THE INFLUENCE OF MYCORRHIZA AND EDTA APPLICATION ON HEAVY METAL UPTAKE BY DIFFERENT MAIZE VARIETIES

ANNA JURKIEWICZ¹, ELŻBIETA ORŁOWSKA¹, TERESA ANIELSKA¹, BARBARA GODZIK², AND KATARZYNA TURNAU¹*

¹Institute of Botany, Jagiellonian University, 
ul. Lubicz 46, 31-512 Cracow, Poland
²Institute of Botany, Polish Academy of Sciences, 
ul. Lubicz 46, 31-512 Cracow, Poland

*e-mail: ubturnau@cyf-kr.edu.pl

Received September 18, 2003; revision accepted February 6, 2004

This study investigated whether mycorrhizal colonization influences heavy metal uptake by maize. Two experiments were carried out. In the first, 15 commercially available maize varieties cultured on industrial waste substratum and inoculated or not with Glomus intraradices, were treated one week before harvest with EDTA, a chelating agent known to mobilize heavy metals in soil. Estimation of mycorrhizal parameters indicated differences between varieties, but differences between treatments of the same variety generally were not statistically significant. Although EDTA treatment strongly decreased the activity of fungal alkaline phosphatase (indicator of fungal viability), the treatment did not totally eliminate arbuscular mycorrhizal fungi (AMF) from the soil. The appearance of AMF structures within roots was modified in plants cultivated in EDTA-treated soil. Among the heavy metals studied, the highest impact of EDTA treatment on heavy metal uptake in shoots was found in the case of Pb. In most cases, EDTA treatment significantly increased the Pb level in shoots of mycorrhizal plants. Among the samples treated with EDTA, mycorrhizal plants of 6 cultivars showed higher Pb content in shoots than did nonmycorrhizal plants. Significant differences in heavy metal content in plant material were demonstrated between the varieties tested. In the second experiment, one selected cultivar was subjected to high soil Pb concentrations and to EDTA for one week, following cultivation in nonpolluted substratum. In this case, EDTA treatment more strongly influenced Pb uptake by nonmycorrhizal than by mycorrhizal plants. The results indicate the need to carefully screen cultivars as well as microorganism strains to be used in phytoextraction procedures. 

Key words: Maize varieties, mycorrhiza, EDTA, heavy metals, phytoremediation, metal uptake.

RESPONSE OF ARMERIA MARITIMA (MILL.) WILLD. TO CD, ZN AND PB

GRAŻYNA SZAREK-ŁUKASZEWSKA¹, AGNIESZKA SŁYSZ², AND MAŁGORZATA WIERZBICKA<sup>2

1</sup>W. Szafer Institute of Botany, Polish Academy of Sciences, 
ul. Lubicz 46, 31-512 Cracow, Poland
²Environmental Plant Pollution Laboratory, Warsaw University, 
ul. Miecznikowa 1, 02-096 Warsaw, Poland

Received October 16, 2003; revision accepted March 9, 2004

The response of Armeria maritima to heavy metals was examined in plants from two populations, one from metalliferous and the other from non-metalliferous soil. Concentrations of metals (Cd, Zn, Pb) were determined in organs of plants growing on 100-year-old calamine spoil (S Poland) and in a “clean” area (central Poland), and in the first generations of those plants grown in hydroponic culture with addition of metals. Large differences between root and green leaf concentrations of metals were found, indicating restriction of transport of metals from roots to aboveground parts in A. maritima, and an exclusion strategy. Even under full availability of metals (hydroponic culture), adult plants effectively limited the flow of metals to their aboveground parts. In A. maritima from calamine populations, part of the metals transported to aboveground plant organs were directed to the oldest leaves. Plants from the “clean” population growing in a non-metalliferous area did not accumulate heavy metals in ageing leaves. It seems that the ability to accumulate metals in withering leaves characterizes plants growing under strong environmental pressure from metal contamination, in which one may expect intensification of metal detoxification processes. 

Key words: Armeria maritima, heavy metals, calamine spoils, hydroponic culture.

MYCORRHIZAL AND SAPROBIC MACROFUNGI OF TWO ZINC WASTES 
IN SOUTHERN POLAND

PIOTR MLECZKO

Institute of Botany, Jagiellonian University, 
ul. Lubicz 46, 31-512 Cracow, Poland

Received September 30, 2003; revision accepted March 10, 2004

Ectomycorrhizal and saprobic fungi of two industrial wastes in southern Poland (calamine spoil in Bolesław and zinc waste in Chrzanów) were studied. Pine (Pinus sylvestris) accompanied by birch (Betula pendula) were present in the investigated area. Fruitbodies of 68 species were recorded, but only 10 were common to both sites. Mycorrhizal species were the most common group on the zinc waste, whereas saprobic fungi prevailed on the calamine spoil. The differences in species composition between sites might be due to differences in plant cover, but also to the toxicity of the material at the sites. Among mycorrhizal species, members of Cortinariaceae and Tricholomataceae were most frequently recorded. Most ectomycorrhizal species had a broad host range, and only a few species known to be associated exclusively with pine or birch were found. Analysis of ectomycorrhizas by classical and molecular (PCR-RFLP) methods revealed that the fungi forming the most abundant fruitbodies were also present in the form of ectomycorrhizas. A few ascomycete and basidiomycete fungi not recorded as fruitbodies were present as pine symbionts. 

Key words: Industrial waste, calamine spoil, Pinus sylvestris, ectomycorrhizal fungi, saprobic fungi, ectomycorrhizas.

