Observation Of Pollen Tube Growth Research Articles

Breakdown of Self-Incompatibility in Citrus by Temperature Stress, Bud Pollination and Polyploidization

Self-incompatibility (SI) is present in around half of all species of flowering plants. SI limits endogamy and contributes to increased genetic diversity. SI is a very important trait in citrus because, when coupled with parthenocarpy, it allows seedless fruit production. Otherwise, SI is an impediment to genetic studies and breeding programs. Temperature stress, bud pollination and polyploidization can induce the breakdown of the SI mechanism in several species. In this work, we investigated how the SI mechanism can be broken down in two self-incompatible diploid citrus genotypes: ‘Fortune’ mandarin and ‘Clemenules’ clementine. The influence of temperature stress on the SI mechanism was assessed in self-pollinated flowers of ‘Fortune’ mandarins subjected to 2 temperature regimes (10 °C and 30 °C), whereas the bud pollination effect was investigated in the same genotype and in ‘Clemenules’ clementines cultivated under field conditions. The tetraploid ‘Clemenules’ clementine cultivated under field conditions was used to study if tetraploidization can bypass the SI reaction. Histological observations of pollen tube growth and seed production in self-pollinated flowers were used to evaluate the breakdown of SI, while the genetic analysis with SSR and SNP markers confirmed that all recovered plants were zygotic and had been originated by selfing. Our results confirm that the SI reaction can be surpassed by temperature stress, bud pollination and tetraploidy. To our knowledge, this is the first report in citrus in which the SI reaction breakdown by these three different strategies is demonstrated by molecular markers.

Self-(in)compatibility, S-RNase allele identification, and selection of pollinizers in new Japanese plum-type cultivars

Japanese plum (Prunus salicina Lindl.) exhibits gametophytic self-incompatibility, a prezygotic reproductive barrier that inhibits the growth of self-pollen tubes in the style, preventing self-fertilization. This reaction of incompatibility is genetically controlled by a multiallelic locus (S). When the S allele in the pollen grain is the same as either of the two S alleles expressed in the pistil, the pollen tube arrests its growth in the style preventing fertilization. Most Japanese plum-type cultivars are self-incompatible and need cross-pollination with compatible cultivars coincident at flowering. In the last years, an important renewal of plant material is taking place, with the introduction of a number of new cultivars from breeding programs of different countries whose objectives include self-compatibility. In South Africa, the ARC Infruitec/Nietvoorbij program has released a series of new cultivars that are being introduced in the main producing countries. However, their adaptability to other areas with different climatic conditions is unknown. In this work, the pollination requirements of twelve cultivars from ARC-Infruitec program is determined, analyzing their behavior in Extremadura (Spain), one of the main producing areas in Europe. For this purpose, for each cultivar, pollen viability has been evaluated through in vitro pollen germination, self(in)compatibility has been determined by the observation of pollen tube growth in self-pollinated flowers under the microscope, and S-alleles have been identified by PCR analysis. The time of flowering has been monitored over five years. The selection of adequate pollinizers were done by assigning the cultivars to their corresponding incompatibility group and comparing their time of flowering. These results can be extrapolated to other areas where these cultivars are newly introduced, although additional data of flowering dates may be required for each zone.

Identification of Self-Incompatibility Alleles by Specific PCR Analysis and S-RNase Sequencing in Apricot.

Self-incompatibility (SI) is one of the most efficient mechanisms to promote out-crossing in plants. However, SI could be a problem for fruit production. An example is apricot (Prunus armeniaca), in which, as in other species of the Rosaceae, SI is determined by an S-RNase-based-Gametophytic Self-Incompatibility (GSI) system. Incompatibility relationships between cultivars can be established by an S-allele genotyping PCR strategy. Until recently, most of the traditional European apricot cultivars were self-compatible but several breeding programs have introduced an increasing number of new cultivars whose pollination requirements are unknown. To fill this gap, we have identified the S-allele of 44 apricot genotypes, of which 43 are reported here for the first time. The identification of Sc in 15 genotypes suggests that those cultivars are self-compatible. In five genotypes, self-(in)compatibility was established by the observation of pollen tube growth in self-pollinated flowers, since PCR analysis could not allowed distinguishing between the Sc and S8 alleles. Self-incompatible genotypes were assigned to their corresponding self-incompatibility groups. The knowledge of incompatibility relationships between apricot cultivars can be a highly valuable tool for the development of future breeding programs by selecting the appropriate parents and for efficient orchard design by planting self-compatible and inter-compatible cultivars.

