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The composition of pollenkitt and its role in the progamic phase of reproduction are poorly understood. With the aim of extending knowledge on these topics, we chose to study two monocotyledons rich in pollenkitt, with bi-celled and long-lived pollen and dry-type stigma: Crocus vernus Hill subsp. vernus and Narcissus poeticus L. Fatty acids of pollenkitt were assayed with gas chromatography. Germination tests were performed in vivo by pollinating the stigmas with a beard hair under a stereomicroscope, and in vitro in liquid culture medium using pollen, either treated or not, with carbon disulphide to remove pollenkitt. The pollen tube percentages were evaluated using fluorescence microscopy techniques. Scanning electron microscopy was used to examine pollen and to follow the early post-pollination stages. Pollenkitt forms bridges between pollen grains but not between grains and stigma papillae. It consists of a mixture of 25 fatty acids, most with long and unsaturated chains, among which are some omega acids. The same acids with different percentages persist on the peritapetal membrane. After its removal, the pollen loses adhesiveness and dries quickly, but retains full capacity for germination on the papillae and can even trigger germination in contiguous pollen grains that do not touch the papillae. The results, while confirming the key role of pollenkitt in protecting pollen and favouring pollination, suggest secondary roles in the progamic phase, and highlight the interactive ability of the pollen regardless of lipid shell. The predominance of fatty acids with 18:3 and 16:0, as already noted in Brassica napus pollenkitt, suggests their hierarchy independent of plant species.

Spring saffron (lat. Crocus vernus (L.) Hill) is a perennial from the Iridaceae family. Purple or white coloured flowers bloom in the early spring. Saffron is commonly used in cookery, but because of its healing properties, crocus is also used in medicine for improving blood circulation, treating the cardiovascular diseases, preventing tumour and healing skin diseases. This research aimed to determine the influence of solvents (water, ethanol and acetone) on antioxidative, antimicrobial activity and the content of biologically important compounds, such as phenols and flavonoids in saffron. FRAP and DPPH methods were used to examine antioxidative activity. Antibacterial activity was analysed by using diffusion technique on bacterial strains of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes , and Pseudomonas aeruginosa . Antifungal activity was examined on Candida albicans . The results showed that the ethanol extracts have the highest antioxidant activity and the aqueous extracts have the lowest one. None of the tested extracts showed antibacterial activity, but aqueous extracts and ethanolic extract of saffron, obtained by maceration, showed antifungal activity.

A method based on Sequence-Characterized Amplified Regions (SCARs) was applied to 24 different food products composed by or containing different amounts of saffron (Crocus sativus L.), in order to detect adulteration/contamination with seven common bulking agents, namely Arnica montana L., Bixa orellana L., Calendula officinalis L., Carthamus tinctorius L., Crocus vernus L. (Hill), Curcuma longa L. and Hemerocallis sp. The food products screened included ground saffron, mixed seasonings and spices, ready-made dishes and herbal formulations. The method allowed a very good performance in both DNA extraction and amplification stages regardless of the presence of lipidic, sugary or polyphenolic substances and despite the presence of dried, stored and processed material. Two samples evidenced a contamination with C. vernus, four with Carthamus, one with Curcuma and one with Hemerocallis, while a product was found to contain no C. sativus. The recourse to SCAR markers may represent a fast, reliable and low-cost screening method for the authentication of a wide range of dried food products containing saffron. Despite the great attention paid by the food industry to the authentication of saffron, some common bulking agents can be present in retail foods, as the 21% of the screened samples was not composed by pure saffron.

