Pollen structure and germination of Crocus thomasii Ten. (Iridaceae)

Abstract

Mature pollen of C. thomasii Ten. (Iridaceae) has been studied from a morphological and physiological point of view and compared to that of C. sativus L., C. thomasii pollen is roundish, with a 83 μm diameter and a 14% of anomalous grains. Percentage of the positive alcoholdehydrogenase (ADH +) pollen grains is about 90% whereas the in vitro germinated rate reaches 55%. Exine is homogeneously thickened (2.5 μm) with randomly distributed thinner zones and 5 μm thick intine. Cytoplasm of vegetative and generative cells is very rich of small and large smooth vesicles and vesicles coated by ribosomes. The generative cell shows a thin ondulated pectocellulosic wall. Its nucleus is intensely fluorescent after treatment with the 4,6-diamidino-2-phenylindole (DAPI) fluorochrome. Although many ultrastructural aspects of the C. thomasii pollen are common in C. sativus. L., C. thomasii pollen is smaller, more regularly structured and germinates in vivo and in vitro in higher percentage than that of C. sativus.     

Pollen dimorphism in threeTeucrium species (Lamiaceae)

In the Iberian Peninsula and Balearic Islands pollen dimorphism was found inTeucrium fruticans L.,T. pseudochamaepitys L., andT. rotundifolium Schreber. Generally, this dimorphism shows two sizes of pollen grains, the smaller more or less collapsed. The percentage of pollen viability is calculated. Differences in size between viable and nonviable pollen grains are similar, being about 40% inT. fruticans andT. pseudochamaepitys, and about 26% inT. rotundifolium. With regard to pollen viability, the percentage of male sterility is higher inT. fruticans, in which from male sterile (ms) plants, 100% nonviable pollen was obtained from every flower observed. InT. pseudochamaepitys andT. rotundifolium with rare exceptions, the percentage of nonviable pollen does not seem to be significant.     

Developmental stages of the pollen wall and tapetum in some Crocus species

The developmental stages of the pollen wall and tapetum, together with exine morphology were studied in a number of Crocus species, by light and scanning electron microscopy. Gametogenesis was characterized by: 1) development of a thick intine, 2) single mitosis, and 3) terminal amylolysis. The tapetum was of the secretory type. In C. cartwrightianus cv. albus, abnormal sporogenesis and gametogenesis produced vacuolate pollen grains with a reduced-or no intine layer, and rich with starch granules; the tapetum was either of the parietal-or amoeboid type. The exine was echinate and the pollen grains had different types of aperture: furrows, colpi or pores. The ornamentation varied from microreticulate to irregularly perforate. The exine framework was overlaid by a pellicle resistant to chloroform-carbon disulphide, on which a layer of pollenkitt was deposited. The results are discussed from both cytological and evolutionary viewpoints.     

Pollen evolution and systematics inAnnonaceae with special reference to the disulcate Australian endemic genera

All genera ofAnnonaceae endemic in Australia (Ancana, Fitzalania, Haplostichanthus) show almost exactly the same type of disulcate (disulculate) pollen with intact exine extending over the sulci. Tetrad stages inHaplostichanthus andAncana reveal a latudinal subequatiorial orientation of the two sulci at the proximal hemisphere. Sometimes they fuse into a ±zonosulcate aperture.Fissistigma pollen grains are ±globose and have a flattened pole with a central elevation and a concentric groove, covered by a somewhat reduced exine. This palynological characters give further support for separating the generaAncana andFissistigma. Germination was observed inHaplostichanthus where the pollen tube emerges at one of the two sulci and inFissistigma where the flattened part breaks up during germination. The aperture types described here are obviously transitional stages between aperturate and inaperturate pollen grains and are discussed in regard to pollen evolution.     

In vitro pollen germination and transient transformation of<Emphasis Type="Italic">Zea mays</Emphasis> and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. An erratum to this article is available at .     

