ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 17-22, 2005

Major Proteins in Plant and Animal EggsMajor Proteins in Plant and Animal Eggs*

Takashi Okamoto1 and Erhard Kranz2

1Department of Biological Sciences, Tokyo Metropolitan University,
Minami-osawa 1-1, Hachioji, 192-0397 Japan
2Biozentrum Klein Flottbek und Botanischer Garten, Entwicklungsbiologie und Biotechnologie, Universität Hamburg, Ohnhorststr 18, 22609 Hamburg, Germany

Received December 14, 2004: revision accepted May 13, 2005

In most higher plants, the female gametophyte is deeply embedded in the ovule. In an earlier work we isolated egg cells from maize ovule tissues and analyzed egg cell lysates by polyacrylamide gel electrophoresis and mass spectrometry-based proteomics technology, and identified the major protein components expressed in these cells. The identified proteins included three cytosolic enzymes for the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, triosephosphate isomerase), two mitochondrial proteins (ATP synthase β-subunit and adenine nucleotide transporter), and annexin p35. Our data indicate that the plant egg cell is rich in an enzyme subset for energy metabolism. This article provides a short overview of major proteins in animal eggs, reports on major protein components of maize egg cells, and compares the major proteins between animal and plant eggs.

Key words: Fertilization, egg cell, zygote, protein, polyacrylamide gel electrophoresis, mass spectrometry-based proteomics, maize.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1:23-29, 2005

Secreted Molecules and their Role in Embryo Formation in Plants: 
a Mini-ReviewSecreted Molecules and their Role in Embryo Formation in Plants: a Mini-Review

Elisabeth Matthys-Rochon

Reproduction et développement des plantes (RDP), UMR 5667 CNRS/INRA/ENS/LYON1, 46 Allée d’Italie, 69364 Lyon Cedex 07, France

e-mail: elisabeth.Matthys-Rochon@ens-lyon.fr

Received January 15, 2005; revision accepted April 2, 2005

This short review emphasizes the importance of secreted molecules (peptides, proteins, arabinogalactan proteins, PR proteins, oligosaccharides) produced by cells and multicellular structures in culture media. Several of these molecules have also been identified in planta within the micro-environment in which the embryo and endosperm develop. Questions are raised about the parallel between in vitro systems (somatic and androgenetic) and in planta zygotic development. A view of exchanges between embryonic and nonembryonic multicellular structures in vitro is presented, and several facts about embryo and endosperm molecular interactions in planta are reported. Analysis of in vitro mechanisms may help in understanding what happens during zygotic embryogenesis.

Key words: Secreted stimulating factors, signaling molecules, tissue culture, zygotic, somatic, androgenetic embryogenesis, endosperm-embryo interrelations. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1:31-36, 2005

Role of Non-Zygotic Parental Genes in Embryogenesis and
Endosperm Development in Flowering PlantsEndosperm Development in Flowering Plants

Val Raghavan*Val Raghavan*

Department of Plant Cellular and Molecular Biology,The Ohio State University,
318 West 12th Avenue, Columbus, Ohio 43210, U.S.A.

*e-mail: raghavan.1@osu.edu 

Received October 15, 2004; accepted March 31, 2005Received October 15, 2004; accepted March 31, 2005

After a prolonged period of uncertainty about the precise role of maternal genes in initiating embryogenesis in flowering plants, considerable evidence for the involvement of maternal genes in embryo and endosperm development in Arabidopsis thaliana has accumulated in recent years. Much attention has centered on a group of mutants known as fis, which display an ability to initiate partial embryogenesis and endosperm development in the absence of double fertilization. This article presents a brief overview of our current understanding of the role of non-zygotic parental genes in the development of these products of double fertilization in A. thaliana. Evidence shows that the expression of paternal alleles of some genes is frequently delayed during embryogenesis and endosperm development, and that the silencing occurs at the transcriptional level by genomic imprinting.

Key words: Arabidopsis thaliana, embryogenesis, endosperm development, fis class mutants, genomic imprinting.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1:37-49, 2005

Epigenetic Aspects of Sexual and Asexual Seed Development

Julio C.M. Rodrigues1,2,3 and Anna M.G. Koltunow2

1Discipline of Wine and Horticulture. Waite Campus, University of Adelaide, 
Glen Osmond, South Australia 5064, Australia
2Commonwealth Scientific and Industrial Organisation Plant Industry, 
Horticultural Research, Glen Osmond, South Australia 5064, Australia
3Embrapa Genetic Resources and Biotechnology, 70770-900, Brasilia, Brazil

