POLLEN PRODUCTION IN SELECTED SPECIES OF ANEMOPHILOUS PLANTS

In the study, structural features of fl owers of the following allergenic plant species were analysed: Betula verrucosa, Secale cereale, Rumex acetosella, Plantago major and Artemisia vulgaris. Pollen production was established by calculating the number of pollen grains produced by the stamen, fl ower and infl orescence. The dates of occurrence and pollen grains concentration in the air of Lublin were determined. A positive correlation was found between the length of anthers and the number of pollen grains produced. The largest number of pollen grains per anther is produced by Secale cereale (22 360), whereas the smallest one by Plantago major (5 870). The other species produced intermediate numbers of pollen grains in the anther: Betula verrucosa – 11 160, Rumex acetosella – 10 850, Artemisia vulgaris – 9 580. The birch pollen season in Lublin lasts about a month, and pollen of this taxon reaches the highest airborne concentrations among the studied taxa. Low values of pollen concentrations are characteristic for rye and plantain, whereas slightly higher values are recorded for sorrel pollen. Mugwort pollen reaches high concentrations which are noted at the beginning of August.


INTRODUCTION
Every year anemophilous plants release to the atmosphere huge amounts of pollen.The abundance of pollen is related to the number of pollen grains produced by the stamen and the fl ower.Levels of pollen production also depend on the number of fl owers and infl orescences on a plant, as well as the conditions in which it grows.For example, it is important whether a plant occurs solitary or forms clusters with other individuals (F a e g r i and I v e r s e n , 1978; M o l i n a et al. 1996).For pollen to perform its basic function, anemophilous plants must produce it in very large amounts.The volume of this production depends primarily on the length

MATERIALS AND METHODS
In the study, structural features of fl owers of the following allergenic plant species were analysed: Betula verrucosa Ehrh.(syn.Betula pendula Roth.), Secale cereale L., Rumex acetosella L., Plantago major L. and Artemisia vulgaris L. Measurements were made of particular fl ower elements, the number of stamens in the androecium as well as the average number of fl owers per infl orescence and per plant were determined in twenty herbaceous plants of each species.In the case of birch, the number of fl owers per infl orescence was established based on the examination of 30 infl orescences sampled from different branches of three trees.The number of pollen grains produced per anther was determined and the obtained results were then recalculated per fl ower and per infl orescence, and in the case of the herbaceous plants, also per plant.Mature stamens were sampled for the investigation before anther dehiscence.Pollen grains were washed out of the thecae with 70% alcohol onto a microscope slide using a stereoscopic microscope.Six replications were made for each species.
The size of pollen grains of the studied plants was determined based on the dimensions of their polar (P) and equatorial (E) axes.200 pollen grains were measured for each species.
The period of occurrence of airborne pollen -the atmospheric pollen season -and pollen concentrations were established using a Lanzoni VPPS 2000 pollen trap.
The study was conducted in Lublin in the years 2001-2007.The pollen trap was located on the roof of the building of the University of Life Sciences (51 0 14'37'' N and 22 0 32'25'' E) at a height of 18 m above ground.

RESULTS
Silver birch (Betula verrucosa Ehrh.) is one of tree species most frequently used for landscape plantings due to its decorative values and low habitat requirements.The common occurrence of this species is however adverse to many people allergic to birch pollen which contains one of the strongest allergens.Male infl orescences of silver birch are set in the summer of the previous year.In August they are already clearly visible.The measurements show that at that time they grow up to a height of 1.5 -2 cm.Immediately before fl owering, these infl orescences reach a length of about 4 cm.During fl owering, the infl orescence axis elongates up to about 7 cm (10 cm at a maximum).On the average, 150 three-fl owered cymes are clustered in male infl orescences; they grow spirally on the infl orescence axis (Fig. 1) and form secondary catkin infl orescences.Each cyme is subtended by a scaly, sparsely haired outer bract with the dimensions of 2.0 mm x 1.6 mm (Fig. 2).Particular fl owers grow in the axils of the inner bracts reaching the dimensions of 1.0 mm x 1.2 mm (Fig. 3).The male infl orescences investigated in the present study had an average of 450 fl owers.