The aim in view was investigation ofthe regenerative potential of rhizomes, bulbs and corms of ten monocotyledonous plant species from four families: Amaryllidaceae (Haemanthus katharinae, Crinum abyssinicum, Leucojum vernum), Araceae (Spathiphyllum wallisii), Iridaceae (Crocus vernus, Iris germanica), Liliaceae (Hosta lancifolia, Muscari racemosum, Scilla laxiflora, Veltheimia viridifolia) under conditions of in vitro culture. All the investigated species were capable of buld or, alternatively, bud and root regeneration. Different morphogenetic potential was noted between the particular families. A high regenerative potential under the conditions of culture applied was characteristic for plants of the Liliaceae and Araceae families, it was lower in plants belonging to Amaryllidaceae and lowest in those of the Iridaceae family. Plants from the Liliaceae family exhibited also the highest ability of callus formation, whereas Amaryllidaceae and Iridaceae plants possessed this ability in only a low degree. The influence of growth regulators of the auxin group (NAA, IAA, 2,4-D) and of cytokinins (K) on the initiation and course of organogenesis was tested. The results of the experiments indicate that auxins in interaction with kinetin gave the highest percentage of regenerating explants and also a large number of buds on the latter. Stimulation of callus tissue was highest under the influence of 2,4-D, and weaker when NAA IAA were used.

In the climatic conditions of Lublin, Crocus vernus blooms from the third decade of March until the third decade of April. The flowers live for 3-4 days. Micromorphological investigations of the tepals and perianth tube as well as of the stamens and pistil were carried out using scanning electron microscopy. The anatomical features of the perianth segments and flower stem were examined with the use of light and fluorescence microscopy. Pollen grains were observed under a light microscope. It was found that the adaxial and abaxial epidermal cells formed papillae in the middle and lower part of the tepals. Much smaller papillae are formed in the upper region of the perianth tube. Elongated non-glandular and glandular trichomes, with spherical protrusions at the tip, are found at the base of the filaments. The filaments are distinguished by epidermal cells with a longitudinally striated cuticle and by the presence of papillae of varying number and size. The cylindrical papillae on the stigma of the pistil are covered by a wrinkled cuticle. The style is surrounded by the perianth tube along a significant length. A narrow space is formed between these organs, and it is filled by the nectar. Stylar channels are found in the style. We observed calcium oxalate crystals and numerous plastids in the parenchymal cells surrounding these channels. Inaperturate, spherical pollen grains are covered by numerous tryphine (or pollenkitt) droplets. This paper also discusses the functions of the papillae found in the epidermis of the perianth and filaments.

Maly's crocus (Crocus malyi Vis., family Iridaceae) is a stenoendemic that so far has been noticed on few localities only, in the middle and south section of Mt Velebit. This research has shown that C. malyi is a relatively common species of rugged thermophilous forests, thermophilous grasslands and other open thermophilous habitats along the littoral slopes of Mt. Velebit. On the other hand, C.vernus (L.) Hill is a species of mesophytic and colder Fagus sylvatica L. forests. Both species grow next to each other on a number of localities forming hybrids, probably hybrid swards as well. In this work we have presented the morphological and ecological characterization of the newly found hybrid C. × velebiticus Randić et Kremer, nothospecies nova. The C. × velebiticus is characterized with several macro morphological traits: plant height 145.4 ± 48.1 mm ; outer perigon leaves length and width 32.6 ± 6.3 mm and 9.3 ± 3.2 mm, respectively ; stamens and anthers length 21.2 ± 3.7 mm and 14.6 ± 3 mm, respectively ; corm length and width 13.8 ± 3.3 mm and 13.4 ± 4.5 mm, respectively. The perigon and perigon´s tube colours are quite variable, while typical specimens are mainly white with a blue-violet 1 to 4 mm wide stripe on the abaxial side of the outer perigon leaves. The stripe extends from the base of perigon leaves up to ½, ⅔ or throughout the perigon leaves length. The inner perigon leaves usually have violet spots on the first ⅓ of the abaxial side length. The perigon throat has a slightly developed yellowish spot and hairs. The stigma is pinnate, usually arranged in 2-3 levels, reddish-orange and in most specimens higher than anthers. The perigon colour of some specimens is violet as in C. vernus, but violet C. × velebiticus specimens possess bracteoles like C. malyi. The existence of bracteoles is a constant trait in all investigated C. × velebiticus specimens, whatever the colour or other variable traits. More or less expressed stunted growth of stamens and/or pistil in some specimens, also indicate the hybrid character of C. × velebiticus. The habitat of C. × velebiticus are usually rocky grasslands with Sesleria juncifolia, Carex humilis, and Bromus erectus s. l. as dominant species, and in most examples it is situated in close proximity to forest patches. The C. × velebiticus specimens chosen for holotypes and isotypes were found on the stony and grassy fire site in the Radlovac area in the middle section of Mt. Velebit, a locus classicus of this hybrid.