e-mail: Anna.Koltunow@csiro.au

Received February 1, 2005; revision accepted April 25, 2005

In angiosperms, seed development initiates after a double fertilization event in the female gametophyte, in which one male sperm cell fuses to the central cell to form the endosperm and the other to the egg cell to form the embryo. Sexually-derived seed is thus characterized by maternal and paternal contributions to the progeny. Some plant species have the capacity to form seeds asexually, a process known as apomixis. This mode of reproduction is characterized by a bypass of meiotic reduction and the absence of paternal contribution to the embryo, resulting in a seed with an embryo genetically identical to the mother. Little is known about the molecular events that regulate apomictic development. Recent findings show that the apomictic and sexual developmental programs share molecular components, suggesting that apomixis is a deregulated sexual program. Furthermore, the identification of apomictic developmental features in fertilization-independent seed (fis) mutants in the sexual model plant Arabidopsis has also shed light on the molecular events that control sexual seed development, and has opened new questions as to the molecular nature of autonomous seed development. FIS-class genes are homologues of the Polycomb Group (PcG) chromatin remodelling factors conserved in Drosophila and humans, where they have been implicated in gene repression and control of cell fate throughout development. fis phenotypes are affected by DNA methylation, a DNA alteration associated with heterochromatin formation and gene silencing. Thus, the chromatin environment can be manipulated to make certain regions of the genome more or less susceptible to transcription; this form of control, in which gene expression patterns are altered without a change in the DNA sequence itself, is defined as epigenetic regulation. Different aspects of plant development have been shown to be controlled by epigenetic regulation. This review will highlight recent advances in understanding the epigenetic control of seed development. They are discussed in light of a model whereby altered epigenetic mechanisms might lead to complete maternal control of reproductive development as seen in apomixis.

Key words: Apomixis, epigenetics, seed development.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 51-60, 2005

Sexual and Asexual Processes in Reproductive Systems of Flowering Plants

Tatyana Batygina

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

Received December 17, 2004; revision accepted March 30, 2005

In understanding ontogenesis it is important to elaborate some aspects of the theory of flowering plant reproduction. As a result of critical analysis of the literature and original data, the unification of some concepts and terms is proposed. The terms “sexual” and “asexual” in reference to flowering plants should be used to characterize only the mode of formation of new individuals (with or without the participation of meiosis and gamete fusion). The concept of “multiplication” is defined; it should denote an increase of the number of descendants, whereas the formation of a new individual, regardless of mode, is the meaning of the term “reproduction.” The various modes of formation of the individual (sexual, asexual), morphogenetic pathways (embryogenesis, embryoidogenesis, gemmorhizogenesis), types of multiplication (seed, vegetative) and the correlations between them are described. Special attention is paid to the attributes of stem cells and their role in plant reproduction. Theoretical questions concerning such phenomena as polyembryony and genetic heterogeneity of seeds in plants are examined.

Key words: Sexual and asexual processes, morphogenesis, stem cells, seed and vegetative multiplication, vivipary, embryoidogeny, polyembryony, modes of reproduction. 

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 61-64, 2005

Interactions with the Environment During Plant Sexual Reproduction and Dispersal. Consequences for the Concept of Sexual Reproduction and Life

Michiel T.M. Willemse*

Laboratory of Plant Cytology and Morphology, Wageningen University,
Arboretumlaan 4, 6703 BD Wageningen, The Netherlands

* e-mail: MTM.Willemse@hccnet.nl

Received November 13, 2004; revision accepted February 12, 2005

The interaction between seed plants and animals during pollination and fruit and seed dispersal is well known, and marks the sexual reproduction process. During the history of the plant kingdom, the development of sexual reproduction has been governed by changes in the environment of the plant, together with the increasing complexity of organisms. The interactions between gametes and the environment are prepared during gametogenesis, and therefore reproduction and dispersal are related from the beginning. The dynamic environment should be considered as an interactive partner. The more intensive interactions in multicellular organisms make the interaction in seed plants far more complex.
	Sexual reproduction plays a key role in the progress of the interaction between the dynamic environment and the biosphere. Sexual reproduction embodies the renewal and dispersal of organisms. This means that the interactions between organisms and their environment are not only an essential element of sexual reproduction but also a characteristic of life, based on the unity of organism and environment. The driving force of the increasing complexity of life is the dynamic environment and the persisting organism.

Key words: Pollination, sexual reproduction, environment, interaction.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 65-73, 2005

Morphogenesis of Lilium martagon L. Explants in Callus Culture

Magdalena Kędra1 and Anna Bach2Magdalena Kędra1 and Anna Bach2

1Department of Plant Physiology and Biochemistry, Jagiellonian University,1Department of Plant Physiology and Biochemistry, Jagiellonian University,
ul. Gronostajowa 7, 30-387 Cracow, Polandul. Gronostajowa 7, 30-387 Cracow, Poland
2Department of Ornamental Plants, Agricultural University,2Department of Ornamental Plants, Agricultural University,
al. 29 Listopada 54, 31-425 Cracow, Polandal. 29 Listopada 54, 31-425 Cracow, Poland

