Microevolutionary Changes in Ecotypes of Calamine Waste Heap Vegetation near Olkusz, Poland: A Review

Małgorzata Wierzbicka¹ and Adam Rostański<sup>2

1</sup>Environmental Plant Pollution Laboratory, Department of Morphogenesis,
Institute of Plant Experimental Biology, University of Warsaw, 
ul. Miecznikowa 1, 02-096 Warsaw, Poland
²Department of Plant Systematics, Faculty of Biology and Environmental Protection, 
University of Silesia, ul. Jagiellońska 28, 40-032 Katowice, Poland

Received December 30, 2001; revision accepted May 27, 2002

This paper reviews several studies dealing with the specific traits of plants growing on calamine waste heaps in the vicinity of Olkusz. The waste heaps here contain very high amounts of zinc, lead and cadmium. Particular traits distinguishing the calamine forms of the species Silene vulgaris, Dianthus carthusianorum and Biscutella laevigata make calamine plants potentially very useful for recultivating land polluted by heavy metals in Poland. Utilization of natural biological processes would seem to be the best approach to the problem of recultivation. Despite this, methods using local plant resources to reclaim areas polluted by high concentrations of heavy metals are still rarely used. The natural vegetation of areas characterized by high concentrations of heavy metals may be a valuable source of genetic material (ecotypes) ideally adapted for growth under the harsh and pioneering conditions of calamine waste heaps.

Key words: Calamine waste heap, tolerance, heavy metals, Silene vulgaris, Dianthus carthusianorum, Biscutella laevigata, recultivation.

Lead Tolerance in Plants Growing on Dry and Moist Soils 

Małgorzata Wierzbicka¹ and A. Potocka<sup>2

1</sup>Environmental Pollution Plant Laboratory, Department of Morphogenesis, Institute of Plant Experimental Biology, 
²Laboratory of Lipids and Biological Membranes, Institute of Biochemistry, Warsaw University, ul. Miecznikowa 1, 02-096 Warsaw, Poland

Received September 17, 2001; revision accepted February 25, 2002

This study addresses what underlies the high tolerance of some plant species to lead. The tolerance to lead of six species differing in their water requirements (xerophytes, mesophytes and hydrophytes) was determined. Seedlings were treated with lead (2.5 mg/dm3 Pb2+ from PbCl2) for 8 days in a hydroponic culture. Tolerance to lead (on the basis of root growth, i.e., index of tolerance), lead concentration in tissues and lead transport to stems (using AAS) were studied. The presence of lead in organs, tissues and cells was determined by the rhodizonate method. Using the results, we classified the tested plant species according to lead tolerance, in the following order: Berteroa incana < Helichrysum sp. < Leontodon hispidus < Cucumus sativus < Dianthus carthusianorum < Rumex aquaticus.
The lead tolerance of these species correlated with their water requirements. Plants from dry stands demonstrated the lowest tolerance to lead (Berteroa incana IT = 10%, Helichrysum sp. IT = 15%), those from damp stands had higher tolerance, and those from wet stands had the highest (Rumex aquaticus IT = 60%). This dependence was corroborated by field observations showing that mesophyte species dominate calamine waste heaps (55%) despite the drought conditions and strong insolation that prevail there. 

Key words: Tolerance, lead localization, rhodizonate method, lead concentration, lead transport. 

Concentration of Alkaline and Heavy Metals in Biscutella Laevigata L. and Plantago Lanceolata L. Growing on Calamine Spoils (S. Poland)

Grażyna Szarek-Łukaszewska¹*, Maria Niklińska<sup>2

1</sup>Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31-512 Cracow, Poland 
²Institute of Ecological Sciences, Jagiellonian University, ul. Gronostajowa 1, Cracow, Poland

Two plant species growing on calamine spoils (vicinity of Olkusz, S. Poland) were examined for their ability to accumulate metals. The plants in these pilot studies were Biscutella laevigata L., a rare plant that occurs in lowlands on zinc-lead mine spoils only in the vicinity of Olkusz, and Plantago lanceolata L. which is a common species in Poland. Concentrations of alkaline metals (Ca, K, Mg) and heavy metals (Cd, Fe, Mn, Pb, Zn) in soil and plants (shoots, roots) from two locations of calamine spoils 100 and 30 years old and control areas were determined. Soils from the mine spoils were alkaline (pH >7.4), with large concentrations of Ca and Mg. Concentrations of Cd, Pb and Zn in soil were up to 224 mg kg-1 3100 mg kg-1 and 78,000 mg kg-1, respectively. The amounts of exchangeable metals (1 M NH4NO3) were up to 9.51 mg kg-1 Cd, <0.1 mg kg-1 Pb, and 24.5 mg kg-1 Zn. Concentrations of heavy metals in plants from the calamine spoils and their distribution within plants depended on the species. B. laevigata accumulated heavy metals in roots (Cd) or shoots (Fe, Mn, Zn). P. lanceolata accumulated heavy metals mainly in roots. Maximum concentrations in roots and shoots of B. laevigata were 14.3 mg kg-1 Cd, 111 mg kg-1 Pb and 410 mg kg-1 Zn. P. lanceolata contained up to 65.6 mg kg-1 Cd, 157 mg kg-1 Pb and 2540 mg kg-1 Zn. Our data suggest that both species tend to exclude Cd, and P. lanceolata also Zn, present in large concentrations in the soil.

