ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 77-82, 2005

RAPD Analysis of Genetic Relations Between Büzgülü Grape Cultivars 
(Vitis vinifera) Grown in Different Parts of Turkey

Sümer Aras1*, Janset Burcin Polat1, Demet Cansaran1, and Gökhan Söylemezoglu2

1Department of Biology, University of Ankara, 06100, Turkey
2Department of Horticulture, Faculty of Agriculture, University of Ankara, 06110, Turkey

*e-mail: aras@science.ankara.edu.tr

Received March 7, 2005; revision accepted June 28, 2005

The genetic relations between Büzgülü grape ecotypes (Vitis vinifera L.) grown in different regions of Turkey were investigated by RAPD analysis. Of 50 decamer primers tested, 20 were used in the study, and 17 yielded clear and reproducible polymorphic bands. Differences between ecotypes were analyzed statistically. The similarity index was lowest (0.601) between Etli Büzgülü (collection no. 619, from Antalya province) and Büzgülü (no. 572, Eskişehir prov.), and highest (0.879) between Sik Büzgülü (no. 516, Konya prov.) and Büzgülü (no. 738, Kütahya prov.). The data matrix indicated genetic distances ranging up to 0.038, although most were under 0.03. Phylogenetic trees obtained by UPGMA and neighbor-joining methods basically resembled each other, with some differences. The results from RAPD analysis correlated with some morphological characteristics. This study of Turkish grape germplasm suggests that the geographical and ecological distribution of the plants contributes to higher genetic similarity. 

Key Words: Vitis vinifera L., Büzgülü, polymorphism, RAPD, Turkey.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 33-40, 2005

Morphological and Anatomical Variability of Isozymatically Identified Clones of Pinus mugo Turra

Alina Bączkiewicz* and Wiesław Prus-Głowacki**

Department of Genetics, Adam Mickiewicz University
ul. Międzychodzka 5, 60-371 Poznań, Poland

*e-mail: alinbacz@main.amu.edu.pl
**prusw@main.amu.edu.pl

Received February 15, 2004; revision accepted April 1, 2005

This study examined the anatomical and morphological variability of 10 needle traits in isozymatically identified clones of three peatbog populations of Pinus mugo, focusing on variation within and between clones, and the relation between isozyme variation and morpho-anatomical characters of needles. In each peatbog there were clones exhibiting high and low plasticity of the anatomical and morphological traits studied. In general, three types of variation within clones were distinguished: (1) clones with ramets very similar to each other, (2) clones with extensive intra-clone variability, and (3) clones with intermediate variability. The differences in phenotypic variability within clones may be explained by differences in the reaction norm of ramets in particular clones and by somatic mutations. In respect to anatomical, morphological and isozymatic traits, clones from the same peatbog showed more similarity to each other than to clones from other peatbogs. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 89-96, 2005

Distribution of Microtubules During Regular and Disturbed Microsporogenesis and Pollen Grain Development in Gagea lutea (L.) Ker.-Gaw.

Jerzy Bohdanowicz*1, Ewa Szczuka**2, Joanna Świerczyńska1, Jolanta Sobieska3,
and Maria Kościńska- Pająk4 

1Department of Genetics and Cytology, University of Gdańsk,
 ul. Kładki 24, 80-822 Gdańsk, Poland
2Department of Plant Anatomy and Cytology, Maria-Curie Skłodowska University,
ul. Akademicka 19, 20-033 Lublin, Poland,
3Department of Biology and Horticulture, University College of Arts and Natural Science, 
ul. Krakowska 26, 27-600 Sandomierz, Poland
4Department of Plant Cytology and Embryology, Jagiellonian University,
 ul. Grodzka 52, 31-044 Cracow, Poland

*e-mail: jurboh@biotech.univ.gda.pl
**e-mail: plantrep@biotop.umcs.lublin.pl

Received January 20, 2005; revision accepted June 30, 2005

The microtubular cytoskeleton in dividing microsporocytes and developing pollen grains of Gagea lutea (L.) Ker.-Gaw. (Liliaceae) was investigated with a modified indirect immunofluorescence method. Meiotic and mitotic stages were identified by DAPI staining. The microtubular cytoskeleton was compared in plants originating from natural localities and others grown in the laboratory. In natural conditions, microsporocytes and pollen grains of wild early-spring Gagea lutea plants are subjected to abiotic factors including cold exposure and lack of water. The persistent influence of these factors can disturb microtubular cytoskeleton functioning. The following disturbances were observed in the course of microsporogenesis and pollen development: abnormal chromosome configurations in the metaphase of meiosis I; abnormally divided dyads with irregular, radial microtubule systems around the nuclei; the formation of differently sized microspores with irregular shapes, and irregular division; and the formation of pollen grains with vacuoles abnormal for their development stage. Similar kinds of disturbances were observed after 1.5 months of cold treatment (4C) and drying in the laboratory. These abiotic factors simulated in laboratory conditions caused more disturbances in the course of microsporogenesis and produced more frequent defective pollen grains than in the sample that had experienced cold and drying in natural conditions. 

