Genetic diversity within cultivated teas based on nucleotide sequence comparison of ribosomal RNA maturase in chloroplast DNA

The wild tuber-bearing Solanumspecies represent a genetic pool of enormous diversity, embracing high variability for many agronomic traits. S. kurtzianum (ktz), S. chacoense(chc) and S. ruiz-lealii (rzl) are wild diploid self-incompatible relatives of the common potato, Solanum tuberosumssp. tuberosum, with resistance to adverse biotic and abiotic factors. Pollen-pistil/style compatibility relations and seed set in intra- and interspecific crosses among ktz, chc and rzl were investigated. Pre- and post-zygotic hybridization barriers were detected in the three species, with variable incidence because the percentage of genotypic combinations that produced seeds was15–30% and 7–28% in intra and interspecific crosses, respectively. In pollen-pistil/style incompatible combinations, either pollen grains did not germinate or pollen tube growth was arrested at different sites of the style, but mainly in its upper third. Unilateral incompatibility was detected in both intra-and interspecific crosses, as well as bilateral incompatibility in certain combinations. The incompatibility relations observed could be explained by a genetic model with dominant genes in pistils that interact with corresponding dominant genes in pollen, that are independent of the S-locus. The complex arrangement of compatibility relationships found could not be directly related to taxonomic hypothesis or geographic distribution. These results suggest that divergence between the three species has occurred recently, and that internal barriers of reproductive isolation have not been an important factor in their evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen–pistil interaction in the inter-specific crosses of Sesamum sp

The wild species of Sesamum have long been recognized as an important source of many valuable pest and disease resistance genes and other novel traits such as male sterility and plant type for the cultivated taxon. Utilization of these species in crop improvement is marginal because of their inherent low crossability with the cultigen. An understanding of the biological nature of incompatibility systems that impede hybridization offers tools for successful introgressions into cultivated sesame. The objective of this investigation was to observe pollen germination and pollen tube growth in cross pollinated S. indicum pistils as an indicator of pre-zygotic barriers operating in wide crosses involving S. indicum. Three wild species: S. alatum, S. radiatum and S. laciniatum were used as pollen donors in hybridization with S. indicum. In the cross S. indicum × S. alatum 85.23% of pollen grains germinated and pollen tubes reached ovaries within 8 h after pollination. In other two crosses, S. indicum × S. laciniatum and S. indicum × S. radiatum the level of incompatibility was high as evidenced by a reduced pollen germination and slow rate of pollen tube growth. Measures to circumvent incompatibility in these crosses to produce interspecific hybrids are discussed.     

Effect of mentor pollen on pistil-pollen incongruities among species of Cucumis L.

Summary The merits of mentor pollen in certain interspecific hybridizations in the genus Cucumis L. were evaluated. African wild cucumber species Cucumis metuliferus Naud. and C. africanus L. and Asian species C. sativus var. hardwickii Alef and var. sikkimensis Hook, were reciprocally crossed. Pollen tube growth was arrested halfway down the style in all combinations except in C. sativus × C. africanus. Mentor pollen irradiated with 100 and 200 krad grew through the style and into the ovules in all species. Fruitset was obtained in all cases when mentor pollen was used either alone or in mixtures. Fruits set after self pollination with mentor pollen yielded only seeds without embryos.In several crosses, aided by mentor pollen, a number of relatively large seeds (with enlarged embryosac) were obtained. A sample of these embryosacs contained a globular structure like an embryo. Embryosacs with embryo-like structures were explanted on several different media but no development was obtained.     

Diversity of penicillarian millets (Pennisetum glaucum and P. purpureum) as for the compatibility between their gynoecia and pollens from some other Poaceae

