Petunia Integrifolia Research Articles

Bioactive Potentials of Petunia Integrifolia (Hook.) Schinz & Thell. Against Culex Quinquefasciatus Larvae, Artemia Salina Nauplii and Poecilia Reticulata Adults

Petroleum ether (pet. ether), chloroform (CHCl3) and methanol (CH3OH) extracts of Petunia integrifolia (Hook.) Schinz & Thell. Whole plant was subjected to assess cytotoxicity against the brine shrimp Artemia salina L. nauplii, larvicidal activity against the mosquito Culex quinquefasciatus Say larvae and piscicidal activity against the Poecilia reticulata Peters adults. The P. integrifolia extract in pet. ether showed the highest cytotoxic activity against A. salina nauplii by giving the minimum LC50 value of 49.25 parts per million (ppm) at 48 h, while in case of CHCl3 and CH3OH extracts, the values were 63.37 and 342.55 ppm, respectively. Against C. quinquefasciatus larvae the three extracts gave LC50 values of 4.92, 5.51 and171.26 ppm, respectively all after 48 h of exposure. Against P. reticulata the LC50 values were 21.70 ppm for pet. ether extract at 48 h, and 31.91 ppm for CHCl3 extract after 42 h of exposure but CH3OH extract offered no mortality. According to the intensity of activity, therefore, the extracts could be arranged in the descending order of: pet. ether > CHCl3 > CH3OH extracts; and the sensitivity of the test organisms to the extract of P. integrifolia could be arranged in the following descending order: C. quinquefasciatus larvae (in Pet. ether; LC50 = 4.92 ppm) > P. reticulata (in pet ether; LC50 = 21.70 ppm) >A. salina nauplii (in pet. ether; LC50 = 49.25 ppm) J. Bio-Sci. 31(1): 99-108, 2023

Bumblebee electric charge stimulates floral volatile emissions in Petunia integrifolia but not in Antirrhinum majus

The timing of volatile organic compound (VOC) emission by flowering plants often coincides with pollinator foraging activity. Volatile emission is often considered to be paced by environmental variables, such as light intensity, and/or by circadian rhythmicity. The question arises as to what extent pollinators themselves provide information about their presence, in keeping with their long co-evolution with flowering plants. Bumblebees are electrically charged and provide electrical stimulation when visiting plants, as measured via the depolarisation of electric potential in the stem of flowers. Here we test the hypothesis that the electric charge of foraging bumblebees increases the floral volatile emissions of bee pollinated plants. We investigate the change in VOC emissions of two bee-pollinated plants (Petunia integrifolia and Antirrhinum majus) exposed to the electric charge typical of foraging bumblebees. P. integrifolia slightly increases its emissions of a behaviorally and physiologically active compound in response to visits by foraging bumblebees, presenting on average 121 pC of electric charge. We show that for P. integrifolia, strong electrical stimulation (600–700 pC) promotes increased volatile emissions, but this is not found when using weaker electrical charges more representative of flying pollinators (100 pC). Floral volatile emissions of A. majus were not affected by either strong (600–700 pC) or weak electric charges (100 pC). This study opens a new area of research whereby the electrical charge of flying insects may provide information to plants on the presence and phenology of their pollinators. As a form of electroreception, this sensory process would bear adaptive value, enabling plants to better ensure that their attractive chemical messages are released when a potential recipient is present.

Phytonutrients and Antioxidant Activity of Extracts from Five Edible Flowers Used in Culinary

The aim of the current study was to evaluate the natural pigments (chlorophylls, carotenoids and anthocyanins), phenolic compounds and antioxidant activity of water and 95% ethanol extracts from five edible flowers - snapdragon (Antirrhinum majus), carnation (Dianthus plumarius), garden geranium (Pelargonium hortorum), petunia (Petunia integrifolia) and dahlia (Dahlia decorative). It was found that water extracts gave higher extractive yields. The highest carotenoids and total anthocyanins content were detected in snapdragon and dahlia flowers. The total phenolic content dominated in water extracts of petunia, garden geranium, carnation and snapdragon - above 30 mg GAE/g of dry extract. Sinapic acid was detected in all edible flowers. Gallic, caffeic, chlorogenic, p-coumaric, rosmarinic, sinapic and ferulic acid were detected only in carnation. Antioxidant activity was the highest in ethanol extracts of petunia, garden geranium, carnation by FRAP method. The current study demonstrated the future application of edible flower extracts as a source of antioxidants for culinary purposes.