Key words: Gagea lutea, microtubular cytoskeleton, microsporogenesis, disturbances, abiotic factors. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 153-156, 2005

Antimicrobial Studies on Three Endemic Species of Sideritis From Turkey

Basaran Dulger*, Ahmet Gonuz, and Tulay Bican

Department of Biology, Canakkale Onsekiz Mart University, 17100, Canakkale, Turkey

*e-mail: dbasaran@comu.edu.tr

Received October 5, 2004; revision accepted September 25, 2005

The antimicrobial activity of several extracts and fractions of some Sideritis species (S. albiflora, S. brevibracteata and S. pisidica) was investigated by disc diffusion and broth microdilution methods against Escherichia coli ATCC 11230, Staphylococcus aureus ATCC 6538P, Klebsiella pneumoniae UC57, Micrococcus luteus La 2971, Micrococcus flavus ATCC 14452, Proteus vulgaris ATCC 8427, Pseudomonas aeruginosa ATCC 27853, Corynebacterium xerosis CCM 7064, Mycobacterium smegmatis CCM 2067, Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 9730, Kluyveromyces fragilis NRRL 2415 and Rhodotorula rubra CCY. The methanol extract and chloroform fraction of S. pisidica, as well as the methanol extracts, butanol and chloroform fractions of both S. albiflora and S. brevibracteata showed good antimicrobial activity against some bacteria and all yeasts tested, with growth inhibition area diameters in the range 10-20 mm, and MIC values between 0.03 and 0.38 µl/ml. The results of the study support the use of these species in traditional medicine.

Key Words: Sideritis, antimicrobial, antifungal activity. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 69-75, 2005

Diversity of Agronomic Traits and Total Seed Protein 
in Black Gram  Vigna mungo (L.) Hepper 

Abdul Ghafoor and Zahoor Ahmad

National Institute of Agri Biotechnology and Genetic Resources,
 National Agricultural Research Centre, Islamabad, Pakistan

Received July 29, 2004; revision accepted June 27, 2005

This study evaluated agronomic traits of 111 genotypes of black gram Vigna mungo (L.) Hepper, mainly from Pakistan, to determine the extent of genetic diversity. Seed proteins were analyzed by SDS-PAGE. High genetic variance was observed for days to flowering, days to maturity, number of branches/plant, number of pods/plant, biomass/plant, grain yield/plant and harvest index, whereas low genetic variance was observed for pod length, seeds/pod and 100-seed weight in both years studied. SDS-PAGE of seed proteins showed low inter-accession diversity and no clear differentiation on the basis of origin or source. The genotypes in one cluster with similar agronomic characters did not necessarily belong to the same origin or source. The lack of a geographic pattern of germplasm collected from Pakistan was probably due to exchange of germplasm between neighbouring regions, or because germplasm represented progenies of the same ancestors. Clustering of advanced breeding lines along with an approved variety in one group revealed that only a portion of the genetic diversity has been exploited for improvement of black gram. It is suggested that the genetic base of cultivated black gram should be broadened by involving diverse parents in the breeding programme. Expansion of the genetic base for black gram breeding might be accomplished by systematic use of germplasm that differs in protein profiles and has better quantitative traits. 

Key words: Black gram, Vigna mungo (L.) Hepper, cluster analysis, gel electrophoresis, genetic diversity, SDS-PAGE.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 121-128, 2005

Organogenesis in Endosperm of Actinidia deliciosa cv. Hayward 
Cultured In Vitro 

Grzegorz Góralski1*, Marzena Popielarska1, Halina Ślesak1, Dorota Siwińska2, and Mariola Batycka1

1Department of Plant Cytology and Embryology, Jagiellonian University,
 ul. Grodzka 52, 31-044 Kraków, Poland
2Department of Plant Anatomy and Cytology, University of Silesia, ul. Jagiellonska
26/28, 40-032 Katowice, Poland

*e-mail: g.goralski@iphils.uj.edu.pl

Received April 20, 2005; revision accepted July 25, 2005

An efficient procedure for shoot regeneration was obtained by endosperm culture in Actinidia deliciosa cv. Hayward. Mature endosperm cultured on MS medium supplemented with 2 mg/l 2,4-D and 5 mg/l kinetin developed callus with 80% efficiency. Callus was transferred on MS medium containing different plant growth regulators (2,4-D, TDZ, IAA, NAA, BAP, kinetin, 2iP) for regeneration. There were significant differences in regeneration response between medium supplemented with TDZ and medium with other hormones. Only medium containing TDZ stimulated shoot induction. The highest efficiency of shoot regeneration (avg. 6.2 shoots per culture) was on medium supplemented with 0.5 mg/l TDZ. The ploidy of callus and organs formed in endosperm culture was examined by flow cytometry. The results, peaks corresponding to 3C DNA amounts, confirmed the endospermal origin of callus, roots and shoots. Aneuploid and polyploid cells were found in endosperm-derived callus and regenerated organs. 