Key words: Calamine spoils, soil, plant, Biscutella laevigata, Plantago lanceolata, heavy metals, S. Poland.

Influence of Selenium on Lead Absorption and Localization in Meristematic Cells of Allium Sativum L. and Pisum Sativum L. Roots

Sława Glińska* And Barbara Gabara

Department of Plant Cytology and Cytochemistry, University of Łódź, 
ul. Banacha 12/16, 90-237 Łódź, Poland

The effect of different concentrations (80-640 uM) of sodium selenate and sodium selenite on lead absorption by Allium sativum (selenium absorber) and Pisum sativum (selenium nonaccumulator) roots treated with 10 uM Pb(NO3)2 was investigated. Lead alone and with either of the selenium compounds was supplied in aqueous solutions for 24 h. Selenite diminished lead content in the two plant species' roots more effectively than selenate. The rhodizonate method showed the absence of lead in the presence of only 160 uM sodium selenite and only 640 uM sodium selenate. The effects of selenium compounds at 80 uM concentration on lead localization in meristematic cells were also investigated. While selenite in garlic cells reduced the number of electron-dense deposits in the cell wall and diminished their size in vacuoles, it increased their number in cytoplasm and plastids, and enlarged them in mitochondria. In pea cells it caused the disappearance of electron-dense sediments from the Golgi apparatus, endoplasmic reticulum, vacuoles and nucleus, or reduced their number in mitochondria, cytoplasm and plastids (lack of large deposits). On the basis of literature data we assume that selenium reduces the lead concentration in Allium sativum and Pisum sativum roots due to the formation of Pb-Se complex in the incubation medium.

Key words: Allium sativum, Pisum sativum, lead, selenium, roots.

Accumulation of Zinc and Lead in Selected Taxa of the Genus Viola L.

Monika Jędrzejczyk¹, Adam Rostański¹, and Eugeniusz Małkowski<sup>2

1</sup>Department of Plant Systematics, ² Department of Plant Physiology, 
Faculty of Biology and Environmental Protection, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland

Received November 12, 2001; revision accepted February 26, 2002

This study investigates the effect of high concentrations of Pb and Zn on biomass production and accumulation of both metals in zinc violets (Viola calaminaria and Viola guestphalica) and two species of violets indigenous to Poland (Viola lutea sudetica and Viola tricolor). The influence of the plants on soil pH also was assessed. The soil used in the experiments, containing 183 mg kg-1 Zn and 53.4 mg kg-1 Pb, was spiked with Zn and Pb at 1000 mg/kg soil (Treatment 1) or Zn at 10,000 mg/kg and Pb at 1000 mg/kg (Treatment 2). Zn concentrations tested higher in roots than in shoots in all investigated species. In zinc violets, a Zn content in the soil correlated with Zn concentrations in plant tissue, both roots and above-ground parts. There was no such dependence among indigenous violets. Since all confirmed hyperaccumulators accumulate metals preferentially in shoots, with lower concentrations in roots, our results suggest that zinc violets should not be classified as Zn hyperaccumulators. V. l. sudetica occurs naturally on soils with low heavy metals content. Its roots accumulated the highest amount of Zn (6498 mg/kg) observed in the current study with no toxic effects. Roots were able to change the soil pH, but the differences were not significant. V. l. sudetica and V. tricolor increased biomass significantly in Treatment 1 versus the control, but in Treatment 2 neither species differed in biomass from the control. In Viola guestphalica no influence of treatments on dry weight was observed. The results suggest that zinc violets should not be treated as zinc hyperaccumulators. We suggest that zinc violets can be useful for phytorestoration of contaminated sites.

Key words: Zinc, lead accumulation, genus Viola.

Thallium Contamination of Selected Plants and Fungi in the Vicinity of the Bolesław Zinc Smelter in Bukowno (Southern Poland). Preliminary Study

Krzysztof Dmowski ¹* and Marta Badurek <sup>2

1</sup>Department of Ecology, Faculty of Biology, Warsaw University, ul. Banacha 2, 02-097 Warsaw, Poland
²Botanical Garden - Center for Biological Diversity Conservation, Polish Academy of Sciences, ul. Prawdziwka 2, 02-973 Warsaw, Poland

High thallium concentrations were found in plants and fungi growing 0.5-2 km from the flotation waste reservoir serving the Bolesław Mining and Metallurgical Works in Bukowno. Rinsed pine needles contained 2.20 ą 0.72 mg/kg d.w. thallium, moss Pleurozium schreberi 4.89 ą 2.00, moss Catharinea sp. 12.65, lichen Cladonia sp. 2.80 ą 1.01 and edible mushrooms 3.48-4.76. Vegetables from a village (Starczynów) closest to the reservoir contained 1.28-3.70 mg/kg d.w. thallium. The inhabitants are threatened by thallium pollution. In natural conditions the element concentrations in biological samples usually do not exceed 0.0X-0.X mg/kg d.w. Only fruit samples from the studied area and all the control samples were devoid of thallium.

Key words: Thallium, plants, fungi, zinc ores, fruits, vegetables, pollution.

