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.     

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.     

Inter-varietal pollen tube growth and ovule penetration in the avocado

Summary Results are presented showing number of pollen tubes in the pistil, and ovule penetrations in crosses involving the avocado varieties Edranol, Ryan, Hass, Reed, Talbot, Jalna, Fuerte, Bacon and Sharwil. Between 5% and 100% of the ovules in each cross were penetrated by a pollen tube. The female parent exerted more control than the male on numbers of tubes and ovule penetrations and the results for type A varieties were more consistent than those for type B varieties. Penetration of an ovule by two tubes was observed in all varieties except Reed and Fuerte and penetration of an ovule by three tubes was observed in the variety Edranol only. With the possible exception of the variety Bacon as female parent there was no difference between pollination with pollen from type A or B varieties.     

Genotypic variation and polymorphism of 2S albumins of sunflower

Summary An early postfertilization ovule culture technique in Alstroemeria was described in which young ovules were cultured in vitro two days after pollination. This resulted in the direct production of seedlings in a normally incongruous cross combination. A histological study of the in vitro cultured ovules revealed the development of the embryo inside the ovule in the absence of normal endosperm. The in vivo grown ovules showed a total breakdown of the developing seed within four weeks after pollination due to a postfertilization barrier. This ovule culture technique was applied in a diallel cross of seven Alstroemeria species, five from Chile and two from Brazil. The selfings and the reciprocal crosses between the Brazilian species A. inodora and A. brasiliensis were congruous and gave seed set. The cross A. inodora x A. pelegrina gave viable seeds. In all other 39 combinations no viable seeds were harvested. In those cross combinations both pre-and postfertilization barriers were found. After early ovule culture hybrid plants were obtained in 27 of the 39 incongruous species combinations of the diallel.Abbreviations DAP days after pollination - MS medium medium according to Murashige & Skoog (1962)     

An embryological study of a Pisum sativum x Vicia faba cross

Summary Flowers of Pisum sativum plants grown in the field and greenhouse were self-pollinated and pollinated with Vicia faba. Germination of pollen and pollen tube growth were slower in the Pisum x Vicia cross. About 2% fertile ovules in the field and 8% in the greenhouse were obtained from the Pisum x Vicia cross, compared to 26% and 48% for Pisum x Pisum. Development of the Pisum x Vicia embryo was normal in form, though much slower than the Pisum x Pisum embryo, and about the sixth day it began to collapse. The greatest difference between the two types of crosses was observed in the endosperm. While the Pisum x Pisum endosperm nuclei divided normally and endosperm developed throughout the ovule cavity and around the embryo, the Pisum x Vicia endosperm nuclei formed only scattered masses of densely stained nuclei which were not always in close proximity to the hybrid embryo. It was concluded that faillure of some ovules to develop following Pisum sativum x Vicia faba cross pollination was the result of slow germination of pollen and slow growth of the Vicia pollen tubes, with subsequent lack of fertilization. Collapse of fertilized ovules was associated with abnormal and limited development of the hybrid endosperm, possibly leading to lack of nourishment and eventual collapse of the hybrid embryo.     

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.     

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.     

Embryo rescue in Rosa hybrida L.

Summary Two crosses between Rosa hybrida L. cultivars, that fail to produce progenies by conventional means, were carried out. In one of them, total hip abscision occured seven weeks after pollination; in the other one, only 2% of the fertilized hips remained on the plants after eleven weeks. Four- and five-week-old fertilized ovules were isolated in the first cross and six-week-old embryos in the second. The ovules were cultured on six media which differed in their mineral salt and sucrose concentrations while the embryos were cultured on only one of these media and eventually cold treated for one month at 4° C before being placed in a culture room set at 23° C. The embryos that had been isolated in ovulo when they were still not visible under a binocular lens developed atypically. The embryos isolated in ovulo when they were heart-shaped and on average 0.27 mm long performed in ovulo germination on some media and/or enlargement on all of them after two weeks; after another two week culture, once isolated from the ovule, all of them germinated. The cultured isolated embryos, that were exposed to cold, enlarged and germinated more rapidly when placed at 23° C than those not exposed to cold. Furthermore, the plantlets resulting from untreated embryo germination were characterized by large cotyledons which were only partially green. These results are discussed in regard to embryogenesis, precocious germination and dormancy.     

Overcoming unilateral breeding barriers between Lycopersicon peruvianum and cultivated tomato,Lycopersicon esculentum

Summary Treatment of Lycopersicon peruvianum stigmas with an artificial medium analogous to stigmatic exudate allowed pollen germination and growth on immature pistils. Growth of Lycopersicon esculentum pollen tubes to L. peruvianum ovules, an otherwise incompatible cross, was achieved following such treated bud-pollinations. No plantlets were recovered, although a few embryos from this cross at the globularity heart stage of development were excised at 22 days after pollination, indicating the presence of crossing failures as severe as in the reciprocal cross. Hybrid plants were obtained from the reciprocal cross, using as pollen parent an L. peruvianum line selected for congruity with L. esculentum. Bud pollinations to L. peruvianum, using these interspecific F₁ hybrids as the pollen parent, allowed viable embryo development and plantlet recovery. Resulting backcross plants, which possess approximately 1/4 L. esculentum genome in a L. peruvianum cytoplasm, may facilitate further introgression of the L. esculentum nuclear genes into in foreign cytoplasm.     

