Crossability of Brassica tournefortii and B. rapa, and morphology and cytology of their F1 hybrids

The crossability between Brassica tournefortii (TT, 2n = 20) and Brassica rapa (AA, 2n = 20) and the cytomorphology of their F₁ hybrids were studied. Hybrids between these two species were obtained only when B. tournefortii was involved as a female parent. The hybrid plants were intermediate for most of the morphological attributes and were found to be free from white rust under field conditions. The F₁ plants showed poor pollen fertility, although occasional seed set was achieved from open pollination. Self-pollination or backcrosses did not yield any seeds in these plants. The occurrence of chromosome association ranging from bivalents (0–7), trivalents (0–2) to a rare quadrivalent (0–1) in the dihaploid hybrids indicates pairing between the T and A genomes. The homoeologous pairing coupled with seed set in the F₁ plants offer an opportunity for interspecific gene transfers from B. tournefortii to B. rapa and vice-versa through interspecific hybridization. Received: 3 July 2000 / Accepted: 22 September 2000     

An analysis of interspecific hybrids and phylogenetic implications inCucumis (Cucurbitaceae)

Investigations on interspecific crossability in 8Cucumis species (2n = 24) and chromosome pairing and pollen fertility of their hybrids from 15 combinations have been utilized for tracing the phylogenetic relationships among these taxa and factors responsible for their differentiation. A collective evaluation of data suggests that there are three broad groups of species, one of the spiny fruited interfertile species, whose hybrids show varying degree of chromosome associations and low to high pollen fertility; the second of species with non-spiny fruits, which are completely incompatible with the former but weakly compatible with the cultivated species,C. melo L. to produce partly developed seeds, and the third group ofC. metuliferus E. Mey. exSchrad. andC. melo and its different botanical varieties. The species with spiny fruits can be further divided based on karyomorphological similarities and/or on relative genomic affinity, indicated by chromosome pairing and hybrid pollen fertility.Cytogenetics inCucumis III.     

Breeding system and crossability in JapaneseEpimedium (Berberidaceae)

A series of experimental pollinations involving six species ofEpimedium provided strong evidence for an outbreeding system and no internal (postmating, postpollination) barrier to hybridization in the taxa concerned. Self-pollinations of four species,E. diphyllum, E. trifoliatobinatum, E. grandiflorum andE. sempervirens indicated high self-incompatibility (at most 5.4%, usually 0% in capsule-set). Field experiments in a population revealed the occurrence of cross-pollination. On the other hand, interspecific cross-pollinations showed high crossability at any combination of species (33.3%–100% in capsule-set). Furthermore, the interspecific F₁s obtained germinated at a high rate (usually more than 20%) and three of them, which bloomed, are highly fertile (more than 68.6% in pollen viability). The results were discussed in connection with the isolating mechanisms between the species ofEpimedium.     

A reconsideration of relationships among Japanese Trillium species based on karyology and AFLP data

To reexamine the relationships among the Japanese Trillium species that form a polyploid series, we performed principal-coordinates analysis (PCOA) based on proposed karyotypic compositions and on amplified fragment length polymorphism (AFLP) analysis. A hexaploid species, T. smallii, whose karyotypic composition had been hypothesized as K₂K₂SSUU, with hybridization between tetraploid T. apetalon (SSUU) and a presumed K₂K₂ diploid species, showed a genotype corresponding to T. yezoense (K₁SU). Accordingly, T. smallii appears to be an allopolyploid of T. yezoense, with the karyotypic composition K₁K₁SSUU. Trillium channellii, a recently described tetraploid species whose origin and genealogical position remains unclear, showed a genotype corresponding to K₁K₁K₂T. We conclude that T. channellii may be derived from hybridization of T. camschatcense (K₁K₁) as the seed parent and T. tschonoskii (K₂K₂TT) or hexaploid T. hagae (K₁K₁K₂K₂TT) as the pollen parent.     