ENDOSPERM DEVELOPMENT IN SEEDS OF ECHIUM VULGARE L.
(BORAGINACEAE) FROM POLLUTED SITES

ANITA BISKUP AND ROMANA IZMAIŁOW*

Department of Plant Cytology and Embryology, Jagiellonian University, 
ul. Grodzka 52, 31-044 Cracow, Poland

*e-mail: izmailow@iphils.uj.edu.pl

Received September 15, 2003; revision accepted March 19, 2004

Analysis of endosperm development in plants from contaminated sites (vicinity of the Żelazny Most copper post-flotation reservoir in the Legnica-Głogów Copper District, and the zinc spoil in Katowice-Wełnowiec) showed general similarities in the pattern of endosperm formation in Echium vulgare, but also deviation from typical haustorium structure (~23% frequency), premature degeneration of the haustorium, or degeneration both of the haustorium and endosperm proper (~40%). The most significant irregularities of endosperm development included lower or higher number of nuclei in two cells of the lateral part of the endosperm, lower ploidy level of haustorium nuclei, and cellularization (instead of coenocytic structure) in the lateral cells of the endosperm.
Irregularities and degenerative processes presumably resulted from the stress of environment conditions. Because of the nutritive function of those structures, degeneration or atypical structure of the endosperm and its haustorium in some maturing seeds may reduce the fertility of plants colonizing contaminated sites.

Key words: Echium vulgare L., endosperm, endospermal haustorium, disturbances in endosperm, polyploidization, heavy metals, pollution.

LOCALIZATION OF LEAD IN ROOT TIP OF DIANTHUS CARTHUSIANORUM

AGNIESZKA BARANOWSKA-MOREK AND MAŁGORZATA WIERZBICKA

Environmental Plant Pollution Laboratory, Institute of Plant Experimental Biology, University of Warsaw, 
ul. Miecznikowa 1, 02-096 Warsaw, Poland 

e-mail: abmorek@biol.uw.pl 
wierzbicka@biol.uw.pl

Received October 6, 2003; revision accepted March 30, 2004

The distribution of lead in root tips of Dianthus carthusianorum was compared in populations from a zinc-lead waste heap in Bolesław near Olkusz and from a natural stand in the Botanical Garden in Lublin. The analyses were made at two developmental stages: seedlings (after 8 days of incubation in 5 mg/dm³ Pb⁺² from PbCl₂ in 1/8 Knop medium) and mature plants (after 23 days of incubation in 10 mg/dm³ Pb⁺2 from PbCl₂ in 1/2 Knop medium). Histochemical methods (rhodizonate and dithizonate) of lead detection revealed significant accumulation of this metal on the root surface of the examined plants. The site of next-strongest lead accumulation in root tips of plants from both populations was in cell walls of the pericycle. The layer of meristematic pericycle cells seemed to be a strong barrier against penetration of lead to the deepest cells of the procambium. Histochemical methods and tissue sections revealed no differences in lead distribution between root tips from the waste heap and from the natural population, but differences were detected on the ultrastructural level. There were numerous lead deposits in the cytoplasm of cells from ground meristem in the natural population, and none in specimens from the waste heap, indicating that lead had a higher toxic effect on the natural population of D. carthusianorum.

Key words: Dianthus carthusianorum, lead, pericycle, roots, ultrastructural localization.

UPTAKE AND LOCALIZATION OF CADMIUM BY BISCUTELLA LAEVIGATA, A CADMIUM HYPERACCUMULATOR

MARIA PIELICHOWSKA AND MAŁGORZATA WIERZBICKA

Environmental Plant Pollution Laboratory, University of Warsaw,
ul. Miecznikowa 1, 02-096 Warsaw, Poland

e-mail: mpiel@biol.uw.edu.pl; wierzbicka@biol.edu.pl 

Received October 21, 2003; revision accepted April 10, 2004

Biscutella laevigata has recently been recognized as a species able to accumulate large amounts of cadmium. The experiments reported in this paper were conducted on two geographically isolated populations of B. laevigata in Poland. Both populations grow on metalliferous soils: a lead-zinc (calamine) waste heap in Bolesław near Olkusz (189 mg Cd/kg d.m.) and limestone rock in the West Tatra Mts (1.4-6.1 mg Cd/kg d.m.). The two populations were compared after cultivating them in medium containing cadmium salt (2-120 mg/dm³) for 3-30 days. Root-to-shoot transport of cadmium was higher in the waste-heap population than in the mountain population. In the waste-heap population, large amounts of cadmium were transported to the oldest leaves, reaching levels even twice those of the mountain population. This shows that the ability to hyperaccumulate metals may be a property of a population, not an entire species, and that the ability to accumulate cadmium in the oldest (withering) leaves may be a way the plant eliminates the toxic metal. Histochemical detection of cadmium (with dithizone) in tissues showed that it was taken up by the root hairs and then transported through vascular bundles to the leaves. The surface cells of the leaves, the epiderm and hairs accumulated particularly large amounts of cadmium. The leaves of the B. laevigata waste-heap population are much more thickly covered by hairs than those of the mountain population; we suggest that the ability to accumulate cadmium in leaf hairs may be a mechanism of detoxifying and hyperaccumulating cadmium in the shoots of that population. 

Key words: Biscutella laevigata, cadmium, dithizone, detoxification of cadmium, hyperaccumulation.

