Fertility of Brachiaria ruziziensis in interspecific crosses with Brachiaria decumbens and Brachiaria brizantha: meiotic behaviour,pollen viability and seed set

Summary Colchicine induced tetraploid (2n=4x=36) Brachiaria ruziziensis were used as female parent in crosses with apomictic tetraploid species (2n=4x=36) Brachiaria decumbens and Brachiaria brizantha. Tetraploid B. ruziziensis pollinated with B. decumbens set significantly more seed than selfed or crossed with B. brizantha. The crossability between B. ruziziensis and B. decumbens is also better than between B. ruziziensis and B. brizantha. In addition, hybrid seedlings obtained in crosses involving B. brizantha are more frequently lethal. All the viable F₁ hybrids are tetraploid with 36 chromosomes. Meiotic chromosome behaviour suggest that the three species belong to the same genomic group and therefore the same agamic complex. Chromosome associations at metaphase I do not allow to identify fertile and sterile hybrids. The interspecific hybrids averaged a lower fertility than their female parent, but some hybrids were more fertile than their apomictic male parent.     

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.     

Meiotic behavior in interspecific hybrids between Brachiaria ruziziensis and Brachiaria brizantha (Poaceae)

The meiotic behavior of two half-sib interspecific tetraploid (2n = 4x = 36) promising hybrids, a sexual and an apomictic one, from crosses B. ruziziensis and B. brizantha, was evaluated. Although chromosome paired predominantly as bivalents, a few tri- and quadrivalents were recorded. Results suggest that B. brizantha and B. ruziziensis are closely related and genetic recombination is expected in hybrids. Introgression of specific target genes from B. ruziziensis into B. brizantha and vice-versa may be foreseen. However, abnormalities such as irregular chromosome segregation, chromosome stickiness and abnormal cytokinesis reported in these hybrids affect pollen fertility. More than 65% of pollen grains are sterile. Since the distinctive cytological feature of these hybrids is abnormal cytokinesis, this fact suggests that both parental genomes are unable to coordinate their activities with regard to this cytological phenomenon. Deployment of such hybrids in the process of developing varieties is discussed.     

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.     

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.     

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.     

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.     

Meiotic behavior in Brachiaria humidicola (Poaceae) hybrids

In the genus Brachiaria, genetic variation can be exploited directly from germplasm collections or released using sexual reproduction in normally apomictic polyploids. The discovery of a natural sexual polyploid accession H031 of Brachiaria humidicola collected in Africa, opened new opportunities to exploit the genetic variation in this species. This accession was crossed with an apomictic cultivar BRS Tupi with the same chromosome number (2n = 36) and 361 F₁ hybrids were obtained. Following visual selection for leafiness, vigor, growth habit, and the mode of reproduction, 50 hybrids were selected for further agronomic evaluation. The parents and 45 of the 50 selected hybrids were evaluated for the regularity of meiosis. In the female parent (H031), meiosis was somewhat irregular, with 16.3% of abnormal tetrads, whereas the male (cv. BRS Tupi) meiosis was very regular, with only 3.1% of abnormal tetrads. Among hybrids (sexual and apomictic), the percentage of abnormal tetrads ranged from 15.8 to 98.3%. The abnormalities included irregular chromosome segregation, chromosome stickiness and the absence of cytokinesis. Considering that apomixis in the genus Brachiaria is pseudogamic, and that meiotic aberrations can compromise pollen viability, the results of this study present another parameter to aid selection for more stable microsporogenesis. Apomictic derivatives with stable meiosis are candidates for new cultivars whereas sexual hybrids can be retained in breeding for another round of recombination.     

Novel genotypes of the subtropical grass Eragrostis curvula for the study of apomixis (diplospory)

The aim of this work was to obtain a series of genetically related lines of Eragrostis curvula with different ploidy levels and reproductive modes, which can be used for the discovery of genes associated to diplospory. E. curvula is a widely cultivated forage grass native to Southern Africa that reproduces naturally by obligate diplosporous apomixis (asexual reproduction by seeds) of the Antennaria type. In vitro culture of immature inflorescences of the apomictic cultivar Tanganyika led to the regeneration of a diploid plant with a particular morphology (plant UNST1122). Embryo sac analysis and progeny tests determined that plant UNST1122 reproduces by sexuality. UNST1122 was cloned by dividing its tillers and a R ₁ generation was obtained from seeds after open pollination among the diploid clones. A set of 500 seeds from a diploid R ₁ plant was treated with colchicine and two plants showing a duplicated chromosome number (2n = 4x = 40) were obtained (plants UNST1112 and UNST1131). Progeny tests using RAPDs indicated that these plants reproduce also by sexuality. The availability of sexual tetraploid genotypes of E. curvula will allow the production of hybrids with new interesting combinations of agronomic traits and the developing of mapping populations segregating for diplospory. Besides, these genetically related plants with different reproduction modes constitutes an excellent model for the identification of gen(es) involved in diplosporous apomixis by mRNA profiling as well as to study the genomic rearrangements and gene expression alterations associated to changes at ploidy levels.     