e-mail: mkedra@mol.uj.edu.pl 

Received July 30, 2004; revision accepted March 25, 3005

The effect of two plant growth regulators on the initiation of callus and regeneration of L. martagon was studied on modified Murashige and Skoog’s (MS) medium. The cultures were initiated using seeds. The explants were isolated from different parts of seedlings (hypocotyls, seedling bulb, root) and adventitious bulblets. The growth regulators stimulated various types of callus. 4-amino-3,5,6-trichloropyridine-2-carboxylic acid (Picloram) induced a yellow, friable, granular callus, whereas benzyladenine (BA) alone induced a cream-white, compact callus. MS medium containing Picloram plus BA stimulated a yellow, compact, granular callus. The most useful explants for callus initiation were seedling bulbs and adventitious bulblet scales. The most efficient embryogenic callus was obtained on MS medium containing 5 μM Picloram and 5 μM BA. Histological studies showed that the embryogenic callus was formed at the epidermal cells and near vascular bundles of explants. Somatic embryos were solitary, whereas adventitious bulblets were closely connected with vascular tissue. No differences in the amount of DNA between scale tissue and callus were observed. Lilium martagon L. is an endangered species in Poland. In vitro culture techniques can play an important role in the future protection of this rare plant species.
	
Key words: Lilium martagon L., plant growth regulators, callus, regeneration, somatic embryogenesis.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 75-83, 2005

Cytological Features of Various Microspore Derivatives Appearing During Culture of Isolated Maize Microspores

Grzegorz Góralski1,*, Frédérique Rozier2, Elisabeth Matthys-Rochon2, 
and Leslaw Przywara1

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

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

Received August 1, 2004; revision accepted December 20, 2004Received August 1, 2004; revision accepted December 20, 2004

Microspore derivatives occurring in culture of maize microspores were studied by light and electron microscopy. The cultures showed a high degree of heterogeneity throughout the whole period of cultivation. Several types of androgenic structures at different developmental stages were observed, indicating a high level of asynchrony among microspores following the androgenic pathway. During the first 5 to 7 days of culture, multicellular structures developed inside the exine. At later stages of culture the developing microspores ruptured the pollen wall and formed structures variable in cellular characteristics. Three main types were distinguished: (i) one-domain structures consisting of small or large cells only; (ii) two-domain structures with large and small cells, the domains differing in size and cellular characteristics; (iii) callus-like structures. The two-domain structures seem to resemble zygotic proembryos which also contain two different parts, the suspensor and the apical region. The observed variability is most likely related to differences in gene activity during the inductive stage of androgenesis and the genetic properties of the microspores themselves, rather than to the conditions of in vitro culture.

Key words: Zea mays L., maize, microspore embryogenesis, androgenesis, cytological analysis, ultrastructure.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 85-92, 2005

In Vitro Pollination of Isolated Ovules of Sunflower (Helianthus annuus L.)

Marzena Popielarska* Marzena Popielarska* 

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

*e-mail: m.popielarska@iphils.uj.edu.pl

Received October 20, 2004; revision accepted March 1, 2005

A model system for in vitro self-pollination of isolated sunflower (Helianthus annuus L.) ovules was developed. The pollinated ovules were cultured on modified MS medium (Murashige and Skoog 1962) supplemented with 3% sucrose, glycine, casein hydrolysate, and growth regulators including indole-3-acetic acid (IAA), kinetin (KIN) and gibberellic acid (GA3). The frequency of developed seedlings and dissected embryos after in vitro pollination was rather low, 2.2% and 2.3%, respectively. Gynogenic seedlings were not produced in culture of unpollinated ovules. Embryological study revealed abnormalities in embryo and endosperm development after in vitro pollination. In vitro culture did not stimulate egg cell induction and autonomous endosperm development in unpollinated ovules cultured on the same media. 

Key words: Helianthus annuus, in vitro pollination, ovules, embryos, endosperm, germination, pollen.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 93-99, 2005

Interphase Structure of Endoreduplicated Nuclei in Diploid 
and Tetraploid Brassica oleracea L.

Predrag Šesek, Bojka Kump, and Borut Bohanec*

Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

*e-mail: borut.bohanec@bf.uni-lj.si  

Received November 10, 2004; revision accepted April 20, 2005Received November 10, 2004; revision accepted April 20, 2005

Endoreduplication of nuclei is a common phenomenon in plants. Cells with multiplied DNA content are usually termed endopolyploid or polysomatic. The study was aimed at clarifying the chromosomal status of endoreduplicated nuclei. So far it has been generally assumed that endoreduplicated nuclei represent a form of polyploidy. We propose a distinction between true polyploid nuclei, possessing multiple sets of chromosomes, and chromosomes formed within endoreduplicated nuclei. We supposed that chromosomes within endoreduplicated nuclei are not separated, as in true polyploids, but rather are bound together. To clarify these two alternatives, we subjected interphase nuclei exhibiting different degrees of endoreduplication and ploidy to fluorescent in situ hybridization, using diploid and tetraploid cabbage root tips containing nuclei of four different sizes (2C-16C or 4C-32C, respectively) as revealed by flow cytometry. Nuclei were hybridized with an rDNA probe (pTA71), which showed four rDNA hybridization sites on diploid metaphase chromosomes. The number of hybridization sites was constant for both diploid and tetraploid samples (3-6 per diploid and 7-12 per tetraploid nuclei) irrespective of endoreduplicated nuclear size. The results showed more signals only in the tetraploid compared to the diploid genotype, but not between different nuclear sizes within a genotype of the same ploidy. We suggest that these results indicate that chromosomes within endoreduplicated nuclei are actually bundles of sister chromatids. The consequences of this understanding of the bundled chromosomal status of endoreduplicated nuclei are discussed.

