ANALELE TIINIFICE

ALE

UNIVERSITII Alexandru Ioan Cuza

DIN IAI (SERIE NOU)

SECIUNEA II a. BIOLOGIE VEGETAL

TOMUL LIV, FASCICULA 2

Editura Universitii Alexandru Ioan Cuza Iai

2008

Editura Universitii Alexandru Ioan Cuza din Iai

ANALELE TIIN IFICE ALE UNIVERSITII Alexandru Ioan Cuza DIN IAI (SERIE NOU ), SEC IUNEA II a. BIOLOGIE VEGETAL Comitetul de redac ie:
Profesor Profesor Profesor Profesor univ. dr. Constantin TOMA Universitatea Alexandru Ioan Cuza din Ia i univ. dr. Toader CHIFU Universitatea Alexandru Ioan Cuza din Ia i univ. dr. Mihai MITITIUC Universitatea Alexandru Ioan Cuza din Ia i univ. dr. Maria Magdalena ZAMFIRACHE - Universitatea Alexandru Ioan Cuza din Ia i Profesor univ. dr. C t lin T NASE Universitatea Alexandru Ioan Cuza din Ia i Conferen iar univ. dr. L cr mioara IV NESCU Universitatea Alexandru Ioan Cuza din Ia i

Comisia de referen i tiin ifici:

Academician Valeriu COTEA Academia Romn , Filiala Ia i Profesor univ. dr. Constantin TOMA Universitatea Alexandru Ioan Cuza din Ia i, membru corespondent al Academiei Romne, Filiala Ia i Profesor univ. dr. Leontin tefan PTERFI Universitatea Babe -Bolyai din Cluj-Napoca, membru corespondent al Academiei Romne, Filiala Cluj-Napoca Profesor univ. dr. Jean Pierre AUQUIRE Universitatea Catolic din Louvain-la-Neuve, Belgia Dr. Mary GREGORY Jodrell Laboratory, Royal Botanic Gardens, Kew, Anglia Profesor univ. dr. Ioana POPESCU Drury University, Springfield, Missouri, SUA Profesor univ. dr. Andrei MARIN Universitatea din Bucure ti Profesor univ. dr. Ioan BURZO Universitatea Agronomic i de Medicin Veterinar din Bucure ti Profesor univ. dr. Toader CHIFU Universitatea Alexandru Ioan Cuza din Ia i Profesor univ. dr. Mihai MITITIUC Universitatea Alexandru Ioan Cuza din Ia i Profesor univ. dr. Ursula ST NESCU Universitatea de Medicin i Farmacie Gr. T. Popa din Iai Profesor univ. dr. C t lin T NASE Universitatea Alexandru Ioan Cuza din Ia i Redactor responsabil: Profesor univ. dr. Constantin TOMA , membru corespondent al Academiei Romne Secretar de redac ie: Preparator dr. Ramona Crina GALE Tehnoredactare computerizat: Preparator dr. Ramona Crina GALE

CONTENTS
M. ANDREI, ROXANA MARIA PARASCHIVOIU- Anatomical researches on the overground vegetative organs of Saxifraga mutata L. subsp. demissa (Schott & Kotschy) D.A. Webb and Saxifraga paniculata Miller

RAMONA GALE, C. TOMA, LCRMIOARA IVNESCUMorphological and histo-anatomical aspects regarding the floral morphogenesis in Euphorbia cyparissias L. (Euphorbiaceae Juss.) 16 GENIANA MIHAELA IULIA PREDAN, IRINA GOSTIN Microsporogenesis and the male gametophyte at Ephedra distachya L. ANCA HEMCINSCHI, RAMONA GALE, C. TOMA Vegetative anatomy of two Galium L. species (Rubiaceae) 25 31

MARIA MAGDALENA ZAMFIRACHE, C. TOMA, MARIA DUCA, SIMONA DUNCA, ZENOVIA OLTEANU, M. TEFAN, RAMONA GALE, CLAUDIA PDURARIU -A comparative study regarding the morphology and anatomy of the vegetative apparatus in two Ocimum basilicum L. breeds 39 IRINA STNESCU, GABRIELA VASILE - A comparative numeric analysis of the secretory trichomes belonging to the foliar limb in some Drosera species 49 CLARA APROTOSOAIE A., URSULA STANESCU, ANCA MIRON, VIOLETA FLORIA, OANA CIOANCA, MONICA HANCIANU Morphological researches regarding the influence of Topsin M treatments on Mentha longifolia (L.) Huds. species 56 LUMINIA HUANU-BASHTAWI, C. TOMA - A histo-anatomical study of the modifications induced by thiophanate methyl on the Calendula officinalis L. strain 64 NICOLETA IANOVICI - The analysis of daily concentrations of airborne pollen in the west and southwest of Romania 74 SILVICA PDUREANU - Variability of some pollen morphological traits of certain taxa in the bordering area of the Ceahlu National Park 80

I. BURZO, V. CIOCRLAN, ELENA DELIAN, AURELIA DOBRESCU, LILIANA BDULESCU - Research as regard to essential oil composition of some Artemisia L. species 87 ALEXANDRINA MURARIU, GIANINA BERECHET, ANIOARA STRATU, CAMELIA IFRIM- The ecophysiological peculiarities of some species of the Acer genus from the Botanical Garden of Iai 93 MARIA ANIOARA STRATU, ALEXADRINA MURARIU,, MAGDALENA ZAMFIRACHE, ZENOVIA OLTEANU, LCRMIOARA OPRIC, C. TNASE, V. C. CHINAN,, C. BRSAN - Physiological and biochemical aspects in the lignicolous species Gloeophyllum odoratum (Wulfen) Imazeki and Fomitopsis pinicola (Sw.)P. Karst. (Fungi, Basidiomycota) collected from Climani National Park (the Oriental Carpathians) 98 SMARANDA VNTU - Somatic embryogenesis in Rubus caesius L. suspension cultures 105 ROXANA-IULIANA TEODOR, AL. YEPHREMOV Genes of Arabidopsis thaliana involved in wax metabolism 110 M. A. PORUMB, M. COSTIC Characteristics of planktonic algoflore from Dorobanti, Aroneanu, Ciric I, II and III lakes (Iasi county) 115

V. SURUGIU - On the occurrence of Zostera noltii Hornemann at the Romanian coast of the Black Sea 122 OANA ZAMFIRESCU, C. MNZU, T. CHIFU - Contributions to the study of128 the ruderal vegetation from the Ceahlu mountain IRINA IRIMIA - Contributions to the vegetation study from the Vaslui river basin (II) 136

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

ANATOMICAL RESEARCHES ON THE OVERGROUND VEGETATIVE ORGANS OF SAXIFRAGA MUTATA L. SUBSP. DEMISSA (SCHOTT & KOTSCHY) D.A. WEBB AND SAXIFRAGA PANICULATA MILLER M. ANDREI*, ROXANA MARIA PARASCHIVOIU*
Abstract: This paper presents the structure of the flowering stem, the rosette and cauline leaves of Saxifraga mutata L. subsp. demissa (Schott & Kotschy) D.A. Webb and S. paniculata Miller. The vegetal material was collected from Bucegi Natural Park. The structural features of the aerial vegetative organs are correlated with the environmental conditions of these plants. The flowering stem of S. mutata subsp. demissa has sclerenchymatous sheaths around the isolated vascular bundle as well as around groups of two or three vascular bundles. The flowering stem of S. paniculata presents some supplementary collateral vascular bundles among the initial bundles as a result of the enlarging of the stem. We observed isolated phloem groups among the collateral vascular bundle in the flowering stem of both plants. There is a multilayered sclerenchymatous pericycle with a mechanical role in the plants in the flowering stem of both analysed taxa. The presence of the tracheid hydathodes in the rosette and cauline leaves of S. mutata subsp. demissa and S. paniculata is an adaptation to the high temperature on the limestone rocks in the subalpine and alpine areas. The existence of the endodermis (with Caspary strips in S. mutata subsp. demissa) and the monolayered parenchyma pericycle around the vascular bundle in the leaf structure makes the foliar vascular bundle similar to a monobundled central cylinder. The flowering stem and the cauline leaf have multiseriate glandular hairs and a few multiseriate and uniseriate non-glandular hairs. The rosette leaves are hairless. The leaves are amphistomatic, with anomocytic stomata in S. mutata subsp. demisa and tetracytic in S. paniculata. Key words: sclerenchymatous pericycle, tracheid hydathode, guttation, epithem, multiseriate glandular hairs, endodermis with Caspary strips in the leaf vein, amphistomatic, collateral vascular bundle.

Introduction Both studied taxa of the Saxifraga genus are included in the Section Ligulatae Haworth, but in two distinct subsections: Aizoonia (Tausch) Schott. (includes S. paniculata Miller) and Mutatae (Engler & Irmscher) Gornall (with S. mutata L. subsp. demissa) [18]. Saxifraga mutata L. subsp. demissa (Schott & Kotschy) D.A. Webb is a rare and vulnerable plant, endemic to Romania, already included in the Red List of vascular plants [13]. S. paniculata Miller is a common plant, widespread in Romania. Both taxa are perennial, but S. mutata subsp. demissa is monocarpic, usually solitaire or found in small groups, while S. paniculata is a policarpic, cespitose plant. S. paniculata preferes sunny limestone rocks while S .mutata subsp. demissa preferes shady places, inside rocks crevices. The leaves of the two analised taxa are placed alternately on the flowering stem as well as in a basal rosette.
*

Faculty of Biology, Bucharest University, Romania

Material and methods The plants were collected in the anthesis from Bucegi Mounts (1530m on 12 of July 2005 for S. mutata subsp. demissa and 1900m on 29 of June 2006 for S. paniculata). The vegetal material was fixed, preserved, sectioned and coloured according to the usual plant histoanatomical research technique. The cross sections and the tangential/longitudinal ones of the aerial vegetative organs were analyzed and photographed with a DOCUVAL microscope. Results and discussions I. Saxifraga mutata L. subsp. demissa (Schott & Kotschy) D.A. Webb 1. The flowering stem structure. The outline of the flowering stem cross section is circular (Pl.I:Fig.1). The epidermis, covered by a relatively thick cuticle, is composed of one layer of isodiametrical cells having thickened walls. Some of the epidermis cells are transformed in stomata and some in hairs. The stomata have a small air space beneath them and are located slightly above the rest of the epidermis. The hairs are multiseriate glandular and multiseriate and uniseriate non-glandular type (Pl.I: Fig.2; Fig.3; Fig.4). The cortex has 8 to 10 layers of parenchymatous cells with a few intercellular spaces. The innermost layer of the cortex is the starch sheath having different sized cells with cellulose walls (Pl.I: Fig.1). The central cylinder presents a multilayered (7-8 layers) sclerenchymatous pericycle, with a mechanical role. The pericycle cells are small, without intercellular spaces and have very thick, lignified walls (Pl.I: Fig.1; Fig.4). The pericycle surrounds numerous vascular bundles (14-16) set in a ring (eustele) (Pl.I: Fig.1; Fig.4). The vascular bundles are collateral. There are pith rays between the vascular bundles with large lignified cells. The mechanical cells surround each vascular bundle or groups of two or three bundles forming sclerenchymatous sheaths (Pl.I: Fig.1; Fig.4). We also observed isolated groups of phloem between the vascular bundles (Pl.I: Fig.4). The pith has parenchymatous cells (Pl.I: Fig.1). 2. The leaves structure. The structural analysis of the leaves pointed out the following features: Both basal rosette leaves and the cauline ones have a bifacial dorsiventral structure with the two epidermis (upper and lower) and a mesophyll differentiated in palisade tissue, located under the upper epidermis, and spongy tissue, on the lower half of the lamina. The palisade tissue has 3-4 layers of hetero-diametrical cells with numerous chloroplasts. The rosette leaves epidermis is hairless while the cauline leaves epidermis has multiseriate glandular hairs with a multicellular spherical head. The rosettes leaves as well as the cauline ones are amphistomatic. The epidermis of the two types of leaves have many anomocytic type stomata, observed in the tangential (superficial) sections (Pl.II: Fig.1). The limits between the subsidiary cells can be seen.

The leaves don't have an obvious petiole. The tapered base of the leaves have a homogenous mesophyll. In the mesophyll veins there are collateral vascular bundles. Sometimes there are one or two small vascular bundles close to the large vascular bundle of the midvein (Pl.II: Fig.2). The vascular bundles are surrounded by a multilayered collenchymatous sheath. A specific feature of the leaf vascular bundle is the presence, under the collenchymatous sheath, of an endodermis whose cells have Caspary strips on their radial walls. This endodermis surrounds a monolayered parenchymatous pericycle and thus the mesophyll veins have a similar structure to a monobundle vascular cylinder. On both sides of the leaves, near the cartilaginous margin, many hydathodes set in a row can be observed. These tracheid hydathodes have an epithem with numerous heterodiametric parenchymatous cells without chloroplasts and with thin walls (Pl.II: Fig.3; Fig.4). This parenchyma is supplied with water from a group of tracheids which are connected to the vascular leaf bundles. The epithem opens on the leaf surface with one large aquifer stoma similar to the other epidermis stomata but without closing movements. This stoma remains permanently opened. The water eliminated from the hydathodes in the guttation process represents the adaptation found by these plants in order to create around them a wet atmosphere, very useful against the high temperature of the limestone rocks in the subalpine area. II. Saxifraga paniculata Miller 1. The flowering stem structure. The outline of the flowering stem cross section is almost circular (Pl.III: Fig.1). The epidermis is composed of one layer of isodiametric cells and covered by cuticle. In the epidermis there are few stomata (Pl.III: Fig.5) with a small air space beneath them, located slightly above the other epidermis cells, similar to those of the other analysed plant. The flowering stem epidermis has some glandular hairs usually with a multiseriate stalk and a terminal multicellular ovoid gland (Pl.III: Fig.2). It has also very few uniseriate non-glandular hairs (Pl.III: Fig.6). The separating walls of adjacent cells of the hair stalk have simple punctuations and may be simple or divaricated (Pl.III: Fig.4). The cortex has 7 to 9 layers of isodiametric parenchymatous cells with different sized intercellular spaces. The innermost layer of the cortex is the starch sheath (Pl.III: Fig.1). The central cylinder has a multilayered (6-7 layers) sclerenchymatous pericycle, with small cells, without intercellular spaces (Pl.III: Fig.1; Fig. 3). The central cylinder is a eustele having many vascular bundles set in a ring (Pl.III: Fig.1, Fig.3). The vascular bundles are collateral. Their size varies because of the presence of supplementary vascular bundles which are formed at the same time with the widening of the stem. We indicate the presence of some phloem bundles separated from the collateral bundles, but derived from the latter, which amplifies the possibility of conducting the photosynthetic products. This observation leads us to the same conclusion as K. Esau (1965) that conducting tissues are formed whenever they are needed.

The mechanical cells from around the vascular bundles make up an external cap which extends also on the sides of the phloem (Pl.III: Fig.3). There are pith rays with large cells (the external ones present lignified walls) set in 4 or 5 rows between the vascular bundles. The pith has parenchymatous cells larger than those of the cortex (Pl.III: Fig.1). 2. The leaves structure. The cross sections of the basal rosette leaves and the cauline ones indicate a bifacial dorsiventral structure. The leaf consists of two epidermis (upper and lower) and of the mesophyll, differentiated in palisade tissue (with 4-5 layers of heterodiametrical cells) and spongy tissue. The leaves of the S. paniculata Miller are almost sessile, the lamina has a tapered base which has a petiole-like structure, with an homogenous mesophyll. The mesophyll differentiation begins in the distal half of the leaf. Both rosettes and cauline leaves have an amphistomatic lamina, with many stomata of tetracytic type (Pl.IV: Fig.1). The rosette leaves are hairless while the cauline leaves have multiseriate glandular hairs with a terminal, ovoid, multicellular gland. The epidermis of the rosette leaf margins is ciliated at the base. We observed some punctuations in the epidermis and mesophyll cell walls on the cross sections of the leaf. Thus, the mesophyll can be interpreted as an aquifer tissue. The vascular bundles of the mesophyll veins are collateral (Pl.IV: Fig.2). The mesophyll veins have a similar structure to a monobundle vascular cylinder, the vascular bundle being surrounded by a monolayered parenchymatous pericycle, an endodermis and two or three layers of colenchymatous cells. Some of the secondary veins stop on the sides of the leaf at the base of a passive hydathode (Pl.IV: Fig.3). This tracheid hydathode has an epithem formed by numerous elongated cells, with thin walls (Pl.IV: Fig.4). In a cross section through the tip of a cauline leaf we observed a terminal hydathode with numerous small cells and without intercellular spaces (Pl.IV: Fig.5). The hydathode opens with one large aquifer stoma, on the upper surface of the leaf, near the margins, at the base of the marginal teeth, in a pit (Pl.IV: Fig.6). The excretion of water and calcium carbonate through the hydathode is an adaptation of these plants to their environmental conditions. The presence, number and localisation of hydathodes represent very important taxonomical criteria in the Saxifraga genus. Conclusions The structure analysis of the aerial vegetative organs of Saxifraga mutata L. subsp. demissa (Schott & Kotschy) D.A. Webb and S. paniculata Miller underlined many similarities between the two taxa, as following: 1. The presence in the flowering stem of a sclerenchymatous multilayered pericycle, with mechanical role, can be correlated to the position of this plant on the rocks, ensuring the plant support. 2. The endodermis and the parenchymatous pericycle present around the leaf vascular bundle makes the leaf vein similar to a monobundle central cylinder.

3. The tracheid hydathodes existent in the rosette leaves and in the cauline leaves represent an adaptation of this plant to the environmental condition. The intense guttation is induced by the high temperature on the limestone substrate during the summer. The water evaporates on the leaves surface and creates a cool atmosphere around the plant. 4. The large number of vascular bundles in the flowering stem can be correlated to the tracheid guttation. We also observed isolated phloem groups which amplify the conducting of the photosynthetic products. 5. The central cylinder is a eustele with collateral vascular bundle. 6. The cauline leaves and the flowering stem are covered with multiseriate glandular hairs and uniseriate non-glandular hairs. 7. The leaves are amphistomatic. We also observed several distinct features in the anatomy of the two analysed taxa: 1. In the flowering stem of S. mutata subsp. demissa we observed a few multiseriate nonglandular hairs and in S. paniculata we indicate uniseriate glandular hairs, with divaricated walls between adjacent cells of the stalk. 2. There are sclerenchymatous sheath around isolated vascular bundles as well as around groups of two or three vascular bundles in the flowering stem of the S. mutata subsp. demissa. 3. The pith rays of the flowering stem are lignified in S. mutata subsp. demissa and parenchymatous except for the external ones which are sclerified in S. paniculata. 4. We observed some supplementary collateral vascular bundles among the initial bundles in the flowering stem of S. paniculata, as a result of the enlarging of the stem. 5. The stomata of the leaves of S. mutata subsp. demissa are anomocytic but those of S. paniculata are tetracytic. 6. In S. paniculata the hydathodes are found at the base of each marginal tooth while in the other studied plant they are set in a row, near the cartilaginous margin. In a cross section of the cauline leaf tip from S. paniculata we observed a hydathode whose epithem is made up of many cells without intercellular spaces. 7. We observed Caspary strip in the cell wall of the endodermis of the leaves of S. mutata subsp. demissa. REFERENCES
1. 2. 3. 4. 5. 6. ANDREI M., 1978 - Anatomia plantelor, Edit. Did. .Pedag., Bucureti ANDREI M., PARASCHIVOIU M. R., 2008 - Protecia i conservarea speciilor genului Saxifraga din Romnia. In Natura. Seria III, Nr. 2, Bucureti ANDREI M., PARASCHIVOIU M. R., 2003 - Microtehnic Botanic, Edit. Niculescu, Bucureti BLAND B., 2000 - Silver Saxifrages, A guide to Encrusted Saxifrages for Gardeners and Botanists, The Alpine Garden Society, The Friary Press, Dorchester, Great Britain BOLDOR O., TRIFU M., RAIANU O., 1981 - Fiziologia plantelor, Edit.Did. Pedag., Bucureti CIOCRLAN V., 2000 - Flora ilustrat a Romniei. Pteridophyta et Spermatophyta, Edit. Ceres, Bucureti

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

CUTLER D. F., BOTHA C.E.J., STEVENSON D.W., 2008 - Plant Anatomy. An applied approach, Blackwell Publishing Ltd., USA CUTLER D. F., GREGORY M., 1998 - Anatomy of the Dicotiledons, second edition, Vol. IV. Saxifragales, Clarendon Press, Oxford ESAU K., 1965 - Plant Anatomy, second edition, John Wiley & Sons, Inc., USA FAHN A., 1982 - Plant Anatomy, Ed. III, Pergamon Press, Oxford, England GRINESCU I., 1985 - Botanica, Ed. II, Edit. tiinific i Enciclopedic, Bucureti HARDING W. 1992 - Saxifrages, A gardeners Guide to the Genus, The Alpine Garden Society, The Friary Press, Dorchester, Great Britain OLTEAN M., NEGREAN G., POPESCU A., ROMAN N., DIHORU, G., SANDA V., MIHILESCU S., 1994 Lista roie a plantelor superioare din Romnia. Studii, Sinteze, Documentaii de Ecologie,.1, Academia Romn, Institutul de Biologie, Bucureti RVRU M., 1956 - Fam. Saxifragaceae, In Flora Republicii Populare Romne, Vol. IV, p. 85-128, Edit. Acad. R.P.R., Bucureti TOMA C., 1995 - Anatomia plantelor I. Histologia i 1977. Anatomia plantelor II. Structura organelor vegetative i de reproducere, Centrul de multiplicare al Univ. Al. I. Cuza, Iai ERBNESCU-JITARIU, Gabriela, TOMA, C., 1980 - Morfologia i anatomia plantelor, E.D.P., Bucureti WEBB D. A., 1964 - Saxifragaceae, In TUTIN T. G., HEYWOOD V. H., BURGES, N. A., VALENTINE D. H., WALTERS S. M., WEBB D. A. Flora Europaea, Vol. I. Licopodiaceae to Plantaceae, University Press, Cambridge WEBB D. A., GORNALL R. J., 1989 - Saxifrages of Europe, Cristopher Helm Ltd., Great Britain.

The explanation of figures: Plate I. S. mutata subsp. demissa: Fig.1. Cross section of the flowering stem showing the epidermis, cortex and the eustele with sclerenchymatous pericycle (Oc.12,5x; Ob. 25); Fig.2. A multiseriate glandular hair in a cross section through the flowering stem; Fig.3. One multiseriate non-glandular hair in a cross section through the flowering stem (Oc.12,5x; Ob. 40); Fig.4. Detail of the flowering stem structure in a cross section showing the collateral vascular bundles surrounded by sclerenchymatous sheath; one isolated phloem group can be seen (Oc.12,5x; Ob.40); Fig.5. Tangential section of the flowering stem with a multiseriate glandular hair and a uniseriate non-glandular hair (Oc.12,5x; Ob. 40). Plate II. S. mutata subsp. demissa: Fig.1. The lower epidermis of the rosette leaf in an apical view. The simple punctuations and the stomata shape can be observed (Oc.12,5x; Ob.40); Fig.2. Cross sections through the basal rosette leaves indicate the midvein structure having a collateral vascular bundle surrounded by a pericycle, an endodermis with Caspary strips and collenchymatic rings. A large vascular bundle and a smaller one can be seen in the midvein of the leaf (Oc.12,5x; Ob. 40); Fig.3. Cross section through the basal leaf rosette indicates the tracheid hydathode (Oc.12,5x; Ob.25); Fig.4. Detail of the tracheid hydathodes with a large aquifer stoma and the epithem (Oc.12,5x; Ob.40). Plate III. S. paniculata: Fig.1. Cross-section of the flowering stem (Oc.12,5x; Ob.25); Fig.2. Multiseriate glandular hair in the flowering stem epidermis (Oc.12,5x; Ob.25); Fig.3. Detail of the collateral vascular bundle along with an independent phloem bundle in the flowering stem cross-section (Oc.12,5x; Ob.40); Fig.4. Uniseriate with divaricated walls glandular hair in the flowering stem epidermis (Oc.12,5x; Ob.40); Fig.5. Stoma in the flowering stem epidermis (Oc.12,5x; Ob. 40); Fig.6. One uniseriate non-glandular hair in the flowering stem epidermis (Oc.12,5x; Ob.40). Plate IV. S. paniculata: Fig.1. Tetracytic stomata of the upper epidermis, in a tangential section through the rosette leaf (Oc.10x; Ob.40); Fig.2. Detail of the midvein of the rosette leaf (Oc.12,5x; Ob.40); Fig.3. Cross section of the rosette leaf indicates the connection between the secondary vein and the tracheid hydathode (Oc.12,5x; Ob. 10); Fig.4. Cross section through the rosette leaf showing a tracheid hydathode which opens on the upper surface of the leaf, in a pit, towards the margin (Oc.12,5x; Ob.40); Fig.5. Detail of the parenchyma epithem surrounded by a tannin cells sheath in a cross section through the tip of the cauline leaf; the epithem cells of this terminal hydathode don't have intercellular spaces (Oc.12,5x; Ob.25); Fig.6. Tangential section of the cauline leaf margin; it can be seen the opening of a hydathode, with an aquifer stoma, at the base of marginal tooth (Oc.12,5x; Ob.10).

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M. ANDREI, ROXANA PARASCHIVOIU

PLATE I

4 5

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M. ANDREI, ROXANA PARASCHIVOIU

PLATE II

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M. ANDREI, ROXANA PARASCHIVOIU

PLATE III

2 1

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M. ANDREI, ROXANA PARASCHIVOIU

PLATE IV

1 2

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

MORPHOLOGICAL AND HISTO-ANATOMICAL ASPECTS REGARDING THE FLORAL MORPHOGENESIS IN EUPHORBIA CYPARISSIAS L. (EUPHORBIACEAE JUSS.) RAMONA GALE*, C. TOMA*, LCRMIOARA IVNESCU*
Abstract: Euphorbia cyparissias L is an herbaceous species, wide-spread in the flora of Romania, often founded in plant cultures and roadsides. This report describes the floral morphogenesis in the cypress spurge. The vegetative activity of the shoot apex early ends by forming the inflorescence bracts, which have a different morphology and structure than the nomophylls. Each branch of the dichasial inflorescence ends with a single cyathium. The order of appearance of the cyathium components is as follows: monostaminate male flowers, involucre, nectary glands and female flower. Key words: Euphorbia cyparissias, shoot apex, morfogenesis, dichasium, cyathium

Introduction Despite the diversity of Euphorbia species, their inflorescence has an homogeneous organization, whose rendering during the time have launched to contradictory hypothesis. Tournefort (1700), Linnaeus (1753), Payer (1857) and Baillon (1858) conclude that the true status is that of a single hermaphroditic flower [4]. Le Maout (1842) adduces a new interpretation of this curios flower, named cyathium by Warming (1912), the notion being a long time discussed [7]. According to the actual conception the cyathium represents a contracted inflorescence, usually constructed by 5 uniparous, scorpioide cymes of monostaminate male flowers, which encircle a central nude female flower, all being surrounded by gamophyllous and nectary involucre [2]. Haber [4] analyzes the vascular origin of the cyathium in some Euphorbia species and adduces an argument to uphold the conception according to which the cyathium is a high specialized inflorescence. Other researchers [1], [6], [8], [9] investigate the several aspects of the embryogenesis and the structure of cyathium nectary glands in some representative Euphorbia species. The lack of information regarding the floral morphogenesis in Euphorbia species in the Romanian literature encouraged us to carry this work. The present paper analyzes the successive stages of the inflorescence development in Euphorbia cyparissias L. Material and methods

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, Iasi, 700506, Romania, ramona.gales@uaic.ro

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To observe the morphological features of the vegetative shoot apex during its transformation into the reproductive one, the Euphorbia cyparissias L. plants were analyzed in vivo at the end of the vegetation period (April), in successive stages of floral morphogenesis. The material used for the histo-anatomical analysis was represented by the shoot apices and inflorescences of Euphorbia cyparissias L., which were fixed in FEEA mixture and preserved in 70% ethylic alcohol. The tissular and cellular modifications of the shoot reproductive apex and the development of the inflorescence were analyzed on serial crosssections, which were performed using the standard paraffin-embedded protocol applied in plant histo-anatomical researches. Fixed samples were dehydrated by a passage through ethanol/water solutions and then embedded in paraffin at 65C for 24 hours. The embedded material was cut into 13 m thick sections with a rotator microtome. The dried serial sections were deparaffinized, rehydrated in serial dilutions of ethanol (100%, 90%, and 70%), coloured with metilen-blue and ruthenium-red, and finally mounted in Canada balsam. All permanent slides were analyzed in light microscopy, using a Novex (Holland) microscope; the microphotographs were made at the same microscope with a Sanyo digital camera and the sketches were drawn on a Romanian MC1 microscope with Projektionszeichenspie gel. Results and discussions Morphological changes of the shoot apex related to the transition from the vegetative to the reproductive phase. The plastochronic functioning of the shoot apex of Euphorbia cyparissias L. is very short (approximately one month). During the vegetative phase, the shoot apex forms numerous leaves with winding disposition. Towards the middle of April, the tip of the foliated stem becomes convex as a result of a high number of bracts loosely clustered around the shoot apex, such as the leaves of a scaly bulb. The bracts form the outside of this bulb, which have approximately the same shape as the nomophylls, but are smaller than these, will form the involucre of the composed cymose inflorescence. The inner bracts, which differ from the outer ones by their smaller size and rhomboidal shape, will form the involucres of the dichasial inflorescences. The vegetative activity of the shoot apex ends by forming the inflorescence bracts; from this moment the reproductive apex will form the flower primordium, so that the stem axis will terminate with a cyathium. The axillary buds will give birth to dichasial branches which may ramify once or twice, each branch ending with a cyathium. Structural changes of the shoot apex related to the transition from the vegetative to the reproductive phase. The initial ring of the shoot apex is consumed by forming the inflorescence bracts. During this stage, the shoot apex becomes dished as a result of numerous mitotic divisions which take place in the apical-axial zone.

16

The structure of the stem tip is exclusively given by the bases of the bracts, being represented by a cellulosic-parenchymatous tissue in which laticifers could be observed alongside the procambium cordons. The bract consists of homogenous mesophyll of meatic type, formed by round parenchymatous cellulosic cells, in which cordons of procambium and laticifers pervade. Each bud is formed in the axil of a bract, as a result of the numerous divisions of the dedifferentiated cells of the upper epidermis and of some hypodermic layers from the bract structure. At the beginning of the floral initiation, the shoot apex enlarges as a result of intense divisions of the apical-axial meristem, being formed by a wide zone of meristematic cells (with central nucleus and voluminous nucleolus) which covers a central zone with vacuolizated cells (with central nucleus and numerous small vacuoles). Immediately below the shoot apex, the cells of the central-axial zone are disorganized, resulting large aeriferous cavities. The ontogenesis of the dichasium (Pl. I). In Euphorbia species, the compound inflorescence is due to the dichasial branching. Each primary axis situated in the axil of an involucral bract represents, in its first stages, a very simple cymose inflorescence, consisting of three flowers- a central and two lateral ones with which the secondary axes end [4]. During the reproductive phase, the apex will give rise to a single cyathium. Subsequently from each axillary bud a primary axis of a dichasium will be formed. In its first stages, the primary axis presents a circular contour in cross-section; from its outer to its inner part, the following histo-anatomical zones could be distinguished: 1. a single-layered epidermis consisting of isodiametrical parenchymatous-cellulosic cells; 2. cortical parenchyma of meatic type formed by 7-8 layers of cellulosic, thin-walled cells; 3. vascular bundles embedded in a parenchymatous matrix and exhibiting meristematic tissue (procambium) between a few xylem vessels and phloem elements; 4. cellulosic-parenchymatous pith, which presents numerous big aeriferous cavities. During the dichasium ontogenesis, the vascular system of the primary axis undergoes several transformations as follows: new vascular elements appear in the external zone of the cortex (being destined to vascularize the involucre); two meristematic-vascular rings surrounding a disorganized pith are formed in the lateral parts; the central vascular bundles are disposed in two opposed arches, the pith being included in their concavities. In an advanced stage of dichasium development, two axilary buds will be formed on the primary axis, which will end with a ciathium. Each axilary bud will give rise to a single ciathium. The ontogenesis of the cyathium (Pl. II- IV). The cyathium appears in the tip of a branch such as a bud, having at its base two bracts and being covered by the involucre of the dichasial inflorescence. Laticifers of the branch pervade in the primordium of the cyathium. On the flanks of the reproductive apex, meristematic protuberances appear which will successively form the primordia of stamens. In the first stages of male flower development (Pl. II), the stamen primordium differentiates a stalked basal region, which will give rise to the

17

pedicel and filament and a trapezoidal upper region, which becomes the anther. The filament extends concomitantly with the individualization of the two locules of the anther. In the next stages of the stamen development, the median longitudinal septum and then the two lateral ones are formed, which will separate each locule into two polinic sacs. In the same time, in the median part of the anther, the connective begins to form. It should be mentioned that the forming of the two polinic sacs is not concomitant in the two locules. The wall of the polinic sacs are not yet completely developed when the sporogenous tissue is forming. Concomitantly with the development of the monostaminate male flowers, the involucre of the cyathium starts to form. Thus, before the forming of the female flower, the cyathium presents at its external part an involucre of polygonal contour in cross-section and a central axis surrounded by stamens (in different stages of development). The apex of the central axis gives rise to the first carpel which closes itself simultaneous with the forming of a single ovule, which initially has a vertical position. In the next stage, the stile of the first carpel is formed concomitantly with the appearing of the second carpel whose evolution is similar with the first one. In the same way the third carpel is formed. The three separately closed carpels grow together by their edges to form a 3-carpellar, syncarpous, 3locullar ovary with central-marginal placentation (Pl. III). The central axis of the cyathium grows in length to form the pedicel of the female flower. Thus, the incompletely developed female flower turns out from the involucre. Each ovule becomes anatropous, consisting of a funicle, two integuments and nucellus, in which the embryo sac is not yet formed. Concomitantly with the development of female flower, primordia of nectary glands appear in the upper level of the involucre. The development of the four nectary glands is not simultaneous. As a result of the intense divisions of meristematic cells, the primordium of gland grows in half-moon shape; the cells of the external layer radialy elongates and will form a single-layered cellulosic parenchymatous epidermis; the first two layers situated just beneath the epidermis will form a glandular tissue, and the other ones will form a parenchymatous tissue, in which some vascular elements of the involucre pervade (Pl. IV). Conclusions 1. The vegetative activity of the Euphorbia cyparissias shoot apex is early ended by the forming of the inflorescence bracts, which exhibit different morphology and structure from that of nomophylls 2. In the axil of the bracts, floriferous buds are formed, which will give rise to the dichasial inflorescences.

