Flowering biology and pollen production of four species of the genus Rosa L

Wild growing rose species are of great importance as a source of pollen for insects. Oil extracted from the petals of various Rosa species is used in perfumery, cosmetic industry, and therapeutics. In our study, we compared the flowering duration and flower lifespan, the number of stamens and pistils, the mass and size of pollen grains as well as the anatomical features of the petals of four Rosa species: R. canina, R. ×damascena, R. gallica, and R. rugosa. Moreover, we examined the pollen loads collected by bumblebees foraging on rose flowers in order to determine the attractiveness of pollen of this genus to insects. We showed the flower lifespan to vary (3.5–8 days) in the roses studied and revealed high variation in the number of stamens (82–260) and pistils (17–65) as well as in the mass of pollen produced. The flowers of R. rugosa produced the highest amount of pollen (26.7 mg per flower), while the flowers of R. canina the least (3.3 mg per flower), which is associated with differences in the number of stamens developed in the flowers between these species. The largest pollen grains were found in R. ×damascena and R. gallica. We demonstrated that R. ×damascena produces the thickest petals and that scent-emitting papillae found on the adaxial surface of the petals differ in size and shape in the rose species investigated.


Introduction
The genus Rosa includes about 200 species growing only in the northern hemisphere.According to some authors, 16 species of this genus are found in the wild in Poland [1], while some other ones mention 32 species [2].Due to their androecium of numerous stamens, wild rose species provide food resources for insects as a rich source of pollen [3].Rosa canina L. belongs to the most common species found in Poland, whereas Rosa gallica L. is a very rarely encountered species that is fully protected [2,4].
In the world, rose oil is currently obtained for industrial purposes from several rose species.Rosa gallica L., R. ×damascena Mill., and R. rugosa Thunb., inter alia, are mentioned among them [5].The two latter species belong to introduced plants in Poland, while R. rugosa has the status of invasive plant in Europe [6][7][8].Rose oil is used in perfumery, cosmetic industry, and therapeutics.It exhibits antiseptic and antiinflammatory activity, and accelerates healing of wounds [5,9,10].Rose oil is also used in aromatherapy, since it has a sedative and mentally stabilizing effect [11].The scent of the flowers of individual rose species may significantly vary.
Dobson et al. [12] showed the scent of the R. rugosa flower to be determined by qualitatively different oils emitted by the sepals, petals, stamens, and pollen.Bergougnoux et al. [13] found that in Rosa ×hybrida both surfaces of the petals emit odorous substances.Similar results were obtained for R. rugosa by Sulborska et al. [14].The above-mentioned authors demonstrated the presence of essential oils both on the adaxial surface of the petals, covered with papillae, and on the abaxial surface on which flat epidermal cells were found.
The aim of our study was to evaluate the apicultural value of 4 Rosa species, including three species grown for oil.The floral morphology, including the number of stamens, and the mass of pollen produced by the stamens was compared.The petal structure was analyzed, with special attention to the micromorphology of papillae emitting odorous substances which are found on the adaxial surface of the petals, to show whether an intense emission of scent is associated with some special structural features of the epidermis.Microscopic analysis of pollen loads collected by bumblebees visiting the flowers of R. rugosa and R. ×damascena was performed in order to determine the share of Rosa pollen in them.

Flowering biology
The phenology of flowering was investigated using the method by Łukasiewicz [15].The time when 10% of flowers on a shrub were open was considered to be the beginning of flowering for each species, full flowering was when about 25% of flowers were open, while the end of flowering when 75% of flowers were found to be senescent (the end of the effect of mass flowering).The date of senescence of the last flowers was considered to be the end of flowering.Flower diameter was measured in 30 flowers.The number of petals, stamens and pistils was estimated in 10 flowers.The stamens were excised from the flowers in order to determine pollen production.