Flow cytometric analysis of nuclear DNA inCrocus sativus and allies (Iridaceae)

The genome size and base composition of the triploidCrocus sativus and its two diploid most probable ancestors,C. cartwrightianus andC. thomasii, was investigated and compared inter- and intra-specifically by means of flow cytometry. There was little variation inC. sativus and little difference fromC. cartwrightianus. Crocus thomasii was significantly different from the others.Crocus cartwrightianus is the most probable diploid ancestor ofC. sativus.     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

Pollen morphology inParis and its related genera

Pollen grains ofParis, Medeola, andScoliopus have been examined with light, scanning electron, and transmission electron microscopies.P. quadrifolia L.,P. verticillata Bieb.,P. delavayi Franch. andP. polyphylla Smith have monosulcate pollen characterized by psilate, foveolate or reticulate exine sculpture. In contrast to the former species,P. japonica (Fr. et Sav.) Franch. andP. tetraphylla A. Gray have monosulcate pollen with gemmate (rarely rugulate) exine.Medeola has monosulcate pollen with reticulate exine that is distinct from that ofParis. Scoliopus has monosulcate pollen characterized by a peculiar reticulate exine pattern. The palynological evidence suggests thatParis andTrillium are closely related to each other, andMedeola andScoliopus should be separated fromParis andTrillium.     

Embryology ofCrocus thomasii (Iridaceae)

The embryology ofCrocus thomasii is described. Male meiosis is of simultaneous type, and gives rise to starchy microspores which develop into lipoid pollen grains; these are two-celled and show a spinulate acolpate, abaculate exine lacking apertures. The tapetum is glandular and its cells become bi- or sometimes multinucleate. The ovule is anatropous and bitegmic; the inner integument forms the micropyle. Megasporogenesis is heteropolar with starch accumulation in the functional chalazal megaspore. Embryo sac development conforms to thePolygonum type. The endosperm development is nuclear. The embryo develops according to the Caryophyllad type. In the ripe seed it is differentiated and enveloped by a starchy cellular endosperm. The embryological characters observed strongly favour a close relation betweenC. thomasii andC. sativus.     

Optimization of conditions for germination of cold-stored <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> pollen

One of the rare weak points of the model plant Arabidopsis is the technical problem associated with the germination of its male gametophyte and the generation of the pollen tube in vitro. Arabidopsis pollen being tricellular has a notoriously low in vitro germination compared to species with bicellular pollen. This drawback strongly affects the reproducibility of experiments based on this cellular system. Together with the fact that pollen collection from this species is tedious, these are obstacles for the standard use of Arabidopsis pollen for experiments that require high numbers of pollen tubes and for which the percentage of germination needs to be highly reproducible. The possibility of freeze-storing pollen after bulk collection is a potential way to solve these problems, but necessitates methods that ensure continued viability and reproducible capacity to germinate. Our objective was the optimization of germination conditions for Arabidopsis pollen that had been freeze-stored. We optimized the concentrations of various media components conventionally used for in vitro pollen germination. We found that in general 4 mM calcium, 1.62 mM boric acid, 1 mM potassium, 1 mM magnesium, 18% sucrose at pH 7 and a temperature of 22.5°C are required for optimal pollen germination. However, different experimental setups may deviate in their requirements from this general protocol. We suggest how to optimally use these optimized methods for different practical experiments ranging from morphological observations of pollen tubes in optical and electron microscopy to their bulk use for molecular and biochemical analyses or for experimental setups for which a specific medium stiffness is critical. F. Bou Daher and Y. Chebli contributed equally to this study.     

Production of fertile tobacco pollen from microspores in suspension culture and its storage for in situ pollination

Summary A simple procedure is described for the in vitro production of tobacco (Nicotiana tabacum L.) pollen from microspores isolated just before entering mitosis. During a 3-day culture period in a liquid medium containing pyrimidine nucleosides these microspores develop into young pollen grains to the stage of starch deposition. Pollen maturation and transition to dormancy is achieved during a further 2- to 3-day culture period in the same medium stepwise supplemented by a concentrated solution of sucrose and l-proline. Upon transfer of the pollen to a simple germination medium containing sucrose and boric acid, up to 40% of the grains were observed to produce relatively long tubes. The in vitro-matured pollen grains can be stored at-20° C either suspended in 1.17 M sucrose and 100 mM l-proline or separated from the medium on filter paper discs. The stored pollen germinated both in vitro and on the stigma, the pollen tubes grew through the style into the ovary and pollination produced up to 300 viable seeds per pod. The procedure is of interest for pollen developmental studies and various fields of pollen manipulation, such as in vitro pollen selection.     

