MICROMORPHOLOGY OF NECTARIES OF RHODODENDRON CATAWBIENSE MICHX . AT DIFFERENT FLOWER DEVELOPMENT STAGES

The analysis of the structure of fl oral nectaries of Rhododendron catawbiense Michx. was performed using stereoscopic, light and scanning electron microscopy. Nectaries were sampled at different development stages: closed bud, budburst and full bloom. The nectary gland exhibits clear ribbings corresponding to fi ve small ribs of the ovary. In the top part of the gland, unicellular and multicellular non-glandular trichomes occur in great density. The upper surface of the nectary differs from its lateral surface by a stronger degree of cuticle development. Stomata are evenly distributed on the upper surface and in the higher regions of the lateral wall. The cuticle forms clear striae on the surface of stomatal cells. Stomata at different development stages were observed, as well as the beginning of nectar secretion which takes places already in the closed bud.

The structure of nectaries of a dozen or so species from the genus Rhododendron was investigated by Philipson (1985) and he proved that a particular category of the nectary structure corresponds to respective subdivisions.The characteristics of the nectary structure in This paper study is a continuation of comparative studies of fl oral nectaries of different species from the genus Rhododendron.In the study, the micromorphology of the fl oral nectary of Rhododendron catawbiense was determined using scanning electron microscopy.Changes in the structure of the nectary epidermis were also investigated at three fl ower development stages.

MATERIALS AND METHODS
The studies conducted in the years 2006-2007 covered fl oral nectaries of Rhododendron catawbiense Michx.Shrubs of this taxon grew in the Botanical Garden of the Maria Curie-Skłodowska University in Lublin, Poland.Flowers with the nectaries were sampled for microscopic observations at the closed bud, budburst and open fl ower stage (the fi fth day of the fl ower life).Morphometric examination was performed under a stereoscopic microscope.
The micromorphology of the structure of the secretory epidermis was analysed using scanning electron microscopy (SEM).Flower fragments with the ovary were fi xed in 4% glutaraldehyde and 0.1 M phosphate buffer with a pH of 7.0 at room temperature for 4 hours.After being dehydrated in acetone, the plant material was dried at critical point in liquid CO 2 and coated with gold using the CS 100 Sputter Coater.Observations of the nectaries were made using the BS -340 Tesla electron microscope.

