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.     

Chromosome numbers and microsporogenesis in Brachiaria brizantha (Gramineae)

The genus Brachiaria, native to the African tropical savannas, has achieved significance as a pasture grass in many tropical and subtropical countries, including Brazil. Many species and accessions are polyploid and apomictic, which complicates the improvement of breeding stocks through hybridization. In support of breeding programs, cytogenetic characterization, including chromosome counts and evaluation of the meiotic behavior in the accessions of the Brachiaria has been undertaken at the Embrapa Beef Cattle Center. In this study, 22 accessions of B. brizantha were analyzed of which one was found to be diploid (2n = 2x = 18), 18 were tetraploid (2n = 4x = 36) and three were hexaploid (2n = 6x = 54). The meiotic chromosome behavior was slightly irregular in the diploid and in some tetraploid accessions, and highly irregular in most tetra- and hexaploid accessions. Meiotic abnormalities were those common to polyploidy, i.e., multivalent chromosome association at diakinesis and irregular chromosome segregation leading to micronuclei formation in the tetrad stage. Low frequencies of multivalent chromosome associations among polyploids suggest that they may be segmental allopolyploids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Feulgen-DNA densitometry of embryo sacs permits discrimination between sexual and apomictic plants in Paspalum simplex

The chromosomes of sexual diploid plants of Paspalum simplex were colchicine-doubled and the plant obtained were crossed with their aposporous natural tetraploid counterparts to generate a F1 population segregating for apomixis. Analysis of the DNA content during megagametogenesis indicated that although the nuclei of nucellus and developing embryo sacs were in both the G1 and G2 phases, polar nuclei and egg cells of mature embryo sacs tended to remain in the G1 phase. Because both meiotic and aposporous mature embryo sacs are of the 8-nucleated-type in P. simplex and are barely distinguishable, nuclear DNA content of polar nuclei was used to distinguish apomictic and sexual phenotypes and confirmation obtained by progeny testing. This revised version was published online in August 2006 with corrections to the Cover Date.     

A synthetic hexaploid (2n = 42) oat from the cross of Avena strigosa (2n = 14) and domesticated A. magna (2n = 28)

A synthetic hexaploid oat was produced by chromosome doubling of a sterile triploid hybrid between cultivated Avena strigosa (2n = 14) cv. Saia and a domesticated form of A. magna (2n = 28). The synthetic hexaploid was intermediate between its parents in panicle shape and lemma color, similar to the tetraploid parent in spikelet structure, and to the diploid parent in having a single, albeit partially shriveled seed per spikelet, and low protein content. By the third generation, plants with yellowish lemmas, mostly two seeds per spikelet and better filled grains had been selected. Rust resistance of the diploid parent was retained in the synthetic hexaploid, but not tolerance to barley yellow dwarf virus disease (BYDV). Chromosome associations at meiosis in the triploid hybrid was low, with over 60% of them being univalents. Bivalent association was the rule in the synthetic hexaploid with an occasional one or two quadrivalents. Regular meiosis with 21 bivalents was observed in further generations. The preferential pairing of homologous chromosomes in the synthetic hexaploid was probably contributed by the A. strigosa genome which exhibits this tendency in artificial allopolyploid situations. Selection of yellow lemma color and two seeds per spikelet suggests that the genes controlling these traits are located on the chromosomes involved in quadrivalents in the synthetic hexaploid. The potential and limitations of utilizing the synthetic hexaploid in oat research and breeding are briefly discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chromosome studies in the genus Cucumis

Summary Cytological investigations of 50 wild Cucumis introductions revealed the presence of three tetraploid species with 2n=48 chromosomes, and one hexaploid species with 2n=72 chromosomes, while all other species are diploid containing 2ns=14 or 2n=24 chromosomes. Two of the tetraploid species, C. heptadactylus (P.I. 282446), and a species related to C. zeyheri (P.I. 273192, 299570, 299571, and 299572) are natives of South Africa, while C. aculeatus (P.I. 193967, 196844, 273648, 273649, and 273650) is found in Ethiopia. The hexaploid, C. figarei (P.I. 343699, 343700, 343701), is a native of Nigeria. All polyploids are perennial, have efficient vegetative reproduction systems, and may have originated from the spontaneous formation of polysomatic cells.Supported by the Colorado State University Experiment Station and published as Scientific Series Paper No. 2089.     