The male birch fl ower is subtended by two perianth segments (1.8 mm x 0.8 mm) enclosing 2 stamens (Fig. 3), with the anthers composed of two separate thecae (Fig. 4).That is why it seems that there are 4 stamens in the fl ower.The anthers reach the dimensions of 1.44 mm x 0.73 mm.Birch pollen grains are triporate and they are classifi ed as small.Their dimensions were 21.34 μm x 18.17 μm (Fig. 5).The calculations show that there are, on the average, 900 stamens in one infl orescence, and each of them produces 11 160 pollen grains, thus the number of pollen grains per infl orescence is 10 044 000 (Tab. 1).
Female birch catkins grow in quite large density on short lateral shoots, below their apex.They are erect or slightly hanging.They are characterised by much smaller dimensions than male catkins, since they reach 1.52 x 0.17 cm.Similarly to male infl orescences, they contain three-fl owered cymes (Fig. 6).A single female  fl ower is devoid of the perianth; it is composed of one pistil growing in the axil of the inner bract.The bracts are fused forming 3-lobed scale protecting the winged nutlet.
Common rye (Secale cereale L.) belongs to cereal plants often grown in Poland.Pollen grains of this plant exhibit a high degree of allergenic activity.
The spikes of Secale cereale cv.Dańkowskie Złote were 9.4 long, on the average.Each of them had an average of 60 fl owers.The anthers reach large dimensions of 9.50 mm x 0.97 mm (Fig. 7) and they are borne on a fi lament which initially is relatively short, as it reaches 1.6 mm, but before pollen shedding, during lemmae and paleae opening, it rapidly increases its length up to about 9.2 mm.
Pollen grains are shed in batches through elongated slits, starting from the apical part of the theca.Pollination starts before the stamens come out completely from the lemmae and paleae.Particular stamens of the fl ower mature successively.During the gradual elongation of the successive stamens, each of them touches the adjacent stamen, provoking pollen shedding.The process of elongation and coming out of three stamens from between lemma and palea takes about 2 minutes.Within about 5 minutes, the thecae empty half of their content, and after 1.5 up to 2 hours they are empty.The apical portion of the thecae forms characteristic bowl-shaped fragments in which pollen is caught in windless weather (Fig. 8).
Spikelets in the middle part of the spike fl ower fi rst.The further opening of the anthers takes place in both directions.In sunny weather, pollen shedding from one rye spike lasted 4 days, whereas the rye canopy bloomed for 14 days.
Rye pollen grains are oval, heteropolar, monoporate (Fig. 9).In the examined material, they reached the dimensions of 48.55 μm x 34.92 μm.It was calculated that one stamen of Secale cereale produced an average of 22 360 pollen grains, whereas the spike produced 4 024 800 of them (Tab.1).
The pistil of Secale has two stigmas with a length of 3.5 mm.The feathery stigmas developed an increased catching surface for sporomorphs borne by the wind.Each fi liform element of the stigma has outgrowths facilitating pollen capture (Figs 11,11).Two membranous lodicules, haired at the apex, adhere to the ovary (1.2 x 0.9 mm) (Fig. 10).
In Poland several sorrel species are found whose pollen causes allergies.One of them is Rumex acetosella L., fi eld sorrel, which is a unisexual, dioecious plant (Fig. 12) growing up to a height of 25 -55 cm.Severalfl owered clusters grow on the stem alternately in three sections.The stalk base is subtended by a small bract and membranous, fi mbriate-laciniate ochrea (Fig. 12).
Male fl owers usually grow in clusters of 5 -6, at an average distance of 4.6 mm from each other.The un-differentiated green-red perianth forms tripartite whorls: the outer whorl with smaller segments (1.3 mm x 0.6 mm), fused at their bases, and a more impressive inner whorl (1.6 mm x 1.2 mm).The fl ower pedicle grows up to 2.7 mm.In the fl ower, six stamens grow whose yellow-coloured anthers reach the dimensions of 1.13 mm x 0.64 mm.The thecae are not joined from the top down to almost half of their length (Fig. 14).During anthesis, the fl owers are directed downwards and the perianth segments straighten up, being arranged in a horizontal plane and forming a protective "roof" over the freely hanging stamens on the fi laments about 1 mm long.After pollen release, the anthers drop off (Fig. 13).
Tricolporate pollen grains of fi eld sorrel can be classifi ed as small, since their dimensions reached 22.1 x 20.0 μm.Inside the cells of pollen grains, large reserve starch grains were visible (Fig. 15).