RESULTS
The location of the nectary surrounding with a wide layer the base of the ovary superior the fl ower of Rhododendron catawbiense can be already noticed at a small magnifi cation (Fig. 1a).Average dimensions of the ovary in this species are 4.6 mm in height and 2.5 mm in width.The nectary reaches the height of 1.3 mm, which is about 1/3 of the ovary height, and its thickness measured along the radius is 0.8 mm.In the longitudinal section of the fl ower, the nectary forms a quite large, rounded convexity protruding in its upper part from the ovary (Fig. 1c).Numerous vascular bundles run in the nectar-secreting tissue.In the cross section of the nectary, 5 poorly noticeable ribs can be observed.However, the nectary gland exhibits clearer ribbing corresponding to particular ribs of the ovary (Figs 1a, d).
The top part of the nectary is densely covered with non-glandular trichomes (Figs 1a, d, e), which are absent on the gland surface adjacent to the ovary (Fig. 1e).These trichomes are different sized and shaped.Among them, there are unicellular and multicellular trichomes, sharpended or rounded at the top (Figs 2 a-e).Some of them grow beside each other in groups (Fig. 1a).The surface of the trichomes is covered by the cuticle with clear, different ornamentation.Light microscopy observations show that the trichomes are live and marked by a quite thin cell wall and the presence of chloroplasts (Figs 2f-i).Unlike the nectary, sparsely distributed non-glandular and secretory trichomes occur on the ovary surface (Figs 1 a, b).
The secretory epidermis of the nectary is composed of polygonal cells with convex or slightly wavy outer walls (Figs 1f, g).The cuticular ornamentation shows great differentiation.In the closed bud, the horizontally situated upper surface of the nectary is characterised by the presence of clearly developed, wavy cuticular striae (Fig. 3a).In the fully-developed fl ower, the micromorphology of the walls of these cells is very similar (Fig. 3e).The lateral walls of the nectary are covered by differently-structured epidermis in which groups of cells lying next to each other have a smooth cuticle (Figs 3c,d) or clear parallel running striae (Fig. 4e).Such a differentiation of cuticular sculpture of the lateral region of the nectary also remains after fl ower opening.The epidermal cells located on the surface of the ribs are generally characterised by more strongly developed cuticular striae than the cells situated in the hollows between the ribs.Better developed striae are also observed on the walls of cells from the higher regions of the nectary.
Anomocytic stomata are distributed quite evenly on the nectary surface and they are located at the level of other epidermal cells (Figs 1f, g).An exception is the basal part of this gland where there are many fewer stomata.In the closed and bursting bud, not fully developed stomata were frequently observed, with the pore completely or partially covered by the cuticle layer (Fig. 3a).These stomata were different sized and had relatively poorly developed outer cuticular ledges (Figs 3a-c).At full bloom stage, most stomata were marked by a stronger development of outer cuticular ledges and a large aperture of the pore (Figs 3d, e).Circularly running cuticular striae were observed on the surface of many stomatal cells (Figs 3 a-e).
The microscopic observations show that nectar secretion in Rhododendron catawbiense starts in the closed, fully coloured bud.This fact is confi rmed by images from the scanning microscope, indicating stomatal nectar secretion.In the case of numerous stomata, the beginning of secretion could be observed, associated with the release of nectar forming different-sized droplets at the outer aperture of the stoma (Figs 4a-g).Similar observations were noted at the opening bud stage.But at full bloom stage, no presence of nectar droplets by the nectary stomata was found.At this stage, most stomata were characterised by an open outer and central aperture (Fig. 3e).In addition to well-developed stomata, not numerous, non-functioning stomata were observed which had reduced dimensions.

DISCUSSION
The size of the nectary gland in fl owers of Rhododendron catawbiense much exceeds the dimensions of nectaries occurring in fl owers of Rh. luteum and Rh.japonicum studied by us previously (Tab.1).
The height of the nectary of Rh. catawbiense was on the average 28% of the ovary height, whereas in Rh. luteum the nectary gland reached the height amounting to 20% of the ovary height (W e r y s z k o -C h m i el e w s k a and C h w i l , 2005), and in Rh. japonicum 11% of the ovary height (W e r y s z k o -C h m i e l e ws k a and C h w i l , 2007).Also the width of nectariferous tissue of Rh. catawbiense, measured along the radius, was much larger than in the two mentioned species, what is a result of a more convex shape of this gland.
Based on studies of over a dozen species from the genus Rhododendron, which did not relate to Rh. catawbiense, P h i l i p s o n (1985) distinguished three main types found within this genus.It seems that the nectary of Rh. catawbiense can be classifi ed as type A which is marked by the formation of large, rounded bulges at the   base of the ovary.The abovementioned author observed such nectary type in Rh. jasminifl orum.
The top part of the nectary of Rh. catawbiense was marked by the presence of densely growing non--glandular trichomes of different structure.The presence of trichomes on the whole surface of the nectary was recorded by P h i l i p s o n (1985) in Rh. konori.But in most Rhododendron species studied by Philipson (1985), the nectary surface was covered in its upper part with unicellular trichomes which occupied an area from one third to half of the nectary height.However, we did not fi nd the presence of trichomes on the nectaries of the species previously studied by us (Rh.luteum and Rh.japonicum).
Both in Rh. catawbiense and in other studied species of Rhododendron (P h i l i p s o n , 1985; W e r y s zk o -C h m i e l e w s k a and C h w i l , 2005; 2007) nectar secretion took place through the stomata.A recurring feature in the species from this genus studied by us is the asynchronous development of stomata on the nectary surface which manifested itself in the presence of a certain number of not fully developed stomata, even at the end of fl owering.At the end of the fl owering stage, most stomata had open outer and central apertures.In some other plant species, an uneven rate of stomata maturation was also observed in nectaries (D a v i s , 1997; 2006; G a f f a l et al. 1998).
We observed the beginning of nectar secretion in closed buds only in Rh. catawbiense, which was associated with the appearance of different-sized nectar droplets at the aperture of the stoma.
Another characteristic of the nectaries of Rhododendron is the occurrence of cuticle with different micromorphology, depending on the place on the nectary surface.The upper surface areas and those located on the convex parts of the nectary were marked by a well-developed, strongly folded cuticle with striated ornamentation, whereas areas of smooth cuticle were observed in the hollows between the ribs and in the lower part of the nectary in Rh. catawbiense and Rh.japonicum.It follows from that that a well-developed cuticle layer accompanied the upper parts of the nectary exposed to the effect of solar radiation, what is probably connected with its protective function.It is known from studies of other authors that the rich cuticular sculpture causes a stronger dispersion of sun rays and protects against the harmful effect of ultraviolet rays (M i r o s l a v o v , 1974; J u n i p e r and J e f f r e e , 1983).