Fluorescence microscopy as a tool for determining self-incompatibility in apricot cultivars

Fluorescence microscopy is a relatively rapid and reliable method to determine self-incompatibility in fruit-tree species. It is based on observation of pollen-tube growth in the pistils. Pollen tubes stained with fluorochromes show fluorescence when exposed to ultraviolet light. Testing of the self-compatibility trait was carried out in 123 apricot cultivars using fluorescence microscopy. In self-compatible cultivars, in the majority of pistils (60-100%), the pollen tubes reached the ovary. In contrast, in self-incompatible cultivars, pollen tubes growth ceased in the style, with plugs forming at their tips. In these cultivars, pollen tubes rarely (0-30%) reached the base of the style. Although apricot cultivars of the European eco-geographical group are traditionally considered self-compatible, we identified many self-incompatible cultivars, especially among those originating from new North American and West European breeding programs. About half (62) of the studied cultivars were self-incompatible. Given that self-incompatibility occurs frequently among new apricot cultivars, special care should be taken when considering cultivar composition in new orchard plantings.

Characterization of self-compatibility in sweet cherry varieties by crossing experiments and molecular genetic analysis

Self-compatibility is a major breeding objective in sweet cherry. The identification and characterization of new sources of self-compatibility will be useful for breeding and research purposes. In this work, self-compatibility of four local Spanish sweet cherry varieties was investigated by crossing experiments and molecular genetic analysis of two self-incompatibility loci. Crossing experiments included self- and cross-pollinations in the laboratory followed by microscopic observation of pollen tube growth and fruit set assay in the field. After crossing experiments, two accessions, ‘Son Miro’ and ‘Talegal Ahin’, were self-compatible while the other two were self-incompatible. Inheritance of S-locus and microsatellite EMPaS02 (linked to self-compatibility, Sc) were investigated in self-pollination progeny of both self-compatible genotypes. Results indicate that self-compatibility in ‘Talegal Ahin’ is similar to self-compatibility described in sweet cherry ‘Cristobalina’ and may be caused by the same mutation. That is a pollen part mutation not linked to the S-locus but linked to microsatellite EMPaS02 in cherry LG3. In ‘Son Miro’ self-compatibility seems more complex, affecting pollen and style function, and probably involving more than one mutation not described previously in sweet cherry. Together with ‘Cristobalina’, the newly described self-compatible varieties ‘Son Miro’ and ‘Talegal Ahin’ confirm the existence of unique self-compatible plant material in local germplasm from Spain that should be conserved and characterized for its use in breeding and research.

Pollen tube growth and self-incompatibility in three Ziziphus species (Rhamnaceae)

Self-incompatibility and synchronous protandrous dichogamy have previously been reported in Ziziphus species. In this work, we conducted a comparative analysis of fluorescence microscopy observations of pollen tube growth following controlled cross pollinations of emasculated flowers and self-pollinations of non-emasculated flowers in three Ziziphus species, Z. jujuba, Z. mauritiana and Z. spina-christi, with the aim to determine the type of the self-incompatibility system of each species. In addition, to test whether autonomous self-pollination, parthenocarpy or agamospermy occurs, flowers were emasculated (or not) and covered. Fruit set and seed viability were monitored. The presence of binucleate pollen grains and the cessation of pollen tube growth in the style suggest that the self-incompatibility system operating in the studied Ziziphus species is gametophytically controlled. Controlled self-pollination in Z. mauritiana resulted in fruits that dropped off before maturation, whereas in Z. spina-christi the flowers dropped off one or two days after pollination. Following controlled self-pollination, small fruits lacking viable seeds were obtained in Z. jujuba, probably due to the stimulus provided by pollination (stimulative parthenocarpy). In the cultivar Tamar of Z. jujuba, the relatively high percentage of seedless fruits obtained in emasculated bagged flowers without hand pollination suggests that this cultivar can set seedless fruits without any pollination stimulus.