Fruitlet Development in the Genus Potamogeton (Potamogetonaceae)

Cezary Toma*

Department of Biological Sciences, Academy of Physical Education,
ul. Raciborska 1, 40-074 Katowice, Poland

Received May 21, 2001; revision accepted November 8, 2001

The development of the pericarp and its taxonomic value were studied in Potamogeton lucens, P. pusillus, P. crispus, P. pectinatus. The fruitlet of Potamogeton is drupaceous. Anatomical study and image analysis of developmental stages in the pericarps confirmed differences between species. The different degrees of pericarp sclerification, different positions of lignified cells in the pericarp, and numbers and types of pericarp layers have great diagnostic value. The vascular system of the Potamogeton flower and fruitlet, and the centrifugal direction of pericarp sclerification, do not have taxonomic significance within the genus Potamogeton. There are no significant differences in cell and layer size in Potamogeton fruitlets between the four species studied.

Key words: Potamogeton, development, anatomy, pericarp, seed, fruitlet.

Plant DNA Isolation from Differently Preserved Thalictrum Leaf Tissues and its Use in RAPD Analysis

Zlatko Liber*, Toni Nikolić, and Božena Mitić

Department of Botany, Faculty of Science, University of Zagreb, Marulićev trg 20/2, 
HR-10000 Zagreb, Croatia

Received July 7, 2001; revision accepted December 5, 2001

There are many unsolved taxonomic problems at the intraspecific level in the genus Thalictrum (Ranunculaceae), which may be clarified using molecular systematics methods. No molecular systematics methods have been applied yet to the genus Thalictrum; this study analyzes different leaf tissue preservation and DNA isolation techniques, and applicability of RAPD. A modified DNA isolation procedure using solution of laundry detergent as a detergent buffer system was the most suitable, especially for dealing with large samples. Since the use of differently preserved leaf tissues simultaneously with fresh leaf tissue may improve sampling in taxonomic research, and because the RAPD technique is sensitive to different factors, the possible drawbacks of using such tissues in RAPD analyses were checked. Of the four preserved leaf tissues, only DNA from silica gel-preserved leaf tissue gave suitably reliable RAPD results to be used with fresh leaf tissue in more extensive taxonomic research. Differently preserved leaf tissues are very problematic starting materials for simultaneous use with fresh leaf tissue in the same RAPD analysis. If differently preserved leaf tissues are to be used and reliable results are to be obtained, research techniques similar to those used in this paper should be applied.

Key words: Thalictrum, tissue preservation, DNA isolation, RAPD, taxonomy.

Ovule and Seed Study in Capsella bursa-pastoris (Brassicaceae) with Peculiar Endothelium Formation Pattern 

Ivan I. SHAMROV*

Department of Embryology and Reproductive Biology, Komarov Botanical Institute, Prof. Popov St., 2, St. Petersburg, 197376 Russia

Received September 5, 2001; revision accepted March 6, 2002

The paracarpous gynoecium in Capsella bursa-pastoris is characterized by postgenital fusion of two carpels into a single structure representing a false two-loculated ovary. The ovular primordium is initiated by periclinal cell divisions of both the subdermal and third layers of the placenta. The ovule is ana-amphitropous, medionucellate, funicular and bitegmic, with the micropyle formed by both integuments. During development the cells of the micropylar and middle nucellar zones degenerate and the persisting chalazal zone assumes a column shape (the postamento-podium). The integuments develop according to Dermal type and Variation C (author's term). In the mature ovule the inner integument consists of three layers, and the outer integument is two-layered except on the abaxial side of the ovule where the integuments are more massive. At the two-nucleate megagametophyte stage the inner epidermis cells of the inner integument begin to divide periclinally. These divisions are followed by differentiation, giving rise to cells that differ in their form, structure, substance accumulation and participation in seed coat organization. The inner layer, consisting of cytoplasm-rich cells with marked radial expansion, represents the endothelium. The cells of the other layer become vacuolate and extend tangentially. They form the middle layer. The cells of the outer epidermis and middle layer are destroyed (in the latter only partly) during seed development. The endothelium becomes the endotegmen (pigment layer), composed of thick-walled cells that contain tannins and possibly lipids. The outer integument gives rise to the testa, composed of an epidermal mucilaginous layer and a sclerotic (mechanical) layer consisting of cells with thickened radial and inner tangential walls and containing starch. The hypostase is differentiated at the base of the nucellus and integuments in contact with the chalaza. The vascular bundle of the ovule reaches the hypostase, which is preserved also in the mature seed and represented by 3-5 layers forming a cup. The cells of the hypostase accumulate proteins and dextrins during the late stages of ovule development, and starch after fertilization. Later, at the early globular embryo stage, the cell walls begin to lignify, the cell contents showing tannin-like substances.

Key words: Capsella bursa-pastoris, ovule, seed, development, histochemistry.