The transfer of triazine resistance from Brassica napus L. to B. oleracea L. III. First backcross to parental species

Summary Atrazine resistant Brassica napus × B. oleracea F₁ hybrids were backcrossed to both parental species. The backcrosses to B. napus produced seeds in both directions but results were much better when the F₁ hybrid was the pollen parent. Backcrosses to B. oleracea failed completely but BC₁s were rescued by embryo culture both from a tetraploid hybrid (2n = 4x = 37; A₁C₁CC) and sesquidiploid hybrids (2n = 3x = 8; A₁C₁C). Progeny of crosses between the tetraploid hybrid and B. oleracea had between 25 and 28 chromosomes. That of crosses between the sesquidiploid hybrid and B. oleracea had between 21 and 27. A few plants that had chromosome counts outside the expected range may have originated from either diploid parthenogenesis, unreduced gametes or spontaneous chromosome doubling during in vitro culture. Pollen stainability of the BC₁s ranged from 0% to 91.5%. All the BC₁s to B. oleracea were resistant to atrazine.     

Tetraploid bahiagrass hybrids: breeding technique,genetic variability and proportion of heterotic hybrids

Manipulating apomixis for fixing tetraploid bahiagrass (Paspalum notatum Flüggé) hybrids exhibiting superior agronomic characteristics would be a valuable tool for the genetic improvement of this species. The objectives were to create a second generation of hybrids by crossing sexual first-generation tetraploid hybrids (FGTH) and unrelated apomictic FGTH or ecotypes, determine the segregation for mode of reproduction, and estimate the resulting genetic variation and heterosis for several agronomic characteristics. The segregation for mode of reproduction was analyzed using mature embryo sac observations. Field measurements and visual ratings were used to evaluate the growth habit, production of inflorescences, cool-season growth and freeze resistance of hybrids. A ratio of 4.6:1 between sexual and apomictic hybrids was observed. Only 3% of the progeny was classified as highly apomictic. The low proportion of highly apomictic progeny restricts the feasibility of this breeding approach. Large genetic variation was observed among hybrids for growth habit, cool-season growth and freeze resistance. The proportion of hybrids exhibiting heterosis was high for plant height, cool-season growth, and freeze resistance. The tetraploid bahiagrass germplasm contains a large diversity for traits of agronomic importance that can be exploited for the genetic improvement of this species.     

Pachytene Chromosomes of Brachiaria ruziziensis Germain et Evrard

Brachiaria ruziziensis is a sexual diploid tropical grass species of wide adaptation and good forage quality. Pachytene chromosomes were studied in morphological detail and all nine chromosomes were identified by their length, centromere position, and size and distribution of chromomeres. The idiogram prepared for the first time in this species consists of four median and five submedian chromosomes.     

Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture

Summary Interspecific crosses were made to introduce the scent of flowers of C. purpurascens into C. persicum cultivars and ovule culture was used to rescue the abortive hybrid embryos. Cultivars of C. persicum diploid (CPD, 2n=2×=48) and C. persicum tetraploid (CPT, 2n=4×=96) were the pistillate parents and wild species of C. purpurascens (CP, 2n=34) were staminate parents. After pollination, crossed ovaries were collected periodically and examined using paraffin sections. Histological observations suggested that both hybrid ovules of CPD x CP and CPT x CP should be transferred to culture medium 35 days after pollination. Based up on this observation, crossed ovaries were collected 28 days after pollination and ovules with placenta were transferred to MS (1962) medium containing 3% sucrose. These ovules were cultured in the dark at 25° C. The hybrids (2n=41) derived from CPD x CP had the scent of C. purpurascens, whereas the hybrids (2n=65) derived from CPT x CP had the scent of C. persicum. Although both hybrids had complete genomes from the parents and produced a few viable pollen grains, they failed to yield viable seeds by self- and cross-pollination with fertile pollen grains of C. persicum cultivars.Abbreviations CPD C. persicum diploid - CPT C. persicum tetraploid - CP C. purpurascens     

Crosses between Beta vulgaris L. and Beta lomatogona F. et M.