Summary The compatibility between gynoecia of different penicillarian millets (Pennisetum glaucum and P. purpureum) and pollens from some other Poaceae was evaluated in order to determine whether different penicillarian pistils showed the same pistil-pollen interactions with a same non-penicillarian pollen and to determine whether compatibility was dependent on the genetic divergence or the degree of sympatric evolution of the mating partners. Ten pearl millet lines (P. glaucum subsp. glaucum), six wild pearl millet accessions (P. glaucum subsp. monodii) and one P. purpureum accession were pollinated with five other Pennisetum species: P. pedicellatum, P. polystachyon, P. schweinfurthii, P. squamulatum, P. ramosum, and with three species from other genera: Cenchrus ciliaris, Panicum maximum and Zea mays. Each male species was represented by a unique accession. Pollen grain germination on the stigmata and pollen tube growth in the gynoecia were monitored by means of fluorescence microscopy after aniline blue staining. Significant compatibility differences were observed between a given pollen and gynoecia from different P. glaucum accessions. But there was no evidence of compatibility differences between the three female taxonomic groups. The eight pollen parents can be classified into three groups according to their mean degree of compatibility with the different penicillarian gynoecia. P. ramosum, P. schweinfurthii and P. squamulatum showed a high compatibility: more than 50% of penicillarian gynoecia had pollen tubes at the micropyle, six hours after pollination. Zea mays, Cenchrus ciliaris and Panicum maximum showed a low compatibility: pollen tubes were arrested in the style. P. pedicellatum and P. polystachyon showed nil compatibility: few pollen grains germinated on the stigmata and very rare tubes reached the entry of the style, where they stopped. It must be noted that such a strong gametic barrier has been observed only with pollinator species sympatric to pearl millet.     

Sterility in some species related to the sweet potato

Summary The kinds and sites of sterility and inviability were noted in induced tetraploids of threeIpomoea species, and of hybrids ofIpomoea lacunosa withI. trichocarpa. Tetraploids were somewhat less fertile than diploids, but seeds were normal appearing and ususally viable. Hybrids were much less fertile. Hybrid sterility occurred as pollen abortion, pollen germination failure, and in failure of pollen tube growth. Small and underdeveloped seeds failed to germinate. The sterility of hybrids (but not of tetraploids) is thus very similar to that of the hexaploid sweet potato. Sterility suggests developmental inbalance, which is probably due to genic and perhaps minor chromosomal differences among the parent species of the sweet potato. Hybrid sterility of the sweet potato may have been fixed by polyploidy, and thus may be impossible to eliminate.     

Barriers to interspecific hybridization in Ulmus americana

Summary Crossability barriers between the tetraploid Ulmus americana and five other diploid elm species were investigated using both fluorescence and scanning electron microscopy. Both pollen germination and pollen tube development were inhibited in all interspecific crosses. Pollen tube behavior on the stigma of U. americana was unchanged when pollen of a colchicine-induced U. pumila tetraploid was substituted for diploid U. pumila pollen.Self-incompatibility was observed in one of two American elms studied and manifested itself via reduced pollen germination and inhibited pollen tube growth on the stigma surface.The results indicate that crossing barrier(s) between U. americana and other elm species occur at the stigma surface and operate independently of ploidy level. This conclusion is discussed in relation to current breeding efforts to transfer Dutch elm disease resistance into the American elm via interspecific hybridization with Eurasian elms.     

Pollination systems, hybridization barriers and meiotic chromosome behaviour in Nemesia hybrids

The ability of 13 Nemesia species (six annual and seven perennial) to sexually hybridize was investigated. Six of the perennial Nemesia species investigated were inter-fertile with one another. Two of the annual species, N. macroceras and N. strumosa, were inter-fertile. Thirty three crosses were successful and resulted in viable seeds. The analysis of meiotic chromosome behaviour in interspecific hybrids indicated that Nemesia chromosomes in different parental species were homeologous. No evidence of chromosome inversions or chromosome translocations was observed during meiosis in interspecific hybrids between the six perennial Nemesia species. In the hybrids produced between N. macroceras and N. strumosa, a quadrivalent was observed during meiotic metaphase I, indicating that these two species differ by a reciprocal translocation. A successful hybridization was made between N. anisocarpa (annual) and N. foetans (perennial), producing two triploid hybrids. In the unsuccessful crosses, pollen tubes were observed entering ovaries and ovules, suggesting that post-fertilization barriers were preventing sexual hybridization. Many of these crosses produced nonviable, shrunken, empty seeds, suggesting that endosperm breakdown and embryo abortion prevent interspecific hybridization in unsuccessful crosses. The manipulation of ploidy levels in N. fruticans and N. strumosa and tissue culture of N. strumosa × N. fruticans ovules failed to overcome post-fertilization barriers between these species.     

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.     