Petal anatomy
Sections from newly open flowers (0.5 × 0.5 cm from the middle part of petal) were fixed in 2% glutaraldehyde with 2.5% paraformaldehyde in 0.75 M phosphate buffer with a pH of 6.8 at a temperature of 4°C for 12 h.Next, the samples were dehydrated in an ethanol series, dried at the critical point in liquid CO 2 and coated with gold using an EMITECH K 550x sputter coater.The preparations were observed under a TESCAN/VEGA LMU scanning electron microscope at an accelerating voltage of 30 kV.

Examination of pollen
Pollen viability was estimated using acetocarmine slides.For each species, viable (staining pink) and non-viable (colorless) pollen grains were counted under a microscope in 4 successive longitudinal transects of the slides, up to a total of 100 grains in each transect.
The size of pollen grains was determined based on measurement of the polar (P) and equatorial (E) axes in semipermanent slides with glycerol-gelatin mounting medium.The size was estimated using light microscopy with an eyepiece micrometer in 30 replicates for each species.

Pollen production
Pollen production was estimated using the ether method by Warakomska [16] with the alcohol modifications of Szklanowska [17].The study material consisted of buds picked at stamen maturity before pollen release (excluding the first anthers beginning to shed pollen).A hundred anthers were placed in each of previously tared glass microcylinders.The microcylinders with fresh anthers were inserted in the drier at a temperature of about 30°C.After 14 days the dried anthers were immersed twice in dimethyl ether.A 96% alcohol was then instilled into the microcylinders to thoroughly wash out the remnants of pollen from the anthers and subsequently the remains of the anthers were removed.After the alcohol evaporated, the pollen samples were placed in the laboratory oven, dried to constant mass, and then weighed.Four samples of 100 anthers were collected for each species.The mass of pollen obtained was calculated per 100 stamens and per 10 flowers.

Analysis of pollen loads
To determine the mass of pollen loads formed by bumblebees, five individuals were captured.Under field conditions, pollen packages were removed from the baskets on the tibiae of the third pair of legs and the insects were released to the environment.After several days the pairs of air-dried pollen loads were weighed and their color was determined using A dictionary of color by Maerz and Paul [18].Preparations were then prepared following the recommendations of Smaragdova [19].For this purpose, each pollen load was ground with 1 ml of distilled water with glycerol at a ratio of 1:1 until fine.Subsequently, permanent microscope slides were prepared.The pollen spectrum of each sample was examined under a Nikon Eclipse E600 light microscope at a magnification of 40×15.At least 300 grains were counted in each slide, according to the recommendations of Moar [20].These grains were identified and assigned to species, genus, type of structure, or family.During the microscopic pollen analysis, reference slides and available keys were used [21][22][23][24][25].The standard deviation (SD) was calculated in an Excel spreadsheet.

Flowering biology
The rose species studied flowered at similar times over the period from May 21 to July 3 (Tab.1).Rosa rugosa bloomed first (May 21), followed by R. canina (June 2), while the other two species entered the flowering period latest (June 8).The flowering of R. canina and R. gallica lasted slightly less than 3 weeks, in the case of R. ×damascena the flowering period was almost 4 weeks, whereas for R. rugosa it was as much as 6 weeks.Among the taxa investigated, the flowers of R. canina had the shortest lifespan (3.5 days), the flowers of R. gallica and R. rugosa were characterized by a medium lifespan (4.5-5 days), while the flowers of R. ×damascena bloomed longest (8 days; Tab. 1).
In the case of three species studied, the number of petals was fixed and it was 5; only in the hybrid Rosa ×damascena the number of petals ranged between 22 and 35, with a mean number of 28 (Tab.2, Fig. 2a).The mean number of stamens was in the range of 83-260, whereas the number of pistils in the range of 17-65 (Tab.2).The flowers of R. rugosa had the most stamens and pistils.The flowers of R. canina produced the least generative parts.Compared to the R. rugosa flowers, they produced 3 times fewer stamens and almost 4 times fewer pistils.The measurements and photographs included in this paper show that the flowers of the species studied differ not only in size, but also in shape and petal color (Fig. 1a, Fig. 2a, Fig. 3a, Fig. 4a,b).We also found differences in the type of scent emitted by the petals.The R. ×damascena flowers were characterized by the strongest scent with a rosy-fruity note.The flowers of R. gallica and R. rugosa emitted a medium strong scent, while the flowers of R. canina had the weakest scent.