The investigated male plants formed few branches (4 -7) on which 70 fl owers develop, on the average, irrespective of the plant height.One fi eld sorrel plant, depending on the number of branches, produced 280 -490 fl owers and 18 233 040 -31 907 820 pollen grains (Tab.1).
In the female plants, the infl orescences usually bore 7 fl owers at an average distance of 3 mm between the nodes.The female fl owers are triangular shaped (Fig. 16), with this shape being formed by the three-edged ovary of the pistil.Similarly to the male fl owers, the double, three-segmented perianth is composed of smaller greenish-coloured outer segments and larger green-or red-coloured inner segments.The dimensions of fl owers are 1.1 mm x 0.9 mm.Three stigmas of the pistil grow on very short styles fused to the ovary edges (Fig. 17).Single stigmas resemble a small star with over a dozen arms and a diameter of 1 mm.
Greater plantain, i.e. common plantain (Plantago major L.), is one of the most commonly found ruderal plants in Poland.Its pollen causes allergies in sensitive people.
Leafl ess plantain shoots ended with spikelike infl orescences reach different heights.They are short in trodden places and grow up to 60 cm under favourable conditions.Generally, the infl orescence stem is slightly longer than the spike.
Bisexual plantain fl owers grow in the axils of the bracts similar to calyx sepals (Fig. 18).The membranous-edged sepals (1.6 x 1.1 mm) with a green band of assimilation tissue in the middle are free and they grow in two whorls in twos (Fig. 18).The fl ower corolla, with fused petals, is greenish and transparent, reaching 22 mm in length.It produces four small bent lobes.The fi laments of 4 stamens are fused to the corolla tube in interopositus way.Their anthers are claret-coloured (major variety) (Fig. 19), sometimes yellow-coloured (sulphurea variety) (Fig. 20).Before the stamens come out, the anthers tightly hold the style and the long fi la-    ment (3.5 mm) is folded in two (Fig. 20).At this stage, the anthers are longer and narrower -1.25 mm x 0.55 mm, but right before pollen shedding their dimensions change to 0.91 mm x 0.74 mm (Figs 21,22).The pistil has a long fi liform style ended with an undivided, elongated stigma (Figs 18,19).
Plantain pollen grains are polyporate, spherical (Fig. 23).Their diameter was 21.15 μm, on the average.In the case of my study, the spikes of the plants reached a length of from 2 up to 35 cm.It was determined that an average of 18 fl owers developed per 1 cm of infl orescence length.It was calculated that 5 870 pollen grains were produced in one anther, thus a spike with an average length of 15 cm can produce 6 339 600 pollen grains (Tab.1).
Plantain fl owers are protogynous.In accordance with the sequence of development of racemose infl orescences, the blooming process fi rst takes place in the lowest located fl owers.One spike fl owers 6 days, on the average.Particular infl orescences grow and bloom successively.One plant can produce a maximum of about 20 spikes.
Mugwort (Artemisia vulgaris L.) is a permanent component of vegetation accompanying man.It occurs in ruderal sites, on roadsides, in wasteland.It is found in all the housing estates of Lublin, and on the outskirts of the city this weed infests meadows and non-agricultural land, often forming dense canopies.Mugwort pollen is one of the most frequent causes of pollinosis in Poland Under favourable conditions, mugwort produces strongly branched plants, growing up to 2 m.Depending on the degree of branching and plant height, it produces a different number of capitula.By way of example, a mugwort plant 140 cm high and with 23 primary branches produced 4 987 infl orescences.It was calculated that the investigated plants produced an average of 8 fl ower heads per 1 cm of a branch with fl owers.A plant with 4 987 fl ower heads can produce 3 192 926 750 pollen grains (Tab.1).
Closed capitula are egg shaped; shortly before fl owering, their size is 2.6 mm x 1.8 mm, whereas during fl owering they reach the dimensions of 4.0 mm x 2.5 mm.The infl orescences are covered with membranousedged bracts of the involucre arranged in three 5-leaved whorls.The outer side of the bracts is strongly haired (Fig. 24), the inner side is smooth.In the present study, it was calculated that one fl ower head was composed, on the average, of 9 female ray fl owers situated on the circumference (Fig. 24, 26) and inside 13 bisexual disc fl owers (Figs 25,27) borne on the convex infl orescence receptacle.