Identification, occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

AbstractA petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.

HD-AGPs as Speciation Genes: Positive Selection on a Proline-Rich Domain in Non-Hybridizing Species of Petunia, Solanum, and Nicotiana.

Transmitting tissue-specific proteins (TTS proteins) are abundant in the extracellular matrix of Nicotiana pistils, and vital for optimal pollen tube growth and seed set. We have identified orthologs from several species in the Solanaceae, including Petunia axillaris axillaris and Petunia integrifolia. We refer to TTS proteins and their orthologs as histidine domain-arabinogalactan proteins (HD-AGPs). HD-AGPs have distinctive domains, including a small histidine-rich region and a C-terminal PAC domain. Pairwise comparisons between HD-AGPs of 15 species belonging to Petunia, Nicotiana, and Solanum show that the his-domain and PAC domain are under purifying selection. In contrast, a proline-rich domain (HV2) is conserved among cross-hybridizing species, but variant in species-pairs that are reproductively isolated by post-pollination pre-fertilization reproductive barriers. In particular, variation in a tetrapeptide motif (XKPP) is systematically correlated with the presence of an interspecific reproductive barrier. Ka/Ks ratios are not informative at the infrageneric level, but the ratios reveal a clear signature of positive selection on two hypervariable domains (HV1 and HV2) when HD-AGPs from five solanaceous genera are compared. We propose that sequence divergence in the hypervariable domains of HD-AGPs reinforces sympatric speciation in incipient species that may have first diverged as a consequence of pollinator preferences or other ecological factors.

Genome-wide identification of quantitative trait loci for important plant and flower traits in petunia using a high-density linkage map and an interspecific recombinant inbred population derived from Petunia integrifolia and P. axillaris

Petunia is a very important flower in the global floriculture industry and has played a critical role as a model in plant genetic studies. Owing to limited genetic variability in commercial germplasm, development of novel petunia phenotypes and new varieties has become increasingly difficult. To enrich petunia germplasm and facilitate genetic improvement, it is important to explore genetic variation in progenitor species that may contain highly valuable genes/alleles. In this study, an interspecific recombinant inbred population (168 recombinant inbreds) derived from Petunia integrifolia × P. axillaris were phenotyped for days to anthesis (DTA), flower count (Flower_C), flower diameter (Flower_D), flower length (Flower_L), plant height (Plant_H), plant spread (Plant_S), and plant size (Plant_Z) in 2014 and 2015. Transgressive segregation was observed for all traits in both years. The broad-sense heritability on a 2-year basis varied from 0.38 (Flower_C) to 0.82 (Flower_L). Ten QTL were consistently identified in both years and by two mapping strategies [multiple QTL mapping (MQM) in MapQTL and inclusive composite interval mapping (ICIM) in IciMapping]. Major QTL explained up to 30.2, 35.5, and 47.1% of the total phenotypic variation for Plant_S, Flower_L, and Flower_D, respectively. These findings should be of significant values for introgression of desirable genes from wild petunias into commercial varieties and future genetic improvement of this important flower.

Multiple markers, niche modelling, and bioregions analyses to evaluate the genetic diversity of a plant species complex

BackgroundThe classification of closely related plants is not straightforward. These morphologically similar taxa frequently maintain their inter-hybridization potential and share ancestral polymorphisms as a consequence of their recent divergence. Under the biological species concept, they may thus not be considered separate species. The Petunia integrifolia complex is especially interesting because, in addition to the features mentioned above, its taxa share a pollinator, and their geographical ranges show multiple overlaps. Here, we combined plastid genome sequences, nuclear microsatellites, AFLP markers, ecological niche modelling, and bioregions analysis to investigate the genetic variability between the different taxa of the P. integrifolia complex in a comprehensive sample covering the entire geographical range of the complex.ResultsResults from molecular markers did not fully align with the current taxonomic classification. Niche modelling and bioregions analyses revealed that taxa were associated with different ecological constraints, indicating that the habitat plays an important role in preserving species boundaries. For three taxa, our analyses showed a mostly conserved, non-overlapping geographical distribution over time. However, for two taxa, niche modelling found an overlapping distribution over time; these taxa were also associated with the same bioregions.ConclusionscpDNA markers were better able to discriminate between Petunia taxa than SSRs and AFLPs. Overall, our results suggest that the P. integrifolia complex represents a continuum of individuals from distant and historically isolated populations, which share some morphological traits, but are established in four different evolutionary lineages.