18

3. During the dichasium ontogeny, the conducting system of the primary axis undergoes several transformations, in order to vascularize the two axilary buds; each of the latest ones which will give birth to a single ciathium just like the primary axis. 4. The cyathium appears in form of a bud at the base of two bracts. 5. During the ontogeny of the cyathium, the first formed elements are the monostaminate male flowers, which appear successively on the flanks of the reproductive apex. 6. The involucre is formed from the central axis of the cyathium concomitantly with the development of the male flowers. 7. In the upper part of the involucre, four nectary glands are formed concomitantly with the development of the female flower, which is formed from the central axis of the cyathium. 8. The ovary is 3-carpellar syncarpous, 3-locullar with central-marginal placentation; the carpels, each of it with a single ovule, are formed successively. 9. Initially, the ovules have a vertical position, subsequently they become anatropous. 10. The incompletely developed female flower turns out from the involucre as a result of the elongation of its pedicel. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. CARMICHAEL J. S., SELBO S. M., 1999 - Ovule, embryo sac, embryo and endosperm development in leafy spurge (Euphorbia esula), Can. J. Bot., 77, 4: 599-610 EMBERGER L., 1960 - Les vgtaux vasculaires. t. II, Trait de Botanique systmatique (de M. Chadefaud et L.Emberger), Libraires de LAcadmie de Medicine , Paris GORI P., 1987- The fine structure of the developing Euphorbia dulcis endosperm, Ann. Bot., 60: 563-569 HABER M. J., 1925 - The anatomy and the morphology of the flower of Euphorbia, Ann. Bot., 39:656-707 KLEIMAN C. 2001 - La Reproduction des angiosperms, Ed. Belin, Paris PAPP N., 2004 - Nectar and nectary studies son seven Euphorbia species, Acta Bot. Hung., 46, 1-2: 225-234 PARROT G., 1947 - Quelques remarques sur linflorescence dEuphorbia peplus L., Bull. Soc. Bot. France, 94, 9: 424-427 RAJA RAJESWARI RAO K., PRAKASA P. S., 1975 - Embryo development in Euphorbia peplus L., Current Science, 44, 1: 57-59 WENIGER W., 1917 Development of embryo sac and embryo in Euphorbia preslii and E. splendens, Bot. Gaz., 63, 4: 266-281

The explantion of figures: Plate I. A, B, C, D, E, F, G- Serial cross-sections through the dichasial inflorescence of Euphorbia cyparissias (successive stages of the dichasium ontogeny) (original diagrams) Abreviations: br.= bract; pr. cyt.= primordium of cyathium; pt. = pith; mt. ts.= meristematic tissue; vs. bd.=vascular bundle Plate II. Fig. 1-5. The ontogenesis of a male flower of the Euphorbia cyparissias cyathium: longitudinal sections through the cyathium in successive stages of the flower morphogenesis (original microphotographs) (scara= 50m). Plate III. Fig. 1-5. The ontogenesis of female flower of the Euphorbia cyparissias cyathium: longitudinal (2, 3, 5) and transverse (1, 4) sections through the cyathium in successive stages of the flower morphogenesis (original microphotographs) (scara= 50m). Plate IV. Fig. 1-5. The ontogenesis of the nectary glands of the Euphorbia cyparissias cyathium: longitudinal sections through the cyathium in successive stages of the flower morphogenesis (original microphotographs) (scara= 50m).

19

RAMONA GALE et al. pt. vs. bd.

PLATE I

pr. cyt.

A
mt. ts.

br.

E D

20

RAMONA GALE et al.

locules

PLATE II

male flower primordium

1 2
polinic sacs

385

filament

articulation pedicel mature anther

21

RAMONA GALE et al.

PLATE III
carpel ovule

2
ovule ovule

anatropous ovule

3 5

22

RAMONA GALE et al.

nectary gland primordium

PLATE IV

1 2

glandular tissue

Epidermis

parenchimatous-cellulosic tissue

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

MICROSPOROGENESIS AND THE MALE GAMETOPHYTE AT EPHEDRA DISTACHYA L. GENIANA MIHAELA IULIA PREDAN*, IRINA GOSTIN**
Abstract: This study describes the microsporogenesis and the male gametophyte in Ephedra distachya L., only one species of the Ephedraceae family growing spontaneous in Romania. Ephedra distachya L. is a dioecious plant. Light microscopy (lm) is used to examine the early developmental stages of the male cones and combined light (lm) and scanning electron microscopy (sem) were used to analyze the mature pollen grains. The pollen cones are clustered at nodes either sessile or stalked. They are oval-oblong, each is composed of 2-8 decussate pairs of membranous bracts, proximal bracts empty; each distal bract subtending a male flower composed of 2 basally fused, orbicular or obovate scales (false perianth); sessile anthers on staminal column. Ephedra distachya pollen is particularly abundant. Ephedra distachya has the polyplicate pollen type characterized by a pointed-oval shape and 6 longitudinal ridges and 6 valleys, due to alternation of thicker and thinner exine regions, inaperturate. In polar view, ridges are low and triangular. On the SEM analysis the pollen wall of Ephedra distachya is simple, so the SEM closely resembles the LM observation. Key words: Ephedra distachya L., male cones, pollen development

Introduction Genus Ephedra L. contains 35-45 species, most of them populating the desert or arid regions. In Romania a single species of Ephedraceae family occurs - Ephedra distachya L. Its status is rare [12]. Genus Ephedra L. has been much studied from the morphological and anatomical point of view, information was summarized and included in synthesis works [8, 10, 14, 15, 16]. The histology of the flowers and strobili of the Ephedra distachya L. plants was examined by Van Tieghem (1869), Thoday and Berridge (1912) [10], later by Favre-Duchartre M. [5]. Baranec T. Rehorek and V. studied the reproductive cycle of the Ephedra distachya L. plants, spontaneous in Slovakia [2] and P. Mehra N. analysed dimensions of the male nuclei of the representatives in the Ephedra genus [10]. Allison S. D. et al. have studied all the important stages of the Ephedra pollen development using both photonic and the scanning and transmission electronic microscope [3]. Gamal El-Ghazaly et al. were concerned about pollen grain polarity, the aperture situation and the pollen tube at 5 species of Ephedra including E. distachya L. [6]. The microsporogenesis and the description of the pollen and the male gametophyte
*

University of Bucharest, Faculty of Biology, Department de Botany and Microbiology, Aleea Portocalelor, nr. 1-3, 060101, Bucureti, Romania; ggentiana@yahoo.com ** Al. I. Cuza University, Faculty of Biology, Bd. Carol I, no. 20A, 700506, Iai, Romania; irinagostin@yahoo.com

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development in Ephedra distachya L. provides interesting characterization in this species. Material and methods Our study was carried out on samples of Ephedra distachya L. from different places in Romania: Agigea Marine Dune Reserve (Constanta County), Culmea Pricopanului (Tulcea County), the Botanical Garden of Iai (cultivated plants) and from Bulgaria (Balchik) [7, 8]. Ephedra distachya L. is a dioecious plant. Male cones were collected during the three spring months (March, April and May), in successive phaenological phases, from the cones initiation up to the pollen shedding. The biological material (male cones) was fixed in 70% alcohol or FAA (formalin 40%, 5 cc: alcohol 70%, 90 cc: glacial acetic acid 5 cc). After being fixed, the material has been processed in accordance with the paraffin embeding protocol [3], sectioned (transverselly and / or longitudinally) with a Minot type microtome at 12-15 m thick, colored with Ehrlichs haematoxylin and embedded in Canada balm. To highlight microspores and young pollen grains microscopical preparations squash type have been made, that have been colored with acetic carmine. All obtained sections were analyzed using light microscope (LM) Docuval type and photographed with a digital photo camera slr Canon EOS 400D. For the analysis of the pollen grains at electronic scanning microscope (SEM) a Vega - TESCAN LSH microscope was used in The Faculty of Biology of The Al I. Cuza University from Iai [12]. Male cones were considered from the morphological point of view with a Belphotonics stereomicroscope and photographed with the same digital camera. The description of the pollen grains has been made in accordance with the terminology adopted by Faegri [4]. Results and discussions The male cones, either sessile or stalked, are found clustered at nodes (fig. 1). The male cones are green, oval-oblongue, each of them being composed of 2-8 descussate pairs of membranous bracts, like cups (Fig. 2, 3). The lower pair bracts is sterile, each distal bract subtending a male flower composed of 2 basally fused, orbicular or obovate scales (primitive periant) (Fig. 4, 7), an axis (microsporangiophore or anterophore) terminal bearing a number of anthers, each anther with two micro sporangia (pollen sacs) (Fig. 4). A group of archespores subepidermal cells in the mature cones is observed in March (fig. 5). In April, following meiosis I, macrospore dyads have resulted. After meiosis II numerous tetrads of microspores appear, the wall formation taking place after each stage of meiosis. Soon, the wall surrounding the 4 microspore cells dissolve and the microspores in the pollen sac are

25

released. The microsporangia wall is composed of three known layers: epidermis, the median layer and the tapetum layer. The median layer is composed of a single row of parenchimatic cells and, as the pollen sac grows, its cells are flattened so that they ultimately disappear. The tapetum has signs of degeneration after the reductional division (Fig. 6) and no longer in the stage of mature pollen (Fig. 8). At mature pollen sac epidermis persists. Its cells have a regular appearance and their walls are thickened (Fig. 9). Only in a small portion of the pollen sac upper part a few parenchimatic cells with thin walls remain. By their breaking a hole will form that will be issued pollen (poricide opening). In May, the pollen is divided and forms a small prothalian cell and a large, central one (Fig. 10). The last divide and formed the second prothalian cell and initial anteridial cell. The last divide and give a vegetative larger cell, and a generative cell. The generative cell will form the stalk cell and spermatogene cell, the latter giving rise to gametes. Regarding pollen sacs, the pollen has 5 cells [8]. The mature pollen grains are yellow, fusiforme and obvious poliplicate: they have 6 longitudinal ridges (plicae) (sometimes slightly corrugated) and 6 valleys, formed due to the alternation of thicker and thinner exine regions (Fig. 9, 10). On a polar view, the ridges are short and triangular. Pollen grains have 53 m long axis and 20 m short axis. At the electronic scanning microscope (SEM), the pollen surface is no obvious regulate and no aperture was noticed eather, so the pollen is inaperturate (Fig. 11, 12). Conclusions In March, a group of archespore cells appears in the pollen sacs, subepidermaly. In April the anther wall is composed of an epidermis and a scrap tapetum and the microspores are free. In May the anther wall is only represented by the epidermis and numerous grains of fusiforme and yellow pollen are found inside the pollen sac, in our observation (in 17 May 2007 on Culmea Pricopanului) the pollen grains are uninucleate. According to the SEM observations, the pollen grains of Ephedra distachya L. have a simple structure, the SEM observations being very similar to those made on an optical microscope. REFERENCES
1. 2. 3. 4. ANDREI M., RDULESCU D., 1972 - Caiet pentru tehnica preparrii i conservrii materialului biologic. Tipogr. Univ. din Bucureti BARANEC, T., V. REHOREK, et al., 1994 - Generative reproduction of ephedra (Ephedra distachya L.) in Slovakia. Biologia Bratislava, 49 (1): 65-67 DOORES ALLISON S., OSBORN J. M., GAMAL EL-GHAZALY., 2007 - Pollen Ontogeny in Ephedra americana (Gnetales). International Journal of Plant Sciences 168 (7): 985-997 FAEGRI K., IVERSEN J., 1966 - Textbook of pollen analysis. 2nd Ed. Munksgaard, Copenhagen

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5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

FAVRE-DUCHARTRE M., 1959 - Contribution ltude de la reproduction sexue chez Ephedra distachya. C. R. Acad. Sci. Paris. 249: 1551-1553 GAMAL EL-GHAZALY, ROWLEY J., HESSE M., 1998 - Polarity, aperture condition and germination in pollen grains of Ephedra (Gnetales). Plant Systematics and Evolution, 213 (3-4): 217-231 GRINESCU G. P., 1952 - Ephedra L. In: T. SVULESCU (red. princip.). Flora Romniei. 1. Bucureti: Edit. Academiei Romne JOHRY B. M., BISWAS C., 1997 - The Gymnosperms. Narosa Publishing House, New Delhi KRSSMANN G., 1985 - Manual of cultivated conifers. B.T. Batsford Ltd. London LEHMANN-BAERTS M., 1967 - La morphologie du sporophyte dans le genre Ephedra. La Cellule (Belg.), 67 (5): 7-56 MEHRA P. N., 1950 - Inequality in size of the male nuclei in the genus Ephedra. Ann. Bot., July 1950; 14: 331339 OLTEAN M., NEGREAN G., POPESCU A., ROMAN N., DIHORU G. SANDA V., MIHILESCU S., 1994 Lista roie a plantelor superioare din Romnia. Studii, sinteze, documentaii de ecologie, 1 PLOAIE G. P., PETRE ZOE. 1979 - Introducere in microscopia electronica cu aplicatii in biologia celulara si moleculara. Edit. Acad. Romane, Bucuresti SCHNARF K., 1933 - Embryologie der Gymnospermen. In: Linsbauer K. Handbuch der Pflanzenanatomie, II Abt., 2 Teil, Bd. 2. Verlag Gebruder Borntraeger, Berlin SINGH H., 1978. - Embryology of gymnosperms. Berlin-Stuttgart SMARANDACHE D., 2005 - Embriologia plantelor. I. Embriologia arhegoniatelor. Edit. Univ. din Bucureti, Bucureti. STEEVES M. W., BARGHOORN E. S., 1959 - The pollen of Ephedra. J. Arnold Arboretum, 40: 221-255

The explanation of figures: Plate I Fig. 1 Ephedra distachya L. - Branch with male cones (1 May 2008; Orig) Fig. 2 Ephedra distachya L. Male cone (20 May 2007) (Oc. 10x; Amplific. 2; Orig.) Fig. 3. Ephedra distachya L. - Longitudinal section of a male cone - fragment (13 April 2008) (Oc 12.5x; ob. 3.2x; Amplific. 8; Orig.) Fig. 4. Ephedra distachya L. - Male flower (20 May 2007) (Oc. 10x; Amplific. 3; Orig.) Fig. 5. Ephedra distachya L. - Longitudinal section through the anther that outlines the archaesporal cells (17 March 2007) (Oc 12.5x.; ob. 25x; Amplific. 160; Orig.) Fig. 6. Ephedra distachya L. - Cross-section through the male flower (13 April 2008) (Oc. 12.5x; ob. 10x; Amplific. 6.3; Orig.) Fig. 7. Ephedra distachya L. - Longitudinal section through the male flower (13 April 2008) (Oc. 12.5x; ob. 10x; Amplific. 6.3; Orig.) Plate II Fig. 8 Ephedra distachya L. - Longitudinal section through the male flower (29 May 2001) (Oc. 12.5x; ob. 3.2x; Amplific.16; Orig.) Fig. 9. Ephedra distachya L. - Longitudinal section through the anther - anteral wall and young pollen grains (29 May 2001) (Oc. 12.5x; ob. 40x; Ampific. 16; Orig.) Fig. 10. Ephedra distachya L. - Unicellular pollen grain (17 May 2007) (Oc. 12.5x; ob. 40x; Amplific.16; Orig.) Fig. 11. SEM micrograph of Ephedra distachya L. poliplicate pollen (21 May 2008; Orig.) Fig 12. SEM micrograph of Ephedra distachya L. pollen (21 May 2008; Orig.)

27

GENIANA PREDAN, IRINA GOSTIN

PLATE I

28

GENIANA PREDAN, IRINA GOSTIN

PLATE II

10

11

12

29

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

VEGETATIVE ANATOMY OF TWO GALIUM L. SPECIES (RUBIACEAE) ANCA HEMCINSCHI*, RAMONA GALE**, C. TOMA**
Abstract. The structure of two medicinal Galium species (G. album Mill. and G. verum L.) was studied to determine the range of variation in certain histo-anatomical characters of the vegetative organs. The study revealed several interesting anatomical data that have not previously been reported in these species, e.g. the structure of the rhizome, the presence of the trichomes on the adaxial face of G. verum foliar limb. Key words: anatomy, vegetative organs, Galium

Introduction Following-up our histo-anatomical investigations on Rubiaceae genre and species [22], the present study compares the anatomical structure of two perennial species belonging to Galium genus: Galium album Mill. (syn. G. mollugo L. ssp. erectum (Huds./Briq.) and G. verum L. The two species are considered medicinal plants [13], [19] and are distinguished from each other by several morpho-anatomical characters, e.g. the colour of the flowers, the presence, the frequency and the length of the trichomes. Among the 37 Galium species from the Romanian flora, G. album and G. verum are common in the entire country, from the durmast to the subalpine belt [4], [12]. The existing literature on the structure of the representative members of Rubiaceae family is quite abundant in results from studies dedicated exclusively to them [14], [15], [17], [18], [21] or from the synthesis treatises on anatomy of Dicotyledons [9] or Angiosperms, in generally [10]. Some authors investigate the medicinal Galium species [1], [2], [6], [8], others analyze several aspects regarding the foliar venation [5], the infraspecific variability [7], the stomata ontogeny [11], the stipules structure [16], the number of stomata in different ecological conditions [20], [3]. Material and methods The research material is represented by two species of Galium genus: G. album Mill. and G. verum L. from the Romanian flora. The material was fixed and preserved in 70% ethylic alcohol. Cross-sections of the rhizome, aerial stem and leaf were performed using a manual microtome, coloured with iodine-green and ruthenium-red and embedded in glicero-gelatine. The superficial sections through the foliar limb were coloured with iodine-green. The obtained
* **

Pharmacy AncaFarm, Botosani, Romania Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, 700506, Iasi, Romania

30

permanent slides were analyzed on a Novex (Holland) microscope and photographed at the same microscope with a digital photo camera. Results and discussions The aerial stem The contour of the transverse section through the aerial stem is quadratic rhomboid, with rounded ribs, less prominent towards the basis of the organ. In G. verum, the epidermis presents unicellular trichomes, with very thick walls, whose frequency decreases towards the upper and the lower level of the stem. In the four ribs there are cordons of tangential collenchyma and the cortex ends with a Casparyan endodermis on the entire stem length. In G. verum, between the stem ribs, the outer cortical layer is collenchymatous. The stellum presents secondary structure, the conducting tissues being of annual type. The xylem ring is thicker than the phloem one, comprising numerous libriform fibres with very thick and intensive lignified walls. The rhizome The outline of the transverse section through the rhizome is circular. In both analyzed species, the rhizome presents secondary structure, resulted only from the cambium activity (in G. album) or from the activity of both lateral meristems (in G. verum). In the latest case, the phellogen is differentiated from the Casparyan endodermis; the epidermis and the cortex are exfoliated. In G. album, the cortex is relatively thin (5-6 layers); this histological feature is unusual for a subterranean stem. In the thickness of the cortical parenchyma, crystalliferous cells with crystal sand and raphides are often present. The stele is very thick and comprises: 1) a thinner (in G. album) or a thicker (in G. verum) phloem ring, some of the collenchymatous parenchyma cells containing crystal sand and 2) a single very thick (in G. album) or three (in G. verum) xylem rings, whose thickness increases from the pith to the secondary phloem. The secondary xylem consists of much libriform, in which the vessels are irregularly disposed. Only in G. verum, there are islands of xylem cellulosic parenchyma, some cells containing crystal sand. The pith is thin, parenchymatous-cellulosic of meatic type, with very big cells in G. verum, many of them containing crystal or raphides (in G. album). The leaf The sessile leaves are hipostomatic, with stomata of paracytic type. The epidermis (in front view) presents cells of irregular outline, their lateral walls being moderately (in G. verum) or mighty (in G. album) wavy. In cross-section, the cells are isodiametric, being much bigger in the upper epidermis.

31

At epidermis level there are unicellular, short trichomes with very thick wall, more numerous on the abaxial face. Our observations, in concordance with the anterior published papers [22], quash the data from Flora of Romania [12], according to which the adaxial face of G. verum leaves is glabrous. The foliar limb has bifacial heterofacial structure, the mesophyll being differentiated in one (in G. album) or two (in G. verum) layers of palisade tissue and 4-5 layers of lacunous tissue; in the thickness of the latest, cells with crystal sand may be observed. In both species studied, the outer wall of the epidemic cells is thicker than the others and covered by a thin cuticle. On the margins of the foliar limb, all the walls of the epidermic cells are thick; a collenchymatous hypodermis is present. The median and lateral vascular bundles are surrounded by parenchymatous theca with isodiametric cells containing slightly chloroplasts. Conclusions The structure layout is similar in both analyzed Galium species. The structural differences between the two Galium species are quantitative, reffering to the frequency, dimensions and localization of the trichomes, the frecquency of the crystalliferous cells, the lignification degree of the pith, the number and thickness of the secondary xylem rings, the sclerification degree of the libriform fibres, the number of the palisade cells layers, the amplitude of the undulations of the lateral walls of foliar epidemic cells. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. BORYSOV M. I., 1965- tude de la structure anatomique de Galium ruthenicum Willd., Farm. Zh. Ukrajin. R. S. R., 39, 5: 59-63 BUTTLER K. P., BRESINSKY A., 1966 Beitrag zur Zitologie von Galium ser. silvatica. Ber. bayer. bot. Gesellsch Erforsch. heim. Flora, 39: 25-28 CHERMEZON, H., 1910- Recherches anatomiques sur les plantes littorales, Ann. des Sci. nat., Bot., sr. 9.,12: 117-313 CIOCRLAN V., 2000- Flora ilustrat a Romniei. Pteridophyta et Spermatophyta. Ed. Ceres., Bucureti DARWIN S. P., 1980- Leaf venation and the classification of certain Rubiaceae. ICSEB-II 2ed Internat. Congr. Syst. and Ecol. Biol., Vancouver (Canada), Jully 17-24, 1980, Abstracts, 176 FISCHER F., 1937- Beitrge zur Pharmakognosie der Plataginalen and Rubialen. Anatomie des Laubblattes. Thsis. Basel HENDRICH R., 1977- Bemerkungen zur Variabilitt von Cruciata glabra (syn. Galium vernum). Preslia, 49: 193-201 KOHLMNZER S., 1964- Recherches botaniques et chimiques sur lespce collective Galium molugo L. en considrant les caryotypes croissant en Pologne. II. Recherches anatomiques. Dissert. Pharm., Pologne, 16, 3:381392 METCALFE C. R., CHALK L., 1972- Anatomy of the Dicotyledons. 2. Clarendon Press, Oxford

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10. NAPP-ZINN, KL., 1973, 1974 - Anatomie des Blattes. II. Angiospermen, In Handbuch der Pflanzenanatomie, Bd.VIII, A 1-2, Gebrder Borntraeger, Berlin, Stuttgart 11. PANT D. D., BHARATI M., 1965- Ontogeny of stomata in some Rubiaceae. Phytomorphology (Indic), 15, 3: 300-310 12. PAUC A., NYRDY E. I., 1961- Rubiaceae. In Flora R. P. Romne, 8: 524-589, Ed. Acad. Romne, Bucureti 13. PERROT E., PARIS R., 1971- Les plantes mdicinales. 1., dit. Presses Universitaires des France, Paris 14. ROBBRECHT E. (ed.), 1994 - Advances in Rubiaceae macrosystematics. Opera Botanica Belgica, Bruxelles, vol. 6 15. ROBBRECHT E., PUFF C., SMET E. (eds.), 1996- Second international Rubiaceae Conference. Proceedings 16. RUTISHAUSER R., 1984- Blattquirle, stipeln und Kollateren bei den Rubieae (Rubiaceae) in Vergleich mit andern Angiospermen. Beitr. z. Biol. Pflanz., 59, 3:375-424 17. SAINT-JUST S., 1904- Recherches anatomiques sur lappareil vgtatif arien des Rubiace. Thse, Paris 18. SOLEREDER H., 1893- Ein Beitrag zur anatomischen Charakteristik und zur Systematik der Rubiaceen. Bull. Herb. Boissier, 1: 167-199 19. STNESCU U. i colab., 2004 Plante medicinale de la A la Z, monografie ale produselor de interes terapeutic, 1, Ed. Gh. T. Popa, UMF Iai 20. TOKARZ H. T., SULMA T., BUJEWICZ M., 1969- The number of leaf stomata in Asperula odorata L. plants derived from ecologically different forest communities and from garden cultivation. Acta biol. med. Soc. Sci. Gedau, 14: 443-466 21. TOLLE H., 1913- Beitrge zur vergleichenden Anatomie der Rubiaceen. Thesis, Gttingen 22. TOMA C., GOSTIN I., 2000- Anatomical structure of some Rubiaceae species. An. t. Univ. Al. I. Cuza, Iai, ser. II a (Biol. veget.) 46: 1-10

The explanation of figures: Plate I. Galium album L. Fig. 1, 2. Cross-sections through the rhizome (x200). Fig. 3 (x40), 4(x200). Cross-section through the upper third of the aerial stem. Fig. 5, 6. Cross-sections through the middle third of the aerial stem (x200). Plate II. Galium album L. Fig. 1. Cross-section through the lower of the aerial stem. (x200). Fig. 2, 3. Cross-sections through the foliar limb (x200). Fig. 4, 5. Superficial sections through the folair limb (x200): 4. lower epidermis; 5. upper epidermis. Plate III. Galium verum L. Fig. 1, 2. Cross-sections through the rhizome (x100). Fig. 3 (x40), 4(x200). Cross-section through the upper third of the aerial stem. Fig. 5. Cross-sections through the middle third of the aerial stem (x200). Fig. 6. Cross-section through the lower of the aerial stem (x200). Plana IV. Galium verum L. Fig. 1(x40), 2-4 (x200). Cross-sections through the foliar limb Fig. 5. Superficial sections through the folair limb: lower epidermis (x200).

33

ANCA HEMCINSKI et al.

PLATE I

34

ANCA HEMCINSKI et al.

PLATE II

1 2

35

ANCA HEMCINSKI et al.

PLATE III

36

ANCA HEMCINSKI et al.

PLATE IV

2 3
Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

A COMPARATIVE STUDY REGARDING THE MORPHOLOGY AND ANATOMY OF THE VEGETATIVE APPARATUS IN TWO OCIMUM BASILICUM L. BREEDS MARIA MAGDALENA ZAMFIRACHE*, C. TOMA*, MARIA DUCA**, SIMONA DUNCA*, ZENOVIA OLTEANU*, M. TEFAN*, RAMONA GALE*, CLAUDIA PDURARIU*
Abstract. This study compares the structure and morphology of the vegetative subterranean and aerial organs in two Ocimum basilicum L. breeds cultivated in Turkey. The object of this study is to emphasize the infraspecific variation of the plant morphology and anatomy of these two breeds, underlining the importance of the secretory hairs that produce the volatile oils and concede this species the virtue of medicinal herb and aromatic plant. Key words: Ocimum basilicum L., morphology, anatomy, vegetative organs, secretory hairs

Introduction Ocimum basilicum L. of the Family Lamiaceae is a herb grown as a perennial in warm, tropical climates. Basil is originally native to Iran, India and other tropical regions of Asia and it is cultivated in greenhouses or in the field in many other regions too. Ocimum basilicum L. is an annual species with a vegetataive apparatus composed of a well ramified fibrous root, a strongly ramified, 60 cm long, four edged erect stem and many pointy ovate-lanceolate opposite leaves with atenuate serrate edges [1]. The flowers are quite big, white in colour and arranged in a terminal spike. The four stamens and the pistil are not pushed under the upper lip of the corolla, but lay over the inferior. Materials and methods The material of our investigations is represented by two Ocimum basilicum L. breeds cultivated in Turkey (to mark out the two species, they were noted with two numbers: 1 nonflowering specimen and 2 flowering specimen). The utilised methods are those currently used for vegatal anatomy investigations. Cross-sections through the vegetative organs using a botanical razor and a manual microtome have been executed. These sections have later been jewelised and tinted using iodine-green and ruthenium-red. The superficial lamina sections have been tinted using iodine-green. The sections were later analysed and photographed using a photonic microscope (NOVEX, Holland).
*

**

Al. I. Cuza University, Faculty of Biology, Carol I Bd., no. 20A, Iasi, 700506, Romania University of State, Faculty of Biology and Pedology, Chisinau, Moldavia

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Results and discussions The morphology of the vegetative apparatus Both basil breeds studied present a variable number (5 to 7 for the first breed and 8 to 9 for the second one) of strongly ramified aerial stems of various thickness (those of the first breed are thicker) that start out at the base of the root. There are leaves on the branches and the main stems, more numerous on the second breed plants and larger ones on the first breed plants (Pl. I: Fig. 1). The structure of the vegetative apparatus The structure of the subterrranean vegetative organs The histo-anatomical analysis of the subterranean vegetative apparatus of the two basil breeds revealed the fact that the main axis which is in the scientific literature considered to be the main root, has a series of tipical subterranean stem (rhizom) structural features: 1. Lignified pith in the center of the organ, composed of polygonal cells without meatuses between them and 4 primary ligneous bundles surounded by few ligneous parenchyme cells also with lignified walls, between them (Pl. I: Fig. 3c); 2. The secondary xylem which is entirely lignified forms two annual clearly visible rings (Pl. I: Fig. 3d): - the libriform fibers are prevalent in the first ring (the inner ring). These fibers have thick and slightly lignified walls. The radial range disposed, small diameter, scarce vessels are surrounded by few lignified ligneous parenchymatous cells. - the second ring (the external ring) is thicker, composed of: numerous variable diameter vessels (bigger than the ones in the first ring); these radial range disposed vessels are surrounded by few lignified ligneous parenchyme cells, and libriform fibers; these fibers have strongly lignified thiner walls (compared to those of the first ring). The felogene formation does not rely on the pericycle (which is the case of many dicotyledonous plants) but on the differentiation of different layers of the cortex, producing many layers of often stratified cork, composed of bigger cells than those of the pheloderme and those of the cortical parenchyme that is still unexfoliated or still persisting between the periderma successively formed (Pl. I: Fig. 3b). A genuine ritidoma that exfoliates along the organ is thus formed (Pl. I: Fig. 3a). Therefore, considering this organ as being a rhizom, the roots that form on it are endogenous formed adventive roots (Pl. I: Fig. 3e). The structure of these roots is secondary, as a single result of the cambium activity. The diarche type stellum has a primary structure (Pl. I: Fig. 3f). The structure of the aerial vegetative apparatus The stem of both basil breeds has a primary structure only in the upper third part and a secondary structure, as a result of the cambium activity, in the other two.

39

The outline of the cross section (Pl. II: Fig. 4, 5) differs in each third of the stem (upper, middle and lower) and in the two breeds as well. In the upper third part, the outline of the cross section is rectangular-quadrangular shaped, with 4 generally attenuate costas, some of them more evident than others, with deeper and narrower valecules berween them in the second breed. The valecules progresivly grow wider and they become less deep with the thickening of the organ, so that in a cross-section they show an allmost circular outline. The epidermis protects the entire surface of the stem (Pl. III: Fig. 6, 7) and it is composed of sligtly tangentially elongated isodiametrical cells (in the lower third) that have pericline walls, thicker than the others, a thin cuticle covering the external one. There are very few stomata present. There are two types of hairs: 1. uniseriate pluricellular trichomes that are present on the entire outer surface of the stem; 2. secretory hairs composed of a unicellular or bicellular hinge, a unicellular pedicle and an unicellular or bicellular gland (Pl. V: Fig. 10a, b, c). The number of secretory and tectorial hairs per suface unit decreases from the top to the base of the stem. There are more hairs (tectotial and secretory also) on the first breed stems than on the second breed stems (Pl. II: Fig. 4, 5). The parenchymatous-cellulosic cortex, meatus type in the upper third, is thin and slightly colenchymatic in a hypodermic position (in the first basil breed especially near the costas) and does not have a special type of endoderma on the exterior. The cortex cells tend to become tangentially elongated and the air spaces between them become larger as the stem grows thicker. The anticline division walls are visible inside the cortex inner cell layers in the lower third of the first basil breed stem. The primary structure stellum (Pl. III: Fig. 6, 7) follows the general outline of the cross section and has four large collaterally open type bundles in the four costas and one very small bundle composed only of phloemic elements between them. The large bundles have radial ranges of ligneous vessels separated by uniseriate or pluriseriate areas of parenchymatouscellulosic cells and the liberian tissue is composed of pierced tubes and annexe cells. Belts of sclerenchyma fibers can be observed at the end of the large phloem vascular bundles in the second basil breed (Pl. III: Fig. 7a) that have in this developing state less thickened but still cellulosic walls. The fiber walls get progressively thicker and lignified from the base up to the top of the stem (Pl. III: Fig. 6, 7). The thickening of the stem is based on the cambium activity that produces a thin phloem ring on the exterior and a thicker xylem one on the interior. The phelogen becomes differentiated based on an inner cortical layer at the base of the second breed stem, producing a single layer of cork (composed of very large cells that have thin walls and little cork) and 1-2 noncolenchymatous pheloderma layers. The cambium activity is initialy unequal in the circumference of the organ, producing more secondary ellements (phloem and xylem) in the large bundles (near the costas); thus the secondary vascular tissue rings are sinuous during this developing stage (Pl. II: Fig. 4b, Fig.

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5b). The cambium produces subsequently many vascular ellements between the costas so that both rings (secondary phloem and secondary xylem) become circular (Pl. II: Fig.4c, Fig. 5c). The secondary xylem ring is almost entirely lignified at the base of the stem (composed of vessels, libriform fibers, lignified ligneous parenchymatous cells, horizontaly lignified parenchymatous cells) in the second breed or it has a thick tangential cellulose ligneous parenchymatous belt only on one side of the organ circumference so that the pith is not situated in the center anymore but on the side, in the first basil breed. The pith is a meatus type cellulose-parenchymatous thick pith (Pl. II: Fig. 4a, b; Fig. 5a, b); the composing cell walls are lignified in the base of the stem (Pl. II: Fig. 4c, Fig. 5c). The amphystomatic type lamina presents very small dyacitic stomata situated on top of the epidermis. Its structure is heterofacial bifacial and the mesophyll is differentiated as a very elongated cell unistratified palisadic tissue and a pluristratified lacunous tissue (4 to 5 layers). The middle nervure (Pl. IV: Fig. 8a, Fig. 9a, b) is visibly prominent on the inferior side of the lamina and has a single vascular bundle inside the noncloroplastic noncolenchymatic fundamental parenchime, which is larger in the first basil breed. The relatively short uniseriated pluricellular trichomes can be found only on the lamina middle nervure (Pl. IV: Fig. 9b). There are two types of secretory hairs: a) located in a very small depression of the upper epidermis, having a bicellular gland (Pl. IV: Fig. 9c; Pl. V: Fig. 10d); b) located in a very large excavation of the lower epidrmis, having a four celled gland (Pl. IV: Fig. 8b; PL. V: Fig. 10e). Conclusions Investigating the vegetative apparatus of the two Ocimum basilicum L. breeds, an infraspecific variation regarding some morphological and anatomical features has been observed. The two basil breeds studied are distinguished by the following morphological features: 1) the size and density of the aerial stem leaves; 2) the developing stage of the subterranean vegetative apparatus. The presence of some subterranean stem (rhizom) specific features among the main subterranean axis indicates that the two basil breeds may be perennial, the species being considered annual according to the scientific literature. The structure of the vegetative apparatus in the two basil breeds differes according to the following features: a. regarding the stem: 1) the outline of the croos-section in the upper third; 2) the number and density of the trichomes on organ surface unity; 3) the lignification stage of the secondary structure stellum; 4) the absence or the presence of the protecting secondary tissues; b. regarding the lamina the developing stage of the vascular tissue in the middle nervure.