MINERAL COMPOSITION IN RHIZOSPHERE OF PLANTS GROWN IN THE VICINITY OF A ZN-PB ORE FLOTATION TAILINGS POND. PRELIMINARY STUDY 

JERZY CABALA¹*, EWA TEPER¹, LESLAW TEPER¹, EUGENIUSZ MAŁKOWSKI², 
AND ADAM ROSTAŃSKI<sup>3 

1</sup> Faculty of Earth Sciences, University of Silesia, 
ul. Będzińska 60, 41-200 Sosnowiec, Poland
² Department of Plant Physiology, Faculty of Biology and Environmental Protection, University of Silesia, 
ul. Jagiellońska 28, 40-032 Katowice, Poland
³ Department of Plant Systematics, Faculty of Biology and Environmental Protection, University of Silesia, 
ul. Jagiellońska 28, 40-032 Katowice, Poland

*e-mail: cabala@us.edu.pl

Received September 27, 2003; revision accepted June 8, 2004

Mineral forms in rhizosphere and bulk substrate were examined for selected plant species (Cardaminopsis arenosa, Calamagrostis epigeios, Deschampsia caespitosa, Festuca ovina, Silene vulgaris, Viola tricolor) growing spontaneously in the close vicinity of a tailings pond and in a zone of eolian transmission of waste particles. Samples of substrates and plant roots were taken from sites in the Bolesław orefield near Olkusz. The substrates together with plant roots were studied by scanning electron microscopy coupled with energy dispersive spectroscopy. Apart from quartz and carbonates, the most frequent mineral phases found were crystalline and amorphous Fe oxides, as well as primary Fe, Zn and Pb sulphides (marcasite, pyrite, sphalerite, galena). Hydrated Ca, Mg and Fe sulphates (gypsum, epsomite and melanterite) and other secondary minerals (smithsonite, cerussite, otavite, and Fe, K and Mg aluminosilicates) were found at larger concentrations in the rhizosphere than in bulk soil, suggesting that plant roots can change the mineral composition of the soil.

Key words: Flotation tailings pond, mineral components, rhizosphere, bulk soil, hyperaccumulator and nonaccumulator plants.

UPTAKE OF CADMIUM, LEAD, NICKEL AND ZINC FROM SOIL AND WATER SOLUTIONS BY THE NICKEL HYPERACCUMULATOR BERKHEYA CODDII

JOLANTA MESJASZ-PRZYBYŁOWICZ¹, MIROSŁAW NAKONIECZNY², PAWEŁ MIGULA², MARIA AUGUSTYNIAK², MONIKA TARNAWSKA², WOLF UWE REIMOLD⁴, CHRISTIAN KOEBERL⁵, WOJCIECH PRZYBYŁOWICZ¹ AND ELŻBIETA GŁOWACKA<sup>3

1</sup>Materials Research Group, iThemba Laboratory for Accelerator Based Sciences,
P.O. Box 722, Somerset West 7129, South Africa 
²Department of Animal Physiology and Ecotoxicology; 
³Department of Zoology, University of Silesia, 
ul. Bankowa 9, 40-007 Katowice, Poland 
⁴School of Geosciences, University of Witwatersrand, 
Johannesburg 2050, South Africa 
⁵Department of Geological Sciences, University of Vienna,
Althanstraße 14, A-1090 Vienna, Austria
 
e-mail: mesjasz@tlabs.ac.za
migula@us.edu.pl
reimoldw@geosciences.wits.ac.za
christian.koeberl@univie.ac.at

Received October 15, 2003; revision accepted July 7, 2004

Berkheya coddii Roessler (Asteraceae), an endemic herbaceous and perennial nickel-hyperaccumulating plant growing on Ni-enriched ultramafic soils in South Africa, is perceived as a promising species for phytoremediation and phytomining due to its large biomass production and high Ni content. Total concentrations of a number of elements in mature leaves, soil and related bedrock were obtained. The average Ni concentration in leaves was 18,000 mg × g^-1 dry mass, whereas in soil and bedrock the total amount of Ni was 1,300 mg × g^-1 and 1,500 mg × g^-1, respectively. Exceptionally high average Ni concentrations (55,000 + 15,000 mg × g^-1, n = 6) were found in B. coddii leaves from Songimvelo Game Reserve, including the highest-ever reported concentration of Ni in leaves (76,100 mg × g^-1 - maximum value in a single sample). Young plants grown in pots with ultramafic soil accumulated small quantities of Cd, Pb or Zn, but the concentrations of these elements increased after the addition of metal solutions to the soil. Excised shoots immersed in concentrated solutions of Cd, Ni, Pb or Zn accumulated large amounts of these metals in the leaves. 

Key words: Berkheya coddii, hyperaccumulation, nickel, zinc, lead, cadmium, phytoextraction, phytoremediation, caulofiltration.

EMBRYOLOGICAL OBSERVATIONS ON UNPOLLINATED OVARY CULTURE OF MULBERRY (MORUS ALBA L.)

T. DENNIS THOMAS*

Postgraduate and Research Department of Botany, St. Thomas College Pala, 
Arunapuram-686574, Kottayam (Dt), Kerala, India

*e-mail: den_thuruthiyil@yahoo.com

Received May 14, 2003; revision accepted January 18, 2004

A three-step procedure was adopted for the induction of gynogenesis in two cultivars of mulberry (Morus alba L.). This includes in vitro flowering, inflorescence segment culture and isolated ovary culture. In the third step, that is, isolated ovary culture, the cultured ovaries burst and an embryo emerged from the ovary. The present paper investigates the ontogeny of the developing gynogenic embryo. The study confirmed that the gynogenic embryo emerged from the egg cell. Before the onset of division of the egg, all other cells of the embryo sac degenerated in the majority of ovules, but in exceptional cases the polar nuclei will be retained along with the dividing egg cell. The presence of the gynogenic embryo along with free-nuclear autonomous endosperm is the most important feature of the present investigation. Autonomous endosperm is formed from either the polar nuclei or secondary nucleus. In both cultivars used for the experiment, the percentage of ovaries showing proembryo induction during inflorescence segment culture is much higher than that of ovaries producing gynogenic plants during isolated ovary culture. This suggests the degeneration of some gynogenic embryos during the initial stages of induction. 