Pollen germination,pollen tube growth and fertilization following self and interspecific pollination of Paspalum species

Summary Crossability between most Paspalum species is very low. This study was undertaken to identify the impediments to hybridization. Accessions of P. intermedium Munro. ex Morong, P. jurgensii Hackel and P. dilatatum Poir were self-pollinated and crossed with one anther. Paspalum intermedium is essentially self-sterile but P. jurgensii and P. dilatatum are highly self-fertile. Following pollination, pollen germination and tube growth were studied by observing the pollinated pistils with fluorescent microscopy. Examination of self-pollinated pistils revealed that the pollen germinated shortly after contacting the stigmas. Germination was over 80% for the P. intermedium and P. dilatatum accessions but only 57% for P. jurgensii. Pollen tubes grew to the micropyle within 45 minutes after pollination in P. dilatatum and 1 hour and 15 minutes in P. jurgensii. However, in the P. intermedium accessions most tubes did not grow beyond the stigma and very few penetrated the style and ovary. Apparently stylar-incompatibility is the reason for the low selfed seed set. In the cross-pollinations, pollen germinated shortly after pollination and germination ranged from 57 to 88% for the different crosses. In all crosses the pollen tubes grew to the micropyle within 30 minutes to 2 hours after pollination indicating that a cross-incompatibility system is not the cause for low crossability among these species. By examining embryo sacs from P. intermedium × P. dilatatum, its reciprocal and P. intermedium × P. urvillei crosses, it was determined that gametes failed to unite in some crosses and this is a major reason for low crossability.     

Pollen tube growth and fertilization after different in vitro pollination techniques of Aechmea fasciata

The effect of cut style and placental pollination on fertilization efficiency was studied for compatible Aechmea fasciata plants. These alternative in vitro pollination techniques resulted in lower penetration rates of the ovules by pollen tubes in comparison to pollination on the stigma. An explanation was found in the intervention of the normal pollination process,through which less pollen-pistil interactions were built up. After cut style pollination the percentage of ovule penetration by pollen tubes increased when a longer style part was left at the ovary. Probably fewer factors that control pollen tube growth are present in the lower style part. Pollen germinated on the ovules after placental pollination but only rarely penetration of the micropyle by a pollen tube occurred. Activation of the ovary, induced by in vivo prepollination for 6 hours, and pollination two days after anthesis did not increase the fertilization percentage. Grafting a style with active growing pollen tubes to ovules on the placenta (placental grafted style pollination) resulted in a higher fertilization percentage. Pollen tube growth through the style was essential for pollen tube guidance to the ovules and penetration of the micropyle. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pollen grain germination and pollen tube growth following in vivo and in vitro self and interspecific pollinations in annual Cicer species

Summary Germination of pollen grains and growth of pollen tubes were studied to determine the cause of barreness in crosses among annual Cicer species. In vivo and in vitro time-course studies and fluorescent microscopy revealed no pollination incompatibility among the selfs, crosses and reciprocals of C. arietinum L., C. reticulatum Lad. and C. cuneatum Rich. In general, Cicer pollen grains germinated and grew on styles of Cicer species. Pollen tube growth was characterized by irregularly spaced and intermittent callose deposits. Failure of seed formation in interspecific pollinations may be attributed to the slowness of pollen tube growth or collapse of fertilized ovules. In addition to these causes, shortness of stamens and sparsity of pollen grains were responsible for flower drop in natural selfs. Although the number of pollen tubes entering the micropyle in interspecific pollinations was low, it may be possible to grow the fertilized ovules on an artificial medium to obtain F₁ plants.     

Pollen and pistil morphology, in vitro pollen grain germination and crossing success of Lens cultivars and species

Morphological and in vitro germination characteristics of pollen altogether with pistil and style length were analysed, so as to carry out a morphological comparison between different lentil (Lens culinaris Medik.) cultivars and wild species, as well as to correlate the morphological and functional statistics with data regarding crossing success. Pollen length and width in the different genotypes were found to have a positive and highly significant correlation with pistil and style length and in vitro pollen tube length. A cluster analysis detected two different plant groups, namely, (i) large‐seeded L. culinaris (macrosperma), and (ii) a second group formed by two subgroups, a first subgroup with L. ervoides standing alone and a second subgroup including small‐seeded L. culinaris (microsperma) and the wild L. culinaris ssp. orientalis, L. odemensis, L. nigricans. A high and significant correlation was detected between crossing success and phenotypic similarity (r > 0.98; P < 0.05), therefore implying that phenotypic similarity, based on pollen morphology and in vitro pollen length together with pistil and style length, is a good predictor of hybridization success.     