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The number of secretory hairs on organ surface unit and the number of cells that compose their gland concede this species the virtue of aromatic and medicinal plant. Our research has shown that the number of secretory hairs on the vegetative apparatus in the first basil breed is greater compared to the second one. Both breeds show more numerous secretory hairs on the lamina and on the tip of the stems, most of the four celled gland hairs being situated on the lamina. REFERENCES
1. 2. 3. 4. 5. CIOCRLAN V., 2000 - Floara ilustrat a Romniei, ed. a II-a. Edit. Ceres, Bucureti GUULEAC M., 1961 Ocimum L. n Flora R. P. R., Edit. Acad. Rom., Bucureti, 8: 390-393 RUGIN R., TOMA C., 1995 Histo-anatomical researches of some medicinal plants V. Labiatae. An. t. Univ. Al. I. Cuza Iai, s. a II-a (Biol. veget), 41: 17-22 TOMA C., RUGIN R., 1998 Anatomia plantelor medicinale. Atlas. Edit. Acad. Rom., Bucureti TOMA-GOSTIN I., TOMA C., IVNESCU L., 2002 Histo-anatomical aspects of Ocimum basilicum L., an important medicinal plant. 2-nd Conference on Medicinal and aromatic Plants of Southeast european Countries. Chalkidiki-Greece (Book of abstracts): 140

Explanation of figures: Fig. 1. The morphology of the aerial vegetative apparatus in Ocimum basilicum L.: first breed (on the left); second breed (on the right) (original macrophotographs). Fig. 2. The morphology of the subterranean vegetative apparatus in Ocimum basilicum L.: first breed (on the left); second breed (on the right) (original macrophotographs). Fig. 3. The structure of the subterranean vegetative apparatus in Ocimum basilicum L. second breed: a-e. rhizom cross sections: a. general view (unjewelised and untinted section) (x40); b. ritidoma detail (x200); c. pith and secondary xylem detail(x200); d. secondary xylem detail the border between the two annual rings (x200); e. the formation of an adventive root (x100). f. adventive root cross section (general view) (unjewelised and untinted) (original microphotographs). Fig. 4. The structure of the aerial stem on different levels in Ocimum basilicum L. first breed: aerial stem upper third (a), middle third (b) and lower third (c) cross sections (x40) (original microphotographs). Fig. 5. The structure of the aerial stem on different levels in Ocimum basilicum L. second breed: aerial stem upper third (a), middle third (b) and lower third (c) cross sections (x40) (original microphotographs). Fig. 6. The structure of the aerial stem on different levels in Ocimum basilicum L. first breed (details): aerial stem upper third (a), middle third (b) and lower third (c) cross sections (x200) (original microphotographs). Fig. 7. The structure of the aerial stem on different levels in Ocimum basilicum L. second breed (details): aerial stem upper third (a), middle third (b) and lower third (c) cross sections (x200) (original microphotographs). Fig. 8. The structure of the lamina in the Ocimum basilicum L. first breed: - middle nervure cross sections a)- unjewelised and untinted (x100) and mesophillun (b, c ) (x200) (original microphotographs). A secretory trichome and its four celled gland located inside a lower epidermis excavation (b) and a stomata in the superior epidermis (c) may be observed. Fig. 9. The structure of the lamina in Ocimum basilicum L. second breed : - middle nervure cross sections a)- unjewelised and untinted (x100) and mesophillun (b) (x200) (original microphotographs). A tectorial trichome on the adaxial side of the middle nervure (b) and a secretory trichome and its bicellular gland located inside a small excavation in the superior epidermis (c) may be observed. Fig. 10. Secretory hairs in an aerial stem cross section in Ocimum basilicum L. (a, b, c) and in a lamina superficial section (d, e) a. unicellular gland secretory trichome; b. bicellular gland secretory hairs; c. bicellular gland secretory trichome; the secretory product eliminated between the wall and the cuticle can also be observed; d. bicellular gland secretory trichome; e. four celled gland secretory trichome (x800) (original microphotographs).

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MARIA MAGDALENA ZAMFIRACHE et al.

PLATE I

3. a

3. b

3. c

3. f
43

3. e

3. d

MARIA MAGDALENA ZAMFIRACHE et al.

PLATE II

4. a

4. b

4. c

5. a

5. b

5. c

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MARIA MAGDALENA ZAMFIRACHE et al.

PLATE III

6. a

6. b

6. c

7. a

7.b

7.c

45

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PLATE IV

8. a

8. b

8. c

9. a

9. b

9. c

46

MARIA MAGDALENA ZAMFIRACHE et al.

PLATE V

10. a

10. b

10. c

10. d

10. e

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

A COMPARATIVE NUMERIC ANALYSIS OF THE SECRETORY TRICHOMES BELONGING TO THE FOLIAR LIMB IN SOME DROSERA SPECIES IRINA STNESCU*, GABRIELA VASILE**
Abstract. The paper is focused on a comparative numeric analysis of the secretory pedicelate (tentacular) trichomes of the upper epidermis of the leaf in 16 Drosera species and of the sessile secretory trichomes belonging to both upper and lower epidermis in the same species. The bigger is the number of the secretory trichomes, in both epidermises, the more efficient is the plant in capturing and retaining different organisms. This way, the carnivorous plants can completely benefit by the nutritive compounds resulted from the preys. Key words: Drosera, secretory trichomes, comparative analysis

Introduction Drosera species are characterized by the presence of the secretory pedicelate (tentacular) and sessile trichomes in the foliar limb. Their structure has been discussed in many anterior papers [2, 3 and 4]. We proposed to present a comparative numeric analysis, underlining the importance of the secretory trichomes in the normal manifestation of their carnivorous character. Material and methods We counted the secretory pedicelate trichomes of the upper epidermis and the sessile trichomes of both upper and lower epidermis belonging to mature leaves (from the basis of the rosette) in all 16 Drosera species (D. aliciae Hamlet, D. binata Labill, D. brevifolia Pursh, D. burkeana Planch, D. capensis L.- with three forms: D. capensis Alba L., D. capensis Narrow Leaf L. and D. capensis Rubra L., D. capillaris Poir, D. cuneifolia Thunb, D. dielsiana Exell et Laundon, D. intermedia Hayne, D. lovella T. N. Bailey, D. montana St. Hill and D. spatulata Labill, all belonging to the collection of Alexandru Borza Botanical Garden, Cluj-Napoca, and Drosera rotundifolia L. from the Natural Reservation Grdinia Meadow, Suceava district). We have separately noted the number of the trichomes of 20 microscopic areas, randomly chosen (belonging to the same leaf, to different leaves of the same individual or to various individuals). We noted: A=microscopic area, D=diameter, r=radius. If D = 860 m, r = 430 m, A = x r2 (m)2 , A = 0.580586 mm2 First we noted the number of trichomes depending on their type, location (upper epidermis or/and lower epidermis) and species and then we calculated their average for the analyzed area (A = 0.580586 mm2). The next step was to calculate the number of trichomes corresponding to A= 1 mm2, as follows: Averrage/0.580586
* **

Al. I. Cuza University of Iasi, Botanical Garden Anastasie Fatu, Romania, irinastanescu2005@yahoo.com Al. I. Cuza University of Iasi, Faculty of Biology, Biochemistry and Molecular Biology Laboratory , Romania

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Results and discussions In order to completely analyze the efficiency of Drosera leaves regarding the capturing of various preys, we considered of great importance the number of the secretory trichomes of the foliar limb. All investigated Drosera species present pedicelate (tentacular) secretory trichomes in the upper epidermis and sessile secretory trichomes in both upper and lower epidermis. A tentacle consists of a various length stalk, longitudinally traversed by fine tracheids ending in large groups in the glands head. The outer part of the tentacles heads is composed of 2-3 layers of glandular radial elongated cells (especially those from the external layer) surrounded by a thin cuticle. The tentacles attract the preys by secreting sticky sweet mucilage, which contains enzymes too, in order to decompose the preys body; then, the tentacles, helped by the sessile trichomes, absorb the nutritive compounds. The sessile secretory trichomes consist of two parallel rows of two cells each, on top of the epidermal cells, and two cells between those of the epidermis. These trichomes absorb the substances secreted by the tentacles, substances which seep on the leaf surface. Comparing the number of the sessile secretory trichomes of the lower epidermis (Fig. 1), we sow that D. capensis Alba and D. intermedia: 31.09 and 31.52, respectively. D. aliciae registrated the lowest value (9.13); D. montana and D. spathulata presented other similar values: 13.18 and 13.09, respectively. Comparing the number of the pedicelate (tentacular) secretory trichomes of the upper epidermis (Fig. 2), we evidenced that the D. binata presented the highest number of tentacles (14.04), while D. montana registrated the lowest number (3.27). D. capillaris and D. cuneifolia showed similar values: 8.01 and 7.75, respectively. An attentive analysis of the Fig. 3 gives us information upon the highest number of sessile secretory trichomes of the upper epidermis, registered in D. intermedia (25.06), and the lowest number, in D. binata (6.20). D. capensis and D. capensisNarrow Leaf present similar values: 21.01 and 20.23, respectively, in one category, in other category being D. brevifolia and D. burkeana: 13.43 and 13.26, respectively. In order to check the possible differences or similarities occurring, numerically, between both types of secretory trichomes belonging to the upper epidermis in all investigated species, the Anova test - the bifactorial model (without interaction) with an equal number of observations in the cell, has been applied [1 and 5]. The test alows us calculate the square sums - based on the variability sources, the factor value and its critical value. Starting with the experimental results obtained, the null (H0) and the alternative (H1) hypothesis of the test have been formulated. F = factor; Crit. F = Critical factor Observation: F < Crit. F - H0 is accepted and H1 is refused; F > Crit. F - H0 is refused and H1 is accepted. The hypothesis of Anova test - the bifactorial model (without interaction): 1. H01: Da = Db = Dbr = Dbk = Dc = DcA = DcNL = DcR = Dcp = Dcn = Dd = Di = Dl = Dm = Dr = Ds

49

H11: Da Db Dbr Dbk Dc DcA DcNL DcR Dcp Dcn Dd Di Dl Dm Dr Ds, = trichomes averrage, Da = D. aliciae, Db = D. binata, Dbr = D. brevifolia, Dbk = D. burkeana, Dc = D. capensis, DcA = D. capensis Alba, DcNL = D. capensis Narrow Leaf, DcR = D. capensis Rubra, Dcp = D. capillaris, Dcn = D. cuneifolia, Dd = D. dielsiana, Di = D. intermedia, Dl = D. lovella, Dm = D. montana, Dr = D. rotundifolia, Ds = D. spathulata 2. H02: tt = st H12: tt st, where tt = tentacular trichomes, st = sessile trichomes The results of our study have been presented in Table I and Figure 4. A comparison between the values of the two (calculated and statistical) factors led to the following conclusions: 1. the calculated value of the factor on columns (0.804) is lower than its critical value (2.403), so the alternative hypothesis (H11) is refused and the null hypothesis (H01) is accepted (considerable numeric differences were not registered between the secretory trichomes in the investigated species); 2. the calculated value of the factor on rows (33.729) is significantly higher than its critical value (4.543), so the null hypothesis is refused (H02) and the alternative hypothesis is accepted, that is why we can conclude: notable numeric differences between both types of secretory trichomes belonging to the upper epidermis exist. In order to check the possible differences or similarities occurring between the number of sessile secretory trichomes belonging to both epidermis of the leaf, the Anova test - the bifactorial model, with an equal number of observations in the cell, has been applied [5]. The hypotheses are: 1. H01: H01: Da = Db = Dbr = Dbk = Dc = DcA = DcNL = DcR = Dcp = Dcn = Dd = Di = Dl = Dm = Dr = Ds H11: Da Db Dbr Dbk Dc DcA DcNL DcR Dcp Dcn Dd Di Dl Dm Dr Ds , where = averrage of trichomes, Da = D. aliciae, Db = D. binata, Dbr = D. brevifolia, Dbk = D. burkeana, Dc = D. capensis, DcA = D. capensis Alba, DcNL = D. capensis Narrow Leaf, DcR = D. capensis Rubra, Dcp = D. capillaris, Dcn = D. cuneifolia, Dd = D. dielsiana, Di = D. intermedia, Dl = D. lovella, Dm = D. montana, Dr = D. rotundifolia, Ds = D. spathulata 2. H02: upp.ep. = lr.ep. H12: upp.ep. lr.ep, where upp.ep. = upper epidermis, lr.ep. = lower epidermis The results of our study have been presented in Table II. Comparing the values of both (calculated and statistical) factors, we can affirm: 1. the calculated value of the factor on columns (6.330) is higher than the critical one (0.00047), so the alternative hypothesis (H11) is accepted, or, in other words, significantly differences were registered between the 16 Drosera species, regarding the number of the sessile secretory trichomes in both epidermis of the limb; 2. the calculated value of the factor on rows (14.609) is higher than the critical one (0.0016), which alows us to refuse the null hypothesis (H02) and accept the alternative one; so, we can admit that there are considerable differences between the two epidermis of the limb, regarding the number of sessile secretory trichomes.

50

The graphical representation of the number of sessile secretory trichomes belonging to both (upper and lower) epidermis in the investigated species may be observed in Fig. 5. Conclusions The small number of sessile secretory trichomes of the upper epidermis in D. binata is balanced by the great number of pedicelate (tentacular) trichomes; the tentacles secrete mucilage rich in sugars and protheolitic enzymes and, then, absorb the compounds resulted from the degradation of the pray, helped by the sessile trichomes. In all the other investigated species, the number of sessile trichomes is higher than that of the tentacles, due to their size differences (a great number of sessile trichomes is required to absorb the substances accumulated in the upper epidermis and maintain it clean, proper for respiration). The lower epidermis presents a higher number of sessile trichomes than the upper epidermis; in the upper epidermis, the absorbtion of the nutritive compounds is realized by both categories of trichomes, while in the lower epidermis there are only the sessile trichomes to absorb the droplets of substances. The greater the number of trichomes, the better can the plants beneficiate the substances obtained by the preys body and more, the better can the plants grow and develop. REFERENCES
1. 2. 3. 4. 5. FOWLER J., COCHEN L., JARVIS P., 2000 - Practical statistics for field biology, Second Edition, Ed. by John Wiley & Sons, Ltd., England, 186 207 METCALFE C. R., CHALK L., 1972 - Droseraceae (1: 581-585). In Anatomy of the Dicotyledons, Clarendon Press, Oxford STNESCU IRINA, TOMA C., 2008 - Secretory structures of the carnivorous plants belonging to the Droseraceae family. Proceedings of the 1st International Conference: Environment - Natural Science - food industry in European Context, Baia Mare, 1: 323-326 TARNAVSCHI I. T., 1957 - Adaptrile morfologice ale plantelor carnivore. Natura, 4: 76-92 VARVARA M., ZAMFIRESCU T., NEACU P., 2001 - Lucrri practice de ecologie, Ed. Univ. Alexandru Ioan Cuza Iai

51

no. of sessile trichomes in the lower epidermis

no. of tentacular trichomes

10

15

20

25

30

35

.a li c ia e
9.13

10

12

14

16

.a li c ia e

D .b in at a
15.42

3.88

D .b

D .b re v ifo l ia
10.51

D .b
14.04

in at a

re v

D .b ur ke a
11.97

D .b
5.08

ifo l ia na
5.77

D .c .c

ur ke a

D .c

.c

.c

26.52

5.77 7.06

31.09

Fig.1. Graphical representation of the number of sessile secretory trichomes of the lower epidermis (in A=1 mm2) in the 16 Drosera species

Fig.2. Graphical representation of the number of pedicelate (tentacular) secretory trichomes of the upper epidermis (in A=1 mm2) in the 16 Drosera species

52
4.48 5.77 8.01 7.75 3.36 6.55 7.49 3.27 5.51 4.65

24.37

19.72

15.59 19.29 11.80 31.52 29.28 13.18 27.90

ap en .c ap si ap en s en si s si "A s "N lb a ar D ro .c w ap Le en af si s "R ub r a D .c ap il l ar D is .c un ei fo l ia D .d ie ls ia D na .i nt er m ed ia D .l ov el D la .m on ta D n .r a ot un di fo D li a .s pa th ul at a

na ap en ap si en ap s si en s si "A s l "N ba ar ro D .c w ap Le en af si s "R ub ra D .c ap il l ar D is .c un ei fo li a D .d ie ls i a D na .i nt er m ed ia D .l ov el la D .m on t an D .r a ot un di fo D l ia .s pa th ul at a

13.09

no. of sessile trichomes in the upper epidermis

no. of trichomes
D

10

15

20

25

30

.a li c ia e
9.99

10
D .b in at a
6.20

15

20

25

30

0
D .b re v ifo l ia
13.43

5
D .b ur ke a
13.26

D .c

.c

.c

21.01 19.46 20.32

Fig.3. Graphical representation of the number of sessile secretory trichomes of the upper epidermis (in A=1 mm2) in the 16 Drosera species

Fig.4. Comparative representation of the number of both pedicelate (tentacular) and sessile secretory trichomes of the upper epidermis in the 16 Drosera species

53
tentacular trichomes sessile trichomes

11.37 13.69 15.76 10.68 25.06 16.45 8.96 17.14

D. al ic i D. ae b in D. br at a e D. vifo bu lia rk ea D. D D ca . ca . ca na pe pe pe ns n n is s is s is "A D. "N lb a ca r a pe row ns Le is a "R f ub D. r a ca D. pill cu ari ne s D. ifo l ia d D. iels ia in n te rm a ed D. ia lo ve D. l l m on a D. ta ro na tu nd D. if sp oli a at hu la ta
na ap en ap si ap en s en si s si "A s "N lb a ar D ro .c w ap Le en af si s "R ub r a D .c ap ill ar D is .c un ei fo lia D .d ie ls i an D .i a nt er m ed ia D .l ov el la D .m on ta D na .r ot un di fo D li a .s pa th ul at a
13.00

35 30 25 20 15 10 5 0
at a vi fo bu l rk ia D. D. D. e a n ca ca c a pe ap pe ns en ns s is is is D. "N "A lb c a ar r pe o w a ns is Lea "R f D . ub c a ra D. pill cu ari s n D. e ifo li d D . i e ls a ia in n te rm a ed D. ia l D. ov e l D . mo la ro nta na tu D. n di s p f ol a t ia hu la ta D. br e
upper epidermis lower epidermis

no. of trichomes

ic i al D.

Fig.5. Comparative representation of the number of sessile secretory trichomes belonging to both epidermis Table I. Calculated and critical values of the factors Source of variability SS g. l. Calculated F SS

D.

D.

bi n

ae

Critical F

Rows (type of trichome) 589.446 1 589.446 33.729 4.543 Columns (species) 210.910 15 14.060 0.804 2.403 Total 1062.493 16 SS = sum of squares, g. l. = degree of freedom (g. l. = n-1, where n = values),

SS = average sum of squares

Table II. Calculated and critical values of the factors Source of variability SS g. l. Calculated F SS Critical F

Rows (type of epidermis) 173.911 1 173.911 14.609 0.0016 Columns (species) 1130.431 15 75.362 6.330 0.00047 Total 1482.901 16 SS = sum of squares, g. l. = degree of freedom (g. l. = n-1, where n = values),

SS = average sum of squares

54

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

MORPHOLOGICAL RESEARCHES REGARDING THE INFLUENCE OF TOPSIN M TREATMENTS ON MENTHA LONGIFOLIA (L.) HUDS. SPECIES CLARA APROTOSOAIE A.*, URSULA STNESCU*, ANCA MIRON*, VIOLETA FLORIA**, OANA CIOANCA*, MONICA HANCIANU*
Abstract. Topsin M is a common systemic fungicide used as protective/curative substance for alimentary and medicinal plants. That is why it is relevant to evaluate the influence of topsin M upon the morphological features of Mentha longifolia, a volatile oil producing medicinal plant and parental species for the hybrid Mentha piperita. We noticed statistical significant variations (oneway Anova) of the dimensional features of the leaves from topsin M treated plants comparing to the control. Key words: Mentha longifolia, topsin M, morphological features

Introduction The abusive and disordered employment of pesticides has the potential to harm human health and the environment and pose the ecosystems to ecological risk. Because of possible health effects, widespread use and insufficient data, pesticide monitoring in plants is necessary. Some of the pesticide monitoring aspects refere to the investigation of the pestide influence on the morphological features of medicinal plants. Data from literature report that pesticides may interfere with morphological features of plants, as following: smaller and fewer leaves, smaller axial organs (carbamate and amido-type erbicides) [5]; depigmentations along the nervures, necrosis at edge of the leaves (urea and sulphonylurea derivates; alchyl N-phenylcarbamates and alchyl-N-phenylthiocarbamates) [1, 2, 3]; variation of the lenght of the lamina of the leaf and the lenght of the whole plant (fungicides) [4]. Topsin M is a common systemic fungicide used as a protective/curative substance for alimentary and medicinal plants. That is why it is relevant to evaluate the influence of Topsin M upon the morphological features of Mentha longifolia, a volatile oil producing medicinal plant and parental species for the hybrid Mentha piperita. Material and methods

* Faculty of Pharmacy, University of Medicine and Pharmacy Gr.T.Popa, Iasi, Romania **Botanical Gardens Anastasie Fatu, Iasi, Romania

55

Plant materials were brought from the experimental lots in Anastasie Fatu Botanical Garden, Iasi. In this experimental area, parallel cultures of Mentha longifolia (L.) Huds have been made in the period 2001-2003. Thus, every year there have been two experimental fields: a field which had no pesticide treatment (control area) and a field which had been treated with pesticide. The antifungal treatment was achieved in vegetative phase by spraying a wettable powder of Topsin M 70 PU (TM) (Oltchim Rm. Valcea-Romania) as 0,1% and 0,4% aqueous solutions. Investigated samples of Mentha longifolia are presented in table I: Table I: Samples of Mentha longifolia
Nr. 1. 2. 3. 4. 5. 6. 7. Sample Control 2001 Treatment TM 0,1% 2001 Control 2002 Treatment TM 0,4% 2002 Control 2003 Treatment TM 0,1% 2003 Treatment TM 0,4% 2003 Codification M.l. M 2001 M.l. TM 0,1% 2001 M.l. M 2002 M.l. TM 0,4% 2002 M.l. M 2003 M.l. TM 0,1% 2003 M.l. TM 0,4% 2003

The morphological study has been achieved through the analysis of the parameters concerning aspect, colour and dimensions of the leaves (officinal product) from both treated and untreated plants, as following: - the shape; - the edges, base and top; - covered hairs for the both faces of leaves; - the length and breadth (cm), depending on the leaf insertion: top, middle or the base of the stem. For each type of leave, for each variant of treatment and for each control, there have been made 10 measurements of the studied parameter. The dimensional parameters data were statistically analysed using the Oneway Anova method and the J.M.P. Programme 5.0.1.2. (SAS Institute, Cary N.C., S.U.A). Results and discussion Mentha longifolia leaves from both untreated (Fig. 6) and TM 0,1% (fig. 7) and TM 0,4% (fig. 8) treated plants have the some morphological features of the leave. The lanceolate or oblong leaves were sessile and arranged opposite on the stem. The tip of the limb is sharp and the base is narrowed. The edges of the leaves are convexe and almost parallel and have triangular teeth, 1-4 mm distanced orientated forward. The nervation is pennate and proeminent on the lower surface. Leaves have covered hairs placed especially along the nervures on the lower surface. The leaves are dark green on the upper surface and lighter on the lower surface. The powdered leaves have an aromatic, characteristic smell.

56

The dimensional parameters of both untreated and treated vegetal material are shown in Table II, as minimal and maximal registered for each type of leave. Table II. The dimensional parameters (cm) of the lamina from Mentha longifolia
Top leaves Sample lenght M.l. M 2001 M.l. TM 0,1% 2001 M.l. M 2002 M.l. TM 0,4% 2002 M.l. M 2003 M.l. TM 0,1% 2003 M.l. TM 0,4% 2003 2.0 2.5 2.4 2.9 2.3 2.9 1.9 2.9 1.7 2.3 2.3 3.3 2.4 4 breadth 0.9 1.7 1.4 - 1.9 1.1 1.6 0.8 1.4 0.7 1.2 1.2 1.9 1 - 1.9 lenght 3.0 4.2 3.2 4.2 3.5 4.3 3.6 4.2 3.5 4.3 3.8 4.4 3.9 4.8 breadth 1.2 1.6 1.3 1.8 1.2 1.8 1.1 1.6 1.4 1.6 1.7 2.0 1.2 2.1 lenght 2.8 4.1 3.0 4.4 3.0 4.3 3.0 4.1 3.0 5.0 2.9 3.8 2.7 3.6 breadth 1.0 1.7 1.2 1.9 1.3 1.6 1.2 1.6 1.3 1.9 1.3 1.8 1.3 1.9 Middle leaves Basal leaves

The dimensional parameters data were statistically analysed using the Oneway Anova method and the J.M.P. Programme 5.0.1.2. (SAS Institute, Cary N.C., S.U.A) [6]. Statistical data (x -mean; SD standard deviation; p*- indicator foe evaluation of statistical significance) were presented in Tables III and IV: Table III. Biometrical data of Mentha longifolia leaves the length of the lamina (cm)
Top leaves Sample x 2.23000 2.57000 2.61000 2.49000 2.10000 SD 0.176698 0.188856 0.228279 0.317805 0.240370 0.18409 0.3450 p* 0.0006 x 3.71000 3.78000 3.82000 3.92000 4.02000 SD 0.369534 0.339280 0.342540 0.225093 0.274064 0.16502 0.4504 3.39000 3.43000 4.39000 p* 0.6643 x 3.22000 3.32000 SD 0.456557 0.545283 0.472464 0.535516 0.645411 0.47821 0.8614 p* 0.6619 Middle leaves Basal leaves

M.l. M 2001 M.l.TM 0,1% 2001 M.l. M 2002 M.l.TM 0,4% 2002 M.l. M 2003

57

M.l. TM 0,1% 2003 M.l. TM 0,4% 2003

2.77000

0.394546

4.14000

0.206559

3.39000

0.369534

2.94000

0.602218

0.35409

4.46000

0.283627

0.15498

3.17000

0.333500

0.69821

*It is considered that the results are statistically significant if p* is lower than 0.05, in case of 2001 and 2002 samples, or if p* has a positive value for 2003 samples. Table IV. Biometrical data of Mentha longifolia leaves the breadth of the lamina (cm)
Top leaves Sample x 1.24000 1.63000 1.36000 1.15000 1.01000 1.44000 1.34000 SD 0.250333 0.176698 0.171270 0.177951 0.172884 0.18511 0.195505 0.279682 0.08511 1.84000 1.72000 0.107497 0.234758 0.02982 1.66000 1.61000 0.142984 0.246982 0.14197 0.0150 p* 0.0008 1.57000 1.48000 1.40000 1.52000 0.182878 0.161933 0.200000 0.063246 0.14982 0.3386 1.52000 1.42000 1.47000 1.69000 0.278089 0.113529 0.125167 0.196921 0.19197 0.3618 x 1.41000 SD 0.137032 p* 0.0400 x 1.30000 SD 0.262467 p* 0.0855 Middle leaves Basal leaves

M.l. M 2001 M.l.TM 0,1% 2001 M.l. M 2002 M.l.TM 0,4% 2002 M.l. M 2003 M.l. TM 0,1% 2003 M.l. TM 0,4% 2003

The statistical data reveal that the following dimensional variations compared to the corresponding controls are significant: - increase in lenght and breadth of the top lamina at the 0,1% treated plants; - increase in breadth of the middle lamina at 0,1% treated plants as well as an increase of lenght and breadth of the same leave at 0,4% treated plants; - decrease in lenght of basal lamina at TM 0,1% and TM 0,4% treated plants; - increase in breadth of the basal leaves at TM 0,1% treated plants. Conclusions Antifungal treatment wih Topsin M did not affect morphological features of Mentha longifolia leaves, except dimensional features. We noticed statistical significant variations (Oneway Anova) of the dimensional features of the leaves from Topsin M treated plants comparing to the control, as following: - lenght and breadth of top lamina increase at 0,1%TM treated plants (fig. 1, 2); - lenght and breadth of middle lamina increase at 0,4% TM treated plants (fig. 3, 4); - only the breadth of middle and basal lamina increase at TM 0,1% treated plants; - the lenght of the basal lamina decreases at TM0,1% treated plants (fig. 5).

58

REFERENCES
1. 2. 3. 4. 5. 6. GOODWIN S., AHMAD N., 1998 - Relationship between azinphosmethyl usage and residues on grapes and in wine in Australia. Pesticide Science, 53 (1): 96-100 GUANG-GUO Y., WILLIAMS B., 1999 - Herbicides residues in grapes and wine. J. Environ. Sci. Health B, 34 (3): 397 411 ERSEN F., KRALOVA K., MACHO V., 2000 - New findings about the inhibitory action of phenylcarbamates and phenylthiocarbamates on photosynthetic apparatus. Pesticide, Biochemistry and Physiology, 68 (2): 113-118 NITA M,1997. Teza de doctorat, Iasi SLONOVSCHI V., NITA M., NECHITA A., 2001 - Prezent i viitor n combaterea buruienilor, Edit. Ion Ionescu de la Brad, Iasi WAYNE D., 1999 - Biostatistics: a foundation for analysis in the health sciences - 7th ed.

*It is considered that the results are statistically significant if Oneway Anova diagrames of the samples have a reduced level of superposition
4.5 4 L varf 2003 3.5 3 2.5 2 1.5 Martor tratam 0.1% Proba tratam 0.4%

All Pairs Tukey-Kramer 0.05

With Control Dunnett's 0.05

Samples

TM 0,1%

TM0,4%

Fig.1. Oneway Anova diagrame for the lenght of the lamina of the Mentha longifolia top leaves

59

2 1.75 D varf 2003 1.5 1.25 1 0.75 0.5 Martor tratam 0.1% Proba tratam 0.4%

All Pairs Tukey-Kramer 0.05

With Control Dunnett's 0.05

Samples

TM 0,1%

TM 0,4%

Fig. 2 Oneway Anova diagrame for the breadth of the lamina of the Mentha longifolia top leaves

4.5 L tulp 2003

3.5 All Pairs Tukey-Kramer 0.05 With Control Dunnett's 0.05

Martor

tratam 0.1% Proba

tratam 0.4%

Samples

TM 0,1%

TM 0,4%

Fig. 3 Oneway Anova diagrame for the lenght of the lamian of the Mentha longifolia middle leaves

60

2.2 2 1.8 1.6 1.4 1.2 1 Martor tratam 0.1% Proba tratam 0.4%

tulp 2003

All Pairs Tukey-Kramer 0.05

With Control Dunnett's 0.05

Samples

TM 0,1%

TM 0,4%

Fig.4.Oneway Anova diagrame for the breadth of the lamian of the Mentha longifolia top leaves

5.5 5 Lbazal 2003 4.5 4 3.5 3 2.5 Martor tratam 0.1% Proba tratam 0.4%

All Pairs Tukey-Kramer 0.05

With Control Dunnett's 0.05

Samples

TM 0,1%

TM 0,4%

Fig.5. Oneway Anova diagrame for the lenght of the lamina of the Mentha longifolia basal leaves

61

Fig. 6: Control

Fig. 7: Treatment TM 0,1%

Fig. 8: Treatment TM 0,4%

62

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

A HISTO-ANATOMICAL STUDY OF THE MODIFICATIONS INDUCED BY THIOPHANATE METHYL ON THE CALENDULA OFFICINALIS L. STRAIN LUMINIA HUANU-BASHTAWI, C. TOMA
Abstract. The effect of the Topsin M fungicide (thiophanate methyl) on the anatomical structure of the Calendula officinalis L. strain is evidenced, comparatively with an untreated sample. The histological modifications caused by this treatment, applied in two different concentrations (of 0.1% and 0.5%, respectively) have the same stimulation effects as those observed in similar experiments. The crosssections, made at three different levels, were performed through the main branches occurring, in the moment of fungicide application, in different stages of development - once known that the morpho-histoanatomical type manifestations of pesticides depend on the extent of tissular differentiation as well as on the development stage of the treated organ. Key words: Calendula, Topsin M, cytokinin hormone-type action, strain, histo-anatomical modifications.

Introduction In recent years, considering the secondary effects of pesticides, drastic measures for reducing their application have been taken, the existing methods for controling different diseases and pests involving elimination of highly-toxic fungicides, a lower concentration of those still present on the market, and introduction of some biologically-active principles [10]. In spite of the success recorded with certain biopreparations, in most cases, total substitution of fungicides with biological agents did not work [2], integration of chemical control with the biological one representing the most promising strategy to combat plant pathogens, with minimum effects on the natural environmental equilibrium [4]. In everyday practice, thiophanate methyl, which is a benzimidazolic fungicide, may be applied in combination with certain biological agents, such as Pseudomonas fluorescens or Trichoderma harzianum [5, 12], in which selected mutant strains are resistant to the action of carbendazime (MBC) the main metabolite of thiophanate methyl and the active substance in the plant [9]. In their experiments made on Nicotiana tabacum, Garcia et al. [7] demonstrated that the toxicity of carbendazime is coordinated by the dose-response relation: at a 50% lower concentration (1.3mM) than the recommended value (2.6mM), the dry mass, the concentration of carotenoids and some mineral elements (N, K) show a positive resnet, comparatively with the reference, while a higher concentration (5.2mM) is phytotoxic. Besides the advantages referring to biomass production, and especially to a reduced fungicide amount (which is actually a condition of the new regulations on the integrated system of plant protection), application of a lower dose induces an increase in the concentration of polyphenolic compounds, comparatively with the reference, and, especially, higher than that of the treatment applied in the recommended concentration; at a
Al. I. Cuza University of Iai, Faculty of Biology, Bd. Carol I, no. 20A., Iai, Romania bashtawibio@yahoo.com

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higher fungicide dose, inhibition of their synthesis may be observed, in spite of the lowest concentration of polyphenolic compounds recorded in the reference sample [6]. Considering the importance of the phenolics in the host-pathogen interaction, their ability of granting resistance to pathogens either directly or indirectly, by mediating the plants Systemic Acquired Resistance as well as reducing the amounts of applied fungicide is especially interesting for reducing the polluting effects of such substances upon the environment [13]. Such beneficial effect - of the cytokynin hormone-type action of the benzimidazolic fungicides - has not been always observed in either practical or experimental studies, even if they had been applied in recommended doses [1]; more than that, the utilization of carbendazime, benomyl or thiophanate methyl fungicides was compromised by the manifestation of some secondary phytotoxic effects [3, 11]. On the basis of such observations, as well as of the idea that most of the investigations devoted to thiophanate methyl involve mainly study of the metabolism, toxicity or estimation of the maximum residual potential from the vegetal material, and considering the influence that fungicides might exert on the morpho-anatomy of the treated plants [8], the present study analyzes the histo-anatomical modifications possibly induced in the Calendula officinalis strain by the thiophanate methyl and/or its metabolite (MBC), comparatively with a non-treated sample, with a well-known structure [14]. Material and methods The experimental material, cultivated in the Anastasie Ftu Botanical Gardens of Iasi, was obtained from seeds of the Petrana kind, provided by the Research Station for Medicinal and Aromatic Plants of Fundulea. Besides the treated plants (TM70 0.1% and TM70 0.4%), a sample batch, formed of nontreated plants, was prepared for comparative purposes. The administration of fungicide, as a moisty powder, was made three times (at intervals of 7 and 10 days), in the moment of branching or of the first anthodium formation, the plants possessing 30-35 nomophyles. The vegetal material, harvested 10 days after the last treatment, was fixed and conserved in 70% ethanol, then processed according to the methods commonly applied in studies of vegetal anatomy. The light micrographs were performed on a Novex (Holland) microscope, using a Canon A95 camera. In this paper we used the following abbreviations: Ca. of. M - Calendula officinalis, control (untreated plants); Ca. of. TM 0,1% - Calendula officinalis, treated with Topsin M 0,1%; Ca. of. TM 0,4% - Calendula officinalis, treated with Topsin M 0,4%. Results and discussions Cross-sections in the upper third of the main branches The reference contour of the cross-section, intensely ribbed (12-13 ribs and an equal number of valecules), numerous secretory hairs. The bark, cholenchymatized in the ribs and parenchymatous-assimilatory in the rest, includes 5-6 layers of cells with slightly thickened walls. Conducting fascicles: 12-13 large or intermediary and 7, respectively, small ones, each evidencing a primary structure and a girdle of periphloemic fibers with thin, cellulosic walls (Fig. 1, 2, 3). The medullary rays are parenchymatic-cellulosic, only in

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front of a few of them occurring small islands (2-3), with very few phloem elements. The pith is thick, parenchymatous-cellulosic. TM 0.1% treatment the outline of the cross-section (larger than in the reference) shows less ribs (9-10), of various size, while the cholencymatic cells have thicker walls (Fig. 1). The epidermal cells, with thicker external walls, are covered by a thicker cuticle. On the margin of the bark, tangentially-elongated aeriferous cavities may be observed. Unlike the reference, more numerous and larger conducting fascicles are present, as follows: 18 large or intermediary and 8 small ones, all underlining a primary structure (Fig. 1, 2); more than that, more numerous, very small conducting fascicles (6-7), possessing exclusively liberian elements (Fig. 2, 4), are observed. The cambium is more active, being formed of more layers (3-4). Many of the xylem vessels appear in process of formation (Fig. 3). The bark evidences 2-3 small, circular conducting fascicles, each with a thin girdle of sclerenchymatic fibers at the periphery of the liber (Fig. 4). Some fascicles are collateral type, structurally similar to those from the central cylinder, others (observed in the main strain) evidencing liberian elements and even incipien ligneous vessels on the internal side of the conducting fascicle, as well, actually representing a form of transition towards the circular hadrocentric vascular bundles, surrounded either partially or totally by liber, at the periphery. Such abnormal fascicles, resulted from the stimulation activity induced by thiophanate methyl, have been noticed in different positions at this level of the strain: in the bark, in the vicinity or in the thickness of the girdles of periphloemic fibers and even inside the liber from the conducting fascicles of the central cylinder (Fig. 5). TM 0.4% treatment diameter of the cross-section larger than in the other 2 samples, more prominent ribs (7), alternating with smaller ones (6-7), while the cholenchyma evidences cells with less thick walls, comparatively with the TM 0.1% treatment. The same aeriferous cavities as in the TM 0.1% sample appear, yet the secretory hairs are extremely numerous comparatively with the first treatment, and, especially, with the reference. The central ring contains 18 large or intermediary and 10 small conducting fascicles, each with primary structure and with a girdle of periphloemic fibers with thin, cellulosic walls (Fig. 1, 2); on the lateral side of the conducting fascicles, more liberian islands (16-17) may be observed (Fig. 2, 4). The cambium is as thick (3-4 layers) and as active as in the TM 0.1% treatment, numerous xylem vessels being still in the process of formation; however, the diameter of the vessels is smaller, although the number of vessels is higher than in the reference (Fig. 3). The bark evidences only one, small conducting fascicle, of collateral type, similar to those observed in the TM 0.1% treatment. Cross-sections through the median third of the main branches The reference the ribs contain a very low amount of cholenchyma, the cell forming it having slightly thickened walls. Small aeriferous cavities some of them tangentially-elongated are presented at the periphery of the bark (Fig. 6). The central cylinder includes 17 large or intermediary conducting fascicles, of open collateral type, have only a primary structure; the ligneous vessels, separated by cells of cellulosic ligneous parenchyma, have thickened, yet weakly-lignified walls, a few of them being still under edification (Fig. 6, 7). The fibers of the periphloemic girdles show slightly cellulosic walls, while the medullary rays are parenchymatic-cellulosic. The very small conducting fascicles (8) evidence only liberian elements (Fig. 9).