Summary A population of 2x^ms sugar beets was crossed with 4x Beta lomatogona F. et M. The 3x F₁-plants were male sterile and were backcrossed with 2x and 4x sugar beets and multiplied without pollination as well. After the 1st backcross mainly 3x apomict types arose again and, among others, a small number of successful 4x backcrosses. After pollination by 4x sugar beets this 4x F₁ B₁ produced. besides predominatly apomictically multiplied 4x plants, also about 7% haploid 2x hybrids. The latter probably possess 1 genome from B. vulgaris and 1 genome from B. lomatogona. In the meiosis of the PMC's a certain amount of homeology between a number of chromosomes of both species could be established. The amphihaploid hybrids can be used as breeding parents for the creation of types in which introgession can occur. During hybridization in addition to 2x and 4x B. vulgaris types a number of 2x-, 3x-, 5x- and 6x-hybrids arose. This is presumably caused by the presence of gametes with the somatic number of chromosomes and the occurrence sometimes of haploid apomictic multiplication.The presence of large numbers of bolters in the F₁ and F₁ B₁ suggests that the bolting tendency of both species is based on different genes.     

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.     

Natural triploid hybrids between Solanum acauleBitter and S. infundibuliformePhilippi in the province of Jujuy,Argentina

Summary The occurrence of natural triploid hybrids between tetraploid Solanum acauleBitter subsp. acaule and diploid S. infundibuliformePhilippi both tuber-bearing Solanums, in the Puna region of Jujuy, is reported. The natural hybrids, which are morphologically intermediate between their putative parents and completely male sterile, have a somatic chromosome number of 36. The artificial hybrids, obtained by crossing both parental species, closely resemble the natural ones thereby confirming their parentage. It is proposed to designate the natural hybrids Solanum x viirsooi hybr. nov.     

Natural hybridization between Solanum acaule Bitt. and S. megistacrolobum Bitt. in the province of jujuy,Argentina

Summary Evidence is provided that hybridization of the tetraploid (2n=4x=48), self-fertile tuber-bearing species Solanum acaule Bitt. with the diploid (2n=2x=24), self-incompatible, tuber-bearing S. megistacrolobum Bitt. takes place in several localities of the province of Jujuy in the high, cold plateaux of the Argentine Puna. The triploid hybrids (2n=3x=36) closely resemble S. acaule in growth habit, leaf morphology and floral structures and for this reason they can be easily overlooked for that species in the field. Experimental data show that S. acaule can be crossed with S. megistacrolobum though the crossability is rather low and variable according to the particular cross considered. The artificial hybrid obtained compares well with the natural hybrid in morphology and chromosome number. The hybrids, though almost completely male sterile, are successful colonizers of disturbed areas around farmers' dwellings, cattle enclosures and other areas where the soil is rich in organic matter.There is some evidence that the Tilcara material of S. acaule subsp. aemulans and the hybrids of S. acaule x S. megistacrolobum have some characters in common, which can be interpreted as having a similar origin.It is postulated that S. acaule subsp. aemulans, in Jujuy at any rate, is not a primitive form of S. acaule as thought by Hawkes and Hjerting, but rather a fertile hybrid derivative of S. acaule x S. megistacrolobum through the functioning of 2n gametes.We also provide evidence that S. bruecheri Correll should not be considered a hybrid of S. acaulle x S. megistacrolobum but a synonym for S. gourlayi Hawkes. The new name, S. x indunii Okada et Clausen, is proposed to designate this hybrid.     

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC₁hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC₂ hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC₁ progeny did not show viralinfection even in the graftingexperiments.     

Transfer of resistance to race 2 of Plasmodiophora Brassicae from Brassica napus to cabbage (B. oleracea var. Capitata). I. Interspecific hybridization between B. napus and B. oleracea var. Capitata

Summary Interspecific hybridization between Brassica napus L. (2n=38, a₁a₁c₁c₁) and B. oleracea var. capitata L. (2x- and 4x-cabbage; 2n=2x=18, cc and 2n=4x=36, cccc) was carried out for the purpose of transferring clubroot disease resistance from the amphidiploid species to cabbage. Nineteen hybrids with three different chromosome levels (2n=28, a₁c₁c; 2n=37, a₁c₁cc and 2n=55, a₁c₁cccc) were obtained. The F₁ plants were mostly intermediate between the two parents but as the number of c genomes in the hybrids increased, the more closely the hybrids resembled the cabbage parent. All F₁ hybrids were resistant when tested against race 2 of Plasmodiophora brassicae wor. The complete dominance of resistance over susceptibility suggested that the gene(s) controlling resistance to this particular race of the clubroot pathogen is probably located on a chromosome of the a genome in Brassica.Contribution No. J654.     

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.