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 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–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.     

Factors affecting haploid production in wheat using the Hordeum bulbosum system. 1. Genotypic and environmental effects on pollen grain germination,pollen tube growth and the frequency of fertilization

Summary A quantitative examination of pollen grain germination and pollen tube growth within the overy wall was made in selected wheat × tetraploid H. bulbosum hybridizations, to investigate the effect of the wheat and the H. bulbosum genotype on these characters. As expected, variation at the known crossability loci had no effect on pollen grain germination. The frequency of pollen tube penetration of the ovary wall was, however, severely reduced when the dominant alleles were present. Pollen tube penetration was nevertheless observed in 3 of the 110 ovaries examined of the non-crossable cultivar Highbury. The H. bulbosum genotype had a much smaller effect on these characters, but significant differences between the clones were observed in the numbers of pollen tubes initially penetrating the ovary wall. Although two H. bulbosum genotypes showed no significant differences in the number of ovaries with pollen tubes at the base of the ovule, significant differences in the frequency of fertilization were observed. The possible cause of this discrepancy is discussed.The frequency of fertilization in crossable wheat × H. bulbosum hybridizations was improved by the application of gibberellic acid within 10 minutes of pollination, and reduced by an increase in the ambient temperature from 20°C to 26°C. Fertilization following the pollination of non-crossable wheat genotypes was not affected by either of these factors.     

A procedure suitable for in vitro pollen selection inBrassica oleracea

Summary Experiments to determine whetherBrassica oleracea pollen could effect fertilisation following incubation in liquid culture medium are reported. Pollen was incubated in vitro, collected by centrifugation and used to pollinate compatible pistils. While retaining viability and the capacity to germinate such pollen was unable to penetrate the stigma papillae. However, incubated pollen produced functional tubes following pollination of styles from which the stigmas had been removed. These tubes grew through the style to the ovary and viable seed was obtained. The potential application of this procedure in pollen selection is discussed.     

Investigations into the Barrier(s) to Interspecific Hybridization between Cicer arietinum L. and eight other Annual Cicer species

Barrier(s) to interspecific hybridization between the cultivated chickpea, Cicer arietinum L., and eight other annual wild species, i.e. C. reticulatum Lad., C. echinospermum Dav., C. pinnatifidum J. and S., C. judaicum Boiss., C. bijugum Rech., C. chorassanicum (Bge) M. Pop., C. yamashitae Kit. and C. cuneatum Rich., were investigated. In general, good pollen germination and pollen tube growth were observed in all eight crosses and their reciprocals. En spite of a few pollen cube growth abnormalities in most crosses, pollen tube penetration into the ovule and, thus, fertilization was observed in all cross combinations. However, differences were observed in the time from pollination to fertilization, not only between different interspecific crosses but also between reciprocals of a particular interspecific cross. The crossability barrier is, therefore, believed to be due to factor(s) operating after fertilization.     

Studies of pollen grain germination,pollen tube growth,micropylar penetration and seed set in intraspecific and intergeneric crosses within three Cruciferae species

Summary Pollen grain germination, pollen tube growth and micropylar penetration were investigated in intraspecific and intergeneric crosses involving Brassica napus L. ssp. oleifera (Metzg.) Sinsk. (oil-seed rape or fodder rape), xBrassicoraphanus Sageret (Raparadish) and diploid (2x) and tetraploid (4x) accessions of Sinapis alba L.(white mustard). For the reciprocal intergeneric crosses between B. napus and xBrassicoraphanus no effective barriers to pollen tube growth on stigmata or in styles were observed. The resulting low frequency of hybrid plants was mainly associated with a low rate of ovules with micropylar penetration per siliqua or with embryo abortion. Hybrid plants could be obtained without use of embryo rescue. In reciprocal crosses between B. napus and S. alba 2x or 4x incongruity barriers were observed on the stigma, in the style, and in the ovary resulting in a low frequency of ovules with micropylar penetration per siliqua. Open flower-pollination compared to bud-pollination generally was the more favourable procedure for pollen grain germination and pollen tube growth in crosses involving S. alba, but for micropylar penetration and seed set no differences were observed. Crosses between S. alba 2x () and B.napus () were found to result in a higher frequency of ovules with micropylar penetration as compared to reciprocal crosses or crosses with S. alba 4x. All reciprocal crosses between B. napus and S. alba 2x or 4x were unsuccessful when no embryo rescue was applied. Embryo rescue shortly after pollination, i.e. 2 to 5 days, however, resulted in hybrid seeds and plants, but only when applied to crosses between S. alba 2x () and B. napus (). The possible effects of the genome constitution, taxonomic distance and the parthenogenetic and parthenogenesis inducing ability of the parental genotypes on the observed malfunctions at the pre-and/or post-zygotic stage of the pollen grain-pistil interactions are discussed.Abbreviations DAP Days After Pollination - IAA Indole-3-Acetic Acid - kin Kinetin     