Key words: Endopolyploidy, polysomaty, Brassica oleracea, fluorescence in situ hybridization.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 101-107, 2005

A Strategy for Overaccumulation of Scopolamine in Datura innoxia 
Hairy Root Cultures

Celine Dechaux, and Michele Boitel-Conti *

Université de Picardie Jules Verne, Faculté des Sciences, Laboratoire Androgenèse et Biotechnologie, 33, rue Saint Leu, F-80039 Amiens Cedex 01, France

*e-mail : michele.boitel@u-picardie.fr
	
Received November 20, 2004; revision accepted April 18, 2005

Hairy root culture plays an important role in investigation of alkaloid production in culture in vitro. The complexity of scientific work and the production capacity limitations associated with whole plant systems and unorganized cells can be overcome using culture of hairy roots. This paper presents an example of a strategy to produce secondary metabolites from plants, in which we investigated the alkaloid pathway, modified gene expression, and cultivated and optimized hairy root growth in a bioreactor. Datura innoxia whole plant was transformed by Agrobacterium rhizogenes, and the obtained hairy roots were studied for their tropane alkaloid production. Optimization of medium led to an increase of scopolamine production in hairy root culture of Datura innoxia by increasing the produced biomass, but genetic engineering seems to be the best way to increase the accumulation of scopolamine. These root cultures were not able to stably overaccumulate scopolamine. The involvement of putative enzymatic regulation of the tropane alkaloid pathway is discussed.

Key words: Hairy roots, Datura innoxia, tropane alkaloid, bioreactor, genetic engineering, h6h gene.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 109-113, 2005

Development of the Wall Labyrinth in Pavement Epithelium Hairs of Some Utricularia Species

Bartosz Jan Płachno 1*, Andrzej Jankun 1, and Jadwiga Faber 2

1 Department of Plant Cytology and Embryology, Jagiellonian University
ul. Grodzka 52, 31- 044 Cracow, Poland
2 Department of Cytology and Histology, Institute of Zoology, Jagiellonian University,
 ul. Ingardena 6, 30-060 Cracow, Poland

*e-mail: bartek78pl@poczta.onet.pl

Received November 20, 2004; revision accepted February 10, 2005

Sodium hypochlorite-digested material and scanning electron microscopy was used to study the morphology of wall ingrowths in pavement epithelium hairs of Utricularia species from the primitive section Pleiochasia (U. volubilis) and the advanced section Utricularia (U. stygia, U. intermedia). Wall ingrowths were reticulate-type in all examined species. Wall ingrowth development started with the formation of small papillae, which later lengthened and eventually fused and branched, forming a network. The sequence of wall deposition in plant hairs is given for the first time with SEM. The wall labyrinth in transfer cells of pavement epithelium hairs was found to be far from static. Different stages of wall ingrowth development were observed within the same cell. 

Key words: Utricularia, transfer cell, wall labyrinth, hairs, carnivorous plants, scanning electron microscopy.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 115-122, 2005

Microtubular Cytoskeleton During Microsporogenesis 
of Dactylorhiza majalis (Rchb.) Hunt et Summerh.

Irena Giełwanowska1*, Ewa Szczuka2, Maria Kościńska-Pająk3, and Józef Bednara2

1Department of Plant Physiology and Biotechnology, University of Warmia and Mazury
in Olsztyn, ul. Oczapowskiego 1A, 10-957 Olsztyn, Poland
2Department of Plant Anatomy and Cytology, 
Maria-Curie Skłodowska University, ul. Akademicka 19, 20-033 Lublin, Poland 
3Department of Plant Cytology and Embryology, Jagiellonian University,
ul. Grodzka 52, 31-044 Cracow, Poland