Pollination mechanisms in sixCampanula species (Campanulaceae)

Pollination mechanisms within the genusCampanula were studied. Tests were undertaken to examine whether in vitro culture of pollen grains can serve as a useful tool for understanding the self- and cross-pollination mechanisms among species. Characteristics of pollen germination were interpreted in relation to mating system and floral biology. Four annual species (Campanula kremeri, C. dichotoma, C. afra, C. lusitanica), and two perennial species (C. rotundifolia andC. persicifolia) were investigated. In the annual species pollen germinability is controlled by (1) the age of pollen and/or (2) in what position pollen is deposited around the style. Correlations were found between pollen germinability and mating system in three of the four annual species. No correlations were found either between germinability and age of pollen or position on the style in the perennial species. Pollen germinability reached its maximum in the middle of the male phase in all species, except forC. dichotoma, which had a decreasing germinability throughout anthesis. The germinability was lowest at the time of stigma receptivity for all species except forC. persicifolia, where the stigma did not develop as long as pollen remained on the style. The pollen collecting hairs and pollen removal have been found to play an important role controlling the stigma development, thus affecting self-pollination. This was especially pronounced inC. persicifolia. Further studies will be undertaken to elucidate factors influencing pollination within the genusCampanula.     

Palynological study of Cupressus sempervirens L. var. pyramidalis and var. horizontalis

ABSTRACT

Cupressus sempervirens L. var. pyramidalis and C. sempervirens var. horizontalis were examined for their pollen morphometry and ultrastructure to verify whether, in addition to phenotype, pollen could also show characteristics useful to discriminate the two infraspecific taxa. C. sempervirens var. pyramidalis compared to C. sempervirens var. horizontalis has mainly larger, subcircular pollen with a higher percentage of apolar and germinating grains, and a wall with thicker intine; the cytoplasm has a greater number of callose-containing vesicles. C. sempervirens var. horizontalis has pollen distributed in five classes of shape. In addition it is smaller and shows a higher percentage of ruptured grains, and a lower percentage of germination in vitro. The cytoplasm contains vesicles rich in callose and also a higher amount of osmophilic bodies. Pollen grains in both varietas contain only one cell at dispersal. The reported differences in pollen morphometry, structure and biology, together with the phenotypic characteristics of the tree, support the validity of two infraspecific taxa.     

Early in vitro flowering and seed production in culture in <Emphasis Type="Italic">Dendrobium</Emphasis> Chao Praya Smile (Orchidaceae)

Plantlets of Dendrobium Chao Praya Smile maintained in vitro were induced to flower, which produced viable seeds within about 11 months. A two-layer (Gelrite-solidified layer topped with a layer of liquid medium of the same volume and composition) culture system containing benzyladenine (BA) at 11.1 μM induced the highest percent of flowering (45%) in plantlets within 6 months from germination. The percentage of inflorescence induction was increased to 72% by pre-selecting morphologically normal seedlings prior to two-layer culture. Plantlets in culture produced both complete (developmentally normal but smaller than flowers of field grown plants) and incomplete flowers. Pollen and female reproductive organs of in vitro-developed complete flowers were morphologically and anatomically similar to flowers of field grown plants. In addition, 65% of the pollen grains derived from in vitro-developed flower were tetrad suggesting that regular meiosis occurred during microsporogenesis. The percentage of germination of pollen grains derived from in vitro-developed flowers and flowers of field grown plants, incubated on modified Knops’ medium for 8 days, were 18.2 and 52.8%, respectively. Despite a lower percentage of germination of the pollen grains derived from in vitro-developed flowers, flowers induced in culture could be self-pollinated and developed seedpods with viable seeds. Nearly 90% of these seeds developed into protocorms on germination in vitro. These seedlings were grown in culture and induced to flower in vitro again using the same procedure.     