CONCLUSIONS
1.The nectary gland of Rhododendron catawbiense forms a separate structure surrounding the base of the ovary in the form of a wide, ribbed belt.
2. At three fl ower development stages, the differentiation of cuticle ornamentation is visible in different parts of the nectary, with the strongest sculpture occurring in the top part of the nectary.
3. The beginning of nectar secretion took place already in the closed bud where different-sized nectar droplets were observed at the outer aperture of the pore.
Rhododendron catawbienseMichx.comes from the mountains of North America (S e n e t a and D ol a t o w s k i , 2007).It was brought to Europe in the 19th century.This species is classifi ed as ornamental, medicinal and melliferous plants (K o n d r a t o w i c z and C z e k a l s k i , 1984; B a u e r , 1986).It is characterised by good acclimatization and high frost resistance (C z e k a l s k i , 1980; S e n e t a and D o l a t o w s k i , 2007).It is a parent used to obtain ornamental hybrids and rhododendron varieties (C z e k a l s k i , 1980; M ur a s , 1994; S e n e t a and D o l a t o w s k i , 2007).It produces large, bell-shaped, lilac-pink fl owers.In Polish conditions it blooms in May and June.Flowers of various Rhododendron species offer pollen and nectar to pollinators (M a u r i z i o and G r a f l , 1969; B a u e r , 1986; W e r y s z k o -C h m i e l e w s k a and C h w i l , 2005).Floral nectaries in Rhododendron surround the basal part of the ovary.Non-glandular and glandular hairs growing on the ovary form their protective cover from the top (W e r y s z k o -C h m i e l e w s k a et al. 2003; W e r y s z k o -C h m i e l e w s k a and C h w i l , Rhododendron luteum was described in the papers of W e r y s z k o -C h m i e l e w s k a et al. (2003) and in W e r y s z k o -C h m i e l e w s k a and C h w i l (2005), whereas the micromorphology of the nectary of Rh. japonicum was presented in the paper of W e r y s z k o -C h m i e l e w s k a and C h w i l (2007).

Fig. 3 .
Fig. 3. Fragments of the surface of secretory epidermis of nectaries from different fl ower development stages: a, b -closed bud; c -open bud; d, e -open fl ower.Visible cuticular striae on the surface of epidermal cells and stomata with the cuticle covering their outer apertures to a different degree (asterisk) and circularly running cuticular striae on the surface of stomata.a, b, d, e -bars = 10 μm; c -bar = 20 μm.

Fig. 4
Fig. 4. a-g.Fragments of the secretory epidermis of nectaries from closed bud -beginning of nectar secretion; visible different-sized nectar droplets fl owing out of stomata.a -g -bars = 10 μm.