*e-mail:i.gielwanowska@uwm.edu.pl 

Received November 22, 2004; revision accepted March 8, 2005

Changes in the microtubular cytoskeleton of Dactylorhiza majalis (Rchb.) Hunt et Summerh. (Orchidaceae) during microsporogenesis were investigated by the immunofluorescence method. Dactylorhiza microsporocytes in microsporangia divide into tetrads after a regular meiosis, which is completed with simultaneous cytokinesis. Three typical configurations of the microtubular cytoskeleton appear during microsporogenesis: the microtubular system in the cytoplasm and at the nuclear envelope, meiotic spindles, and phragmoplasts. Microtubules of the early prophase I microsporocyte are dispersed throughout the cortical cytoplasm, and later the arrays of microtubules are visible at the nuclear envelope. During metaphase I, the microtubules form the spindle which also acts during anaphase I. At telophase I, interzonal microtubules of the first meiotic spindle disappear, and new microtubular arrays extend from the nuclei towards the equatorial plane of the microsporocyte. There, these microtubules form the phragmoplast, which disintegrates before the second meiotic division. During the second meiotic division, the microtubular cytoskeleton repeats the configurations from the first meiotic division. The microtubular arrays emanating from the telophase II nuclei form interconnections of all non-sister and sister nuclei. During the formation of the cell plates between the future microspores, these microtubular arrays disappear. The results support the view that cytoskeletal configurations participate in the formation of the nuclear-cytoplasmic domains of the dividing microsporocyte in its transition from mononucleate microsporocyte to undivided dyad, and to four microspore domains after the second meiotic division. 

Key words: Dactylorhiza, microtubular cytoskeleton, microsporogenesis.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 123-127, 2005

Polyploidization of Endosperm Chalazal Haustorium 
of Rhinanthus serotinus (Scrophulariaceae)

Joanna Świerczyńska*, Małgorzata Kozieradzka-Kiszkurno,
 and Jerzy Bohdanowicz

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

*e-mail: joannasw@biotech.univ.gda.pl

Received November 30, 2004; revision accepted February 28, 2005

The paper reports a study of karyological differentiation of the chalazal endosperm haustorium of Rhinanthus serotinus (Schönheit) Oborny. Polyploidization began soon after the formation of the haustorium cell. The fully differentiated chalazal haustorium is a large elongated cell containing two huge nuclei. The nuclei are situated halfway along the length of the haustorium cell. Measurements of their nuclear DNA content revealed its degree of ploidy, which could attain a maximum 768C. Nuclei with higher (192C-384C) and the highest (768C) levels of ploidy were found in mature chalazal haustorium cells (from 100- to 500-celled endosperm proper). During polyploidization, the volume of the nuclei increased, and changes in the chromatin structure of polyploid nuclei were noted. With increasing levels of ploidy, polytene chromosomes were observed in haustorium nuclei. The rhythmical increase of DNA content and changes in nuclei size and chromatin structure point to endoreduplication as the mechanism of polyploidization of the haustorium cell.

Key words: Rhinanthus serotinus, DNA cytophotometry, endoreduplication, polyploidization, endosperm, chalazal haustorium.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 129-134, 2005

Artificial Hybridization of Pinus sylvestris L. and Pinus mugo Turra

Andrej Kormuťák1*, Mária Ostrolucká1, Božena Vooková1, Anna Preťová1, and Monika Fečková2

1Institute of Plant Genetics and Biotechnology SAS,
Akademická 2, P.O.Box 39A, SK-950 07 Nitra, Slovakia
2State Veterinary Institute, Akademická 3, SK-949 01 Nitra, Slovakia

e-mail: nrgrkorm@savba.sk

Received November 30, 2004; revision accepted March 2, 2005

Artificial hybridization experiments revealed a relatively high degree of crossability between Pinus sylvestris and P. mugo, ranging between 0.15 and 0.98. Differences between P. sylvestris x P. mugo and reciprocal hybrids of the species were observed. Hybrid seeds of P. sylvestris x P. mugo shared higher germination potential than seeds from selfing, controlled intraspecific crossing, and open pollination. The corresponding value in reciprocal combination was slightly lower. The hybrid nature of the seeds obtained was shown by restriction analysis of chloroplast DNA (cpDNA) using the trnV-trnH/Hinf I primer-enzyme combination.

Key words: Pinus, cross-pollination, seed viability, cpDNA.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 135-138, 2005

Embryological Studies on Ovules of Melandrium album Pollinated In Vitro with Lychnis coronaria Pollen Grains

Maciej Zenkteler, Agnieszka Bagniewska-Zadworna, and Elżbieta Zenkteler

Department of General Botany, Adam Mickiewicz University, 
ul. Umultowska 89, 61-614 Poznań, Poland

Received November 1, 2004; revision accepted February 20, 2005Received November 1, 2004; revision accepted February 20, 2005

Placentae of Melandrium album excised from ovaries of unopened flowers were pollinated in vitro with pollen grains of Lychnis coronaria. Pollen grains germinated, and pollen tubes were found entering the micropyles 18 h after pollination. Double fertilization occurred. Immature and mature hybrid embryos at different stages of development were analyzed up to the third week of culture. Twenty-five days after culture, cotyledonary embryos were isolated from ovules and transferred to MS (Murashige and Skoog 1962) medium supplemented with 0.5, 1 or 2 mg l-1 indole-3-acetic acid (IAA). About 85% of the 115 inoculated embryos died shortly after transfer to medium, due to atrophy of the root and one cotyledon. Eighteen embryos survived; of these, 16 well-formed seedlings with root systems were transferred to soil. The hybrid plants grew normally, and their hybridity was confirmed by morphological study. Female and male organs of the intergeneric hybrid were completely sterile.

