Histo-Anatomical Aspects of the Ajuga Genevensis L

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Analele Ştiinţifice ale Universităţii „Al. I. Cuza” Iaşi http://www.bio.uaic.ro/publicatii/anale_vegetala/anale_veg_index.html s. II a. Biologie vegetală, 2012, 58, 1: 11-18 ISSN: 1223-6578, E-ISSN: 2247-2711

HISTO-ANATOMICAL ASPECTS OF THE AJUGA GENEVENSIS L. AND AJUGA REPTANS L. VEGETATIVE ORGANS

Georgiana GHIŢĂ1*,2, Irina BOZ1, Radu NECULA2,3, Elvira GILLE2, Maria Magdalena ZAMFIRACHE1, Ursula STĂNESCU3

Abstract: The paper aims to achieve a comparative study of the histo-anatomical structure of Ajuga genevensis L. and Ajuga reptans L., species known in our country for their therapeuric properties. In this respect, we analyzed the underground vegetative organs: the root and the rhizome, the aerial vegetative organs: the stem (the upper, lower and medium part), the leaf, to highlighten the possible similarities and differences that may appear in the structures of the two species. At root level, in both the species analyzed, we noticed that the passing from the primary to the secondary structure takes place early, due the cambium activity. In the rhizome structure one may notice that the central cylinder is much thicker than the bark, presenting either arches of conducting tissues or conducting solitary free-xylem fascicles. The stem conducting fascicles are of the collateral type, present in a great number and with different dimensions depending on the species analyzed. In both species studied, the stomatas are of the diacytic type, the tector hairs are uniserial multicells formed of two to seven cells, and the secretory hairs are rare being especially located in the superiour epidermis, with bi-cellular pedicel and bi- or tetra-cellular gland. Keywords: Ajuga genevensis L., Ajuga reptans L., anatomy, secretory hairs.

Introduction

The Ajuga L. genus comprises herbaceous plants, bi-annual or perenial, with an errect or upward stem, being found all over the world. In the flora of our country there are six Ajuga species, all herbaceous. Five are perenial plants - Ajuga salicifolia (L.) Schreber, A. laxmanii (L.) Bentham, A. pyramidalis (L.), A. reptans (L.), A. genevensis (L.) and only one is annual - A. chamaepytis (L.) Schreber. A. reptans L., bugle (synonym Ajuga reptans Host, Bugula densiflora Ten, Teucrium reptans) is a short plant with stolons that start from the radical leaf axilla, producing vegetative multiplication. A. genevensis L. is known as blue bugleweed and has an underground stem in the form of a short, oblique rhizome, with numerous roots but without stolons (Răvăruţ, 1961; Ciocârlan, 2000; Pârvu, 2006). Both species studied contain many active principles, the plants being used especially in traditional medicine in forms of infusions or tinctures, as adjuvants in the treatment of some inflamatory, rheumatic affections, in diarrhoea, leucorrhoea and also for their hepatoprotecting and healing effects. (Bhakuni et al., 1991; Calcagno et al., 1996; Debell et al., 2005; Tomas et al., 1992). The aim of the investigation was to establish the structural similarities and differences between the two species, in relation with the biosynthetic capacity of the active principles as well as the highlighting of the intra and interspecific variability. The objectives of the histo-anatomic study were to determine the structure of the different vegetative organs.

1* Faculty of Biology, “Al. I. Cuza” University, Carol I 22, 700505, Iasi. [email protected] (corresponding author) 2 NIRDBS/„Stejarul” Biological Research Centre, Alexandru cel Bun 6, 610004, Piatra Neamt, Neamt 3 Faculty of Pharmacy, „Gr. T. Popa” University of Medicine and Pharmacy, Universitatii Str. 16, 700115, Iasi Ghiţă, G. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 11-18

Materials and methods

The vegetal material is represented by the two Ajuga species (A. genevensis and A. reptans) harvested in the stage of anthesis in different slightly anthropic areals from the Botosani County (A. genevensis, the de Draslea populations and A. reptans the de Guranda population), on May, 14th 2011. The fixation and preservation of the fresh vegetal material was made in 70% ethylic alcohol. The cross-sectioning was manually performed with a microtom and a botanical razor, on a support of elder tree pith. The obtained cross-sections were submitted to a discolouration process using sodium hypochlorite for 20-35 minutes, then washed with acetic water and distilled water. The sections were coloured with green iodine and red ruthenium and mounted in gel (glycerogelatine). The preparations were photographed by means of photonic microscope NOVEX (HOLLAND), with a Canon A95 photo-digital camera.

