Pollen Tube Growth

The molecular bases of self-incompatibility have been intensively studied in a restricted number of model species, but for most families the expression and distribution of S-proteins is unknown. In this work, pistil cryosections from apple were used for in situ detection of S-proteins. Two specific antibodies, one against the S3-protein and another against all apple S-proteins were used. S-proteins were shown to be localised in the intercellular space of the transmitting tissue, both in the stigma and style, which agrees with the proposed mechanism of action for S-RNases in gametophytic self-incompatibility. Some intracellular labelling was also observed in all ovary sections, confined to one layer of the nucellus surrounding the embryo sac, but this labelling was found to be non-S-allele-specific. Nevertheless, the signal in the ovary was tissue-specific, which may indicate that some component not encoded by the S-locus but similar to S-proteins was detected. To the best of our knowledge this is the first report on the precise distribution of S-RNases in a rosaceous species.

The floral biology and breeding system of pohutukawa (Metrosideros excelsa, Myrtaceae) , a mass-flowering tree of northern New Zealand coastlines, were examined. Trees flower over a pea k period of 2 weeks, and compound inflorescence s contain an average of 14 .3 showy, hermaphrodite , red brush flowers that remain open for 7 days. A brie f female flower stage (mean duration 1 .3 d) is followed by the main hermaphrodite phase that lasts fo r 4 days. Neither dichogamy nor herkogamy is important in preventing pollen and stigma interference. Pollen is highly viable (93 .6%), and stigma receptivity extends for at least 9 days, as indicated by peroxidase activity, pollen germination, pollen tub e length 24 h after pollination, and seed set. Stigmati c exudate production appears to increase up to 5 days post-anthesis. On average, flowers produce 46 p 1 nectar per day, containing 18% (w/v) sucrose. Floral design and display of pohutukawa are consisten t with high levels of autogamous and geitonogamous self-pollination. Controlled pollination experiments were used to assess the effect of self-and crosspollen and a pollen mixture from five unrelated parents on capsule and seed production, and on pollen tube growth in seven trees. Three trees in the experiment were self-incompatible, as quantified by th e index of self-incompatibility (ISI), indicating that natural populations may consist of a mosaic of selfcompatible and incompatible individuals. Self-in-B9900 2

Ca 2+ -calmodulin (Ca 2+ -CaM) is a critical molecule that mediates cellular functions by interacting with various metabolic and signaling pathways. However, the protein expression patterns and accompanying serial cytological responses in Ca 2+ -CaM signaling deficiency remain enigmatic. Here, we provide a global analysis of the cytological responses and significant alterations in protein expression profiles after trifluoperazine treatment in Picea meyeri, which abrogates Ca 2+ -CaM signaling. Ninety-three differentially displayed proteins were identified by comparative proteomics at different development stages and were assigned to different functional categories closely related to tip growth machinery. The inhibition of Ca 2+ -CaM signaling rapidly induced an increase in extracellular Ca 2+ influx, resulting in dramatically increased cytosolic Ca 2+ concentrations and ultrastructural abnormalities in organelles as the primary responses. Secondary and tertiary alterations included actin filament depolymerization, disrupted patterns of endocytosis and exocytosis, and cell wall remodeling, ultimately resulting in perturbed pollen tube extension. In parallel with these cytological events, time-course experiments revealed that most differentially expressed proteins showed time-dependent quantitative changes (i.e. some signaling proteins and proteins involved in organelle functions and energy production changed first, followed by alterations in proteins related to cytoskeletal organization, secretory pathways, and polysaccharide synthesis). Taken together, Ca 2+ -CaM dysfunction induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca 2+ -CaM in the regulation of tip growth machinery.

Gibberellins (GAs) play many biological roles in higher plants. We collected and performed genetic analysis on rice (Oryza sativa) GA-related mutants, including GA-deficient and GA-insensitive mutants. Genetic analysis of the mutants revealed that rice GA-deficient mutations are not transmitted as Mendelian traits to the next generation following self-pollination of F1 heterozygous plants, although GA-insensitive mutations are transmitted normally. To understand these differences in transmission, we examined the effect of GA on microsporogenesis and pollen tube elongation in rice using new GA-deficient and GA-insensitive mutants that produce semifertile flowers. Phenotypic analysis revealed that the GA-deficient mutant reduced pollen elongation1 is defective in pollen tube elongation, resulting in a low fertilization frequency, whereas the GA-insensitive semidominant mutant Slr1-d3 is mainly defective in viable pollen production. Quantitative RT-PCR revealed that GA biosynthesis gen...