Petal anatomy
The petal thickness in the investigated Rosa species ranged from 120 to 374 µm (Tab.3).The thickest petals were found in the flowers of R. ×damascena, followed by R. rugosa.R. canina and R. gallica had a similar petal thickness which was at the same time the smallest.
The height of the papillae found on the adaxial surface of the petals and the width of the cuticular striae on their surface were compared (Tab.3).The largest papillae (28 µm) were found on the petals of R. gallica (Fig. 3c,e,f).In the case of R. ×damascena (Fig. 2d,e) and R. rugosa (Fig. 4e-h), the papilla height reached similar values.The width of the cuticular striae on the papillae also significantly differed among the species (Tab.3).The widest striae formed on the papillae of R. ×damascena (Fig. 2b-d), while the striae of R. canina had an almost twice smaller width (Fig. 1b-d).
It can be concluded based on the SEM analysis that the papillae of Rosa species differ not only in size but also in shape.In R. rugosa they narrow down towards the apex most (Fig. 4c-f,h), whereas in R. canina and R. ×damascena they are the most rounded at the tip (Fig. 1b-d, Fig. 2b-d).The papillae on the petals of R. gallica (Fig. 3b-f) show an intermediate form between the two above-mentioned species.Moreover, in R. canina high variation is observed in the size of the papillae located next to one another on the petals (Fig. 1b-d).The massive structure of the striae in R. ×damascena can also be seen in the SEM images (Fig. 2b-d).A comparison of the petal cross sections for the four Rosa species reveals that the thickest petals, with the most dense arrangement of cells, are produced by R. ×damascena (Fig. 2e).

Characteristics of pollen grains
Rose pollen grains are tricolporate and round in shape, and have a striate exine (Fig. 5).In the species studied, the length of the polar axis in equatorial view was 29.4-33.5 µm, whereas that of the equatorial axis in polar view ranged 29.2-34.2µm (Tab.4).Among the species studied, pollen grains of R. ×damascena and R. gallica were largest.Generally, pollen grains of the investigated species can be considered to be medium.The P/E ratio reached a value close to 1.0, which indicates the round shape of pollen grains.The pollen viability, as estimated using the acetocarmine method, ranged from 40% (R. canina) to 95% (R. rugosa; Tab. 5).

Pollen production
The pollen mass determined for 100 stamens ranged between 4.0 mg (R. canina) and 10.3 mg (R. rugosa; Tab. 5).Significant differences were found when the pollen mass was calculated per flower, which resulted from the varying numbers of stamens in the flowers.Three times more pollen was estimated for R. rugosa flowers (267 mg per 10 flowers) compared to R. ×damascena flowers (89 mg per 10 flowers).Much higher (eightfold) differences were found between the pollen content in the flowers of R. rugosa and R. canina.

Pollen loads
Bumblebees showed interest in pollen from the flowers of Rosa species and formed pollen loads.The mass of a pair of sampled and dried pollen loads ranged from 9.7 mg to 50.5 mg (on average 30.3 mg).The smallest pollen loads were formed on the R. rugosa flowers, while the largest ones on the R. ×damascena flowers.In both cases, the pollen load was sampled from Bombus sylvarum L. Tab. 2 Characteristics of the flowers of the four Rosa species.
The color of the pollen loads sampled, as determined using A dictionary of color by Maerz and Paul [18], ranged between yellow amber and mustard green (T10/H-4 -T11/J-3).
The microscopic analysis of the samples of pollen loads revealed the presence of pollen of 16 taxa, including 10 belonging to nectariferous plants and 6 non-nectariferous plants (entomophilous or anemophilous).From 5 to 11 types of pollen were found in the individual samples.Rosa pollen grains were recorded in each pollen load sample and their participation was dominant, ranging from 88.61% to 97.94% (Fig. 6, Fig. 7).The percentage of pollen belonging to the other taxa was low and corresponded to the groups of single pollen (3-16%) or sporadic pollen (below 3%).