Occurrence of 2n gametes in the F1 hybrids of Oriental × Asiatic lilies (Lilium): Relevance to intergenomic recombination and backcrossing

Cytological modes of the origin of 2n gametes were investigated in six different genotypes of F₁ hybrids between Oriental and Asiatic (OA) lilies (Lilium, 2n = 2x = 24). Chromosome pairing between the parental genomes was very low, the average frequency range from 0.3 to 1.2 bivalents per cell among the genotypes. Within a genotype the frequency of bivalents varied from 0 to 6 in some cases. The normally occurring haploid pollen grains were totally sterile. In contrast, in different genotypes, variable percentages of 2n pollen were found and shown to be fertile as estimated from pollen germination. A cytological analysis of Metaphase I and subsequent stages of meiosis using genomic in situ hybridization (GISH) revealed that there was intergenomic recombination between the alien genomes. Following Metaphase I stage, three different types of abnormal cytological events led to the formation of 2n pollen: (i) Post-Metaphase I division (PMI), (ii) Post-Metaphase II division (PMII) and (iii) Asymmetric Cytokinesis of the pollen mother cell followed by chromosome division. All three cytological events led to first division restitution (FDR) gametes. Based on in vitro pollen germination it was proved for two genotypes that 2n pollen was viable only during the first day of anthesis. It was possible to use 2n pollen successfully for backcrossing. Implications of 2n pollen for intergenomic recombination in BC₁ progenies are discussed.     

Cross compatibility between Abelmoschus esculentus and A. moschatus

Interspecific cross compatibility between cultivated and wild okra (Abelmoschus esculentus and A. moschatus) and pollen tube growth behaviour in the crosses among a local cultivar of A. esculentus, A. moschatus and their F₁s were studied. Fruit set was observed in all the crosses except one and seed setting was absent in two of the crosses which set fruit. All seed produced were shrivelled but F₁ plants were obtained from two crosses where cultivated okra was used as the seed parent. The F₁ plants were perennial in nature with very low pollen viability and seed set. A high percentage of pollen germination and profuse pollen tube penetration in the style were observed in the cross A. esculentus × A. moschatus but low pollen tube penetration with abnormal pollen tubes was observed in the reciprocal cross. The number of pollen tubes was very low but they appeared to be normal in the backcross A. esculentus × F₁, but were generally abnormal in the reciprocal cross. Both pre- and postzygotic barriers seemed to occur in crosses between the two species. The present studies indicate that these barriers can be overcome and desirable characters from A. moschatus transferred to cultivated okra using conventional hybridisation techniques. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of hybrids betweenCajanus platycarpus andCajanus cajan

Summary Cajanus platycarpus a wild species of pigeonpea incompatible with the cultigen has many desirable characters important for the improvement of cultivated pigeonpea. In the present study, barriers to hybridization were studied and were identified as post-zygotic. Efficient embryo rescue techniques were developed. As a result, hybrids were produced. Morphological and cytological studies as well as esterase isozyme band pattern confirmed the hybrid nature of the plants. The F₁ hybrids were completely pollen sterile. Meiotic studies were carried out to check for the cause of pollen sterility.Abbreviations MS Murashige & Skoog's (1962) medium - BAP 6-Benzylaminopurine - IBA Indole-3-butyric Acid - NAA Naphthaleneacetic Acid     

Investigation on pre-zygotic barriers in the interspecific crosses involving Gossypium barbadense and four diploid wild species

Interspecific hybridisation has played significant role in the improvement of economically useful traits such as productivity, earliness, fibre quality and resistance to pests and diseases in cotton. However, wide crosses are often limited by the operation of either pre or/and post-fertilization barriers. The present study on pollen tube behaviour of four diploid wild species viz., G. triphyllum, G. davidsonii, G. thurberi and G. armourianum in the pistils of G. barbadense was taken up to determine the extent of pre-zygotic barriers operating in these crosses. High rate of pollen germination (∼80%) and normal growth of pollen tubes were observed upon selfing of both G. barbadense and four wild species. Pollen tubes reached the pistils and fertilization was accomplished within 8 HAP in all the five parental species. In the interspecific crosses, pollen germination was normal in the cross involving G. armourianum and drastically inhibited in the crosses involving G. triphyllum, G. davidsonii and G. thurberi. Even though delayed pollen tube growth was a general feature in all the four crosses, successful fertilization was observed only in the cross involving G. armourianum. In crosses involving G. triphyllum, G. davidsonii and G. thurberi, pollen tubes failed to reach the ovary even at 24 HAP due to the presence of strong stylar and ovarian incompatibility. Abnormalities in pollen tubes such as swelling, twisting, intense callose plug formation, breakage, branching and reverse orientation were frequently observed among the incompatible crosses.     