Were sea level changes during the Pleistocene in the South Atlantic Coastal Plain a driver of speciation in Petunia (Solanaceae)?

BackgroundQuaternary climatic changes led to variations in sea level and these variations played a significant role in the generation of marine terrace deposits in the South Atlantic Coastal Plain. The main consequence of the increase in sea level was local extinction or population displacement, such that coastal species would be found around the new coastline. Our main goal was to investigate the effects of sea level changes on the geographical structure and variability of genetic lineages from a Petunia species endemic to the South Atlantic Coastal Plain. We employed a phylogeographic approach based on plastid sequences obtained from individuals collected from the complete geographic distribution of Petunia integrifolia ssp. depauperata and its sister group. We used population genetics tests to evaluate the degree of genetic variation and structure among and within populations, and we used haplotype network analysis and Bayesian phylogenetic methods to estimate divergence times and population growth.ResultsWe observed three major genetic lineages whose geographical distribution may be related to different transgression/regression events that occurred in this region during the Pleistocene. The divergence time between the monophyletic group P. integrifolia ssp. depauperata and its sister group (P. integrifolia ssp. integrifolia) was compatible with geological estimates of the availability of the coastal plain. Similarly, the origin of each genetic lineage is congruent with geological estimates of habitat availability.ConclusionsDiversification of P. integrifolia ssp. depauperata possibly occurred as a consequence of the marine transgression/regression cycles during the Pleistocene. In periods of high sea level, plants were most likely restricted to a refuge area corresponding to fossil dunes and granitic hills, from which they colonized the coast once the sea level came down. The modern pattern of lineage geographical distribution and population variation was established by a range expansion with serial founder effects conditioned on soil availability.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0363-8) contains supplementary material, which is available to authorized users.

Phylogeography of thePetunia integrifoliacomplex in southern Brazil

In this study, we evaluated the genetic diversity of the Petunia integrifolia species group using a phylogeographical approach, and attempted to understand better its diversification and taxonomy. Plants from five morphological groups were collected, covering a large part of the geographical distribution of most of the species. Two major clades were found in the phylogenetic tree, and an additional lineage, corresponding to P. inflata, was found in the haplotype network obtained for plastid markers. All three lineages are clearly delimited geographically, but, with the exception of P. inflata, the morphological groups were not genetically distinct. Our results suggest that a population expansion after a size reduction resulted in the establishment of two distinct and allopatric groups c. 0.5 Mya, one group occurring in a geologically ancient area, and the other occurring in areas that were under the influence of a series of marine transgressions/regressions at the end of the Pleistocene. These two clades are evolutionarily significant units with significantly different allele frequencies in their nuclear genome and reciprocal monophyly in maternal, uniparentally inherited markers. All our results suggest that the morphology-based taxonomy in this group does not reflect its evolutionary history, and revision of its species limits should incorporate the distribution of the genetic diversity. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174, 199–213.

ISOLATION BARRIERS BETWEEN PETUNIA AXILLARIS AND PETUNIA INTEGRIFOLIA (SOLANACEAE)

The isolation barriers restricting gene flow between populations or species are of crucial interest for understanding how biological species arise and how they are maintained. Few studies have examined the entire range of possible isolation barriers from geographic isolation to next generation hybrid viability. Here, we present a detailed analysis of isolation barriers between two flowering plant species of the genus Petunia (Solanaceae). Petunia integrifolia and P. axillaris feature divergent pollination syndromes but can produce fertile hybrids when crossed in the laboratory. Both Petunia species are primarily isolated in space but appear not to hybridize in sympatry. Our experiments demonstrate that pollinator isolation is very high but not strong enough to explain the absence of hybrids in nature. However, pollinator isolation in conjunction with male gametic isolation (i.e., pollen-pistil interaction) can explain the lack of natural hybridization, while postzygotic isolation barriers are low or nonexistent. Our study supports the notion that reproductive isolation in flowering plants is mainly caused by pre- rather than postzygotic isolation mechanisms.