A method based on sequence-characterized amplified regions (SCARs) was developed from random amplified polymorphic DNA markers (RAPDs) specific for Arnica montana L., Bixa orellana L., Calendula officinalis L., Carthamus tinctorius L., Crocus vernus L. (Hill), Curcuma longa L., and Hemerocallis sp. to detect these common bulking agents in commercial saffron (Crocus sativus). The method enabled the unequivocal detection of low amounts (up to 1%) of each adulterant, allowing the preemptive rejection of suspect samples. Its enforcement limits the number of samples to be subjected to further evaluation with pharmacognostic or phytochemical analyses, especially when multiple batches have to be evaluated in a short time. The dimension of the amplicons is suitable for the analysis of degraded DNA obtained from dried, stored, processed, and finely ground commercial material. Proper SCAR markers may represent a fast, sensitive, reliable, and low-cost screening method for the authentication of dried commercial saffron material.

A chitinase has been isolated and purified from Crocus vernus corms. N-­terminal amino-acid sequence analysis of the approximately 30 kDa protein showed 33% identity to narbonin, a seed protein from Vicia narbonensis L. The C. vernus chitinase was crystallized by the hanging-drop vapour-diffusion method using PEG 8000 as the main precipitant. The crystal belonged to the monoclinic space group C2, with unit-cell parameters a = 172.3, b = 37.1, c = 126.4 A, β = 127° and two molecules per asymmetric unit. Diffraction data were collected to a resolution of 2.1 A.

In Crocus vernus, a spring bulbous species, prolonged growth at low temperatures results in the development of larger perennial organs and delayed foliar senescence. Because corm growth is known to stop before the first visual sign of leaf senescence, it is clear that factors other than leaf duration alone determine final corm size. The aim of this study was to determine whether reduced growth at higher temperatures was due to decreased carbon import to the corm or to changes in the partitioning of this carbon once it had reached the corm. Plants were grown under two temperature regimes and the amount of carbon fixed, transported, and converted into a storable form in the corm, as well as the partitioning into soluble carbohydrates, starch, and the cell wall, were monitored during the growth cycle. The reduced growth at higher temperature could not be explained by a restriction in carbon supply or by a reduced ability to convert the carbon into starch. However, under the higher temperature regime, the plant allocated more carbon to cell wall material, and the amount of glucose within the corm declined earlier in the season. Hexose to sucrose ratios might control the duration of corm growth in C. vernus by influencing the timing of the cell division, elongation, and maturation phases. It is suggested that it is this shift in carbon partitioning, not limited carbon supply or leaf duration, which is responsible for the smaller final biomass of the corm at higher temperatures.

Spring ephemerals of deciduous forests appear shortly after snow melt and senesce shortly after the overstorey canopy has closed. During this short period, they take advantage of the high light conditions, but also of the low-temperature regime that favours growth in these species. This better growth at cooler temperatures has been attributed to longer leaf life duration. The objective of this study was to test if temperature effect on growth in spring ephemerals can be explained solely by the leaf life duration, or if there is also a direct temperature effect on the underground organ growth. Crocus vernus (L.) Hill was exposed to two temperature regimes, 12/8 and 18/14°C (day/night), for sequential harvests and to four differential air to soil temperature regimes: 12/12, 12/18, 18/12 and 18/18°C (air/soil temperature). Dry mass, leaf nitrogen, starch concentration and cell size of the new corm were determined. Final dry mass and cell size were higher at the lower temperature regime while leaf lasted longer than at the higher temperature regime. Although both air and soil temperatures had an impact on the different growth parameters measured, the impact of soil temperatures was greater and it also influenced leaf life duration. At higher temperature, corm growth stopped before the first visual sign of leaf senescence, suggesting that corm growth controls leaf life duration in C. vernus rather than the opposite and that crocus growth becomes rapidly sink limited at higher temperatures.