EVALUATION OF THE REPRODUCTIVE PROCESS AS THE CAUSE FOR LOW FRUIT SET IN TWO JAPANESE PLUM CULTIVARS

The reproductive process has been evaluated to ascertain the causes of low fruit set in two Japanese plum cultivars, 'Sweet August' and 'Rubirosa'. In order to evaluate pollen germination, pollen-pistil incompatibility and ovule viability, different experiments were performed in orchard conditions. Pollen viability was determined by assessing the percentage of in vitro pollen germination. The self-(in)compatibility of both cultivars and their cross-compatibility with 'Champion' were examined by self- and cross-pollination experiments followed by observation of pollen tube growth under the microscope. Likewise, the S-RNase alleles of each cultivar were determined by PCR amplification of the S-RNase gene. Finally, ovule development was examined under the microscope. While 'Sweet August' behaved as self-incompatible, 'Rubirosa' appears to be self-compatible. The observation of pollen germination and pollen tube growth in the different crosses analysed showed that fruit set was not limited by the absence of compatible pollen. However, a high percentage of degenerated ovules were observed in flowers from both cultivars. This premature ovule degeneration is likely to be the cause of low fruit set in both cultivars.

Breeding system and thrips (Thysanoptera) pollination in the endangered tree Ocotea porosa (Lauraceae): implications for conservation

AbstractOcotea porosa has been extensively exploited over the past few decades because of the quality of its wood. Today, populations are reduced and the species is now included in the Red List of threatened species by the International Union for Nature Conservation. For conservation and management purposes, it is extremely important that we understand its reproductive ecology. Floral morphology was described based on field and scanning electron microscopic examination. The reproductive system was determined through experimentally controlled pollination along with observations of pollen tube growth. Pollinators were identified through field observations. Flowers of O. porosa are small, shallow, inconspicuous, asynchronous and grouped in inflorescences. This species presented self‐compatibility, but did not reproduce through apomixy and spontaneous self‐pollination was very rare (5%). Despite being monoclinal, flowers were protogynic, and the gynoecium was receptive after the first day of anthesis when the anthers were closed. Spontaneous self‐pollination was avoided by the extrorse position of the anthers of the internal stamens. Frankliniella gardeniae (Thysanoptera) was the only pollinating species observed and, after visiting several asynchronous flowers in the same inflorescence, favors geitonogamy. Together the small distance supposedly achieved by thrips in flight and the small population density of O. porosa can reduce the chances of cross‐pollination in this species. If so, conservation measures must include preservation of the current population and possibly planting to increase population density. This would reduce the distance between individuals and increase genetic variability. Thrips as pollinators must be included in conservation planning for O. porosa.

THE EFFECT OF SELF-COMPATIBILITY IN POLLEN GERMINATION AND TUBE GROWTH IN ALMOND

Although self-compatibility is a genetically qualitative trait in almond, its expression has been shown to be variable and dependent on the genotype. Thus, pollen germination and pollen tube growth were studied in the progenies of two different crosses, involving the same self-compatible female parent, ‘Mardia’ (S6Sf), and two self-incompatible male parents ,‘Desmayo Largueta’ (S1S10) and ‘Marcona’ (S11S12). Pollen germination was tested following the Hermosa procedure and pollen tube growth was assessed in the lab after hand self-pollination of pistils following flower emasculation, pistil sampling after 96 hr and pollen tube growth observation by microscopic observation with fluorescence light. The results were statistically evaluated by variance analysis and the means were compared by the Duncan Multiple Range Test (DMRT). Results showed that pollen germination in an artificial medium was good for all progenies, with no significant differences between the individuals. Pollen tube growth, however, showed significant differences (5% level) between the styles of self compatible and self-incompatible seedlings. The observation of pollen tube growth after self-pollination allows not only the differentiation of the self-compatible plants and the determination of the phenotype frequencies, but also the establishment of the quality of the self-compatibility expression and the possible interest of each plant of the breeding program.