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 × 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.

 Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to ensure cross-pollination. To the best of our knowledge, this report provides the first insight into the sexual reproduction cycle in this species. The cork oak flowering season extends from April until the end of May. Our results show that, at anthesis, the pistillate flower is not completely formed and ovules are just starting to develop. Pollen reaching the dry stigmatic surface adheres to the receptive cells, germinates and penetrates the epidermis in aproximately 24 h, and grows through the intercellular spaces of a solid transmitting tissue. In cross-pollination, a sequential arrest of pollen tubes was observed along the style, providing preliminary evidence for a pollen tube competition mechanism. As a consequence, few pollen tubes reach the basal portion of the style. Furthermore, pollen tube growth is a discontinuous process since tubes are arrested in the basal portion of the style about 10–12 days after pollination. While tubes are latent, the ovarian loculus starts to develop from an emerging mass of sporogeneous cells which later will differentiate into the placenta and ovules. One and a half months after pollination ovules complete their differentiation, tubes resume growth and fertilisation occurs. Ovular abortion is frequent at this stage, and only one ovule will successfully mature during autumn into a monospermic seed.

Najas is the largest of the 18 hydrophilous angiosperm genera, however, study of pollination biology in the genus is largely ignored. In this study, the pollination mechanism of Najas marina L. was observed in two freshwater lakes as well as in aquaria. A unique mechanism of rapid pedicel elongation and curvature in the male flower of N. marina that brings the anther to lie opposite to the branches is reported for the first time. This process occurs before anther dehiscence and facilitates dispersal of pollen by water currents. A large number of pollen grains are dispersed in dense "clouds" for 5-10 min after anther dehiscence. Microscopic examination of pollen grains revealed the presence of numerous starch grains in the cytoplasm. These increase the gravity of the pollen grains and provide energy for pollen tube growth to a length of 2 mm in the water. The development of pollen tubes before contact with the stigma enhances the chances of the pollen to be captured by the branched stigmas. In addition, the pollen grains undergo a marked elongation of their polar axes just prior to release. The relatively long period of viability of both precocious and ungerminated pollen grains further enhances pollination. The high pollen loads observed on stigmas could account for the high seed yield in this submerged plant. The precocious pollen grains may be functionally equivalent to the filiform pollen occurring in submarine-pollinated seagrasses. This provides an example of convergent evolution in response to the pollination environment created by strong water currents.

The role of zinc (Zn) in reproduction of lentil (Lens culinaris Medik. cv. DPL 15) and the extent to which the Zn requirement for reproduction can be met through supplementation of Zn at the time of initiation of the reproductive phase have been investigated.

Sexual plant reproduction depends on the production and differentiation of functional gametes by the haploid gametophyte generation. Currently, we have a limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. To unravel such mechanisms, it is necessary to identify transcription factors (TF) that are part of such haploid regulatory networks. Here we focus on bZIP TFs that have critical roles in plants, animals and other kingdoms. We report the functional characterization of Arabidopsis thaliana AtbZIP34 that is expressed in both gametophytic and surrounding sporophytic tissues during flower development. T-DNA insertion mutants in Atb-ZIP34 show pollen morphological defects that result in reduced pollen germination efficiency and slower pollen tube growth both in vitro and in vivo. Light and fluorescence microscopy revealed misshapen and misplaced nuclei with large lipid inclusions in the cytoplasm of atb-zip34 pollen. Scanning and transmission electron microscopy revealed defects in exine shape and micropatterning and a reduced endomembrane system. Several lines of evidence, including the AtbZIP34 expression pattern and the phenotypic defects observed, suggest a complex role in male reproductive development that involves a sporophytic role in exine patterning, and a sporophytic and/or gametophytic mode of action of AtbZIP34 in several metabolic pathways, namely regulation of lipid metabolism and/or cellular transport.