From a recombinant DNA-library from Crocus vernus, two closely related clones of highly repetitive DNA, pCvKB7 and pCvKB8, were sequenced and their genomic distribution and organization were investigated by Southern and in situ hybridization. The lengths of the clones were 181 and 178 bp respectively; the sequences were approximately 85% identical, and thus belonged to a sequence family, named the pCvKB8-family. No homologous sequences were found in the databases (BLAST made may 2004). The presence of pCvKB8 in 52 Crocus species and six species from other genera were analyzed by Southern hybridization. The sequence family was essentially Crocus-specific. However, the distribution of hybridization signal across the genus showed poor agreement with the taxonomic structure of the Crocus genus, suggesting that the subdivision does not follow the phylogeny of this sequence family. The chromosomal distribution on three Crocus species was essentially identical: tandem organization close to all telomeres and most centromeres, with a few additional intercalary sites.

The molecular structure and carbohydrate-binding activity of the lectin from bulbs of spring crocus (Crocus vernus) has been determined unambiguously using a combination of protein analysis and cDNA cloning. Molecular cloning revealed that the lectin called C. vernus agglutinin (CVA) is encoded by a precursor consisting of two tandemly arrayed lectin domains with a reasonable sequence similarity to the monocot mannose-binding lectins. Post-translational cleavage of the precursor yields two equally sized polypeptides. Mature CVA consists of two pairs of polypeptides and hence is a heterotetrameric protein. Surface plasmon resonance studies of the interaction of the crocus lectin with high mannose-type glycans showed that the lectin interacts specifically with exposed alpha-1,3-dimannosyl motifs. Molecular modelling studies confirmed further the close relationships in overall fold and three-dimensional structure of the mannose-binding sites of the crocus lectin and other monocot mannose-binding lectins. However, docking experiments indicate that only one of the six putative mannose-binding sites of the CVA protomer is active. These results can explain the weak carbohydrate-binding activity and low specific agglutination activity of the lectin. As the cloning and characterization of the spring crocus lectin demonstrate that the monocot mannose-binding lectins occur also within the family Iridaceae a refined model of the molecular evolution of this lectin family is proposed.

The physical localization of three tandemly-organized repetitive DNA sequences was investigated byin situ hybridization to metaphase chromosomes of 11 Crocus vernus accessions. The sequences included were the 18S–25S rDNA, the 5S rDNA and a tandemly-repeated sequence cloned from C. vernus(clone pCvKB8). Ten 2n = 8 karyotypes from accessions ranging across the Alps and the Pyrenees could be interpreted as variations of a standard karyotype. Polymorphisms were found involving size of the satellite chromosomes, extra 5S rDNA sites, and extensive differences in size and number of pCvKB8 loci. The 2 n = 16 type did not correspond to any possible tetraploid derived from the 2 n = 8 types.

This study examines the effects of environmental factors on the pollination activity on Crocus vernus by Apis mellifera , and also whether bees discriminate among flowers on the basis of floral display size and colour. Flower density was much more important than temperature, humidity, and time of day and season in explaining variation in bee numbers, the total number of flowers visited, the number of flowers visited by individual bees and the total number of visits per flower (visitation rate) during 10 min observation periods. Although flower density positively influenced bee abundance and the number of flowers visited by individual bees, we found a negative relationship between flower density and visitation rates, suggesting that the pool of available pollinators was saturated at flower densities below maximum. Despite this, visitation rates were high. On average a flower received 3.42 visits during one hour; thus there seems to be little intraspecific competition for pollinators despite saturation of the pollinator pool. There was no significant difference between the size or colour of flowers that were visited, approached, or ignored by bees, and duration of visits was not related to floral display size or colour. Thus, on average A. mellifera did not appear to discriminate between flowers on the basis of floral display. Consequently, the data indicate that there is no pollinator mediated selection on floral display, driven by discriminating pollinators.