Chromosome Maps of Trilliaceae: II. A Study of the Genome Composition in Polyploid Species of the Genus Trillium by Fluorescence Nucleotide Base-Specific Staining of Heterochromatic Chromosome Regions

Chromosome banding with nucleotide base-specific fluorochromes chromomycin A₃ (CMA) and Hoechst 33258 (H33258) was used to study the karyotypes and to construct cytological maps for diploidTrillium camschatcense(2n = 10), tetraploid T. tschonoskii(2n = 20), hexaploidT. rhombifolium (2n = 30), and a triploid T. camschatcense × T. tschonoskii hybrid (T. × hagae, 2n = 15). With H33258, species- and genome-specific patterns with numerous AT-rich heterochromatin bands were obtained for each of the four forms; CMA revealed a few small, mostly telomeric GC-rich bands. In T. tschonoskii, the two subgenomes were similar to each other and differed from the T. camschatcense genome; on this evidence, the species was considered to be a segmental allotetraploid. InT. ×hagae, one T. camschatcense and both T. tschonoskii subgenomes were identified. The subgenomes of T. rhombifoliumonly partly corresponded to the T. camschatcense and T. tschonoskii genomes, in contrast to the morphologically identical Japanese species T. hagae. This was assumed to indicate that allohexaploids T. rhombifolium and T. hagae originated independently at different times; i.e., their origin is polyphyletic. Based on the chromosome maps, a new nomenclature was proposed for theTrillium genomes examined: K₁K₁ for T. camschatcense,T₁T₁T₂T₂ for T. tschonoskii,K₁T₁T₂ for T. × hagae, and K_1RK_1RT_1RT_1RT_2RT_2R for T. rhombifolium.     

Prospects for interspecific hybridization of Lupinus atlanticus Gladst. with L. cosentinii Guss.

Summary Successful crossing is reported between L. atlanticus Gladst. (2n = 38) and L. cosentinii Guss. (2n = 32), using lines of both species selected for crossability followed by selection of relatively fertile progenies. In one cross, 82E75, from a single F₂ segregating plant, 22 F₃ seeds were obtained. Some other less crossable combinations were completely sterile in the f₁ or F₂. Backcrossing to both parent species was successful, but some crosses gave relatively more seed by using F₂ plants for backcrossing rather than F₂'s. It is concluded that potential exists for introgression of useful genes in both directions.     

Gene transferability from transgenic Brassica napus L. to various subspecies and varieties of Brassica rapa

Gene transferability from transgenic rapeseed to various subspecies and varieties of Brassica rapa was assessed in this study. Artificial crossability was studied in 118 cultivars of 7 B. rapa subspecies and varieties with the transgenic rapeseed GT73 (Brassica napus) as the pollen donor. On average 5.7 seeds were obtained per pollination, with a range from 0.05 to 19.4. The heading type of B. rapa L. showed significantly higher crossability than non-heading types of B. rapa. The spontaneous outcrossing rate between B. rapa (female) and the transgenic rapeseed Ms8 × Rf3 (B. napus) (male) ranged from 0.039 to 0.406%, with an average of 0.19%. The fertilization process and the development of the hybrid seeds as shown by fluorescent staining techniques indicated that the number of adhered pollens on the stigma was reduced by 80%, the number of pollen tubes in the style was reduced by 2/3 and the fertilization time was delayed by over 20 h when pollinated with the transgenic rapeseed Ms8 × Rf3 in comparison with the bud self-pollination of B. rapa as control. About 10–70% of the interspecific hybrid embryos were aborted in the course of development. Some seeds looked cracked in mature pods, which showed germination abilities lower than 10%. The spontaneous outcrossing rates were much lower than the artificial crossability, and their survival fitness of the interspecific hybrid was very low, indicating that it should be possible to keep the adventitious presence of the off-plants under the allowed threshold, if proper measures are taken.     

