Cytogenetic analysis of interspecific hybrids in genus Citrullus (Cucurbitaceae)

Investigations on interspecific crossability in two species of the genus Citrullus were studied based on their morphology, chromosome pairing and pollen fertility in their hybrids resulting from 12 combinations(including reciprocals). These have been utilized for tracing the phylogenetic relationship between the taxa and factors responsible for their differentiation. A collective evaluation of data on cross compatibility,morphology, chromosome association and pollen fertility indicated a close phylogenetic relationship between the species C. colocynthis (wild2n = 22) and C. vulgaris (cultivated 2n = 22). Significant differences for crossability and pollen fertility were observed between hybrids of C. colocynthis × C. vulgaris and reciprocals. Meiotic analysis of F₁ hybrids revealed that these two species are freely crossable and provide normal chromosome association, involving an average of 9 to maximum 11 pairs of chromosomes. This indicated the possibility of high recombination and thereby genetic introgression of desirable traits. As both the species are of economic significance, breeding strategies can be developed for the genetic improvement of these species. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosome association and fertility in hybrids of Iris laevigata Fisch. x I. ensata Thunb.

Summary Chromosome association at MI of PMCs in hybrids of I. laevigata x I. ensata was examined and compared with that of parental species. The mean chromosome association per cell in the parental species was 0.02_I+11.99_II for I. ensata (Kachô, 2n=24), 0.11_I+15.94_II and 0.15_I+15.92_II for I. laevigata (Yukidôrô and wild type, 2n=32), respectively. On the other hand, the mean chromosome association per cell in the hybrids (2n=28) was 20.22_I+3.88_II+0.0046_IV for Yukidôrô × Kachô and 20.92_I+3.54_II for wild type × Kachô. Thus, the hybrids indicated a low homology between I. ensata and I. laevigata, and did not exhibit pollen and seed fertility. The utility of the hybrids was discussed.     

Lilium x ‘Black Beauty’—a potential bridging hybrid in Lilium

Summary Lilium Black Beauty, a rare intersectional hybrid previously reported sterile, was re-examined for pollen and ovule fertility. No pollen tubes were detected following incubation of styles pollinated with Black Beauty pollen. However, embryos varying in size from small globular to long boat-shaped and only rarely associated with endosperm, were found in 0.1–1.0% of the ovules in Black Beauty pistils pollinated with Oriental or Aurelian division lilies. Aseptic extraction and culture of these embryos on a nutritive medium resulted in growth, germination, and plantlets. It is apparent that with embryo culture, Black Beauty has unique value as a species bridge in that it accepts pollen of both Oriental and Aurelian lilies, two of the major commercial lily divisions.Scientific Journal Series Paper Number 9197 of the Minnesota Agricultural Experimental Station.     

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.     

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.     

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

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

Estimating pollen fertility in Solanum species and haploids

Summary Three staining methods (acetocarmine, fuchsin and oxidation of benzidine) and germination in vitro and in vivo were applied to estimate pollen fertility in Solanum species and dihaploids. Pollen was divided into six classes based on shape and contents of the grains. With acetocarmine, fuchsin, peroxidase and germination in vitro 4, 3, 2 and 1 classes, respectively, are supposed to be included in the percentage of good pollen as measured by these methods. This percentage therefore, in more than 96% of the cases studied, shows a decrease in the order indicated. Neither aging of pollen at room conditions nor collecting pollen from flowers on 1–9 days after anthesis does influence the percentage of good pollen with acetocarmine and fuchsin, whereas this percentage drops sharply to zero with peroxidase and germination in vitro. The latter two methods apparently measure as good pollen only the grains with living cytoplasm. When pollen is collected at three successive dates from the same flowers the percentage of good pollen drops sharply with all methods used. There is a relation between quantity of pollen per flower and pollen quality (% good), low-quantity pollen containing significantly lower percentages of good pollen than medium- and high-quantity pollen. The latter two are not significantly different in this respect.From calculations of correlation coefficients it is concluded that only germination of pollen in vitro is significantly correlated with berry and seed set and thus gives a reliable estimate of male fertility. This does not hold true for the two staining methods without due reserve. The peroxidase method is not useful for the Solanum material studied.After standardized pollination the average number of haploid pollen grains on diploid stigmata was found to be 1625±127, that of diploid pollen on tetraploid stigmata 2863±98.A. W. B. Janssen 1975.     