A unique mannose-binding lectin, highly specific for terminal Man(alpha1,3)Man groups, was isolated from bulbs of crocus (Crocus vernus All.). The lectin failed to bind to a mannose affinity column and was purified by simple gel permeation chromatography (Sephacryl S200). The purified lectin, obtained in crystalline form, had a molecular mass of 44 kDa on gel filtration and showed a single peptide band with a molecular mass of 11 kDa on SDS-polyacrylamide gel electrophoresis, indicating it to be a tetrameric protein composed of four identical subunits. The N-terminal amino acid sequence analysis of the crocus lectin showed essentially no homology with that of other mannose-binding bulb lectins. The crocus lectin selectively interacted with the wild type Saccharomyces cerevisiae and other mannans carrying terminal Man(alpha1,3)Man but not with those lacking this disaccharide unit. In hapten inhibition studies, methyl alpha-mannopyranoside did not inhibit the mannan-lectin interaction. Of various alpha-mannooligosaccharides, those having the Man(alpha1,3)Man sequence showed the highest inhibitory potency, confirming the strict requirement of lectin for terminal alpha1,3-linked mannosylmannose units. An affinity column of immobilized lectin enabled the complete resolution of yeast mannan and glycogen. The immobilized lectin may provide a useful tool for purification and analysis of biologically important polysaccharides and glycoproteins.

Although dysploidy and polyploidisation events are known to be important drivers in the evolutionary history of the genus Crocus , only a few examples of natural hybrid origins have so far been documented. Here, we describe the phylogenetic affinities of five taxa in the Crocus vernus species complex from the Carpathian Basin in Central Europe. Genetic variability was evaluated using chloroplast DNA sequences of the acc D- psa I intergenic spacer and amplified fragment length polymorphism (AFLP) in combination with karyological observations. Genetic diversity and differentiation of the seven investigated Crocus species were also evaluated using AFLP data. We created a phylogenetic hypothesis using both sequences and AFLP fingerprinting data under maximum parsimony (MP). AFLPs were also analysed by means of multivariate statistics using principal coordinate (PCoA) analysis and Bayesian clustering (BC) to test for hybridity. Both the AFLP and plastid phylogenetic trees separated the taxa into two groups: (1) a ‘Balkan’ clade, and; (2) an ‘Adriatic’ clade. The Balkan clade contained Crocus heuffelianus samples and one Crocus vittatus from Croatia as well as Crocus tommasinianus samples from Hungary; the Adriatic clade included Crocus vittatus and Crocus vernus samples from Hungary and Croatia as well as Crocus neapolitanus from Italy. A hard incongruence was found in the placement of the Slovakian Crocus scepusiensis which clustered inside the Balkan clade on our plastid tree, while it fell in the Adriatic clade on the AFLP tree. The same populations occupied intermediate position on the PCoA plot. The BC analysis assigned all Crocus scepusiensis specimens as F 1 hybrids, while Crocus vernus and Crocus heuffelianus were assigned as parental species. Together with our cytological investigation that determined a 2n=18 chromosome number of Crocus scepusiensis , we conclude an allopolyploid hybrid origin for this Northern Carpathian taxon as the result of a cross between members of the 2n=10 Balkan clade (e.g., Crocus heuffelianus from Transylvania) and the 2n=8 Adriatic clade ( Crocus vernus ). A similar origin is postulated for the North Balkan Crocus vittatus (2n=18), which was clustered as an F 1 hybrid in the BC analysis; thus, parallel evolution may have taken place in the northern and southern part of the Carpathian Basin beginning from the same parental species but leading to different allopolyploid derivatives.

Crocus taxonomy has until now been based primarily on morphology, taking chromosome numbers into consideration. The genetics and genome structure of the genus, the relationships and diversity within the genus are not well known. Amplified fragment length polymorphism (AFLP) is a whole genome approach to study genetic variation that is gaining in popularity for lower-level systematics. The present study employed the AFLP technique for analyzing relationships among taxa of the Crocus genus (particularly the Crocus vernus aggregate) with Carpathian Basin origin. The molecular variance obtained was based on amplification, separation and detection of EcoRI and Tru1I double-digested Crocus spp. genomic DNAs. Our results confirm the relatedness of C. tommasinianus, C. vittatus and C. heuffelianus at the Verni series of the Crocus genus. C. banaticus is taxonomically isolated as the sole member of the subgenus Crociris based on unique morphological features, but the difference is not convincing from AFLP data. The second interesting AFLP analysis result is the position of C. scepusiensis which separated it from the Crocus vernus aggregate.