Key words: Morus alba, mulberry, autonomous endosperm, embryo sac, gynogenesis, unpollinated ovary culture.

FATTY ACID AND TOCOCHROMANOL PATTERNS OF TURKISH PINES

EYUP BAGCI* AND YALÇIN KARAAĐAÇLI

Department of Biology, Firat University, Elazig-Turkey

*e-mail: ebagci@firat.edu.tr

Received July 10, 2003; revision accepted January 19, 2004

The plant family Pinaceae is known to produce a set of unusual fatty acids in the seed oils. In Turkey it has been less studied in respect to the fatty acid and tocochromanol (tocopherols and tocotrienols) composition of the seed oils, particularly in terms of chemotaxonomy. This study examined the fatty acid, tocopherol, tocotrienol and plastochromanol-8 content of Pinus L. taxa naturally growing in Turkey (Pinus nigra subsp. pallasiana, P. nigra subsp. pallasiana var. pyramidata, P. halepensis, P. sylvestris, P. pinea, P. brutia, P. radiata, P. pinaster). The fatty acids (oleic, linoleic, a-linolenic, D5-taxoleic, pinolenic and coniferonic acid) and tocochromanols are valuable components of Pinus seed oils. in the family Pinaceae. Unsaturated fatty acid comprises ~90% of the seed oil. While g-tocopherol was found to be the largest component of all Pinus seed oil, ß-tocotrienol was not found at all. Total tocopherols in the studied taxa was much higher than total tocotrienols. The distribution of fatty acid and tocochromanol patterns in the genus Pinus is discussed, and in particular the presence of D5-UPIFA acids such as taxoleic and pinolenic acids, as well as common and distinguishing features that may contribute to knowledge about the taxonomy and phylogeny of pines. 

Key words: Pinaceae, Pinus, fatty acid, tocochromanol chemotaxonomy, phylogeny.

CHANGES DURING LONG-TERM GROWTH OF DESULFOTOMACULUM ACETOXIDANS DSM 771

RYSZARD PADO* AND LUCYNA PAWŁOWSKA-ĆWIĘK

Department of Microbiology, Pedagogical University of Cracow, 
ul. Podbrzezie 3, 31-054 Cracow, Poland

*e-mail: ibiol@wsp.krakow.pl

Received September 10, 2003; revision accepted January 28, 2004

Growth rate and concentrations of sulfates, sulfides, proteins and glucosamine were analyzed during long-term (over 60 days) incubation of Desulfotomaculum acetoxidans DSM 771. To imitate natural conditions, incubation was done in obligate anaerobic conditions in three series, without stirring or shaking. In the first 2-3 days of incubation (lag phase), only a decrease of the sulfate level occurred. Between days 2 and 7 of incubation (logarithmic increase phase, log phase) the growth rate and levels of proteins and glucosamine increased significantly. Simultaneously the amount of dissimilated hydrogen sulfide began to increase. Hydrogen sulfide content in parallel samples treated with lysozyme was much higher. Between days 7 and 18 a plateau ascribed to the stationary phase was observed. After 2 weeks of incubation a certain reduction of the measured substances was observed, but from days 20 to 24 the growth rate again increased (“post-stationary” phase). The high coefficients of correlation (for individual series 0.6735; 0.7245; 0.8217) between proteins and sulfide levels and control tests done with standards (albumin and Na₂S) suggest that H₂S and probably sulfides react with proteins and presumably with peptidoglycan. This could explain cumulation of sulfide and its decrease in the post-stationary phase.

Key words: Desulfotomaculum acetoxidans, long-term growth, sulfide accumulation.

ULTRASTRUCTURAL CHANGES IN ZYGOTIC EMBRYOS OF GENTIANA PUNCTATA L. DURING CALLUS FORMATION AND SOMATIC EMBRYOGENESIS

ANNA MIKUŁA¹*, TERESA TYKARSKA², MARTA ZIELIŃSKA¹, MIECZYSŁAW KURAŚ², AND JAN J. RYBCZYŃSKI<sup>1

1</sup>Botanical Garden - Center for Biological Diversity Conservation, Polish Academy of Sciences, 
ul. Prawdziwka 2, 02-973 Warsaw, Poland
²Department of Plant Morphogenesis, Faculty of Biology, Warsaw University, 
ul. Miecznikowa 1, 02-096 Warsaw, Poland