The disc fl owers develop asynchronously in the fl ower head and their blooming also starts not simultaneously.The fl owers measured during fl owering were 2.5 -3 mm long.The corolla was initially transparent, greenish-yellow and it tightly shrouded the stamens and pistil.During fl owering, it took on the claret colour.
In their apical portion, the linear thecae, with the dimensions of 1.27 mm x 0.28 mm, have membranous, pointed outgrowths which probably perform a protective role for the pistil and the fl ower inside, since, being bent inwardly, they tightly close the entry to the corolla tube (Figs 28,29).The anthers become fused with one another right before anthesis.It points out to the fact that particular stamens can be isolated without any problem from the fl owers at the bud stage.Following the petal fall stage, the anthers remain joined.The bisexual fl owers have a pistil with a sigma 0.4 mm long, ended with two pollen presenters with glistening hairs.They function like a piston, pushing out pollen from the anthers and the stamen tube and raising it above the corolla level (Fig. 27).
The ligulate corolla of the female fl owers is transparent, with an elongated tube tightly adhering to the style (Fig. 21).The ligule reaches a length of 0.2 mm; two lobes are sometimes found in the top part of the corolla.The young perianth is greenish-yellowish-coloured; at a later stage of development, its upper part as well as the stigmas and apical fragments of the style become claret-coloured.The stigmas of the pistil reach large dimensions.Their length can be as much as 0.7 mm, whereas the whole fl ower reaches a length of 3 mm.
The studied species produced a different number of pollen grains per stamen.A clear correlation was found between anther length and number of pollen grains per stamen.The results relating to pollen production in the studied plant species are presented in Table 1.
Based on the results of pollen monitoring conducted under the seven-year-long study in Lublin, the average start and end dates of pollen seasons of the studied plant genera were established; they also included species other than those mentioned in the table due to the fact that it was impossible to distinguish their pollen.In Figure 31, the average duration of the pollen season is marked in dark colour, and in bright colour -the extreme start and end dates of the season over the seven-yearlong period of study.Birch pollen grains are present in the air of Lublin, on the average, from the middle of April until the middle of May.The birch pollen season was recorded earliest from 3 April in 2002.Birch pollen reaches very high concentrations in the air.The highest average concentration from the seven-year-long study is 2 364 pollen grains per day (Fig. 32).The rye pollen season lasts relatively short, on the average, from 23 May to 17 June.It started earliest on 13 May, also in 2002 (Fig. 31).The maximum pollen concentration of this taxon appears at the end of May or at the beginning of June.These are relatively low values (Fig. 33).no.days from January 1st.
The dates of Rumex and Plantago pollen seasons coincide to a large extent, but sorrel pollen grains are mostly recorded a week earlier than plantain pollen.The average duration of the sorrel and plantain pollen seasons is similar -118 and 119 days, respectively.The average sorrel pollen season lasted from 14 May to 8 September, whereas for plantain from 21 May to 16 September (Fig. 31).Plantago pollen reaches low airborne concentrations (Fig. 33).In July (on the average, 18 July), the mugwort season starts and it lasts until 26 September.The maximum airborne pollen concentration for this taxon in the air of Lublin occurs at the beginning of August (Fig. 33).

DISCUSSION
The number of airborne sporomorphs is of special signifi cance in aerobiology.The plant species causing allergic reactions, and the same time frequently occurring in the conditions of Lublin, were selected for our observations.Adaptations to anemophily are associated with a particular structure of fl owers.Among the studied plants, the Longistaminae type infl orescence (Secale, Rumex, Plantago) was predominant.d i , 1986; M o l i n a et al. 1996).Also We r y s z k o -C h m i e l e w s k a and B a r t y ś (2000) found that the number of pollen grains was positively correlated with anther length in entomophilous meadow plants.The size of pollen grains is also of essential importance.Molina et al. (1996) demonstrated that Acer negundo produces, in an anther which is twice longer than the anthers of common olive and with medium-sized pollen grains (27,2 x 30,5 μm), a similar number of pollen grains as Olea europaea producing small pollen grains (22 μm).But Juglans regia produces 18 times less pollen grains than common olive in the anther of small length, as it is slightly shorter than the anther of common olive, and with medium-sized sporomorphs (45 μm).