Key words: Actinidia, endosperm culture, regeneration, tissue culture, ploidy. 

Abbreviations: MS – Murashige and Skoog (1962) medium; 2,4-D – 2,4-Dichlorophenoxyacetic acid; TDZ – 1-Phenyl-3-(1, 2, 3- thiadiazol-5-yl) urea; IAA – Indole-3-acetic acid; IBA – 3-indolebutyric acid, NAA – alpha-Naphthaleneacetic acid; BAP – 6-Benzylaminopurine; 2iP – 6-(gamma, gamma-dimethylallylamino) purine. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 61-67, 2005

Nuclear ribosomal DNA ITS Paralogs as Evidence of Recent Interspecific Hybridization in the Genus Ophrys (Orchidaceae)

Gergely Gulyás*1, Gábor Sramkó1, Attila V. Molnár1, Szabolcs Rudnóy2, 
Zoltán Illyés2, Tímea Balázs2, and Zoltán Bratek2

1Department of Botany, University of Debrecen, H-4010, P. O. Box 14, Debrecen, Hungary
2Department of Plant Physiology, Eötvös Loránd University, 
H-1117, Pázmány Péter str. 1/C, Budapest, Hungary

*e-mail: compo2@freemail.hu

+Present address: Biological Research Center, Hungarian Academy of Sciences; Institute of Genetics, Molecular Biodiversity Group; H-6726 Szeged, Temesvári krt. 62.

Received March 29, 2005; revision accepted June 16, 2005

Ophrys holubyana Andrasovszky (Orchidaceae), distributed in the Carpatho-Pannonian region, is generally believed to be of hybrid origin. Although its hybrid origin is broadly accepted by many authors, no molecular evidence has been found to support the hypothesis. The nuclear ribosomal DNA internal transcribed spacer (nrDNA ITS) region was sequenced from Ophrys holubyana, and from the presumed progenitor taxa: Ophrys fuciflora (Cr.) Rchb. and Ophrys bicornis Sadler ex Nendtvich. Nearly all the known populations in the Carpatho-Pannonian region were sampled, and the first data on the nrDNA ITS sequence of O. holubyana and O. bicornis are presented. Aligning the ITS sequences revealed no differences among the ten samples. After cloning the amplified ITS regions, eight discrete ITS paralogs with regularly appearing nucleotide differences could be partitioned, differing in only 6 base pairs. 
	Paralog sequences were detected not only in O. holubyana but also in some populations of the two parent species, suggesting that O. fuciflora and O. bicornis in the Carpatho-Pannonian region are also partially of hybrid origin themselves, or influenced by introgression. The study suggests that nrITS regions can be generally useful in the study of Ophrys systematics and phylogeography and for analyzing the hybrid zones of related Ophrys species groups. 

Key words: Ophrys, Carpatho-Pannonian region, cloning, sequencing, hybridization, hybrid speciation, nrITS paralogs.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 41-52, 2005

Development of Flower Organs in Common Lilac 
(Syringa vulgaris L.) cv. Mme Florent Stepman

Agata Jędrzejuk and Władysław Szlachetka

Department of Ornamental Plants, Warsaw Agricultural University,
ul. Nowoursynowska 166, 02-787 Warsaw, Poland

Received March 5, 2004; revision accepted May 30, 2005

Studies on the development of common lilac cv. Mme Florent Stepman inflorescence buds and flowers were carried out in 2001–2002 in order to observe the development of flower organs before and after winter dormancy during the following phenological phases: inflorescence bud swelling, inflorescence elongation, flower bud whitening, flower bud swelling and flowering anthesis.
	The hypogynous, actinomorphic and perfect flower conforms to the general pattern of the Oleaceae. The calyx is four-lobed, and the four-lobed corolla is gamopetalous with a cylindrical basal tube. Two stamens are basally fused with the corolla. The anthers are tetrasporangiate, with a uniseriate epidermis, bilayered endothecium, single middle layer, and secretory, binucleate tapetum. Microspore tetrads are isobilateral and the pollen grains are spherical, three-colpate and two-celled. The ovary is superior and bilocular. Four anatropous, unitegmic and tenuinucellar ovules are formed. The female gametophyte development is Polygonum-type. Flower primordia differentiation starts in midsummer and lasts until autumn. Before entering dormancy, microspore meiosis was discernible in flowers in the lower part of the inflorescence. The ovular primordia differentiate after winter dormancy. During inflorescence bud breaking after winter dormancy, pollen mother cells in anthers and ovule primordia were observed. During inflorescence elongation, pollen mother cell meiosis and microspore tetrads occured in anthers, and the archesporial cell of the megaspores was present in ovules. During flower bud whitening and flower bud breaking, young microspores were observed in anthers, and developing megaspores were visible in ovules. In the last phenological phase, anthers dehisced and the female gametophyte was organized. 