Key words: Melandrium album (Silene latifolia), Lychnis coronaria, hybrid embryo development, intergeneric crosses, in vitro culture.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 139-146, 2005

Immunodetection of Arabinogalactan Proteins 
in Different Types of Plant Ovules

Barbara Chudzik, Barbara Zarzyka, and Renata ŚnieżkoBarbara Chudzik, Barbara Zarzyka, and Renata Śnieżko

Department of Cell Biology, Maria Curie-Skłodowska University, 
ul. Akademicka 19, 20-033 Lublin, Poland

e-mail: cellbiol@biotop.umcs.lublin.pl

Received December 1, 2004; revision accepted March 10, 2005

Arabinogalactan proteins (AGPs) are a diverse class of highly glycosylated plant cell surface proteoglycans. They are discussed as signal molecules participating in cell-cell interaction or cellular signalling during morphogenetic processes. AGPs are abundant in the stigma and transmitting tissue of different flowering plants, and are supposed to play an important role in pollen tube adhesion and guidance. In the present work we localized two epitopes of AGPs using the monoclonal antibody JIM 8 and JIM 13 in ovules of anatomically different types. Although some differences in the localization of these epitopes in different plant species were observed, in all the studied ovules they were present on the pathway of pollen tube growth. In particular plant species they appeared at the time of the ovule’s highest receptivity. 

Key words: Galanthus nivalis, Galtonia candicans, Oenothera, Sinapis alba, arabinogalactan proteins (AGPs), JIM 8 Mab, JIM 13 Mab, ovules.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 147-157, 2005

Cellular Accumulation of Protein Bodies and Changes in DNA Ploidy Level During Seed Development of Lathyrus tuberosus L.Cellular Accumulation of Protein Bodies and Changes in DNA Ploidy Level During Seed Development of Lathyrus tuberosus L.

Sławomira Knake-Sobkowicz and Kazimierz Marciniak

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

Received December 1, 2004; revision accepted april 20, 2005

Mitotic activity, changes in nuclear DNA content and accumulation of protein bodies during endosperm and embryo development of Lathyrus tuberosus L. (2C DNA 15.1 ± 0.08 pg) were analyzed. A gradual decrease in mitotic activity of embryo cells was observed during seed development. At the final stages of embryogenesis, mitotic activity was noted only in tip meristems and provascular tissue. The decrease in mitotic activity in endosperm was accompanied by an increase in amitosis frequency. Cytophotometry of nuclear DNA revealed that in endosperm the nuclei underwent six endoreplication rounds, reaching 192C. The C DNA level was much higher at the chalazal pole than at the micropylar pole, where it was only 12C. In the cotyledon the nuclear DNA level reached 64C (five endoreplication rounds), while in the hypocotyl-root axis it increased from 2C to 4C. Cells of all Lathyrus tuberosus embryo organs are involved in the synthesis of storage proteins and their accumulation in the form of protein bodies. This process starts ˜21 days after fertilization (DAF). Cells containing protein bodies are capable of division. The authors characterized the central and basal zones of cotyledons according to the amount of protein bodies per 400 µm2 of cytoplasm area. Double staining (Feulgen/Coomassie Brilliant Blue R250) was used for measurement of nuclear DNA and storage protein content in the same cells. The ploidy of nuclei correlated with total protein body volume (r = 0.97) in Lathyrus tuberosus cotyledon cells.

Key words: Lathyrus tuberosus, seed development, embryo, cotyledon, endosperm, endoreplication, protein bodies.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 159-165, 2005

SEED COAT PATTERNS IN RAPID-CYCLING BRASSICA FORMS 

Jagna Karcz1*, Tomasz Ksiazczyk2, and Jolanta Maluszynska2

1Laboratory of Scanning Electron Microscopy, University of Silesia, 
ul. Jagiellońska 28, 40-032 Katowice, Poland
2Department of Plant Anatomy and Cytology, University of Silesia,
 ul. Jagiellońska 28, 40-032 Katowice, Poland

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

Received December 3, 2004; revision accepted April 15, 2005Received December 3, 2004; revision accepted April 15, 2005

The micromorphological typology of seed surfaces was investigated in rapid-cycling Brassica (RCBr) forms, using scanning electron microscopy. Four types of basic ornamentation pattern were recognized: reticulate (B. rapa, B. juncea), reticulate-foveolate (B. nigra), randomly reticulate (B. oleracea, B. napus) and reticulate-rugose (B. carinata). The seed coats showed variation in the shape and size of the testa epidermal cells and the structure of the outer periclinal and anticlinal cell walls. The surface patterns in RCBr seeds were less exposed and were moderately reticulated compared to cultivated Brassica species. The micromorphological characteristics of the seed coats may provide valuable additional diagnostic criteria for delimitation of RCBr forms, and can be used in identification of seeds.

