Morphological and Histo-Anatomical Study of Bryonia alba L . ( Cucurbitaceae )

The purpose of this study consisted in the identification of the macroscopic and microscopic characters of the vegetative and reproductive organs of Bryonia alba L., by the analysis of vegetal material, both integral and as powder. Optical microscopy was used to reveal the anatomical structure of the vegetative (root, stem, tendrils, leaves) and reproductive (ovary, male flower petals) organs. Histo-anatomical details were highlighted by coloration with an original combination of reagents for the double coloration of cellulose and lignin. Scanning electronic microscopy (SEM) and stereomicroscopy led to the elucidation of the structure of tector and secretory trichomes on the inferior epidermis of the leaf. Micrographic analysis of the powder obtained from aerial parts revealed segments of each organ (e.g. stomata, trichomes) and confirmed furthermore the results obtained by the histo-anatomical studies. Sections achieved through vegetative organs reveal typical anatomical structures: a transition to the secondary structure for the root, a typical dicotyledons’ structure with bicollateral vascular bundles for the stem, a bifacial structure of the leaf and stem-like structures for the tendrils and petioles, which prove they are metamorphosis of the stem. Anatomical structure of reproductive organs was performed hereby for the first time and revealed a typical anatomical structure for the 3-lodged ovary and a leaf-like structure of the male flower petal. Some of the results obtained confirm existing data from the scientific literature and additional information have been provided, outlining features that were not previously reported, such as SEM analysis of the leaf trichomes and histo-anatomical structure of the reproductive organs.


Introduction
Family Cucurbitaceae (order Cucurbitales) comprises 90 genera, with about 700 species, distributed mostly in the tropical regions (Tutin et al., 2010) and is one of the most important plant families over the world, especially from the economic point of view (Kocyan et al., 2007).It comprises species as Cucurbita pepo L. (pumpkin), Cucumis sativus L. (cucumber), Citrullus lanatus (Thunb.)Mansf.(watermelon), which represent the basis of a large number of industries, including food and cosmetic industry (Ali and Al-Hemaid, 2011).Despite its economical importance, Cucurbitaceae family comprises less known species, but which were found to have promising importance, especially from the therapeutic point of view (Manvi and Ganesh, 2011).
Bryonia genus comprises a total of 10 species, which are distributed from the Mediterranean to Central European region (Tutin et al., 2010), Northern Africa and Central Asia (Kocyan et al., 2007;Schaefer and Renner, 2011).
Recent classification of the Cucurbitaceae family includes Bryonia genus in the Cucurbitoideae subfamily, Bryonieae tribe (Kocyan et al., 2007).Phylogeny studies (Kocyan et al., 2007;Volz and Renner, 2008) revealed that Bryonieae tribe comprises only the Ecballium elaterium (L.) A. Rich.species and the 10 Bryonia species, of which seven are dioecious and three are monoecious.B. alba L. is a normally monoecious species which, according to Volz and Renner (2008), ranges from Central Europe to Kazakhstan, while B. cretica L. and B. dioica Jacq.are dioecious species, difficult to separate but having different area distribution, especially Southern, Central and Eastern part of Europe, where B. dioica Jacq.appears to be spreading.Geographic distribution of the major chloroplast haplotypes of the Bryonia species (Volz andRenner, 2008, 2009) suggest that B. alba L. might be the only Bryonia species found in Romania and also the most widespread Bryonia species in the world.
B. alba L. is known especially for the use in homeopathy, for its antipyretic, anti-inflammatory, antibacterial, laxative-purgative and smooth muscle relaxant properties (Demarque et al., 2007).At the same time, there is scientific evidence of other therapeutic activities of B. alba L. such as citotoxic (Baek Ni et al., 1995;Konopa et al., 1974), antiinflamatory (Park et al., 2004), hepatoprotective (Manvi and Ganesh, 2011), anti-diabetic (Singh et al., 2012), which have been shown to be promising for the treatment of various diseases.
Morphological and histo-anatomical studies have a great importance in the Bryonia genus since they are essential for the correct description of the species, of its vegetal products, for the identification of substitutions and adulterations with species without biological activity, but also for the correct localization of the active principles in different parts of the plants (Manvi and Ganesh, 2011).
Data about the histo-anatomical characterisation of B. alba L. were first published by Toma and Rugină (1998) and revealed the histo-anatomical structure of the vegetative organs (root, stem, leaves).Other histo-anatomical researches on the species were published by Manvi and Ganesh (2011), describing the anatomy of the root and localization of some active principles (starch grains).Existing data regard mostly the structure of root, which is the organ of the plant that contains the most important active principles, cucurbitacins (Konopa et al., 1974).Anatomical structures of other organs are still poorly described.
The lack of a description of important morphological and histo-anatomical characters, important for both the taxonomy and for the species identity, was noticed.Cucurbitaceae family is widely known for the presence of trichomes on the inferior surface of the leaves, which represent an important taxonomic significance on the species belonging to this family (Ali and Al-Hemaid, 2011).At the same time, since Bryonia genus was proved to be an important model system for the study of plant sex chromosome evolution (Volz and Renner, 2008), the correct histo-anatomical description of the reproductive organs appears to be of great importance.
Therefore, the aim of the present study was to analyse the main morphological and histo-anatomical characters of B. alba L. by scanning electronic micoscopy on the leaf's inferior epidermis and optical microscopic analysis of the reproductive organs.Given the fact that this information is missing from the scientific literature, the present study may improve the knowledge in the ability of understanding the taxonomic importance of the Bryonia genus in the Cucurbitaceae family, but may also be very useful for the correct and complete determination of the identity of a species that proved promising for therapeutics.