Reproductive pathways of seed development in apomictic guinea grass (<Emphasis Type="Italic">Panicum maximum</Emphasis> Jacq.) reveal uncoupling of apomixis components

One hundred and sixty accessions representing global germplasm of guinea grass (Panicum maximum Jacq.), an important apomictic (aposporous) fodder crop, were subjected to study on reproductive diversity in apomictic seed development utilizing ovule clearing and flow cytometric seed screen (FCSS). Single seed FCSS of selected 14 tetraploid and five hexaploid lines demonstrated uncoupling between the three apomixis components, viz. apomeiosis, parthenogenesis and functional endosperm development, in natural as well as experimental populations, though it differed across ploidy levels and genotypes. Reconstruction of reproductive pathways yielded a total of eight different pathways of seed development, arising by uncoupling/recombination between apomixis components. Amongst these, two pathways involving modifications in embryo-sac (ES) (presence of two polar nuclei in aposporous ES that fuse prior to fertilization) and fertilization process (fusion of only one polar nucleus in a sexual ES) have been reported for the first time. Some of the combinations, such as M_I (haploids arising from parthenogenetic development of reduced egg cell), were found viable only in hexaploid background. Germplasm lines with higher expression of individual components were also identified. These components (including autonomous endosperm development) were also experimentally partitioned in hexaploid progenies (derived from a tetraploid parent viz. accession IG 04-164) that showed segregation in their reproductive capacities, and are reported for the first time. Occurrence of several apomixis recombinants (phenotypic) in guinea grass lines suggested their hybrid origin, favors a multigene model for apomixis, with their penetrance affected by modifiers and epigenetic mechanisms, in contrast to earlier reports of single locus control. Implications of partitioning components on apomixis research are discussed.     

Production of 27-, 28-, and 56-chromosome apomictic hybrid derivatives between pearl millet (2n=14) and Pennisetum squamulatum (2n=54)

Summary An interspecific hybridization program designed to transfer gene(s) controlling apomixis from Pennisetum squamulatum Fresen. (2n=6x=54) to induced tetraploid (2n=4x=28) cultivated pearl millet, Pennisetum americanum (L.) Leeke resulted in four offtype plants, two with 27 chromosomes and two with 28 chromosomes. These plants were found among 217 spaced plants established from open-pollinated seed of an apomictic 21-chromosome polyhaploid (2n=21) plant derived from an apomictic interspecific hybrid (2n=41) between tetraploid pearl millet and Pennisetum squamulatum. It appeared that a 21- (or 20-) chromosome unreduced egg from the apomictic polyhaploid united with a 7-chromosome pearl millet (2n=2x=14) gamete to produce a 28- (or 27-) chromosome offspring. Meiotic chromosome behavior was irregular averaging from 3.60 to 4.05 bivalents per microsporocyte in the 27- and 28-chromosome hybrids. The 27- or 28-chromosome hybrids, like the 21-chromosome female parent, shed no pollen, but set from 1.8 to 28 seed per panicle when allowed to outcross with pearl millet. Progeny of the 28-chromosome hybrids were uniform and identical to their respective female parents, indicating that apomixis had been effectively transferred through the egg. In addition, a 56-chromosome plant resulting from chromosome doubling of a 28-chromosome hybrid was identified. Pollen was 68 per cent stainable and the plant averaged 2.3 selfed seeds per panicle. Chromosomes of the 56-chromosome plant paired as bivalents (x=10.67) or associated in multivalents. Three to nine chromosomes remained unpaired at metaphase I. Multiple four-nucleate embryo sacs indicated the 56-chromosome hybrid was an obligate apomict. The production of 27-, 28-, and 56-chromosome hybrid derivatives were the results of interspecific hybridization, haploidization, fertilization of unreduced apomictic eggs, and spontaneous chromosome doubling. These mechanisms resulted in new unique genome combinations between x=7 and x=9 Pennisetum species.     

