Modes of inheritance of two apomixis components,diplospory and parthenogenesis,in Chinese chive (Allium ramosum) revealed by analysis of the segregating population generated by back-crossing between amphimictic and apomictic diploids

To investigate the mode of inheritance of apomixis in Chinese chive, the degrees of diplospory and parthenogenesis were evaluated in F₁ and BC₁ progenies derived from crosses between amphimictic and apomictic diploids (2n = 16, 2x). The F₁ population was generated by crossing three amphimictic diploids 94Mo13, 94Mo49 and 94Mo50 with an apomictic diploid KaD2 and comprised 110 diploids and 773 triploids. All the diploid F₁ plants examined were completely or highly eusporous and completely syngamic. All the triploid F₁ plants examined were highly diplosporous and highly parthenogenetic. KaD2 could not transmit its high level of apomixis via monoploid pollen grains. The BC₁ population, generated by crossing 94Mo49 with apomictic triploids found in the F₁ offspring, exhibited heteroploidy; it comprised haploid, diploid, triploid, tetraploid and various aneuploid individuals. In this generation, clear segregation was observed between diplospory and parthenogenesis. Analysis of the BC₁ population suggests that diplospory and parthenogenesis are each controlled by single dominant genes, D and P, respectively. However, all the BC₁ plants characterized as parthenogenetic were diplosporous. The absence of phenotypically eusporous parthenogenetic plants can be explained by assuming that the presence of diplospory gene is a prerequisite for the parthenogenesis gene expression in Chinese chive.     

Seasonal changes in the incidence of apomixis of diploid,triploid, and tetraploid plants of Paspalum cromyorrhizon

Summary Changes in incidence of apomixis were determined at different intervals of the flowering period in a highly sexual diploid cytotype of Paspalum cromyorrhizon Trin., a natural tetraploid cytotype of the same species, and in the triploid and tetraploid intraspecific hybrids that were produced by pollinating the 2x cytotype with pollen of the tetraploid. Reproductive behaviour was determined from observations of embryo sacs in mature ovules. Gametophytic apomixis in this species in characterized by aposporous embryo sacs which differ from normal sacs by their number, orientation inside the ovule, their dimensions and shape, and the absence of antipodal cells.The potential for apomictic reproduction increased in relation to the ploidy level, suggesting a gene dosage effect in the incidence of apomixis. In the three ploidy levels, the highest incidence of apomixis was observed when the plants reached the maximum number of flowering heads. These findings suggest that the same environmental conditions that favour flowering should also be responsible for increasing the incidence of apomixis. An additional experiment under controlled conditions indicated that the light regime should be one of the factors that govern the amount of apomictic reproduction. Thus, polyploidy and apomixis should be of special significance in the dispersion and evolution of this grass species. The 4x facultative apomictic cytotype is far more frequent in nature than the highly sexual 2x. Moreover, most of the seeds produced in a flowering season should arise asexually. So, apomixis rapidly increases the number of plants bearing the successful maternal genotype, and sexual reproduction becomes important in adverse environmental conditions.     

Reproductive diversity and strategies for breeding in St. John's wort (Hypericum perforatum L.)

St. John's wort (Hypericum perforatumL.) is a medicinal crop plant that reproduces by apomixis. To study the reproductive diversity within the initial breeding material, 656 plants from 92 accessions of the species were analysed by flow cytometric seed screening (FCSS) and compared to 66 plants of cv. ‘Topaz’ as a control. Among plants from the 92 accessions, 16 were obligate sexuals, nine were obligate apomicts and all remaining ones were facultative apomicts. Among controls, there were six obligate and 60 facultative apomicts. Tests of reproductive pathways indicated that, in the investigated collection, all apomictic plants were tetraploid and all sexual plants were diploid. A strategy for a cross breeding program in St. John's wort is illustrated by crossing one diploid obligately sexual mother plant with six tetraploid facultative apomicts. All 91 F₁ progeny obtained were triploid with 58% being obligate sexuals and 42% facultative apomicts. To quantify the different routes of seed formation within facultatively apomictic individuals, the reproductive pathways were reconstructed from about 30 single seeds from each of five tetraploid and two triploid plants using FCSS. The frequency of seed formed by apomixis ranged from 54% to 67% among four tetraploid genotypes. One triploid and two tetraploids formed 23% to 73% of the so-called B_III hybrids (fertilization of unreduced eggs) and one triploid was obligately sexual. The results show that while the mode of reproduction in H. perforatum is highly variable, tetraploid obligate apomicts as well as diploid and triploid obligate sexuals can be selected as starting material for breeding purposes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation of chromosome numbers and essential oil components of plants derived from anther culture of the diploid and the tetraploid in Pelargonium roseum