Stomata Size Variability in the Caltha palustris Complex (Ranunculaceae) from Poland

Elżbieta Cieślak¹* and Sławomir Florjan<sup>2

1</sup>W. Szafer Institute of Botany, Polish Academy of Sciences, ul. Lubicz 46, 31-512 Cracow, Poland
²Institute of Botany, Jagiellonian University, ul. Lubicz 46, 31-512 Cracow, Poland

Received October 11, 2001; revision accepted February 17, 2002

The results of variability analysis of basal leaf epidermis stomata in the Caltha palustris L. complex are presented. Stoma length and width were measured in material from 17 Polish localities. In C. palustris subsp. laeta the stomata are clearly elongated and bigger (mean length 56.52 ą 8.41 um) than those of subsp. palustris (mean length 46.80 ą 6.73 um). Within the latter subspecies, in the sample determined as var. radicans they are only slightly longer (mean length 51.46 ą 5.46 um) than wide (mean width 46.20 ą 5.25 um), while in var. cornuta they are distinctly longer (mean length 52.45 ą 5.17 um) than wide (mean width 45.00 ą 3.86 um); both, however, are more circular than in subsp. laeta. 

Key words: Caltha palustris complex, stomata variability, taxonomy.

Ultrastructural and Autoradiographic Study of Chara vulgaris Manubria

Maria Kwiatkowska*, Agnieszka Wojtczak, and Katarzyna Popłońska

Department of Cytophysiology, University of Łódź, ul. Pilarskiego 14, 90-231 Łódź, Poland

Received October 11, 2001; revision accepted April 10, 2002

Ultrastructural changes of manubria of Chara vulgaris during spermiogenesis were studied. The changes (as compared to the antheridial filament proliferation stage) involve thickening of the content of secretive vesicles and changes in their consistency, the disappearance of rough endoplasmic reticulum (ER) and appearance of smooth ER, increases in the number and size of plastoglobules in plastids, condensation of mitochondria, and significant cell vacuolization. Autoradiography with 3H-fucose pulse incubation showed that during spermatogenesis it was incorporated into the manubria. Their radioactivity decreased after 2 h postincubation in nonradioactive fucose during spermiogenesis. This was not observed during proliferation of antheridial filament cells. It is suggested that polysaccharide secretion into the internal antheridial space increases during spermiogenesis, perhaps connected with the thickening of secretive vesicle content observed by electron microscopy. 

Key words: Chara, antheridium, 3H-fucose, manubrium, spermatogenesis, spermiogenesis.

In Vitro Propagation of Dianthus gigantheus ssp. croaticus

Marija Prolić*, Sandra Radić, and Branka Pevalek-Kozlina

Department of Botany, Faculty of Science, University of Zagreb, Rooseveltov trg 6, HR-10000 Zagreb, Croatia

Received November 2, 2001; revision accepted March 4, 2002

Micropropagation of Dianthus gigantheus D’Urv. ssp. croaticus (Borbás) Tutin, a Croatian neoendemic plant species, was investigated. Shoots from aseptically germinated seeds were used for culture initiation. The highest multiplication rate (3.3 shoots per explant) was achieved on basal MS medium containing 2.9 uM gibberellic acid and 0.5 uM 6-benzylaminopurine. The rooting percentage was high on all media tested (basal MS medium supplemented with different concentrations of indole-3-acetic acid, indole-3-butyric acid and ?-naphthalene acetic acid or without them), with slight suppression on media containing higher concentrations of ?-naphthalene acetic acid. Plantlets were successfully acclimatized to outdoor conditions. 

Key words: Dianthus, multiplication, rooting, acclimatization, endemic plant.

Polymorphism of Heterochromatin Bands on Chromosomes of Rye Secale vavilovii Grossh. Lines

Stanisława Maria Rogalska¹*, Magdalena Achrem¹, Renata Słomińska-Walkowiak¹, Ewa Filip¹, Lidia Skuza¹, Jadwiga Pawłowska¹, and Barbara Apolinarska<sup>2

1</sup>Department of Cell Biology, University of Szczecin, ul. Wąska 13, 71-415 Szczecin, Poland
²Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyńska 34, 60-479 Poznań, Poland

Received November 5, 2001; revision accepted February 26, 2002

The distribution of heterochromatin in mitotic chromosomes was studied in five inbred lines of rye Secale vavilovii Grossh. (nos. 52, 109, 116, 121 and 225). An additional heterochromatin band was found on the long arm of one of the chromosomes of pair 2R, at an average distance of 2.08 m from the centromere. After the plants with an additional band on 2R were reproduced, plants with two chromosomes with an additional band were obtained, as well as those with one chromosome with an additional band and with chromosomes without that band. Distinct differences were observed in 1R in terms of the presence/absence of a band in the vicinity of the NOR constriction, and the size of the telomere bands. Telomere band size also differed in chromosomes 2R, 4R, 6R and 7R. Modifications of the heterochromatin fragment consisted in deletion of a telomeric heterochromatin segment and in the presence of different numbers of interstitial heterochromatin bands. The content of telomeric heterochromatin was the highest in 3R (18.86%) and the lowest in 4R (6.90%). During telophase, daughter nuclei connections in the form of a chromatin bridge were observed in a number of cells.

Key words: Chromosomes, heterochromatin, C-banding, Secale vavilovii Grossh.