Genotypic control of chromosome pairing in amphiploids involving the cultivated oat Avena sativa L.

Summary A genotype of the diploid species Avena longiglumis (Cw 57) has been shown to modify the genetic control of diploid-like chromosome pairing in the cultivated oat, A. sativa (2n=6x=42) leading to increased homoeologous chromosome pairing in 4x hybrids between the two species (Rajhathy & Thomas, 1974). The Cw 57 genotype has a similar effect in increasing homoeologous chromosome pairing in amphiploids combining diploid and hexaploid genomes including associations between alien chromosomes and their corresponding pairs in hexaploid species. The effect of the Cw 57 genotype is probably in altering the specificity of chromosome pairing in the early stages of meiosis. The use of the Cw 57 genotype to induce homoeologous chromosome pairing as a technique for the transfer of desirable alien variation into the cultivated oat is discussed.     

Interfering with regular meiotic behaviour in Avena sativa as a method of incorporating the gene for mildew resistance from A. barbata

Summary The regular meiotic behaviour of the cultivated oat Avena sativa (2n=6x=42) is genetically controlled. The factors which control the diploid-like meiotic behaviour also restrict the amount of pairing that occurs between alien chromosomes and their homoeologues in A.sativa, and hence increases the difficulties of introducing desirable variation from wild species into the cultivated oat. A genotype of the diploid species A.longiglumis which interferes with the regular meiotic behaviour of A. sativa can be used to induce pairing between alien chromosomes and their corresponding chromosomes in A. sativa. Using this procedure the dominant gene conferring mildew resistance has been transferred from the tetraploid weed species A. barbata into the cultivated oat.     

Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

Analysis of meiotic behavior in selecting potential genitors among diploid and artificially induced tetraploid accessions of Brachiaria ruziziensis (Poaceae)

Sexuality is correlated with diploidy and apomixis with polyploidy in the Brachiaria genus. Brachiaria ruziziensis is a key species in Brachiaria breeding due to its obligate sexuality and intrinsic agronomic qualities. Interspecific crosses in the genus became feasible only when a few diploid accessions of B. ruziziensis were artificially tetraploidized and remained sexual. Hybridization has been done since, using natural tetraploid apomictic accessions of B. brizantha or B. decumbens as pollen donors. Twenty two accessions of B. ruziziensis from the Embrapa Beef Cattle germplasm collection (Campo Grande, MS, Brazil) were cytologically analyzed: 16 are natural diploids (2n = 2x = 18) and six are artificially induced tetraploids (2n = 4x = 36). The meiotic behavior in the 16 diploid accessions varied. The mean of meiotic abnormalities per accession ranged from zero to 24.46%. Meiotic behavior in the induced tetraploid accessions also varied with the mean of meiotic abnormalities ranging from 5.20% to 54.71%. The most common abnormalities observed in both the diploid and the tetraploid accessions, were those related to irregular chromosome segregation. In one tetraploid accession, with a high frequency of those, other irregularities involving chromosome orientation at metaphase plate and chromosome convergence to the poles, a meiotic mutation known as divergent spindle, were recorded. Meiotic behavior should be considered in selecting potential genitors for breeding.     

