Identification of barriers to interspecific crosses in the genus Trifolium

Summary A study of pre- and post-fertilisation barriers after interspecific crosses of diploid and tetraploid Trifolium pratense L. and wild species T. alpestre L., T. medium L. and T. sarosiense Hazsl. was aimed at finding of a promising cross combination for obtaining hybrids. The growth of pollen tubes was arrested in interspecific crosses mainly when T. pratense was at a diploid level. To investigate the post-fertilisation barriers in detail, the hybrid embryo viability was traced by two clearing treatments of immature seeds: (1) using chloral hydrate (which proved to be most appropriate); and (2) a mixture of benzyl benzoate and dibutly phthalate. In interspecific combinations T. pratense (4×) × either T. alpestre or T. sarosiense, enlargement of immature seeds occurred, but no hybrid embryo was traced. Of the wild species used as a male parent for crosses, T. medium was the only exception from the point of view of fertilisation. Globular, heart and the early torpedo stages of hybrid embryos were observed 7 days after pollination (DAP) but only when T. pratense was at a tetraploid level. When T. pratense (2×, 4×) was used as a male parent for interspecific crosses with T. alpestre, T. medium and T. sarosiense, strong defects in various stages of embryogenesis were observed, particularly wrinkled and narrowing embryo sacs caused by an expansion of endothelial cells. We conclude with the following finding: (1) to make crosses only in one direction with T. pratense as a female parent and T. medium as a male; (2) to use tetraploid plants of T. pratense; (3) and to excise hybrid embryos at an early torpedo stage, about 7 DAP.     

Effects of aminoethoxyvinylglycine (AVG), environment,and genotype in overcoming hybridization barriers between Cucumis species

Summary It was attempted to overcome crossing barriers between the cucumber (Cucumis sativus L.), the melon (C. melo L.) and two wild Cucumis spp. (C. metuliferus Naud. and C. zeyheri 2x Sond.) by application of aminoethoxyvinylglycine (AVG) and by limiting the amount of rooting substrate. The reciprocal crosses between both wild species were used as a model system.Crosses between C. metuliferus and C. zeyheri 2x succeeded well if the maternal plants were grown in containers of 10 or 25 1 instead of in open soil. This treatment also improved crossability in the cross C. metuliferus x C. melo. It strongly enhanced the number of female flowers per plant in C. metuliferus. Application of AVG to pollinated flowers initially seemed also to improve crossability in these cross combinations, but later on this effect was not found again.Significant genetic variation for crossability was found in C. zeyheri 2x. Rare plants yielded numerous fruits with embryos after crosses with C. metuliferus and also with C. sativus. Clones from these plants gave similar results.Excessive temperature and radiation decreased crossability especially in crosses with C. metuliferus and C. sativus as maternal parent.     

Variation of prezygotic barriers in the interspecific hybridization between oilseed rape and wild radish

Escape of transgenes from genetically modified oilseed rape, Brassica napus, into wild radish, Raphanus raphanistrum, depends on sexual compatibility. The variation in prezygotic barriers of two different cultivars for interspecific hybridization with a population of wild radish was investigated by hand crossing and fluorescence microscopy of pistils. Significant differences were observed between oil seed rape cultivars in their ability to accept wild radish pollen germinating onto their stigma and the rate of fertilization of ovules. Some differences among the pollen donor plants were also detected. These results suggest that the rate of interspecific hybridization in the field would depend upon the oilseed rape cultivar and the genotype composition of the local wild populations. The implication of S-related genes, as revealed through identification by pistil tissue prints of class I and II S-types of SLG (S-Locus Glycoprotein) and SLR1 (S-Locus Related),and immuno-IEF, was not significant. This revised version was published online in August 2006 with corrections to the Cover Date.     