Results and discussions

The root The passing over from the primary to the secondary structure is rather early at the level of the central cylinder, the proof being the wooden massif formed both of elements of primary xylem and of elements resulted from the early activity of the cambium; the liberian tissue, partly of secondary origin, too, is quantitatively reduced and fragmented from place to place into medular rays. The rhizoderm has isodiametric cells with thin celulose walls. From place to place, the rhizoderm exfoliates, cell left-overs that formed the adsorbant hairs being noticed. The cortex is thick (13-15 layer), of meatic type, its exteriour layer having cells with the walls moderately colenchymatized. The exoderm is of the primary type, the cells having visible Caspary thickenings in the radial walls. The central cylinder, greatly thinner than the bark, has tangentially prolongued cells of the pericycle, and the phloem and the secondary xylem are fragmenteed by some large medular rays, parenchymatically cellulosed, that goes to the limit of the primary wood. The pith is parenchymatic-lignified and the component cells have maderately thickened walls.

The rhizome A. genevensis has a secondary structure characterized only by the central cylinder, visibly thicker than the bark. The epidermis presents isodiametric cells, but of different sizes, with an external wall slightly more thickened than the others. The cortex is relatively thick (8 layers), the cell sizes increasing from the exterior to the interior of the area. The endoderm is of the primary type, with cells of different sizes, slightly radially prolongued, with casparian thickening visible in the lateral walls. The central cylinder is devided by two very large parenchymatic rays, in the thickness of which one may notice very few elements with thickened and lignified walls: some represent vessels, others libriform fibres. At the edge of these xylem elements, the medular rays have parenchymatic cells with a strictly radial disposition. The 2 great arches of conducting tissues, with a face to face disposition, have between them a relatively thick pith, parenchymatic-cellulosed, of the meatus type; in the pith, part of the cells get disorganized, resulting in an air cavity of irregular outline.

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In case of A. reptans, the plan of the general structure is the one mentioned for A. genevensis, with the difference that on the basis of a differentiated phellogen in hypodermic position a thin subarea was already formed, which exfoliates from place to place. Another differentiating element is the presence in the thickness of the bark of some solitary vascular bundles. Likewise, we noticed that in case of this species the secondary wood is much thicker, with many vessels on the unit surface, separated by libriform fibres; in the thickness of the wood there are visible 2-3 areas in which parenchymatic-cellulosed cells appear, fact that leaves the impression of a number of annual areas that mark the age of the rhizome.

The stem For A. genevensis, at the level of the superior third of the stem, the cross section outline is square shaped, with very prominent ribs, while for A. reptans the cross section outline at this level is circular, modified by two pairs of overlaped ribs that limit each a large and superficial ditch. The epidermis presents isodiametric cells, with the external wall slightly thicker than the others and covered by a thin cuticle. From place to place there are multicellular uniserial tector hairs, formed of 2 to 7 cells, the ultimate one being greater and with a sharp tip. The frequency of these hairs is higher between the ribs for A. genevensis. Besides the tector hairs, we also found secretory hairs, greater than the firs, very short, with unicellular pedicel and a gland often bicellular. The bark is thick (11-13 layers), parenchymatic-cellulosed, of the meatus type, the size of the cells growing towards the centre of the area and decreasing close to the central cylinder. The bark/cortex does not end in a special type of endoderma. For A. genevensis the central cylinder is very thick and comprizes: conducting fascicles, perimedular area and a very wide central air cavity. The fascicles of the colateral type, in great numbers (approx. 25), have different dimensions, the greatest being located near the ribs. The greatest fascicles, as well as the intermediary ones, are of the open colateral type with a multistratified meristematic area, having the cells disposed in radial series. At the outskirts of these fascicles there is a relatively thin belt/ring of mechanical elements with the walls evenly thickened, but cellulosed. In the structure of these fascicles the phloem is thinner and formed of sieved tubes and companion cells, and the xylem, visibly thicker, presents solitary vessels, or more often disposed in radial lines, all having thickened and lignified walls, separated by few wooden parenchymatic-cellulosed cells, at the level of the protoxylem the vessels having a much smaller diameter and are separated by many wood parenchymatic cells. Compared to A. genevensis, A. reptans has the majority of the conducting fascicles great alternating with few small fascicles, all disposed in a circle. At the level of the middle third, for A. genevensis the outline of the cross section is square shaped, with mitigated ribs. One may notice that the hairs are less numerous on the surface unit, the number of conducting fascicles is greater, they are closer so that the wood forms a continuous area, but obviously of different thickness. The external elements of the wood in the great fascicles are of secondary origins, the vessels being separated by libriform fibres, while the primary wooden vessels are separated by many parenchymatic- cellulosed cells, and the central air cavity is much wider.