In the present study, consistent results were obtained with data given by other authors with respect to the number of pollen grains contained in the rye infl orescence and the birch fl ower.According to literature data, a single fl ower of Betula verrucosa produces 20 145 pollen grains, whereas Secale cereale -57 310.The number of pollen grains produced by the infl orescence is estimated at 5 450 000 for Betula verrucosa, and for Secale cereale -4 250 000 (P o h l , 1937 following E r dt m a n , 1954; D y a k o w s k a , 1959; M a u r i z i o and G r a f l , 1969; S z a f e r and Wo j t u s i a k o w a , 1969).A similar number of pollen grains per rye infl orescence, amounting to 4 200 000, was obtained by D e V r i e s (1971).No literature data has been found relating to pollen production by Rumex acetosella, Plantago major and Artemisia vulgaris.D e Vr i e s (1971) as well as A g n i h o t r i and S i n g h (1975) stress the variation in numbers of pollen grains produced between species of the same genus, and even between the varieties.It has been shown that different wheat varieties may produce in one anther from 856 up to 3867 pollen grains (D e Vr i e s , 1971).Signifi cant differences in the number of pollen grains produced by different species of the same genus were also found by S u b b a R e d d i and R e d d i (1986).
The period of birch pollen shedding is relatively short; there is generally only one high peak on the curve presenting the pollen season pattern.It is attributable to the fact that 70-80% of pollen is released from the anthers of birch within 2-3 days (S u s z k a , 1979).The intensity of the birch pollen season differs signifi cantly between particular years and depends, inter alia, on the number of infl orescences produced in the previous year (L a u k k a n e n et al. 2003).The occurrence of airborne rye pollen is characterised by great regularity.Maximum concentrations in Lublin in the years 1994-1997, likewise in the years 2001-2007, were found at the end of May or at the beginning of June (P i o t r o w s k a , 1999).In Lublin plantain pollen was noted from 21 May, on the average.A comparison of the pollen seasons in Lublin, Poznań and Rzeszów shows that Plantago pollen appeared earliest in Rzeszów (at the beginning of May) and latest in Poznań (at the end of May), whereas the dates of occurrence of Rumex pollen in the three cities were similar (P i o t r o w s k a , 2007).In Lublin the mugwort pollen season starts about the middle of July.When comparing the dates of occurrence of pollen of the abovementioned taxon in the air of 8 cities, it was found that it appears in the western part of Poland earlier than in the eastern part (We r y s z k o -C h m i e l e w s k a et al. 2006).In the recent years, an ever increasing number of people allergic to mugwort pollen has been recorded.R a p i e j k o and We r y s z k o -C h m i e l e w s k a (1999) note that it can be associated with an increased share of pollen of these plants in aeroplankton.

Figs
Figs 12-17.Rumex acetosella Fig. 12. Fragments of male and female plants, bract (arrow) and ochrea (arrow head) visible on the stem, x 10.Fig. 13.Male fl owers at different development stages.On the left, the fl ower after anthesis without anthers, perianth segments and fi laments are visible, x 12. Fig. 14.Schematic diagram of the stamen structure, x 16.Fig. 15.Pollen grain -LM x 1100.Fig. 16.Female fl owers with green and red inner parianth segments, x 30.Fig. 17.Pistil with star-shaped stigmas (two out of three are visible), x 25.

Figs
Figs 18-23.Plantago major Fig. 18.Flowers in the pistil stage, x 23.Fig. 19.Flowers in the stamen stage, x 20.Fig. 20.Flowers at different development stages.In the upper part, a fl ower with stamens coming out, the fi lament folded in two (arrow), x 18. Fig. 21.Schematic diagram of the stamen structure before pollination, x 16.Fig. 22.Schematic diagram of the stamen structure during pollination, x 16.Fig. 23.Pollen grain -LM x 1020.

Fig. 31 .
Fig. 31.The mean and extreme terms of the pollen seasons beginning and end of the selected taxa plants (averages from 2001-2007).
Many authors note the abundance of pollen produced by anemophilous plants (S u b b a R e d i and R e d d i , 1986; M o l i n a et al. 1996).The number of pollen grains in one anther of the studied plants was within the range of 5 870 -22 360.The largest amount of grains per anther was found in Secale cereale, whereas the least amount was in Plantago major, which is closely related to anther length.This correlation was confi rmed by many authors (D y a k o w s k a , 1959; D e V r i e s , 1971; A g n i h ot r i and S i n g h , 1975; S u b b a R e d d i and R e d -