SUMMARYKaryotypical analysis of Crocus vernus (L.) Hill subsp. vernus (2n = 16 + 2B) and of Crocus etruscus Pari. (2n = 8) is reported. B-chromosomes in Crocus vernus (L.) Hill subsp. vemus are found for the first time. The karyological comparative analysis points out a true micromorphic differentiation between the two species considered.

Crocus sativus Linnaeus var. vernus Linnaeus, Species Plantarum 1: 36. 1753. RCN: 284. Type not designated. Original material: Herb. Burser III: 64-68 (UPS). Current name: Crocus vernus (L.) Hill (Iridaceae)., Published as part of Jarvis, Charlie, 2007, Chapter 7: Linnaean Plant Names and their Types (part C), pp. 370-473 in Order out of Chaos. Linnaean Plant Types and their Types, London :Linnaean Society of London in association with the Natural History Museum on page 459, DOI: 10.5281/zenodo.291971

The micropylar secrete of the ovules of Crocus vernus ssp. vernus was analyzed for the Ca+2 content by atomic absorption, and its capacity to germinate and attract pollen was tested by pollinating explanted ovules, and incubating in absence of culture medium. The results display a Ca+2 concentration of 28.9 mM in the micropylar secrete. On this secrete both compatible- and incompatible pollen germinates with a mean percentage of 53.7%, and their pollen tubes enter the micropylar canal with percentages of 32.3% to 21.0%. In situ the ovules fail to attract tubes of incompatible pollen. The results are discussed in relation to the ovule receptivity and the guided growth of pollen tubes, substantiating the model of the tropic growth towards increasing calcium concentrations.

Virus like particles were detected in leaves of Crocus vernus hybrids showing pale green and yellowish narrow stripes as well as broken lines on the leaves. The virus was mechanically transmissible only to Chenopodium quinoa. It does not seem to be identical or related to other viruses hitherto described in Crocus spp.

The hydration and germination of Crocus vernus ssp. vernus pollen were examined when placing pollen on the stigma papillae under 10% to 100% relative humidity (RH). Following adhesion of the pollenkitt to the papilla, the pollen grain hydrates and swells up to emit a pollen tube, and its adjoined pollen grains may bypass contact with the papilla for hydrating and germinating. The hydration capacity increases in parallel to both the pollen dryness and the RH increase, but dry pollen does not reach full hydration. Without being placed on the stigma, dry pollen shows a moderate capacity to absorb water from the moist air, and slightly dehydrated pollen tends to lose water. Germination on the stigma shows the main increment in the interval of 20-40% RH, with a linear increase from 35.2% to 82.5%. Under 20% RH, the germination is low and inhomogeneous, while at over 40% RH it stabilizes, until it increases again at over 70% RH, with slightly different peaks for dry (86.3%) and undehydrated (94%) pollen at 90-1...

No records have previously been available for the genus Crocus in the Paklenica National Park. In 1997 and 2000 three taxa from this genus were found in the Park area and at its fringes. They are: Crocus weldenii Hoppe et Fiirnrohr. C. malyi Vis., and C. vernus (L.) Hill. C. weldenii was found in mixed populations of its yellow-flowered and white-flowered plants (f. lutescens and f. weldenii). Paklenica is the most southern locality of the C. malyi so far.

Eight clones of repetitive DNA were isolated from Crocus vernus Hill. The genomic organization of the clones was analyzed by in situ hybridization to C. vernus and Southern hybridization to a range of Crocus and other species. Seven clones were used for in situ hybridization. Sequence analysis showed that all eight clones were nonhomologous, and thus represented eight different sequence-families. In situ hybridization showed that six were dispersed in high copy numbers on all chromosomes of the C. vernus genome, whereas one was localized proximal to the secondary constriction, at the NOR (nucleolar organizer region) and was not further analyzed, as it was considered part of the 18S-25S rDNA repeat. Except for short palindromes, none of the sequences showed notable internal structures. Clone pCvKB4 showed homology to the reverse transcriptase gene of Ty1-copia-like retrotransposons; the others showed no homology to known sequences. When used as probes for Southern hybridization, four showed a ladder of 3-4 bands superimposed by irregular patterns, indicating organization in short tandem arrays. Each clone had a unique distribution among Crocus species (12-16 species analyzed with each clone) and six species of Iridaceae, Liliaceae, and Amaryllidaceae; all seven investigated sequences were Iridaceae specific and four were Crocus specific. The species distribution of these seven clones showed notable discrepancies with the taxonomic subdivision of the genus at the subgenus, section, and series levels. The results suggest that the phylogeny and taxonomic structure of the genus Crocus might need reconsideration. The analysis of repetitive DNA as a major and rapidly evolving part of the genome could contribute to the study of species relationships and evolution.Key words: phylogeny, evolution, in situ hybridization, sequence analysis, dispersed elements.