Biological containment of potato (Solanum tuberosum): outcrossing to the related wild species black nightshade (Solanum nigrum) and bittersweet (Solanum dulcamara)

The biological containment of the potato (Solanum tuberosum) was assessed by establishing the crossability of this tuberous crop with the related wild non-tuberous species in The Netherlands, black nightshade (S. nigrum) and bittersweet (S. dulcamara). To circumvent crossability barriers, genotypes with different ploidy number were employed and crosses were performed under different environmental conditions. S. dulcamara was shown to be incongruent with potato at all ploidy levels, while S. nigrum displayed unilateral incompatibility. If S. nigrum was emasculated and used as female, fertilization by potato pollen resulted in berry set and seed development. Emasculation of S. nigrum was essential in this cross, because analysis of the fertilization process demonstrated that this species is highly self-compatible and potato pollen was outcompeted by pollen of S. nigrum. The hybrid seeds derived from this cross did not mature and appeared not to be viable. By application of the technique of embryo rescue of immature embryos, hybrid plants could be obtained. However, these hybrid plants proved to be sterile. These data demonstrate that gene flow by pollen dispersal from potato to its most common wild relatives in Western Europe is highly unlikely. The potato is thus a naturally contained species in this part of the world.     

Experimental evidence on the affinities ofPapaver somniferum (Papaveraceae)

Results obtained from crossing experiments betweenP. somniferum subsp.somniferum (2n = 22) and subsp.setigerum (2n = 44),P. glaucum (2n = 14) andP. gracile (2n = 14) and from the observation of meiotic chromosome pairing in the various hybrids obtained do not provide straightforward evidence for the hypothesis thatP. somniferum originated as a triploid hybrid between taxa similar toP. glaucum andP. gracile (Kadereit 1986a, b).—On the one hand, the pattern of crossability found reflects the closer similarity of subsp.somniferum toP. glaucum and of subsp.setigerum toP. gracile, which was interpreted as segregation of parental characters, and the high frequency of 2n = 28 chromosomes among F₂-progeny from the hybrid subsp.somniferum × subsp.setigerum (2n = 33) might reveal n = 7 as the base number also ofP. somniferum. On the other hand, however, the general difficulty of obtaining hybrids, and the low incidence of bivalent formation in their meiosis, probably indicating a lack of chromosome homology between the different species, do not fit the above hypothesis.—These results are in marked contrast to the morphological similarity between the three species involved.     

A single dominant gene for Fusarium wilt resistance in protoplast-derived tomato plants

Summary Autotetraploids were established from 8 diploid wild species of section Arachis. In all the autotetraploids the chromosomes paired largely as bivalents even though they possess the ability to pair as multivalents. Pollen and pod fertility in the C₁ generation were not directly associated with chromosome pairing. The C₂ generation autotetraploids showed a gradual increase in bivalent associations and pollen and pod fertility. The identification of two genomes, A and B, in the diploid species and in the tetraploid, A. hypogaea, of the section Arachis, a fairly good crossability, and the type of chromosome associations observed in hybrids between A. hypogaea and the autotetraploids of wild Arachis species indicated good prospects of utilizing autotetraploids as genetic bridges in transferring desired traits from these taxa into groundnut.Submitted as Journal Article No. 516 by International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Spontaneous hybridization between a male-sterile oilseed rape and two weeds

Spontaneous interspecific hybrids were produced under natural conditions (pollination by wind and bees) between a male-sterile cybrid Brassica napus (AACC, 2n = 38) and two weeds Brassica adpressa (AdAd, 2n = 14) and Raphanus raphanistrum (RrRr, 2n = 18). After characterization by chromosome counts and isozyme analyses, we observed 512 and 3 734 inter-specific seeds per m² for the B. napus-B. adpressa and B. napus-R. raphanistrum trials respectively. Most of the hybrids studied had the expected triploid structure (ACX). In order to quantify the frequency of allosyndesis between the genomes involved in the hybrids, their meiotic behavior was compared to a haploid of B. napus (AC). For the B. napus-B. adpressa hybrids, we concluded that probably no allosyndesis occurred between the two parental genomes, and that genetic factors regulating homoeologous chromosome pairing were carried by the B. adpressa genome. For the B. napus-R. raphanistrum hybrids, high chromosome pairing and the presence of multivalents (in 9.16% of the pollen mother cells) indicate that recombination is possible between chromosomes of different genomes. Pollen fertility of the hybrids ranged from 0 to 30%. Blackleg inoculation tests were performed on the three parental species and on the interspecific hybrids. BC₁ production with the weeds and with rapeseed was attempted. Results are discussed in regard to the risk assessment of transgenic rapeseed cultivation, F₁ hybrid rapeseed variety production, and rapeseed improvement.     