Pollen storage of Fragaria and Potentilla

Summary Low temperature and humidity were used for the storage of pollen of four species of Fragaria (2n=14, 42 and 56) and two species of Potentilla (2n=14). The stainability of Fragaria pollen was assessed over a number of years by use of aceto-carmine stain and its viability by cross-pollinations; stainability of Potentilla pollen was assessed by acetocarmine. Fragaria pollen so stored remained stainable for seven years and showed the ability to set seed for three years. over the latter period, aceto-carmine staining gave a reasonable prediction of seed set produced by the stored pollen of Fragaria and so is of value in estimating pollen viability in that genus. Potentilla pollen remained stainable for three years.     

Wheat pollen dispersal under semiarid field conditions: potential outcrossing with Triticum aestivum and Triticum turgidum

Isolation distance is the main barrier to crop-to-crop gene-flow. A 3-year study assessed the maximum potential outcrossing under field conditions between two wheat cultivars (Triticum aestivum L.) and between wheat and durum wheat (Triticum turgidum L. var. durum). Outcrossing was measured by seed set on emasculated recipient plants placed at four sides with different distances from a 3 m × 3 m T. aestivum (cultivar Chinese Spring) pollen source. Frequencies of seed set at 0 m distance were 45% (37–56%) for T. aestivum cultivars and 18% (5–30%) with T. turgidum. These values agree with hybridization in non-limiting pollen conditions measured by manual crosses in greenhouse. The number of pollen grains and the outcrossing frequencies decreased at increasing distances influenced by the prevailing wind direction. Under semiarid conditions of this assay, viable pollen was found 14 m from the pollen source, with a maximum distance of 8 m at which cross-pollination decreases below 1%. Ambient conditions affect pollen viability, hybridization and pollen dispersal. Data presented in this paper emphasize the major role played by environmental conditions in outcrossing. Data obtained in one area may therefore not coincide with the prevailing situation in different locations and climates.     

The origin of polyploids via 2n gametes in Vaccinium section Cyanococcus

Summary The production of 2n pollen (pollen with the sporophytic chromosome number) was evaluated in 4x and 6x taxa of Vaccinium section Cyanococcus. Mean frequencies of 2n pollen producers were 17.1% and 8.3% in natural 4x and 6x populations, respectively. The frequency of 2n pollen producers in the 4x species ranged from 8.6% (V. angustifolium) to 23.8% (V. pallidum). Level of 2n pollen production was genotypically variable (1% to 37.4%). The widespread occurrence of 2n pollen in 2x, 4x and 6x taxa suggests that sexual polyploidization was widespread and responsible for the origin of the polyploid species found in this genus. The frequency of 2n pollen producers was not significantly different between the 4x species and their putative 2x ancestors. These results support the origin of 4x and 6x taxa as a consequence of sexual polyploidization. Polyploids derived from sexual polyploidization would be expected to have increased fitness and flexibility due to the mode of 2n pollen formation. In blueberry species the predominant mode of 2n pollen formation is genetically equivalent to a first division restitution mechanism (FDR). FDR 2n pollen transmits a high percentage of the heterozygosity and a large fraction of the epistasis from the 2x parent to the 4x offspring.     

Interspecific hybrid of Helianthus annuus × H. simulans: Characterization and utilization in improvement of cultivated sunflower (H. annuus L.)

The first success at interspecific hybridization between cultivated sunflower(H. annuus) and a diploid perennial species, H. simulans is reported. The F₁s from both direct and reciprocal directions exhibited dominance of the wild species phenotype and were pollen sterile. Meiosis was irregular in the F₁ hybrids and both univalents and multivalents were observed. Multiplication of the interspecific hybrids was achieved through in vitro culture of nodal sections on Murashige & Skoog medium supplemented with 0.5 mg l^-1 benzyladenine. Fertility of the interspecific hybrids was improved by subjecting the in vitro proliferating shoots to 0.001% colchicine incorporated in the shoot multiplication medium. The amphiploids serve as fertile bridges and facilitate interspecific gene transfer through conventional breeding. This revised version was published online in July 2006 with corrections to the Cover Date.