Female reproductive success in animal pollinated plants may be limited by the amount of suitable pollen that reaches stigmas, but little knowledge exists on how the extent of pollen limitation varies among plants of the same population. Here we ask; (I) if there is pollen limitation at the whole population level in a Norwegian population of an early spring blooming herb, Crocus vernus, (2) if so, if a variation in pollen limitation exists among plants, (3) over which plant traits this variation occurs, and (4) if there is phenotypic selection on plant traits caused by pollen limitation. Pollen limitation on seed number and seed:ovule ratio occurred at the whole population level. By measuring flowering time, plant size, and various aspects of floral display on both supplementary hand-pollinated plants and control plants, we found a significant relationship between reproductive success and petal size in the control plants, but not in plants that received additional pollen. Reproductive success of plants with white stigmas were more pollen limited than that in orange-stigma plants. It is likely that plants with small petals and white stigmas have lower chances of being recognised by pollinators (Apis mellifera), and that this causes the pollen limitation on reproductive success in such plants. Multivariate selection analyses showed significant linear selection on petal size in control plants, but not in extra-pollinated plants, suggesting pollinator mediated selection on petal size.

Western and eastern Carpathian populations ofCrocus heuffelianus s. lat. (incl.C. scepusiensis) have 2n = 18 but differ in karyotype. While western populations are chromosomally monomorphic, eastern populations exhibit geographical karyotypic differentiation.

Intact floral tubes of Crocus vernus grown under controlled conditions elongated 50 mm in 8 days. Mitoses of the epidermal cells did not occur during the growth of the intact tube; however, cells did elongate from 50 gm to 150 gm, a three-fold increase in cell length. When the floral buds were excised and maintained in distilled water, elongation of floral tubes was inhibited by 46%. The presence of the ovary or the addition of nutrients had no significant effect upon the elongation of the floral tubes of excised buds. When the excised floral buds were placed in l0-6 M indoleacetic acid, the final tube lengths exceeded that of the water controls by 30% and achieved 91% of the elongation of the intact tubes. Gibberellic acid and kinetin had no effect on floral tube elongation. As with the intact floral tubes, mitoses of the epidermal cells did not occur during the elongation of the excised floral tubes. CROCUS, A SPRING FLOWERING ORNAMENTAL PLANT of the family Iridaceae, is a typical cormose plant. Flowers and leaves are initiated in the fall, and growth is suspended until a cold treatment has taken place. In very early spring, the leaves and flowers undergo extensive elongation. However, nothing is known about the relative contributions of cell division and elongation during the formation of the large showy purple flowers from small floral buds. After the flowers wither, new corms are produced asexually above the old corm which eventually shrivels. If a young corm is formed near the soil surface, fleshy contractile roots will pull the corm into the soil (Rockwell and Grayson, 1977). Physiological studies of the saffron crocus (Crocus sativus) revealed that treatment of dry corms with gibberellin and kinetin reduced the time required for the transition from vegetative growth to generative development (Azizbekova, et al., 1978). Leaf and root length increased, and additional floral buds were initiated from undifferentiated primordia. A further anatomical investigation of Crocus sativus described the transformation of vegetative apices into floral apices (Milyaeva and Azizbekova, 1978). Other anatomical studies of floral tube formation within the Solanaceae have described the fusion of floral parts to form the corolla tube (Daniel and Sattler, 1978; Nishino, ' Received for publication 18 July 1981; revision accepted 11 March 1982. This study was sponsored by the Watkins Scholarship Program at the University of Montana and could not have been accomplished without the direction and encouragement of Dr. David Bilderback and the invaluable support of the Botany Department. 1978). However, floral tube elongation has not been investigated in any previous studies. MATERIALS AND METHODS-Corms of Crocus vernus Wulfen (Bailey, 1949) were purchased from the George W. Park Company, Greenwood, S.C. The giant-flowered, earlyblooming Dutch varieties, Paulus Potter and Striped Beauty, were selected for flower size and ease in induction of floral development. Six corms were planted 7 cm deep in a soil mixture of equal parts peat, loam, and vermiculite in 15-cm plastic pots. The pots were watered thoroughly and placed in a cold-room at 0-10 C in constant darkness to insure adequate root growth and floral development. The plants were examined every 2 wks and watered from below when the soil became dry. After 8-10 wks, roots appeared through the drain holes of the pots, and the embryonic leaves with surrounding bracts extended approximately 3 cm above the soil surface. The pots were then transferred to a growth chamber with a 12-hr photoperiod and a constant temperature of 12.5 C. The radiant density of the growth chamber was 10 watts/M2. To measure the growth of intact floral tubes, the enclosing bracts were removed, and the tubes were marked at 2-mm intervals with India ink (Fig. 1). The distance between the marks was observed daily for eight days. Floral buds were removed from the plant below the ovary and trimmed to the base of the ovary (Fig. 2). After the pedicel was removed, the buds were measured and inserted through individual holes in a plastic lid on a 120-ml culture jar containing the appropriate solution. Each culture jar held five floral buds