Key words: Syringa vulgaris L., inflorescence, flower bud, ovule, female gametophyte, pollen grains. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 97-110, 2005

Pollen Morphology and Two-Dimensional Patterns of Pollen Coat and Protoplast Proteins in Aegilops kotschyi x Secale cereale Amphiploids

Andrzej Kalinowski1*, Małgorzata Klimko2 and Barbara Wojciechowska1

*1 Institute of Plant Genetics, Polish Academy of Sciences, 
ul. Strzeszyńska 34, 60–479 Poznań, Poland
2 Department of Botany, August Cieszkowski Agricultural University,

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

Received November 7, 2004; revision accepted June 30, 2005

Amphiploid pollen of Aegilops kotschyi x Secale cereale was compared to the parental form by SEM, and comparatively measured using light microscopy.Amphiploid pollen of Aegilops kotschyi x Secale cereale was compared to the parental form by SEM, and comparatively measured using light microscopy.Amphiploid pollen of Aegilops kotschyi x Secale cereale was compared to the parental form by SEM, and comparatively measured using light microscopy.Amphiploid pollen of Aegilops kotschyi x Secale cereale was compared to the parental form by SEM, and comparatively measured using light microscopy. Pollen grains of amphiploids were larger and more variable in total and pore diameter than the parents. Amphiploid pollen was prolate, subprolate and prolate-spheroidal in shape. The exine of Ae. kotschyi AK-2 and AK-3 had a delicate verrucate surface, whereas Secale cereale S14 exine had a verrucate surface. Amphiploid pollen grain surfaces were more or less similar to those of the parents: delicate verrucate, verrucate and well verrucate. The sculpture of parental and amphiploid pollen grains showed conspicuous granulation. All amphiploids produced pollen with one pore with an operculum, surrounded by a well-defined annulus.
	Sporophytically produced peptides from the pollen coat and gametophytically produced peptides from the protoplast were analyzed separately by two-dimensional gel electrophoresis. Two accessions of Ae. kotschyi (AK-2, AK-3) showed differences in the 2-D patterns of peptides from both the pollen coat and the protoplast. The majority of pollen coat and protoplast peptides of the parents were detected in the amphiploids, but a number of parental peptides were absent. All the amphiploids possessed peptides in their pollen coat and protoplast not detected in the corresponding pollen fractions of the parents. No relation between colchicine and in vitro amphiploid production and its 2-D patterns was observed. The results of pollen morphology and pollen protein analysis are convergent.

Key words: Amphiploids, Aegilops kotschyi, Secale cereale, pollen morphology, protein, pollen coat, pollen protoplast.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2

Molecular evidence of natural hybridization between  Pinus mugo
and P. sylvestris as revealed by a chloroplast DNA marker

Andrej Kormuťák1,  Božena Vooková1, and Monika Fečková2

1Institute of Plant Genetics and Biotechnology Slovak Academy of Sciences, Akademická 2,  
 P.O.Box 39A, SK-950 07 Nitra, Slovakia; nrgrkorm@savba.sk
2State Veterinary Institute, Akademická 3, SK-949 01 Nitra, Slovakia

The existence of spontaneous hybrids between Pinus sylvestris L. and P. mugo Turra has been postulated since the second half of the nineteenth century (Christ, 1864). The most recognized places in Europe where hybrid swarm populations of P. mugo x P. sylvestris have been reported to occur are Rila Planina and Rodopy in Bulgaria (Dobrinov, 1965),  Nowotarska Valley in Poland (Staszkiewicz and Tyszkiewicz, 1969), Swiss Alps (Net-Sarqueda et al., 1988) and Orava region in northern Slovakia  (Viewegh, 1981).
Molecular evidence has been provided illustrating continuation of introgressive hybridization between P. mugo and P. sylvestris on the locality „Medzi Borami“ near Habovka (830 m a.s.l.), northern Slovakia where both species occur sympatrically.
Molecular evidence for introgression was based on a previous finding of the species-specific Hinf  I restriction profiles of cpDNA trnV-trnH region in P. sylvestris and P. mugo (Kormuťák et al., 2002) as well as on the original finding of the paternal inheritance of cpDNA in conifers (Wagner et al., 1987). Simultaneous analysis of the respective cpDNA region using needles of a given tree along with the megagametophytes and embryos of individual seeds has accordingly enabled to score either the P. mugo or P. sylvestris haplotypes in the embryos illustrating gene flow between both species. The P. mugo  haplotype consisted of 750, 650, 320, 300 and 190 bp fragments and essentially similar profile was also characteristic for P. sylvestris hyplotype except for the 320 bp fragment which was replaced in the species by the 350 bp fragment. Restriction profiles of needles and megagametophytes of a given tree were identical, whereas the embryos exhibited either the intra- or interspecific nature of their haplotypes.    Of the total number od 35 individuals of the putative hybrid population, 18 trees exhibited P. mugo haplotype and 17 trees P. sylvestris haplotype as evidenced by their needle and megagametophyte restriction profiles. In a group of 18 trees of P. mugo haplotype, 22 embryos shared the same haplotype as mother tree and 15 embryos were of P. sylvestris haplotype representing interspecific hybrids of P. mugo x P. sylvestris. Similar situation has also been found within a group of 17 trees of P. sylvestris haplotype with 17 embryos of P. sylvestris haplotype and 19 embryos of hybrid nature representing reciprocal hybrid P. sylvestris x P. mugo. Introgression between P. mugo and P. sylvestris trees seems to be rather extensive on the locality as evidenced by the 40% share of the hybrid embryos of P. mugo x P. sylvestris among 18 trees of P. mugo haplotype and 52% proportion of the hybrid embryos of reciprocal combination among 17 trees of P. sylvestris haplotype.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 7-13, 2005