*e-mail: obpan@ikp.atm.com.pl

Received June 9, 2003; revision accepted February 10, 2004

Imbibed immature zygotic embryos of Gentiana punctata L. were cultured on MS (Murashige and Skoog) medium consisting of 4.5 mM dicamba, 0.54 mM NAA (naphtaleneacetic acid), 8.88 mM BAP (6-benzylaminopurine) and 0.43 mM AS (adenine hemisulfate). The primary response of explants consisted in thickening of the subcotyledon and hypocotyl root (HR) zone. Cotyledons and the seminal root did not show any response. Ultrastructural analysis of the initial stages of callus formation revealed numerous changes in cells of explants. Dedifferentiation of the explant tissues was associated with separation of cells resulting from thickening and folding of walls, destruction of plasmodesmata, and enlargement of intercellular spaces. At the same time, the number of lipid bodies decreased and starch appeared. Indicative of changes in 3-day cultures, the first cell divisions were observed to occur in the HR zone, including cells of the primary cortex, endodermis and pericycle. The dividing cells contained small vacuoles, large, centrally located, layered nuclei with vacuolated nucleoli, amyloplasts with starch, lipid bodies, numerous active Golgi structures, mitochondria and rough endoplasmic reticulum. Actively dividing cells formed callus tissue in which three zones of cells could be distinguished after 14 days of culture: (I) outer (starch) layer, (II) middle layer with actively at dividing small cells, and (III) inner layer containing large vacuolated cells. As the result of cell divisions, at about the fourth week of culture the starch zone formed meristematically active centers of small cells, with dense cytoplasm and large amounts of starch. Among them were small cellular complexes consisting of three cells, with the cell wall structure typical for pre-embryos. By the fifth week of culture, numerous globular and early heart-shaped somatic embryos which formed cotyledons were observed, and further mature somatic embryos showing conversion ability.

Key words: Gentiana punctata L., zygotic embryo, ultrastructure, dedifferentiation, callus development, somatic embryo.

SEED DEVELOPMENT IN SOLANUM MURICATUM AITON

JOANNA KOPCIŃSKA¹, BARBARA ŁOTOCKA¹, KATARZYNA KOWALCZYK², AND JOLANTA KOBRYŃ<sup>2

1</sup>Department of Botany, Warsaw Agricultural University, 
ul. Nowoursynowska 159, 02-776 Warsaw, Poland
²Department of Vegetable and Medicinal Plants, Warsaw Agricultural University, 
ul. Nowoursynowska 166, 02-787 Warsaw, Poland

Received September 15, 2003; revision accepted March 8, 2004

In Solanum muricatum Aiton the development of cellular endosperm preceded the divisions of the zygote, and 5 days after pollination (dap) it consisted of several cells. The mature endosperm accumulated lipid and protein bodies. The separation and secretion zone on the embryo/endosperm interface was noted at 20 dap. The first division of the zygote occurred at 7 dap. The apical cell of the 2-celled proembryo produced the embryo proper, and the basal one gave rise to the suspensor, the central part of the root cap (columella) and the connecting layer of the embryonic root meristem. Four-celled proembryos were observed with the cells in linear arrangement at 8 dap. The first globular embryos were found at 16 dap, heart-stage embryos at 26 dap, torpedo-shaped embryos at 30 dap, and circinate embryos at 33 dap. After 56 dap no further changes were observed in the anatomical structure of the embryos. The testa of mature seeds was composed of the outer epidermis of the integument and a layer of obliterated parenchyma and endothelium.

Key words: Solanaceae, embryo, endosperm, pepino, seed, testa.

CLEARED-OVULE TECHNIQUE USED FOR RAPID ACCESS TO EARLY EMBRYO DEVELOPMENT IN SECALE CEREALE X ZEA MAYS CROSSES

ALEKSANDRA PONITKA* AND AURELIA ŚLUSARKIEWICZ-JARZINA 

Institute of Plant Genetics, Polish Academy of Sciences,
ul. Strzeszyńska 34,60-479 Poznań, Poland

*e-mail: apon@igr.poznan.pl

Received November 29, 2003; revision accepted March 11, 2004

Nine rye genotypes and pollen mixture of three maize cultivars were evaluated for embryo formation in rye x maize crosses. Based on cleared ovule preparations, the development of embryo and endosperm were observed at 48, 72 and 96 h after pollination. Embryos were formed in eight cross combinations (3.3% to 23.3% depending on rye genotype), but in five combinations ovules had both embryos and endosperm nuclei. In a sample of 344 cleared ovules, 28 (8.1%) had both embryo and endosperm, 15 (4.4%) had only embryo, and 13 (3.8%) had only endosperm. 

Key words: Secale cereale, Zea mays, embryo formation, endosperm, cleared-ovule technique, wide crosses.

IMPROVED TRANSFORMATION OF ALFALFA SOMATIC EMBRYOS USING A SUPERBINARY VECTOR

SLAVICA NINKOVIĆ¹*, JOVANKA MILJUŠ-ĐUKIĆ², BRANKA VINTERHALTER¹ and MIRJANA NEŠKOVIĆ<sup>1

1</sup>S. Stanković Institute for Biological Research, University of Belgrade, 
29 novembra 142, 11060 Belgrade, Serbia and Montenegro
²Institute of Molecular Genetics and Genetic Engineering, 
Vojvode Stepe 444a, 11000 Belgrade, Serbia and Montenegro

*e-mail: slavica@ibiss.bg.ac.yu

Received October 10, 2003; revision accepted March 17, 2004

Increased transformation efficiency of alfalfa (Medicago sativa L. cv. Zaječarska 83), was achieved using Agrobacterium tumefaciens LBA4404 carrying the superbinary vector pTOK233 and the hpt gene for hygromycin resistance. Evidence for transformation was based on GUS activity and the presence of a fragment of the uidA gene in transformed embryos and regenerated plants, as demonstrated by the polymerase chain reaction. Efficiency was 1% (LBA4404/pBI121) to 14% (LBA4404/pTOK233) higher than that of some other vectors used in previous work with the same cultivar. The higher efficiency may be due to the presence of an extra set of vir genes in pTOK233. Moreover, the presence of the hpt gene, enabling the use of hygromycin instead of kanamycin for selection, was better for the development of secondary somatic embryos, thus contributing to a higher final number of transgenic plants.

Key words: Agrobacterium tumefaciens, Medicago sativa L., hygromycin, somatic embryogenesis, superbinary vector.