Soybean [Glycine max (L.) Merr.] reproduction is sensitive to temperatures . 35°C. Two studies were conducted to determine temperature effects on soybean pollen germination (PG) and to detect genotypic differences. Pollen collected from 44 genotypes (Maturity Groups III to VI) grown outdoors was subjected to in vitro temperatures from 15 to 50°C at 5°C intervals. Genotypes differed significantly for in vitro PG percentage (mean of 81%) and tube length (mean of 437 mm). Mean cardinal temperatures (T min , T opt , and T max ) were 13.2, 30.2, and 47.2°C for PG and 12.1, 36.1, and 47.0°C for pollen tube growth. Genotypes differed for leaf cell membrane thermostability (CMTS), but CMTS did not correlate with pollen parameters. Cumulative temperature response index, CTRI (unitless), of each genotype calculated as the sum of eight individual stress responses (ISRs) derived from maximum PG, maximum pollen tube length (PTL), and the maximum (T max ), minimum (T min ), and optimum (T opt ) temperatures for PG and for PTLs was used to group genotypes for temperature tolerance. Heat-tolerant genotype (DG 5630RR) was less sensitive to high temperature (38/30°C) compared with heat-intermediate (PI 471938) and heat-sensitive (Stalwart III) genotypes that had deformed pollen, with reduced apertures and collumellae heads. Hence, pollen can be used as a screening tool for heat tolerance. Most sensitive to temperature was D88-5320 with a CTRI of 6.8, while AG 4403RR was most tolerant with a CTRI of 7.5. Elevated [CO 2 ] did not modify reproductive parameters or CTRI. The study also revealed that heat tolerance of vegetative tissue had little or no relationship with the heat tolerance of reproductive tissue. Maturity groups lacked a specific trend for tolerance to high temperature. The identified high temperature-tolerant genotypes and temperature-dependent pollen response functions might be useful in soybean breeding and modeling programs, respectively.

The effective pollination period (EPP) determines the success of seed set in Citrus. In this research, the EPP was studied in three citrus species with different seed set abilities, C. clementina Hort. ex Tanaka, C. unshiu Marcovich and C. sinensis (L.) Osbeck, so as to identify the factors affecting seed set and flower receptivity. The objective of this research was to enhance agronomical efforts [C. Mesejo, A. Martínez-Fuentes, C. Reig, F. Rivas, M. Agustí, The inhibitory effect of CuSO 4 on Citrus pollen germination and pollen tube growth and its application for the production of seedless fruit, Plant Sci. 170 (2006) 37-43] to prevent seed presence in fruits given that seedlessness is the main factor of quality for fresh citrus fruit consumption.

The reproductive biology of Grevillea robusta growing under exotic conditions in Kenya and Australia is reported. The species showed both protandry and a self-incompatibility mechanism. The stigma was wet and papillate with a distinct groove in the middle. The anthers dehisced prior to anthesis, when the perianth opened. Stigmatic receptivity began 1 d after anthesis, with the greatest pollen germination rates and longest pollen tubes obtained 2 d after anthesis. Nectar secretion commenced with pollen dehiscence and was abundant at anthesis. Most stigmatic grooves opened widely 1±2 d after anthesis and stigmas showed taller papillae and abundant secretion. Controlled pollinations gave a greater fruit set from cross-pollination (5 . 9 % in April and 17 . 5 % in July) than open-pollination (0 . 1 % in April and 3 . 3 % in July). No fruit set from self-pollination was obtained in April, and very few fruit set for geitonogamous (two out of 1622; 0 . 1 %) or for autogamous (one out of 2707¯owers; 0 . 04 %) pollination treatments in July. Following self-pollination, growth of pollen tubes was poorer than in other treatments, and was generally arrested in the upper style. Cross-pollinated¯owers produced normal and straight pollen tubes, while self-pollen tubes had growth abnormalities. Most of the open-pollinated¯owers were found without pollen or with only selfpollen on their stigmas indicating that the amount of cross-pollen reaching the stigma under open-pollination may be a factor limiting seed production. Flowers shed soon after the fertilization phase were those with ungerminated pollen or no pollen. Although a very low rate of sel®ng may occur, G. robusta presents a self-incompatibility system and allogamy is its primary breeding behaviour.

Pollen germination and pollen tube growth was quantified among various native Iranian wild almonds (P. dulcis (Mill.) D. A. Webb, P. eleaegnifolia Mill., P. orientalis Mill., P. lycioides Spach, P. reuteri Bioss. et Bushe, P. arabica Olivier, P. glauca Browick and P. scoparia Spach in order to identify differences in the tolerance of pollen to temperature variations. Pollen germination and pollen tube growth were observed after incubation in darkness in a germination medium for 24 h at 10–50°C at 5°C intervals. Maximum pollen germination of the wild almond species and specify that 60% was obtained for P. orientalis pollen and 98% for P. scoparia. Pollen tube length ranged from 860 μm was obtained in P. lycioides and 1490 μm in P. scoparia. A modified bilinear model best described the response to temperature of pollen germination and pollen tube length. Almond species variation was found for cardinal temperatures (T min, T opt and T max) of pollen germination percentage and pollen tube growth. Mean cardinal temperatures averaged over eight almond species were 14.7, 24.2, and 43.7°C for maximum percentage pollen germination and 14.48, 25.3, and 44.4 °C for maximum pollen tube length. The principal component analysis (PCA) identified maximum percentage pollen germination and pollen tube length of the species, and T max for the two processes as the most important pollen parameters in describing a species tolerance to high temperature. PCA also classified Prunus L. spp. into four groups according to the tolerance of pollen to temperature variations. The T min and T opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating species for high temperature tolerance.