Relations between Betula pendula Roth (Betulaceae) Leaf Morphology and Environmental Factors in Five Regions of Croatia

Sanja Kovačić* and Toni Nikolić

University of Zagreb, Faculty of Science, Botanical Department with Botanical Garden, 
Marulićev trg 9, 10000 Zagreb, Croatia

*e-mail: sanja@botanic.hr

Received December 4, 2003; revision accepted March 3, 2005

In this research we used numerical analysis of environmental and morphometric data sets to study the possibility of environmental control of leaf morphology in Betula pendula stands from five regions of Croatia. The correlations were poor between leaf variables and the ecological gradients of the stands, as well as between the ecological, vegetational and biometric characteristics of the two morphologically most similar populations. This being so, we viewed the populations in the historical context. As two morphologically closest B. pendula populations grow in disjoined natural marshland vegetation with different environmental features, their similarity might be found in the genetic background, which should be further investigated. Possibly, at least in these two areas of its Croatian distribution range, B. pendula grows in its primary habitats from preglacial times, and is of older (relict) origin than in the rest of its recent Croatian (secondary) area of distribution, where it recolonized after the Ice Ages. 

Key words: Betula pendula, morphometry, leaf morphology, ecology, clinal variability, Croatia.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 23-26, 2005

Breaking Seed Dormancy in Black Mulberry (Morus nigra L.) by Cold Stratification and Exogenous Application of Gibberellic Acid 

Fatma Koyuncu*

Department of Horticulture, Süleyman Demirel University, 32260 Isparta, Turkey

*e-mail: fkoyuncu@ziraat.sdu.edu.tr

Received October 10, 2004; revision accepted March 30, 2005

Experiments were done to determine the effects of cold stratification (0, 20, 40, 60, 80 or 100 days), application of gibberellic acid (GA3) (0, 250, 500, 1000 or 2000 mg/l) and the combination (GA3 + stratification) on seed germination of black mulberry. Application of 1000 mg/l GA3 proved more effective than any of the other concentrations of GA3 applied. Seeds stratified for 100 days showed 88% germination. The combined treatment of 250 mg/l GA3 and 100 days of stratification yielded 96% germination of seeds. The relationships between GA3 concentration and seed germination (r = 0.93), and between stratification duration and seed germination (r = 0.91) of black mulberry were linear.

Key words: Dormancy, black mulberry, germination, gibberellic acid, stratification.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 137-144, 2005

Individual Differentiation of Abies alba Mill. Population From the Tisovik Reserve. Part I. Variability Expressed in Morphology and Anatomy of Needles 

Ewa M. Pawlaczyk1*, Jacek Grzebyta2, Maria A. Bobowicz1**,
and Adolf F. Korczyk3***

1Department of Genetics, Adam Mickiewicz University, 
ul. Międzychodzka 5, 60-371 Poznań, Poland
2Department of Genetics, University of Warmia and Mazury, 
ul. Plac Łódzki 3, 10-967 Olsztyn, Poland
3 Natural Forest Department, Forest Research Institute, 
Park Dyrekcyjny 6, 17-230 Białowieża, Poland

*e-mail: ewapaw@amu.edu.pl
** mabwa@amu.edu.pl
***e-mail: akorczyk@las.ibl.bialowieza.pl

Received May 2, 2005; revision accepted July 30, 2005

One-year-old needles from 20 silver fir trees in the Tisovik Reserve (Belarus) were characterized in respect to 12 morphological and anatomical traits, and the data were analyzed statistically to determine variability between trees. Individuals within the population generally were homogenous for those traits. Needle length was the most variable trait within the studied population. The width/height ratio of the hypodermic cell exerted the weakest differentiating effect between trees. The results that the Tisovik Reserve population is weakly differentiated. 