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TM 0.1% treatment diameter of the cross-sections is much larger than in the reference, while the secretory hairs are more frequent. The aeriferous cavities occurring at the periphery of the bark are very large and considerably elongated in tangential direction (Fig. 6). The girdle of conducting tissues includes 19 large or intermediary and 5 small fascicles, all evidencing only a primary structure; during this treatment, more numerous small fascicles (5) and liberian islands (12) are formed (Fig. 6, 7, 9). Lignification of the ligneous vessels is weaker, while the fibers of the periphloemic girdles have thin, cellulosic walls. The cambium, especially the intrafascicular one, is extremely thick (6-7 layers); only here and there, formation of the interfascicular cambium begins. Cambial activity is much more intense than in the untreated sample and, consequently, more numerous vessels hardly appear in a process of edifications (Fig. 8, 9). TM 0.4% treatment comparatively with the other two samples taken into study, the walls of the epidermal cells are thicker, being covered by a thicker cuticle. In the ribs, mention should be made of the presence of typical angular cholenchyma, the cells having thicker walls than both in the TM 0.1% treated and in the untreated samples. More numerous conducting fascicles, all with a primary structure, are formed: 22 large or intermediary and 5-6 small ones, but on the lateral side of the conducting fascicles more liberian islands (22) may be observed (Fig. 6, 7, 9). The highly active cambium, both intraand interfascicular, includes 6-7 cell layers, which form an almost continuous ring (Fig. 8, 9), while the periphloemic fibers have cellulosic walls, too. In the bark, 1 or 2 neighbouring conducting fascicles, surrounded by meristematic tissue, are present (Fig. 7). Cross-sections in the inferior third of the main branches The reference the ribs are more attenuated while, in the peripheral area, in front of the more prominent ribs, largely tangentially, elongated aeriferous cavities may be observed. The bark includes 4-5 layers of cells and the central cylinder has 19 big or intermediary, 6-8 small conducting fascicles and 8-10 liberian islands (Fig. 10, 15); the first two categories, of open collateral type, show, each, a thick girdle of sclerenchymatic fibers with moderately thick, yet intensely lignified walls, at the periphery of the liber. The vessels of the primary xylem are separated by cells of cellulosic parenchyma, while those of secondary xylem, by a lignified parenchyma; the libriform elements have slightly thickened, yet intensely lignified walls (Fig. 10). In some fascicles, the procambium is quite sufficiently thick (6-8 layers), to become soon an intrafascicular cambium, which is continuing with the interfascicular cambium, resulted from the dedifferentiation of the medullary rays (Fig. 12, 15). All conducting fascicles are separated by large medullary rays, parenchymatically-lignified at xylem level and parenchymatically-cellulosic at liberian level (Fig. 15). The pith is thick, parenchymatous-cellulosic, the cells showing very thin walls. TM 0.1% treatment although the diameter of the cross-sections is larger than in the reference sample, the small ribs are equally attenuated, with thinner cholenchyma girdles and cells with less thick walls. The secretory hairs are more frequent and, at the periphery of the bark, smaller aeriferous cavities may be observed, the cortical parenchyma being thicker (7-9 layers of cells) (Fig. 10). The treatment stimulates cellular division at cortical level and even in the medullary rays (only up to the level of the liber), almost all cells from the bark evidencing more division, both anticline and pericline walls (2, 3 or

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even 4) (Fig. 14). The large or intermediary conducting fascicles (19-20) are very close to one another, evidencing much larger sizes, while the phloemic girdles show fibers with thicker and more intensely lignified walls; more than that, small conducting fascicles (13) and islands of liberian elements (20) occur among these conducting fascicles (Fig. 10, 11, 15). The secondary xylem shows more libriform, the perimedular parenchyma being moderately sclerified and lignified. The meristematic tissue forms a continuous ring (8-10 layers), the tracheogenesis process developing still (Fig. 12). The conducting fascicles xylem, along with the moderately sclerified and lignified medullar rays, form a much thicker ring than in the reference (Fig. 10). It should be also mentioned that in the bark there are some atypical conducting fascicles present (2-3) some of which, relatively large or very small, of open collateral type, surrounded by a plurilayered meristematic tissue, others being concentric hadrocentric (cortical), each with a thin girdle of sclerenchymatic fibers at the periphery of the liber, or leptocentric vascular bundles (on the inner side of the ring of conducting fascicles), surrounded either exclusively by vessels or by vessels and libriforme fibers (Fig. 16, 17). TM 0.4% treatment the diameter of the cross-sections exceeds by far that of the previous samples, yet the cuticle is thinner, while the secretory hairs are more numerous (Fig. 10). In the much attenuated ribs, less cholenchyma is present, yet the hypodermal layer is tangentially cholenchymatized along the whole circumference of the strain (Fig. 13). The bark contains approximately 8-10 cell layers, no aeriferous cavities being present at its periphery, so that the tissues are much more compact. Division of the cortical cells is intensely stimulated, more numerous division walls being present in the same cell, especially in those from the periphery of the bark (Fig. 15). The conducting fascicles are very large or intermediary (20-21) and small (17-19), the last of them having no girdles of periphloemic fibers; on the lateral side of the conducting fascicles more liberian islands (20) are occurring. Sclerification and lignification of the libriform elements and of the ligneous vessels are moderate, while those of the periphloemic fibers are more intense. The medullary rays are parenchymatically lignified, the cells having slightly thickened walls. The meristematic tissue forms a much thicker ring (of 10-12 cell layers); more than that, the cambium is still to be divided, its cell being highly active, which explains why the process of tracheogenesis is still under development (Fig. 12, 15). The bark evidences the same peculiar type of fascicle, with the xylem surrounded by plurilayered meristematic tissue. Conclusions The most important histo-anatomical reactions from the part of the strains treated with thiophanate methyl involve a more intense cambial activity and the occurrence of some liberian-xylemic nodules, in various places of the strains thickness: from outside the bark (at the bottom of the cholenchymatized ribs) up to the central cylinder (inside the liber). The intrafascicular cambium is very active, being formed of several cell layers, while the interfascicular one tends to form, when treated, a continuous, much thicker ring. The size of the conducting fascicles is much larger than in the reference, their number exceeding those of the reference; generally, more small conducting fascicles and more liberian islands result after the treatment. The process of tracheogenesis is still under

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development, which explains the presence of more immature vessels with thin, celullosic walls. The treatment with thiophanate methyl stimulates cellular division at cortical level and, consequently even in the medullary rays (yet only up to the level of the liber) more division walls (2-3, sometimes 4), usually anticlynic or periclynic, may be observed in the cells of the bark. As a result of the stimulating action, the much larger-sized conducting fascicles form, together with the medullary rays, a much thicker ring than that of the reference, while the secretory hairs are more numerous than in the untreated samples. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
BAYMAN P., GONZALEZ E.J., FUMERO J.J., TREMBLAY R.L., 2002 - Are fungi necessary? How fungicides affect growth and survival of the orchid Lepanthes rupestris in the field. J. Ecology, 90(6): 1002 1008 BENITEZ T., RINCON M. A., LIMON M. C., CODON A. C., 2004 - Biocontrol mechanisms of Trichoderma Strains. International Microbiology, 7(4): 249-260 BEZO M., HRASKA S., CAGAN L., 1980 - Cytogenetic effect of Derosal 60 WP and Topsin M 79 on Vicia faba L.. Pol' nohospodarstvo, 26(6): 534-538 DORNER, J.W. 2006 - Combined effects of biological control formulations, cultivars, and fungicides on preharvest aflatoxin contamination of peanuts. Peanut Science. 31:79-86 EL-MOHAMEDY R.S.R., 2005 - Integration between biological and chemical treatments to control Fusarium root rot of some citrus rootstocks under saline soil conditions. Ann. Agric. Science Cairo, 49(1): 357-375 GARCIA P.C., RIVERO M. R., LOPEZ-LEFEBRE L.R., SANCHEZE., RUIZ J.M., ROMERO L., 2001 Direct action of the biocide carbendazim on phenolic metabolism in tobacco plants. J Agric Food Chem., 49(1): 131-137 GARCIA P.C., RUIZ J. M., RIVERO M. R., LOPEZ-LEFEBRE L.R., SANCHEZ E., ROMERO L., 2002 Is the application of carbendazim harmful to healthy plants? Evidence of weak phytotoxicity in tobacco. J. Agric. Food Chem., 50(2): 279-283 HUTANU-BASHTAWI L., TOMA C., 2008 Contributionsto the histo-anatomical study of the Calendula officinalis L. leaves treated with thiophanate methyl (Topsin M). An. t. Univ. "Al. I. Cuza" Iai, s. II a , fasc.1 (Biol. veget.), 54: 22-32 JAYARAJ J., RADHAKRISHNAN N.V., 2003 - Development of UV-induced Carbendazim-resistant mutants of Trichoderma harzianum for integrated control of damping-off disease of cotton caused by Rhizoctonia solani. J. Plant Diseases Protection, 110(5): 449460 KHAN M. R., KHAN M. S., MOHIDDIN F. A., 2004 - Biological control of Fusarium wilt of chickpea through seed treatment with the commercial formulation of Trichoderma harzianum and/or Pseudomonas fluorescens. Phytopathol. Mediterr., 43 (1): 20-25 KOZERA W., KLEIN M., 1980 - The influence of the fungicides Benlate and Topsin M on the mitotic process in root meristems of onion setts (Allium cepa L.). Acta Agrar. Silvest., Ser. Agrar., 19: 117-132 MALATHI P., VISWANATAN R., PADMANABAN P., MOHANRAJ D., 2002 - Compatibility of biocontrol agents with fungicides against red rot disease of sugarcane. Sugar Tech., 4(3-4): 131-136 MOLINA A., HUNT M.D., RYALS J.A., 1998 - Impaired fungicide activity in plants blocked in disease resistance signal transduction. Plant Cell, 10: 1903-1914 RUGINA R., TOMA C., 1989 - Recherches histo-anatomiques sur quelques plantes mdicinales de la famille des Composes. An. t. Univ. "Al. I. Cuza" Iai, s. II a (Biol.), 35: 15-18

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 1. Cross-sections main branch, upper level (Oc.10x Ob.2)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 2. Cross-sections conducting fascicle, upper level (Oc.10x Ob.20)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 3. Cross-sections intrafascicular cambium, upper level (Oc.10x Ob.40x3)

Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 4. Cross-sections upper level, cortical, colateral conducting fascicles

Fig. 5. Cross-sections strain, upper level, TM 0.1 % treatment, concentric conducting fascicles, present in the bark, in the periliberian fibers and in the liber

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 6. Cross-sections main branch, middle level (Oc.10x Ob.2)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Ca. of. TM 0.4% Fig. 7. Cross-sections middle level normal (a) and cortical (b) conducting fascicle

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 8. Cross-sections conducting fascicle, cambium, middle level (Oc.10x Ob.40)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 9. Cross-sections interfascicular cambium, middle level (Oc.10x Ob.20)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 10. Cross-sections main branch, basal level (Oc.10x Ob.2)

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Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 11. Cross-sections conducting fascicle, basal level (Oc.10x Ob.10)

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 12. Cross-sections conducting fascicle, cambium, basal level (Oc.10x Ob.40)

b
Ca. of. M Ca. of. TM0.1% Ca. of. TM0.4% Ca. of. TM0.4% Fig. 13. Cross-sections angular (a) and tangential (b) cholenchyma, basal level

Ca. of. M Ca. of. TM 0.1% Ca. of. TM 0.4% Fig. 14. Cross-sections bark, basal level (Oc.10x Ob.40)

Ca. of. M

Ca. of. TM 0.1%

Ca. of. TM 0.4%

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Fig. 15. Cross-sections interfascicular cambium, basal level (Oc.10x Ob.20)

Fig. 16. Cross-sections basal level, TM 0.1% treatment - conducting fascicles of open collateral type, surrounded by a plurilayered meristematic tissue, occurring in the bark

Fig. 17. Cross-sections - basal level, TM 0.1% treatment - concentric, hadrocentric conducting fascicles, with ligneous vessels inside, situated in the bark (a), in the vicinity (b) or in the thickness (c) of the periliberian fibers; concentric, leptocentric conducting fascicles, surrounded by large xylem vessels and by libriforme elements, present on the inner part of the conducting fascicles (d) and collateral conducting fascicles, occurring in front of the medullary rays at the level of the liber (e)

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

THE ANALYSIS OF DAILY CONCENTRATIONS OF AIRBORNE POLLEN IN THE WEST AND SOUTHWEST OF ROMANIA NICOLETA IANOVICI*
Abstract. This analysis is meant to determine the way the pollen grains are disseminated in the atmosphere in Western and South-Western Romania in the year 2004, by using the volumetric method of collecting, identifying and quantifying data. The monitoring period started on the 16th of February and ended on the 10th of October 2004 (238 days). 23 pollen types were identified. The early spring spring period was dominated by the pollen coming from anemophilic trees. In the late summer autumn period the airplankton was dominated by the airpollen coming from herbaceous plants. For the investigated area the maximal quantity of airpollen occurred in April. The daily concentration of airpollen for each pollinic type is expressed in number of pollen grains on m of air (PG/ m). Other parameters taken into account were: the total daily concentration of airborne pollen, the total monthly variation, and the total annual variation. The highest daily concentrations were those of Ambrosia (August and September), Artemisia (August), and Poaceae (May, June, and July). The main airborne polluter was the pollen of Ambrosia artemisiifolia. Key words: airpolynic spectrum, pollen types, anemophilic taxa

Introduction The changes that have occurred in the floristic composition immediately influence the airplankton composition [24, 17]. The studies of the airpolynic spectrum are of interest to biologists, but they mainly have an impact on physicians and allergic patients, since chronological correlations may be established between airpollen concentrations and certain symptoms of asthmatic and pollinosis patients. To better manage such diseases, pollinic calendars have been devised in many countries [1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 15, 19, 23, 25, 26, 16, 27, 28, 29]. In Romania, such yearly calendars have been devised for the West and the Southwest of the country [12, 13, 14]. The aim of this analysis is to describe the dynamics of the airpollen in Timioara in the year 2004, by taking into consideration the data obtained using the volumetric method of collecting, identifying and quantifying. Materials and methods The monitoring aeropalynologic station (the only one in Romania) belonging to the Biology Department of The University of Timioara, uses a VPPS 2000 Lanzoni volumetric pollen-trap. The trap allows the evaluation of the airborne dynamics of the polen in the city and its surroundings; the results are important for the plains in the west and southwest of Romania. The apparatus is placed on the roof of the west University building, approximately 20 meters from the ground, far from industrial areas and the barriers which might prevent the circulation of air currents. The apparatus applies the principle initially
*

Department of Biology, Faculty of Chemistry-Biology-Geography, West University of Timisoara, Romania

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presented by Hirst (1952) and the identification is made with a photonic microscope. The siliconed bands inside the volumetric trap were changed and analyzed weekly [8, 18, 20, 22] using fuxine for coloring. The identification of the pollen was performed on morphological bases at x400. The daily concentration of pollen is expressed in the number of pollen grains on m of air (pg/ m). Our bulletins were provided weekly on the following websites: http://www.pollinfo.ini.hu, http://www.nspolen.com,http://www.polleninfo.org. Results and discussions In 2004 the monitoring lasted for 238 days, starting on the 16th of February and ending on the 10th of October. 23 types of airpollen were identified (Tab. I). The plants that produce allergenic pollen are woody plants, grasses and herbs. The herbs belong to the Hamamelidae subclass (Urticaceae family), Asteridae (Asteraceae, Plantaginaceae), Caryophylidae (Chenopodiaceae, Amaranthaceae, Polygonaceae). Most of the anemophilic woody plants that have an allergenic potential belong to the Hamamelidae subclass (Platanaceae, Juglandaceae, Moraceae, Urticaceae, Fagaceae, Betulaceae i Corylaceae) and to the Dileniidae (Salicaceae, Tiliaceae), Rosidae (Aceraceae) and Asteridae (Oleaceae) subclasses. The Pinophyta (Pinaceae, Taxaceae/ Cupressaceae) are also anemophilic. The pollinic range of the month of February included five types of pollen coming from the following taxa: Corylus (27.75%), Alnus (27.36%), Ulmus (15.91%), Acer (4.81%) and Taxaceae/Cupressaceae (24.05%). The day with the highest concentration was the 20th of February (40 PG/m3). In March we noticed the presence of the following five pollen types in the airplankton: Populus, Salix, Fraxinus, Betula and Carpinus. The pollen of the Taxaceae/Cupressaceae represented 26.47% of the total monthly concentration. The day with the highest concentration was the 30th of March (224 PG/m3); the airpollen of Populus represented 34% of this concentration. April 2004 was the month displaying the highest quantity (4017 PG/m3) correlated with the largest number of taxa which were in the flowering phenophase (16). The highest concentrations were established for the airpollen of Betula (22.5%), Populus (16.23%), Salix (15.23%), Fraxinus (10.08%), Carpinus (8.36%). The day when the concentration was the highest was the 1st of April (318 PG/m3), that can be explained by the presence of the 9 pollen-producing taxa (Fig.1). In May, in spite of the presence of 15 pollinic types, the total monthly concentration of pollen was much lower than the one in the previous month (1489 PG/m3). The pollinic range was dominated by the pollen of Poaceae (46.4%). As to woody plants, the highest concentration was that of Pinaceae (13.7%). The highest daily concentration was noted on the 30th of May (132 PG/m3), the airpollen of Poaceae representing 43%. In June, eight pollinic types were identified: Pinaceae (4.78%), Poaceae (32.43%), Urtica (39.33%), Plantago (7.86%), Rumex (6.55%), Tilia (6.84%), Chenopodiaceae/ Amaranthaceae (1.48%) and Ambrosia (0.22%). The pollinic range was dominated by the airpollen coming from herbaceous plants. The total monthly concentration was higher than

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the one noted in the previous month (1746 PG/m3). The day with the highest concentration was the 23rd of June (88 PG/m3), the Urtica type airpollen representing 50% of the total.
Tabel I. Monthly pattern of airborne pollen (%), Timisoara, Romnia, 2004 (airpolynic spectrum)
II ULMUS CORYLUS ALNUS TAXACEAE/CUPRESSACEAE ACER POPULUS SALIX FRAXINUS BETULA CARPINUS QUERCUS PLATANUS JUGLANS MORUS PINACEAE URTICA POACEAE PLANTAGO RUMEX TILIA CHENOPODIACEAE/AMARANTHACEAE AMBROSIA ARTEMISIA 41,75% 26,22% 25% 7,61% 5,51% 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 III 58,25% 72,73% 71,62% 55,74% 16,95% 28,04% 35,80% 18,07% 8,20% 8,60% 0 0 0 0 0 0 0 0 0 0 0 0 0 IV 0 1,05% 3,04% 34,66% 77,54% 71,96% 64,20% 79,57% 89,30% 90,60% 73,30% 79,50% 87,30% 79,90% 2,40% 0,04% 1,80% 0 0 0 0 0 0 V 0 0 0,34% 1,99% 0 0 0 2,36% 2,50% 0,80% 26,70% 20,50% 12,70% 20,10% 69,20% 5,86% 31,70% 28% 27,10% 17,70% 0 0 0 VI 0 0 0 0 0 0 0 0 0 0 0 0 0 0 28,40% 27,30% 25,70% 28,40% 33% 81,60% 5% 0,10% 0 VII 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 22,30% 26,50% 27% 37% 0,70% 15,50% 4,80% 8,60% VIII 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 37,50% 10,70% 14,70% 2,90% 0 36% 33% 54,40% IX 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7% 3,40% 1,90% 0 0 42,50% 61% 36,40% X 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0,20% 0 0 0 1% 1,10% 0,60%

The Poaceae (33.21%) and the Urtica (31.9%) also dominated the pollinic range of July. The other anemophilic taxa in the flowering phenophase were: Plantago, Rumex, Chenopodiaceae/Amaranthaceae, Ambrosia and Artemisia. The highest daily concentration was noted on the 11th of July (119 PG/m3). In August the second highest quantity of the year was determined (3240 PG/m3). Only the herbaceous plants were in the flowering phenophase; the pollen of Ambrosia (31.9%), Urtica (29.2%) and Artemisia (22.6%) dominated the airplankton. The highest daily concentration was noted on the 31st of August (185 PG/m3), the airpollen of the Ambrosia type representing 61% of it (Fig.2). In September, five pollinic types were identified, the highest concentrations belonging to Ambrosia and Artemisia. On the 8th of September the total concentration reached 287 PG/m3, the airpollen produced by Ambrosia representing 77%.

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During the two monitored weeks in October, four pollinic types were identified, with low concentrations (Ambrosia, Artemisia, Chenopodiaceae/ Amaranthaceae, Poaceae).

Fig. 1. Concentrations of airpollen (PG/m3) measured in April 2004

Fig. 2. Concentrations of airpollen (PG/m3) measured in August 2004

If we consider 30 pg/m3 as a threshold value for the onset of the pollinosis symptoms for the pollen of all anemophilic plants with an allergenic potential, 11 out of the 23 identified pollen types did not reach this value at any time: Corylus, Alnus, Ulmus, Acer, Quercus, Pinus, Platanus, Plantago, Rumex, Tilia, Chenopodiaceae/Amaranthaceae. Even if Urtica produces much pollen, the involvement of this pollen type in the monosensitization to allergens wasn not proved. For the west and south west of Romania a real danger is represented by the allergenic airpollen produced by Ambrosia (in August and September) Artemisia (in August) and Poaceae (in May, June, and July). The number of days when the daily concentration for these taxa exceeded 30 pg/m3 was of 29 for Ambrosia, 21 for Artemisia, and 17 for Poaceae. The high daily concentrations are

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accompanied by the high number of days these pollinic types were present in the airplankton: 174 days for Poaceae, 111 days for Ambrosia, and 99 days for Artemisia. In the SW of Romania the most important taxa with an allergenic potential are: Ambrosia, Artemisia, Poaceae and Betula. Conclusions The early spring spring period was dominated by the pollen coming from anemophilic trees: Acer, Alnus, Betula, Carpinus, Juglans, Morus, Pinaceae, Platanus, Corylus, Fraxinus, Populus, Salix, Quercus, Taxaceae/Cupressaceae, Tilia, Ulmus (38.6% of the total annual concentration). During the late summer autumn period the airplankton was dominated by the airpollen coming from herbaceous plants: Ambrosia, Artemisia, Chenopodiaceae/ Amaranthaceae, Plantago, Rumex, Urtica (48.7% of the total annual concentration). The Poaceae produced 12.7% of the total annual concentration. In the investigated area the daily concentrations reached two highs: in april and in august. The highest daily concentrations were those of Ambrosia (in August and September) Artemisia (in august) and Poaceae (in May, June, and July). The pollen season of the plants in the continental climate ends in october. The main airborne polluter was the pollen of Ambrosia Artemisiifolia which represented 18.11% of the annual pollinic range. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. ABREU I., RIBEIRO H., CUNHA M., 2003 - An aeropalynological study of the Porto region (Portugal), Aerobiologia, 19: 235241 BICAKCI A., AKYALCIN H., 2000 - Analysis of airborne pollen fall in Balikesir, Turkey, 1996-1997, Ann Agric Environ Med, 7: 510. BICAKCI A., AKKAYA A., MALYER H., UNLU M., SAPAN N., 2000 - Pollen calendar of Isparta, Turkey. Israel J Plant Sci, 48: 67-70 BICAKCI A., TATLIDIL S., SAPAN N., MALYER H., CANITEZ Y., 2003 - Airborne pollen grains in Bursa, Turkey, 19992000. Ann Agric Environ Med, 10: 3136 CARAMIELLO R., POLINI V., SINISCALCO C., MERCALLI L., 1990 - A pollen calendar from Turin (19811988) with reference to geography and climate. Grana 29: 239249 D'AMATO G., SPIEKSMA F.T.M., 1990 - Allergenic pollen in Europe. Grana, 30, 67-70 EMBERLIN J., MULLINS J., CORDEN J., JONHS S., MILLINGTON W., BROOKE M., SAVAGE. M., 1999 - Regional variations in grass pollen seasons in the UK, long-term trends and forecast models. Clin. Exper. Aller. 29: 347356 FAEGRI K., IVERSEN J., 1992 - Textbook of Pollen Analysis, Ed. John Wiley and Sons GALN C., TORMO R., CUEVAS J., INFANTE F., DOMNGUEZ E., 1991 - Theoretical daily variation patterns of airborne pollen in the southwest of Spain. Grana 30, 201209 GARCIA-MOZO H., PEREZ-BADIA R., FERNANDEZ-GONZALEZ F., GALN C., 2006 - Airborne pollen sampling in Toledo, Central Spain, Aerobiologia, 22: 5566 GUVENSEN A., OZTURK M., 2003 - Airborne pollen calendar of Izmir - Turkey. Ann Agric Environ Med, 10, 3744 IANOVICI N., FAUR A., 2004. Seasonal distribution of airborne pollen in Timioara, Proceeding of VIth International Symposium Young People and Multidisciplinary Research, 23-24 September 2004, 426-436 IANOVICI N., FAUR A., 2005a - Quantitative and qualitative study of the atmospheric pollen in 2001, Annals of West University of Timioara, ser. Biology, 7: 35-44 IANOVICI N., FAUR A., 2005b - Monitoring the allergenic pollen from the airplancton in 2000, Annals of West University of Timioara, ser. Biology, 5-6: 197-206

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15. JAGER S., SPIESKMA F.TH.M., NOLARD N., 1991 - Fluctuations and trends in airborne concentrations of some abundant pollen types, monitored at Vienna, Leiden and Brussels. Grana 30: 309312 16. KAPLAN A., 2004 - Predominant aeroallergen pollen grains in the atmosphere of Ankara, Turkey, Allergy: 59:670672 17. LATORRE F., BIANCHI M. M., 1997 - Relacin entre aeropolen y vegetation arbrea en Mar del Plata (Argentina). Polen 8, 4359 18. MANDRIOLI P., COMTOIS P., DOMINIQUEZ-VILCHES E., GALAN SOLDEVILLA C., SYZDEK L.D., ISSARD S.A., 1998 - Sampling: Principles and Techniques. In: Mandrioli P, Comtois P, Levizzani V (Eds): Methods in Aerobiology, Pitagora Editrice, Bologna: 47-112 19. NARDI G., DEMASI O., MARCHEGIANI A., 1986 - A study of airborne allergenic pollen content the athmosphere of Ascoli Piceno. Ann Allergy, 57, 193-197 20. OGDEN E.C., RAYMOR G.S., HAYES G.V., LEWIS D.M., HAINES J. H., 1974 - Manual for sampling airborne pollen, H. Y. Hafner Press 21. PETERNEL R., ULIG J., MITI B., VUKUI I., OSTAR Z., 2003 - Analysis of airborne pollen concentrations in Zagreb, Croatia, Ann Agric Environ Med, 10, 107112 22. RADII P., SIKOPARIJA B., JUHSZ M., IANOVICI N., 2003 - Betula pollen season in the DanubeKris-Mures-Tisa Euroregion (2000-2002), ISIRR: 389-393 23. RODRIGUEZ-RAJO F.J., JATO V., AIRA M. J., 2003 - Pollen content in the atmosphere of Lugo (NW Spain) with reference to meteorological factors (19992001), Aerobiologia 19: 213225 24. ROMANO B., 1988 - Pollen monitoring in Perugia and information about aerobiological data. Aerobiologia 4: 2026 25. ROMANO M. L. G., CANDAU P. MINERO F.G.G., 1992 - Pollen calendar of Seville and its relation to allergies. J. Invest. Allergol. Clin. Immunol. 2: 323328 26. SAVEROVA E., POLEVOVA S., 1996 - Aeropalynological calendar for Moscow 1994. Ann Agric Environ Med, 3: 115-119 27. SPIEKSMA F.T.M., NOLARD N., JAGER S., 1991 - Fluctuations and trends in airborne concentrations of some abundant pollen types, monitored at Vienna, Leiden and Brussels. Grana, 30: 309-312 28. SYED M. HASNAIN, KHATIJA F., ABDULRAHMAN AL-FRAYH, SULTAN T. AL-SEDAIRY., 2005 One-Year pollen and spore calendars of Saudi Arabia: Al-Khobar, Abha and Hofuf, Aerobiologia, 21: 241 247 29. WERYSZKO-CHMIELEWSKA E., PIOTROWSKA K., 2004 - Airborne pollen calendar of Lublin, Poland. Ann Agric Environ Med, 11: 9197

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

VARIABILITY OF SOME POLLEN MORPHOLOGICAL TRAITS OF CERTAIN TAXONS IN THE BORDERING AREA OF THE CEAHLU NATIONAL PARK SILVICA PDUREANU*
Abstract: This study presents the variability of four pollen morphological traits from three taxons: Ranunculus polyanthemos L., Veronica chamaedrys L. and Plantago media L., sampled from four stationaries found in the bordering area of the Ceahlu National Park. Among the four stationaries, three of them were affected by polluting noxa agents (dust powder from the cement factory of Taca and escapement gases), and one was used as control. The four pollen morphological traits taken into account were: shape of the pollen grain, exine ornamentations, size of the pollen grain, number of germinative pores/pollen grain. For a real evaluation of these traits, determinations were carried out on 1000 grains/taxon. Being qualitative traits, the shape of pollen grain and exine ornamentations were estimated by description and microphotographs. As quantitative traits, the size of pollen grain diameter and the number of germinative pores/pollen grain were determined by biostatistic methods. Measuring the diameter of the pollen grains was a safe way to separate the diploid against self-tetraploid or mixoploid plants. On the other hand, there was a tight correlation between the number of germinative pores/pollen grain and the plant polyploidy degree. The shape of the pollen grain, next to the exine ornamentations did not show variability from one individual to another of the same taxon, no matter if the stationary was or not polluted; this proved the genetic strengthen of these two qualitative traits. The biostatistic indices of the two quantitative traits shaped the limits of pollen variability in the three taxons, which depended not only on genotype but also on environment. Key words: pollen grain, exine ornamentation, germinative pores/pollen grain

Introduction The pollen morphology has a strong taxonomic and phylogenetic significance in the world of plants. The knowledge of the evolution of pollen morphology brings an important contribution to plant phylogenetic interpretation. The constant or changing environment conditions where plants develop are determinant for the appearance of new ecotypes, which are reflected by pollen morphology [2]. The palinological studies have shown that the pollen morphology has expressed the polyploidy degree of a certain taxon, which determined the variability of morphopolinic traits. A polyploidy degree higher than the diploid one is responsible for the formation of greater pollen grains, with differently placed germinative pores and a diminished fertility caused by meiosis disturbance [3, 4, 5]. This scientific paper focuses on the limits of the variability of pollen morphology, characterized by many traits, at certain taxons belonging to different botanic families. We had in view the influence of polluting noxa agents from the environment on the variation of morphopalinological traits. Material and methods
* University of Agricultural Sciences and Veterinary Medicine Ion Ionescu de la Brad Iasi, Romania

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The biological material consists in three taxons: Ranunculus polyanthemos L., Veronica chamaedrys L. and Plantago media L. They were sampled from four different stationaries found in the bordering area of the Ceahlu National Park, Neam County. Three of these stationaries (1, 2 and 4) are affected by polluting noxa agents, while the stationary 3 is used as a control, not being exposed to pollution. Therefore, stationaries 1 and 2 are polluted with dust powder from the cement factory of Taca, stationary 4 is polluted with escapement gases from Taca, while the unpolluted stationary 3 is represented by Potoci Village. Pollen was sampled from these taxons in the anthesis stage. It was used to investigate four traits: shape of the pollen grain, exine ornamentations, size of the pollen grain and number of germinative pores/pollen grain. To determine the shape of the pollen grain and exine ornamentations, we have used the Tesla electron-scan microscope and microphotographs have been taken [6]. For the determination of the pollen grain size, micromeasurements have been carried out on 1000 pollen grains/taxon/stationary. According to the shape of the pollen grains, we have measured a diameter for the round shaped and two diameters (longitudinal and equatorial ones) for those ovoid or elliptic shaped. These data helped us to determine the arithmetical mean and the height of variation. To analyse the variability of the pollen grain size, the biostatistical indices have been used: standard deviation (S), variation coefficient (S%) and mean error of the arithmetical mean (S x ) [1]. For establishing the number of germinative pores/pollen grain, determinations on 1000 pollen grains/taxon/stationary have been executed. The method consisted in introducing the pollen in a mixture of concentrated sulphuric acid (one part) and acetic acid (two parts) and 3% methylene blue [7]. Results and discussion The shape of the pollen grains and the exine ornamentations Ranunculus polyanthemos L. has spherical pollen grains, with a scabrat type exine ornamentation (fig. 1). Veronica chamaedrys L. has elliptic pollen grains, with rugulated exine ornamentation (fig. 2). In Plantago media L., the pollen grains are approximately spherical and the exine has a claviform ornamentation (fig. 3) [8]. The pollen sampled from the three taxons was yellow-coloured, no matter what stationary it has been collected from. In these taxons belonging to distinct families, the shape of pollen grains and the exine ornamentations were different. Size of the pollen grains Measuring the diameter of the pollen grains is a safe way to separate diploid from self-tetraploid or mixoploid plants. This was due to the fact that doubling the number of chromosome sets was also correlated to the increase in pollen grain size and to the rate of non fertile pollen grains.

