined markers linked to diplospory and parthenogenesis. In addition, three additional hybrids combined markers linked to the two functional regions but produced only aborted embryos. The apomicts varied in percentage of diplosporous ovules (4.7–95.3% of all ovules produced) and in percentage of ovules that developed into seed (3.8–58.0%). These results support the hypothesis that apomeiosis and autonomous seed formation are genetically distinct, and that the traits can be separated and recombined to create hybrids exhibiting apomixis at near wildtype levels.     

Callose in cell walls during megasporogenesis in angiosperms

Summary Callose was detected by fluorescence microscopy in megasporogenesis in all investigated species with mono- and bisporic embryo-sac development. Callose occurs first in the meiotic prophase in the chalazal part of the megasporocyte wall and by the first meiotic metaphase the whole cell is enveloped in a callose-containing wall. Later, there is a marked decrease of callose fluorescence, usually at the chalazal end of the megasporocyte. In Oenothera, where the micropylar megaspore is active, decrease of fluorescence takes place at the micropylar pole of the megasporocyte. Callose appears centrifugally in the cell plates forming eventually the walls dividing the megaspores. It disappears from the walls of the megaspores during degeneration and differentiation.     

SYSTEMATICS OF TRIPSACUM DACTYLOIDES (GRAMINEAE)

Tripsacum dactyloides (L.) L. extends across the range of this genus from about 42°N to 24°S latitude in the New World. It is recognized to include T. dactyloides var. dactyloides (North America), var. meridonale deWet et Timothy (South America), var. hispidum (Hitchc.) deWet et Harlan comb. nov. (Mesoamerica) and var. mexicanum deWet et Harlan var. nov. (Mesoamericana). The genus is divided into sections Tripsacum and Fasciculatum. Mesoamerican members of section Tripsacum are classified into T. bravum Gray, T. dactyloides (L.) L., T. intermedium deWet et Harlan spec, nov., T. latifolium Hitchc., T. manisuroides deWet et Harlan spec. nov. and T. zopilotense Hern,*** et Randolph. A key to the species of section Tripsacum is presented.     

Cell number,cell growth,antheridiogenesis, and callose amount is reduced and atrophy induced by deoxyglucose in <Emphasis Type="Italic">Anemia phyllitidis</Emphasis> gametophytes

Fluorescence staining and morphometrical measurements revealed that callose was a component of newly formed cell plates of symmetrically dividing cells and asymmetrically dividing antheridial mother cells during gibberellic acid-induced antheridiogenesis as well as in walls of young growing cells of Anemia phyllitidis gametophytes. Callose in cell walls forms granulations characteristic of pit fields with plasmodesmata. 2-deoxy-d-glucose (DDG), eliminated callose granulations and reduced its amount estimated by measurements of fluorescence intensity. This effect was accompanied by reduction of antheridia and cell numbers as well as size and atrophy of particular cells and whole gametophytes. It is suggested that inhibition of glucose metabolism and/or signalling, might decrease callose synthesis in A. phyllitidis gametophytes leading to its elimination from cell plates of dividing cells and from walls of differentiating ones as well as from plasmodesmata resulting in inhibition of cytokinesis, cell growth and disruption of the intercellular communication system, thus disturbing developmental programs and leading to cell death.     

Mechanism of male sterility in Petunia: The relationship between pH,callase activity in the anthers,and the breakdown of the microsporogenesis

Summary In the locules of fertile Petunia hybrida anthers the in vivo pH during meiosis is 6.8–7.0 and no callase activity can be detected. Towards the end of the tetrad stage, the pH drops to 5.9–6.2 followed by a burst of callase activity. Subsequently, callose in the tetrad walls is digested and the quartets of microspores are released into the anther locules and develop into pollen grains. In the anther locules of one cytoplasmic male sterile (cms) Petunia type the pH drop and strong callase activity are already evident at early meiotic stages. Consequently, the callose already accumulated in the pollen mother cell (PMC) walls is digested and the PMC's cease to develop and are degraded. In another sterile genotype, the pH of the locule remains high (6.8–7.0), no callase activity is detected at the end of tetrad stage and the callose walls remain intact until a very late stage. It is suggested that the timing of callase activity is critical for the normal development of the male gametophyte and that faulty timing may result in male sterility. Measurements of pH in vivo and assays for callase activity in vitro indicate that the low pH is a precondition for the enzyme activity. Furthermore, it is suggested that the activation of callase in vivo is in some way connected with the changes in the pH of the locule.Contribution from The Volcani Institute of Agricultural Research, 1970 Series, No. 1709-E.Supported in part by grant No. FG-Is-171 from the United States Department of Agriculture, under P. L. 480.     

MEGASPOROGENESIS AND GAMETOPHYTE SELECTION IN PAEONIA CALIFORNICA

Walters , James L. (U. California, Goleta.) Megasporogenesis and gametophyte selection in Paeonia californica. Amer. Jour. Bot. 49(7): 787–794. Illus. 1962.—In the ovules of Paeonia californica, a massive archesporium produces numerous (estimated at 30–40) megasporocytes, many of which complete meiosis. Several continue into gametophyte development, which is of the Polygonum type, and at the time of fertilization there are from 1 to 4 gametophytes per ovule. Rarely does more than 1 seedling per seed appear in germination. This species is characterized by extensive translocation heterozygosity, and other meiotic irregularities, in its natural populations. It shows a complete range from plants forming only pairs to those with all their chromosomes in rings at meiosis. The latter types have as high as 90% bad pollen. The course of events in the ovules is compared with the “Renner-effect” found in Oenothera. The multiple megasporocytes and subsequent events are seen as a mechanism which insures each ovule a high probability of containing a viable egg in spite of meiotic behavior which can produce 90% sterility, and thus insures high seed set in the translocation heterozygotes.     

CYTOLOGY OF MAIZE-TRIPSACUM INTROGRESSION

The American Maydinae genera Zea and Tripsacum cross readily when not isolated from each other by gametophytic barriers, and it has been suggested that intergeneric introgression played a role in the evolution of maize. Four Zea chromosomes pair with members of at least one basic genome of tetraploid Tripsacum, and in hybrids involving octaploid Tripsacum all 10 chromosomes of the basic maize genome frequently compete successfully in synapsis with Tripsacum chromosomes. Hybrids that combine 36 Tripsacum and 10 maize chromosomes are female fertile. When they are pollinated by maize their offspring have 36 Tripsacum and 20 maize chromosomes, or again have 36 Tripsacum and 10 maize chromosomes, but the 10 Zea chromosomes are contributed by the new pollen parent. Later backcross generations also include plants with 36 Tripsacum and 12, 14, 16, or 18 maize chromosomes. Individuals with 2n = 56 produce an abundance of offspring with 18 Tripsacum and 20 maize chromosomes when backcrossed with maize. Further backcrossing results in elimination of Tripsacum chromosomes, and eventually plants with 2n = 20 Tripsacum-contaminated maize chromosomes are obtained. Two generations of selfing restore full fertility to these 2n = 20 plants and eliminate all obvious traces of Tripsacum morphology.