Key words: European silver fir, variability, needle, multivariate statistical analysis.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 53-59, 2005

Clonal Structure of Small Isolated Populations of Pinus mugo Turra from Peatbogs in the Tatra Mts

Wiesław Prus-Głowacki*, Alina Bączkiewicz**, and Dorota Wysocka

Department of Genetics, Adam Mickiewicz University, 
ul. Międzychodzka 5, 60-371 Poznań

*e-mail: prusw@main.amu.edu.pl
**alinbacz@main.amu.edu.pl 

Received February 15, 2004; revision accepted June 2, 2005

This study examined genetic variability at 11 allozyme loci and the clonal structure of peatbog populations of dwarf mountain pine (Pinus mugo Turra) from three small isolated stands in the Tatra Mts, Poland. The percentage of clones in the peatbog populations was high, 48.45% on average. Genetic variability was lower in these populations than in populations from the continuous range of the species. The mean number of alleles per locus (A) was 2.2. Estimates of total (HT = 0.231) and within-population (HS = 0.224) genetic diversity were very similar. Differentiation between populations was low (DST = 0.7%; GST = 2.1%;). Gene flow (Nm = 8.11) was also relatively low because of their geographic isolation. Wright's fixation index (F) for all examined enzymatic loci demonstrated that the populations were in Hardy-Weinberg equilibrium.

Key words: Pinus mugo Turra, genetic variability, isozymes, clones, isolated populations, gene flow.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 145-151, 2005

Influence of Cultivar, Explant Source and Plant Growth Regulator on Callus Induction and Plant Regeneration of Cannabis Sativa L.

Aurelia Ślusarkiewicz-Jarzina*, Aleksandra Ponitka, Zygmunt Kaczmarek

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

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

Received March 28, 2005; revision accepted August 10, 2005

The effects of different combinations of plant growth regulators (PGRs) on callus induction and plant regeneration were investigated in five cultivars of Cannabis sativa L. Callus was induced from different explant sources (young leaves, petioles, internodes, axillary buds) on MS basal medium with various concentrations of PGRs (2,4-D, DICAMBA, KIN, NAA). The highest frequency of callus induction (avg. 82.7% of eight medium combinations) was exhibited by petiole explants of cv. Fibrimon-24. Plant regeneration was obtained from all studied cultivars. The highest number of plants was regenerated from callus tissue of petiole explants on MS medium containing DICAMBA. A total of 46 plants (1.35% of callus) were regenerated: 16 (0.47%) from cv. Silesia petioles, 7 (0.20%) from cv. Novosadska petioles, 6 (0.18%) from cv. Fedrina 74 petioles, 12 (0.35%) from cv. Fibrimon 24 axillary buds, and 5 (0.15%) from cv. Juso 15 internodes. Significant improvement of hemp plant regeneration in vitro was achieved.

Key words: Cannabis sativa, axillary buds, callus induction, plant growth regulators, internode, leaf, petiole, plant regeneration. 

Abbreviations: 2,4-D – dichlorophenoxyacetic acid; DICAMBA – 3,6-dichloro-o-anisic acid; KIN – 6-furfurylaminopurine; NAA – 1-naphthaleneacetic acid. Abbreviations: 2,4-D – dichlorophenoxyacetic acid; DICAMBA – 3,6-dichloro-o-anisic acid; KIN – 6-furfurylaminopurine; NAA – 1-naphthaleneacetic acid.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 111-119, 2005

Ultrastructure of the Nectary Spur of Platanthera chlorantha (Custer) Rchb. (Orchidaceae) During Successive Stages of Nectar Secretion

Malgorzata Stpiczyńska1*, Claudio Milanesi2, Claudia Faleri2, and Mauro Cresti2 

1Department of Botany, University of Agriculture, 
ul. Akademicka 15, 20–905 Lublin, Poland
2 Department of Environmental Science “G. Sarfatti”, Siena University, 
via P.A. Mattioli 4, 53 100 Siena, Italy 

*e-mail: malgorzata.stpiczynska@ar.lublin.pl

Received March 24, 2005; revision accepted: July 5, 2005

In Platanthera chlorantha, nectar is secreted into the lumen of the nectary spur by numerous unicellular hairs. These hairs arise from epidermal cells lining the spur. The nectary was studied in the presecretory (closed bud, 4 days before anthesis), secretory (day 2 of anthesis) and resorption (days  14–16 of anthesis) stages to compare the ultrastructure of the cells during various stages of nectary activity. Plant tissue was fixed for conventional TEM or prepared by freeze-substitution. Secretory cell ultrastructure was observed to change significantly during the various stages of nectary activity. During the presecretory stage, the cells have a large nucleus, dense, granular cytoplasm with numerous mitochondria and ER, and several small vacuoles. Plastids within secretory epidermal cells or subepidermal parenchyma enclose starch, but starch is absent throughout nectar secretion and resorption. During the secretory stage, plastids with a dense stroma contain tubules enclosing osmiophilic material. Abundant dictyosomes and secretory vesicles occur in the cytoplasm, particularly near the plasmalemma, indicating that granulocrine secretion operates in this species. During the resorption stage, most cells still have dense cytoplasm with numerous mitochondria and ER, but dictyosomes are uncommon. Vesicles are present adjacent to the cell wall; they are similar in size and occur in similar numbers to those seen during the secretory stage. They are probably formed by endocytosis. The ultrastructure of the cells prepared by freeze-substitution compared favorably with that seen in cells subjected to conventional chemical fixation.