In vitro germination and transient GFP expression of American chestnut (Castanea dentata) pollen

The development of the male reproductive structures of American chestnut (Castanea dentata) is described to advance our understanding of its reproductive behavior. This information has been vital in the development of a strategy to collect pollen grains from male catkins suitable for in vitro germination and transformation experiments. Cutting male catkins into small segments and rolling them over a culture plate resulted in evenly dispersed and large amounts of pollen with minimal unwanted accessory floral parts. To optimize pollen viability, the effect of various storage conditions on in vitro germination was examined. Our results showed that initial storage at 4°C for 2 weeks significantly increased percent germination as compared to freshly collected pollen and those stored directly at −20°C or −80°C. This also means that for long-term storage of American chestnut pollen, the catkins should first be kept at 4°C for a couple of weeks and then at −80°C. The use of pollen grains with high viability is necessary for the transformation of American chestnut pollen. To optimize pollen transformation via particle bombardment, the effects of target distance, target pressure, and pollen developmental stage were examined. Statistical analysis showed that bombardment of ungerminated pollen at 1,100 psi resulted in the highest percent transient GFP expression (4.1%).     

Pollen-pistil interaction in Brassica oleracea

Quantitative studies of the adhesion of pollen grains to the stigma in Brassica oleracea revealed that self-pollen is initially less firmly bound than cross-pollen. The pollen grain tryphine, believed to be important in the adhesion process, has been shown to differ in mobility following self- and cross-pollination when observed using fluorescent probes. The hydration of the pollen grains has been investigated in vitro by measuring the changes in shape, volume and fresh weight of the imbibing grains. Whilst little change in volume could be detected there was a considerable increase in fresh weight together with a change of shape. The significance of these events, which occur prior to pollen germination, is discussed in relation to their effect upon subsequent germination and expression of self-incompatibility.Abbreviations RH relative humidity - SI self-incompatibility - ConA concanavalin A - I-ANS I-anilino-napthyl-sulfonic acid     

Pollen-ovule patterns in tribeTrifolieae (Leguminosae)

Relative pollen and ovule production in the genera of the legume tribeTrifolieae is explored particularly as to howMedicago with its explosive pollination mechanism compares with its allies.Medicago produces much larger, although much fewer pollen per ovule than the other five genera in the tribe; this is interpreted as a consequence of its highly specialized, irreversible pollination mechanism, which allows only one effective exchange of pollen with pollinators.Melilotus andTrifolium produce a comparatively large quantity of pollen; this can be related to their floral characteristics requiring abundant pollen to achieve fertilization. InMedicago andTrigonella, annuals convert a higher proportion of pollen grains and ovules into seeds than perennials.     

Embryo culture and taxane production inTaxus spp

Summary We have previously shown (Flores and Sgrignoli, 1991) that immature embryos ofTaxus brevifolia andT. X media are capable of precocious germination and can grow into seedlings in vitro. The cultural and environmental parameters for embryo germination and conversion into seedlings have been optimized and extended toT. baccata andT. cuspidata. A 14-h photoperiod improved embryo germination and growth into seedlings. A pregermination cold treatment of the seeds had a positive effect on both the onset and percentage of germination. Embryos from cold-treated seeds germinated earlier and at a higher frequency than those from control seeds. Boron was necessary for embryo germination, and levels of this micronutrient were established for optimal growth and germination ofT. brevifolia andT. X media cv. Hicksii embryos. Gupta and Durzan’s medium was superior to White’s for embryo germination and root formation. Naphthaleneacetic acid stimulated root formation in embryo-derived seedlings. We also found that immature embryos could be induced to form callus with embryogenic potential. Taxol and related taxanes were detected in embryo- derived seedlings.     

Effects of γ-ray treatment onCannabis saliva pollen viability

The viability and thein vitro germination capability of hemp pollen (cv. Carmagnola) were studied. Viability tests were based on the microscopic observation of the fluorescence of loaded fluorescein diacetate (FDA), while, for germinability tests, five different media were tested. The effects of irradiation with γ-rays on pollen viability and germination and on seed set were also studied, at three different irradiation doses (20, 60 and 100krad). The results show that in one of the media tested, about 85–90% of the pollen grains are viable and able to germinate in control samples, and that while viability measured by FDA test is not affected by increased γ-ray doses, the pollenin vitro germinability drops to about one-half of the controls at the maximum γ-ray dose employed, 100krad. Seed set of hemp plants pollinated with the irradiated pollen dropped to less than 1% of that of plants pollinated by untreated pollen for the higher dose used. The different media suitable forin vitro germination of hemp pollen, and the observed lack of correspondence between viability and germination capacity tests are discussed.     

Intergeneric hybridization between Diplotaxis siifolia,a wild species and crop brassicas

Summary Attempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F₁ hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.