Summary Anthers of the diploid (2n=77) and the colchi-tetraploid (2n=154) Pelargonium roseum were cultured in vitro. In both ploidy level anthers containing uninucleate or binucleate microspores were incubated on a modified White's medium. Calli formed were then subcultured on Murashige and Skoog's medium for organoid differentiation. Plants developed from organoids were transferred to filter paper bridges and after that transplanted into pots. Plants derived from anthers of the tetraploid had diploid chromosome number. Wide variation of their essential oil components suggested their genetic heterogeneity. Further, high correlations between different seasons in the rate of essential oil components showed that the wide variation was due to genetic differences. Therefore, these plants probably originated from pollen grains. On the other hand, plants derived from anthers of the diploid had diploid chromosome number. Small variation and low correlations between different seasons in essential oil components indicated their genetic homogeneity. Their origin was ascribable to the somatic tissues of the mother plant. It is concluded that in plant species in which usual sexual reproduction is difficult, anther culture of chromosome-doubled plants will give a useful method for obtaining genetic variation.     

Cytology and reproductive behavior of diploid, tetraploid and hexaploid germplasm accessions of a wild forage grass: Paspalum compressifolium

Summary Fourteen germplasm accessions of Paspalum compressifolium native from southern Brazil were cytologically and embryologically analysed. The study revealed that one accession was diploid (2n=20), twelve were tetraploid (2n=40) and one was hexaploid (2n=60). This is the first report of diploid and hexaploid cytotypes for this species. Studies on microsporogenesis, megasporogenesis, and embryo sac development indicated that the diploid cytotype had regular meiotic behavior and reproduces sexually. Tetraploid cytotype usually had an important proportion of chromosomes that associated as quadrivalents during meiosis and reproduced by mean of aposporous apomixis. The hexaploid cytotype showed irregular meiotic behavior with about one third of the chromosomes associated as multivalents and reproduced by aposporous apomixis. Thus, P. compressifolium could be an agamic complex. Breeding in this complex is possible due to the presence of diploid sexuals which can be treated by colchicine and pollinated by apomicts.     

Inheritance and genetic diversity of some enzymes in the sexual and diploid pool of the agamic complex of Maximae (Panicum maximum Jacq., P. infestum Anders. and P. trichocladum K.Schum.)

Summary The tribe Maximae (Panicum maximum Jacp., P. infestum Anders., P. trichocladum K.Schum.) includes two sympatric pools with different modes of reproduction and ploidy levels: an apomictic and tetraploid pool on the one hand, and a smaller, sexual and diploid pool on the other.In the latter, the sexuality allowed crosses and hence it was possible to gather evidence of the structure (monomeric or dimeric) of nine enzymatic systems and of their inheritance (16 loci). Some segregation distortion was observed in the malatedehydrogenase and esterase systems.The genetic diversity of the diploid pool is large, in agreement with its mode of reproduction (allogamy and anemophily). The diversity is divided into three groups corresponding to the geographical origin of populations. This organisation is very similar to that observed from morphological traits.     

Chromosome doubling through adventitious shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids

Summary Nearly 450 plantlets were produced from 51 diploid Solanum etuberosum x S. pinnatisectum F₁ hybrids through adventitious shoot formation on in vitro cultivated rachis and petiole explants.On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7% of the plants were scored as doubled or doubled twice. A cytological analysis of ploidy in the three layers L₁.L₂ and L₃ of 112 plants revealed 83.9% complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy chimeras were detected. Doubled plants were obtained from all 51 clones.Various flower colours and epinastic leaves (in 1 clone) may be indications of mutagenesis through the treatment.     