Key words: Platanthera chlorantha, Orchidaceae nectary, nectar secretion, nectar resorption, ultrastructure, freeze-substitution. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 129-135, 2005

Variability of Christmas Rose (Helleborus niger L.) Populations 
and Its Potential Use in Genetic Breeding

Andrej Šušek1,2+*, Anton Ivančič1, Marie-Claude Lemoine2, Jean Philippe Guillemin3, Jacques Caneill3, Metka Šiško1, and Franc Janžekovič4, 
And Lidija Praprotnik1

1Faculty of Agriculture, University of Maribor, Vrbanska 30, 2000 Maribor, Slovenia 
2INRA/Agri Obtentions Unité mixte de Prédévelopement in vitro, INRA, 
17 rue Sully, B. P. 86510, 21065 Dijon Cedex, France
3ENESAD, UMR BGA ENESAD/INRA/University of Burgundy, BGA, 
26 Bd Docteur Petitjean, BP 87999, 21000 Dijon, France
4Department of Biology, University of Maribor, 
Koroška 160, 2000 Maribor, Slovenia 

+Present address: University of Maribor, Faculty of Agriculture, Vrbanska 30, 2000 Maribor, Slovenia. 
*e-mail: andrej.susek@uni-mb.si

Received December 30, 2004; revision accepted  July 25, 2005

The Christmas rose (Helleborus niger L.) is widely distributed in Southern and Central Europe, and Slovenia is probably one of the centers of its diversity. The investigation was aimed at evaluating phenotypic diversity among selected wild populations in Slovenia and neighboring Croatia. Some of the analyzed traits expressed relatively very high variation (e.g., number of leaves per plant, number of inflorescences per plant). The variation ranges of some of the analyzed traits were found to be wider than those mentioned in the literature. Statistical analysis indicated that variability was more expressed within than between populations. In terms of present market requirements, the most valuable populations are Žumberak (Croatia), containing plant materials with the longest peduncles and largest flowers, and Peca, containing genotypes with the highest number of inflorescences per plant.

Key words: Helleborus niger, Christmas rose, geographical distribution, phenotypic variation, population diversity. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 15-22, 2005

Structure of Trichomes from the Surface of Leaves of Some Species 
of Kalanchoë Adans.

Elżbieta Weryszko-Chmielewska* and Mykhaylo Chernetskyy** 

Department of Botany, Agricultural University, ul. Akademicka 15, 20-950 Lublin, Poland

e-mail: *ewbot@agros.ar.lublin.pl
**mczerneckyj@yahoo.com

Received June 10, 2004; revision accepted March 29, 2005

The paper presents the results of studies on the structure of non-glandular and glandular trichomes occurring on the surface of leaves of eight species of the genus Kalanchoë Adans. (Crassulaceae DC.). Stereoscopic, light and scanning electron microscopy were used to observe the length and number of trichomes on a 1 mm˛ area of both surfaces of the leaf blade, the shape and number of their cells, the content of anthocyanins or tannins in the cells, cuticle sculpture, the presence of surface waxes, and protuberances of the cell walls of the mentioned structures. Considerable differentiation of trichome structure was observed. Trichomes were divided into eight types. Four were for non-glandular trichomes: bushy three-branched type (K. beharensis Drake et Castillo, K. millotii Hamet et H. Perr., K. tomentosa Bak), peltate stellate type (K. hildebrandtii Baill.), peltate sagittal-hastate type (K. hildebrandtii, K. orgyalis Bak.), and peltate rhomboid type (K. rhombopilosa Mann. et Boit.). The remaining four were for glandular trichomes: clavate type (K. gastonis-bonnieri R. Hamet et H. Perr.), capitate type (K. beharensis, K. manginii R. Hamet et H. Perr., K. millotii), peltate type (K. millotii) and truncate type (K. orgyalis). The presence of different types of dead non-glandular and glandular trichomes on the surface of the leaves, the morphology of which was studied in the present work, may be considered an important adaptation to the conditions of the natural environment.