Materials and methods
The vegetal material was harvested from the spontaneous flora of Cluj County, (North-Western Romania) and identified at the Department of Pharmaceutical Botany of "Iuliu Haţieganu" University of Medicine and Pharmacy Cluj-Napoca, where voucher specimen nr.105.3.1.1-7was deposed.

Stereomicroscopy
For the identification of macroscopic characters, photos of the vegetative and reproductive organs were taken with a Motic K-500L stereomicroscope, connected to a MoticCam Pro 205A digital camera.

Optical microscopy -classical technique
Preserved fragments of vegetative organs (root, stem, petioles) were cross-sectioned using a Nahita 501 manual microtome.Sections were stained using alum carmine Alfa Aesar® (specific staining reagent for cellulose, which is stained purple-red) and malachite green Merck® (specific staining reagent for lignin, which is stained green), included in gelatin and studied using an Olympus CX31 optical microscope equipped with a digital camera.In order to better observe the trichomes on the inferior face of the leaves, superficial sections were performed (Crişan et al., 2013).

Optical microscopy -histological tehnique
Fragments of the preserved vegetal material (leaf lamina, tendrils, ovary and male flower petals) were fixed and dehydrated.Vegetal material was then passed into xylene and poured in paraffin cubes, which were sectioned using a Microtec CUT 4050 microtome.Sections were stained with toluidin blue (staining reagent which stains cellulose in purple-blue and lignin in green) and studied with the optical microscope.
All vegetal material containing compounds that might interfered with the microscopic analysis (e.g.root sections containing a large amount of starch grains, leaves sections containing chlorophyll) were previously clarified, using a chloralhydrate solution, which, at the boiling temperature, allowed the precipitation of undesirable compounds.
Optical microscopy -study of the powder Samples of vegetative organs and of aerial parts were dried and grinded using a Grindomix knife mill to obtain the powder, which was studied with the optical microscope, using chloralhydrate as inclusion medium (European Pharmacopoeia 8 th edition, 2014).

Scanning electronic microscopy
Analysis were performed on fresh vegetal material, namely on the inferior epidermis of the leaves, in order to investigate the type of trichomes present on this part of the leaf.The samples were studied under vacuum, using a JEOL JSM 5510 LV Scanning Electronic Microscope, after a vacuum metalizing technique (Nagy et al., 2014).

Tendrils
Tendrils had a stem-like structure (Fig. 3.a), being a stem metamorphosis.Almost the same parts of the anatomical structure of the stem were observed on the section.These zones and their coloration with the toluidine blue reagent were: epidermis (purpleblue) (Fig. 3  Furthermore, in order to elucidate the structure of each type of trichome, SEM analysis was performed on the inferior surface of the leaf.Images (Fig. 6.a-f) revealed stomata (Fig. 6.b) and the structure of each type of trichome (Fig. 6.c-f).Tector trichomes had a pluricellurar body and a cup-like basis, also formed by

Powder
Analysis of the powder confirmed the morfological and histoanatomical structure of the vegetative and reproductory organs.Parts of the organs could be found in the powder of the root, leaves, fruit and in the aerial part of the species.Powder of the roots showed the presence of cortex and cork cells (Fig. 10.A.) and xylem vessels (Fig. 10.B).In the leaf powder, parts of tector trichomes (Fig. 10.C-D.) and of epidermis with stomata (Fig. 10.E) were found.The powder of the fruit showed parts of fundamental parenchyma (Fig. 11.I-J) and the powder of the aerial parts (Fig. 11.F-H) contained each of the parts of the analysed structures (tector trichomes, parenchyma cells etc.).