Meiotic Behavior of Chromosomes in PMCs in Plants of Secale vavilovii Grossh. Lines with Additional Heterochromatin in Chromosome 2R

Stanisława Maria Rogalska¹, Barbara Apolinarska^2, Magdalena Achrem^1, Renata Słomińska-Walkowiak¹, Lidia Skuza¹, and Ewa Filip<sup>1

1</sup>Department of Cell Biology, University of Szczecin, ul. Wąska 13, 71-415 Szczecin, Poland
²Institute of Plant Genetics, Polish Academy of Sciences, ul. Strzeszyńska 34, 60-479 Poznań, Poland

Received November 5, 2001; revision accepted March 18, 2002

Meiosis was observed in pollen mother cells in plants of four rye Secale vavilovii Grossh. lines (nos. 52, 109, 116, 225). The behavior of 2R chromosomes with additional heterochromatin, and of other chromosomes, was observed in diakinesis, metaphase I, anaphase I and anaphase II. 2R chromosomes with additional heterochromatin formed ring and rod bivalents without disturbances. Some other chromosomes formed heterobivalents and trivalents as well as chromosome bridges and chromosome fragments, indicating structural modifications. 

Key words: Meiosis, heterochromatin, Secale vavilovii Grossh.

Flower Organs of Solanum muricatum Aiton. I. Perianth and Stamen

Joanna Kopcińska¹*,Barbara Łotocka¹, Katarzyna Kowalczyk², and Jolanta Kobryń<sup>2

1</sup>Department of Botany, Faculty of Agriculture, Warsaw Agricultural University, ul. Rakowiecka 26/30, 02-528 Warsaw, Poland
²Department of Vegetable and Medicinal Plants, Faculty of Horticulture and Landscape Architecture, ul. Nowoursynowska 166, 02-787 Warsaw, Poland

Received November 6, 2001; revision accepted February 23, 2002

In Solanum muricatum Aiton the general flower structure was typical for Solanaceae. The anther wall comprised the epidermis, endothecium (restricted to the anther tip), 3-4 middle layers and secretory tapetum. Placentoids developed in the anther loculi. Tapetum degeneration was noted in buds with the corolla shorter than the calyx, while loculi were filled with microspore tetrads. At the next stage (corolla even with calyx) pollen grains were visible. The anthers opened with tip pores in the still-closed buds, and then at anthesis the stomium split along the hypodermal row of idioblasts. Inhibition of pollen tube growth in vivo was not observed under self- or cross-pollination.

Key words: Pepino, anther dehiscence, pollen grain ultrastructure, pollen germination.

Flower Organs of Solanum muricatum Aiton. II. Pistil

Joanna Kopcińska¹*, Barbara Łotocka¹, Katarzyna Kowalczyk², Jolanta Kobryń<sup>2

1</sup>Department of Botany, Faculty of Agriculture, Warsaw Agricultural University, ul. Rakowiecka 26/30, 02-528 Warsaw, Poland
²Department of Vegetable and Medicinal Plants, Faculty of Horticulture and Landscape Architecture, ul. Nowoursynowska 166, 02-787 Warsaw, Poland

Received November 6, 2001; revision accepted February 23, 2002

The pistil of Solanum muricatum Aiton was composed of two carpels joined in a folded condition. The ovary was bilocular with a central placenta. The ovules were anatropous, unitegmic and tenuinucellar. The megaspore mother cell underwent meiosis in buds with the corolla shorter than the calyx, producing a linear tetrad. Embryo sac development was Polygonum-type. The embryo sacs were examined when corolla and calyx were the same length. In older ovules the hyposthase became visible. The cells in the center of the style formed a solid column of transmitting tissue. The papillate stigma was of the wet type. No anatomical or ultrastructural disturbances that would prevent seed set were observed in floral organ development.

Key words: Pepino, embryo sac development, synergid, egg cell, antipodals, transmitting tissue.

A Protocol for Quantitative Analysis of Green Fluorescent Protein -Transformed Plants, Using Multiparameter Flow Cytometry with Cluster Analysis

Borut Bohanec*, Zlata Luthar and Katarina Rudolf

Centre for Plant Biotechnology and Breeding, University of Ljubljana, 
Jamnikarjeva 101, 1000 Ljubljana, Slovenia

Received November 19, 2001; revision accepted March 26, 2002

Green fluorescent protein (GFP) is a particularly important reporter gene used in various transformation studies. Expression of GFP fluorescence can be visually monitored under UV or blue excitation in transformed cells. However, quantifications of fluorescence expression using fluorimetric methods are limited to average expression in tissues and cannot be assessed in single cells. An improved protocol to determine quantitative single cell fluorescence was developed using GFP-transformed tobacco leaf protoplasts measured by multiparameter flow cytometry. It was shown that a Percoll density gradient or sucrose flotation are essential for optimal separation. Fluorescent protoplasts and those expressing only background autofluorescence were successfully separated using three-parameter analysis. For clustered subpopulations, relative fluorescence intensity and proportions of cells with expressed or non-expressed fluorescence can be measured. Further applications of this novel procedure are discussed. 