DNA fingerprint of F1 interspecific hybrids from the Triticeae tribe using ISSRs

Inter-simple-sequence repeat (ISSR) were used to fingerprint three kinds of F1 interspecific hybrids: the Portuguese durum wheat cultivar ‘Candial’ × tritordeum, the Portuguese bread wheat cultivar ‘Barbela’ × tritordeum and the Portuguese triticale cultivar ‘Douro’ × tritordeum. Among the 30 primers tested, the number of discriminative primers varies from 13 to 20 in each hybrid combination, the total number of ISSR loci analysed ranged from 101 to 183, and the number of discriminative ISSR loci varied from 50 to 71. ISSR-PCR technique revealed to be a valuable fingerprint system for all F1 hybrid combinations and respective parents analysed here. The complementary use of molecular cytogenetic techniques and molecular markers could allow a more accurately evaluation and characterisation of different plant species.     

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.     

Comparison of different pollen viability assays to evaluate pollen fertility of potato dihaploids

Summary The main obstacle in breeding potato (Solanum tuberosum L.) dihaploids is the severe limitation of male fertility. To determine pollen viability assays that correlate well to fertility in crosses, results of five different pollen viability assays were compared by correlation analysis with fruit and seed set characters in test crosses, and to pollen tube growth in situ (PL-test). The methods used were: staining the pollen cells with carmino acetic acid (CAA-test); in vitro pollen germination (PG-test); and detection of pollen staining rates after incubation with fluoresceine diacetate (FDA-test), 2-3-5-triphenyle tetrazolium chloride (TTC-test), and 5-bromo-4-chloro-3-indolyle--galactoside (X-Gal-test).The results of test crosses and pollen tube growth in situ correlated with the results of all other assays with the following ranking, from highest to lowest: enzyme activity assays (X-Gal-test, FDA-test, TTC-test), in vitro pollen germination (PG-test), and pollen staining by CAA. The newly developed X-Gal-test for monitoring -galactosidase activity showed the least variation of all assays investigated. Thus, this highly reproducible simple procedure is recommended for male fertility screening.Abbreviations B/F Berries obtained per 100 flowers - CAA Carmino acetic acid - FDA Fluoresceine diacetate - PG Pollen germination rate in vitro - PL Pollen tube growth in situ - S/B Seeds per berry - S/F Seeds per pollinated flower - TTC 2-3-5-triphenyle tetrazolium chloride - X-Gal 5-bromo-4-chloro-3-indolyle--galactoside     

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.     

Cytology and pollen grain fertility in creeping bentgrass interspecific and intergeneric hybrids

Creeping bentgrass (Agrostis stolonifera L. or A. palustris Huds.) is a highly outcrossing allotetraploid species. It can form hybrids with a number of other Agrostis species and Polypogon genus. However, cytology and pollen grain fertility of the creeping bentgrass interspecific and intergeneric hybrids are not well known. In this research, chromosome pairing behaviors during meiosis I in F₁ and pollen viability of F₁ hybrids, as well as seed set rate and seed germination rate of backcrosses were studied in hybrids between creeping bentgrass, and other bentgrass species and three species of Polypogon genus. Abnormal chromosome pairing, laggard chromosomes, and premature segregation in F₁ hybrids were found. Pollen viability ranged from 1.6 to 48.5% amongst F₁ hybrids, significantly lower than that of the parents (85.5–94.1%). Some hybrids produced pollens of different sizes within the same anther. Seed set following backcrosses using F₁ hybrids as the male parent and creeping bentgrass as the recurrent parent was significantly lower than their parents. The study of chromosome paring behaviors and progeny fecundity are important in utilizing the alien genes to improve bio-stress and abio-stress resistance, and in assessing the potential transgene risks of creeping bentgrass.     

Interspecific hybridization between Coffea arabica L. and tetraploid C. canephora P. Ex Fr. II. Meiosis in F1 hybrids and back crosses to C. Arabica

Summary Melosis was studied in Coffea arabica, in induced tetraploid C. canephora, in their F₁ hybrid (arabusta hybrid) and in backcross generations of the hybrid with C. arabica as recurrent parent. Irregularities were observed, consisting of univalents (especially in the arabusta hybrid), multivalents (especially in tetraploid C. canephora) and uneven distribution of chromosomes at first anaphase. Chromosome distribution was improved by backcrossing. Meiotic irregularities wer negatively correlated with pollen fertility.     