Discussions
Microscopic analysis allowed the identification of the anatomical structures of the vegetative and reproductive organs: secondary anatomical structure of the root, primary anatomical structure of the stem, petiole and tendril anatomical structure, bifacial anatomical structure of the leaf, anatomical structure of the ovary and male flower petals.
Scientific approaches on the morpho-anatomic study of B. alba L. species (Toma and Rugină, 1998) provide fragmentary and relatively old data concerning morpho-anatomy of the vegetative organs.Few other approaches on the topic exist, but most of them concerning the anatomy and morphology of the root, which has proved to be the most important part of the species, containing the largest amount of active principles, cucurbitacins, which exibited cytotoxic activity (Baek Ni et al., 1995;Konopa et al., 1974).Manvi and Ganesh (2011) revealed the anatomical structure of the root of the species and the localization of the strach grains in the fundamental parenchyma of the root.Same results were obtained in the present study, both concerning the morpho-anatomical structure of the root and the large amount of starch grains, which was removed in order to observe better the structure.In the same direction, the studies of Burrows and Shaik (2014) on the anatomy of the taproot of Citrullus colocynthis (L.) Schrad., Citrullus lanatus (Thunb.)Mansf.and Cucumis myriocarpus E. Mey.ex Naud.proved a similar structure of the root to the one found for B. alba L. in this study, supporting the relationship between the species of the same family and representing, at the same time, one more argument in sustaining the taxonomic position of B. alba L. In the same direction of sustaining the relationship between the species of the Cucurbitaceae family, comes the study of Christodoulakis et al. (2011), which revealed similar leaf structure of Ecballium elaterium (L.) A. Rich., the closest species to B. alba L. in taxonomy.Other existing data on the morpho-anatomy vegetative organs of the species were not found.
On the other hand, new important data is brought to support taxonomic position of the species.Microscopic and SEM analysis of the leaf epidermis, in order to elucidate the structure of the tector and secretory trichomes, have not been carried out before.different types, depending on their basis (multi-seried trichomes with one secretory cell on top, having unicellular basis and pluricellular basis), while the ones in E. elaterium (L.) A. Rich.have unicellular basis and a number of four secretory cells on top.Other SEM analysis confirming the relationship of B. alba L. with species in the same family were provided by Kolb and Müller (2004), which revealed the four types of trichomes in Cucurbita pepo L. subsp.pepo var.styriaca, of which type II resembles most with one type of secretory trichomes of B. alba L., multi-seried trichomes with one secretory cell on top, having unicellular basis.Furthermore, Ali and Al-Hemaid (2011) performed the analysis on the trichomes of some species of the family and revealed similarities with the ones found for B. alba L., highlighting the taxonomical importance of the trichomes in authentication of medical cucurbits.
Micrographic analysis gave supplementary arguments to confirm the results obtained in this study, which is the first that approaches this analysis for the species.
Not least, another element of novelty included in the study is the mixture of colorants used for the coloration, which is an original recipe and has not been used before for the double coloration of cellulose and lignin on the walls of the cells.

Conclusions
This study is bringing scientific evidence to support existing data on the morpho-anatomical structure of the root, stem and leaves belonging to the B. alba species, but also provides new data on the structure of reproductory organs (male flower petals, ovary).Also, by the SEM analysis, the study is bringing, for the first time, scientific evidence which might be helpful for the correct and complete identification of the tector and secretory trichomes found on the inferior surface of the leaf.The obtained data are important criteria for the macroscopic and microscopic characterization of the species.The results represent a first step towards the localization of the main active substances of this species.At the same time, data in this study represent important arguments to sustain taxonomic position of the B. alba L. species in the Cucurbitaceae family and can help establishing the relationships between species inside the family.

Fig. 2 .
Fig. 2. Cross section of the stem colored with alum carmine and malachite green -general view 40x (a) and structural details (b, d -200x; c, e -400x) Fig. 3. Cross section of the tendril colored with toluidine bluegeneral view 40x (a) and structural details (b -200x; c -400x)