Key words: Flow cytometry, genetic transformation, GFP, mosaic fluorescence expression, protoplast, tobacco.
*e-mail: borut.bohanec@bf.uni-lj.si

Genetic Relatedness Among Basil (Ocimum spp.) Accessions Using Rapd Markers

Zlatko Satovic¹*, Zlatko Liber², Ksenija Karlovic³, and Ivan Kolak¹

1Department of Seed Science and Technology, Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia
2Department of Botany, Faculty of Science, University of Zagreb, Marulicev trg 9a, 10000 Zagreb, Croatia
3Department of Ornamental Plants and Landscape Architecture, Faculty of Agriculture, University of Zagreb, Svetosimunska 25, 10000 Zagreb, Croatia

Received November 19, 2001; revision accepted March 4, 2002

Genetic relatedness among basil accessions including six species of Ocimum (O. basilicum L., O. americanum L., O. x citriodorum L., O. minimum L., O. gratissiumum L., O. tenuiflorum L.) and six botanical varieties or cultivars of O. basilicum L. (var. basilicum L. cv. Genovese, var. basilicum L. cv. Sweet Basil, var. difforme Benth., var. purpurascens Benth., cv. Dark Opal, and var. thyrsiflorum /L./ Benth.) were analyzed by random amplified polymorphic DNA (RAPD) markers. Five primers were used for amplification. They yielded a total of 102 easily scorable polymorphic markers. Jaccard indices were calculated and phylogenetic relationships were determined by neighbor- joining cluster analysis. Different Ocimum species were clearly divided into separate clusters with the exception of O. minimum accessions, which clustered together with O. basilicum accessions. In addition to morphological, chemical and crossability data, RAPD analysis can be a useful tool for resolving existing problems in identification and classification of basils.

Key words: Basil, Ocimum, germ plasm characterization, molecular taxonomy, molecular marker.

Effect of BAP, TDZ and CPPU on Multiple Shoot Formation in Pea (Pisum sativum L.) in Culture In Vitro 

Suzana Tulac, Dunja Leljak-Levanic, Marijana Krsnik-Rasol, and Sibila Jelaska*
Department of Molecular Biology, Faculty of Science, University of Zagreb, 
Rooseveltov trg 6, 10000 Zagreb, Croatia

Received November 19, 2001; revision accepted March 15, 2002

Shoot regeneration in five pea (Pisum sativum L.) cultivars (Atlas, Avola, Karina, Mali provansalac and Tristar) was achieved by direct culture of mature seeds on MSB5 medium supplemented with either N6-benzylaminopurine (BAP), N-phenyl-N’(-1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ) or N-(2-chloro-4-pyridyl)-N’-phenylurea (forchlorfenuron, CPPU). Multiple shoots formed de novo without an intermediary callus phase at the cotyledonary notch, in the axillary meristem regions of the seedlings, and in the hypocotyl subepidermal tissues within two to three weeks of culture initiation. Bud formation began after 5 to 7 days of treatment and the number of buds increased with the duration of culture and increasing concentration of growth regulators. Transient exposure to plant growth regulators (24-28 h) was sufficient to induce bud formation. CPPU was the most effective and BAP the least effective for the induction of regeneration. Separated shoots (1-2 cm) were rooted (60%) on MSB5 medium supplemented with 1.1 uM indole-3-acetic acid (IAA) and 2.0 uM ?-naphthaleneacetic acid (NAA) and developed into flowering plants.

Key words: Pisum sativum, 6-benzylaminopurine, phenylurea derivatives, pea, shoot regeneration.

Antibacterial and Antifungal Activity of Extracts and Quercetagetin Derivate Isolated from Centaurea rupestris L. (Asteraceae)

Gordana Rusak¹*, Neil Robinson², Stjepan Pepeljnjak<sup>3

1</sup>Department of Biology, Faculty of Science,University of Zagreb, Marulićev trg 20/II, Zagreb, Croatia
²Molecular Nature Limited, 12 Crawford Rise, Maidenhead, SL6 7LS, United Kingdom
³Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, A. Kovacica, Zagreb, Croatia

Received December 15, 2001; revision accepted March 31, 2002

The antibacterial and antifungal activity of Centaurea rupestris L. ethanolic and etheric leaf and inflorescence extracts and of quercetagetin 3’-methyl ether 7-O-ß-D glucopyranoside isolated from inflorescences was investigated. Inflorescence extract in 45% ethanol showed significant antifungal activity against the dermatophytes Epidermophyton floccosum, Microsporum gypseum and Trichophyton mentagrophytes (fungicidal zones between 18 and 23 mm, minimum fungicidal concentration 5-7%). The same extract showed weak bactericidal activity against Bacillus anthracis (bactericidal zone 9 mm, minimum bactericidal concentration 90%). In contrast, leaf extract in 90% ethanol possessed significant antibacterial activity against Streptococcus faecalis (bactericidal zone 23 mm, minimum bactericidal concentration 9%). The antimicrobial spectrum of quercetagetin was broad but weak for G- bacteria and moderate for dermatophytes. Namely, it showed antibacterial activity against Klebsiella pneumoniae, Escherichia coli, Salmonella java and Serratia sp. (bactericadal zones 10 mm, minimum bactericidal concentrations 5-6 mg/ml). The fungicidal zones created by quercetagetin against the dermatophytes Epidermophyton floccosum, Trichophyton mentagrophytes and Microsporum gypseum ranged from 14 to 18 mm (minimum fungicidal concentrations 3 mg/ml).

Key words: Centaurea rupestris L., flavonoids, quercetagetin, antibacterial activity, antifungal activity. 