LeAbsi1 gene expression regulation by various abiotic and oxidative stresses

A full-length LeAbsi1 cDNA was isolated from the tomato (Lycopersicon esculentum L.) plant. Phylogenetic analysis was performed, based on the deduced amino acid sequence of the LeAbsi1 cDNA and revealed high amino acid identity (AAID) to unknown proteins in Solanum melongena (87% AAID) and Petunia integrifolia (80% AAID). LeAbsi1 shares high sequence identity with a zinc finger (C2H2-type) family protein from Arabidopsis (53% AAID) and the CaAbsi1 protein in Capsicum annuum (49% AAID). Expression of the LeAbsi1 gene under abiotic and oxidative stresses was investigated in this study, including exposure to the conditions of 200 mM NaCl, 200 mM mannitol, cold temperature (4°C), 100 µM abscisic acid (ABA), 10 mM hydrogen peroxide (H2O2), and 50 µMM methyl vilogen (MV). LeAbsi1 was significantly induced after a 3 h exposure to NaCl, mannitol and low temperature treatment. Its transcripts also accumulated strongly after plant hormone ABA treatment for 1 h. Moreover, LeAbsi1 was significantly induced by MV treatments after 6 h. These observations suggest that the LeAbsi1 gene plays a role in both abiotic and oxidative stress responses.Key words: Abiotic stress, LeAbsi1, tomato, oxidative stress

Single Gene–Mediated Shift in Pollinator Attraction inPetunia

Animal-mediated pollination is essential in plant reproductive biology and is often associated with pollination syndromes, sets of floral traits, such as color, scent, shape, or nectar content. Selection by pollinators is often considered a key factor in floral evolution and plant speciation. Our aim is the identification and characterization of the genetic changes that caused the evolution of divergent pollination syndromes in closely related plant species. We focus on ANTHOCYANIN2 (AN2), a well-defined myb-type transcription factor that is a major determinant of flower color variation between Petunia integrifolia and Petunia axillaris. Analysis of sequence variation in AN2 in wild P. axillaris accessions showed that loss-of-function alleles arose at least five times independently. DNA sequence analysis was complemented by functional assays for pollinator preference using genetic introgressions and transgenics. These results show that AN2 is a major determinant of pollinator attraction. Therefore, changes in a single gene cause a major shift in pollination biology and support the notion that the adaptation of a flowering plant to a new pollinator type may involve a limited number of genes of large effect. Gene identification and analysis of molecular evolution in combination with behavioral and ecological studies can ultimately unravel the evolutionary genetics of pollination syndromes.

Molecular Heterogeneity of the Chalcone Synthase Intron in Petunia

A method was developed to characterize the genetic heterogeneity of the chalcone synthase gene intron within the Petunia integrifolia (Hook.) Schinz & Thell. species complex. The DNA from wild species collected from known locations was used to amplify the chalcone synthase gene intron through the polymerase chain reaction (PCR). The resulting PCR product was then characterized by Rsa 1 restriction, revealing a degree of heterogeneity that could be used to characterize the species genetically. Of the four different species that were characterized, two could be placed in the same genetic grouping. This study shows that the variation in the intron of the Chs A gene may be species-specific.