Discussion
In the flowers of the genus Rosa, the perianth is situated at the apex of a green hypanthium (cup-shaped) that surrounds the gynoecium.The hypanthium in Rosa has to be interpreted as "a fusion product of leaf structures" on the basis of the vascular bundle system [26].Among the Rosa species investigated, three species have 5 petals (R. canina, R. gallica, and R. rugosa), whereas R. ×damascena produces many more petals (28) in several whorls.In addition to the petals of the corolla in this species, numerous petaloids can be found, which are intermediate forms of the stamens.The number of stamens in the flowers of the Rosa species investigated greatly varied, ranging 83-260 stamens.In terms of the increasing number of stamens, the species in question can be ranked as follows: R. canina, R. ×damascena, R. gallica, R. rugosa.
Flower scents are most frequently secreted diffusely by the corolla epidermis.However, scent can also be produced by other parts of the flower.In R. rugosa and R. canina, the scent emitted by the flowers is dominated by terpenoid and benzenoid alcohols produced by the petals.Other parts of the flowers of these species produce scents with a different chemical composition.Sesquiterpenes have a major contribution to the scent of the sepals, while in the case of the anthers and pollen grains it has been found that there is a diversity of compounds, largely similar to those produced by the corolla [27,28].
Among the Rosa species investigated in our study, R. ×damascena was characterized by the most intense floral scent.It was classified as a rosy-fruity scent.Góra and Lis [5] report that among various floral scents of roses grown for essential oil, the scent of R. ×damascena, which is described as a beautiful floral scent with a warm tea/honey undertone, is most appreciated.The floral scent of R. ×damascena is considered to be a standard for rose scent.According to these authors, in the petals of R. damascena var.kazanlik (Bulgaria) the average essential oil content is 0.035%, which is one of the highest values found for the genus Rosa.In the oil obtained from R. ×damascena flowers, the following monoterpenes predominate: citronellol and geraniol [5].The above data concerning the type of scent of Rosa flowers correspond to the findings of Proctor et al. [28] who claim that the flowers that are more specially adapted to bees have a sweet or honey-like scent.The scents of R. gallica and R. rugosa were determined in our research to be medium intense and this is also reflected in the data on the essential oil content in R. gallica petals (0.017%) [5], which is much lower than in the case of R. ×damascena.The R. canina flowers were characterized by the least intense scent.It is worth noting that this species is not mentioned among the roses used for the production of rose oil [5].
In the case of the R. ×damascena flowers, the large number of petals and their considerable thickness as well as their intense scent confirm their usefulness as oil material.It has been shown that the highest content of rose oil, located mainly on the petal surface, is found in roses at the bud stage in early morning hours [5].
The present study found that each of the four Rosa species produces papillae of different shape and size.It is recognized that the role of the papillae produced in the petal epidermis is adaptation to entomophily as a foothold or as a light trap and petal reflexing [28,29].
The length of the polar axis of pollen grains in the rose species studied was in the range of 23-43 µm, which proved to be wider compared to that given by Kirk [30] for various rose varieties (24-36 µm).A wider size range was also obtained in the case of R. canina pollen grains (23.2-33.5 µm) compared to the data reported by Ricciardelli d' Albore [24] (31.4-32.6 µm), but it was smaller than that given by Beug [31] (30.5-40.5 µm).In our study, the dimensions of the polar axis in R. gallica were in the range of 25.8-31.0µm, whereas Beug [31] found a range of 28.1-38.6µm for the pollen of this species.
In this research, the shape of pollen grains was determined to be round or close to round, as indicated by the P/E ratio ranging from 0.99 to 1.00.The ratio of 0.99 obtained for R. canina was slightly higher compared to the value of 0.96 that is reported for this species by Ricciardelli d' Albore [24].
In terms of apicultural usefulness, the flowers of wild roses are treated as polleniferous flowers, since they do not produce nectar [3,32].Dobson et al. [33] emphasise that in the case of R. rugosa the main stimulus that attracts bumblebees is the pollen odor and the visual attractiveness of the anthers, whereas the petal color and scent play a secondary role.Cultivated roses are not important as a source of food for insects due to numerous petals and a low number of stamens.Some authors report that the rose flowers produce small amounts of nectar [3,30,34].
A large number of stamens in the flower frequently determines an abundant mass of pollen [28,35].We found that the R. rugosa flowers, which had the highest number of stamens (260) among the species investigated, produced the highest mass of pollen (26.7 mg per flower).Very diverse results concerning the abundance of pollen in the latter mentioned species can be found in the literature.Jabłoński [36] reports that R. rugosa produces 19.1 mg of pollen per flower, whereas Lipiński [3] mentions a pollen mass of 45.9 mg per flower.The significant divergence in the results obtained by individual authors may arise from different soil nutrient availability and from the impact of weather conditions on the plants before and during flowering [36,37].We found the lowest pollen mass in the flowers of R. canina (3.3 mg per flower).Rawski [34] also concluded that the flowers of R. canina are poor in pollen, giving them the lowest value on a three-level scale.A much lower mass of pollen was shown for R. multiflora (2.0 mg per flower) whose flowers had a similar number of stamens as the flowers of R. canina, but a much smaller corolla diameter [37].
The presence of Rosa pollen grains in samples of insect pollen loads [38,39] and in honey samples [40,41] is a confirmation of the attractiveness of pollen rewards to insects.The pollen loads formed by bumblebees which were sampled in this study had different shades of color, from yellow amber to mustard color.According to Hodges [21] and Kirk [30], bee loads of Rosa pollen are dark orange or even brownish.Ricciardelli d' Albore [24] reports that insects forage on Rose form slightly pink loads.Lipiński [3] finds that pollen loads collected from the same plant species can have a different color, depending whether the bee gathered pollen from open anthers or extracted it by gnawing through the anther walls.Their color can also be affected by the color of honey used by bees to moisten the pollen [42].