Barriers to hybridization of Solanum bulbocastanum Dun. and S. Verrucosum Schlechtd. and structural hybridity in their F1 plants

Summary Results of reciprocal crosses between Solanum verrucosum (2n=2x=24) and S. bulbocastanum (2n=2x=24) are described in terms of pollen tube behaviour in styles, of berry and seed set, of fertility and of meiotic behaviour of the F₁ hybrids. Pollen tube growth of S. verrucosum is strongly inhibited in styles of S. bulbocastanum, whereas no inhibition is observed in the reciprocal cross. Therefore S. bulbocastanum x S. verrucosum fails to set berries or seeds, whereas the reciprocal cross produces both berries (54.4% berry set) and seeds (0.3 per berry). Only 14.6% of the seeds germinate. Both the diploid and corresponding tetraploid hybrid plants are vigorous, flower abundantly, have a rather regular meiosis (mainly rod bivalents), but show a high degree of cytoplasmic-genic male sterility. Crossability of the diploid hybrid plants is nil when used as pollen parents and near to zero when used as pistillate parents. The barriers to hybridization of the parent species (unilateral inhibition of pollen tube growth, somatoplastic sterility, cytoplasmic-genic male sterility and structural differences of the parental chromosomes) are discussed and methods are suggested to overcome these barriers.     

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.     

Effects of Temperature on Pollen Tube Growth and Fruit Set in Reciprocal Crosses Between Cucumis sativus and C. metuliferus

Interspecific hybridization between Cucumis sativus and C. metuliferus may be used to introduce disease resistance into the cultivated cucumber. Hybrid plant development, however, is restricted by strong barriers to crossing. The present study was undertaken to examine temperature effects on pollen tube growth and fruit set in reciprocal crosses between C. sativus and C. metuliferus. When crossing C. sativus×C. metuliferus, at 20°C, pollen tube growth was arrested in the stylar region of the pistil and no fruits containing embryos were found. However, at 23° and 26°C, C. metuliferus pollen tubes penetrated into the C. sativus ovules. The enhanced pollen tube penetration was correlated with improved embryo development. Although pre-fertilization barriers were overcome, post-fertilization barriers caused abortion of hybrid embryo development at the globular stage. No effects of higher temperatures were found in the cross C. metuliferus×C. sativus; Pollen tubes were generally arrested in the stylar region of the pistil.     

Interspecific hybridization among cultivars of hardy Hibiscus species section Muenchhusia

Rose mallows belong to the Muenchhusia section of the Hibiscus genus. They represent a small group of cold tolerant North American plants and are popular ornamentals mainly because of their abundant, large and colorful flowers. Due to their geographical origin they are well suited for garden use in temperate regions worldwide. The aim of the study was to investigate hybridization barriers in crosses among cultivars of Hibiscus species from the Muenchhusia section: H. coccineus, H. laevis and H. moscheutos. Crossing barriers were identified as both pre- and post-zygotic. The analysis of pollen tube growth revealed inhibition of pollen tubes and their abnormal growth. In specific crosses the fertilization success was low. The pre-fertilization barriers did not cause a complete reproductive isolation between the hybridization partners. In relation to post-fertilization barriers, the occurrence of hybrid incompatibilities such as unviability, chlorosis, necrosis, stunted growth and albinism were the main drawback in production of hybrids. The appearance of symptoms of hybrid incompatibilities was dependent upon specific parental plants. The obtained progeny had intermediate leaf morphology and flower morphology compared to parental plants. Hybridity state was verified by morphological analysis and RAPD markers. Based on the overall plant morphology, 472 hybrid progenies were obtained.     