Allelism of endosperm balance number (EBN) in Solanum acaule Bitt. and other wild potato species

The inheritance of endosperm balance number (EBN), a genetic, dose-dependent crossability system functioning in tuber-bearing Solanum (potato) species, was investigated for certain wild potato species having an EBN equal to one half of their ploidy. The EBN of Solanum acaule, a disomic 4(2EBN) South American species, was investigated by producing F₁ and F₂ hybrids with artificial 4x(2EBN) S. commersonii. This allowed assessment of recombination among the two genomes of disomic S. acaule and that of S. commersonii. When crossability of the hybrids with 1EBN, 2EBN and 4EBN standards was tested, no variation for EBN was detected. The apparent lack of recombination and segregation for EBN in these hybrids indicates that the genomes of S. acaule and S. commersonii carry EBN in a genetically-similar way. Combined with previous reports, these data indicate that the inheritance of EBN is similar in widely-separated taxa from South America and Mexico.     

Successful crosses between Festuca arundinacea Schreb. and Dactylis glomerata L.

Summary Five F₁ plants have been obtained after extensive crossing between different ecotypes or varieties of Festuca arundinacea Schreb. and Dactylis glomerata L. The success did not appear to depend on specific treatments (spraying with -aminocaproic acid or gibberellic acid or pre-pollination with killed pollen from the seed parent), but the crossability is limited to exceptional plants.F₁ hybrids showed characteristics of both the parents. In four hybrids various developmental disturbances were observed (low viability, aneusomaty, absence of development of inflorescences). Only one hybrid consistently showed 2n=35 chromosomes, good viability and growth, however, it was sterile. After clonal propagation, attempts for polyploidization were started.     

Cytogenetic studies in Trifolium Spp. related to berseem

Summary Seed formation by large-scale hybridization within and between Trifolium species related to T. alexandrinum L., was studied. The twelve species studied were: a. T. alexandrinum L., b. T. berytheum Boiss., c. T.salmoneum Mout., d, T. apertum Bobr., e. T. Meironense Zoh. et Lern., f. T. echinatum M.B., g. T. latinum Seb., h. T.carmeli Boiss., i. T. scutatum Boiss., j. T. plebeium Boiss., k. T. vavilovi Eig. and 1. T. constantinopolitanum Ser. Hybridization was done either by emasculation and fertilization by hand, or in nature, by utilizing the existence of natural stands and to serve as pollen source the self-incompatibility of some of the species concerned. Results of the two methods were highly comparable although seedset was much higher when crossing was done manually.Crossability, as estimated by seedset, varied in specific cross combinations and ranged between 0 and 70%. Based on the pattern of crossability, five crossability groups were identified with > 20% seedset in interspecific—intra-group cross combinations, and usually less than 5% in inter-group cross combinations. Species a, b, c, d and e form the first crossability group; f, g and h form the second one; h, i, j belong to the third crossability group, while k and l appear as unispecific fourth and fifth crossability groups. The high level of seedset in interspecific hybridization is discussed.Publication of the Agricultural Research Organization No. 2195-E. The work reported here was financed by Grant FG-IS-222 from the U.S. Department of Agriculture under P.L. 480.     

Interspecific hybridization of perennial Medicago species using ovule-embryo culture