Petuniasterones from Petunia parodii and P. integrifolia; unusual ergostane-type steroids

From leaves of Petunia parodii were isolated four epoxy group-bearing petuniasterones. These were shown to be (22 R,24 R)-7α-acetoxy-4β,5β-epoxy-17β,22,24,25-tetrahydroxyergost-1-en-3-one 22,24,25-[(methylthio)carbonyl]orthoacetate (petuniasterone I); (22 R,24 R)-7α,12α-diacetoxy-4β,5β-epoxy-22,24,25-trihydroxyergost-1-en-3-one 22,24,25-orthoacetate (petuniasterone J); (22 R,24 R)-7α-acetoxy-16β,17β-epoxy-22,24,25-trihydroxyergosta-1,4-dien-3-one 22,24,25-orthoacetate (petuniasterone K); and (22 R,24 R)-7α-acetoxy-16β,17β-epoxy-22,24,25-trihydroxy-ergosta-1,4-dien-3-one 22,24,25-[(methylthio)carbonyl]orthoacetate (petuniasterone L). From leaves of Petunia integrifolia were isolated (22 R,24 R)-7α,12ξ-diacetoxy-11β,22,24,25-tetrahydroxyergosta-1,4-diene-3-one 22,24,25-orthoacetate (12ξ-acetoxy-11-β-hydroxypetuniasterone D 7-acetate); (22 R,24 R)-7α,12ξ-diacetoxy-11β,22,24,25-tetrahydroxyergosta-1,4-dien-3-one 22,24,25-orthopropionate (12ξ-acetoxy-11β-hydroxypetuniasterone M 7-acetate); (22 R,24 R)-7α,22,24,25-tetrahydroxyergosta-1,4-dien-3-one 22,24,25-orthopropionate (petuniasterone M); and (22 R, 24 R)-12α-acetoxy-7α,22,24,25-tetrahydroxyergosta-1,4-diene-3-one 22,24,25-orthopropionate (12α-acetoxypetuniasterone M). From leaves of Petunia hybrida were isolated 30-hydroxypetuniasterone A 7-acetate and petuniasterone C 7-acetate.

On the origin of anthocyanin methyltransferase isozymes of Petunia hybrida and their role in regulation of anthocyanin methylation

Four genes controlling anthocyanin methylation in flowers of Petunia hybrida have been described. Three of them, Mt2, Mf1 and Mf2, caused a dosage effect on anthocyanin methyltransferase activity and degree of methylation of anthocyanins. Antiserum raised against partially purified Mf2-enzyme precipitated three of the four anthocyanin methyltransferases. In two subspecies of one of the ancestral species of P. hybrida: Petunia integrifolia, different anthocyanin methyltransferases were found as determined by immunoprecipitation. The methyltransferase isozymes showed no differences in subcellular or tissue location, and had no physiologically important difference in time course of activity during bud development. The methylation-system in Petunia is discussed with regard to anthocyanin methylation in other plant species.

Microspore-derived haploid, diploid and triploid plants in Petunia violacea lindl

Haploid, diploid and triploid plants in Petunia violacea have been produced by culturing anthers of early binucleate pollen on kinetin supplemented basal medium. Cytological studies reveal that non-haploid embryoids developed due to nuclear fusion at early stages of pollen embryogenesis. The significance of plants of various ploidy levels in this commonly cultivated species of Petunia is discussed.

Laboratory Feeding Preferences of the Banded Woollybear, Isia isabella12

Larvae of Isia isabella (J. E. Smith) were offered cuttings of 95 plant species representing 57 families. The plants included many that contain well-known secondary plant substances. Feeding was assayed by fecal pellet count and direct observation in a 24-hour period. Consumption of the various plants varied widely, but many of the biochemically distinctive species were eaten readily. Only 1 plant, Equisetum arvense, failed to elicit any feeding response. Petunia violacea was consumed freely but is toxic to young larvae after 24 hours' exposure; older larvae tolerate the plant.

The inheritance of self-sterility in Petunia Violacea

1. Self-fertilisation of self-sterile types ofPetunia violacea was accomplished by the method of bud-pollination. 2. A series of four multiple allelomorphs for self-sterility was established as a result of the examination of (a) two selfed families and (b) F1 and F2 of crosses between homozygous types. 3. Two families proved unsuitable for genetic analysis probably owing to the segregation of minor factors for self-fertility. 4. The homozygous type S2S2 was lethal; S1S1 was dwarfed with curled leaves; S3S3 and S4S4 were normal in appearance but weak. 5. The phenomenon of hybrid vigour in relation to the selective advantage of heterozygotes against homozygotes is discussed and it is concluded that the hypothesis of superior vigour and selection value of the heterozygote phase of growth factors provides an adequate explanation of heterosis which is not inconsistent with the hypothesis of linked growth factors put forward byJones.