A tobacco pollen tube glycoprotein, p69 is encoded by the pollen-specific gene ntp303 that is transcribed during pollen development and pollen tube growth, but it is abundantly translated only after pollen germination. To investigate the translational repression of ntp303 mRNA during pollen development the compartmentation of ntp303 mRNA was examined and compared against another transcript (ntp52), which is efficiently translated during pollen maturation. Three subcellular fractions were isolated: a post-polysomal fraction enriched with messenger ribonucleoprotein particles, a polysomal fraction and a novel fraction of EDTA/ puromycin-resistant particles co-sedimentating with polysomes (EPP). At all developmental stages studied, ntp303 mRNA was found to be present in all fractions. Surprisingly, most of the translationally inactive ntp303 mRNA was localised in the polysomal fraction and EPPs, whereas ntp52 mRNA was distributed between the post-polysomal fraction and polysomes but was virtually undetectable in EPPs. This differential mRNA distribution pattern may help to explain the developmentally regulated translational repression of the ntp303 gene during pollen maturation, highlighting a potential role of EPPs. A model of how this differential mRNA compartmentation pattern regulates ntp303 mRNA translation is proposed.

We isolated and characterized a pollenpreferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5 and 773 bp of 3 non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3 noncoding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.

For studies of the evolution of development in angiosperms, early-diverging eudicot taxa are of particular interest for comparisons with established core eudicot model plants, such as Arabidopsis. Here we provide a detailed description of shoot and floral development of the basal eudicot California poppy (Eschscholzia californica). Rosette formation in the vegetative phase is accompanied by increased leaf complexity and shoot apex size. The flowering phase is characterized by internode elongation, formation of dissected cauline leaves, terminal flowers, and basipetal inflorescence branching. For developing flowers and fruits, we have defined 14 stages according to important landmark events, from inflorescence primordium initiation through seed dispersal. Floral organ initiation, morphogenesis, increase in floral meristem size, and the surface structure of mature floral organs are recorded in detail. The duration of the later floral stages, as well as the path of pollen tube growth in the gynoecium, is documented. Comparison of California poppy floral development with that of Arabidopsis indicates considerable differences in terms of organ fusion, whorl proliferation, and variability of size and organ number between the two species. Transitions in meristem identity from germination to floral organogenesis were monitored using expression of the developmental control gene EcFLO, the Eschscholzia ortholog of FLORICAULA/LEAFY. We found that the pattern of expression of EcFLO in the flanks of the shoot apex is maintained from late embryogenesis to flower initiation, indicating a continuous role for this gene in meristem function. As flower organs develop, EcFLO expression becomes more restricted to petal and stamen primordia. Development of the gynoecium occurs without EcFLO expression, indicating that EcFLO may not be necessary for the activation of C-class genes.

The generation of novel hybrid lines is critically limited by the compatibility between pollen and pistil and their ability to achieve successful seed formation. Fertilization in flowering plants requires the successful transfer of the male gametes from the pollen grain to the egg contained within an ovule. This occurs via the formation of a tubular protrusion from the pollen grain

Abstract. In this study in vitro pollen viability and germination in intact pollens and pol-len tube growth in germinated pollens of five common pomegranate cultivars grown in Croatia and Bosnia and Herzegovina was investigated first time. Pollen viability varied from 36.73 % (cv. Konjski Zub) to 51.80 % (cv. Barski) in fluorescent diacetat (FDA) test. The average pollen germination percentages were found as the lowest 6.83 % in cv. Kon-jski Zub and the highest 42.51 % in cv Glavas. Among the germination media in general 0.2 agar + 10 % sucrose+5 ppm H3BO3 gave better results to obtain higher pollen germina-tion for all cultivars. In germinated pollens of these five cultivars, significant differences were observed on pollen tube length. Average pollen tube lengths in germinated pollens of five cultivars were measured as minimum 302 μm in cv. Konjski Zub and as maximum 344 μm in cv. Ciparski. The results showed that there were obvious differences in pollen germinability among cultiva...