Comparison of diploid and tetraploid potato families derived from Solanum phureja x dihaploid S. tuberosum hybrids and their vegetatively doubled counterparts

Summary To determine whether in potatoes the tetraploid level is preferable to the diploid level, especially regarding tuber yield, four diploid (2n=2×=24) Solanum phureja x dihaploid S. tuberosum hybrid parents and their vegetatively doubled, tetraploid (2n=4×=48) counterparts were intermated, which resulted in two F₁ hybrid families at both levels of ploidy. The parents and clones of the F₁ families and their offspring were used in crosses in such a way that in addition Sib₁, Sib₂, F₁×Sib₁, BC₁ and Sib₁×Sib₁ families were produced. Of the first clonal generation of the 12 2 x families and their 12 counterpart 4 x families two tubers per clone were planted in three replications in a field experiment at Sturgeon Bay in 1969; of the parents six tubers were planted in each replication. Data were recorded on 16 characters, including plant height at four different times.The ANOVA's showed significant clone effects within each family for all characters. Computed from all family means as well as from the family means per ploidy level, differences due to family were also significant for all characters except one.As at the 2 x level and at the 4 x level of ploidy the mean phenotypic correlations between characters were of similar magnitude, it is concluded that they are independent of ploidy level.With the exception of eye depth, the mean coefficient of variation was greater at the 2 x than at the 4 x level of ploidy, indicating the greatest response to selection for those characters at the 2 x level. From the differences in family means between the 4 x and the 2 x level of ploidy it was apparent that the 4 x families generally had significantly taller plants, later maturity, fewer tubers, higher mean tuber weight, more tuber yield and more dry matter yield than their counterpart 2 x families. In contrast, the 4 x parents had on the average shorter plants, lower mean tuber weight, much lower tuber yield and lower dry matter yield than their 2 x counterparts.The phenotypic correlation and Spearman's rank correlation between the family means of the 2 x and the 4 x level of ploidy were positive for almost all characters and significant for nearly half of them.From the results it is concluded that 1. in potatoes the 4 x level of ploidy is preferable to the 2 x level, and 2. the performance of 4 x families is predictable from the performance of their counterpart 2 x families.Based on results mentioned in the literature and on the present results, a continued use of S. tuberosum dihaploids in potato breeding needs to be dissuaded.     

Bilateral polyploidization inDactylis glomerata L. subsp.lusitanica: occurrence,morphological and genetic characteristics of first polyploids

Summary InDactylis glomerata L. subsp.lusitanica, triploid and tetraploid plants were obtained by bilateral sexual polyploidization in crosses between diploid parents known to produce 2n gametes. The polyploid and diploid progeny were compared for allozyme diversity (allele number and heterozygosity), phenological (pollen fertility, inflorescence emergence date), cellular (stomatic cell size) and morphological characters (vegetative biomass, seed weight, total seed number per plant, panicle number and seed number per panicle length) in four contrasting growing conditions. Stomatal cell size and the total number of alleles were significantly lower in diploids than in polyploids which sometimes recovered more than two distinct alleles at a locus via 2n gametes possessing heterozygous loci. Pollen fertility was similar in diploids and tetraploids, but significantly lower in the triploids. Seed weight was significantly higher in polyploids, although the average number of seeds per panicle length and the total number of seeds was higher in the diploid sibs. No phenological differences occurred among ploidy levels whereas such differences were observed inDactylis in all the natural areas of sympatry between diploids and their related polyploids. Our results provide evidence for an instantaneous effect of polyploidization on certain traits and suggest that natural selection would act subsequently on several other traits to allow the establishment of new polyploids.     