Key words: Rapid-cycling Brassica (RCBr), micromorphology, sculpture, seed surface pattern, testa, SEM.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 167-171, 2005

Regeneration of Haploid Plants after Distant Pollination of Wheat 
Via Zygote Rescue

Ferenc Bakos, Katalin Jäger, and Beáta Barnabás*

Agricultural Research Institute of the Hungarian Academy of Sciences, P.O.B. 19, 
H-2462, Martonvásár, Hungary

*e-mail: bea@mail.mgki.hu

Received December 5, 2004; revision accepted April 2, 2005

Haploid wheat plants have been produced by a new method of zygote rescue carried out after distant pollination. Wheat stigmas were pollinated with maize pollen and subsequently the activated egg cells from the elongated ovaries were rescued for in vitro plant development in single cell culture. As the control, 2-week-old embryos were also dissected and then cultured. The efficiency of both techniques was comparable. Wheat was also pollinated with rice, and the further development of rescued zygotes into multicellular structures is reported here for the first time. Because the lack of a normal endosperm hampers embryo development even in the early stages, early zygote rescue (two days after distant pollination) may represent a more efficient way of producing double haploid (DH) plants in cultivars that are recalcitrant in androgenic cultures, after further optimization of in vitro culture of isolated single cells. 

Key words: Activated egg cell, co-culture, double haploid, embryo rescue, haploid, nurse culture, parthenogenesis, wheat  maize, zygote rescue.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 173-178, 2005

Using Enzyme Polymorphism to Identify the Gametic Origin of Flax RegenerantsUsing Enzyme Polymorphism to Identify the Gametic Origin of Flax Regenerants

Zuzana Bartošová, Bohuš Obert, Tomáš Takáč, Andrej Kormuťák, and Anna Preťová*

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

*e-mail: anna.pretova@savba.sk

Received December 6, 2004: revision accepted April 29, 2005

Anther culture is currently the most successful method for production of doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this contribution we investigated the incorporation of enzyme polymorphism of acid phosphatase and peroxidase as molecular markers for the gametic origin of flax plants derived from anther and ovary cultures.

Key words: Anther culture, ovary culture, isozymes, haploids, flax.

Abbreviations:Abbreviations
RAPD - random amplified polymorphic DNA; ISSR – inter-simple sequence repeat amplification; PCR/RFLP - PCR primers produce a DNA fragment, which after digestion with an enzyme yields the special RFLP (restriction fragment length polymorphism) patterns.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 179-184, 2005

Developmental Switch During Embryo Sac Formation 
From a Bisporic Mode to the Tetrasporic Fritillaria Type
 in Hyacinthoides vincentina (Hoffmanns & Link) Rothm. (Hyacinthaceae)

Irma Ebert and Johann Greilhuber

Department of Systematic and Evolutionary Botany, Faculty Center for Botany, 
University of Vienna, Rennweg 14, A 1030 Vienna, Austria

Received December 6, 2004; revision accepted May 1, 2005

Developmental transitions between the major types of embryo sac formation, in which development starts according to one type and continues according to another type, have been reported seldom in the embryological literature. Here we describe in a sample of the rare taxon Hyacinthoides vincentina (a species of the Hyacinthaceae related to H. italica, sometimes considered conspecific with H. mauritanica) a bisporic start of embryo sac development and its tetrasporic continuation according to the Fritillaria type. The decisive events are the postmeiotic dissolution of the cell wall, which had been formed after the first meiotic division, the assumption of a 1+3 position of the spore nuclei in the germinating coenomegaspore, and the formation of a triploid chalazal spindle during the first embryo sac mitosis (Carano-Bambacioni phenomenon). The endosperm is pentaploid. While the bisporic Hyacinthoides type is considered characteristic for the genus Hyacinthoides, the Fritillaria type has not been found in the family hitherto. These observations add one further and particular example to the remarkable diversity of embryo sac types in the family Hyacinthaceae, in which the Polygonum, Allium, and Drusa-I types have been described, in addition to the Hyacinthoides type.

Key words: Hyacinthoides, embryo sac, Hyacinthoides type, Fritillaria type, developmental plasticity.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 185-192, 2005

FISH and GISH Analysis of Brassica Genomes

Robert Hasterok*, Tomasz Ksiazczyk, Elzbieta Wolny, and Jolanta Maluszynska

Department of Plant Anatomy and Cytology, University of Silesia, Jagiellonska 28, 
40-032 Katowice, Poland 

email: hasterok@us.edu.pl 

Received December 28, 2004; revision accepted May 5, 2005

Fluorescence and genomic in situ hybridization (FISH and GISH) methods were used for discrimination of Brassica genomes. The three diploid and three allotetraploid species of Brassica, known as the “U-triangle,” represent an attractive model for molecular and cytological analysis of genome changes during phylogeny in the genus Brassica. The use of genomic DNA probes enabled unambiguous discrimination of the ancestral genomes in B. juncea and B. carinata, and was only partially successful in B. napus. GISH signals in all genomes were localized predominantly in pericentromeric regions of chromosomes. Simultaneous application of genomic and ribosomal DNA probes in multicolor GISH and FISH allowed identification of a significant number of chromosomes in the B. juncea complement. The study also revealed that species of Brassica possess Arabidopsis-type telomeric repeats which in all genomes occupied exclusively terminal, that is, telomeric, locations of chromosomes.