Resistance to barley yellow-dwarf-virus disease in derivatives of crosses between hexaploid wheat and species of Lophopyrum (Triticeae; Poaceae)

Among the wheatgrasses that are possible sources of genetic resistance for wheat to barley yellow-dwarf-virus disease (BYD) are those that have been commonly subsumed under the name Agropyron elongatum (Host) P. Beauv. Two of these wheatgrass species are the diploid Lophopymm elongatum (Host) Á. Löve (2n = 2x = 14) and the decaploid L. ponticum (Podp.) Á. Löve (2n = 10x = 70). These two species, the addition and substitution lines of L. elongatum chromosomes in hexaploid wheat (Triticum aestivum L.), and derivatives of hybrids between hexaploid wheat and L. ponticum, were screened for resistance to BYD, as defined by visual symptoms in field-grown plants. The two species, an amphiploid derived from L. elongatumבChinese Spring’ wheat, and the derivatives involving L. ponticum chromosomes were all highly resistant. The substitution and addition lines of L. elongatum chromosomes in ‘Chinese Spring’ revealed that the genetic control of resistance in L. elongatum must be complex, with more than one critical locus involved. Chromosomes 2E and 5E are involved and there are lesser contributions to resistance from the remaining wheatgrass chromosomes. One highly resistant derivative was determined to have only three pairs of L. ponticum chromosomes. It has a wheat-like morphology and shows promise for further characterization.     

Aneuploids of perennial ryegrass (Lolium perenne)

Summary Aneuploid plants of perennial ryegrass (Lolium perenne L.) with 2n=15 to 30 chromosomes were obtained by crossing a near-triploid (2n=3x+1=22) with a diploid or on open-pollination with diploids and tetraploids. Aneuploids occurred with a frequency of 83% in near triploid × diploid progeny and 92% on open-pollination with diploid and tetraploid plants. Aneuploid plants with 15 to 18 chromosomes resembled diploids in morphology and those with 19 to 30 chromosomes were akin to tetraploids. Meiotic studies suggested that most aneuploid plants resulted from transmission of aneuploid egg cells (n=8 to 23). Aneuploid plants with 2n=27 to 30 chromosomes in the progeny of 22×14 cross originated from unreduced egg cells. Plants with 19 to 21 chromosomes were recovered only by immature seed culture. Aneuploid plants with 26 to 30 chromosomes and triploids (2n=21) had higher pollen fertility and bigger seeds than plants with 15 to 22 chromosomes.     

Recurrent Addition of the Pennisetum americanum Genome in a P. americanum×P. orientale Hybrid

An interspecific hybrid involving Pennisetum americanum (2 n = 14) and a diploid cytotype of P. orientale (2 n = 18) was backcrossed to P. americanum using the hybrid (2 n = 16, 7‘A’+ 9 ‘O’) as the female parent. Pollen mother cells of 13 BC₁ plants contained a complement of 14‘A’+ 9 ‘O’ chromosomes. Five BC₂ plants obtained through further backcrossing to P. americanum had 21‘A’+ 9 ‘O’ chromosomes revealing another addition of the P. americanum genome. The role of such recurrent additions of parental genomes in the evolution of polyploid species has been discussed.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Crosses between Beta intermedia bunge and B. vulgaris L.

Summary A tetraploid annual male sterile form of Beta vulgaris L. (2n=4x=36) was crossed with the wild beet species Beta intermedia Bunge (2n=36). The resulting F₁-plants were male sterile annuals being two or three times back-crossed to diploid and tetraploid sugar and fodder beets in the next years. Apart from tetraploid material (36 chromosomes) hexaploid (54 chromosomes) and a number of aneuploid plants developed.The results obtained justify the conclusion that, at a tetraploid level the material mostly propagates apomictically after the F₁ generation. The presence of penta-, hexa-, septa-and even octaploid plants might be explained by assuming that no meiosis has taken place in the crossing partners. Triploid plants are sometimes found in the progeny of hexaploid material and may presumably be considered haploids. Moreover some pentaploid plants were found in the progeny of the open pollinated F₁ which after two generations of bagging are still pentaploids although they produce no pollen. This is another clear indication of apomictic reproduction.The tetraploid generation from the cross between the hexaploid material and diploid sugar beets probably contains the best prospects for breeding.     