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     

麻疯树花粉管荧光染色及生长过程的研究

花粉管的生长是完成植物受精的重要生理过程,观察花粉管生长首先需要对雌蕊组织进行软化透明。本研究探索适合麻疯树雌蕊结构的透明方法,并利用苯胺蓝染色技术,在荧光显微镜下观察授粉后麻疯树花粉管的萌发和生长过程。结果表明:麻疯树雌蕊组织可采用NaOH溶液软化透明,较优的条件是2 mol·L-1的NaOH,50℃恒温箱内软化3~4 h;麻疯树授粉2 h后花粉管开始萌发,10 h后花粉管穿过花柱基部,11 h后在子房中观察到花粉管。麻疯树授粉后,花粉管进入子房至少需要11 h,完成受精则需要更长的时间。     

Prezygotic interspecific hybridization barriers between kenaf (Hibiscus cannabinus L.) and four wild relatives

Use of interspecific hybridization in genetic improvement of kenaf (Hibiscus cannabinus), a multipurpose crop, is limited by low cross‐compatibility. Previous hybridization studies suggest that part of incompatibility barrier is prezygotic. Pollen germination, stigma receptivity and growth of alien pollen on kenaf pistil were studied using wild relatives of kenaf. Four wild and semi‐domesticated species, H. surattensis, H. acetosella, H. radiatus and H. vitifolius, were used as pollen donors in the present experiment. H. radiatus exhibited highest pollen germination (65.89%) on H. cannabinus stigma. For closely related species (H. surattensis, H. acetosella, H. radiatus), stylar incompatibility was noticed, while for distantly related species H. vitifolius, the incompatibility was stigmatic. Number of pollen tubes entering ovule of H. cannabinus pistil was higher for H. radiatus (1.30). Alien pollen growth and penetration of pollen tube through ovule were higher in species sharing same genome of H. cannabinus. High callose deposition was observed in incompatible crosses, suggesting a general mechanism for prezygotic incompatibility in Hibiscus section Furcaria. Callose deposition in incompatible crosses increased with time.     

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.     

Compatibility relations between the edible carrot Daucus carota and D. pusillus, a related wild species from the Argentinian Pampas

To establish the feasibility of hybridization between the wild carrot species Daucus pusillus Michx. (2n = 2x = 22; 2n = 2x = 22 and 20), collected in the pampas grasslands of Argentina, and the edible carrot, Daucus carota L. (2n = 2x = 18), controlled pollinations were attempted on the plant. Due to the difficulties encountered, flowers of 12 accesions and three commercial cultivars were excised from individual plants and pollinated in Petri dishes following an incomplete diallel design. After processing, the pollinated pistils (four to six per genotypic combination) were observed under a microscope with UV light. Pollen tubes reaching the ovaries and/or the ovules (compatible relation) were observed in six out of nine D. pusillus × D. pusillus and seven out of 18 D. pusillus × D. carota genotypic combinations. In the eight D. carota × D. pusillus genotypic combinations, only ungerminated pollen, pollen not adhered to the stigmas or pollen tubes overlapping the stylar tissue were observed. Additional flowers were pollinated in a sample of compatible genotypic combinations and the pollinated pistils were in vitro culture to study embryo and endosperm development. Eight out of nine pistils from the intraspecific and nine out of 13 from the interspecific crosses enlarged to form apparently normal schizocarps. Histological analyses revealed normal development of embryo and endosperm. The breeding barriers between the two species are incomplete, making feasible the obtainment of interspecific hybrids by conventional techniques.     

Interspecific compatibility barriers,development of interspecific hybrids through embryo rescue and lineage of Trifolium alexandrinum (Egyptian clover)—important tropical forage legume

Genetic improvement of Trifolium alexandrinum L. is hampered because of narrow genetic base and lack of interspecies compatibility information. The study was envisaged to understand the interspecies incompatibility and the affinity between T. alexandrinum and 22 species of the genus representing different sections and to develop interspecific crosses. Development of interspecific hybrids with T. resupinatum, T. lappaceum, T. subterraneum, T. vesiculosum and T. echinatum, through embryo rescue, revealed their affinity with T. alexandrinum. Failure of pollen germination or restricted pollen tube growth showed its distant relatedness with T. clypeatum, T. balansae, T. purpureum, T. leucanthum, T. hirtum, T. pilulare and T. hybridum. Pollen tubes reached up to ovule among the crosses with T. alpestre, T. repens and T. nigrescens, whereas it travelled up to ovary among the crosses with T. angustifolium, T. grandiflorum and T. dasyurum indicating need of manipulation to overcome barrier. Thus, incompatibility of T. alexandrinum with many Trifolium species could be overcome through embryo rescue with intensive crossing. Egyptian clover ecotype ‘Fahli’ was more compatible with other species than ‘Mescavi’.     

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.     

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.