Conclusions
1. Due to their largest biomass (the number and thickness of petals), the R. ×damascena flowers with an intense scent can be abundant raw material for the production of rose oil. 2. The scent-emitting papillae that are found on the adaxial surface of the petals differ in size and shape in the species studied.
3. Among the Rosa species investigated, R. rugosa is characterized by the highest apicultural value due to the production of the highest mass of pollen, the longest flowering period as well as the greatest flower diameter.4. Rosa pollen predominated (over 90%) in the pollen loads of bumblebees visiting two of the Rosa species investigated, which may be evidence of its attractiveness to insects.

Tab. 1
Flowering phenology and flower lifespan of the four Rosa species.

Fig. 1
Fig. 1 Habit of the flower (a) and petals of Rosa canina (SEM).b-d Different sized papillae on the adaxial epidermis with a striated cuticle.Visible remnants of dried secretion (arrow; c). e Petal cross section showing a loose arrangement of mesophyll cells.

Fig. 2
Fig. 2 Habit of the flower (a) and petal sections of Rosa ×damascena (SEM).b,c Papillae from the adaxial surface of the petals with massive cuticular striae on the outer wall surface and remnants of dried secretion (arrow).d Partial cross section of outer tissues of the petal.e Petal cross section with a visible vascular bundle (vb).

Fig. 3
Fig. 3 Habit of the flower (a) and petal sections of Rosa gallica (SEM).b-d Papillae from the adaxial surface of the petals with a striated cuticle and remnants of dried secretion (arrows).e,f Petal cross sections.

Fig. 4
Fig. 4 Habit of the flowers visited by Apis mellifera and Bombus sp.(a,b) and petal sections of Rosa rugosa (SEM).c-f Papillae narrowing at the apex on the adaxial epidermis of the petals with visible striae and secretion remnants (arrow).g Petal cross section.h Enlargement of a longitudinal section of a papilla.

Fig. 7
Fig. 7 Percentage participation of pollen of the individual taxa in pollen loads from Bombus sylvarum L.

Fig. 6
Fig.6 Pollen frequency and percentages in pollen loads sampled from Bombus sylvarum L.