Relationship between somaclonal variation and type of culture in cucumber

Highly inbred B line of cucumber was used to compare the effect of four types of in vitro culture on somaclonal variation. The plants were regenerated from the following types of culture: twelve- and eighteen-month-old liquid culture of meristematic clumps (LMC₁₂₍₁₈₎), ten-month-old embryogenic cytokinin-dependent suspension (CDS), eighteen-month-old embryogenic cytokinin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (CDS 1.7), twelve-month-old embryogenic auxin-dependent suspension (ADS), thirty six-month-old embryogenic auxin-dependent suspension in medium with modified NH⁺ ₄/NO₃ ^- ratio (ADS 1.7) and recurrent leaf callus regeneration (RLC) – repeated 5 times. The differences in the incidence of the following properties were observed: the ploidy of R₀ plants, the segregation of new morphological traits in R₁ and the germination ability of R₁ seeds. R₁ families with the segregation of new phenotypes were most numerous in CDS (62.5%) and LMC₁₈ (57.9%), next in CDS1.7 (35.7%), while the smallest number was found in LMC₁₂ (11.1%) and RLC (3.4%).Tetraploid and mixoploid plants occurred in ADS1.7 and ADS (100%) whereas CDS and RLC were observed to contain only tetraploids, respectively 33.3% and 55.2%. There were no changes of ploidy after LMC₁₂, LMC₁₈ and CDS1.7. Among new phenotypes there were such that have not been described so far in cucumber: ginkgolike leaf (gll), yellow-green chlorophyll mutants (y-gc), serrate margin of corolla in male and female flowers (smc). This revised version was published online in August 2006 with corrections to the Cover Date.     

Rapid estimation of ploidy levels in in vitro-regenerated interspecific Arachis hybrids and fertile triploids

Summary A significant correlation among chromosome number, chloroplast number and pollen grain size was observed using interspecific hybrids (2n=30,60), Arachis hypogaea (2n=40), A. stenosperma (PI 338280) (2n=20), A. batizocoi (PI 468329) (2n=20) and A. villosulicarpa Hoehne (PI 336984) (2n=20), representing four ploidy groups. Both chloroplast number in guard cells and pollen grain size were found to be positively correlated with ploidy, and provided a reliable method to distinguish diploid, triploid, tetraploid and hexaploid plants from each other regardless of their taxonomic backgrounds. These methods in combination with root-tip chromosome counts enabled us to confirm ploidy determinations on all three dissue layers, L₁, L₂ and L₃ (guard cells, microsporocytes and root meristematic cells, respectively), and to detect the chimeric nature of some colchicine-treated tissue culture-derived plants. Screening pollen grains by size and the detection of highly stainable and viable, 30-chromosome pollen grains have enhanced the use of triploid hybrids in peanut breeding programs.     

Ploidy dependent expression of apomixis and its components in guinea grass (<Emphasis Type="Italic">Panicum maximum</Emphasis> Jacq.)

Panicum maximum (guinea grass) is a model crop for apomixis and polyploidy studies. It is predominantly tetraploid (2n?=?32) and is characterized by gametophytic apomixis, Panicum-type apospory and pseudogamous endosperm development. The three components of apomixis, viz. apomeiosis, parthenogenesis and functional endosperm development, can be uncoupled in this crop. An exhaustive single progenitor-derived ploidy series comprising of 32 accessions representing 3x, 4x, 5x, 6x, 7x, 8x, 9x and 11x cytotypes was utilized in present study to understand ploidy effects on expression of apospory as well on uncoupled components in two phases of progeny formation i.e. in matured ovules (using embryo-sac analysis) and in matured self-pollinated seeds (using Flow Cytometric Seed Screen method). Rise in ploidy enhanced the formation of sexual embryo-sacs (ES) thereby increasing the frequency of facultative accessions at higher ploidy level. Our results suggested that the eventual phenotype depends on relative doses of apospory and sexual factors in the genome. Ploidy level was also found affecting the penetrance and expressivity of uncoupled apomixis components. Formation of B_III hybrids (3n) appeared to be more stabilised and less affected by the ploidy change, however, formation of M₁ (1n) progenies increased with the rise in ploidy. Ploidy effects on traits such as occurrence of multiple ES, autonomous endosperm development, and twin embryos were also studied. Flexibility of guinea grass to tolerate excessive genome burden and successful formation of seeds overcoming endosperm balance number and endosperm imprinting constraints is also discussed.     

A test for obligate apomixis in grain sorghum R473

Summary Segregation patterns in progeny arrays of selfed plants, heterozygous for the Mdh 1 isozyme marker locus, were used in an attempt to confirm the presence of apomixis in the grain sorghum line R473. No evidence for obligate apomictic reproduction was obtained. However, our studies did not rule out the possibility of a low level of facultative apomixis in R473.     