Intravarietal polyploidy in the apple (Malus pumila Mill.) cultivar Hazratbali

Summary A triploid cytotype of cv. Hazratbali (Malus pumila Mill.), was found growing in orchards of diploid trees. Morphological comparisons between the two cytotypes reveal that the triploid is more vigorous, and bears more attractive fruit. The meiotic behaviour of the two cytotypes has been described. The impact of polyploidy on quality characteristics of apple at the intravarietal level is analysed. The possible mode of origin of the triploid cytotype is discussed.     

Cytological and geographical characterizations of Hemarthria

Summary Chromosme numbers were determined for 56 Hemarthria altissima (Poir.) Stapf et C. E. Hubb. introduction, forty of these were diploid (2n=18) and 16 were tetraploid (2n=36). These results, combined with previous findings, show that all but three of the tetraploids in the USDA Hemarthria altissima collection originated north of 20 S latitude. All other introductions collected throughout southern Africa were diploid. Meiosis was regular in a hexaploid, 6 tetraploid and 32 diploid introductions but a low frequency of quadrivalents was found in the teraploids. H. uncinata R. Br. and H. uncinata var. spathacea (Domin) Vickery were found to have 2n=36 chromosomes H. compressa (L.f.) R. Br., introduced from Japan, had 2n=54 chromosomes. No major cytological abnormalities exist in these Hemarthria species. Significant variation exists among the ploidy levels for cold tolerance and in vitro organic matter digestion to provide the basis for a forage improvement program.Joint contribution from the Department of agronomy. Institute of food and Agric. Sciences (IFAS), Univ. of Florida. Gainesville, Fl and, Plant Genetics and Germplasm Institute, USDA, Beltsville, MD Published as IFAS Journal Series No. 2849.     

Regeneration and somaclonal variation in apomictic Paspalum dilatatum Poir

Summary In an attempt to incorporate variation into a uniform obligate apomict, plants of apomictic common dallisgrass, Paspalum dilatatum Poir., were regenerated from callus derived from immature inflorescences. Plants developed through both organogenesis and embryogenesis. A total of 682 regenerants were produced and more than 400 were transplanted into a field nursery and screened for somaclonal variation. Eventually 20 regenerants were selected, increased, and planted into a replicated nursery along with normal common dallisgrass. The characteristics examined were maturity date, plant height, number of racemes per inflorescence, number of spikelets per raceme, pubescence, stigma and anther color, ergot resistance, seed germination, seed set, pollen stainability, method of reproduction, and chromosome number. There were differences among the regenerants and between them and common dallisgrass for all traits except chromosome number, stigma and anther color, and ergot resistance. One of the more important regenerants had significantly higher seed set than common dallisgrass. All regenerants reproduced by aposporous apomixis but some exhibited a high degree of abortion while others had more aposporous embryo sacs per ovule than common dallisgrass. These findings demonstrate that common dallisgrass can be regenerated through tissue culture and that somaclonal variation is expressed in some of the regenerants, even though some of the altered traits are deleterious.     

Nematode Resistance Derived from Wild Beet and its Meiotic Stability in Sugar Beet

Three different karyotypes of sugar beet with resistance against the beet cyst nematode (Heterodera schachtii) have been investigated. These comprised monosomic addition lines (2n = 19) with one complete chromosome from B. patellarris or B. procumbens, one line with a chromosomal fragment added to the normal sugar beet chromosome complement (2n =18 + fragment) and one diploid line (2n = 18). The fragment originated from a B. procumbens chromosome since during meiosis it formed a univalem. It carries the gene for nematode resistance. Meiotic disturbances like univalems. laggards, anaphase I bridges, fragments and micronuclei were observed in all resistant genotypes. These may result in an exclusion of the chromosome fragment carrying the resistance from the rest of the genome. In the diploid resistant line, a chromosome with a translocation could be distinguished from the other B. vulgaris chromosomes. Meiotic irregularities also appeared in diploid resistant types and are one main reason for low transmission of the resistance. Tin-relationship between meiotic stability and the transmission rate of the resistance gene is discussed.