<I>Petunia violacea</I>ノ受精力ニ關スル生理學的研究XI

Temperature is an important factor for self fertilization. Extreme coldness is, of course, unfavourable for fertilization. But in general, cool weather is likely more favourable than hot weather for self fertilization in some plants.In order to study the effect of temperature upon self fertilization, the following experiments with Petunia violacea were made by the author.1. Intra-self pollination (strictly self pollination) was carried on with some self incompatible plants in 1931. As the flower season advanced from the beginning of August toward the end of September, the percentage of seed setting was observed to have gradually increased. But it is not evident that whether this increase in percentage is due to the effect of cool weather or of the so-called “end-season fertility.”2. So, the author divided these materials into two groups, one of which was kept in a hot glass house (27°C-40°C) and the other in a cool glass house kept between 18°C-32°C. After that, they were intra-self pollinated. The results showed exactly that cool weather was more favourable for seed bearing than hot weather.3. However, it is by no means certain that whether these seeds were produced through self fertilization or through parthenogenesis. In order to see which of these is really responsible, about 30 flowers in each group were left unpollinated, with the result that there was no phenomenon of parthenogenesis observed. Thus, it becomes clear that the increase in seed bearing ability in cool weather must be due to the increase in the fertility.4. Does cool weather act on the character of the pistil or on the activity of the pollen? This is a question. So, the author smeared the pistils with the pollen from another plant of the same vegetative line kept under medium condition. In this experiment a similar pollen was applied on to the pistils in each group. Nevertheless, the fertilizing percentage under cool condition was higher than those obtained in hot weather.5. Thus, it becomes clear that cool weather affects the character of pistils making it favourable for self fertilization. Again, the high seed bearing percentage must be due to the changed character of pistils and not to the increase in the fertility on the part of eggs. For, the author observed that the cross fertilization was easier in hot weather than under cool condition.6. The author took notes of the life-duration of each flower, which was intra-self pollinated immediately after the opening of its anthers. The life-duration was about 10-20% longer during cool weather, say, in October, than during hot weather, say, in August. This may suggest that the longer duration gives to the tubes of the selfed pollen an opportunity to reach the ovules. But the tubes of selfed pollen could penetrated into only onefifth part of the length of the style during flower life in summer, as was reported in the author's foregoing papers. Therefore, such a slight increase in life-duration can not be taken as the cause of self fertilization in cool weather.7. The author gathered the flower buds which were to bloom about three days hence, and placed them in two glass bottles containing some amount of water. Each bottle was placed either in a thermostat regulated at 30°C, or in that kept at 22°C. When the flowers opened, their pistils were smeared with the pollen of the same line. After the application of pollen, all bottles were placed in the cooler thermostat. Two days after this pollination, the pistils of these flowers were gathered and fixed, and the length of the pollen tubes penetrated was measured. The results showed that the tube-growth was inhibited more strikingly in the pistiles of the flowers which had been placed, during their bud stages, in a hotter thermostat.

A method of obtaining self-fertilized seeds in the self-incompatible plants

In the course of the writer's experiments with self-incompatible lines of Petunia violacea, it was noticed that the germination of pollen-grains and the growth of pollen-tubes were inhibited by the stigma-secretion, or the tissue-juice of styles, or both. Although it is very interesting to know the exact time when these substances are produced, yet it can not be definitely stated as yet. So far as the writer's experiments go, it may be conjectured that they may be produced shortly before or after the opening of flowers. So, the pollinations between the pistils of the flower-buds of self-incompatible individuals of Petunia violacea and the pollens of open flowers of the same plant was made as well as between the pistils and pollens of open flowers of the same individual. The result of such experiments were that pistils of the flower-buds were easily fertilized, but those of open flowers were utter failure in their fertilization. The seeds thus ontained germinated fairly well. The experiments and their results above noticed show the posibility of obtaining pure-line seeds of self-incompatible plants, at least in petunia violacea, by means of the pollination method above stated, i. e., by carrying on the pollination between the pistils of flower-buds and the pollens of open flowers.