Evidence of sexuality in induced tetraploids of Brachiaria brizantha (Poaceae)

Difficulties in obtaining new breeding lines of Brachiaria (Trin.) Griseb., an important forage grass in Brazil, are mostly related to differences in ploidy among the accessions, and to apomixis, an asexual mode of reproduction. Usually, sexual accessions are diploid while apomicts are polyploid. Induced tetraploids of Brachiaria brizantha (A. Rich.) Stapf have been successfully obtained and this paper presents the results of a study of their reproductive modes and fertility. Despite frequent meiotic aberrations during microspore development, the induced tetraploids produced viable pollen and produced progeny after controlled self-pollination. Similarly to the original diploid sexual progenitor, embryo sacs of the Polygonum type with confirmed meiotic origin were present in the induced tetraploids suggesting chromosome doubling did not alter the reproductive mode. The embryo sac of the Polygonum type was also observed in progenies obtained after self and open pollination. Nevertheless, embryo sacs of the Polygonum and the Panicum types within the same ovule were observed in some progenies obtained after open pollination, probably having resulted from hybridization with tetraploid, apomictic plants. Indeed, the compatibility of the progeny with tetraploid, apomictic B. brizantha was confirmed by the formation of mature caryopses after controlled pollination. Evidence is presented that the induced tetraploids and their progeny are sexual plants and that they are compatible with natural tetraploid B. brizantha. The induced tetraploids will be useful for analyses of apomictic inheritance as well as in the development of sexual tetraploid lines in Brachiaria breeding programs.     

New efforts to overcome apomixis in Poa pratensis L.

Summary By means of a new method, plants of Poa pratensis can be classified rapidly and reliably as to whether they are capable or incapable of parthenogenesis. Parthenogenesis was found to be under strong genetic control, dominant over obligatory fertilization. The selected sexual plants lack all genes/alleles responsible for parthenogenesis, while the polyploid apomictic varieties investigated were heterozygous with one or more dominant alleles. Also dosage effects and/or modifying genes are probably involved. Crosses of sexual individuals with various apomictic varieties resulted in sexual as well as highly apomictic F₁ hyrids. A scheme of recurrent hybridization for breeding of Kentucky bluegrass is proposed.Two other experimental ways to overcome apomixis in Poa pratensis were studied in addition. By application of growth regulators temporary sexuality could not be induced. Attempts of in vitro regeneration of plants from endosperm resulted only in callus and root formation.     

Chemical induction of apomictic seed formation in maize

Summary Silks of 18 maize (Zea may L.) F₁ hybrids were treated with different combinations of 9 growth regulators, colchicine, and dimethyl sulfoxide (DMSO) for the purpose to induce apomixis (agamospermy) in 1988 and 1989. Hybrid K301 × K303 gave the highest (0.36%) average frequency of seed induction among the hybrids. The most effective treatments were DMSO, gibberellic acid plus 6-benzyl aminopurine (6-BA), and DMSO plus methanesulfonic acid. Individually, the highest frequency of seed induction was 1.4% for hybrid K731×K306 when treated with -naphthalene acetic acid (NAA)-zeatin mixture. The frequency of seed induction seemed to depend partially on the interaction between chemicals and hybrids. Cytological observation of root-tip cells indicated that the majority of the seeds obtained were diploid, some were mixoploid, and a few were haploid. Diploid plants from induced seeds from the same parent were morphologically uniform and resembled the parent. Variations in plant and ear heights were comparable to those of the hybrid parent. Cytological and morphological investigations suggested that the chemically induced seeds originated mainly from somatic tissue but occasionally came from reduced cells in the embryo sac, leading to haploids. The results showed that chemical induction of adventitious embryony in maize hybrids is possible, but the more effective chemicals, their concentrations, and ways of application for increasing the frequency of seed induction need to be explored for practical use.     