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In Ranunculus polyanthemos L., the pollen having a spherical shape, it could be characterized from the size point of view by measuring one diameter. On the average, it has 30.36 m in the control. In individuals from the stationary 4, the pollen grain has a size similar to the one of the control ones, while in individuals from the stationary 1, the pollen grain was smaller. The variability of this trait was low in the control, average for the stationary 4 and high in the stationary 1 (Tab. I, Fig. 4). In Veronica chamaedrys L., the pollen has the greatest diameter comprised between 37 and 40 m, with a low variability in the control and average in stationary 4 (tab. II). The equatorial diameter has a low variability (Tab. III, Fig. 5). The ratio between the two diameters is between 2.468 and 2.519 m (fig.6).
control

micrometers

micrometers

35 30 25 20 15 10 5 0 control st. 1 st.4

50 40 30 20 10 0 high diam. equat. diam.

st. 4

Fig. 4 Diameter mean value of pollen grain in Ranunculus polyanthemos L.

Fig. 5 Size mean value of pollen grains in Veronica chamaedrys L.

2,52 micrometers

22,34 micrometers
control st. 4

2,5 2,48 2,46 2,44

22,32 22,3 22,28 22,26 22,24 control st. 4

Fig. 6 Rate high diameter/equatorial diameter of pollen grain in Veronica chamaedrys L.

Fig. 7 Diameter mean value of pollen grain in PLantago media L.

In Plantago media L., the pollen grain has a diameter of aproximately 22 m, with a mean variability (tab. IV, fig. 7). The number of germinative pores/pollen grain It is well known that there is a tight correlation between the number of the germinative pores/pollen grain and the degree of plant polyploidy. The number of germinative pores/pollen grain is also an indicator of the fertility degree of male gametophyte. That is the reason why we have studied this trait on the six taxons sampled from different stationeries found in the bordering area of the National Park of Ceahlu. The characteristic number of germinative pores/pollen grain in Angiospermae, class Magnoliatae was three. According to different factors, defectively structured pollen grains (with a lower pore number) or pollen grains with more pores than normal in the species also appeared, at different rates. These pores are in fact germinative apertures, crevices, allowing the release of the polynic tube. In Ranunculus polyanthemos L., the pollen has generally, three pores/grain. It is surprising that there are 10-15% grains with 4 pores, showing the beginning of polyploidy (tab. V). In Veronica chamaedrys the pollen has generally normal values of the number of pores/pollen grain, namely three. At very low rates (1-2%), pollen has four germinative pores; therefore, there was a slight tendency of polyploidy in this species (Tab. VI). There

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were no significant differences between stationaries concerning the number of pores/pollen grain. In case of the Plantago media pollen, we have found that most of the pollen grains had nine germinative pores (Tab. VII) [8]. This situation was also reported in both stationaries. The increased number of pores/pollen grain showed an advanced polyploidy process at this taxon. Table I. The variability of pollen grains diameter in Ranunculus polyanthemos L. S S% No. Mean Minimum Maximum Variation (m) height value value stationary value Sx (m) (m) (m) (m) (m) Control 30.360 24.150 37.950 13.800 2.377 7.829 0.238 (3) 1 23.137 24.150 41.400 17.250 8.896 38.450 0.889 4 30.136 17.250 39.675 22.425 4.450 14.766 0.445 Table II. The variability of high diameter pollen grains in Veronica chamaedrys L. S S% No. Mean Minimum Maximum Variation (m) height value value stationary value Sx (m) (m) (m) (m) (m) Control 40.124 31.050 48.300 17.250 2.759 6.876 0.687 (3) 4 37.812 20.700 46.575 25.875 4.041 10.687 0.404 Table III. The variability of equatorial diameter of pollen grains in Veronica chamaedrys L. S S% No. Mean Minimum Maximum Variation (m) height value value stationary value Sx (m) (m) (m) (m) (m) Control 16.255 6.600 20.400 13.800 2.007 5.387 0.200 (3) 4 15.011 7.250 23.988 16.738 3.500 9.997 0.350 Table IV. The variability of the pollen grains diameter in Plantago media L. S S% No. Mean Minimum Maximum Variation (m) height value value stationary value Sx (m) (m) (m) (m) (m) Control 22.270 10.350 31.050 20.700 2.742 12.312 0.274 (3) 4 22.322 13.800 25.875 12.075 2.437 10.920 0.243

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Table V. Nnumber of germinative pores/pollen grain in Ranunculus polyanthemos L. No. Mean % pollen grains with germinative pores stationary value 0 1 2 3 4 Control (3) 1 4 (X ) 3.03 3.01 3.01 0 0 0 0 2 1 7 5 12 83 83 72 10 10 15

Table VI. Number of germinative pores/pollen grain in Veronica chamaedrys L. % pollen grains with germinative pores No. Mean stationary value 0 1 2 3 Control (3) 4 (X ) 2.96 2.88 0 0 0 2 5 10 94 86

4 1 2

Table VII. Number of germinative pores/pollen grain in Plantago media L. % pollen grains with germinative pores No. Mean stationary value 0 1 2 3 4 5 6 7 8 9 10 Control (3) 4 (X ) 9.15 9.07 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 3 3 6 78 78 12 7

11 5 6

Conclusions 1. The shape of the pollen grain, next to exine ornamentations, does not show variability from one individual to another of the same taxon, no matter if the stationary was or not polluted, demonstrating the good genetic strength of these two morphological traits. 2. The size of pollen grains is a species trait. This has a certain variation height according to eco-physiological conditions of stationaries. 3. If the pollen grains have an elliptic shape, the greater diameter has a higher variability compared to the equatorial one. 4. In most studied cases, the diameter variability of pollen grains belonging to the control is more balanced as compared to the one of the individuals from stationaries affected by polluting noxa. This could be explained by a greater genetic stability of that trait in areas which are not affected by environment pollution. 5. At Ranunculus polyanthemos L. and Veronica chamaedrys L., the characteristic number of germinative pores/pollen grains is three, but we have also found pollen grains with four germinative pores, at different rates, both at the control and at the individuals

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from pollution affected-stationaries. This proved the presence at these two taxons of mixoploidy, which developed with different intensity according to the stationary. 6. In Plantago media L., most of pollen grains have nine pores. Next to nine pore pollen, at this taxon there are also pollen grains with seven, eight, ten and eleven germinative pores. The increased number of germinative pores shows an advanced polyploidy in that taxon. The value differences concerning the rate of pollen grains with a certain number of pores, according to stationary, are insignificant. 7. In Ranunculus polyanthemos L. and Veronica chamaedrys L., we have found pollen rates with a number of germinative pores lower than three. In the control, these rates are lower as compared to the other stationaries. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. CEAPOIU N., 1968 - Metode statistice aplicate in experientele agricole si biologice. Bucuresti, Edit. Agrosilvica ERDTMAN G., 1969 - Handbook of palynology. Morphology Taxonomy Ecology, Munksgaard PDUREANU S., 1996 - The influence of pollen quality in some vine varieties on the grape yield II .Lucr. st. U.A.M.V. Iasi, ser. Agron., 39-140 PDUREANU S., 2001 - Researches regarding the variability of some morphological characters of the pollen at Feteasca neagra grape-vine variety. An. st. Univ. Al. I. Cuza Iasi, , s. II a, Biol. veget., 47: 103108 PDUREANU S., 2003 - Researches regarding the variability of some morphological characters of the pollen at Ampelopsis aconitifolia Bge. and A. brevipedunculata (Maxim.) Trautv. An. st. Univ. Al. I. Cuza Iasi, s. II a, Biol. veget., 49: 89-94 PLOAIE P.G., PETRE Z., 1979 - Introducere in microscopia electronica cu aplicatii la biologia celula si moleculara. Bucuresti, Edit. Acad. R.S.R.: 177-181 RAICU P., 1962 - Metode noi in genetica. Bucuresti, Edit. Academiei Romne: 107-109 TARNAVSCHI I.T., SERBANESCU-JITARIU G., MITROIU-RADULESCU N., RADULESCU D., 1990 Monografia polenului florei din Romnia, III: 69-70

Explanation of figures: Fig. 1 Pollen grains of Ranunculus polyanthemos L. (2300X) (stationary no. 1) Fig. 2 Pollen grains of Veronica chamaedrys L.(1740X) (stationary no. 4) Fig. 3 Pollen grains of Plantago media (2300X) (stationary no. 3)

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SILVICA PDUREANU

PLATE

Fig. 1

Fig. 2

Fig. 3

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

RESEARCHES REGARDING THE ESSENTIAL OIL COMPOSITION OF SOME ARTEMISIA L. SPECIES I. BURZO*, V. CIOCRLAN*, ELENA DELIAN*, AURELIA DOBRESCU*, LILIANA BDULESCU*
Abstract. The chemical composition of the essential oils of Artemisia sp. L. growing in Romania has been studied. Analysis of the essential oil extracted by hydrodistillation was performed by GC-MS and emphasised the presence of some major similar compounds in Artemisia austriaca Jack, A. lerchiana Weber ex. Stechm and A. santonica L.: eucaliptol, camphor, cis-verbenone and borneol or iso-borneol. The essential oil extracted from another species had a specifically composition. Thus, A. annua L. essential oil contained 60,20% artemisia ketone; A. dracunculus L. contained 42,34% sabinene and 24,92% methyl eugenol; A. vulgaris L. contained 41,46 germacrene D and 11,19% -caryophyllene; A. dzevanovskyi Leonova in Wulf. 57,13% eucaliptol and 20,37% borneol; A. absinthium L. contained 41,65 % -pinene and 12,77 % myrtenil acetate; A. abrotanum L. contained 12,27 % eucaliptol and 9,23 % borneol. Key words: Artemisia sp., chemical composition, essential oils.

Introduction Artemisia genus comprises wild and cultivated species, used in the alimentary and chemical industry. In the literature only one paper that presents a comparatively study regarding the essential oil composition extracted from ten Artemisia sp. in Serbia was found. Knanina et al. [2] noticed a variation of the essential oil depending on specie, the age of the plant and the ecological conditions. Lawrence [3] had stated that the essential oil extracted from A. absinthium L. has two major compounds: cis-sabinil acetate (40,00%) and -thujene (35,00%). Artemisia annua L. contains 35,70% artemisia ketone and 31,50% 1,8-cineole [4]. In the oil of A. dracunculus L. the major compound is methyl carveol [6], and in A.vulgaris L. sabinene and mircene were identified [5]. A. dracunculus L. plants or their extracts are used for food aromatisation, those of A. absithium L. for the preperation of some drinks, while A. abrotatum L., A. annua L. and A. vulgaris extracts are toxic. The aim of the present study, which is carried out for the first time in these plants grown in Romania, is to make a comparative analysis regarding the essential oils composition of nine Artemisia sp. Material and methods The research has been performed on the following Artemisia species: A. annua L. - wormwood, from Botanical Garden of U.S.A.M.V. Bucharest. A. abrotanum L. southernwood, from Botanical Garden of U.S.A.M.V. Bucharest.
*

University of Agronomical Sciences and Veterinary Medicine Bucharest, Romania

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A. absinthium L. absinthium, Iasi area. A. austriaca Jack. Alah-Bair, Dobrogea area. A. dracunculus L. tarragon, from Botanical Garden of U.S.A.M.V. Bucharest. A. lerchiana Weber ex. Stechm Capul Doloman area, Tulcea A. santonica L. tartarian southerwood, Muntele de sare area, Buzu. A. vulgaris L. mugwort, Iai area and a new species in the Romanian flora [1]: A. dzevanovskyi Leonova in Wulf Capul Doloman area, Tulcea. The volatile compounds were extracted by hydrodistillation with a Singer-Nickerson apparatus. The separation and identification of components has been carried out using an Agilent gas chromatograph, equipped with quadruple mass spectrometer detector. A capillary column DB-5 (25 m length x 0,25 mm i.d. and 0.25 m film thickness) and helium as carrier gas were used. The initial oven temperature was 600C, then rising to 2800C at a rate of 40C/min. The NIST spectra bank was used for to identify the volatile compounds, which were verified with the Kovats indices. Results and discussions The obtained results are presented in figures 1-9. The performed analysis permitted us to identify in the obtained mentioned extracts 17 compounds in A. vulgaris sp. and 36 compounds in A. abrotanum, respectively. From the presented analytical data, it can observed that in Artemisia austriaca, A. lerchiana and A. santonica there is a similarity regarding the major compounds: eucaliptol, camphor, cis-verbenone and borneol, or isoborneol, but these are present in different proportions. Thus, A. lerchiana contains 57.13% eucaliptol, while in A. santonica and A. austriaca oils camphor, cis-verbenone and eucaliptol were identified; eucaliptol, camphor and isoborneol, respectively, in aproximately similar amounts (15.8-21.54%). The main difference concerning these species essential oil composition consists in the fact that the essential oil extracted from A. austriaca contains a higher amount of higher molecular weight substances and less volatile: -eudesmol (6,97 %), germacrene D (3,40 %) and relemene (2,64 %). A relatively similar essential oil composition, almost as in the previous mentioned species it was noticed at A. dzevanovskyi, the oil included a higher eucaliptol content (38.42%) and borneol (20.37%) and lower amount of camphen (6.15%) and camphor (5.44%), respectively. The others Artemisia analysed species had a specific essential oil composition, that can alow their identification. So, the A. absinthium oil contained a higher proportion of pinene (41.65 %) and myrtenyl acetate (12.77 %), a small amount of thujonen also being determined (4.60 %). The A. annua oil contained appreciable amounts of artemisia ketone (60.20%), next to a small amount of eucaliptol (13.77%) and -pinene (9.19 %). Artemisia vulgaris oil was characterised by higher amounts of substances with a higher molecularly weight and a lower volatility degree, as for instance germacrene D (41.46%) and -caryophyllene (11.94 %). These substances may assure the characteristic odour persistence for a longer period of time.

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The major components of Artemisia dracunculus L. essential oil were sabinene (42.34%) and methyl eugenol (24.92%). This composition is similar to the one that is characteristic for the Russian A. dracunculus type. The essential oil obtained from A. abrotanum contained a higher amount of eucaliptol (17.59%), but was different compared to the A. lerchiana, A. austriaca, A. santonica i A. dzevanovskyi oils, because camphor was not identified. Next to eucaliptol, -terpinene (13.55 %), borneol (13.24 %) and p-cimene (11.30 %) were determined in a lower amount. Conclusions 1. Artemisia austriaca, A. lerchiana and A. santonica species are characterised by similarities regarding the major essential oil compounds: eucaliptol, camphor, cisverbenone and borneol or isoborneol. 2. The essential A. abrotanum oil contained a higher amount of eualiptol, but varied compared to the Artemisia austriaca, A. lerchiana, A. santonica i A. dzevanovskyi oils, with the lack of camphor. 3. It has been shown that the A. dzevanovskyi oil contains higher amounts of eucaliptol (38.42%) and borneol (20.37%). 4. The other Artemisia species analysed had a specific essential oil composition, which can permit their identification. 5. A. annua essential oil contained a higher amount of artemisia ketone, those of A. absinthium -pinene, those of A. vulgaris - germacrene D, and those of A. dracunculus sabinene. REFERENCES
1. 2. 3. 4. 5. CIOCRLAN V., 2002 - Noi specii n flora Romniei. Buletinul Grdinii Botanice Iai, 11: 97 98. LAWRENCE B.M., 1992 - Chemical Composition of some Warmwood oils produced in North America. Perf. Flavor, 17: 42. LIBBEY L. M., STURTZ G., 1989 - Unusual Essential Oils Grown in Oregon II. Artemisia annua L., Journal of Essential Oil Research, 1: 201-202. MICHAELIS K. et.al., 1982 - Das aetherische Oel aus Blueten von Artemisia vulgaris L. Z. Naturforsch. 37 C: 152-158. TUCKER A. O., MACIARELLO M. J., 1987 - Plant Identification (oil from fresh leaves). In Proceedings of the First National Herb Growing and Marketing Conference, Edit. J.E. Simon and L. Grant, Purdue Univ. Press, West Lafayette: 126-172

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

THE ECOPHYSIOLOGICAL PECULIARITIES OF SOME SPECIES OF THE ACER GENUS FROM THE BOTANICAL GARDEN OF IAI ALEXANDRINA MURARIU *, GIANINA BERECHET*, ANIOARA STRATU*, CAMELIA IFRIM**
Abstract. The paper presents the results of the biochemical and physiological studies carried out in 2005, on 8 species of the Acer genus, cultivated in the Botanical Garden of Iai in order to emphasize the ecophysiological peculiarities of the foliar limb, at the pedoclimatic conditions specific to the area. Two native species (Acer campestre i Acer monspessulanum) and six exotic species (Acer ginnala, A. negundo, A. negundo var. auratum, A. negundo var. variegatum, A.opalus, A. saccharinum) at which the print of the ecological factors is recognized in the main physiological manifestations: photosynthesis, respiration, perspiration, mineral nutrition, was studied. The analysis of the physiological behaviour of the exotic species, as compared to the native ones emphasized the fact that most of them have adapted to the conditions of the temperate climate in our country. Key words: physiological processes, wooden species, adaptation.

Introduction The Acer genus comprises about 115 species of trees, and more seldom shrubs, with falling leaves, spread in Europe, Northern Africa, Asia and North America. The Botanical Garden of Iai is structured on 12 sections, and the themes worked out for each section have a didactic, scientific purpose, the preservation of the genetic background of the plants but also a recreative - cultural and hygienical-sanitary purpose [16]. The species we have analyzed belong to the trees collections of the Flora of the Globe section: some of them native (Acer campestre i Acer monspessulanum) and others exotic (Acer ginnala, A. negundo, A. negundo var. auratum, A. negundo var. variegatum, A.opalus, A. saccharinum), acclimatized and naturalized, of decorative interest. The concerns of the researchers in our country on these species focused especially on the fields: morphology [1], [7], [11], [9], [2], [15], histo-anatomy [14], phenology [5], phytocenology [8], [4], [10], phytopathology [6], phytochemistry [3], [13]. The physiologycal references concerning Acer genus are a few; they were caried out by Papadopol S. (1959) and Sprchez Z. (1962). Starting from these general considerations, the paper presents the results of the biochemical (water content, assimilating pigments and total mineral elements) and physiological studies (the intensity of photosynthesis, respiration and perspiration) on 8 species of the Acer genus, cultivated in the Botanical Garden of Iai. Material and methods
*

**

Al. I. Cuza University, Faculty of Biology, Carol I Bd., No 20 A, 700506, Iai, Romania Anastasie Ftu Botanical Gardens, Dumbrava Roie Street, No 7-9, Iai, Romania

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The vegetal material, represented by leaves, was gathered between 8 and 10, 30 a.m, in the aestival period of 2005, a year characterized from the climatic point of view as showing a hydric deficit and a thermal excess. We analysed the following physiological indicators: the water and dry matter (gravimetrical method), the assimilating pigments (tirban and Frecu spectro-photometrical method), the total mineral elements (ash) (dry calcination at 4500 C method), the intensity of photosynthesis and respiration (IvanovKosovici method), the intensity of perspiration (Huber - Ivanov method). Results and discussions Analyzing the ecophysiological behaviour of the exotic species as compared to the native ones, the data presented in Table I emphasize the fact that these species adapted to the conditions of the temperate climate in our country, but the action of the high temperatures and of the relative low humidity (10-20%) in the atmosphere during the day, produces specific biochemical and physiological changes. Thus the water losses increase through perspiration leading to a disequilibrium between the absorption and the perspiration (310 - 357 mg H2O / dm2 / hour: A. negundo var. auratum, A.opalus and A. ginnala). These results are ecologically explained through the process of adaptation to stress which is never completed, and the fact that these species do not impede the perspiration means that they use a more efficient way, namely satisfying the thermal needs through an increased respiration (1,55 - 2,26 mg CO2 / g / hour). At these species the ecophysiological reaction of adaptation to stress is not made through the intensification of the assimilation but through the intensification of the oxido-reduction reactions, through an increased energetic consumption to the prejudice of the reduction of the biomass and the dry matter, of which the synthesized organic matters are only 0,640,79% (fig.1). The species which are resistant to drought (A. negundo var. variegatum, A. saccharinum ) avoid losing too much water through perspiration (20-30 mg / hour) due to the specific morpho-anatomical and physiological peculiarities, which constitute xeromorphism characters. Regarding the gross photosynthesis, the native species (A. campestre and A. monspessulanum) which live in their ecological optimum have the biggest intensity of the net photosynthesis (1,55- 1,88 mg CO2 / g / hour) and the lowest intensity of the respiration (0,38- 0,47 mg CO2 / g / hour), showing a productive metabolism (14-16% accumulated dry matter). In the exotic species (A. negundo var. auratum, A. negundo var. variegatum, A.opalus) the net photosynthesis is maintained at low quotas (0,69- 0,98 mg CO2 / g / hour) due to the increased respiration (1,54 - 2,45 mg CO2 / g / hour). Although the relation between the assimilating pigment content and the rapport between them has not found yet its mathematical expression, it doesnt mean that it doesnt exist but only that it is not operative due to the complexity of the ecological inter-relations of the factors on the photosynthesis. There is undoubtly the fact that at the exotic species in which the intensity of the net photosynthesis is low, the assimilating pigment content is also diminished (1,63 -2,04 mg / g fresh substance). We can notice the biosynthesis and the lower accumulation of the

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chlorophyll b and of the carotenoidic pigments, which influences the net photosynthesis decrease, with repercussions on the biomass production. Through the percentage values of the amount of total mineral elements, absorbed and accumulated in the cells, most of the species belong to the mesotrophic category (8,04 9,17 %). Conclusions Based on the results obtained we can estimate that the print of the ecological factors specific to 2005 causes differentiated influences in the exotic species, as compared to the native ones. The species well acclimatized (A. negundo, A. ginnala) are favoured through the bigger water content, the total assimilating pigments, the increased intensity of the net photosynthesis and the performances of those which do not integrate in a harmonious assimilation of the microclimate conditions decrease (A. negundo var. variegatum, A. negundo var. auratum, A.opalus, A. saccharinum). REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. BELDIE AL., 1953 - Plantele lemnoase din Romnia. Manual de determinare. Edit. Agro-silvic de Stat, Bucureti : 342-356. CIOCRLAN V.,1990 Flora ilustrat a Romniei. Determinarea i descrierea speciilor spontane i cultivate. vol.I. Edit.Ceres, Bucureti: 416-418. CODREANU A. MARILENA-VIORICA, ISTUDOR D. VIORICA, DINU V. MIHAELA, DOCIU N. NICULINA, 2006 - Physico-chemical studies on Acer negundo L. seed oil. 4 th Conference on Medicinal and Aromatic Plants of South - East European Countries. 28-31mai 2006, Iai: 374 -378. DONI N., PURCELEAN T., 1975 - Pdurile de leau din R.S.R. i gospodrirea lor. Edit. Ceres Bucureti. LUPU I., 1971- Observaii privind comportarea n primii 3 ani a unor specii lemnoase indigene, transplantate la Grdina Botanic Iai . An. t. Univ. Al. I. Cuza Iai, sec. II. XVII, fasc. 1: 162 - 168. MITITIUC M., IACOB. V., 1997 - Ciuperci parazite pe arborii i arbutii din pdurile noastre. Ed. Univ. Al. I. Cuza Iai. NEGULESCU E. G., SVULESCU AL., 1957 - Dendrologie. Edit. Agro-silvic, Bucureti: 415-432. PACOVSKI S., 1967- Succesiunea speciilor forestiere. Edit. Agro-silvic, Bucureti: 71-73. POPA A., GHIU M., OGRUAN I., 1959 - Floricultur i dendrologie. Edit. Agro-silvic de Stat, Bucureti: 324-325. SRBU I., OPREA A., TNASE C., 1997- Vegetaia Pdurii parc Grboavele (jud. Galai). Bul. Grd. Bot. Iai, 6, 2: 311-332. SVULESCU T., 1958 - Flora R.P.R, VI. Edit. Academiei R. P. R., Bucureti: 228-248. TTRANU D.I., 1960 - Arbori i arbuti forestieri i ornamentale cultivai n R.P.R. Edit. Agro-silvic, Bucureti:166-175. TCACENCO V. LUMINIA, TMA N. VIORICA, POMPONIU A. DANIELA, BERTEANU C. ELENA, BOTEZATU I. AURICA, 2006 - Studies for identification of some biological active compounds from indigenous plants with therapeutical value. 4 th Conference on Medicinal and Aromatic Plants of South - East European Countries. 28-31mai 2006, Iai: 78. TOMA C., IVNESCU L., 1998 Cercetri privind unele modificri histo-anatomice induse de poluanii atmosferici asupra aparatului foliar de la specii lemnoase aparinnd familiilor Aceraceae i Oleaceae. Buletinul Grdinii Botanice Iai, 7: 51-58. ZANOSCHI V., SRBU I., TONIUC A., 2004 Flora lemnoas spontan i cultivat din Romnia. III. Edit. Univ. Al. I. Cuza Iai: 138-174. *** - 1993- Grdina Botanic ( ghid ). Ed. Univ. Al. I. L. Cuza . Iai.

14. 15. 16.

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Table I. The ecophysiological characteristics of some species of the Acer genus from the Botanical Garden of Iai
Species Wa ter (%) Perspiration mg /h mg / dm2 /h 101 45 125 357 192 310 322 81 Dry Assimilating pigments (mg /g. Gross photosynthesis (mg CO2 / g / h) mat fresh substances) ter Net Respiration Gross Chl Ch Car Total photosynthesis (g oro lor ote pigments photosynthesis %) phy op noi ll a hyl d l b 2.092 0.760 0.725 3.577 1.55 0.38 1.93 14.15 1.970 2.207 0.977 0.917 1.290 0.705 1.574 0.765 0.429 0.314 0.428 0.286 3.965 4.546 1.720 1.631 2.008 4.292 2.041 1.58 0.94 0.98 0.69 0.82 1.31 1.25 0.47 1.54 1.90 2.45 1.55 2.26 0.99 2.05 2.48 2.88 3.14 2.37 3.57 2.24 16.42 8.98 8.68 9.82 14.37 9.97 15.58 Ash (g %) Orga nic subst ance (g %) 5.60 7.89 0.97 0.64 0.65 8.82 5.47 7.28

Acer campestre L. Acer monspessulanum L. A. negundo L. A.negundo var. auratum (Spaeth) A.negundo var. variegatum (Booth) A.opalus( Mill.) Acer ginnala Maxim A. saccharinum L.

85.85 83.58 91.02 91.32 90.18 85.63 90.03 84.42

67 13 23 33 20 54 87 30

8.55 8.53 8.19 8.04 9.17 5.55 4.50 8.30

1.095 0.518 0.395 2.317 1.190 0.785 0. 818 0.619 0.604

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18 16 14 12 10 8 6 4 2 0
tre es mp a c A. m la s su pe s on m nu do um tu m un rat ga eg au rie n . a r a er r. v ov Ac va nd o u d g n ne gu er ne r Ac e Ac o er Ac lus pa A g cer ala inn er Ac m nu a ri h c c sa

er Ac

dry matter content (g%)

total mineral elements content (g%)

organic substance content (g%)

Fig. 1. The dry matter, total mineral elements and organic substance content in leaves of studied species

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

PHYSIOLOGICAL AND BIOCHEMICAL ASPECTS IN THE LIGNICOLOUS SPECIES GLOEOPHYLLUM ODORATUM (WULFEN) IMAZEKI AND FOMITOPSIS PINICOLA (SW.) P.KARST. (FUNGI, BASIDIOMYCOTA) COLLECTED FROM CLIMANI NATIONAL PARK (THE ORIENTAL CARPATHIANS) ANIOARA STRATU*, ALEXADRINA MURARIU*, MARIA - MAGDALENA ZAMFIRACHE*, ZENOVIA OLTEANU*, LCRMIOARA OPRIC*, C. TNASE*, V. C. CHINAN*, C. BRSAN**
Abstract. The paper presents the results of the determinations of some physiological and biochemical parameters (water content, dry matter, total mineral elements, organic substances and soluble proteins and also the respiration intensity) in the lignicolous species Gloeophyllum odoratum (Wulfen) Imazeki and Fomitopsis pinicola (Sw.)P.Karst. (Fungi, Basidiomycota). The results obtained emphasize, for both species investigated, specific variations of the monitored parameters, the values determined presenting comparable amplitudes for each parameter. Key words: Gloeophyllum odoratum (Wulfen) Imazeki, Fomitopsis pinicola,(Sw.) P.Karst, physiological and biochemical parameters.

Introduction The lignicolous fungi species, because of their particular manner of nutrition, produce extracellular enzymes which mainly decompose the cellulose and the lignin basic components of the cellular walls in the woody species but also different substances resulted from the anthropic activity (hydrocarbons, petrol products, pesticides, residues resulted from sylviculture etc.) being reduced to non-toxic products [6]. The Gloeophyllum odoratum (Wulfen) Imazeki species and Fomitopsis pinicola (Sw.) P.Karst species belong to the Poliporacee family [5], they produce the brown rot and are considered primary decomposers of cellulose, - poly-carbohydrate which, the same as lignin, is generally resistant to the microbial decomposition. The paper presents the results of the determinations of some physiological and biochemical parameters from a more ample study, which focuses on the potential of mycoremediation of some pollutants from the deposits resulted from mining. Material and methods The physiological and biochemical researches were carried out on two macromycetes species which were assayed from Climani National Park, and namely from the Pinus mugo and Pinus cembra Reservation (N 470625; E 251427,3; Alt. 1600 m), which were constituted in control samples.

**

Al. I. Cuza University, Faculty of Biology, I Carol I Bd., No 20 A, 700506, Iai, Romania Anastasie Ftu Botanical Gardens, Dumbrava Roie Street, No 7-9, Iai, Romania

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At the end of June (the first assay) only one species was collected, from the same substratum (coniferous wood), and namely Gloeophyllum odoratum. The fungus was assayed from three different areas of Climani National Park (area I - Go I; area II - Go II; area III- GoIII.). In the last decade of July (the II assay) the sample analyzed was represented by the Fomitopsis pinicola (Fp - II) species. The climatic conditions specific to the analyzed period (the presence of drought) had an unfavorable influence on the growth and development of fungi. The physiological and biochemical parameters studied are: the water and dry matter content (gravimetrical method) the respiration intensity (Warburg method), the content of total mineral elements (dry calcination at 4500 C method), the organic substance content (calculated through the difference between the dry matter content and ash), and also the soluble proteins content (Bradford method). The quantitative determination of soluble proteins was achieved on the species of selected macromycetes, as indicator of the protein metabolism and nucleic acids. The determinations of parameters mentioned above (with the exception of the content of soluble proteins) were carried out gradually (whole sporocarp) and differentiated on component parts of the sporocarp (trama and hymeneal region) Results and discussions The results obtained regarding the physiological and biochemical parameters are presented in figure 1-7. The water content. The presence of water is an indispensable condition for the achievement of the metabolism processes. Although water is one of the final products of the breathing process, the process is only triggered at a certain degree of tissue hydration, and its intensity increases as the water content increases. This is explained through the fact that the enzymatic complex implied in the oxydo-reduction processes characteristic to breath can only function in the aqueous medium. The water content globally determined present moderate values comprised between 33.95 g % (Fomitopsis pinicola) and 50,3 g% (Gloeophyllum odoratum) . Differentially determined on the areas of the sporocarp, the water content presents values comprised between 25.49 g % - 58.18 g % (for the trama) and 36.32 g % - 69.47 g % (for the hymeneal region) (Figure 1-2). This physiological indicator has a strong connection with: the atmospheric humidity, the consistency of fructification body, the sensitivity of sporocarp tissues and the mycelium related to the presence of water in the atmosphere or substratum. In the analyzed lignicolous species, the water content reflects the coniferous wood characteristics which is more porous and absorbs more easily the water from the medium. The dry substance content represents a basic indicator which characterizes the level of organic and mineral constituents. The species analyzed are characterized through a high content of dry matter 49.7 % (Gloeophyllum odoratum) - 66,05 % (Fomitopsis pinicola) and organic substances. The trama is noticed through a higher content of dry matter, compared to the hymeneal region. (Figure 1, 2).

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The content of total mineral elements. According to the specialty literature data [1], the sporocarp body of fungi present a high content of mineral elements (phosphorus, potassium, calcium, magnesium, sulfur, sodium, iron, zinc) which differ according to species, age of the fungus, diameter of the pileus, the component parties of the sporocarp, substratum. In the two analyzed species, the content of total mineral elements globally determined register low values (0,95 g % - 3,03g %) (fig. 3,4). This denotes differences as regards the nature of the substratum and the capacity of exploiting the mineral elements from the substratum that they have at the disposal. The organic substances in the macromycetes are represented by proteins, polyglucides, lipids, amino acids, vitamins, phenol compounds, substances which give the aroma of fungi [1]. The content of organic matter globally determined presents high values in the two analyzed species (fig. 3, 4). This fact emphasizes an intense metabolic substance, specific to the saprophyte mode of nutrition and present a special ecological importance since the contribution of organic matter contributes to the soil formation, to the improvement of its physical-chemical characteristics. The content of soluble protein of the biological sample (Gleophyllum odoratum) assayed from three different areas of Climani National Park, but from the same sublayerconiferous wood, is relatively constant, being comprised between 70,060 mg% and 71,487mg% (Fig. 5). This is fully justified by the fact that it is the same species, assayed from the same region, without pollution, the only difference being the substratum. The concentration of soluble proteins in the samples of Fomitopsis pinicola was lower compared with the Gleophyllum odoratum species (100,132 mg% compared to 70-71 mg%) (fig. 5). This extremely small difference could however be explained through the species specificity of the protein metabolism which could be more intense in Fomitopsis pinicola compared to Gleophyllum odoratum, the samples being assayed from the same area without pollution. The biochemical studies carried out have lead us to the conclusion that the metabolic activity - in the present case the protein metabolism presents a specific manifestation being influenced by a series of external and internal factors. The respiration intensity is an indicator of the metabolic activity and indirectly, an indicator of the climatic stress state. The respiration intensity globally determined has low, but similar values (0.0178- 0,0176 mm3 oxygen/g s.pr./hour).The determinations carried out by us in the mycorrhiza species have emphasizes higher values of respiration intensity compares with these lignicolous species (unpublished data). These differences could be determined by the degree of development of the sporocarp, the cork consistency and its chemical composition, its hydration degree, the abiotic factors. The hymeneal region presents an intense respiration in both species, fact determined by the presence of reproductive structure, located at this level (Fig. 6-7). The data presented from the specialty literature (Li Xiong, 2000) for the fresh edible fungi indicate the fact that under normal conditions, their respiration intensity is high, compared with the one of vegetable species (tomatoes or salad). The biological samples of Gleophyllum odoratum assayed from the II area presents, compared with those assayed from the I, III areas, slightly lower values of the analyzed parameters (with the exception of the respiration intensity and soluble proteins).

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The analyses differentially carried out on regions of the sporocarp emphasize the fact that, compared with the trama, the hymeneal region, which comprises the reproductive formations structures with intense metabolic activity - is characterized through a higher hydration degree, a more intense respiration, a high content of organic substance and low content of mineral elements. Conclusions The analyzed species are characterized through low respiration intensity, water content with moderate values, high content of organic substances and soluble proteins, a low content of total mineral elements, fact which reveals a metabolism specific to the lignicolous fungi. The results obtained emphasize, for both species investigated, specific variants of the monitored parameters, the values determined presenting comparable amplitudes for each parameter. The particular manifestations of the investigated indicators are mostly determined, according to us, by the abiotic conditions of the areas where the biologic material subject to analyses was assayed. REFERENCES
1. 2. 3. 4. 5. 6. BERNAS EMILIA, JVORSKA GRAZYNA, LISIEWSKA ZOFIA., 2006 - Edible mushrooms as a source of valuable nutritive constituents. Acta scientiarum Polonorum tehnologia alimentaria 5 (1): 5-20 BOLDOR O., TRIFU N., RAIANU O., 1981 - Fiziologia plantelor (lucrri practice). Edit. Didactic i pedagogic, Bucureti. BRADFORD, M. M., 1976 - A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-dye binding, Anal. Biochem., 72:248-254 LI XIONG., 2000 - Extend Shelf Life of Mushroom by Using Micro-perforated Film (a research proposal) Department of Food Science, Pennsylvania State University. 5. SLGEANU GH., SLGEANU A., 1985 - Determinator pentru recunoaterea ciupercilor comestibile, necomestibile i otrvitoare din Romnia. Edit. Ceres SING A., WARD O.P. (eds), 2004 - Biodegradation and Bioremediation. Springer- Verlag Berlin Heidelberg: 19 -57.