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 × 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.

Pollen germination and pollen tube growth were studied in the cross Erucastrum abyssinicum × Brassica oleracea. 25 per cent ovules showed pollen tube entry into the micropyle 5 days after pollination. Embryos did not grow beyond the globular or heart-shaped stage. Thus post-fertilization barriers prevent hybridization in this cross. Sequential culture of ovaries and ovules 10 days after pollination helped rescue globular embryos. Three hybrids were obtained. All the hybrids flowered in vitro. Pollen fertility was 6.5 per cent. Leaf esterase and peroxidase isozymes confirmed the hybrid nature. Meiotic metaphase in the pollen mother cells showed 25 chromosomes. The presence of up to 9 bivalents and a quadrivalent indicates high homology between the two genera. The intergeneric hybrids have been multiplied using nodal segment culture. They are a source for obtaining cms cole crops and widening the gene pool of B. oleracea.

Growth of pollen tubes and seed set were compared after hand pollination in situ and in vitro in two self-incompatible species, Trifolium pratense and Trifolium repens. Adhesion of pollen grains to the stigma was greater in vitro for both species. After cross-pollination, in vitro culture gave a significant increase in the cumulative growth of pollen tubes in pistils of T. pratense compared to in situ conditions. After selfing in T. repens, pollen tube growth was significantly increased by in vitro culture of florets. Seed set after crossing in situ and in vitro was similar for both species. Seed set after selfing in vitro was not increased in T. pratense. Several genotypes of T. repens were classified as very good, good and poor sellers based on their capacity for seed set following selfing in situ. In vitro pollination increased self seed formation by 1.7-, 18.0-and 31.0-fold for each class, respectively. Ovules located nearest to the style were fertilized more often after selfing than after crossing.

Pollen tube germination, growth, and guidance (progamic phase) culminating in sperm discharge is a multi-stage process including complex interactions between the male gametophyte as well as sporophytic tissues and the female gametophyte (embryo sac), respectively. Inter-and intra-specific crossing barriers in maize and Tripsacum have been studied and a precise description of progamic pollen tube development in maize is reported here. It was found that pollen germination and initial tube growth are rather unspecific, but an early, first crossing barrier was detected before arrival at the transmitting tract. Pollination of maize silks with Tripsacum pollen and incompatible pollination of Ga1s/Ga1s-maize silks with ga1-maize pollen revealed another two incompatibility barriers, namely transmitting tract mistargeting and insufficient growth support. Attraction and growth support by the transmitting tract seem to play key roles for progamic pollen tube growth. After leaving transmitting tracts, pollen tubes have to navigate across the ovule in the ovular cavity. Pollination of an embryo sac-less maize RNAi-line allowed the role of the female gametophyte for pollen tube guidance to be determined in maize. It was found that female gametophyte controlled guidance is restricted to a small region around the micropyle, approximately 50-100 mm in diameter. This area is comparable to the area of influence of previously described ZmEA1-based short-range female gametophyte signalling. In conclusion, the progamic phase is almost completely under sporophytic control in maize.

In contrast to model species, the self-incompatibility reaction in citrus has been poorly studied. It is assumed to be gametophytically determined and genetically controlled by the S-locus, which in other species encodes for glycoproteins (S-RNases) showing ribonuclease activity. To investigate pollen-pistil interaction, the pollen tube growth of two clementine varieties, 'Comune' (self-incompatible) and 'Monreal' (a 'Comune' self-compatible mutation) was analysed by histological assays in self-and cross-pollination conditions. Cross-pollination assays demonstrated that the mutation leading to self-compatibility in 'Monreal' occurred in the stylar tissues. Similar rates of pollen germination were observed in both genotypes. However, 'Comune' pollen tubes showed altered morphology and arrested growth in the upper style while in 'Monreal' they grew straight toward the ovary. Moreover, to identify genes putatively involved in pollen-pistil interaction and self-incompatibility, research based on the complementary DNA-amplified fragment length polymorphism technique was carried out to compare the transcript profiles of unpollinated and self-pollinated styles and stigmas of the two cultivars. This analysis identified 96 unigenes such as receptor-like kinases, stressinduced genes, transcripts involved in the phenylpropanoid pathway, transcription factors and genes related to calcium and hormone signalling. Surprisingly, a high percentage of active long terminal repeat (LTR) and non-LTR retrotransposons were identified among the unigenes, indicating their activation in response to pollination and their possible role in the regulation of self-incompatibility genes. The quantitative reverse trascription-polymerase chain reaction analysis of selected gene tags showed transcriptional differences between the two genotypes during pollen germination and pollen tube elongation.