Incongruity in the interspecific crosses of Vitis L. Morphological abnormalities in the F2 progeny

Summary The F₂ generation of interspecific crosses of Vitis were examined for symptoms of incongruity, manifested as morphological abnormalities in the seedlings. Dwarfing, achlorophyllic variegation, deformed leaves, yellow mottling, sparse roots, and enation were documented. Most comparisons between classed of crosses were significantly different. The V. riparia x V. vinifera class exhibited the greatest frequency of abnormalities, the V. riparia x V. riparia class displayed the lowest frequency of abnormalities, and the highly-intercrossed V. riparia x French Hybrid class demonstrated an intermediate frequency of abnormal plants. When incongruity was viewed as a syndrome, or collection of symptoms, differences between classed were consistently evident. The highly-intercrossed nature of the French Hybrid ancestry appears to have been responsible for attenuating incongruity.Minnesota Agricultural Experiment Station, Scientific Journal Series No. 20834     

Incongruity in the interspecific crosses of Vitis L. Reproductive expression in the F1 progeny

Summary F₁ progeny from three classes of crosses were studied for indications of incongruity in the form of reproductive dysfunction. Percent fruit set was found to be approximately equivalent to the additive inverse of percent aborted flowers, as the effect of shot berries was small and not significant. In interspecific crosses, barriers were manifested as reduced fruit set and seed number per berry. These two characters were used to create an index of reproductive efficiency. Nine F₁s from crosses of most divergent class, V. riparia × V. vinifera (R × V), displayed significantly tower reproductive efficiency, percent fruit set, and seed number per berry. Four female F₁s from the least divergent class, V. riparia × V. riparia (R × R), had the highest reproductive efficiency, percent fruit set and seed number per berry, when pollinated by two V. riparia pollen sources. Seventeen F₁s from the highly-intercrossed class, V. riparia × French Hybrid (R × FH), displayed intermediate levels of reproductive efficiency, percent fruit set, and seed number per berry. The R × FH class involved V. vinifera, V. rupestris, V. aestivalis, V. labrusca, V. berlandieri, V. cinerea, and V. riparia. It is proposed that the complex intercrossing in the pedigrees of the R × FH class ameliorated incongruity. In the most divergent class, R × V, progenies of one of the V. riparia parents displayed fewer incongruity effects than progenies of the other V. riparia parents. Progeny testing of many proposed parental combinations may be used to uncover useful congruent combinations.Minnesota Agricultural Experiment Station, Scientific Journal Series No. 20835     

Quality characteristics of backcross hybrids between Trifolium repens and Trifolium ambiguum

Two generations of novel backcross hybrids between white clover (Trifolium repens L.) and the related species Trifolium ambiguum,with white clover as the recurrent parent, were analysed for forage quality characteristics. Water soluble carbohydrate (WSC) levels, nitrogen concentrations (%N), and dry matter digestibility (DMD) were recorded for the hybrids and parental species grown in deep soil bins with and without a companion grass. Half the experimental material was subjected to moisture stress by being left unwatered through the four-week period when measurements were made. Four such four -week cycles of drought were imposed. The first and second generation backcrosses had the highest values for WSC, but were lowest for N%. There was no difference between the different legume lines (parents and backcrosses) for DMD. No effects of either moisture stress or presence of a grass companion on quality traits were observed. The WSC and %N of the grass component differed significantly depending on which legume line it was grown with. The importance of these results for the potential use of these hybrids in agriculture is discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of reciprocal translocations observed in several Melilotus species (subgenus Eumelilotus) by interspecific triple crossings

Summary Melilotus alba differs by a reciprocal translocation from 7 other species which are categorized into M. officinalis and M. dentata groups. The two species groups, however, remained to be studied in relation to their cytological relations because of early degeneration of hybrid embryo. Interspecific triple crosses were successfully made in order to examine whether or not the reciprocal translocations observed were of the same origin. When F₁ hybrids between M. alba and M. officinalis group were crossed with M. dentata group, about a half of hybrids plants were heterozygous for a reciprocal translocation while the remaining plants were normal chromosome pairing, showing a segregation ratio of 1:1. This result indicates that reciprocal translocations observed among the three groups are of the same origin. Accordingly, it is expected that M. officinalis group and M. dentata group have the same chromosomal constitutions.     