Acknowledgements The researches were supported through funds from the project Biotech No. 128: The Ecological reconstruction through the processes of mycoremediation of soils degraded by mining activities, financed by the Ministry of Education, Research and Youth of Romania.

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dry matter
100 80 g% 60 40 20 hymeneal hymeneal 0 globally

water

area I

area II

area III

Fig. 1 The content of water and dry matter in Gloeophyllum odoratum

dry matter

w ater

70 60 50 40 g% 30 20 10 0 globally trama hymeneal region

Fig. 2 The content of water and dry matter in Fomitopsis pinicola

101

hymeneal

trama

trama

globally

globally

trama

80 70 60 50 40 30 20 10 0 trama hymeneal region trama hymeneal region trama hymeneal region globally globally globally

1,6 1,4 1,2 1 0,8 0,6 0,4 0,2 0

g%

g% organic substance total mineral elements

area I

area II

area III

Fig. 3 The content of total mineral elements and organic substances in Gloeophyllum odoratum

70 60 50 40 30 20 10 0 globally trama hymeneal region

2 1,5 1 0,5 0 g% organic substance total mineral elements

Fig. 4 The content of total mineral elements and organic substances in Fomitopsis pinicola
soluble proteins
Fp(II.) samples analyzed

g%

Go(I.3)

Go(I.2)

Go(I.1) 0 20 40 60 mg % 80 100 120

Fig. 5 The content of soluble proteins in Gleophyllum odoratum and Fomitopsis pinicola

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respiration intensity (mm3 O2 / g fresh material /hour) 0,045 0,04 0,035 0,03 0,025 0,02 0,015 0,01 0,005 0 globally

trama

trama

hymeneal region

hymeneal region

trama area III

area I

area II

Fig. 6 The intensity of respiration in Gleophyllum odoratum

Fig. 7 Respiration intensity at Fomitopsis pinicola

0,3 0,25 0,2 0,15 0,1 0,05 0 globally trama

respiration intensity ( mm3 O2/ g fresh material / hour)

Fig. 7 The intensity of respiration in Fomitopsis pinicola

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hymeneal region

globally

globally

hymeneal region

Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

SOMATIC EMBRYOGENESIS IN RUBUS CAESIUS L. SUSPENSION CULTURES SMARANDA VNTU*

Abstract: In vitro cultures technologies include many applications: plants improvement, micropropagation, , unconventional alternatives to exploit secondary metabolites. The regenerative potential of Rubus caesis L. was evaluated for the establishment of a propagation protocol. Not only the capacity of differentiation was tested, but also the regenerative potential. The initiation of in vitro cultures at Rubus sanctus Schreb was achieved from axillary buds, cultivated on different variants of MurashigeSkoog medium. The callus cultures obtained by axillary buds dedifferentiation were multiplicated on agar medium and liquid medium. The MS medium supplemented with 1mg/l benzylaminopurine and 0,1 mg/l IAA stimulated somatic embryogenesis at Rubus caesius L. suspension cultures ,whereas the MS medium supplemented with 1mg/ kinetine generated an intensive proliferative reaction and callus development. The regeneration of whole plants was obtained in three steps: callus initiation, suspension cultures initiation, somatic embryogenesis induction. Key words: somatic embryogenesis, Rubus caesius, suspension cultures

Introduction In this paper the procedure for the regeneration of Rubus caesius L. plants based on indirect somatic embryogenesis is presented.The studies are focused on the application of in vitro methods on this species, well known as a medicinal plant. The fruits are a good source of natural antioxidants [1], [5], [6]. Material and methods The initiation of in vitro cultures of Rubus caesius L. was achieved from axillary buds. The axillary buds were sterilized with ethanol 70 % and then sodium hypochlorite 0,5 % 10-15 minutes. After rinsing with sterile distilled water, the explants were transferred to MS medium. 4 variants of MS medium were tested. (Table 1 and 2). The studies of the in vitro behaviour of this species is based on the use of auxins and cytokinins, in different combinations and concentrations [2], [3], [4]. The variants 1 and 3 (Table 2) stimulated callus induction and callus multiplication, whereas the variants 2 and 4 stimulated somatic embryogenesis and variants 5, without growth regulators induced embryos development and maturation. The axillary buds explants were cultivated on the callus induction media at 24 C in complete darkness. After 30 days the callus produced was used to establish cell suspensions on the same basal MS medium. Suspension cultures were established by transferring the section of calli 1 cm3 each, from the exponential growth phase into 250 ml Erlenmeyer flasks containing 100 ml liquid medium. The flasks were rotated on rotatory shaker at 100 rpm. The cell suspensions were periodically subcultivated by filtration with a metallic sieve. The next step was the induction of somatic
*

Al. I. Cuza University, Faculty of Biology, 20 A Carol I Bd, Iasi, 700506, Romania

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embryogenesis. The suspension cultures were transferred on regeneration media and maintained at 24 C under 16 hours light cycle. Table 1. Chemical composition of MS medium
COMPOSITION NH 4 NO 3 KNO 3 MACROELEMENTS CaCl 2 2H 2 O Concentration (mg/l) 1650 1900

440

MgSO 4 7 H 2 O KH 2 PO 4

370 170

H 3 BO 4 MnSO 4 4 H 2 O MICROELEMENTS ZnSO 4 7 H 2 O

6,2 22,3 8,6 0,25

Na 2 MoO 4 2H 2 O CuSO 4 5 H 2 O CoCl 2 6H 2 O KI Nicotinic acid VITAMINES Pyridoxine HCl Thiamine Mezoinozitol SUCROSE pH 0,025

0,025 0,83 0,5 0,5 0,1 100 30 g/l 5,8

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Table 2. Variants of MS medium

VARIANTS BAP 1 2 3 4 5 1 mg/l 1 mg/l GROWTH REGULATORS IAA 1 mg/l 0,1 mg/l NAA 1 mg/l 0,1 mg/l K 1 mg/l 1 mg/l -

Results and discussions The main aim of the investigation was to select different cell lines from suspension cultures for embryogenetic capacity. The axillary buds of Rubus caesius L. were the explants for callus initiation. Five different media are used for plant propagation via somatic embryogenesis. These media differ by the type and level of plant growth regulators. The begin of callus proliferation was achieved on two variants of MS medium: variants 1 with a same concentration of benzylaminopurine and indolilacetic acid and variants 3 with the same concentration of kinetine and naphtalenacetic acid. The first cell proliferation were observed on the cantact surface of the explant with nutritive medium (Photo 1, 2). The primary callus cultures were obtained after 4 weeks from the in vitro cultivation, when the entire explant dedifferentiated (Photo 3). The callus cultures obtained on agar medium were multiplicated on liquid medium (Photo 4). The submerse cultures were periodically transferred on fresh medium. The suspension cultures were cultivated on rotatory shaker at 100 rpm and maintained in the dark. After 2 weeks, they were transferred on somatic embryogenesis induction medium (Photo 4, 5). The main factors for somatic embryogenesis induction were: the selection of cells, the use of growth regulators in some concentrations and combinations, the photoperiode. The embryogenic clusters were selected after each stage of subcultivation on induction media. Early stages of somatic embryogenesis were observed after a week from the initiation of the submerse cultures (Photo 6). The specific steps of the embryo development that were encountered were: globular, cardo, torpedo and cotyledonary stages. Embryogenesis was induced under various ratios of auxin to cytokinin. Despite the selection, the suspension cultures display not only a morphological heterogenity, but also differences in cell reaction: some cells multiplied and formed clusters, without regenerative potential, whereas some cells redifferentiated and developed somatic embryos.

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The conditions that favor initial somatic embryogenesis may inhibit further development of the embryos. That is why the variants 2 and 4 are used for initiation of somatic embryogenesis and variant 5 is utilized to allow somatic embryos development. The use of MS medium supplemented with excess of benzylaminopurine in combination with indolilacetic acid and kinetine in combination with naphtalenacetic acid stimulated the somatic embryo development. The differentiated embryos could be converted into plants by transferring to the MS medium for maturation, without growth regulators. Conclusions Callus induction was stimulated on MS medium with an equal concentration of a cytokinine and auxine. Cell suspensions of Rubus caesius L. were obtained from callus cultures derived from axillary buds. The development and maturation of the somatic embryos occurred after 4 weeks in suspension cultures. The use a combination of a cytokinine in excess and an auxine led to a increase in the frequency of embryos differentiation in suspension cultures. Changing the growth regulators balance in the MS medium has major effects on in vitro dedifferentiation and redifferentiation at Rubus caesius L. REFERENCES
1. 2. 3. 4. 5. 6. BENVENUTI, S., PELLATI, F., MELEGARI, M., BERTELLI, D., 2004-Polyphenols, anthocyans, ascorbic acid and radical scavenging activity of Rubus, Ribes and Aronia, Journal of Food Science 69(3):164-169. BUSBY, A. , HIMELRICK, D.G., 1999- Propagation of blackberries (Rubus ssp.) by stem cuttings using various IBA formulations, Acta Hort, 505: 327-332 FIOLA, J.A., SCHWARTZ, H., 1986-Somatic embryos organogenesis and proliferation in vitro from Rubus embryos, Acta Hort., 183: 91-98 FIOLA, J.A., HASSAN, M.A., SWARTZ, H.J., BORS, R.H. , MCNICHOLS, R., 1990- Effect of thidiazuron light fluence rates and kanamycin on in vitro shoot organogenesis from excised Rubus cotyledons and leaves, Plant Cell, Tissue and Organ Culture, 20: 223-228 MOYER, R.A., HUMMER, K.E., FINN, C.E., FREI, B., WROLSTAD, R. E., 2002- Anthocyanins phenolics and antioxidant capacity in diverse small fruit: Vaccinium, Rubus and Ribes, Journal of Agricultural and Food Chemistry, 50: 519-525 NIKITINA, U.S., KUZMINA, L., MELENTEV, A.I., SHENDEL, G.V., 2007- Antibacterial activity of polyphenolic compounds isolated from plants of Geraniaceae and Rosaceae families, Applied biochemistry and microbiology, 43(6): 629-634

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SMARANDA VNTU

PLATE

Photo 1. Callus development

Photo 2. Cell proliferation

Photo 3. Primary callus cultures

Photo 4. Suspension cultures

Photo 5. Cell cluster

Photo 6. Embryogenic cells cultures

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

GENES OF ARABIDOPSIS THALIANA INVOLVED IN WAX METABOLISM ROXANA-IULIANA TEODOR*, AL. YEPHREMOV*
Abstract: The aerial surfaces of land plants are covered with cuticle that acts as a barrier providing protection against water loss, pathogen invasion and other environmental aggressions. Besides physical and chemical barriers, such as a waxy cuticle, plant defense mechanisms also involve a coordinated activation of cellular responses to limit damage. The eceriferum (cer) mutants of Arabidopsis define multiple genes required for various steps in cuticular wax biosynthesis, transport and regulation of lipid-related pathways. Although the basic biochemistry of wax production has been elucidated, very little is known about its regulation and its contribution to the natural immunity. This review presents recently cloned wax biosynthetic genes and discusses regulatory aspects of wax biosynthesis. Key words: wax, Arabidopsis mutants

Role of cuticle for plants A distinctive characteristic of all epidermal cell types is the presence of cuticle covering their outer surface as a continuous lipophilic layer, which forms a barrier over the aerial organs of land plants during their primary stages of development [7; 14]. Plant cuticle is formed mainly of cutin, a polyester of hydroxy and hydroxy-epoxy fatty acid derivatives generated from cellular fatty acids [11], embedded in and covered with a complex mixture of highly hydrophobic soluble materials (aliphatic compounds comprised mainly of C24-C34 alkanes, alcohols and ketones) called cuticular waxes, as well as other minor compounds, of an extremely diverse nature [30]. As they are physically very closely associated, it is difficult to distinguish between the relative contribution of the cutin matrix and that of cuticular waxes to the physical properties and the biological roles of the cuticle. However, in biochemical experiments, the cutin and cuticular waxes are usually considered and analyzed separately, due to the soluble properties of the waxes versus the cutin polymer, which remains insoluble. Such a structure gives the cuticle a set of highly protective features and these were studied initially as to limiting nonstomatal water loss and gaseous exchanges, controlling the absorption of lipophilic compounds, and providing mechanical strength and viscoelastic properties [2; 25]. Additionally, the cuticle also functions in normal plant developmental processes, including the prevention of postgenital organ fusion and pollenpistil interactions [17; 28], as well as protecting the plant from biotic and non-biotic environmental stress factors [27]. Pruitt et al. [24] and Sieber et al. [28] have suggested that cuticle permeability also influences cell-to-cell communication by enhancing or attenuating the passage of signal molecules. For example, such signals could be required for organ adhesion, when they

Molecular Plant Genetics, Max Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, Cologne, Germany

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would be moving across the cuticle, or for mediating signaling between trichomes and stomata, when moving within the developing epidermis [18; 13]. Arabidopsis mutants offer information on the regulation of cuticle development Mutants with deficient or altered wax coatings have been identified due to their nonglaucous or glossy phenotype. Thus, there are no reports of mutants that lack wax completely, indicating the vital function that waxes play for the normal development of plants. On the other hand, changes in a minor wax component are less likely to lead to a clearly discernable phenotype and therefor have not been reported. Similarly, plants that overproduce waxes are not easily detected by visual screenings. In Arabidopsis, as well as in other species, the mutants that have been identified to be defective in wax and/or cutin formation facilitated the identification of enzymes associated with the cutin and wax pathways. Some of the enzymes catalyzing various steps in the wax pathway have been characterized or their function has been proposed based on the phenotype of the corresponding mutants. The identification of eceriferum (cer) mutant lines described mainly by Koornneef et al. [12] and by McNevin et al. [20] has led to the isolation and characterization of various genes associated with cuticular wax metabolism in Arabidopsis. CER1 intended to encode an aldehyde decarbonylase [1]. Several genes playing a role in the fatty acid elongation pathway that generates very long chain fatty acid (VLCFA) wax precursors have also been characterized. They include the FATTY ACID ELONGATION1 homologs (FAE1) [9], FIDDLEHEAD (FDH) [38; 24], 3-KETOACYL-CoA SYNTHASE [33], CUT1/CER6, and CER60 [21; 5]. CER6 has been suggested to be the key condensing enzyme for wax biosynthesis in Arabidopsis, due to its expression throughout all stages of stem and leaf development, as well as in the inflorescence [8]. CER2 encodes a CoA-dependent acyltransferase, a component of the fatty acid elongase complex, apparently located in the nucleus [37; 14]. The cer2 mutant shows reduced levels of the decarbonylation pathway products and it accumulates C26 and C28 acyl groups, primary alcohols, and wax esters but the precise function of the gene is still unknown. Furthermore, its nuclear localization is very intriguing for a protein of the fatty acid elongase complex. Many of the cer mutants remain still to be characterized and the isolation of their corresponding genes might bring valuable information on the mechanisms of wax metabolism. Several reports have also provided insights into the biosynthesis of cutin monomers in plants. Chen et al. [3] reported the isolation of the WAX2 gene and showed that the protein it encodes for has 32% similarity to CER1 and contains certain regions with homology to sterol desaturases and short-chain dehydrogenases/reductases. It was suggested therefore that WAX2 plays a metabolic role in both wax and cutin synthesis, thus pointing to a link between wax and cutin metabolism. ADHESION OF CALYX EDGES/HOTHEAD (ACE/HTH) is proposed to be an oxidase catalyzing the formation of dioic acids from -hydroxy acyl-CoAs [13; 16]. The Arabidopsis LACERATE (LCR) gene [36] encodes a cytochrome P450; enzyme activity assays using the recombinant LCR protein showed that it could efficiently catalyze the formation of -hydroxy fatty acids (ranging from C12 to C18:1). Expression of LCR gene is predominant in inflorescence and

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siliques, as well as in roots and young seedling tissue and it is the first cytochrome P450 hydroxylase for which a mutant has been isolated. Results of microarray analysis conducted in our group (Yephremov et al., unpublished data) on three independent cuticular mutants has revealed that a palmytoil protein thyoesterase (PPT) is almost ten fold up-regulated in three mutants, as compared to wild type. In humans, PPT is a lysosomal long-chain fatty acyl hydrolase that removes fatty acyl groups from modified cysteine residues in proteins, and the defective enzyme causes infantile neuronal ceroid lipofuscinosis, a recessive hereditary neuro-degenerative disorder [34]. In plants, acyl-acyl carrier protein (ACP) thioesterases play an essential role in chain termination during de novo fatty acid synthesis and in the channeling of carbon flux between the lipid biosynthesis pathways [10]. Epidermal differentiation and implicitly cuticle formation is essential for the general development of the whole plant, starting from the very early embryo stage. This fact is supported by the characterization of the abnormal leaf shape 1 (ale1) mutant of Arabidopsis, which shows impaired cuticle formation, adhesion of endosperm and embryo, as well as fusion of cotyledons and leaves. The corresponding ALE1 gene encodes a member of the subtilisin-like serine protease family and it is preferentially expressed during seed development, showing a weak transcript expression in young embryo and a strong one within the endosperm cells closely surrounding the developing embryo [32]. Three aminoacid residues (aspartic acid, histidine and serine) are consistently conserved in the catalytic regions of subtilisin-like serin proteases. In animals, such proteases activate precursors of hormones, growth factors, or receptors involved in the control of various developmental processes, including embryonic patterning and proper epidermal differentiation. Although many members of this family of proteases were reported in plants [29; 26], little is known, with few exceptions, about their precise role. In addition to the developmental factors controling the synthesis of cuticular lipids, environmental signals such as light intensity, photoperiod [19; 35], humidity [31], chilling [22; 23] and seasonal variation [6; 4] have also been shown to ifluence wax biosynthesis. In 1984, Sutter [31] described the dramatic response of wax production to environmental cues, during tissue culture, observing that when the relative humidity is high, wax production is low. When tissue-culture-grown plants are transfered to an environment with less humidity (growth chabinet or greenhouse), production of wax is stimulated and within a rather short period of time of a few days only, the plant synthesizes a complete protective layer of wax. Conclusion The fact that cuticular wax is ubiquitously present is testimony to its essential role in the adaptation of plants to the aerial environment, with all its implications. On the other hand, the fact that environmental cues have an influence upon wax composition and quantity is evidence that wax production is an actively regulated process. An active regulatory netword is indicated also by the high diversity of proteins that have been shown, through the Arabidopsis mutants, to be involved in the process of wax biosynthesis. Although the biosynthesis of plant cuticular components has been studied for over four decades, we still know little about the factors regulating the partitioning of fatty

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acid precursors and the synthesis of waxes with the synthesis of cutin and with other cuticular compounds. However, the cloning of wax biosynthetic genes and the further characterization of the respective proteins promises to bring valuable insights into the very deep regulatory mechanisms of wax and cuticle development. REFERENCES
1. 2. 3. AARTS, M.G.M., KEIJZER, C.J., STIEKEMA, W.J., and PEREIRA, A., 1995 - Molecular Characterization of the CER1 Gene of Arabidopsis Involved in Epicuticular Wax Biosynthesis and Pollen Fertility. The Plant Cell Online, 7: 2115-2127. BAKER, C.J., MCCORMICK, S.L., and BATEMAN, D.F., 1982 - Effects of Purified Cutin Esterase Upon the Permeability and Mechanical Strength of Cutin Membranes. Phytopathology ,72: 420-423. CHEN, X., GOODWIN, S.M., BOROFF, V.L., LIU, X., and JENKS, M.A., 2003 - Cloning and Characterization of the WAX2 Gene of Arabidopsis Involved in Cuticle Membrane and Wax Production Article, publication date, and citation information can be found at www. plantcell. org/cgi/doi/10.1105/tpc. 010926. The Plant Cell Online, 15: 1170-1185. FAINI, F., LABB, C., AND COLL, J., 1999 - Seasonal changes in chemical composition of epicuticular waxes from the leaves of Baccharis linearis. Biochemical Systematics and Ecology, 27: 673-679. FIEBIG, A., MAYFIELD, J.A., MILEY, N.L., CHAU, S., FISCHER, R.L., AND PREUSS, D., 2000 Alterations in CER6, a Gene Identical to CUT1, Differentially Affect Long-Chain Lipid Content on the Surface of Pollen and Stems. The Plant Cell Online, 12: 2001-2008. GLZ, P.G., AND MLLER, E., 1992 - Seasonal variation in the composition of epicuticular waxes of Quercus robur leaves. Zeitschrift fr Naturforschung. C. A journal of biosciences, 47: 800-806. HOLLOWAY, P.J. (1982). Structure and histochemistry of plant cuticular membranes: an overview. HOOKER, T.S., MILLAR, A.A., AND KUNST, L., 2002 - Significance of the Expression of the CER6 Condensing Enzyme for Cuticular Wax Production in Arabidopsis. Plant Physiology, 129: 1568-1580. JAMES, D.W.J., LIM, E., KELLER, J., PLOOY, I., RALSTON, E., AND DOONER, H.K., 1995 - Directed Tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) Gene with the Maize Transposon Activator. The Plant Cell Online 7, 309-319. JONES, A., DAVIES, H.M., AND VOELKER, T.A., 1995 - Palmitoyl-Acyl Carrier Protein (ACP) Thioesterase and the Evolutionary Origin of Plant Acyl-ACP Thioesterases. The Plant Cell Online, 7: 359371. KOLATTUKUDY, P.E., 2001 - Polyesters in higher plants. Advances in biochemical engineering, biotechnology, 71, 1-49. KOORNNEEF, M., HANHART, C.J., AND THIEL, F., 1989 - A Genetic and Phenotypic Description of Eceriferum (cer) Mutants in Arabidopsis thaliana. Journal of Heredity, 80: 118. KROLIKOWSKI, K.A., VICTOR, J.L., WAGLER, T.N., LOLLE, S.J., AND PRUITT, R.E., 2003 Isolation and characterization of the Arabidopsis organ fusion gene HOTHEAD. The Plant Journal, 35: 501511. KUNST, L., and SAMUELS, A.L., 2003 - Biosynthesis and secretion of plant cuticular wax. Prog. Lipid Res, 42: 51-80. KUNST, L., SAMUELS, A.L., AND JETTER, R., 2005 - The plant cuticle: formation and structure of epidermal surfaces. (Oxford,UK: Blackwell). KURDYUKOV, S., FAUST, A., TRENKAMP, S., BR, S., FRANKE, R., EFREMOVA, N., TIETJEN, K., SCHREIBER, L., SAEDLER, H., AND YEPHREMOV, A., 2006 - Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain -,-dicarboxylic fatty acids and formation of extracellular matrix. Planta, 224: 315 - 329. LOLLE, S.J., HSU, W., AND PRUITT, R.E., 1998 - Genetic Analysis of Organ Fusion in Arabidopsis thaliana. Genetics, 149: 607-619. LOLLE, S.J., BERLYN, G.P., ENGSTROM, E.M., KROLIKOWSKI, K.A., REITER, W.D., AND PRUITT, R.E., 1997 - Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant: a role for the epidermal cell wall and cuticle. Dev. Biol, 189: 311321.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

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19. MACEY, M.J.K., 1970 - The effect of light on wax synthesis in leaves of Brassica oleracea. Phytochemistry 9: 757-761. 20. MCNEVIN, J.P., WOODWARD, W., HANNOUFA, A., FELDMANN, K.A., AND LEMIEUX, B., 1993Isolation and characterization of eceriferum (cer) mutants induced by T-DNA insertions in Arabidopsis thaliana. Genome, 36: 610-618. 21. MILLAR, A.A., CLEMENS, S., ZACHGO, S., GIBLIN, E.M., TAYLOR, D.C., AND KUNST, L., 1999 CUT1, an Arabidopsis Gene Required for Cuticular Wax Biosynthesis and Pollen Fertility, Encodes a VeryLong-Chain Fatty Acid Condensing Enzyme. The Plant Cell Online, 11: 825-838. 22. NORDBY, H.E., AND MCDONALD, R.E., 1991 - Relationship of epicuticular wax composition of grapefruit to chilling injury. Journal of Agricultural and Food Chemistry, 39: 957-962. 23. NORDBY, H.E., AND MCDONALD, R.E., 1995 - Variations in Chilling Injury and Epicuticular Wax Composition of White Grapefruit with Canopy Position and Fruit Development during the Season. Journal of Agricultural and Food Chemistry, 43: 1828-1833. 24. PRUITT, R.E., VIELLE-CALZADA, J.P., PLOENSE, S.E., GROSSNIKLAUS, U., AND LOLLE, S.J., 2000 - FIDDLEHEAD, a gene required to suppress epidermal cell interactions in Arabidopsis, encodes a putative lipid biosynthetic enzyme, 97: 1311-1316. 25. RIEDERER, M., and SCHREIBER, L., 2001 -. Protecting against water loss: analysis of the barrier properties of plant cuticles. Journal of Experimental Botany, 52: 2023. 26. SCHALLER, A., 2004 - A cut above the rest: the regulatory function of plant proteases. Planta 220, 183-197. 27. SCHWEIZER, P., FELIX, G., BUCHALA, A., MLLER, C., AND MTRAUX, J.P. (1996). Perception of free cutin monomers by plant cells. The Plant Journal, 10: 331-341. 28. SIEBER, P., SCHORDERETA, M., RYSERA, U., BUCHALAA, A., KOLATTUKUDYB, P., MTRAUXA, J.-P., AND NAWRATH, C., 2000 - Transgenic Arabidopsis Plants Expressing a Fungal Cutinase Show Alterations in the Structure and Properties of the Cuticle and Postgenital Organ Fusions. Plant Cell, 12: 721738. 29. SIEZEN, R.J., 1997 - Subtilases: The superfamily of subtilisin-like serine proteases. Protein Science, 6: 501523. 30. SUH, M.C., SAMUELS, A.L., JETTER, R., KUNST, L., POLLARD, M., OHLROGGE, J., AND BEISSON, F., 2005 - Cuticular Lipid Composition, Surface Structure, and Gene Expression in Arabidopsis Stem Epidermis 1 [W]. Plant Physiology, 139: 1649-1665. 31. SUTTER, E., 1984 - Chemical composition of epicuticular wax in cabbage plants grown in vitro. Canadian Journal of Botany, 62: 74-77. 32. TANAKA, H., ONOUCHI, H., KONDO, M., HARA-NISHIMURA, I., NISHIMURA, M., MACHIDA, C., AND MACHIDA, Y., 2001 - A subtilisin-like serine protease is required for epidermal surface formation in Arabidopsis embryos and juvenile plants. Development, 128: 4681-4689. 33. TODD, J., POST-BEITTENMILLER, D., AND JAWORSKI, J.G., 1999 - KCS1encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis inArabidopsis thaliana. The Plant Journal, 17: 119-130. 34. VESA, J., HELLSTEN, E., VERKRUYSE, L.A., CAMP, L.A., RAPOLA, J., SANTAVUORI, P., HOFMANN, S.L., AND PELTONEN, L., 2002 - Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature, 376: 584-587. 35. VON WETTSTEIN-KNOWLES, P., AVATO, P., AND MIKKELSEN, J.D., 1979 - Light promotes synthesis of the very long chain fatty acyl chains in maize wax. Biogenesis and Function of Plant Lipids. Elsevier/North Holland Biomedical Press, New York: 271274. 36. WELLESEN, K., DURST, F., PINOT, F., BENVENISTE, I., NETTESHEIM, K., WISMAN, E., STEINERLANGE, S., SAEDLER, H., AND YEPHREMOV, A., 2001 - Functional analysis of the LACERATA gene of Arabidopsis provides evidence for different roles of fatty acid omega-hydroxylation in development. Proceedings of the National Academy of Sciences, 98: 9694. 37. XIA, Y., NIKOLAU, B.J., AND SCHNABLE, P.S., 1996 - Cloning and Characterization of CER2, an Arabidopsis Gene That Affects Cuticular Wax Accumulation. The Plant Cell Online, 8: 1291-1304. 38. YEPHREMOV, A., WISMAN, E., HUIJSER, P., HUIJSER, C., WELLESEN, K., AND SAEDLER, H., 1999 - Characterization of the FIDDLEHEAD Gene of Arabidopsis Reveals a Link between Adhesion Response and Cell Differentiation in the Epidermis. The Plant Cell Online, 11: 2187-2202.

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CHARACTERISTICS OF THE PLANKTONIC ALGOFLORE FROM DOROBANTI, ARONEANU, CIRIC I, II AND III LAKES (IASI COUNTY) M. A. PORUMB* , M. COSTIC**
Abstract:After the setting of Ciric river by building some barrages, the lakes Dorobanti, Aroneanu, Ciric I, II and III were formed, their total surface being of about 123 ha. When the lakes were formed, processes of colmating and accumulating used waters started, these waters were coming from the side localities and from the Ciric recreation area. Water quality from these aquatic mediums was evaluated on these conditions, knowing the diversity of phytoplankton algae, the phytoplankton evolution and the specific structure of algoflore. Researches concerning phytoplankton of the Ciric river area (May, 2006) show the diatoms dominance (BACILLARIOPHYTA) at Dorobanti mijloc and Parau Ciric Pod stations, of the CHLOROPHYTA group at Ciric II, Ciric I and Aroneanu stations and the big heaviness of the CYANOPHYTA group at Dorobanti Dig station (in this moment, this station is attested by the algologic indicators as being very polluted). During the first cold period of the year 2007, biodiversity is reduced (because of the pollution), and the genus analysis shows the dominance of the green algae (CHLOROPHYTA) at the first 4 lakes: at Dorobanti station the biggest numerical heaviness is presented by Monoraphidium tortile (W.et G.S.West) Kom.-Leg. 21.18% of the total algae; at Aroneanu station Monoraphidium contortum (Thur.) Kom.-Leg - 25.81% and Monoraphidium tortile (W.et G.S.West) Kom.-Leg. 18.54% are dominant; at Ciric I, Monoraphidium contortum (Thur.) Kom.-Leg 48.88%, at Ciric II, Monoraphidium contortum (Thur.) Kom.-Leg 55.66%, and at the last lake, Ciric III, the biggest numerical heaviness was observed by Diatoma elongatum (Lyngb.) Agardh. (Bacillariophyta) 47.70% of the total planktonic algae determined at that station. Numerical densities of phytoplankton from the 5 studied lakes varied between the values of 1,105 3,830 exemplars algae/ml in March, 2007, and 15,603 73,759 exemplars algae/ml in November, the same year. These values show an important pollution degree of the investigated ecosystems. As it follows, researches are necessary for a period of several years in order to point out the implied processes and phenomena complexity and evolution in those aquatic ecosystems that are affected by anthropic pollution. Key words: Ciric lacustrine complex, phytoplankton, algal taxons, water quality.

Introduction Ciric recreation complex is made of a series of artificial barrage lakes, in falls, that are alimented by Parau Ciric and by rains, and it has a recreation function: Dorobanti (70.00 ha), Aroneanu (23.00 ha), Ciric I, II, III with a total surface of 30.00 ha and 2.70 m in maximum depth. Ciric Complex, which is situated on Valea Ciricului between Dealul Ciric in the East and Dealul Sorogari in the West, is at 3.5 km up the confluence with Bahlui River. From a physical-geographical point of view, it is placed at the contact between two big subunits of Podisul Moldovenesc (the Moldavian Plateau): Campia Moldovei (Moldavian Plain) and Podisul Central Moldovenesc (Central Moldavian Plateau). This lacustrine Complex is 1.5 km long and its tail is near Aroneanu village.
* **

The Station of Biological Researches Petre Jitariu Piatra Neam, Romania Faculty of Biology, Al. I. Cuza University of Iasi, Romania

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The trophic level of the Ciric lakes surface water is of 3rd category in quality (according to STAS 4706/88), with a more accentuated degradation degree during the hot period of the year when high temperatures determine a massive development of green-blue algae and of euglenophycea, and finally, a more accentuated pollution that has also implications in the underground waters in the area. The main causes of the water quality depreciation come from the diffused pollution and from direct evacuations of used waters that come from the economic agents in the area, the tourist activity and private proprieties, these facts being amplified by the lack of canalization in the area. Phytoplankton indicates the water quality of an aquatic ecosystem by its qualitative and quantitative composition. Season conditions interfere with existent pollution, the result being a phytoplankton that reflects the whole existent conditions. Material and methods Phytoplankton samples were collected each semester, within 2006 2007, from the stations: Parau Ciric (1); Dorobanti (2); Aroneanu (3); Ciric I (4); Ciric II (5) and Ciric III (6). Quantitative results of the samples that were studied at microscope by the above mentioned proceedings, are appreciated by calculi, and a formula that includes the surface of the microscope lamella, the volume of the drop under lamella (0.03 ml a drop that comes from a graded and calibrated dropping glass of 1 ml), the ocular field diameter, the number of analyzed microscopic fields and the quantity of sedimented sample or/and centrifuged. Algal biomass is determined by the establishment of cellular volumes (in microns) of the counted algae and the conversion of these volumes in grams/m3, starting from Dussart raport, 1966 (Limnologie. Letude des Eaux Continentales, Gauthier Villars, Paris): 1,000,000 microns3 = 0,000001 grams. For the algal biomass calculus, the lists of cellular volumes from the literature are used for each genus, lists that are completed with original lists. Microscope observations were made using the phase contrast a technique by which fine details can be identified, these details being difficult to see by common proceedings. In the documentary research regarding the results of planktonic algae there were mainly used the series of Polish determinators Flora Slodkowodna Polski, of authors: SIEMINSKA [13], STARMACH [14, 15, 16, 17, 18]. There were also consulted the works of HINDAK [3,4,5], KOMAREK and AGNOSTIDIS [7], JOHN and collab.[6], that were completed by other determinators and with the latest revisions of some genera and races of different systematic groups of algae. There were also used some works from the ecological literature of specialty [2, 8, 10, 11, 12]. Work methods were applied in a critical manner, according to the necessities of the theme.