Distyly typically involves reciprocal polymorphism in stamen and style lengths and a set of associated polymorphisms of pollen and stigma characters. This flower polymorphism has been regarded as a strategy to reduce the likelihood of self-and intramorph pollination and to promote legitimate intermorph pollination. Although most distylous plants are characterized by a physiological selfand intramorph-incompatibility system, previous work on Pulmonaria affinis and other distylous Boraginaceae species have shown that self-incompatibility was not strict. In this study, we examined pollen deposition rates and the functioning of the distylous breeding system in Pulmonaria officinalis. Flowers exhibited reciprocal herkogamy and several other ancillary features of heterostyly. Controlled pollinations clearly showed weak self-incompatibility, with the LS-morph showing higher rates of seed set following self-, intramorph-and intermorph-pollinations than the SSindividuals. On the other hand, SS-pollen showed higher germination rates and pollen tube growth than that of LSpollen. Under natural conditions, both the proficiency of legitimate pollen transfer and the proportion of deposited pollen were asymmetrical. Although SS-pollen grains showed a higher proficiency for legitimate transfer than that of LS-pollen on a per pollen basis, the ratio of legitimate to illegitimate deposited pollen on stigmas of SS-flowers markedly exceeded that of LS-flowers. The latter was explained by the higher pollen production rates of LSindividuals compared to SS-individuals. High levels of illegitimate pollen deposition on LS-stigmas, on the other hand, were attributed to the limited proboscis length of the principal pollinators and the occurrence of floral hairs at the corolla entrance of LS-flowers. This study thus indicates asymmetrical pollen transport patterns and morph-specific differences in the strength of the incompatibility system; two aspects that may affect female reproductive success and morph ratio variation under pollen limited conditions.

It is well known that a tip-focused intracellular Ca 21 gradient and the meshwork of short actin filaments at the tip region are necessary for pollen tube growth. However, little is known about the connections between the two factors. Here, a novel Ca 21dependent actin-binding protein with molecular mass of 41 kD from lily (Lilium davidii) pollen (LdABP41) was isolated and purified with DNase I chromatography. Our purification procedure yielded about 0.6 mg of LdABP41 with .98% purity from 10 g of lily pollen. At least two isoforms with isoelectric points of 5.8 and 6.0 were detected on two-dimensional gels. The results of N-terminal sequencing and mass-spectrometry analysis of LdABP41 showed that both isoforms shared substantial similarity with trumpet lily (Lilium longiflorum) villin and other members of the gelsolin superfamily. Negative-stained electron microscope images showed that LdABP41 severed in vitro-polymerized lily pollen F-actin into short actin filaments in a Ca 21sensitive manner. Microinjection of the anti-LdABP41 antibody into germinated lily pollen demonstrated that the protein was required for pollen tube growth. The results of immunolocalization of the protein showed that it existed in the cytoplasm of the pollen tube, especially focused in the tip region. Our results suggest that LdABP41 belongs to the gelsolin superfamily and may play an important role in controlling actin organization in the pollen tube tip by responding to the oscillatory, tip-focused Ca 21 gradient. fax 86-10-58807721.

RESEARCH PROBLEM In crop growth and development, boron (B) is an essential trace element. Boron deficiency is common in highly leached, acidic sandy soils of cotton (Gossypium hirsutum L.) growing regions in the world. Boron is particularly important in pollen germination and pollen tube growth resulting in successful fruit setting. Therefore, B deficiency during flowering and fruiting may significantly reduce boll retention, re- sulting in a low yield. A better understanding of the effect of B deficiency during the early growth of cotton on leaf photosynthesis and physiological characters can help us to make cotton production recommendation and to improve yield.

A molecular technique for the identification of S-alleles involved in self-incompatibility has been used to analyse the S-allele reference collection of Brassica oleracea. The reference collection contains nearly 50 different lines each with a different S-alMe present in the homozygous state. The technique consists of amplifying by the polymerase chain reaction (PCR) sequences belonging to the S multigene sequence family using a single pair of conserved primers. PCR products are then analysed further by digestion with six restriction enzymes followed by gel electrophoresis of the digestion products. A simple method of estimating the band sizes of the digestion products is described. The S-locus-related sequences can be distinguished from S-locus glycoprotein and S-receptor kinase genes by the restriction patterns. Furthermore, with any one restriction enzyme, several alleles showed the same restriction pattern. Alleles could therefore be grouped together. With two exceptions, each member of the S-allele reference collection showed a unique set of restriction patterns. Investigation of the exceptions using pollen tube growth tests showed that these accessions represented duplications within the collection. This technique therefore provides a simple and useful method for identifying different S-alleles.