Flower Emasculation as the Cause for Lack of Fruit Set in Japanese Plum Crosses

Flower emasculation is widely used in breeding programs for hybridization of fruit trees. In japanese plum (Prunus salicina), some genetic crosses made by emasculation have resulted in very low or lack of fruit set, but the causes leading to this situation are not clear. In this work, the influence of flower emasculation on fruit set was evaluated in four japanese plum-type cultivars by comparing cross-pollinations performed with and without emasculation. Fruit set and fruit drop in the crosses were characterized until harvest. To ascertain which factors in the reproductive process could be related to the lack of fruit set, compatibility was determined for each cross by the observation of pollen tube growth under the microscope and by polymerase chain reaction. Likewise, the stage of ovule development was observed under the microscope in emasculated and non-emasculated flowers. An analysis of the different pollination treatments and the study of the compatibility relationships helped to dismiss factors that intervene in the reproductive process and to identify flower emasculation as the cause of premature degeneration of ovules and its implication in determining subsequent fruit set.

SELF-INCOMPATIBILITY IN JAPANESE PLUM – S-ALLELE GENOTYPING OF CULTIVARS

Japanese plum exhibits gametophytic self-incompatibility and thus many cultivars are inter-incompatible. In this work, two methods were used to determine the compatibility among 54 Japanese plum cultivars. S-alleles of each cultivar were determined by PCR amplification of the S-RNase and cross-pollinations experiments, followed by microscope observation of pollen tube growth in the style, were done to test cross-compatibility among some of the cultivars. The results allowed grouping the cultivars in their corresponding Incompatibility Groups.

Mechanism of seedlessness in Iranian seedless barberry ( Berberis vulgaris L. var. asperma)

Species of barberry ( Berberis vulgaris L. var. asperma) is cultivated in arid and semi arid areas of Iran (South Khorasan province). It is widely used as a food additive. Fruits of this species are seedless, while wild type barberries produce seeds in the same area. In this study, we investigated the mechanism of seedlessness in seedless barberry by pollen viability test, field pollination experiments and microscopic observation of pollen tube growth in pistil and ovule development. For comparison, we also examined ovule development in wild type barberry ( B. crataegina DC). In seedless barberry pollen germination was about 54%. Seedless barberry produced 20% seeded fruits when pollinated with pollen of wild type barberry. There was a sharp decrease in fruit set in emasculated unpollinated flowers of seedless barberry. In seedless barberry, a large number of pollen grains (about 370) were observed on stigma of each flower at 12 h after balloon stage (ABS). Most of them germinated and penetrated intracellular area of stigma surface, but no pollen tube reached ovary. In seedless barberry, many ovules did not have any embryo sac or had a very small incomplete embryo sac. In addition, unfused polar nuclei were clearly recognized in some cases at 14 days after full bloom (AFB). However, in wild type, double fertilization was accompanied by disappearance of polar nuclei. In seeded barberry, the cellularized endosperm became apparent at seven days AFB. At 21 days AFB, all ovules of seedless barberry were degenerated, while at the same time in wild type, one or two ovules of each flower were normal and were developing into complete seeds. Results showed that self-incompatibility has a main role in seedlessness of seedless barberry. However, the high frequency of abnormal ovules and single fertilization can be considered as two other reasons of seedlessness. Due to our results, fruits of seedless barberry were set by stimulative parthenocarpy.

A new case of late-acting self-incompatibility in Capparis L. (Brassicaceae): C. jacobinae Moric. ex Eichler, an endemic andromonoecious species of the Caatinga, Pernambuco State, Brazil

We studied the reproductive system of Capparis jacobinae Moric ex Eichler (Brassicaceae), based on controlled hand-pollination and observation of pollen tube growth made in a fluorescence microscope. Of 105 self-pollinated flowers only one produced fruits (success = 0.95%), all the other flowers abscised at the same time, between the eighth and tenth day after anthesis. Nevertheless, self- and cross-pollinated pollen tubes reached the micropyle. The rate of penetrated ovules in self-pollinated flowers was lower during the first 24 h after pollination; thereafter this rate was similar between self- and cross-pollinated flowers for treatments of 48 h, 72 h and 96 h after pollination. In addition, we carried out two indirect estimates of the reproductive system, based on pollen/ovule and seed/ovule ratios, which resulted in typical features of xenogamous species. We concluded that C. jacobinae has a late-acting self-incompatibility system. This is the third record of this mechanism for the genus and the first for a species of Capparis endemic to the Caatinga. We suggest that this self-incompatibility system may occur in other species of the same genus and family.