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Results and discussions Researches regarding phytoplankton of the Ciric river area that were made in 20062007 show significant differences between the studied aquatic ecosystems, from an algological point of view. Qualitative data show the following qualitative structure of planktonic algal communities in the 6 stations of assay: CYANOPHYTA- Anabaena flos-aque Breb. ex Born et Flah (4), Anabaena solitaria Kleb. (2,4), Anabaena spiroides Kleb (2,4,5), Anabaena sp. (4), Aphanizomenon flos- aque Ralfs ex Born et Flah (4), Anabaena sp. (4), Gloeocapsa punctata Nag. (4,5), Gloeocapsa turgida (Kutz.) Hollerb. (2,4,6), Gomontiella marthae Claus (2), Merismopedia tenuissima Lemm. (3,4,5), Microcystis incerta (Lemm.)Lemm. (2,5), Microcystis pulverea(Wood) Mig. In Lemm. (5), Oscillatoria limnetica Lemm. (2), Oscillatoria planctonica Wolosz. (3), Oscillatoria granulata Gordner (1), Pseudanabaena sp. (2), Romeria leopoliensis (Rocib.) Koczw. (4), Romeria gracilis Koczw. (2), Spirulina laxissima G.S. West (2,4,5), Spirulina meneghiniana Zanard. (4), Spirulina raphidioides Geitl (2,5), Spirulina sp. (4,5), Synechococcus elongatus (Nag.) Nag. (2,4); CHRYSOPHYTA- Chrysococcus sp. (2,3), Hymenomonas roseola Stein (2), Mallomonas sp. (4,5), Ochromonas sp. (2,4,5,6); XANTHOPHYTA- Tribonema monochloron Pascher et Geitler (1,2) BACILLARIOPHYTA- Achnanthes minutissima Kutz. (1, 2, 3, 5, 6), Achnanthes sp. (1, 2, 6), Amphora ovalis Kutz. (1, 2, 5, 6), Caloneis sp. (2), Coconeis pediculus Ehr. (2, 3, 4), Coconeis placentula Ehr. (2), Cyclotella compta (Ehr.) Kutz. (2,5), Cyclotella meneghiniana Kutz. (5), Cyclotella sp. (2.), Cymbella amphicephala Naeg. (1), Cymbella ventricosa Kutz. (5), Diatoma anceps (Ehr.) Kirchn. (5), Diatoma elongatum (Lyngb.)Agardh (6), Diatoma elongatum var. actinastroides Krieger (6), Diatoma elongatum var. tenue (Agardh) Van Heurck. (3,6), Gomphonema constrictum Ehr. (6), Gomphonema olivaceum (Hornem.)Breb. (1,2,3,4,6), Melosira granulata (Ehr.) Ralfs (2), Navicula cryptocephala Kutz. (1,2,6), Navicula placentula (Ehr.) Grun. (1), Navicula radiosa Kutz. (1,2), Navicula rhynchocephala Kutz. (2), Navicula viridula Kutz. (1,2), Navicula sp. (1,2,5), Nitzschia acicularis W.Smith (1,4,5), Nitzschia capitellata Hust. (4), Nitzschia closterium (Ehr) W.Sm. (5), Nitzschia circumsuta (Bailey) Grunov (1), Nitzschia linearis (Ag.) W.Sm. (1,2), Nitzschia palea (Kutz.) W. Smith (1,2,3,4,5), Nitzschia sigmoidea (Ehr.) W.Smith (1), Nitzschia tryblionella Hantz. (Kutz.) Grun. (1), Nitzschia sp. (3,4,5,6), Rhoicosphaenia curvata (Kutz.)Grun. (2,4,5,6), Rhizosolenia longiseta Zach. (1), Rhizosolenia eriensis H.L.Smith (5), Rhopalodia gibba (Ehr.) O.Muller (4), Surirella angustata Kutz. (1), Surirella ovata Kutz. (1), Surirella sp.(6), Synedra acus Kutz. (3), Synedra nana Meist.(4), Synedra tenera W.Sm. (2), Synedra ulna (Nitz.) Ehr. (3,5), Synedra vaucheriae Kutz. (3), Synedra sp. (3,5); PYRROPHYTA- Chroomonas acuta Utermohl (2), Chroomonas nordstedtii Hansa. (1,2), Peridinium cinctum Ehr. (5); CHLOROPHYTA- Chlorella vulgaris Beijer. (1,2,3,4,5), Chlorogonium tetragamum Bohlin(4), Chlamydomonas sp. (3,5), Cladophora sp. (4), Closteriopsis acicularis (G.M.Smith) Belcher et Swale (2), Closterium acutum Breb. (1), Coelastrum sphaericum

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Naeg. (2,5), Coelastrum sp. (2), Crucigenia rectangularis (Nag.) Gay (1), Crucigenia tetrapedia (Kirchn.) W.et G.S.West (1,2,5), Dictyosphaerium pulchellum Word (5), Didymocystis fina Komarek (5), Golenkinia radiata Chodat (5), Hyaloraphidium contortum Pasch. et Korsch. (4), Keratococcus bicaudatus (A.Br.) Boye.-Pet. (5), Kirchneriella aperta Teiling (2), Kirchneriella contorta (Schm.) Bohl (2, 3, 4), Kirchneriella irregularis (Smith) Korsch. (6), Kirchneriella obesa (W.West) Schm. (1,4), Kirchneriella subcapitata Korsh. (1, 2), Koliella longiseta (Wisl.)Hind. (3, 4), Koliella planctonica Hind. (2, 3, 4, 5, 6), Koliella spiculiformis (Wisch.)Hind. (1, 3, 4, 5, 6), Koliella sp.(3,4), Lagerheimia genevensis (Chodat) Chodat (2, 3), Monoraphidium arcuatum (Korsch.) Hind. (2, 3, 4, 5), Monoraphidium contortum (Thur.) Kom.-Leg. (2, 3, 4, 5, 6), Monoraphidium griffithii (Berkeley) Kom.-Leg. (3, 5, 6), Monoraphidium komarkovae Nygaard (4, 5), Monoraphidium minutum (Nag.) Kom.-Leg. (2, 3, 4, 5, 6), Monoraphidium pusillum (Printz) Kom.-Leg.(2), Monoraphidium tortile (W.et G.S.West) Kom.-Leg. (2, 3, 4, 5, 6), Monoraphidium sp. (2, 5), Nephrochlamys agardhianum Nag.(2), Nephrochlamys sp. (3), Oocystis lacustris Chodat (1, 2, 5), Oocystis marsonii Lemm. (2), Oocystis sp. (2), Pediastrum tetras (Ehr.) Ralfs (1), Scenedesmus acutus Meyen (5), Scenedesmus acuminatus (Langerh.) Chodat. (2,3), Scenedesmus bicaudatus Deduss. (4, 5), Scenedesmus dispar Brebis. (2, 4, 5), Scenedesmus ecornis (Ehr.ex Ralfs) Chodat (5), Scenedesmus linearis Kom. (1, 2, 5, 6), Scenedesmus opoliensis Rich. (2, 5), Scenedesmus quadricauda (Turp.) Brebis. 1, 3, 4, 5, 6), Scenedesmus sp. (3, 5), Schroederia nitzschioides (G.S.West) Korsch. (3, 4, 5), Schroederia spiralis (Printz) Kors. (1, 5), Schroederia sp. (1), Siderocelis ornata (Fott) Fott (2), Staurastrum sp. (4), Stichococcus bacillaris Nag. (1, 2), Tetraedron caudatum (Corda) Hansg. (1), Tetraedron minimum (A. Braun) Hansg. (3), Tetraedron trigonum (Naeg.)Hansg. (1, 2, 4), Tetrastrum glabrum (Roll.) Ahlst.et Tiff. (3), Ulothrix sp. (4); EUGLENOPHYTA- Euglena acus Ehr (4, 5), Euglena clavata Skuja (2, 3), Euglena gasterosteus Skuja (2, 3), Euglena limnophila (2, 3), Euglena matvienkoi Popova (3), Euglena polymorpha Dang. (4), Euglena proxima Dang. (2, 3), Euglena spathirhyncha Skuja (3), Euglena texta (Duj.) Hubn. (2, 3), Euglena tripteris (Duj.) Klebs. (3), Euglena sp. (2, 3, 4), Lepocinclis acuta Prescott (3), Lepocinclis ovum (Ehr.) Lemm. (2, 3), Lepocinclis sp. (2, 5), Peranema sp. (4), Phacus pleuronectes (Ehr.) Duj. (4), Phacus pyrum (Ehr.) Stein (4), Phacus sp. (2), Trachelomonas verrucosa Stokes (2, 3), Trachelomonas volvocina Ehr. (2,5), Trachelomonas sp. (3); The diversity degree of planktonic algoflore is, generally, determined by the water quality existent conditions. Quantitative data (Table I) show the diatoms dominance (BACILLARIOPHYTA) in Dorobanti mijloc and Parau Ciric Pod stations, of the CHLOROPHYTA group in Ciric II, Ciric I and Aroneanu stations and the big heaviness of the CYANOPHYTA group in Dorobanti Dig station (in this moment, this station is attested by the algologic indicators as being very polluted).

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Table I. The phytoplankton of the Ciric lacustrine complex (nr. exempl../ml)

Nr Station/Phylum Parau CiricPod 1 2 3 4 5 Cyanophyta Chrysophyta Bacillariophyta Chlorophyta Euglenophyta Total algae 52 837 8 510 260 639 12 056 51 773 355 8 510 3 901 1 064 5 319 6 028 3 546 4 610 355 10 993 5 886 709 255 674 709 2 482 142 638 5106 Dorobantmijloc Dorobantdig Aroneanudig Ciric I Ciric II

During the samples assay in May, general season conditions and the moment atmospheric situation of the barrages area have determined the dispersal in the phytoplankton mass of some elements from the algal periphyton. In July, 2006 (Table II) some important differences are found in the total number of algae and the distribution of planktonic algae groups, according to the assay station. Table II. The phytoplankton of the Ciric lacustrine complex - July 2006 (nr.exempl../ml)
Nr Station/Phylum Parau CiricPod 1 2 3 4 5 6 7 Cyanophyta Chrysophyta Xanthophyta Bacillariophyta Pyrrophyta Chlorophyta Euglenophyta Total algae 3 793 319 142 425 425 71 2411 71 284 284 248 71 3050 71 4 008 922 2 269 446 1 064 709 355 8 156 14 184 7 801 4 610 22 694 3 901 2 482 20 567 3 546 6 383 532 319 3 191 709 6 028 709 7 092 709 Dorobantmijloc Dorobantdig Aroneanudig Ciric Idig Ciric Ii-dig

Massive algal development is stated in the following stations: Aroneanu dig, Ciric I dig and Ciric II dig, this fact proving the presence of big charges of biogenous substances at these stations, the existence of a very big pollution, respectively. In stations:

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Aroneanu dig; Ciric I dig; Ciric II dig is also stated an important development of bluegreen algae (CYANOPHYTA), which indicates the presence in these stations of an excess of nutrients of organic and inorganic nature, which help these algae to develop and they are represented here by some genera that proliferate in very intense pollution conditions. EUGLENOPHYTA group is a very important indicator of water quality due to its mixotroph nutrition manner. These algae very much develop in water if it is polluted with toxic substances, especially organic ones, in big quantities. So, important numerical development of euglenoides at station 4 - Aroneanu dig, but also at Ciric I dig; Ciric II dig show the fact that important pollution sources are overflowed and collected here. In the studied lakes, in phytoplankton, in the first cold period of the year 2007 (Table III), 53 taxons were identified that belong to 6 phylums; they are distributed as it follows: 19 taxons in Dorobanti lake; 23 in Aroneanu lake, 14 in Ciric I lake, 21 in Ciric II lake and 15 in Ciric III lake. Table III The phytoplankton of the Ciric lacustrine complex - March 2006 (nr.exempl../ml)
Nr. 1 2 3 4 5 6 Station/Phylum Cyanophyta Chrysophyta Bacillariophyta Pyrrophyta Chlorophyta Euglenophyta Total algae 1 105 468 42 489 2 149 64 2 638 3 830 5 275 2 765 3 404 Dorobant 106 106 319 Aroneanu Ciric I 299 42 85 255 42 4 808 425 2 340 Ciric II 170 Ciric III

This determined number of taxons reflects a reduced biodiversity that is mainly owed to pollution. Analysis of dominant genera from the 1st of March, 2007 shows the following: at Dorobanti station Monoraphidium tortile (W.et G.S.West) Kom.-Leg (CHLOROPHYTA) is dominant, it represents 21.18% of the total algae; at Aroneanu station Monoraphidium contortum (Thur.) Kom.-Leg - 25.81% and Monoraphidium tortile (W.et G.S.West) Kom.-Leg 18.54% are dominant; at Ciric I, Monoraphidium contortum (Thur.) Kom.-Leg represented 48.88%, at Ciric II Monoraphidium contortum (Thur.) Kom.-Leg was registered with values of 55.66%, and at the last lake, Ciric III, Diatoma elongatum (Lyngb.)Agardh (BACILLARIOPHYTA) was dominant with 47.70% of the total algae determined at that station. Except for Diatoma elongatum (Lyngb.) Agardh. algae, which is -oligosaprobe, the other 2 dominant genera mentioned above are mesosaprobe, and this fact situates the water quality in that saprobity degree. Algological analysis results show a significant pollution degree in the 5 studied hollows, at Ciric II and Ciric I lakes, especially.

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- In the 5 studied lakes in November, 2007 (Table IV), 51 taxons were identified in phytoplankton. Table IV The phytoplankton of the Ciric lacustrine complex November 2007 (nr.exempl./ml)
Nr. 1 2 3 4 5 Station/Phylum Cyanophyta Chrysophyta Bacillariophyta Chlorophyta Euglenophyta Total algae 6 383 40 425 355 73 759 15 603 51 064 73 404 30 496 2 482 10 993 Dorobant 26 596 Aroneanu 2 218 Ciric I 25 532 2 482 1 064 21 986 Ciric II 50 709 355 9 929 12 411 709 11 347 Ciric III 18 440

This number of taxons reflects a quite reduced algal biodiversity, which is mainly owed to pollution. Conclusions Although Dorobanti Lake presents the biggest algal numerical diversity, the dominance of green algae (CHLOROPHYTA) in detriment of CYANOPHYTA group shows, comparatively, the fact that it is not the most polluted lake of the studied ecosystems. Ciric II Lake is more polluted compared to Dorobanti Lake as, in the conditions of a total algal density of close values, the high number of green-blue algae (CYANOPHYTA) of Ciric II lake shows an important organic charge of water and a significant pollution, respectively. Lakes Ciric I, II and III have also an accentuated pollution level which is demonstrated not by the high numerical algal densities only, but especially by the presence of some algae of the CYANOPHYTA group with a big cellular volume and a high biomass/individual (e.g.: Aphanizomenon flos-aquae Ralfs ex. Born et Flah; Numerical densities of phytoplankton in the 5 studied lakes have oscillated between the values of 1,105 3,830 exemplars algae/ml in March, 2007, and 15,603 73,759 exemplars algae/ml in November, the same year. These values show an important pollution degree of the investigated ecosystems. In comparison, in this sense, we mention that 5 lakes from the course of Bistrita river have phytoplankton values of 383 9,382 exemplars algae/ml [10], Cuiejdel lake had 1,060 10,116 exemplars algae/ml within 2000 2004 [11], and 9 aquatic ecosystems of reduced productivity in the Danube Delta had values between 684 2,271 exemplars algae/ml [9] limits; Although results concerning the pollution spectrum in the area are significant, there are necessary some researches for a period of various years in order to highlight the complexity and evolution of implied processes and phenomena in the respective

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ecosystems and in order to elaborate and apply these ecosystems reconstruction that are affected by the anthropic pollution so badly; The water quality of the 5 lakes which is shown by the dominant indicative genus of algae is of -mesosaprobe type; The measures that will be taken in order to improve water quality will refer to the reduction of pollution sources and to the improvement of canalization, water treatment and general salubrization conditions. REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. DUSSART B., 1966- Limnologie. Letude des Eaux Continentales, Gauthier Villars, Paris GERMAIN H., 1981- Flore des diatomees (Diatomophycees)- eaux douces et saumtres du Massif Armoricain et des contres voisines d,Europe occidentale, Socit Nouvelle des Editions Boube 11, place Saint-Michel, 75006-Paris HINDAK Fr.,1978 (ed)-Slodkovodne rissy, Slovenske Pedagogiske nakladeistvo, Bratislava HINDAK Fr., 1977- Studies on the chlorococcal alge (Chlorophycceae) I, Biologicke Prace, 4, XXIII, Bratislava. HINDAK Fr.,1980- Studies on the chlorococcal alge( Chlorophycceae)II, , Biologicke Prace,6, XXVI, Bratislava. JHON D.M., WITTON B.A., BROOK A.J. (Editors), 2003 (Second Edition) - The Freshwather Algal Flora of the British Isle, University Press, Cambridge KOMAREK J., AGNOSTIDIS K., 1986 Modern approach to the classification System of cyanophytes.2. Chroococcales. Arch. Hydrobiol. Suppl. 73, 2 (Algological Studies 43), 157-226 Sttugart PORUMB M. A., 1986- Clasificarea nivelurilor de troficitatae a 18 iazuri din judetele Cluj, Alba si Bistrita Nasaud pe baza criteriului algologic, Lucr. Simpoz. A III-a Comfrinta Nationala de ecologie Arad, 4-7 iunie (1986), 349-351 PORUMB M. A., 2000 - Cercetri privind algele planctonice din Delta Dunrii n arealul dintre Bra Sulina i Bra Sf. Gheorghe. Studii si Cercetari, Muzeul de t. Naturale Piatra Neam, 10: 35-46. PORUMB M. A., 2006- Cercetari privind algoflora planctonica a lacurilor de baraj din cursul mijlociu si inferior al raului Bistrita, Stud. Si Cercet., Muz. St. Nat. Piatra Neamt, 10: 53-65 PORUMB M.A., 2006- Studiu privind algele din lacul de baraj natural Cuejdel-Neamt, Stud. Si Cercet. Muz. St. Nat. Piatra Neamt, 10: 65-77 PREMAZZI G., DALMIGLIO A., CARDOSO A.C., CHIAUDANI C., 2003- Lake management in Italy: The implications of the Watyer Framework Directive, Lakes Reservoirs: Research and Management, 8: 4159 SIEMINSKA J., 1966- Bacillariophyceae, Flora Slodkow. Polsky STARMACH K., 1966- Cyanophyta. Glaucophyta. Flora Slodkow, Polsky, 2 STARMACH K., 1968- Chrysophyceae. Flora Slodkow. Polsky, 7 STARMACH K., 1972- Chlorophyta III. Zielenice nitkowate. Flora Slodkow. Polsky STARMACH K., 1974- Cryptophyceae, Dinophyceae, Raphidophyceae. Flora Slodkow. Polsky, 4 STARMACH K., 1980- Chrysophyceae, Flora Slodkow. Polsky STARMACH K,. 1983- Euglenophyta. Flora Slodkow. Polsky

Acknowledgements The researches were supported from the funds distributed within the CEEX project no. 634: The terrestrial and aquatic peri-urban ecosystems from Ciric river basin, from the north of Iai municipality, financed by the Ministry of Education and Research of Romania.

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

ON THE OCCURRENCE OF ZOSTERA NOLTII HORNEMANN AT THE ROMANIAN COAST OF THE BLACK SEA V. SURUGIU*
Abstract. The finding of a small Zostera noltii patch (~20 m2) at the Romanian Black Sea coast is reported. The systematics as well as the geographical distribution of this species is briefly reviewed. Some ecological characteristics of this species are also provided. The causes of the declining of Zostera noltii beds at the Romanian coast of the Black Sea are discussed. Key words: seagrass beds, Zostera noltii, distribution, Romanian coast, Black Sea

Introduction The dwarf eelgrass Zostera noltii (Hornem.) Toml. & Posl. belongs to the family Zosteraceae Dumortier, 1829. This family together with the families Cymodoceae, Posidoniaceae, and Hydrocharitaceae forms an ecological group of aquatic angiosperms adapted to live in the marine environment [5]. Representatives of these families are collectively called seagrasses due to their grass-like appearance. At the Romanian coast, there is also present another species of the genus Zostera, the common eelgrass Z. marina L. [6, 12]. In the past both these eelgrass species covered with a lush growth the bottom of marine lagoons Sinoie, Zmeica and Golovia [14]. Isolated patches were reported also at Cape Midia [2] and Agigea [3]. At that time one even spoke about the exploitation of eelgrasses and their use as surrogate for artificial wool, as stuffing material for pillows and mattresses and for packing up eggs, pieces of furniture and other fragile objects [1, 11]. In the last 40 years, due to pollution and eutrophication, seagrasses have declined drastically in abundance, not only at the Romanian coast, but also in the entire Black Sea. The aim of this paper is to reveal the actual status of Zostera noltii beds at the Romanian coast of the Black Sea. Although Zostera noltii is distinguishable from Z. marina, in many cases the authors have cited these plants simply as Zostera, irrespective of whether they refer to one or another species. In order to prevent the confusion between these two species of eelgrass an identification key is provided. Material and methods The dwarf eelgrass patch was identified at Mangalia (4348'18.0"N; 02835'31.9"E), between 1.3-1.9 m deep, on sandy substrate, in a small embayment formed by a dyke (Fig. 1). The patch is approximately 7 m long and 5 m wide. Samples of Zostera
* Al. I. Cuza University of Iai, Faculty of Biology, Bd. Carol I, no. 20A, 700506, Iai, Romania e-mail: vsurugiu@uaic.ro

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noltii were taken by snorkelling on 26th May 2005, 30th June 2005, 4th August 2006, and 8th August 2006. Voucher specimens were herborised and deposited in the Herbarium collection of the Faculty of Biology, Alexandru Ioan Cuza University of Iasi. The in situ photographs were taken with a digital ReefMaster DC 310 underwater camera. All the specimens collected were checked against the species description from the speciality literature [6, 9].

Fig. 1 Location of the Zostera noltii meadow at Mangalia (4348'18.0"N; 02835'31.9"E). Results and discussions Systematics Zostera (Zosterella) noltii Hornemann, Fl. Dan. 12(35): 3, tab. 2041 (1832) (Dwarf eelgrass) Fig. 2, 3 Zostera marina auct. non L.Phucagrostis minor Cavol., Phucagr. Anth. XIV, Pl. 2 (1792) nom. invalid.Zostera trinervis Stokes, in Bot. Mat. Med. 4: 319 (1812) nom. illeg.Zostera nana Mertens ex Roth, Enum. Pl. Phan. Germ. 1: 8 (1827) nom. invalid.Zostera minor (Cavol.) Nolte ex Reichenb., Icon. Fl. Germ. 7: 2 (1845).Zostera pumila Le Gall in Congr. Sc. France 16: 96, 144 (1850). Description.Colour is grass-green. Rhizomes 0.5-2 mm in diameter, with 1-4 roots at each node. Internodes 4-35 mm long. In cross-section the strengthening fibre bundles occur in the innermost layer of the outer cortex of the rhizome. Sterile shoots (leaves) alternately arranged and flattened. The leaf sheath at the base clasps the stem but is not fused into a tube, 4-5 cm long. Leaf blades 6-22 cm long and 0.5-1.5 mm wide, with approximately (1)3

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irregularly spaced veins. The tips of leaves initially rounded, but, as the plant matures, they become notched (emarginate), often asymmetric. Generative shoots lateral, unbranched or with a few branches near the base, shorter and narrower than the sterile shoots; with 1-6 spathes. Spathal sheath 12-20 mm long and 1.3-2 mm wide. Spadix lanceolate with 4-5 staminate flowers and 4-5 pistillate flowers. Fruit ellipsoid, 1.5-2 mm long; pericarp dark brown. Seeds smooth, white in colour, 1.5-2.0 mm long, excluding the style. 2n = 12. Biology.Hermaphrodite, perennial herb. Flowering period extends from June to August [6]. In the British Isles the plant retains its leaves throughout the winter. Main method of reproduction is by vegetative growth. However, seedling germination appears to be also important. In the Black Sea peaks of asexual reproduction occur in spring and autumn, when regrowth of the rhizome system is the most intense [7]. Habitat.Intertidal, between mean high water neap and mean low water neap. In waters with reduced salinity Zostera notii grows deeper and may become permanently submerged. Thus, in the Black Sea the species occurs from 0.6 m down to 8-10 m deep. In Mediterranean the dwarf eelgrass may grow down to 20 m deep. Inhabit sandy or muddy sand substate. Euryhaline species, very tolerant to desiccation. The lower salinity limit is about 15 g l1. The plant is restricted to sheltered sites such as estuaries, salt marshes, bays and lagoons. At Mangalia it was found in association with Cladophora sericea (Fig. 2). Geographical distribution.The species is distributed along the Atlantic coasts of Europe and northern Africa, extending from southern Scandinavia (Norway) to the tropic of Cancer (Mauritania and Senegal). It also occurs around the British Isles, in the Baltic Sea, Mediterranean Sea, Adriatic Sea, Black Sea, Sea of Azov, Caspian Sea and Aral Sea [4]. It the Black Sea dwarf eelgrass was recorded in the Sevastopol Bay, along the Caucasian coast (gulf of Anapa, gulf of Novorosiisk, gulf of Gelendjik), gulf of Karkinit, Tendrovsky, and Burgas Bay [8]. At the Romanian coast the presence of Zostera noltii meadows was previously reported at Mamaia, Agigea, Cape Midia, Mangalia, lake Razim, lake Golovia and Sinoie lagoon [2, 3, 7, 14]. Key to the Zostera species from the Black Sea 1. Rhizome thin, with 1-4 roots at each node. Leaves 6-22 cm long and 0.5-1.5 mm wide, with one principal vein; leaf-sheaths open with open margins overlapping; leaf tip emarginate. Generative shoot lateral. Retinacula present. Seeds smooth ......................... ................................................................................................. Zostera (Zosterella) noltii - Rhizome thick, with numerous roots at each node. Leaves 50-150 cm long and 3.07.0(9.0) mm wide, with 3 to 5(9) principal veins; leaf-sheaths closed, tubular, rupturing with age; leaf tip obtuse to slightly mucronate. Generative shoot terminal. Retinacula absent. Seeds with 16-25 longitudinal ridges ........................... Zostera (Zostera) marina In the Black Sea marine phanerogames are represented by 6 species: Zostera marina, Z. noltii, Potamogeton pectinatus, Ruppia maritima, R. spiralis, and Zannichellia major [8]. However, only the two species belonging to the genus Zostera, commonly

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known as eelgrasses, are considered to be fully confined to the marine environment. The remaining species forms the so-called eurysaline group, an ecological group of flowering aquatic plants tolerant of considerable changes in salinity from full-strength seawater to freshwater [5]. The eelgrass biocoenosis is an important element of shallow-water coastal benthic environments [9]. Seagrass beds are among the most productive marine communities. The standing biomass of dwarf eelgrass in the Black Sea ranges from 126 g m2 (Gulf of Karkinit) to 380 g m2 (Gulf of Anapa) [12]. The production of Zostera noltii is estimated to 15 g fresh weight/kg/day. As key primary producers, the eelgrasses represent an important source of food for many organisms, especially in the form of detritus. Although, Zostera noltii is grazed directly by waterfowl and by some fish species [6]. The dense, matted root system of eelgrasses stabilise the soft sediments, and thus reduce coastal erosion. Also, seagrass beds increase habitat diversity, providing shelter for a wide variety of marine organisms [2, 9]. Zostera noltii occurs on sedimentary substrata, in areas sheltered from water motion (currents and waves). Because Romanian coast is exposed to north-south alongshore current and to strong winter storms, it offers very few suitable conditions for eelgrass grow. Extensive carpets of eelgrasses were present only in the quiet brackish water lagoons. However, sparse Zostera shrubs were reported in front of Agigea at 0.8-1 m depth [3, 7]. Denser mats were found at Cape Midia [7]. Recently Teac et al. [13] indicated on the presence of small areas (approx. 3030 cm) populated by small eelgrass Zostera sp. (most probably Z. noltii) at Mamaia Casino and Mangalia, on a sandy-silty texture substrate, in the proximity of the protective wave-breaking dam. The decline of seagrasses in relatively open areas of the Romanian coast, as well as in the entire Black Sea, is due principally to the eutrophication. The nutrient enrichment of water increased the phytoplankton density, thus decreasing the transparency and diminishing the amount of light that can reach the bottom. The light penetration is also reduced by the siltation of near-shore sediments due to littoral works and to the construction of protective dams. Another cause for the decline of eelgrasses is the collapse of fishing in the Black Sea which reduced grazing on the epiphytes that live on the grass blades. The overgrowth of epiphytes thus prevents or reduces light intensity at the surface of the grass blade. Thus, the blades of the dwarf eelgrass observed at Mangalia were densely covered by various microscopic algae. The freezing of the sea may also have dramatic deleterious effects due to the ice scour [9]. However, the severe frost of the sea occurred during the 2005-2006 winter had no detectable impact upon the size and density of the meadow from Mangalia. As a result of a gradual reduction of salinity in all Romanian littoral lakes from polyhaline to ahaline over the last 40 years, vast fields of eelgrasses have disappeared completely. Because at the Romanian littoral Zostera noltii occurs very sparsely and is threatened to extinction it must be put under protection. .

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Fig. 2 Zostera noltii meadow at Mangalia.

REFERENCES
1. BACALBAA-DOBROVICI N., 1951 - Posibilitatea valorificrii ierbii de mare (Zostera) n R.P.R. Bul. Inst. Cercet. Pisc., Bucureti, 10(1): 25-32. 2. BCESCU M., MLLER G.I., GOMOIU M.-T., 1971 - Ecologie marin. Cercetri de ecologie bental n Marea Neagr. Analiza cantitativ, calitativ i comparat a faunei bentale pontice. Ecologie marin, IV, Ed. Academiei R.S.R., Bucureti 3. BOTEZ M., CRUU S., BOCEC A., CALINICENCO N., 1937 - Le plan de la Station Zoologique Maritime Regele Ferdinand dAgigea Constantza (Roumanie): Topographie, Biotopes et Biocnoses littoraux. AnnalesScientifiques de lUniversit de Jassy, 23(2): 1-4. 4. DEN HARTOG C., 1970 - The Sea Grasses of the World. North-Holland Publishing Company, Amsterdam 5. DEN HARTOG C., KUO J., 2007 - Taxonomy and Biogeography of Seagrasses. In: A.W.D. Larkum, R.J. Orth, C.M. Duarte (Eds.), Seagrasses: Biology, Ecology and Conservation, XVI, Springer Verlag, Berlin 6. GRINESCU I., NYRDY E.I., PAUC A., PRODAN I., ERBNESCU I., YAHARIADI C., 1966 - Flora Republicii Socialiste.Romnia., XI,Ed. Academiei R.S.R., Bucureti 7. MIHNEA P., 1965 - Biocenoza faciesului cu Zostera. Lucrare de diplom, Univ. Al. I. Cuza Iai, Facultatea de Biologie-Geografie 8. MILCHAKOVA N.A., 1999- On the status of seagrass communities in the Black Sea. Aquatic Botany, 65: 2132. 9. PHILLIPS R.C, MEEZ E.G., 1988 - Seagrasses. Smithsonian contributions to the marine sciences, No. 34, Smithsonian Institution Press, Washington, D.C.

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10. ROBERTSON A.I., MANN K.H., 1984 - Disturbance by ice and life history adaptations of the seagrass Zostera marina. Mar. Biol., 80: 131-142. 11. RUDESCU L., 1956 - Cercetri privitoare la exploatarea i prelucrarea ierbii indigene de mare. Rev. Ind. Lemnului, celulozei i hrtiei, Bucureti, 7: 354-359. 12. SKOLKA H., 1977 - Algues macrophytes et Phanerogames des mers saumtres pontocaspiennes. In: E.A. Pora & M.C. Bcescu (eds.), Biologie des eaux saumtres de la mer Noire, pp. 59-69. Institut Roumain de Recherches Marines, Constana. 13. TEAC A., BEGUN T., GOMOIU M.-T., PARASCHIV G.-M., 2006 - The present state of the epibiontic populations to the biocenosis of stone mussels in the shallow water off the Romanian Black Sea coast. GeoEco-Marina, 12: 53-66. 14. TEODORESCU-LEONTE R., LEONTE V., MATEI D., OILEANU B., 1956 - Observaii asupra complexului Razelm-Sinoie n perioada 1950-1952. Ann. Inst. Cerc. Pisc., 1: 1-50. Acknowledgements I am much indebted to my colleague Dr. C. Mnzu for checking the identification of the species and for helpful advice

Fig. 3 The habit of Zostera noltii (Hornem.) Toml. & Posl.

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Analele tiinifice ale Universitii Al. I. Cuza Iai Tomul LIV, fasc. 2, s.II a. Biologie vegetal, 2008

CONTRIBUTIONS TO THE STUDY OF THE RUDERAL VEGETATION FROM THE CEAHLU MOUNTAIN OANA ZAMFIRESCU*, C. MNZU*, T. CHIFU*
Abstract: The study presents four types of ruderal phytocoenoses identified in the Ceahlu Mountain and classified through the analysis of a appropriate number of relevs in the following vegetation associations [4] [8]: Potum supinae Brun-Holl 1962 em. Gutte 1969 and Rumicetum alpini Beger 1922, of all. Rumicion alpini Rbel ex Klika in Klika et Hada 1944, ord. Rumicetalia alpinae Mucina in Karner et Mucina 1993. cl. Mulgedio-Aconietea Hada in Klika 1948, Sambucetum ebuli Felfldy 1942, all. GalioAlliarion (Oberd. 1957) Lohmeyer et Oberd. in Oberd. et al. 1957, ord. Lamio albi-Chenopodietalia bonihenrici Kopecky 1969, and Telekio-Petasitetum hybridi (Morariu 1967) Resm. et Raiu 1974, all. Petasition officinalis Sillinger 1933, ord. Convolvuletalia sepium R. Tx. 1950 em. Mucina 1993, cl. Galio-Urticetea Passarge ex Kopecky 1969. For each association we give the description of the site and climatic conditions, the characteristic and associate species, and the synthetic table based on the analyses of the carried out relevs. Key words: weeds, mountain zone, anthropic impact

Introduction The existence of weed associations in the national park area is related to the grazing activity, especially the station of the animals in a certain space, and to the anthropic impact, which is hard to control because of the numerous touristic paths that cover the mountain. Although not complete, our ruderal formation inventory indicates the presence and the wide distribution of weed communities, and states the need of an action plan for the management of the area. Material and methods The Ceahlu Mountain, whose central part was designated a national park, is located in the centre of the northern part of the Oriental Carpathians [1] [9], in the western extremity of Neam County. The boundaries of the mountain are Bistricioara River towards north, Izvoru Muntelui-Bicaz reservoir towards east, Bicaz River towards south, and the streams Pntec and Bistra towards south-west [5]. The studied communities lay between 530 m and 1200 m of altitude, which corresponds to the mixed forest and coniferous forest zones. We used the classical method for vegetation research, by J. Braun-Blanquet, completed and adapted to the local conditions [2]. The relevs were sampled various conditions in regard to the altitude, exposition, slope, and periods of the vegetation season,

University Al. I. Cuza Iai, Faculty of Biology, Carol I, no. 20A, 700506, Iai, Romania.

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with the aim to achieve the complete picture of the grass layer composition. Consequently, the relevs ware analysed for the identification of the associations to which they belong. The survey has been accomplished form May to September. According to the Phytosociological Code of Nomenclature, the description of the associations resulted from the analyses of five to ten relevs [2]. Results and discussions We found the following associations: Class Mulgedio-Aconitetea Hada in Klika 1948 Order Rumicetalia alpini Mucina in Karner et Mucina 1993 Alliance Rumicion alpini Rbel ex Klika in Klika et Hada 1944 1. Ass. Potum supinae Brun-Holl 1962 em. Gutte 1969 2. Ass. Rumicetum alpini Beger 1922 1. Ass. Potum supinae Brun-Holl 1962 em. Gutte 1969 (tab. I) - the association is spread in the mountain zone, on stripped and compacted grounds where animals are stationed for a long time, around sheepfolds and households. - it grows on soils that are rich in nitrates, and on which the original vegetation still resists. - there are many species of the class Molinio-Arrhenatheretea that stands for the fact that the communities of this association form small patches in the meadows of Festuca rubra and Agrostis tenuis. - the phytocoenoses are dominated by Poa supina, which forms a dense layer together with the species Alchemilla vulgaris agg., Veronica persica, Polygonum aviculare, Stellaria nemorum, Plantago major, P. media, Potentilla anserina, Stellaria media, Geranium pusillum, Cerastium alpinum, Urtica urens etc. - this layer is penetrated here and there by the high stems of some species like: Cirsium vulgare, Festuca rubra, Sisymbrium officinale, Matricaria discoidea, Trifolium pratense, Campanula abietina, Ranunculus acris ssp. friesianus, Rumex acetosa, Cirsium arvense, Galeopsis speciosa etc. 2. Ass. Rumicetum alpini Beger 1922 (tab. I) - it is a nitrophilous mountain association that occurs on grounds stripped for a long time, on sufficiently humid soils. - on these grounds, the original vegetation disappeared almost completely. - these communities grow along some streams, on soils rich in nutrients resulted from the vegetation matter decomposition or brought from higher regions. - in the Ceahlu Mountain, the communities with Rumex alpinus are rare and limited to small areas [6]. - the species richness is small - Rumex alpinus dominates the communities together with Urtica dioica and Ranunculus repens [7].