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In case of A. reptans, at the level of the middle third the structure is similar to that of the previously analyzed level. Regarding A. genevensis, at the level of the inferior third, the outline of the cross section becomes almost eliptical, the ribs being much mitigated. Compared to the middle level, the structure differs by the rarity of the tector and secretory hairs, the lignification of the periphloemic rings and the sclerification and lignification of the medular rays at the wooden level. For A. reptans, at the level of the inferior third, the rings of the periphloemic elements do not have sclerified elements but only slightly lignified here and there, the wooden ring is thicker with visible elements of secondary origin in the great fascicles, the central parenchym is partially disorganized, resulting at least 2 air cavities, of different sizes and irregular outline. The frequency of the secretory hairs remains constant to the stem basis.

The leaf The epidermis seen from the front: is formed of cells of an irregular polygobal outline, with waved lateral walls, the ondulations having a greater amplitude compared to the superiour part of the limb. The stomata of the diacytic type are present in both epidermis, so the limb is amfistomatic. From place to place one may notice tector and scretory hairs, having the general structure similar to those described in case of the stem. In the cross section, for A. genevensis, the median nervure is strongly prominent in the inferior face of the limb, and in the superior face there is a visible but superficial ditch. On the median nervure, the inferior epidermic cells have the external wall extremely thick and covered by a thicker cuticle. In the homogenuous parenchym, greatly colourless, of the median nervure, one may notice conducting fascicle of the colateral type; in its structure there are the same tissues as those mentioned when we made reference to the fascicle structure of the stem with a primary structure. In both epidermis, especially in the inferior one, there are prominent stomata visible over the protecting tissue level. The epidermic cells of both the epidermis have thin walls, only the exterior one is covered by a very thin cuticle. The tector and the secretory hairs are very rare, the later being located especially in the superior epidermis from the median nervure, they are short, with bi-cellular pedicel, and the gland bi- or tetracellular. Between the lateral nervures with much smaller conducting fascicles and surrounded by a colourless parenchymatic sheath, the mesophyll is differentiated into palisade tissue on the superior face and spongy tissue on the inferior one, so the limb has a bifacial- heterofacial structure (dorsoventral). The palisade tissue, often three-layerd, occupy approximately 60% of the mesophyll thickness; the spongy tissue has isodiametric cells or slightly branched, leaving great air spaces among them. For A. reptans, the medium nervure is slightly prominent on the inferior face, the epidermic cells have the external wall slightly thickened and covered by a thin cuticle, and in the fundamental parenchym there is a single conducting fascicle with little phloem and xylem. Between the lateral nervures, the epidermic cells are greater on the superior face of the limb, and the mesophyll is differentiated in stratified palisade tissue with low cells on the superior face and pluristratified spongy tissue with great air spaces among the cells of

14 Ghiţă, G. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 11-18 the inferior face; thus, in this case too, the limb has a bifacial heterofacial structure (dorsoventral).

Conclusions

Our studies highlighted many histo-anatomical similarities, and differences, between the two analyzed Ajuga species. Thus, we noticed that, regarding the structure of the root and rhizome, there are no significant differences between the two species. One may notice that at the level of the stem the cross section ouline is square shaped for A. genevensis and circular in case of A. reptans. In both cases we found both tector hairs, formed of two to seven cells, and secretory hairs with unicllular pedicel and with a mostly bicellular gland. In case of the A. reptans, at the level of the stem, the most conducting fascicles are great alternating with small ones, all disposed on a circle, compared to A. genevensis. At the level of the leaf, the stomata are of the diacytic type present in both epidermis, both for A. genevensis and A. reptans, thus, the limb is amfistomatic. In both the analyzed species, the mesophyll is differentiated in the palisade tissue on the superior face and spongy on the inferior face, thus, the limb presents a bifacial- heterofacial structure (dorsoventral).

Acknowledgements This paper was published with support provided by the POSDRU project “Developing the innovation capacity and improving the impact of research through post- doctoral programmes POSDRU/89/1.5/S/49944”.