TEM INVESTIGATION OF TANNINS AND CHLOROPLAST STRUCTURE IN NEEDLES OF DAMAGED SILVER FIR TREES (ABIES ALBA MILL.)


TOMISLAV BAČIĆ¹, NIKOLA LJUBEŠIĆ², ZVONIMIR UZAREVIĆ¹, LJILJANA GRGIĆ¹ AND JADRANKA ROŠA<sup>3


1</sup>J.J. Strossmayer University, L. Jägera 9, 31000 Osijek, Croatia 
²“Ruđer Bošković” Institute, Bijenička cesta 54, 10000 Zagreb, Croatia
³Croatian Forestry, Vukotinovićeva 1, 10000 Zagreb,Croatia

Received November 5, 2003; revision accepted April 31, 2004

As part of investigations on the effect of air pollution on plant anatomy, we examined the condition of tannins and chloroplast structure in damaged fir trees (Abies alba Mill.) at Risnjak National Park. Two populations were chosen for needles sampling: one at the Risnjak site with trees having 20%, 45% and 85% damage, and the other at the Donja Dobra site (control locality) with relatively healthy trees having 5% to 10% damage. Current and previous-year needles were fixed with glutaraldehyde and osmium tetroxide, and then the condition or ultrastructure of tannins and chloroplasts were examined by transmission electron microscopy (TEM). The tannins were shown to be granular and arranged in thin or thick ribbons. The chloroplasts were first somewhat rounded and then round or irregular in shape, with reduced and swollen thylakoids, especially those of the grana, increased numbers of plastoglobuli, large lipid droplets/accumulations, and vesiculation of the cytoplasm. These symptoms were more frequent in previous-year than in current-year needles. All these alterations can be attributed to air pollution. 

Key words: Tannins, chloroplasts, transmission electron microscopy, air pollution. 

RESPONSE OF ANTIOXIDANT SYSTEMS TO SHORT-TERM NaCl STRESS IN GRAPEVINE ROOTSTOCK-1616C AND VITIS VINIFERA L. CV. RAZAKI

ÖZLEM YILDIRIM¹*, SÜMER ARAS¹, ALI ERGÜL², GÖKHAN SÖYLEMEZOĐLU<sup>3 

1</sup>Department of Biology, University of Ankara, 
06100 Tandođan-Ankara, Turkey, 
²Institute of Biotechnology, and 3Department of Horticulture, University of Ankara,
06110 Dişkapi-Ankara, Turkey

*e-mail: ozlemesn@hotmail.com 

Received November 25, 2003; revision accepted May 20, 2004

The present study examined free-radical scavenging enzyme activity and the levels of lipid peroxide, ascorbic acid, nitric oxide and glutathione in 1616C rootstock and the Razaki cultivar of Vitis vinifera L. under treatment with different concentrations of salt. At day 7, in leaves of both 1616C rootstock and cv. Razaki treated with 12 mM NaCl there were significant increases in glutathione peroxidase and catalase activity, and in the levels of thiobarbituric acid reactive substance and reduced glutathione, measured on a protein basis and fresh weight basis. Superoxide dismutase activity increased under NaCl treatment at day 7 in both samples versus the controls. In the Razaki cultivar, glutathione peroxidase activity was at maximum at day 7 under 12 mM NaCl treatment. Catalase activity was very low, and increased with increasing NaCl concentration in the Razaki cultivar and 1616C rootstock at day 7. In 1616C rootstock the nitrite level was lower than the controls within 4 days.

Key words: Vitis vinifera L., 1616C rootstock, antioxidant enzymes, glutathione, lipid peroxidation, ascorbic acid, nitric oxide.

THE EFFECT OF CARBOHYDRATES AND POLYETHYLENE GLYCOL ON SOMATIC EMBRYO MATURATION IN HYBRID FIR ABIES ALBA X ABIES NUMIDICA

TERÉZIA SALAJ, RADOSLAVA MATڊOVÁ, AND JÁN SALAJ

Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, 
Akademicka 2, P.O. Box 39A, 950 07 Nitra 1, Slovak Republic

e-mail: nrgrtesa@savba.sk

Received December 11, 2003; revision accepted May 8, 2004

Embryogenic cell line AN 72 derived from immature hybrid fir Abies alba x A. numidica zygotic embryos was subjected to different maturation treatments. The effect of the carbohydrates sucrose, maltose and glucose (each at 3%, 6% and 9%) or PEG-4000 (5.0%, 7.5% and 10%) combined with different carbohydrate sources was tested. PEG-4000 stimulated somatic embryo maturation of hybrid fir. This stimulatory effect was dependent on the carbohydrate source used. Culture medium with maltose as carbohydrate source combined with PEG-4000 produced the highest number of cotyledonary somatic embryos. Carbohydrates supplied alone (mainly at 6% and 9%) exerted an unfavorable effect, increasing the frequency of abnormally shaped somatic embryos without regeneration capacity. The structural organization of morphologically well-developed cotyledonary somatic embryos was similar to that of zygotic embryos. In abnormal somatic embryos the shoot apical meristem and root meristem were very damaged. Electrophoretic separation of denatured proteins using SDS-PAGE showed differences in the accumulation of low molecular weight storage proteins in somatic embryos. Storage protein accumulation was dependent on the concentration of PEG-4000 and the carbohydrate source.

Key words: Abies alba x A. numidica, carbohydrates, embryogenesis, PEG, somatic embryos.