Total Phenolic Variations in Leaves of Some Hieracium L. and Pilosella Hill. Species

Faik Ahmet Ayaz* And Kamil Coskuncelebi

Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey

Received January 20, 2002; revision accepted May 16, 2002

Mature leaves of 20 Hieracium L. and 11 Pilosella Hill. species collected from the alpine and subalpine regions of North East Anatolia (Turkey) were examined for total phenolic content. The highest level was found in leaves of H. conicum (22.5 mg phenol equiv/g dry wt) and the lowest H. cardiophyllum (3.7 mg phenol equiv/g dry wt). Total phenolics content varied significantly (p = 0.05) among species of Hieracium and in decreasing order as follows: H. conicum (22.5) > H. hypoglaucum (21.5) > H. amblylepis (18.1) > H. subsilvularum (17.2) > H. jurassicum (15.1) > H. tamderense (14.8) > H. mannagettae (14.7) > H. cardiophyllum (3.7). In Pilosella the level ranged between 5.6 and 25.5 mg phenol equiv/g dry wt, with the lowest in P. grossheimii and the highest in P. hypeuryum. Total phenolics content varied significantly (p = 0.05) among species of Pilosella, in dereasing order: P. hypeuryum (22.5) > P. tephrocephala (17.9) > P. officinarum (15.7) > P. macranthum (13.7) > P. fennica (10.5) > P. macrotricha (7.7) > P. grosheimii (5.6). Within genera, 40% of the Hieracium species and ~64% of the Pilosella species significantly differed in total phenolic content, while ~19.4% of the species significantly differed between genera. The range of differences in total phenolic content in the genera was not wide, clearly due to their taxonomically close relationships between species.

Key words: Hieracium, Pilosella, phenolics, leaf.

In Vitro Culture Techniques in Conservation ofRubus Chamaemorus L.

Barbara Thiem*

Department of Pharmaceutical Botany
Karol Marcinkowski University of Medical Sciences in Poznań
ul. Św. Marii Magdaleny 14, 61-861 Poznań, Poland

Received February 1, 2002; revision accepted April 23, 2002

The study evaluated two methods of conserving Rubus chamaemorus L. shoot cultures: medium-cold storage and encapsulation of shoot buds. Cold-stored explants were transferred every 12 weeks to proliferation medium and their multiplication rate was observed after the first passage. Shoot cultures stored at 4°C under low light intensities for 12 months without intervening subculture survived with 90% viability. Axillary buds obtained from in vitro shoot culture of cloudberry were encapsulated in calcium alginate hydrogel. Regrowth response of encapsulated buds was estimated. Encapsulated buds stored at low temperature in the dark survived for up to 3 months without loss of viability. The fidelity of subcultured plantlets was also evaluated by phytochemical analysis (fingerprinting) of some phenolic compounds and morphological observations. Preliminary trials in the botanical garden showed that plantlets of Rubus chamaemorus derived in vitro can be used for ex situ germplasm conservation.

Key words: Rubus chamaemorus L., shoot culture, axillary buds, in vitro cold storage, encapsulation, germplasm storage.

Polyploidization in the suspensor of Triglochin palustre L. (Juncaginaceae)

Małgorzata Kozieradzka-Kiszkurno, Joanna Świerczyńska, and Jerzy Bohdanowicz

Department of Genetics and Cytology, University of Gdańsk, ul. Kładki 24, 80-822 Gdańsk, Poland

Received February 19, 2002; revision accepted May 13, 2002

Differentiation of the suspensor basal cell was studied in Triglochin palustre (2n = 24). The zygote divides into the smaller apical cell and the bigger basal cell, which becomes the basal cell of the suspensor. The nuclear DNA content of the suspensor basal cell attains a high degree of ploidy, up to 256C. Nuclei with the highest ploidy levels of 128C and 256C were observed in mature basal cells (from 50 to 500-celled embryos). As a result of polyploidization the volume of the nucleus increased and changes in the chromatin structure of polyploid nuclei were noted. Endochromocenters at middle ploidy level and bundle-like aggregations of chromatin at the highest ploidy levels were found. Rhythmic enlargement of DNA content and nuclear volume of the basal cell, as well as the characteristic structure of its chromatin, point to endoreduplication as the mechanism of polyploidization in the suspensor.

Key words: Triglochin palustre, DNA cytophotometry, endoreduplication, suspensor, basal cell.

Autonomous Endosperm development in Unpollinated Ovaries of Brassica napus L. cv. Topas Cultured In Vitro

Joanna Rojek, Eżbieta Kuta*, and Leslaw Przywara

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

Received January 7, 2002; revision accepted March 27, 2002

Autonomous endosperm (AE) was found in 39 (17.3%) of cultured in vitro unpollinated ovaries of Brassica napus L. cv. Topas, with average frequency 1.5 ovule with AE per ovary. AE was induced on MS medium and on MS + 2 mg/l BAP + 0.1 mg/l NAA, MS + 2 mg/l BAP + 2 mg/l 2,4-D, and in ovules exposed to 1 h auxin treatment (MS + 20 or 40 mg/l 2,4-D) and then transferred to hormone-free MS medium. The highest frequency of AE induction occurred after 1 h auxin treatment (34.5%). The structure and size of AE nuclei suggest their origin from secondary nucleus or polar nucleus/nuclei. The control experiment with unpollinated, bagged flower buds left on plants, revealed no AE induction in situ. Gynogenic embryos were not observed in the material studied.