Summary New interspecific hybrids between alfalfa (Medicago sativa L.) and several perennial Medicago species were obtained by embryo rescue techniques. The methodology, designated ovule-embryo culture, involved preculturing the fertilized ovule (10 to 20 days post-pollination) for a period of six to 12 days followed by excision and direct culture of the embryo. Placement of the hybrid embryo directly onto culture medium without the interim ovule culture was unsuccessful. Ovule culture to germination without removing the embryo also was unsuccessful. Ovule-embryo culture was essential for recovering interspecific hybrids between diploid alfalfa (2n=2x=16) and the following diploid (2n=2x=16) species: M. hybrida Traut., M. marina L., M. papillosa Boiss., M. rhodopea Velen. and M. rupestris M.B. In addition, trispecies hybrids between M. sativa x M. dzhawakhetica Bordz. F₁ hybrids (2n=3x=24) and either M. cancellata M.B. (2n=6x=48) or M. saxatilis M.B. (2n=6x=48) were obtained from ovuleembryo culture. Media manipulations using M. sativa x M. rupestris F₁ and first backcross generation embryos demonstrated the optimum concentration of 12.5 mM NH₄ ⁺ for successful embryo rescue; ammonium salt formulation (whether chloride, nitrate or sulfate) was not critical. From a few thousand crosses, hybrids between M. sativa and either M. rhodopea or M. rupestris were recovered relatively efficiently with 157 and 66 hybrids, respectively. However, only 13 hybrids between M. sativa and M. papillosa were obtained from more than 2,000 crosses, and just two hybrids each have been recovered from the combinations M. sativa x M. hybrida and M. sativa x M. marina from 2,000 to 3,000 crosses. The predominant chromosome number between diploid alfalfa and the other diploid perennial species was 2n=2x=16. Morphology of the hybrids was generally intermediate. Electrophoretic analysis of the F₁ hybrids and parental clones on uniform or gradient polyacrylamide gels demonstrated that peroxidase phenotypes could be used to confirm hybridity. For all interspecific combinations there was at least one peroxidase isozyme unique to the wild species that was present in the F₁ interspecific hybrid.     

Inheritance of freezing resistance in interspecific F1 hybrids of Eucalyptus

Summary The inheritance of freezing resistance in interspecific F₁ hybrid families of Eucalyptus encompassing 27 different species combinations and a range of levels of hardiness was examined. Freezing resistance was assessed by determining the temperatures required to cause either 30% (T30), 40% (T40), or 50% (T50) leakage of electrolytes from excised leaf discs subjected to artificial freezing. Highly significant variation in freezing resistance occurred between species; the maximum difference between parents in any specific combination was over 9°C (E. gunnii x E. globulus). Freezing resistance was inherited in a predominantly additive manner in interspecific hybrids, although there was a tendency towards partial dominance toward the more sensitive species in some combinations (e.g., E. nitens x E. Globulus, E. nitens x E. camaldulensis, E. gunnii x E. globulus). The full expression of this genetic variation appeared to increase with hardiness and in some cases appeared to vary with ontogeny. Estimates of individual narrow-sense heritability of freezing resistance for pure E. nitens families were h ² = 0.66±0.44 and 0.46±0.44. Across all species combinations examined, the heritability of F₁ family means estimated from midparent regression was h ² = 0.76±0.06 and h ² = 0.89±0.06 for T40 and T50 values, respectively. The advantage of using selected parents for interspecific hybridization is demonstrated and the implications of these results for breeding for freezing resistance in Eucalyptus are discussed.     

Wide hybridization in Brassica

Summary Crosses were made to obtain interspecific hybrids between B. fruticulosa (wild species , 2n = 16) × B. campestris (cultivar , 2n = 20). Although many pollen grains germinated and their tubes entered the style, only about 30% of the ovules received pollen tubes. Fertilized ovules aborted at various stages of development. A few hybrid seeds resulted from hand pollinations in the field, and they showed poor germination and seedling establishment. The in vitro culture of ovaries, ovules, and seeds increased the frequency of obtaining hybrid seeds and plants: the most effective method was ovary culture followed by ovule culture. The hybrid nature of the plants was confirmed through morphological, cytological, and electrophoretic studies. A meiotic analysis of F₁ hybrids (2n = 18) showed that they had 0–5 bivalents and were completely pollen sterile. Electrophoretic analysis of leaf esterases and acid phosphatases of F₁ hybrids revealed bands derived from each parent. Induced amphidiploids of F₁ hybrids contained mostly bivalents, and had about 50% fertile pollen.     