IN VITRO CLONAL PROPAGATION OF PRIMULA VERIS L.
AND PRELIMINARY PHYTOCHEMICAL ANALYSIS 

MARIA MOROZOWSKA¹*AND MARIA WESOŁOWSKA²**

¹Department of Botany, August Cieszkowski Agricultural University, 
ul. Wojska Polskiego 71C, 60-625 Poznań, Poland
²Department of Pharmaceutical Botany,
Karol Marcinkowski University of Medical Sciences, 
ul. Św. Marii Magdaleny 14, 61-861 Poznań, Poland

* e-mail: mariamor@owl.au.poznan.pl
** e-mail: mwesolow@amp.edu.pl

Received December 10, 2003; revision accepted May 21, 2004

A method of in vitro clonal propagation of cowslip Primula veris L. (Primulaceae) is reported. This is a species of medicinal importance, protected by law in Poland. MS medium with BA (4.44 mM) and 2,4-D (1.13 mM) was found to be optimal for in vitro cowslip propagation from shoot tips under the conditions of culture. Rooting was best in the presence of IBA (2.45 mM). In vitro seedlings were transferred to pots and then acclimated. After transplanting to a garden they showed further growth and development, including flowering and fruiting. Phytochemical analysis (2D-TLC) revealed that the flavonoid compounds in leafy shoots from in vitro culture were similar to those in leaves from field cultivation.

Key words: Primula veris L., cowslip, in vitro clonal propagation, flavonoids.

IN VITRO MANIPULATION OF CUCUMBER (CUCUMIS SATIVUS L.) POLLEN AND MICROSPORES: ISOLATION PROCEDURES, VIABILITY TESTS, GERMINATION, MATURATION

LILIANA VIZINTIN AND BORUT BOHANEC*

Biotechnical Faculty, Centre for Plant Biotechnology and Breeding, 
Jamnikarjeva 101, 1000 Ljubljana, Slovenia

*e-mail: bonec@uni-lj.si

Received January 10, 2004; revision accepted June 2, 2004

In vitro germination of isolated pollen grains and maturation of microspores are needed for several biotechnological manipulations of cucumber pollen. The present study was focused on establishing optimal conditions for monitoring isolation, sterilization and viability. Germination of mature cucumber pollen and maturation of microspores were attempted. Five protocols for testing pollen and microspore viability were tested and three were studied in detail. The viability of fresh or artificially damaged grains was tested using aniline blue in lactophenol, fluorescein diacetate (FDA) and 2,5-diphenyl tetrazolium bromide (MTT). For strong contrast, simple manipulation and non-fluorescence, MTT was found to be the most suitable. Pollen was efficiently isolated by shaking chopped anthers in germination media. Germination of isolated pollen was achieved for the first time using this procedure. The highest germination rate was obtained using 90.4% maltose in germination media at pH 7.0. Using a similar isolation method, microspores cultured on four selected maturation media showed relatively high viability (up to 29.1%) which was not affected by the sterilization procedure, although mature pollen grains did not form.

Key words: Aniline blue in lactophenol, Cucumis sativus L., fluorescein diacetate (FDA), MTT, pollen viability, pollen isolation, in vitro germination.

COMPARISON OF GROWTH PROPERTIES, ALKALOID PRODUCTION AND WATER UPTAKE OF TWO SELECTED DATURA HAIRY ROOT LINES

ARNAUD LANOUE¹, MICHELE BOITEL-CONTI¹*, CELINE DECHAUX¹, JEAN-CLAUDE LABERCHE¹, PHILIPPE CHRISTEN², AND BRIGITTE SANGWAN-NORREEL<sup>1

1</sup>Université de Picardie Jules Verne, Faculté des Sciences, Laboratoire Androgenčse et Biotechnologie, 
33, rue Saint Leu, F-80039 Amiens Cedex 01, France
²Laboratoire de Chimie Analytique Pharmaceutique, Université de Genčve, 
20 Bd d’Yvoy, 1211 Geneva 4, Switzerland

Received January 12, 2004; revision accepted July 4, 2004

Two selected hairy root lines of Datura, D. innoxia and D. candida × D. aurea were compared for their performance in growth and tropane alkaloid productivity in shake flasks. Specific consumption rate and biomass yield on sucrose and nitrates during the phase of maximum growth were calculated versus changes in liquid medium volume during culture. Growth parameters were found to be better in D. innoxia hairy root cultures than in D. candida × D. aurea. Overall productivity of hyoscyamine in D. innoxia (2.1 mg l^-1 d^-1) was double that in D. candida × D. aurea (1 mg l^-1 d^-1). However, hybrid transgenic roots produced 0.5 mg l^-1 d^-1 of scopolamine. The relationship between water uptake and root growth was studied. During the first days of culture, water was released into the culture medium and then reabsorbed in the root tissue. After that, water uptake on a biomass basis in D. innoxia and D. candida × D. aurea transformed root cultures reached 0.74 and 0.53 ml g^-1 FW, respectively. The correlation between biomass accumulation and liquid medium volume could be used as a simple and inexpensive method for indirect estimation of root growth.

Key words: Datura, growth, hairy roots, tropane alkaloids, water uptake.

TRANSFER CELL WALL ARCHITECTURE IN SECRETORY HAIRS 
OF UTRICULARIA INTERMEDIA TRAPS

BARTOSZ J. PŁACHNO* AND ANDRZEJ JANKUN**

Department of Plant Cytology and Embryology, The Jagiellonian University,
ul. Grodzka 52, 31- 044 Cracow, Poland

*e-mail: bartek78pl@ poczta.onet.pl 
** jankun@iphils.uj.edu.pl

Received May 5, 2004; revision accepted July 12, 2004

This paper discusses transfer cell wall deposition and architecture in various trap hairs of the carnivorous plant Utricularia intermedia. Scanning electron microscopy showed that the middle cells of both internal hairs and pavement epithelium hairs have reticulate-type wall ingrowths. The wall ingrowths of the middle cell of both quadrifids and bifids are very well developed. However, in middle cells of pavement epithelium hairs the level of development of wall ingrowths is not uniform. The presence of ruptured cuticles and wall ingrowths in these hairs suggests that water is transported by the pavement epithelium hairs from the trap to the external environment. 