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- other highly constant species are: Chenopodium bonus-henricus, Stellaria media, Veratrum album, Poa pratensis, Myosotis scorpiodes etc. Table I. Associations of the alliance Rumicion alpini Rbel ex Klika in Klika et Hada 1944 Association Altitude (m.s.m.) Orientation Slope (degrees) Vegetation cover (%) No. of surveys Caract. ass. Poa supina Rumex alpinus Rumicion alpini et Rumicetalia alpine Capsella bursa-pastoris Chenopodium bonus-henricus Galeopsis tetrahit Stellaria media Urtica dioica Mulgedio-Aconitetea Milium efffusum Rumex obtusifolius Veratrum album ssp. lobelianum Hypericum maculatum Veronica serpyllifolia Ranunculus repens Achillea distans Alchemilla vulgaris agg. Molinio-Arrhenatheretea Plantago major Festuca rubra Agrostis capillaris Taraxacum officinale Lolium perenne Polygonum aviculare Prunella vulgaris Potentilla anserina Trifolium pratense Ranunculus acris ssp. friesianus Rumex acetosa Potum supinae 650-1200 V-E 0-10 80-100 7 V II V II I I IV I II II I II V I II V IV II II I I I I Rumicetum alpini 920-1200 V 0-10 80-100 5 II V I III III V I II II I I V II I I I I I I

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Association Lotus corniculatus Plantago lanceolata Cynosurus cristatus Poa pratensis Trifolium repens Variae syntaxa Cirsium vulgare Stellaria nemorum Sisymbrium officinale Veronica persica Arctium lappa Veronica chamaedrys Senecio rupestris Elsholtzia ciliata Matricaria discoidea Urtica urens Myosotis alpestris Matricaria recutita Arctium minus Plantago media Cerastium alpinum Campanula abietina Geranium pusillum Malva pusilla Cirsium arvense Polygonum hydropiper Lappula squarosa Galeopsis speciosa Myosoton aquaticum Myosotis scorpioides Geranium phaeum Torillis japonica Stachys sylvatica

Potum supinae I I I IV I III II II II II II I I I II I I I I I I I I I I I -

Rumicetum alpini I III II II I I I I I I I I II I I I I

Class Galio-Urticetea Passarge ex Kopecky 1969 Order Lamio albi-Chenopodietalia boni-henrici Kopecky 1969 Alliance Galio-Alliarion (Oberd. 1957) Lohmeyer et Oberd. in Oberd. et al. 1957 3. Ass. Sambucetum ebuli Felfldy 1942 Order Convolvuletalia sepium R. Tx. 1950 em. Mucina 1993

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Alliance Petasition officinalis Sillinger 1933 4. Ass. Telekio-Petasitetum hybridi (Morariu 1967) Resm. et Raiu 1974 3. Ass. Sambucetum ebuli Felfldy 1942 (tab. II) - occurs in dumping grounds, stream shores near localities, ruderal meadows with soil rich in organic matter. - the characteristic species Sambucus ebulus forms dense clusters, which sometimes are almost pure or it can associate with ecologically different species. - over 70% of the species are mesophilous whereas the rest are mesohygrophilous, hygrophilous, xerophilous, and xeromesophilous. - the frequent species are: Urtica dioica i Galium aparine 4. Ass. Telekio-Petasitetum hybridi (Morariu 1967) Resm. et Raiu 1974 (tab. II) - occurs on stream shores on alluvial deposits, on shaded or partly shaded areas with highly humid substrata. - 25% of the species of the floristic composition are hygrophilous and mesohygrophilous, higrofile i mezohigrofile, but the great majority of the species are mesofilous. - the characteristic species, Petasites hybridus, is also dominant and covers the area with its broad vigorous leaves. - the subconstant species are: Mentha longifolia, Cirsium oleraceum, Telekia speciosa, Urtica dioica, Ranunculus repens etc - we consider that the studied phytocoenoses belong to the subassociation typicum because of the high cover of the characteristic species, Petasites hybridus. Table II. Associations of the alliance Galio-Alliarion (Oberd. 1957) Lohmeyer et Oberd. in Oberd. et al. 1957 and Petasition officinalis Sillinger 1933 Association Altitude (m.s.m.) Orientation Slope (degrees) Vegetation cover (%) No. of surveys Caract. ass. Sambucus ebulus Telekia speciosa Galio-Alliarion et Lamio-Chenopodietalia Aegopodium podagraria Campanula trachelium Cardamine impatiens Chelidonium majus Sambucetum ebuli 550-950 E, N, SE 0-15 80-100 10 V I Telekio-Petasitetum hybridi 530-890 S, V, N, SE 0-10 80-100 8 III I I I -

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Association Geum urbanum Glechoma hederacea Lamium maculatum Lapsana communis Aegopodion podagrariae et Impatienti-Stachyon Anthriscus sylvestris Geranium robertianum Heracleum sphondylium Impatiens noli-tangere Rumex obtusifolius Petasition officinalis et Convolvuletalia sepium Angelica sylvestris Carduus personatus Chaeropyllum hirsutum Cirsium oleraceum Filipendula ulmaria Myosoton aquaticum Petasites hybridus Poa trivialis Balloto nigrae-Robinion Ballota nigra Torilis japonica Galio-Urticetea Carduus crispus Eupatorium cannabinum Galium aparine Salvia glutinosa Urtica dioica Geranium phaeum Artemisietea et Stellarietea mediae s.l. Arctium lappa Artemisia absinthium Tussilago farfara Arctium minus Convolvulus arvensis Echium vulgare Onopordon acanthium Salvia verticillata

Sambucetum ebuli I II I I II II I I I III I IV I I II I II II I I I

Telekio-Petasitetum hybridi I I I II I I I I I I II III II II IV I I I I III -

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Association Cirsium arvense Polygonum convolvulus Bunias orientalis Anthemis tinctoria Nepeta cataria Veratrum album ssp. lobelianum Equisetum arvense Molinio-Arrhenatheretea Daucus carota Ranunculus repens Poa pratensis Medicago lupulina Rumex crispus Potentilla anserina Taraxacum officinale Alchemilla vulgaris agg. Potentilla reptans Elymus repens Mentha longifolia Bromus commutatus Achillea millefolium Equisetum telmateia Poa chaixii Lythrum salicaria Carex hirta Plantago major Plantago lanceolata Trifolio-Geranietea s.l. Origanum vulgare Verbascum lichnytis Inula conyza Veronica chamaedrys Solidago virgaurea Variae syntaxa Lepidium campestre Sambucus nigra Euphorbia cyparissias Poa nemoralis Hypericum maculatum Stachys sylvatica Galeopsis speciosa

Sambucetum ebuli I I I I I I I II II II I I I I I I I I I I I II I I I I I I I I I I I

Telekio-Petasitetum hybridi III II II III II II II I I -

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Association Vicia sepium Hypericum hirsutum Scrophularia nodosa Lysimachia vulgaris Orobanche caryophyllacea Carex remota Campanula rapunculoides Lycopus europaeus Valeriana tripteris Euphorbia amygdaloides Rubus idaeus

Sambucetum ebuli I I I REFERENCES

Telekio-Petasitetum hybridi I II I I I I I I

1. 2. 3. 4. 5. 6. 7. 8. 9.

BURDUJA C., 1968 - Muntele Ceahlu. Flora i vegetaia. Ocrot. nat., Bucureti, 6: 63 92. BRAUN-BLANQUET, J., 1964 Pflanzensoziologie, 3, Aufl., Springer, Wien, 865 CHIFU T., MITITELU D., DSCLESCU D., 1987 - Flora i vegetaia judeului Neam. Mem. Sec. t. Acad. Rom. Seria IV, 10(1): 281 302. CHIFU, T., MNZU, C., ZAMFIRESCU, O., 2006 - Flora i vegetaia Romniei, vol. 2. Vegetaia. Ed. Univ. Al. I. Cuza, Iai. GRINESCU I., 1924 -Consideration gobotaniques sur le mont Ceahlu ( Carpates Orientales). Bul. Soc. St. Cluj, 2(2): 104 112. NYARADY E., 1924 -Contribuii la cunoaterea vegetaiei i florei muntelui Ceahlu. Bul. Grd. Bot. i Muz. Bot. Cluj, 4: 2 3. RVRU M., 1936 - Nouti din flora Muntelui Ceahlu, Distr. Neam. Bul. Grd. Bot. Muz. Bot. Univ. Cluj, 16 (1 4): 78 85. SANDA V., POPESCU A., STANCA D., 2001 -Structura cenotic i caracterizarea ecologic a fitocenozelor din Romnia. Ed. Conphis. ZANOSCHI V., 1971 - Flora i vegetaia masivului Ceahlu. Tez de doctorat. Cluj Napoca

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CONTRIBUTIONS TO THE VEGETATION STUDY FROM THE VASLUI RIVER BASIN (II) IRINA IRIMIA
Abstract: The paper presents 6 vegetal associations, 2 belonging to the Bidentetea tripartiti R. Tx. et al. ex von Rochow 1951 class, 3 to the Galio Urticetea Passarge ex Kopeck 1969 class and 1 to the Epilobietea angustifolii R. Tx. et Preising ex von Rochow 1951 found on the territory of Vaslui river basin. Each association is accompanied by a phytosociological table and an analysis of the bioforms, floristic elements and ecological indices. Key words: phytosociology, bioforms, floristic elements, ecological indices.

Introduction The association taken into account were classified in the following phytocoenosystem, taking into account the recent papers on phytosociological classification [2, 6, 11-16]: BIDENTETEA TRIPARTITI R. Tx. et al. ex von Rochow 1951 BIDENTETALIA TRIPARTITI Br.-Bl. et R. Tx. ex Klika et Hada 1944 BIDENTION TRIPARTITI Nordhagen 1940 em. R. Tx. in Poli et J. Tx. 1960 1. Ass. Polygono lapathifolii Bidentetum tripartiti Klika 1935 2. Ass. Bidentetum cernui Kobendza 1948 GALIO URTICETEA Passarge ex Kopeck 1969 LAMIO ALBI CHENOPODIETALIA BONI HENRICI Kopeck 1969 GALIO ALLIARION (Oberd. 1957) Lohmeyer et Oberd. in Oberd. et al. 1957 3. Ass. Sambucetum ebuli Felfldy 1942 CONVOLVULETALIA SEPIUM R. Tx. 1950 em. Mucina 1993 SENECION FLUVIATILIS R. Tx. 1950 4. Ass. Urtico Convolvuletum Grs et Mller 1969 5. Ass. Galegetum officinalis Dobrescu et Vialariu 1981 EPILOBIETEA ANGUSTIFOLII R. Tx. et Preising ex von Rochow 1951 ATROPETALIA Vlieger 1937 ATROPION Br.-Bl. et Aichinger 1933 6. Ass. Eupatorietum cannabini R. Tx. 1937 Material and methods

Alexandru Ioan Cuza University, Faculty of Biology. B-dul Carol I, 20 A, Iai, Romania

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For the identification of plant associations, we used phytosociological research methods according to the CentralEuropean school. The establishment of the bioforms and floristic elements was made on the basis of Flora ilustrat a Romniei Pteridophyta et Spermatophyta, by V. Ciocrlan (2000). The ecological indices were noted having in mind the works of H. Ellenberg [5]. Results and discussions Ass. Polygono lapathifolii Bidentetum tripartiti Klika 1935 (Syn.: Bidentetum tripartiti Libbert 1932, Bidentetum tripartiti sesu auct.) Chorology: Dneti, Deleni, Fereti, Munteni de Sus (Leocov M., 1972) Moara Grecilor (Mititelu D., 1975), Vaslui, Vleni (Mititelu D. and collab., 1996), Vrriei hill Ecology: It is a nitrophilous association growing in marshes, on the border of the water and in wet micro-depressions where grows on areas rich in decomposing organic materials. The phytocoenological characterization: The characteristic species is Bidens tripartita which covers 65-80%. We also find other characteristic species of the Bidention alliance, of the order and class of: Bidens cernua, Polygonum lapathifolium, Lycopus europaeus etc (tab. I). After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms indicates the dominance of hemicryptophytes (50%), followed by terophytes (38,88%), hydrohelophytes (5,56%) and chamaephytes (5,56%); - the phytogeographical spectrum shows us the predominance of Eurasian elements (55,56%), followed by those circumpolar (27,77%), cosmopolite (11,11%) and European (5,56%); - the spectrum of ecological indices shows the prevalence of heliophilous species (38,88%), amphitolerant to temperature (33,34%), mesohydrophilous (33,34%), amphitolerant to soil reaction (72,22%) and which grow on soils with a high concentration of mineral nitrogen (38,88%). Observations: The association was mentioned as part of the studied area, by M. Leocov (1972) presenting only the floristic composition of the association and by D. Mititelu (1975, 1996) without having a table with floristic surveys. Ass. Bidentetum cernui Kobendza 1948 (Syn.: Bidentetum cernui Slavni 1951) Chorology: Micleti (Mititelu D.,1975), Vaslui Ecology: The association comprises annual hydrophilous species that appear as stripes on the border of the water where it grows on areas rich in decomposing organic materials. The phytocoenological characterization: The characteristic and dominant species is Bidens cernua together with Bidens tripartita, Echinochloa crus-galli, Myosoton aquaticum etc. We may also find characteristic species of the class PhragmitiMagnocaricetea that emphasize the hydrophilic character of the association (tab. II).

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After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms indicates the dominance of hemicryptophytes, (44,45%), followed by terophytes (33,35%), hemiterophytes (5,55%), hydrohelophytes (5,55%), phanerophytes (5,55%) and geophytes (5,55%); - the phytogeographical spectrum shows us the predominance of Eurasian elements (50%), followed by those circumpolar (27,77%), cosmopolite (11,11%), Pontic (5,56%) and European (5,56%); - the spectrum of ecological indices shows the prevalence of heliophilous species (35,29%), mesothermal (35,30%), developing on humid wet soils (29,42%), which grow on soils with a high concentration of mineral nitrogen (41,18%), amphitolerant to soil reaction (52,95%). Observations: The association was quoted by D. Mititelu (1975) without floristic surveys. Ass. Sambucetum ebuli Felfldy 1942 Chorology: Micleti (Mititelu D.,1975), Codeti, Dneti, Soleti, Tcuta, Vaslui, Vleni (Mititelu D. and collab., 1996), Chirceti, Coropceni, Emil Racovi Ecology: This association was encountered under the form of compact clusters, of variable dimensions, near the households, on the place of abandoned sheepfolds, where the animals stood, and the substrate is rich in organic substances in decomposition. We noticed a fast evolution of this associations extension within the territory taken in the study, in the meadows that are frequently grazed by the sheep, appearing under the form of groves, and on the border of the roads, thus forming green, high fences. The phytocoenological characterization: The characteristic and dominant species is Sambucus ebulus, which forms a layer with coverage of 75-100%. Because of the strongly developed system of rhizomes, this species plays an important role in consolidating the eroded lands (tab. III). The floristic composition of the association is relatively rich in species (51 species), noticing that besides the species characteristic to the Galio-Urticetea class, the also appear species from the Festuco-Brometea class and Molinio-Arrhenatheretea class, which come from the neighboring meadows. After the analysis of the surveys undertaken, the following was noticed: - from the spectrum of bioforms we notice the predominance of hemicryptophytes (58,48%), followed by terophytes (15,09%), hemiterophytes (13,21%), geophytes (5,67%), phanerophytes (5,67%) and chamaephytes (1,88%); - the phytogeographical spectrum shows us the predominance of Eurasian elements (49,05%), followed by the European (9,43%), cosmopolite (11,32%), circumpolar (7,54%), continental Eurasian (5,65%), central European (3,78%), adventive (3,78%), Pontic-Mediterranean (3,78%), Pontic-Balkan (1,89%), Asian (1,89%) and Mediterranean ones (1,89%); - from the spectrum of ecological indices we notice that the species which form the association are heliophile (35,55%), amphitolerant species towards the temperature

138

indece (37,77%), being spread in the central Europe (28,88%), which grow on dry up to humid soils (4-28,88%, 5-24,45%), amphitolerant towards the reaction of the soils (60%) and the contents in mineral nitrogen (24,44%). Observations: The association has been mentioned from the area taken in the study, but without having presented a table with floristic surveys. Ass. Urtico Convolvuletum Grs et Mller 1969 (Syn.: Urticetum dioicae Steffen 1931) Chorology: Codeti, Vleni, Soleti, Vaslui, Dneti, Micleti, Tcuta (Mititelu D. and collab., 1996) Ecology: The association was encountered on the places where garbage is thrown, therefore on the lands rich in decomposing organic substances, usually near the villages, parks, and even at the border of forests. The phytocoenological characterization: The characteristic and dominant species is Urtica dioica, which covers a surface of 90-100%, besides which the species Convolvulus arvensis also grows. In the floristic composition, few species are present, accompanying or being characteristic for other classes as well (tab. IV). After the analysis of the surveys undertaken, the following was noticed: - the spectrum of bioforms reveals us the predominance of hemicryptophytes (63,62%), followed by geophytes (13,64%), terophytes (13,64%) and hemiterophytes (9,10%); - the phytogeographical spectrum indicates us the predominance of Eurasian elements (45,45%), followed by the cosmopolite (22,73%), circumpolar (13,64%), European (9,10%), Mediterranean (4,54%) and continental Eurasian ones (4,54%); - from the spectrum of ecological indices we notice that the species which compose the association are heliophile (42,10%), mesothermal (36,84%), spread in central Europe (36,84%), which are developed on dry up to moderately humid soils (31,57%), with a high content of mineral nitrogen (36,85%), amphitolerant towards the soil reaction (52,64%). Observations: The association was mentioned from the area, but without presenting floristic surveys. Ass. Galegetum officinalis Dobrescu et Vialariu 1981 (Syn.: Senecio biebersteinii-Galega officinalis Borza 1960 n.n.) Chorology: Vaslui Ecology: It forms compact phytocoenoses situated at the border of rush-beds or in some slightly lowland, damp-humid or even with excess of humidity places, on glazed soils. The phytocoenological characterization: Besides Galega officinalis, which is the most important one, there are also the species characteristic to the classes Galio-Urticetea, Artemisietea i Stellarietea. Among the species with a high constancy we mention Taraxacum officinale, Tanacetum vulgare, Polygonum dumetorum, Lolium perenne, Stellaria media etc. (tabel V). After the analysis of the surveys undertaken, the following was noticed:

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- the spectrum of bioforms indicates the predominance of hemicryptophytes (58,06%), followed by terophytes (22,58%), geophytes (16,13%) and hemiterophytes (3,23%); - the phytogeographical spectrum ; reveals the preponderance of Eurasian elements (51,61%) and cosmopolite (22,58%), followed by the circumpolar (16,12%), Pontic-Mediterranean (3,23%), Mediterranean (3,23%) and continental Eurasian ones (3,23%); - the spectrum of ecological indices indicates species which love the light (40,74%), which are developed on humid soils (22,22%) and rich in mineral nitrogen (25,92%). The species are amphitolerant as regards the temperature (48,15%) and soil reaction (59,25%). Observations: The association is quoted for the first time from the area taken in the study. Ass. Eupatorietum cannabini R. Tx. 1937 Chorology: Dobrov, Poiana, Slobozia (Dobrescu C., 1978), Brnova, Poiana cu Cetate, Poieni (Mititelu D. and collab., 1995) Ecology: This association in encountered in different biotypes with humid and shadowed substrate: in forest cuts, in glades, forest margins and bushes, parks, along some rivers, confluents of Vaslui river, on soils rich in nutritive elements. The phytocoenological characterization: Besides the characteristic species Eupatorium cannabinum there also appear species from the classes Galio-Urticetea, Artemisietea vulgaris i Molinio-Arrhenatheretea. Among these, we mention Sambucus ebulus, Geum urbanum, Epilobium hirsutum, Urtica dioica etc. (tab. VI). After the analysis of the surveys undertaken, the following was noticed: - from the spectrum of bioforms, we notice the predominance of hemicryptophytes (65,21%), followed by terophytes (13,05%), hemiterophytes (17,39%) and geophytes (4,35%); - the phytogeographical spectrum reveals the predominance of Eurasian elements (65,21%), reflecting the temperate character of climate, followed by the circumpolar (17,39%), cosmopolite (8,70%) and adventive ones (8,70%); - from the spectrum of ecological indices it results that the species comprising the association love the light (33,33%), are mesothermal (42,86%), being spread in the central Europe (66,66%), which are developed on moderately-humid up to humid soils (623,81%, 8-23,81%), with a high content of mineral nitrogen (33,33%), amphitolerant towards the soil reaction (42,85%). REFERENCES
1. 2. 3. BELDIE AL., 1977 - Flora Romniei - Determinator ilustrat al plantelor vasculare. Vol. I-II. Ed. Acad. R.S.R., Bucureti CHIFU T., MNZU C., ZAMFIRESCU O., 2006 - Flora & vegetaia Moldovei (Romnia). Vol. II. Ed. Univ. Al. I. Cuza, Iai: 519-551, 666-687 CIOCRLAN V., 2000 - Flora ilustrat a Romniei - Pteridophyta et Spermatophyta, Ed. Ceres, Bucureti

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4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

DOBRESCU C., 1978 - Completri la cercetrile fitocenologice din Podiul Central Moldovenesc. An. t. Univ. Al. I. Cuza Iai, s. II a (Biol.), 24: 11-13 ELLENBERG H., 1974 - Indicator values of vascular plants in Central Europe. Scripta Geobotanica, Vol. IX, Verlag Erich Goltze K.G., Gttingen: 1-97 GEIELBRECHT TAFERNER L., MUCINA L., 1993 - Bidentetea tripartiti In: MUCINA L., GRABHERR G., ELLMAUER T. Die pflanzengesellschaften sterreichs, Gustav Fischer Verlag Jena Stuttgart New York, Bd. I: 90-109 LEOCOV M., 1972 - Contribuii la studiul agro- i geobotanic al buruienilor din bazinul Vasluie. Tez de doctorat, Instit. Agron. Ion Ionescu de la Brad Iai, Facultatea de Agricultur MITITELU D., 1975 - Flora i vegetaia judeului Vaslui. St. i Com. Muz. t. Nat. Bacu, Biol. veget.: 67162 MITITELU D., CHIFU T., SCARLAT A., ANIEI L., 1995 - Flora i vegetaia judeului Iai. Bul. Grd. Bot. Iai, 5: 99-124 MITITELU D., HUANU Mariana, 1996 - Noi contribuii la flora i vegetaia judeului Vaslui. St. i Cerc. Muz. Piatra-Neam, 8: 193-211 MUCINA L., 1993 - Galio Urticetea In: MUCINA L., GRABHERR G., ELLMAUER T. Die pflanzengesellschaften sterreichs, Gustav Fischer Verlag Jena Stuttgart New York, Bd. I: 203-251 MUCINA L., 1993 - Epilobietea angustifolii In: MUCINA L., GRABHERR G., ELLMAUER T. Die pflanzengesellschaften sterreichs, Gustav Fischer Verlag Jena Stuttgart New York, Bd. I: 252-270 MUCINA L., 1997 - Conspectus of classes of European vegetation. Folia Geobot. Phytotax., Praha, 32, 2: 117-172 SANDA V., 2002 - Vademecum ceno-structural privind covorul vegetal din Romnia. Ed. Vergiliu, Bucureti SANDA V., POPESCU A., BARABA N., 1997 - Cenotaxonomia i caracterizarea gruprilor vegetale din Romnia. St. i Com. Muz. t. Nat. Bacu, Biol. veget., 14: 2-365 SANDA V., POPESCU A., STANCU D., 2001 - Structura cenotic i caracterizarea ecologic a fitocenozelor din Romnia. Ed. Conphis, Bucureti Tab. I. Ass. Polygono lapathifolii Bidentetum tripartiti Klika 1935
Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Bidens tripartita Polygonum lapathifolium Bidention tripartiti Bidens cernua Polygonum mite Polygonum hydropiper Rumex conglomeratus Bidentetalia et Bidentetea Lycopus europaeus Echinochloa crus-galli Myosoton aquaticum Mentha longifolia Ranunculus sceleratus Phragmito-Magnocaricetea Ranunculus repens Alisma plantago-aquatica Epilobium hirsutum Molinio-Arrhenatheretea Juncus inflexus Agrostis stolonifera Lysimachia nummularia Mentha pulegium 1 94 80 10 9 3 + + + + 1 + + 1 2 330 70 10 9 4 + + + + + + + + 3 94 80 10 5 4 1 1 + 4 90 50 10 11 3 + + + + + + + + + + + 5 220 80 10 8 4 + + + + 1 + + -

K V III II II I I III III III III I IV II II III II I I

141

Place and date of the surveys: 1,3. Vaslui, 20.08.2003, 12.08.2003; 2. Vrriei hill, 23.08.2003; 4. Moara Grecilor, 12.08.2003; 5. Fereti, 08.2001 Tab. II. Ass. Bidentetum cernui Kobendza 1948
Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Bidens cernua Bidention tripartiti Bidens tripartita Polygonum hydropiper Bidentetalia et Bidentetea Echinochloa crus-galli Myosoton aquaticum Polygonum lapathifolium Ranunculus sceleratus Rorippa austriaca Phragmiti-Magnocaricetea Alisma plantago-aquatica Lythrum salicaria Typha latifolia Molinio-Arrhenatheretea Agrostis stolonifera Juncus inflexus Cichorium intybus Variae syntaxa Arctium lappa Lathyrus tuberosus Salix alba 1 + + + + + 2 + + IV I I I I I 1 94 100 10 11 5 + + + + + 2 94 80 10 4 5 + + 3 94 60 10 7 3 + + + + 4 94 65 10 6 3 + + + 1 + 5 94 70 10 12 4 + + + + + + + +

K V V II IV III II II I II II I

Place and date of the surveys: 1-5. Vaslui, 12.08.2003 Tab. III. Ass. Sambucetum ebuli Felfldy 1942
Number of survey 1 2 3 Altitude (m.s.m.) 190 210 260 Exposition NE S S Slope () 1 1-2 1 Cover of the vegetation (%) 100 100 75 Surface of survey (m) 25 25 25 Number of species 18 14 12 Associations characteristics Sambucus ebulus 5 5 4 Galio-Alliarion et Lamio albi-Chenopodietalia Aristolochia clematitis + Veronica chamaedrys + Galio-Urticetea Galium aparine + Glechoma hederacea + Geum urbanum + + Onopordetalia Berteroa incana + + 1 Tanacetum vulgare + Artemisia absinthium + Carduus nutans 4 260 SV 2 100 25 18 5 + + + + 5 180 NV 1-2 100 25 23 5 + + + + 6 210 90 20 11 4 1 1 90 20 12 4 + + + 7 210

K V III III III III III III II I I

142

Artemisia vulgaris Arctium tomentosum Urtica dioica Ballota nigra ssp. nigra Elymus repens Carduus acanthoides Conium maculatum Rumex obtusifolius Erigeron annuus Helianthus tuberosus Leonurus cardiaca ssp. villosus Carduus crispus Torilis arvensis Conyza canadensis Polygonum aviculare Cardaria draba Cirsium arvense Lathyrus tuberosus Atriplex tatarica Sonchus oleraceus Achillea setacea Eryngium campestre Salvia nemorosa Galium humifusum Poa angustifolia Galium verum Scabiosa ochroleuca Convolvulus arvensis Bromus inermis Erodium cicutarium Galium mollugo Cichorium intybus Lolium perenne Centaurea jacea Plantago lanceolata Lotus corniculatus Juncus inflexus Achillea millefolium Lysimachia nummularia Dactylis glomerata Poa pratensis Prunus spinosa Rosa canina Elaeagnus angustifolia

Artemisietea vulgaris + + 1 + + + + Stellarietea mediae + + + + + + + + + + + Festuco-Brometea + + + + + + + + + + + + Molinio-Arrhenatheretea + + + + + + + Variae syntaxa + + + + + + -

+ + + + + + + + + + + + + + + + + + +

+ 1 + + + + + + -

+ 2 + + + + + + -

III III III III III III II I I I I I II II II II II II I I III II II II I I I I I I I III III III II II I I I I I II I I

Place and date of the surveys: 1. Chirceti, 6.08.2003; 2. Coropceni, 27.07.2003; 3,4. Emil Racovi, 6.08.2003; 5. Codeti, 07.2002; 6,7. Dobrov, 1.07.2004 Tab. IV. Ass. Urtico Convolvuletum Grs et Mller 1969
Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Urtica dioica 1 150 100 25 9 5 2 220 100 15 10 5 3 160 100 20 12 5 4 150 100 10 10 5 5 94 100 20 12 5

K V

143

Petasition officinalis et Convolvuletalia sepium Galium aparine + + + Cucubalus baccifer + + + Aristolochia clematitis + + Sambucus ebulus + + Galio-Urticetea Convolvulus arvensis + + + Veronica chamaedrys + + + Glechoma hederacea + + Artemisietea vulgaris Taraxacum officinale + + + Carduus acanthoides + + Elymus repens + + Elymus hispidus + Arctium tomentosum Leonurus cardiaca ssp. villosus Stellarietea mediae Capsella bursa-pastoris + + Lathyrus tuberosus + Lepidium ruderale + Molinio-Arrhenatheretea Bellis perennis Lolium perenne Verbena officinalis Geum urbanum Agrimonia eupatoria + + + + Variae syntaxa + + + + -

+ + + + + + + + +

IV III III II IV III II IV II II I I I III II II

+ +

III III II I I

Place and date of the surveys: 1, 2. Codeti, 5.08.2005; 3. Vleni, 5.08.2005; 4. Soleti, 5.08.2005; 5. Vaslui, 5.08.2005 Tab. V. Ass. Galegetum officinalis Dobrescu et Vialariu 1981
Number of survey 1 2 Altitude (m.s.m.) 94 94 Cover of the vegetation (%) 90 100 Surface of survey (m) 10 10 Number of species 12 14 Associations characteristics Galega officinalis 5 5 Senecion fluviatilis et Convolvuletalia sepium Polygonum dumetorum + + Aristolochia clematitis + Calystegia sepium + Galio-Urticetea Urtica dioica + Ranunculus repens Convolvulus arvensis + Artemisietea vulgaris Taraxacum officinale + + Tanacetum vulgare + Lathyrus tuberosus + Elymus repens + Artemisia vulgaris Verbena officinalis Cannabis sativa ssp. spontanea 1 Equisetum arvense + Stellarietea mediae Stellaria media + Linaria vulgaris 3 94 95 10 13 5 + + + + + + + + + 4 94 70 10 12 4 + + + + + + + + + 5 94 55 10 16 3 + + + + + + + + +

K V III II I II II I V IV III III III III II II III II

144

Cirsium arvense Lolium perenne Lotus corniculatus Mentha longifolia Agrostis stolonifera Trifolium repens Inula britannica Polygonum hydropiper Bidens tripartita Lycopus europaeus Xanthium strumarium Polygonum aviculare

Molinio-Arrhentheretea + + + + + Bidentetea tripartiti + + + Variae syntaxa + + -

+ + + -

+ + -

+ + + + + 1 -

I III III III II II I II I I I I

Place and date of the surveys: 1-5. Vaslui, 20.08.2003 Tab. VI. Ass. Eupatorietum cannabini R. Tx. 1937
Number of survey Altitude (m.s.m.) Cover of the vegetation (%) Surface of survey (m) Number of species Associations characteristics Eupatorium cannabinum Atropion et Atropetalia Sambucus ebulus Cirsium vulgare Epilobietea angustifolii Conyza canadensis Galio-Urticetea Urtica dioica Epilobium hirsutum Erigeron annuus Artemisietea vulgaris Geum urbanum Myosoton aquaticum Sambucus ebulus Lapsana communis Arctium lappa Dipsacus fullonum Molinio-Arrhenatheretea Ranunculus acris Lythrum salicaria Symphytum officinale Agrostis stolonifera Prunella vulgaris Mentha pulegium Equisetum telmateia Stachys sylvatica Scrophularia nodosa 1 370 70 50 7 4 + + + + + 2 350 75 50 9 4 1 + + + 3 393 100 50 9 5 + + + + + + + + 4 393 100 25 10 5 + + + + + + + + + 5 393 100 25 12 5 + + + + + + + + + + +

K V III II II IV III I IV II I I I I III II II II I I III II I

+ + + + Querco-Fagetea + -

Place and date of the surveys: 1. Dobrov, 12.07.2003; 2. Poieni, 08.2002; 3-5. Brnova, 08.200

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INSTRUCTIONS TO AUTHORS The Journal Analele tiinifice ale Universitii Al. I. Cuza din Iai (serie nou), Seciunea II a. Biologie vegetal, includes original articles of cytology, morphoanatomy, physiology, taxonomy, phytosociology, mycology, phytopathology, along with book reviews and anniversary announcements. All papers must be submitted to our redaction address (Dr. Ramona GALE, Al. I. Cuza University, Faculty of Biology, Department of Biology, Bd. Carol I., no. 20A, 700506, Iasi, e-mail: ramona.gales@uaic.ro) both as printed manuscripts and electronic format. For the graphic uniformity of the volume, please consider followings: PAGE FORMAT: paper A4; margins settings: 5,8 cm top, 6 cm bottom, 4 cm left, 4 cm right. TEXT: the papers will be printed in English language; the text must be typed (in a PC compatible text editor) with Times New Roman 10, single-spaced; the Abstract and the Key words must be typed in English, using the font Times New Roman 8 points; the title must be typed in Times New Roman 10 bold capitals; authors' names and surnames must be typed in caps; male surnames must be abbreviated; the address of each author must be provided in footnote; the text will be partitioned as follows: Introduction, Material and methods, Results and discussions, Conclusions, References. All subtitles must be centred typed in Times New Roman, bold 10 ex.: Introduction the scientific names must be typed in italics; the text references of tables and figures (included in plates) must be typed between round brackets: ex: (fig. 2, Pl. I), (tab. II); the text references of the cited bibliography must be typed between square brackets: ex: [5]; the References subtitle must be centred and typed in bold 10 points caps:. ex.: REFERENCES bibliography references must be alphabetically ordered and typed in Times New Roman 8 point font, as follows: for books: 1. BELDIE Al., 1972 - Plantele din Munii Bucegi. Edit. Acad. Rom., Bucureti

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for articles: DAUB E. M., 1981 - Cercosporin, a photosensitizing toxin from Cercospora species. Phytopathology, 72: 370 374 3. REDZI S., TUKA M., PAJEVI A., 2006 - Research into microscopic structure and essential oils of endemic medicinal plant species Satureja subspicata Bartl. ex. Vis. (Lamiaceae). Bosn. J. Basic Med. Sci., 6, 2: 25-31 4. RUGIN R., TOMA C., IVNESCU L., 2007 Morphological and histo-anatomical aspects at some dicotyledonate seedlings related to the vascular transition. An. t. Univ. Al. I. Cuza Iai, s. II a., Biol. veget., 52: 133- 142 FIGURES (colour or black and white photos, drawings) all figures must be grouped in plates on separated pages; the number of the plate (ex. PLATE I) must appear in the top-right position and the names of the authors (Times New Roman 10, caps, bold) must appear in the top-left position of each plate; The explanation of figures (including figures) must be typed on separated page (after References) the figures must be printed on tracing paper (photocopies are not acceptable, just original materials) and must not exceed 18 x 12,7 cm; all materials must be accompanied by graphical scale. TABLES the tables must be printed on separated pages and must be numbered with roman digits (Table I, Table II,). For Book Reviews: - mention the followings: author (name, surname, in caps), coma, year, title with italic bold characters, coma, place of publishment, number of pages, ISBN. Leave a blanc line and write the text of the reviews (paragraphs as few as possible) single spaced, on A4 paper with Times New Roman 10 font. Recommendations: The submitted paper must not exceed 10 pages (illustration included) and must have an even number of pages (including an even number of pages with colour plates). The papers will be further submitted to the reference comity and will be published for a charge in Analele tiinifice ale Universitii Al. I. Cuza din Iai, Sec. II a. Biologie vegetal. The editorial comity reserves the right to: reject certain papers (one paper as first author and another one in collaboration would be acceptable) reduce the number of figures, in case they are to many Only papers presented in the Plant Biology section of the scientific congress organised by our Faculty will be published. Responsibility upon the articles content belongs to the author (s). 2. Papers that do not meat these rules will be returned to the author (s). Editorial comity

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