Chromosome location of genes controlling the content of dietary fibre and arabinoxylans in rye

Differences in fruit-set success reputed to exist between Heveabrasiliensis genetic clones were verified by an analysis of breeding recordsfor the clones PB 5/51, RRIM 600 and PR 107. Although the bestfruit-set success was obtained with PB 5/51 following artificial (hand)pollination, this clonal trait was not reflected in terms of greater numbersof pollen tubes developing it the styles of hand-pollinated PB 5/51 femaleflowers. It was observed, however, that more pollen tubes reached theovules in PB 5/51 than in RRIM 600 or PR 107. Significantly, PB 5/51female flowers required fewer pollen tubes to effect penetration of all threeof its ovules. As fruit formation in Hevea is dependent on all threeovules of the flower being successfully fertilised, PB 5/51 female flowershave hence a greater propensity for successful fruit-set. In all the threeclones studied, the frequency distribution of female flowers with 0, 1, 2 or3 of the ovules penetrated by pollen tubes did not conform to binomialexpectations. Flowers with no ovule penetrated and flowers with all threeovules penetrated were greatly over-represented. One explanation for thisnon-random distribution is the existence of `receptive' female flowers thatfavour successful fertilisation whereas `non-receptive' flowers tend to remainunfertilised even when hand-pollinated.     

Effects of γ-radiation on vitality and competitive ability of Cucumis pollen

Summary Pollen of 4 Cucumis sativus genotypes and of the related species C. metuliferus and C. zeyheri 2x was irradiated with four doses of -rays: 1, 2, 4 and 5 kGy. Higher radiation doses were found to inhibit germination and pollen tube growth in vitro with C. sativus, the most sensitive species.Irradiated pollen of cucumber never performed normal fertilization. However, after radiation with 1 kGy, cucumber pollen was able to occupy all available ovules, of which about 50% developed into empty seeds without embryo. This makes 1 kGy-irradiated maternal pollen less suitable to serve as mentor pollen in interspecific hybridizations. Doses of 2 kGy and more appeared to eliminate the ability to compete for ovules, while fruit set after a 2 kGy radiation was still high.     

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     

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.     

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.     

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.     

Gamma-irradiation of cacao (Theobroma cacao L.) pollen: Effect on pollen grain viability,germination and mitosis and on fruit set

Summary Theobroma cacao pollen fertilization capability was studied after 0, 50, 100, 200, 500 and 1000 Gy gamma-irradiation. For all irradiation doses, no alteration of pollen grain viability and in vitro germination was observed. In situ, for all doses, pollen tubes penetrated into the styles and reached the ovules 20 hours after pollination. In vitro observations of the pollen grain nuclei after 20 hours incubation showed that pollen irradiation causes inhibition of the division of the generative nucleus. Fruit survival rate 30 days after pollination decreased as irradiation doses increased from 0 to 100 Gy, and over 100 Gy no fruit set was obtained. The possibility of using irradiated pollen as a method for obtaining haploid cacao plantlets is discussed.     

The effect of ovule age on ovary-slice culture and ovule culture in intraspecific and interspecific crosses with Tulipa gesneriana L.

The efficiency of two embryo rescue techniques, direct ovule culture and ovary-slice culture followed by ovule culture, is studied in crosses with Tulipa gesneriana as maternal genotype. The germination percentages increased, in most cases, significantly with increasing ages of te ovary-slices and ovules at the start of the cultures. The low number of embryos recovered at early culture dates is caused by a higher rate of embryo abortion and by retarded embryo development. The germination percentages for ovary-slice culture followed by ovule culture was mostly comparable to direct ovule culture. Unique hybrids have been obtained from the crosses T. gesneriana × T. agenensis and T. gesneriana × T. praestans. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Use of Ovule Culture in Reciprocal Hybridization between B. campestris L. and B. oleracea L.

Ovule culture was used to overcome postzygotic interspecific incompatibility in reciprocal crosses between Brassica campestris and B. oleracea. Hybrid plants were obtained from 82 % of all reciprocal pollinations, representing 91 % of the attempted combinations. Comparisons of the embryo culture and the ovule culture indicated a significantly higher germination rate after ovule culture.     

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.     

Production of intergeneric hybrid plants between <Emphasis Type="Italic">Sandersonia aurantiaca</Emphasis> and <Emphasis Type="Italic">Gloriosa rothschildiana</Emphasis> via ovule culture (Colchicaceae)

Intergeneric hybrid plants between Colchicaceous ornamental plants, Sandersonia aurantiaca and Gloriosa rothschildiana, have successfully been produced via ovule culture. After 5 days of reciprocal cross-pollination, a few pollen tubes were observed in the ovary. Although seeds were obtained in both reciprocal cross-combinations, they did not germinate under ex vitro conditions. Ovules with placental tissues isolated 14 days after cross-pollination of S. aurantiaca × G. rothschildiana were cultured on a medium containing 0.01 mg l^–1 each of -naphthaleneacetic acid (NAA) and 6-benzyladenine (BA), on which 41.5% of ovules swollen and produced callus-like structures within 10 weeks. When such swollen ovules were transferred to a medium containing 0.1 mg l^–1 each of NAA and BA, 7.5% of the initially cultured ovules produced rhizome-like structures within 6 weeks. Among the rhizome-like structures, those derived from two independent ovules (3.7% of the initially cultured ovules) produced multiple shoots following transfer to a medium containing 0.25 mg l^–1 NAA and 2.5 mg l^–1 BA. Multiple shoot-derived plantlets were established on a plant growth regulator-free medium, and they were successfully transplanted to pots. Early verification of their hybridity was accomplished by flow cytometry (FCM) analysis, chromosome observation and rDNA analysis.