A modifier locus affecting the expression of the S-RNase gene could be the cause of breakdown of self-incompatibility in almond

Self-compatibility has become the primary objective of most almond (Prunus amygdalus Batsch) breeding programmes in order to avoid the problems related to the gametophytic self-incompatibility system present in almond. The progeny of the cross 'Vivot' (S(23)S(fa)) x 'Blanquerna' (S(8)S(fi)) was studied because both cultivars share the same S(f) allele but have a different phenotypic expression: active (S(fa)) in 'Vivot' and inactive (S(fi)) in 'Blanquerna'. In addition, the microscopic observation of pollen tube growth after self-pollination over several years showed an unexpected self-incompatible behaviour in most seedlings of this cross. The genotypes of this progeny showed that the S(fi) pollen from 'Blanquerna' was not able to grow down the pistils of 'Vivot' harbouring the S(fa) allele, confirming the active function of this allele against the inactive form of the same allele, S(fi). As self-compatibility was observed in some S(8)S(23) and S(8)S(fa) individuals of this progeny, the S(f) haplotype may not always be linked to the expression and transmission of self-compatibility in almond, suggesting that a modifier locus may be involved in the mechanism of self-incompatibility in plants.

S‐RNase genotyping and incompatibility group assignment by PCR and pollination experiments in Japanese plum

AbstractMost Japanese plum‐type cultivars are self‐incompatible and cross pollination is necessary to ensure fruit set. In this study, the S‐RNase genotype and the incompatibility group of 68 Japanese plum‐type cultivars were determined by PCR amplification of the S‐RNase gene. The S‐RNase genotype of 50 cultivars is first reported here and five new Japanese plum S‐RNase alleles (So, Sp, Sq, Sr, Ss) were identified. The results obtained, together with information compiled from previous studies, allowed describing 12 new incompatibility groups (VIII–XIX). The self‐incompatibility of several cultivars and the cross‐compatibility among different incompatibility groups were verified by self‐ and cross‐pollination experiments followed by observation of pollen tube growth. Five cultivars behaved as self‐compatible, but two of them do not have the Se allele, which has been correlated with self‐compatibility. Thus, additional sources of self‐compatibility different from Se appear to be involved in Japanese plum self‐compatibility.

Genomic characterization of self-incompatibility ribonucleases (S-RNases) in loquat (Eriobotrya japonica Lindl.) (Rosaceae, Pyrinae)

Loquat (Eriobotrya japonica Lindl.) is a fruit tree species of the Pyrinae subtribe of the Rosaceae that behaves as self incompatible. Since self-incompatibility in the Rosaceae is of the gametophytic type where a stylar ribonuclease (S-RNase) controls the female function of pollen–pistil recognition, consensus primers derived from the alignment of S-RNase sequences from other Pyrinae species were used to search for S-RNases in loquat. As a result, the first four S-RNases were sequenced for this species. The genomic sequences obtained showed the structural features of Pyrinae S-RNases. Moreover, microscopic observations of pollen tube growth in the style confirmed the inter-(in)compatibility relationships predicted from the molecular analyses. Phylogenetic analysis of the deduced amino acid sequences with other Pyrinae S-RNases confirmed that divergence of S-alleles in loquat and the Pyrinae predated speciation. This study reports for the first time the genomic characterization of S-RNases in loquat, providing a sound basis for an appropriate selection of pollinator cultivars and an adequate design of breeding programs.

High Outcrossing in the Annual Colonizing Species Ambrosia artemisiifolia (Asteraceae)

Variation in mating patterns may be particularly evident in colonizing species because they commonly experience wide variation in plant density. Here, the role of density for the mating system of Ambrosia artemisiifolia (common ragweed), a wind-pollinated annual colonizing species previously reported as self-compatible, is explored. The effect of population density on the proportion of self- and cross-fertilized seeds was examined using allozyme markers and experimental arrays conducted over two seasons in the field. Also the reproductive success of isolated plants located in diverse habitats was measured. The potential occurrence of a physiological mechanism preventing self-fertilization, i.e. self-incompatibility, following controlled self- and cross-pollinations in the glasshouse was examined. Outcrossing rates estimated using allozyme markers were uniformly high, regardless of the spacing between plants. However, when single plants were isolated from congeners they set few seeds. Observations of pollen-tube growth and seed set following controlled pollinations demonstrated that plants of A. artemisiifolia possess a strong self-incompatibility mechanism, contrary to earlier reports and assumptions. The maintenance of high outcrossing rates in colonizing populations of A. artemisiifolia is likely to be facilitated by the prodigious production of wind-borne pollen, high seed production and extended seed dormancy.