Key words: Brassica napus L., rape, autonomous endosperm, in vitro culture, unpollinated ovaries, unpollinated ovules. 

Influence of Sugars on Isolated Microspore Development in Maize (Zea mays L.)

Grzegorz Góralski¹*, Claude Lafitte², Lamia Bouazza³, 
Elisabeth Matthys-Rochon³ and Leslaw Przywara<sup>1

1</sup>Department of Plant Cytology and Embryology, Jagiellonian University,
ul. Grodzka 52, 31-044 Cracow, Poland 
²Centre de Biologie et Physiologie Végétales, UMR CNRS, UPS 5546,
Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex 04, France 
³Reproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon,
UMR 5667 CNRS-INRA-UCB Lyon 1, 46 Allée d’Italie, 69364 Lyon cedex 07, France

Received April 5, 2002; revision accepted June 7, 2002

Carbon source and osmotic pressure are known to be of great importance in cultures in vitro. We compared the effectiveness of sucrose, maltose and mixtures of glucose with fructose in androgenic culture of isolated maize microspores. Also we examined changes in media during culture, focusing on osmotic pressure and sugar composition. The results suggest that osmotic pressure and kind of sugar have a great influence on androgenesis induction but much less influence on the number of macroscopic structures formed. In media containing sucrose the osmotic pressure rises significantly due to sucrose hydrolysis. In other media tested, changes in osmotic pressure are much smaller or not significant. These results suggest that the factors involved in androgenesis induction are different from those responsible for macroscopic structure formation. Changes in sugar composition and osmotic pressure increase in sucrose-based medium might decrease the effectiveness of androgenesis in maize microspore culture.

Key words: Androgenesis, maize, Zea mays, isolated microspores, pollen embryogenesis, sugars, osmotic pressure.

In vitro Culture of Immature Zygotic Embryos of White Clover (Trifolium repens L.) 

Wojciech Hałdaś and Lesław Przywara

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

Received April 16, 2002; revision accepted May 16, 2002

On the basis of EC6 medium, specific conditions were established to improve its properties as a nutrient medium for isolated, immature zygotic embryos of white clover. The highest frequency of embryo development occurred on medium with 117 mM sucrose concentration and 30% (v/v) coconut water for globular-stage (23.3%) and heart-stage embryos (78.3%). The development of globular-stage embryos was abnormal, however. The embryos callused after two weeks of culture. From the callus produced, plantlets and then plants were regenerated. A double-layer culture system, with the top layer having higher osmolarity than the bottom layer, enabled proembryos smaller than 60 um to be cultured. For in ovulo embryo culture, Nitsch medium supplemented with 10% (v/v) cucumber juice proved most suitable. On this medium, approximately 13% of the ovules containing few-celled embryos germinated and produced seedlings.

Key words: Trifolium repens, white clover, immature zygotic embryos, in vitro culture.

Ultracytochemical Localization of Calcium in Rice Central Cell Before and After Fertilization

Jun Yang, Jie Zhao, Shi-Ping Liang, and Hong-Yuan Yang*

Key Laboratory of MOE for Plant Developmental Biology, Wuhan University, Wuhan 430072, China

Received April 16, 2002; revision accepted June 18, 2002

Temporal and spatial changes of calcium distribution in rice central cells was studied ultracytochemically by potassium antimonate precipitation. Before anthesis the calcium level was quite low. Calcium increased remarkably just after pollination and then decreased again gradually. Calcium precipitates were localized in the polar nuclei, including the nucleolus and nucleoplasm, and the cytoplasm surrounding polar nuclei. In the large central vacuole the calcium amount was negligible, though pollination-independent calcium elevation occurred in the chalazal region of the vacuole. During fertilization, abundant calcium was found in the fusing sperm and polar nuclei. Fertilization-induced calcium accumulation in the micropylar region of the central vacuole appeared after fertilization and during early mitosis of free endosperm nuclei. No obvious change of calcium level was observed in emasculated but unpollinated central cells, and in this case the level was much lower than in pollinated central cells. Calcium was also localized in the antipodal cells, embryo sac wall and nucellus, and no change was observed in these places before and after pollination.

Key words: Ultracytochemical localization, calcium, central cell, fertilization, rice.

In vitro Storage  of Polypodium vulgare L. Rhizome Shoot Tips Using ABA Treatment Before Dehydration - Encapsulation Technique

Agnieszka Bagniewska-Zadworna* and Elżbieta Zenkteler**

Laboratory of General Botany, Institute of Experimental Biology, 
Adam Mickiewicz University, Al. Niepodległości 14, 61-713 Poznań, Poland

An efficient protocol for storage of Polypodium vulgare rhizome shoot tips was developed. The best result was observed when the rhizomes were pretreated with 2 mg × l-1 abscisic acid/24 h and dehydrated 10 h in hypertonic solution of 20% mannitol before shoot tip isolation. Those explants were encapsulated and stored for 2 months. Plantlets of good quality regenerated after the capsules were transferred to Murashige fern multiplication medium.

Key words: Polypodium vulgare, rhizome shoot tips, medium-term storage, abscisic acid, mannitol dehydration, calcium alginate encapsulation.