New interspecific hybrids in the genus Agropyron

Summary Fifteen species from the genus Agropyron were crossed together. Fourteen of the crosses did not produce fruits. Twenty crosses produced varying numbers of caryopses. Of the fruits produced, 22% contained no embryos. For a variety of reasons it was possible to obtain only 2 plants from the remaining 140 embryos. The crosses which yielded viable plants were between A. trachycaulum cv. Primar (2n=28) and A. intermedium cv. Chief (2n=42) as well as between A. trachycaulum from Lethbridge (2n=28) and A. desertorum cv. Nordan (2n=28). The somatic chromosome numbers for the hybrids are 37 and 28. respectively. Studies of vegetative plant character are presented.     

Crossability relationships among Cajanus,atylosia and Rhynchosia species and detection of crossing barriers

Summary Crossability of two cultivars of Cajanus cajan, eight species of Atylosia and one of Rhynchosia was investigated. Of the 73 combinations attempted, success was achieved in 12 cases. C. cajan crossed successfully with A. albicans, A. cajanifolia, A. lineata, A. scarabaeoides, and A. trinervia. Within the genus Atylosia, A. lineata crossed with A. albicans and A. scarabaeoides, and A. scarabaeoides with A. sericea. Three species A. platycarpa, A. volubilis and R. rothii did not cross with any other one. In most of the unsuccessful combinations, although the pollen germinated on the receiving stigmas, the pollen tube growth was inhibited inside the stigma or in the stylar tissue.     

Pre- and post-fertilization barriers to backcrossing the interspecific hybrid between Allium fistulosum L. and A. cepa L. with A. cepa

Summary To facilitate the introgression of desirable traits of Allium fistulosum into the genome of A. cepa, several accessions of the hybrid between these species were pollinated with A. cepa as the recurrent parent, and in vitro ovary and ovule culture were performed to obtain an increase in the recovery of backcross progeny. Compared to the results obtained from seed development in planta, the increase in the number of backcross progeny was generally very limited, and in some cases even a decrease was found. Raising the sucrose concentration in the ovary culture medium resulted in a higher frequency of ovules developing back seed coats but this was not followed by an increase in the number of backcross progeny obtained. Pollen tube growth of A. cepa was disturbed in the styles of the interspecific hybrids. Per ovule, frequencies of micropylar penetration exceeded frequencies of backcross progeny only to a limited extent. Hence, it was concluded that in the tested interspecific hybrid accessions the attainable gain in viable backcross progeny by the application of in vitro culture techniques is limited by strong pre-fertilization barriers acting at the level of stylar incongruity.     

Genetic diversity in advanced derivatives of Brassica interspecific hybrids

Genetic diversity among the 88 entries including eighty F₄ derivatives i.e., 20 each selected from Brassica crosses viz., B. juncea × B. napus, B. juncea × B. rapa var. toria, B. juncea ×B. rapa var. yellowsarson and B. tournefortii × B. juncea, and eight parent genotypes was assessed through multivariate analysis (D² statistic). Significant differences among the family groupsas well as within the family were recorded for all the 14 characters studied. The D² analysis revealed enormous diversity among the interspecific cross derivatives. The genetic distances calculated among different Brassica species revealed that B. tournefortii had maximumdiversity with B. juncea followed by B. napus, B.rapa var. toria and B. rapa var. yellow sarson.Amongst interspecific crosses, maximum diversity was noticed indescendants of cross B. tournefortii × B. juncea followed byB. juncea × B. napus, B. juncea × B.rapa var. toria and the least in the cross B. juncea ×B. rapa var. yellow sarson. These results indicated that the derivatives selected from cross of diverse parents revealed greater diversity. The clustering pattern showed that many derivatives of the cross fell into the same cluster but in many cases in spite of common ancestry many descendants of the cross spread over different clusters. The characters, namely, plant height, secondary branches per plant, days to flowering and1000-seed weight were contributed maximum towards genetic divergence. This revised version was published online in August 2006 with corrections to the Cover Date.     

Increasing biodiversity of irrigated rice in Africa by interspecific crossing of Oryza glaberrima (Steud.) × O. sativa indica (L.)