The generation of novel hybrid lines is critically limited by the compatibility between pollen and pistil and their ability to achieve successful seed formation. Fertilization in flowering plants requires the successful transfer of the male gametes from the pollen grain to the egg contained within an ovule. This occurs via the formation of a tubular protrusion from the pollen grain

This study, combining the use of fluorescence and electron microscopy, is devoted to the behaviour of the Populus deltoides male gametophyte following compatible crosses but prior to fertilization. Close association between the vegetative nucleus and the generative cell, initiated at pollen maturity, has been observed from the time of pollen activation at the stigma surface through early pollen tube growth and after generative cell division to form sperm cells. The elongated generative cell contains the normal set of cytoplasmic organelles with the exception of plastids. Microtubules are orientated along its longitudinal axis. In actively growing pollen tubes at the lower end of the style sperm cells are frequently observed. Pairs of interconnected sperm cells derived from generative cell division are detected alone or in close association with the vegetative nucleus, forming a male germ unit. The two sperm of a pair are apparently not dimorphic. In this bicellular system, the male germ unit association is apparently initiated at pollen maturity and maintained during pollen tube growth within the style.

The floral biology and breeding system of Alpinia blepharocalyx were studied in Yunnan province, southwest China. Our results indicate that populations of A. blepharocalyx have two floral morphs, which differ in flowering behaviour: the cataflexistylous morph in which the stigma is held erect above the dehiscent anther when anthesis begins in the morning and becomes curved under the anther at afternoon, and the anaflexistylous morph in which the receptive stigma is curved under the indehiscent anther first and moves into a reflexed superior position above the anther as it begins to shed pollen in the afternoon; the flowering period of both floral morphs is 12 hours; the stylar movements in the two floral morphs are synchronous, and they have similar traits to those found in other Alpinia species previously reported. Also, on average, cataflexistylous flowers are larger than anaflexistylous flowers, especially the labellum and corolla tube length; moreover, the P/O ratio of the two floral morphs is significantly different: the cataflexistylous morph has more pollen grains and fewer ovules than the anaflexistylous morph; the evolutionary significance of this comparison is discussed. Cataflexistylous flowers were observed to produce a lower concentration of nectar than that of anaflexistylous flowers before 11:00 a.m., but they then have higher nectar concentration; cataflexistylous flowers always have more nectar than anaflexistylous flowers. Flowers of A. blepharocalyx were visited by fewer insects at a lower frequency, including honey bees (Apis cerana cerana) and two species of carpenter bees (Xylocopa spp.). No significant differences were found in the fruit set among the hand-pollination treatments regardless of selfing (geitonogamy and autogamy) or crossing (intermorph and intramorph); but all hand-pollination treatments exhibited much higher fruit set than the controls; meanwhile, no fruit set occurred in the unpollinated bagged plants. Pollen tube growth was examined using fluorescence microscopy following self, intramorph, and intermorph hand pollinations at different times of the day; the pollen tubes of both floral morphs have the same growth rate and the capability to reach the ovary. Both the results of pollen tube growth and fruit sets under different pollination conditions show that A. blepharocalyx is self-compatible and dependent upon insects for pollination. The distinctive flexistyly mechanism of A. blepharocalyx is likely a floral mechanism that promotes effective intermorph pollen transfer.

The wild tuber-bearing Solanum species 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 selfincompatible relatives of the common potato, Solanum tuberosum ssp. 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 was 15-30% 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.

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since about 10% of the genes are expressed in the male gametophyte and about 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 kb to 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

The callose synthase (CalS) activity of membrane preparations from cultured Nicotiana alata Link & Otto pollen tubes is increased several-fold by treatment with trypsin in the presence of digitonin, possibly due to activation of an inactive (zymogen) form of the enzyme. Active and inactive forms of CalS are also present in stylar-grown tubes. Callose deposition was first detected immediately after germination of pollen grains in liquid medium, at the rim of the germination aperture. During tube growth the 3-linked glucan backbone of callose was deposited at an increasing rate, reaching a maximum of 65 mg h−1 in tubes grown from 1 g pollen. Callose synthase activity was first detected immediately after germination, and then also increased substantially during tube growth. Trypsin caused activation of CalS throughout a 30-h time course of tube growth, but the degree of activation was higher for younger pollen tubes. Over a 10-fold range of callose deposition rates, the assayed CalS activity was sufficient to account for the rate of callose deposition without trypsin activation, implying that the form of CalS active in isolated membranes is responsible for callose deposition in intact pollen tubes. Sucrose-density-gradient centrifugation separated a lighter, intracellular membrane fraction containing only inactive CalS from a heavier, plasma-membrane fraction containing both active and inactive CalS, with younger pollen tubes containing relatively more of the inactive intracellular enzyme. The increasing rate of callose deposition during pollen-tube growth may thus be caused by the transport of inactive forms of CalS from intracellular membranes to the plasma membrane, followed by the regulated activation of these inactive forms in this final location.

Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2 , which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.

Due to its widespread industrial use, chromium is considered a dangerous environmental pollutant. It is known to inhibit plant growth and development. The present study provides the first evidence of the toxicity of this metal on the male haploid generation of a higher plant. Both Cr(III) and Cr(VI) species, supplied as CrCl 3 and CrO 3 , respectively, exerted a strong dose-dependent inhibitory effect on kiwifruit pollen tube emergence and growth. Cr(III) resulted more effective than Cr(VI) in the 16-75 lM interval; moreover, complete inhibition of germination was attained at much lower doses than Cr(VI). Also tube morphology was affected. While the plasma membrane was still undamaged in the large majority of the treated pollen grains, dramatic ultrastructural alterations were induced by chromium including chromatin condensation, swelling of mitochondria, cytoplasmic vacuolization, and perturbed arrangement of endoplasmic reticulum cisternae. Thus, it seems that the impact of the two chromium species on kiwifruit pollen may result in severe compromission of both essential structures and functions of the male gametophyte.

The basis of low seed set was investigated in a diploid hybrid population with germplasm from the cultivated speciesSolanum tuberosum spp.tuberosum andS. tuberosum ssp.phureja and the wild speciesS. chacoense. Controlled crosses were performed following an incomplete diallel mating design which included the hybrid population and the parental species. Pollen grain germination and pollen tube growth were observed with fluorescence microscopy in 174 intrapopulation and interspecific combinations of genotypes. Fifty percent of the combinations of genotypes within the hybrid population and 46% of those between this population and the parental species were incompatible; overall, 71% of the interspecific combinations of genotypes were compatible when plants of the hybrid population were used as females, vs. 5% when used as males. Although the site of reaction could vary in a given intrapopulation or interspecific cross with the combinations of genotypes, pollen tube inhibition occurred in the first third of the style in 42% of them. Since the gene pool of the hybrid population had been widened with germplasm from other geographic areas and the genotypes ofS. tuberosum ssp.tuberosum, S. chacoense andS. tuberosum ssp.phureja used in this study were not the ones involved in the original crosses, it is concluded that the S-locus is not controlling the incompatibility reaction but rather that a cross-incompatibility system, possibly governed by more than one locus, is acting. Se investigó la base de la producción baja de semilla en una población híbrida diploide con germoplasma de las especies cultivadasSolanum tuberosum ssp.tuberosum yS. tuberosum ssp.phureja y la especie silvestreS. chacoense. Los cruzamientos controlados se realizaron siguiendo un diseño dialélico incomplete, el cual incluyo la población híbrida y las especies parentales. Se observa la germinacién del grano de polen y el crecimiento del tubo de polen con un microscopio de fluorescencia en 174 combinaciones intrapoblacionales interespecificas de genotipos. El 50% de las combinaciones de genotipos en la población híbrida y el 46% de aquéllas entre esta población y las especies parentales fueron incompatibles; en total, el 71% de las combinaciones interespecíficas de genotipos fueron compatibles cuando las plantas de la población híbrida se usaron como hembras vs. el 5% cuando se usaron como machos. Aunque el lugar de reacción podría variar en en cruzamiento intrapoblaciónale interespecifico dada con las combinaciones de genotipos, la inhibición del tubo de polen se dio en el primer tercio del estilo en el 42% de ellas. Desde que el acervo de genes/de la población híbrida se amplió con germoplasma de otras areas geográficas y los genotipos deS. tuberosum ssp.tuberosum, S. chacoense y S. tuberosum ssp.phureja empleados en este estudio no fueron los involucrados en los cruzamientos originales, se concluye que el locus S no controla la reacción de incompatibilidad sino que esta un sistema de incompatibilidad cruzada, posiblemente controlado por mas de un locus.

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 ) 9 '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 selfcompatibility 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.