Key words: Kalanchoë species, leaf, trichomes, structure, adaptation.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 27-32, 2005

Efficient In Vitro Plant Regeneration of Pinellia ternata (Thunb) Breit

Tiefeng Xu1, Lei Zhang2, Xiaofen Sun2 and Kexuan Tang1,2,*

1Plant Biotechnology Research Center, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, School of Agriculture and Biology, Shanghai Jiao Tong University, 
Shanghai 200030, China 
2State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan-SJTU-Nottingham Plant Biotechnology R&D Center, Morgan-Tan International Center for Life Sciences, Fudan University, Shanghai 200433, China 

e-mail: kxtang1@yahoo.com or kxtang1@163.com )

Received November 10, 2004; revision accepted March 30, 2005

An efficient plant regeneration system for Pinellia ternata (Thunb) Breit, an important wild Chinese herb, was established. The investigated factors influencing callus induction and plant regeneration included sterilizing agents, explant type, culture medium, and combinations of plant growth regulators in the medium. Ten min treatment of explants with 0.1% HgCl2 was more suitable for sterilization of tuber explants than NaClO and H2O2. Tubers as explants were superior to leaves and petioles, and MS medium was better than N6 and White’s media. It was found that 9.1 µM 2,4-dichlorophenoxyacetic acid (2,4-D) plus 4.4 µM 6-benzyladenine (BA) in the medium was the best hormone combination for promoting callus induction. The highest shoot regeneration frequency (99%) was achieved from callus cultured on MS medium supplemented with 9.3 µM kinetin (KN) and 2.3 µM 2,4-D. The rooted shoots were successfully transplanted in soil, with over 95% survival. Establishment of an efficient plant regeneration procedure provides a basis for rapid in vitro propagation of P. ternata, which is in great demand in China, and represents an excellent tool for further work on its genetic manipulation.

Key words: Pinellia ternata, in vitro culture, callus, plant growth regulators, regeneration, tuber explants.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 157-164, 2005

Effects of Some Heavy Metalson Content of Chlorophyll, Proline and Some Antioxidant Chemicals in Bean (Phaseolus vulgaris L.) Seedlings

Fikriye Kirbag Zengin*, Omer Munzuroglu

Department of Biology, Faculty of Science, Firat University, Elazig 23119, Turkey

e-mail: fzengin@firat.edu.tr

Received January 6, 2005; revision accepted September 30, 2005

The effects of lead, copper, cadmium and mercury on the content of chlorophyll, proline, retinol, α-tocopherol and ascorbic acid were investigated in 17-day-old bean seedlings (Phaseolus vulgaris L.) grown in Hoagland solution spiked with various concentrations of Pb, Cu, Cd and Hg. Control and heavy metal-treated plants were grown for 10 days in Hoagland solution. Content of total chlorophyll (a and b), proline, retinol, -tocopherol and ascorbic acid was measured in ten-day-old primary leaves by High Performance Liquid Chromatography (HPLC). Total chlorophyll content declined progressively with increasing concentrations of heavy metals. A significant increase of proline, β-carotene, α-tocopherol and ascorbic acid content was detected in in primary leaves after ten-day exposure to heavy metals. The strongest effect on total chlorophyll, proline, retinol, α-tocopherol and ascorbic acid content was found in plants exposed to mercury, followed by the sequence Cd++ > Cu++ > Pb++.

Key words: Chlorophyll, heavy metal, proline, retinol, α-tocopherol, ascorbic acid.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/2: 83-87, 2005

Micropropagation of Rhodiola Kirilowii Plants Using Encapsulated 
Axillary Buds and Callus

Monika Zych1* Mirosława Furmanowa1, Anna Krajewska-Patan2, Anna Łowicka2, Mariola Dreger2, and Sylwia Mendlewska2 

1Department of Biology and Pharmaceutical Botany, Medical University of Warsaw,
ul. Banacha 1, 02-097 Warsaw, Poland
2 Research Institute of Medicinal Plants, ul. Libelta 27, 61-707, Poznań, Poland

*e-mail: zych_monika@yahoo.de

Received March 5, 2005; revision accepted June 29, 2005

This study assessed regrowth of micropropagated Rhodiola Kirilowii buds after encapsulation of axillary buds and differentiating callus in calcium alginate hydrogel and low-temperature preservation. This method of micropropagation was applied to obtain enough plant material for studies on chemical compounds of Rhodiola Kirilowii, a plant difficult to obtain from its natural environment. Axillary buds and differentiating callus were encapsulated in calcium alginate and stored at 4C for 1 to 15 weeks and then transferred to hormone-free MS medium. The best results were obtained after six weeks of preservation: 100% of the encapsulated explants survived and developed into shoots and plantlets after subculture on basal MS medium. Longer storage of encapsulated axillary shoot buds decreased their regrowth, but the duration of storage of encapsulated differentiating callus had no significant influence on its survival.

Key words: Rhodiola Kirilowii, axillary buds, differentiating callus, encapsulation, cold storage, plant regeneration.

Abbreviations: MS - Murashige and Skoog (1962) medium; MS/2 - Murashige and Skoog medium with half amounts of all components; BA - benzyl adenine; IAA - indole-3-acetic acid; SA - sodium alginate; CA - calcium chloride; NAA - alpha-naphtaleneacetic acid; AC - adenine chloride.