The African rice Oryza glaberrima, traditionally cultivated since more than 3.500 years, is of poor agronomic performance but resistant/tolerant to various stresses and diseases. The introduction of these characters into O. sativa cultivars is difficult since crossing barriers cause spikelet sterility in F1. Backcrossing can restore fertility and recently facilitated the development of fertile O. glaberrima × O. sativa ssp. japonica hybrid progenies for rain fed systems. With the objective to gain access to African rice germplasm for improvement of irrigated rice, crosses were performed with eighteen O. glaberrima and twenty O. sativa ssp. indica accessions. In total about one hundred F1-hybrid grains were obtained. The F1 plants were all completely sterile and backcrossing (BC) to O. sativa was performed in order to restore spikelet fertility. Monitoring of Tog5681 × IR64 hybrid progenies under field conditions revealed a broad genetic diversity within the BC₁ and BC₂ populations. Some BC₁ and BC₂ progeny plants outperformed the O. sativa parent, indicating that the heterozygocity level and complementary gene action after two backcrosses are still sufficient to positively influence plant vigor. Spikelet fertility of progenies was highly variable, but almost complete fertility was already observed within the BC₁F₂ population. High spikelet fertility was preserved in one out of two analyzed BC₁F₃ families and inmost of the BC₂F₃ families. The ability to restore spikelet fertility within few generations and the potential of the genetic diversity present in interspecific progenies facilitates the development of plant types specifically designed for the African irrigated and lowland environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Failure in an interspecific cross between Solanum khasianum Clarke and Solanum mammosum L.

Summary Attempts to obtain a successful interspecific cross between Solanum khasianum Clarke and Solanum mammosum L. were unsuccessful, and the probable reasons for this failure were investigated. A reduced number of viable pollen grains and low receptivity of the stigmas of S. mammosum L. and heterostyly in both species are the probable reasons for the failure of the attempted interspecific crosses between S. khasianum Clarke and S. mammosum L.     

Cytogenetics of interspecific hybrids in the genus Capsicum L.

Summary Crosses between a wild species C. chacoense and three cultivated species of chili pepper viz. C. annuum, C. frutescens and C. chinense yielded hybrids when C. chacoense was the seed parent but the reciprocal crosses were unsuccessful. C. chacoense × C. annuum F₁ hybrids were partly fertile and therefore an F₂ population could be raised; the other two F₁ hybrids were totally sterile. Chromosome pairing in the F₁ plants resulted largely in bivalents and a few multivalents and univalents. The genomes of the four species share large homologies and the role of chromosome structural changes in genome differentiation is suggested. Hybrid sterility is the major reproductive isolation mechanism.     

Pollen pistil interactions and interspecific incompatibility in Pennisetum

Summary The causes of low crossability between Pennisetum species were studied. In the compatible cross P. typhoides x P. violaceum, the pollen tube growth was normal and there was good seed set. In P. typhoides x P. cenchroides, pollen tubes reached the ovary but no seed set was obtained indicating ovarian incompatibility. The stylar incompatibility was found in the crosses of P. typhoides with P. schweinfurthii, P. hohenackeri, P. orientale and P. squamulatum. In crosses P. typhoides x P. polystachyon and P. typhoides x P. pedicellatum, mostly pollen tubes did not grow beyond stigmatic surface and only few reached the style. Stigmatic as well as stylar incompatibilities seem to be the reasons for no seed set in these crosses.     

Heterosis for symbiotic nitrogen fixation in Phaseolus interspecific hybrids

Summary Interspecific hybrids of Phaseolus vulgaris and Phaseolus acutifolius or Phaseolus filiformis have been evaluated for evidence of heterosis for symbiotic nitrogen fixation activity. Hybrids showing composite leghemoglobin profiles were selected for comparison. Rooted cuttings from hybrids and their respective parents were used to estimate nitrogen fixation (acetylene reduction) rates and to determine nitrogen accumulation during the growth period. Some hybrids of Phasolus vulgaris × Phaseolus acutifolius and Phaseolus vulgaris × Phaseolus filiformis had significantly (P<0.01) higher nitrogen fixation rates and in the latter case, accumulated more total nitrogen, than either parent. The interspecific hybrids also showed more complex leghemoglobin profiles than either parent.     