Key words: transfer cell, wall ingrowths, hairs, Utricularia, carnivorous plants.

VARIATIONS IN ABSCISIC ACID, INDOLE-3-ACETIC ACID, GIBBERELLIC ACID AND ZEATIN CONCENTRATIONS IN TWO BEAN SPECIES SUBJECTED TO SALT STRESS 

FUSUN YUREKLI¹*, Z. BANU PORGALI¹ AND ISMAIL TURKAN<sup>2

1</sup>Department of Biology, Art and Science Faculty, Inönü University, Malatya, Turkey
²Department of Biology, Science Faculty, Ege University, Izmir, Turkey

*e-mail: fyurekli@inonu.edu.tr 

Received November 10, 2003; revision accepted July 10, 2004

Common bean (Phaseolus vulgaris L.) is sensitive to drought and salinity, while an ancestral legume, tepary bean (P. acutifolius A. Gray) is cultivated successfully where high temperature and drought are common (Lazcano-Ferrat and Lovatt, 1999). Hence, P. acutifolius is a potential source of stress tolerant traits for P. vulgaris through interspecific hybrids. This study comparatively evaluated the effects of salt stress on leaf relative water content (RWC), soluble protein, the phytohormones indole acetic acid (IAA), gibberellic acid (GA3), zeatin, and abscisic acid (ABA) levels in P. vulgaris and P. acutifolius. With the exception of ABA, stress-induced changes in hormonal levels putatively related to stress tolerance have not been investigated previously in either species. Treatment with 50 mM, 100 mM and 150 mM NaCl reduced relative water and protein content in P. vulgaris, but did not affect relative water content and increased protein content in P. acutifolius. Varietal differences between P. vulgaris and P. acutifolius were also observed in hormonal content during the stress period. ABA levels in salt-treated plants of P. vulgaris increased but did not change in P. acutifolius versus the controls. Both IAA and gibberellin levels increased in leaves of P. acutifolius but decreased in P. vulgaris under salt stress. In salt-treated plants, zeatin concentrations in leaves of P. vulgaris decreased within 24-72 h after treatment. However, P. acutifolius responded to all tested NaCl levels by increasing zeatin concentrations after short-term NaCl treatment. 

Key words: Relative water content, abscisic acid, indole acetic acid, gibberellin, zeatin, NaCl-stress, Phaseolus, protein.

LOCALIZATION OF NITROGENASE AND NITRATE REDUCTASE IN THE CYCAS-ANABAENA CYCADEAE ASSOCIATION

ANITA SHARMA¹* PANKAJ MISHRA²**, AND ASHOK KUMAR<sup>3

1</sup>Department of Microbiology, C. B. S. & H., G. B. Pant University of Agriculture and Technology, 
Pantnagar, 263 145, U. S. Nagar, U. P., India
²Department of Botany, R. B. S. College, Agra, 282002, U. P., India
³School of Biotechnology, B. H. U., Varanasi, India

*Corresponding author
** e-mail: misrapank12@rediffmail.com

Received October 20, 2003; revision accepted July 30, 2004

Induction of nitrate reductase (NR) activity in coralloid roots of Cycas revoluta was observed after 8 h incubation in 0.02 M KNO₃. Other plants growing near Cycas showed a higher level of NR immediately when incubated in KNO₃. In contrast to NR, intact coralloid roots showed very high nitrogenase activity (~1.2 to 1.6 mmol C₂H₄ g fresh wt^-1 h^-1) under both light and dark conditions as compared to transverse sections of roots. Localization of NR and nitrogenase was tested in coralloid roots using different sets of roots and also in the endophyte. Our results showed that NR activity was mainly due to the endophyte (Anabaena cycadeae); coralloid roots lacked it, as no NR activity was observed in chloramphenicol-treated intact root samples. 

Key words: Nitrate reductase, Anabaena cycadeae, coralloid root, nitrogenase activity.

MEIOTIC BEHAVIOUR OF CHROMOSOMES IN PMCS AND KARYOTYPE OF TRIFOLIUM REPENS L. FROM DARJEELING HIMALAYA

SUDIPTA MAJUMDAR, SOMENATH BANERJEE, AND KALYAN KUMAR DE*

Post Graduate Department of Botany, Darjeeling Govt. College, 
P.O. and Dist. Darjeeling 734 101, West Bengal, India

*e-mail: kalyannet2003@yahoo.co.in

Received March 22, 2004; revision accepted July 30, 2004

Detailed analyses of the chromosome meiotic behaviour and of mitotic metaphase chromosomes (2n = 32), as well as stainability studies of pollen fertility, were carried out in order to determine the cytological status of amphidiploid Trifolium repens L. (Dutch white clover). In amphidiploid (allotetraploid) Trifolium repens L., diploid-like meiotic behaviour of chromosomes was found, with no multivalent formation, and a normal karyotype with a single pair of chromosome having a secondary constriction was observed. These characteristics indicate favourable genetic and cytological stability in nature, and high pollen fertility further enhances its usefulness in breeding. 

Key words: Trifolium repens L., meiotic chromosome, karyological analysis.