Comparison of Self- and Cross-pollination on Pollen Tube Growth, Seed Development, and Germination in Crapemyrtle

Crapemyrtle (L. indica and L. indica × L. fauriei hybrids) is one of the most popular flowering landscape plants in the U.S. Although many cultivars have been developed through breeding efforts, little has been published on the reproductive biology of the genus. The objective of this study was to evaluate barriers to successful self-seed production in crapemyrtle. Self-compatibility was assessed by comparing pollen tube growth, fruit and seed production, and seed germination following controlled self- and cross-pollinations. Observations of pollen tube growth at intervals up to 24 hours after self- and cross-pollination indicated no barriers to self-fertilization acting at the stigmatic or stylar level in L. indica, L. fauriei or cultivars derived from inter-specific hybrids of these two species. Self-pollinations of `Catawba', `Whit IV', `Tonto' and `Tuscarora' had lower percent seed pod set and seed germination than did cross-pollinations of these cultivars. The number of seeds per pod was lower when `Catawba', `Whit IV' and `Tuscarora' were self-rather than cross-pollinated, but no difference between `Tonto' self- and cross-pollinations was observed. When decreased pod set is combined with much lower seed germination for self-pollinations, selfing of crapemyrtle is extremely unproductive when compared to cross-pollination. A late-acting self-incompatibility system or inbreeding depression is indicated for L. indica and inter-specific crosses with L. fauriei.

Distyly and variation in floral traits in natural populations of Psychotria ipecacuanha (Brot.) Stokes (Rubiaceae)

Psychotria ipecacuanha is a perennial, medicinal herb that grows in clusters in the understory of humid, shady areas of the Atlantic Rain Forest of southeastern Brazil. The present study characterized the variation in floral traits among 35 clusters from three natural populations of this plant species. Field observations showed that the clusters are isomorphic, that is, a given cluster will either set long-styled or short-styled flowers. Stigmas and anthers are reciprocally placed in each morph, a dimorphism characteristic of distyly. The populations are isoplethic, that is, a given population exhibits an equilibrium 1:1 ratio of floral morphs. Morphometric analyses revealed that anther length, stigma length, corolla diameter, and pollen grain diameter were consistently greater in short-styled flowers, regardless of the population investigated. Significant differences for floral traits in the short-styled morph were found among populations. Floral traits in the long-styled morph also showed some significant differences among populations, but not for stigma height and corolla length. Controlled pollinations carried out in natural populations showed that fruit production was higher after inter-morph pollination. Nevertheless, observations of pollen tube growth in style, and also fruit production after spontaneous self-pollination and intra-morph pollination, indicated partial intramorph compatibility in this plant species.

Pollination Biology of Hydrangea macrophylla

Little information is available on the reproductive behavior of Hydrangea macrophylla (Thunb. Ex J.A. Murr.) Ser. The objectives of this study were to investigate time of stigma receptivity, viability of pollen from sterile flowers, and self-incompatibility in this popular ornamental shrub. Pollen germination and pollen tube growth in styles were examined using fluorescence microscopy. Stigma receptivity was examined in cross-pollinations made from 1 day before anthesis to 8 days after anthesis. Maximum stigma receptivity for the two cultivars examined occurred from anthesis to 4 days after anthesis. Viability of pollen from sterile flowers was evaluated through pollen staining and observations of pollen tube growth. No significant difference in percent stainable pollen between fertile and sterile flowers was observed in any of the six taxa examined. Pollen germination and pollen tube growth were studied in cross-pollinations made using pollen from fertile and sterile flowers of two cultivars. For both cultivars, pollen tubes from fertile and sterile flowers grew to the same length and had entered ovules by 72 hours after pollination. Self-incompatibility was evaluated by comparing pollen germination and pollen tube growth in cross- and self-pollinations. In the five taxa examined, self pollen tubes were significantly shorter than cross pollen tubes in flowers that were examined 72 hours after pollination. This finding indicates the presence of a gametophytic self-incompatibility system in H. macrophylla.