Trifolium ambiguum as a source of variation in the breeding of white clover

Summary Hybrids between T. ambiguum and T. repens were successfully established after ovule culture. The hybrids were male sterile but one of the hybrids produced a single seed when backcrossed to T. repens, while the other produced a significantly greater number of seeds. The majority of the backcross hybrids from the latter had 48 chromosomes, being produced from the fertilisation of unreduced gametes of the F1 hybrid by T. repens pollen. Regular meiosis in the BC1 hybrid resulted in the production of balanced gametes containing the full complement of T. repens chromosomes and the polyhaploid set of T. ambiguum chromosomes. There was no difficulty in establishing a substantial population of BC2 hybrids. The meiotic behaviour of the BC1 hybrid indicated that it should be possible to transfer genes from T. ambiguum into white clover T. repens. The significance of the results in terms of the use of alien variation in the improvement of white clover is discussed.     

Chromosome association and pollen fertility in some interspecific hybrids of Lilium

Summary Meiotic observations in PMCs were made in eight kinds of interspecific hybrids in Lilium. Three hybrids, 6134-S × L. cernuum. L. regale × L. leichtlinii maximowiczii and L. auratum platyphyllum × L. henryi showed respectively the mean chromosome association frequencies of 1.4_II, 0.8_II and 1.5_II per cell at MI which were the lowest values hitherto reported in Lilium hybrids. In L. longiflorum × L. henryi, 33.3° of the cells had 1 or 2 multivalents of three to five chromosomes in addition to uni- and bivalents. 12° bivalents and 12 univalents were invariably observed in the triploid hybrid L. longiflorum × L. cernuum. The hybrids between the species belonging to the different sections of the genus generally showed high pollen sterility, with some exceptions. A more or less remote genomic homology was found between the different sections in Lilium.     

Triple fusion of the primary endosperm nucleus as a cause of interspecific cross-incompatibility in Avena

Summary Using five diploid, two tetraploid and three hexaploid Avena species (x=7) 78 reciprocal crosses were made. Of these 74 were successfull, though the frequency of seed set varied greatly. The development of hybrid seeds differed widely between cross combinations, especially in reciprocal crosses. Hybrid seeds were grouped into four types, (1) normal kernels, well developed and germinative (designated as D+); (2) shrivelled-empty kernels, which do not germinate (E–); (3) small viable kernels (Rd+) and (4) small inviable kernels (Rd–). Some rare intermediate types were found in certain crosses.The results are well interpreted in terms of a hypothesis of polar-nuclei activation in which the strength of the activating action of the male nucleus, and the reaction of the female nucleus at double fertilization are expressed as activating value(AV) and response value(RV), respectively. The degree of seed failure is closely related to the activation index(AI) of the polar nuclei, AV/2RV (or × 100). In a selfed plant the activation index is always 1/2=0.5 (or50%), and this usually results in the formation of normal seeds. If AI deviates from 50% the endosperm often stops developing or degenerates.Based on the degree of abnormality of hybrid seeds, activating values of the ten species were arbitrarily assigned from 0.4 in A. ventricosa (2x) to 3.0 in A.sterilis (6x). For comparison A.strigosa (2x) was used as a standard and set at 1.0. Among the 74 succesful interspecific crosses the AI of the polar nuclei varied widely, from 7 to 375% Generally speaking, AI values of less than 20, 20–30, 30–80, and more than 80% show the R-, Rd+, D+ and E-kernel types, respectively. In other words, if the AV of the pollen parent is < 40 or >160% that of the maternal parent hybrid seeds fail to develop. Thus in double fertilization of Avena or probably angiosperms, the triple fusion of two polar nuclei with a secondary male nucleus is apparently a sexual isolation mechanism, being a barrier to interspecific hybridization.Professor Emeritus of Kyoto University, Faculty of Agriculture, Kyoto.