This paper is concerned with the opening and closing processes in the flowers of Crocus and Tulipa. As is well known from the work of Pfeffer, the flowers of Crocus and Tulipa are only slightly photonastic. They are, however, extremely thermonastic. As regards this thermonastic response Crocus is much more sensitive than Tulipa. In the case of Crocus, the temperature may be so low that the flowers cannot open, yet the flowers may develop fully, nevertheless. Thus Pfeffer found that in temperatures below 8° C. the flowers developed but did not open. He found that the flowers of some Crocus plants opened to an extent with fluctuations of only 0.5° C. He made this thermonastic movement more evident by means of a fine silver wire which projected beyond the flower division and which was fastened to the latter by means of lacquer. Others have found that a very fine glass capillary will serve this purpose equally well. My experiments show that the flowers of one species of Crocus are capable of responding to a somewhat smaller temperature change than 0.5° C. This is the case with the flowers of Crocus vernus which has been found to

Acid phosphatase has been localized in the wall of the pollen grain of Crocus vernus Wulf at the electron-microscope level by a method using 2-naphthyl thiol phosphate as substrate in a simultaneous coupling reaction with fast blue BBN at pH 50, the product being given electron opacity by osmication. Activity was found to be concentrated mainly in the intine, and to be associated with ribbon-like or filamentous inclusions believed to be proteinaceous on the basis of other criteria. Some activity was also detectable in the interstices of the exine. The observations confirm the general interpretation of the distribution of wall-held enzyme based upon light-microscopic cytochemistry, and provide the resolution necessary to establish unambiguously that they are associated with protein layers inserted during intine growth.

The distribution of all the different cytotypes of the C. vernus aggregate is reviewed and mapped. C. vernus subsp. albiflorus, 2n = 8, is shown to have a constantly heteromorphic karyotype. C. siculus, 2n = 8, and C. napolitanus, 2n = 8, are shown to have the same karyotypes as C. vernus subsp. vernus, 2n = 8. C. napolitanus, 2n = 16, of previous workers may by synonymous with C. vernus subsp. vernus, 2n = 16, reported here. C. scepusiensis, 2n = 18, is shown to have a karyotype distinct from C. heuffelianus, 2n = 18. C. heuffelianus exhibits infraspecific variation with chromosome numbers of 2n = IO, 12, 18, 19, 20, 22 & 23. C. vernus subsp. albiflorus (2n = 8) x C. heuffelianus (2n = 18) and C. vernus subsp. vernus (2n = 16) x C. tommasinianus (2n = 16) are described.