REFERENCES

Bhakuni, R.S., Shukla, Y.N., Thakur, R.S., 1991. Chemical constituents of Ajuga bracteosa. Indian J. Pharm. Sci. 49: 225-226. Calcagno, M.-P., Camps, F., Coll, J., Melé, E., Sánchez-Baeza, F., 1996. New phytoecdysteroids from roots of Ajuga reptans varieties. Tetrahedron 52: 10137-10146. Ciocârlan, V., 2000. Flora ilustrată a României. Edit. Ceres, Bucureşti. Debell, A., Makonnen, E., Zerihun, L., Abebe, D., Teka, F., 2005. In vivo antipyretic studies of the aqueous and ethanol extracts of the leaves of Ajuga remota and Lippia adoensis. Ethiop. Med. J. 43, 2: 111-118. Pârvu, C., 2006. Universul Plantelor, ed. a IV-a. Edit. ASAB, Bucuresti. Răvăruţ, M., 1961. Ajuga L., in: Săvulescu, T. (Ed.). Flora Republicii Populare Române, VIII. Edit. Acad. R.P.R.: 86-97. Tomas, J., Camps, F., Claveria, E., Coll, J., Melé, E., Messenguer, J., 1992. Composition and location of phytoecdysteroids in Ajuga reptans in vivo and in vitro cultures. Phytochemistry 31, 5: 1585-1591.

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Explanation of the plates:

Plate I: Cross section of the vegetative organs of Ajuga genevensis L.

Fig. 1. (x100 µm) Cross section through the root Fig. 2. (x300 µm) Cross section through the root – detail Fig. 3. (x100 µm) Cross section through the rhizome Fig. 4. (x200 µm) Cross section through the rhizome – detail Fig. 5. (x100 µm) Cross section through the stem aerial – superior third Fig. 6. (x400 µm) Cross section through the stem aerial – superior third, detail Fig. 7. (x100 µm) Cross section through the stem aerial – middle third Fig. 8. (x200 µm) Cross section through the stem aerial – middle third, detail Fig. 9. (x200 µm) Cross section through the stem aerial – third inferior Fig. 10. (x400 µm) Cross section through the stem aerial – third inferior, detail Fig. 11. (x100 µm) Cross section through the leaf Fig. 12. (x400 µm) Cross section through the leaf – detail Fig. 13. (x200 µm) Cross section through the leaf – seen from the front of the superior epidermis Fig. 14. (x200 µm) Cross section through the leaf – seen from the front of the inferior epidermis Fig. 15. (x400 µm) Secretory hairs the leaf Fig. 16. (x400 µm) Secretory hairs the middle stem

Plate II: Plate I: Cross section of the vegetative organs of Ajuga reptans L.

Fig. 17. (x100 µm) Cross section through the root Fig. 18. (x400 µm) Cross section through the root – detail Fig. 19. (x100 µm) Cross section through the rhizome Fig. 20. (x200 µm) Cross section through the rhizome – detail Fig. 21. (x100 µm) Cross section through the stem aerial – superior third Fig. 22. (x200 µm) Cross section through the stem aerial – superior third, detail Fig. 23. (x100 µm) Cross section through the stem aerial – middle third Fig. 24. (x400 µm) Cross section through the stem aerial – middle third, detail Fig. 25. (x100 µm) Cross section through the stem aerial – third inferior Fig. 26. (x200 µm) Cross section through the stem aerial – third inferior, detail Fig. 27. (x100 µm) Cross section through the leaf Fig. 28. (x400 µm) Cross section through the leaf – detail Fig. 29. (x400 µm) Cross section through the leaf – seen from the front of the superior epidermis Fig. 30. (x400 µm) Cross section through the leaf – seen from the front of the inferior epidermis Fig. 31. (x400 µm) Secretory hairs the middle stem Fig. 32. (x400 µm) Secretory hairs the inferior stem

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PLATE I

Fig.1. Fig.2. Fig.3. Fig.4.

Fig.5. Fig.6. Fig.7. Fig.8.

Fig.9. Fig.10. Fig.11. Fig.12.

Fig.13. Fig.14. Fig.15. Fig.16.

17 Ghiţă, G. et al., 2012/ An. Stiint. Univ. Al. I. Cuza Iasi, Sect. II a. Biol. veget., 58, 1: 11-18

PLATE II

Fig.17. Fig.18. Fig.19. Fig.20.

Fig.21. Fig.22. Fig.23. Fig.24.

Fig.25 Fig.26. Fig.27. Fig.28.

Fig.29. Fig.30. Fig.31. Fig.32.