Interspecific hybrids and possible phylogenetic relations in grain amaranths

Summary Phylogenetic relations among the three species in grain amaranth need investigation to provide information for breeding experiments germplasm conservation efforts, and decision on evolutionary patterns in the grain types. Hybrid development from crosses between species was studied to find out genetic relationship between them. Interspecific crosses were made among Amaranthus hypochondriacus, A. caudatus and A. cruentus in the glasshouse. The F₁ plants were relatively easy to obtain but had low pollen fertility (10.3–15.1%) and low seed set. A few of these hybrids did not produce seeds. Only a few F₁ seeds obtained in crosses between A. cruentus and A. caudatus. All the F₁ plants from these crosses died at the seedling stage. Crosses between A. cruentus and A. hypochondriacus produced few seeds. Most of the F₁ plants obtained from the seeds died at the seedling stage with only four plants growing to maturity but were sterile. Based on hybrid development, it was suggested that A. hypochondriacus and A. caudatus were genetically closer than the other two combinations of species studied. A. cruentus seemed to be genetically closer to A. hypochondriacus than it was to A. caudatus.Research was supported by Grand No. AMA-KE-4-83-22. (CRG GRANT) from the National Academy of Science, U.S.A.     

Doubling the chromosome number of bahiagrass via tissue culture

Crop improvement in bahiagrass (Paspalum notatum Flüggé) is limited by apomixis in most natural tetraploids, however, diploid sexual types occur. Production of sexual tetraploids by chromosome doubling will allow hybridization with apomictic tetraploids. Diploid bahiagrass (Paspalum notatum Flüggé) embryogenic callus tissue was exposed to three concentrations of three antimitotic chemical agents, colchicine, oryzalin and trifluralin. Callus was generated to plants and ploidy was evaluated by stomata size, mitotic chromosome counts, and flow cytometry. A total of 310 plants were verified as tetraploid of 1,432 plants that reached transplanting size. All treatments yielded 4x plants. The mean percentage success over all treatments was 22%, with means of 31% for oryzalin, 24% for colchicine and 16% for trifluralin. The high rates of success indicate that all agents can be successfully used to double chromosome numbers in bahiagrass. The percentage of 4x plants ranged from 9% (20 μM trifluralin) to 43% (20 μM oryzalin). Several treatments adversely affected regeneration. Mitotic chromosome counts are difficult and labor intensive in bahiagrass. Therefore, leaf stomata measurements were used as a preliminary screen. Data gave a bimodal distribution with overlapping tails and based on chromosome counts would have given an error rate of 12%. Flow cytometry analysis of regenerated plants resulted in mean nucleus fluorescence distributions consistent with control diploid or tetraploid values. These values agreed with chromosome counts, and this method is recommended for determining bahiagrass ploidy level. Research goals and available resources should be taken into consideration when selecting a treatment for chromosome doubling in bahiagrass.     

Breeding Panicum maximum in Brazil. 1. Genetic resources,modes of reproduction and breeding procedures

Summary Apomixis is widely distributed among tropical forage grasses, and has long been merely regarded as an impediment to breeding. Panicum maximum is presented as the first opportunity for Brazilian geneticists to develop and test original breeding schemes adapted to an apomictic species.A large and representative germplasm of P. maximum has been introduced and is currently being evaluated. Basic knowledges on biology and reproduction are also available, which demonstrate an easy manipulation of apomixis and sexuality. Several limiting traits have already been detected during evaluation, which justify breeding attempts. An ideal scheme is given to transfer new qualities to already selected varieties.     

Race specific resistance against Phytophthora infestans in potato is controlled by more genetic factors than only R-genes

Summary For RFLP mapping of R-genes, determining resistance to specific races of Phytophthora infestans in tetraploid potato, it is necessary to develop well segregating populations at the 2x level. During mapping studies, evidence was obtained that more genetic factor(s) are involved in the expression of R-genes than conventionally believed. Two experiments are described in which such an additional genetic factor was suppressing or enhancing the expression of unknown R _nand R _ifactors. R _nand R _iappeared to be present in the investigated plant material, containing R4 and R10, or in one of the susceptible crossing parents. In a third experiment, the expression and the segregation of the well known R1 gene was influenced by an additional genetic factor. In that case there were indications for a dominant suppressor. This was established by the selection of susceptible plants carrying a RFLP allele of probe GP21 closely linked to R1. In three of the four F₁ populations, resulting from crosses between such susceptible plants and susceptible tester plants, resistnat progenies were found. The resistance appeared to be R1-specific. This clearly indicates that in three of the four investigated susceptible plants, the R1 gene was still present but not expressed.