Fertility restoration of hybrids between Solanum melongena L. and S. aethiopicum L. Gilo Group by chromosome doubling and cytoplasmic effect on pollen fertility

Reproductive fertility traits were studied in the reciprocal hybrids of the eggplant(Solanum melongena L.) and S. aethiopicum L. Gilo Group, and in synthetic amphidiploids to discover whether fertility in these reciprocal hybrids was restored by chromosome doubling. Isozyme and RAPD analyses confirmed hybridity of the hybrids and amphidiploids. Analyses of chloroplast and mitochondrial DNAs confirmed that the cytoplasm of each of the hybrids and amphidiploids was from the maternal parent. Pollen sterility of S. melongena × S. aethiopicum Gilo Group [F₁ (Mel × Aet)] was restored by chromosome doubling, while the reciprocal hybrid S. aethiopicum Gilo Group ×S. melongena [F₁ (Aet × Mel)]and its amphidiploid did not produce any pollen grains; their microspores degenerated without being released from tetrads. Hence the cytoplasm of S. aethiopicum Gilo Group seems to beresponsible for their pollen-non-formation type sterility of the hybrid. Both the F₁ hybrids did not set any fruits by either selfing or backcrossing, while their amphidiploids set fruits after pollinating with pollen from the amphidiploid of F₁ (Mel × Aet). Seeds obtained from both the amphidiploids germinated normally. Chromosome doubling has been effective in restoring fertility of the hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

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

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

Pollen fertility among BC2 offspring of Impatiens interspecific hybrids of New Guinea and Indonesian ancestry

BC_T2 seedlings, derived from the cross Tangeglow (I. hawkeri Bull. × I. aurantiaca Teysm.) × 7851-1 (I. hawkeri Bull. × I. platypetala Lindl.) with 7851-1 as recurrent parent, were tested for presence of pollen fertility. Of 59 BC₂ seedlings, 11 were capable of in vitro pollen germination and also of effecting fertilization and subsequent seed set as pollen parent when crossed with Tangeglow as the seed-parent tester. The Spearman's rank correlation coefficient [r_s] of + 0.63 indicated a moderately good correlation of seedling ranking, based on pollen germination percent in vitro and seed set in vivo. Pollen germination of the 11 pollen-fertile BC₂ seedlings varied from a mean low of 4% to a mean high of 41%. The 3 highest pollen-germinating BC₂ seedlings also had the highest seed sets, but siblings showed a wide range in pollen-germinating ability. The best pollen germination for a seedling was 8% in the BC₁ and 41% in the BC₂. Pollen fertility in the BC₂ is discussed in relation to using interspecific hybridization in an Impatiens breeding program. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chromosome pairing and fertility of interspecific hybrids between Trifolium repens L. and T. occidentale Coombe

Trifolium occidentale is a diploid wild relative with the potential to improve the drought and salt tolerances of white clover (Trifolium repens). Previous work has shown that it is possible to efficiently produce large T. repens × T. occidentale breeding populations using colchicine‐doubled (4×) T. occidentale. For effective introgression (backcross) breeding, it is also essential that interspecific chromosome pairing and recombination occur. In this study, it was apparent that chromosome pairing was occurring not only between T. occidentale and T. repens subgenomes, but also between the ancestral subgenomes of T. repens. Thus, interspecific hybridization has the potential for major genome recombination and opens the way for introgression of traits from T. occidentale into white clover.     

Interspecific hybridization between Coffea arabica L. and tetraploid C. canephora P. ex Fr. I. Fertility in F1 hybrids and backcrosses to C. Arabica

Summary The interspecific (C. arabica × tetraploid C. canephora) F₁ hybrid showed a low but highly variable fertility. An almost complete restoration to normal fertility was observed in individual trees of BC₁ and BC₂ generations with C. arabica as recurrent parent. From a comparison of various tests of fertility, it was found that pollen viability is a satisfactory measure of fertility.