Relationships of pollen size,pistil length and pollen tube growth rates in Rhododendron and their influence on hybridization

Summary Pollen size and pistil length data have been collected for 93 species of Rhododendron (Ericaceae) belonging to a number of different subgeneric taxa. For a sample of eight species in section Vireya, pollen tube growth in the style after selfor interspecific pollination has been quantified. Pollen volume and the time taken for pollen tubes to reach the ovary were both related to pistil length. Pollen-tube growth rates were generally greater for species with longer pistils and larger pollen. Increasing temperature increased the rate of pollen-tube growth. There was no detectable effect of pollen tube density on tube growth rate in the style. After interspecific pollinations tube growth rates in foreign styles could be faster or slower than in self styles. A semisterile individual with two viable pollen grains per tetrad and a plant grafted as scion to a longer-styled stock both showed more rapid pollen-tube growth than expected on the basis of pistil size. Data collected for 26 species in section Vireya showed that where extreme disparity of pollen/pistil size causes failure of interspecific crosses, one or more bridging species with intermediate pollen/pistil size can generally be selected.     

Cytogenetics of double cross hybrids between Pennisetum americanum — P. purpureum amphiploids and P. americanum x Pennisetum squamulatum interspecific hybrids

Summary Pearl millet, Pennisetum americanum L. Leeke-napiergrass, Pennisetum purpureum Schum. amphiploids (2n=42) were crossed with pearl millet X Pennisetum squamulatum Fresen. interspecific hybrids (2n=41) to study the potential of germplasm transfer from wild Pennisetum species to pearl millet. These two interspecific hybrids were highly cross-compatible and more than two thousand trispecific progenies were produced from 17 double crosses. All doublecross hybrids were perennial and showed a wide range of morphological variations intermediate to both parents in vegetative and inflorescence characteristics. Some crosses resulted in sublethal progenies. Chromosomes paired mainly as bivalents (¯x15.88) or remained as univalents. At metaphase I, trivalents, quadrivalents, an occasional hexavalent and a high frequency of bivalents indicated some homeology among the genomes of the three species. Delayed separation of bivalents, unequal segregation of multivalents, lagging chromosomes, and chromatin bridges were observed at anaphase I. Although approximately 93% of the double-cross hybrids were male-sterile, pollen stainability in male-fertile plants ranged up to 94%. Seed set ranged from 0 to 37 seed per inflorescence in 71 plants under open-pollinated conditions. Apomictic embryo sac development was observed in double-cross progenies when crosses involved a pearl millet x P. squamulatum apomictic hybrid as pollen parent. These new double-cross hybrids may serve as bridging hybrids to transfer genes controlling apomixis and other plant characteristics from the wild Pennisetum species to pearl millet.     

Reproductive interactions between hybridizing irises: analyses of pollen-tube growth and fertilization success

Pollen-tube growth and seed siring ability were measured in crosses between the Louisiana iris species Iris fulva and Iris hexagona and their F₁ and F₂ hybrids. Flowers of the parental species were pollinated with self, outcross intraspecific, and interspecific pollen. Pollen-tube lengths were similar for all three pollen types in I. fulva, but in I. hexagona interspecific pollen tubes were longer than intraspecific pollen tubes. Pollen-tube lengths also differed for F₁ and F₂ flowers pollinated with I. fulva, I. hexagona, and hybrid pollen. For both hybrid classes I. fulva pollen tubes were the shortest while pollen tubes from I. hexagona and hybrids grew the furthest. Mixtures of genetically marked pollen were used to assess the seed siring ability of intra- and interspecific pollen in the parental species by varying the proportion of each pollen type in a replacement series design. For both species, the observed proportions of hybrid seeds were lower than the expected based on the frequency of each pollen type in the mixtures across all treatments. Flowers of I. fulva produced less than 10% hybrid progeny even when 75% of the pollen applied to stigmas was derived from interspecific flowers. The frequency of hybrid seed formation was somewhat greater in I. hexagona, but was still significantly lower than expected across all mixture treatments. Seed set per fruit remained constant across the mixture treatments for both species, but in I. fulva fruit set decreased with an increase in the proportion of interspecific pollen. The data indicate that both pre- and postfertilization processes contribute to discrimination against hybrid seed formation.