Key words: 5S rDNA, 25S rDNA, Brassica, FISH, GISH, telomeres.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 193-198, 2005

Differentiation of adventitious buds from Capsicum annuum L. hypocotyls after co-culture with Agrobacterium tumefaciens

Monika Delis1, Grażyna Garbaczewska2, Katarzyna Niemirowicz-Szczytt1

1 Department of Plant Genetics, Breeding and Biotechnology, Warsaw Agricultural University, ul. Nowoursynowska 166, 02-787 Warsaw, Poland
2 Department of Botany, Warsaw Agricultural University,
ul.  Nowoursynowska 166, 02-787 Warsaw, Poland

Received January 2, 2005; revision accepted April 10, 2005

In vitro organogenesis in hypocotyl explants of the pepper cultivar ‘Bryza’ was induced on MS medium (Murashige and Skoog, 1962) containing 5 mg/l 6-benzyloaminopurine (BAP) and 1 mg/l indole-3-acetic acid (IAA). The hypocotyl explants were then inoculated with Agrobacterium tumefaciens LBA4404(pBI121). After 2 days of culture the first cell divisions were observed in the epidermis and cortex. After 6-7 days, numerous adventitious bud primordia appeared in 58.4% of the explants. In further stages of culture, buds developed into shoots in 8.4% of the explants. Histological analysis revealed hypertrophy and the presence of necrotic cells in the cortex. Necrotic changes were also observed in the vascular bundles. It is likely that culture on a selective medium containing kanamycin and co-culture with Agrobacterium tumefaciens strongly affected the organization of the hypocotyl meristematic tissue, and in consequence brought about necrosis and isolation of the adventitious buds from the vascular bundles.

Key words: Genetic transformation, adventitious buds, anatomical structure of hypocotyl, kanamycin.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 199-204, 2005

Superoxide Dismutase Activity in Organs of Mesembryanthemum crystallinum L. Plant at Different Stages of CAM Development

Marta Libik1*, Robert Konieczny2, Ewa Surówka1, and Zbigniew Miszalski1,3

1Department of Plant Physiology, Polish Academy of Sciences,
ul. Niezapominajek 21, 30-239 Cracow, Poland
2Department of Plant Cytology and Embryology, Jagiellonian University,
ul. Grodzka 52, 31-044 Cracow, Poland
3Pedagogical Academy, Institute of Biology, ul. Podbrzezie 3, 31-054 Cracow, Poland

e-mail: libik@ifr-pan.krakow.pl

Received January 7, 2005; revision accepted March 3, 2005

Fluctuations of acidity were found in leaves, shoots and roots of two groups of Mesembryanthemum crystallinum plants: watered and salt-treated. This suggests that the functioning of CAM (related to malate accumulation) in the tested organs can differ in "early CAM" in watered plants versus "full CAM" in NaCl-irrigated plants. Among the different organs analyzed, shoots exhibited the highest acidity level at the beginning of the day in both groups of M . crystallinum plants. Superoxide dismutase forms (MnSOD, CuZnSOD and FeSOD) differed in activity depending not only on their localization in the plant tissue but also on the stage of CAM development. Plant exhibiting "full CAM" and showing clear diurnal Δ-acidity had much higher total SOD activity during evening hours than in plants following "early CAM" photosynthesis. Shoots showed the highest activity of SOD. 

Key words: Mesembryanthemum, ice plant, oxidative stress, salinity, SOD.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 205-212, 2005

Increased Sensitivity to, and Reduced Production of, Ethylene 
in an ABA-Overproducing Tomato MutantIncreased Sensitivity to Ethylene and Reduced Production of it in an ABA-Overproducing Tomato Mutant*

Martin Fellner1,+, Jennifer A. Franklin2, David M. Reid2, Vipen K. Sawhney1*

1Department of Biology, University of Saskatchewan, 
112 Science Place, Saskatoon, SK, S7N 5E2, Canada
2 Department of Biological Sciences, University of Calgary, 
2500 University Drive NW, Calgary, AB, T2N 1N4, Canada

*e-mail: sawhney@admin.usask.ca
 +Present address: Palacky University, Department of Cell Biology and Genetics, Šlechtitelů 11, Olomouc – Holice 783 71, Czech Republic, and Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Laboratory of Growth Regulators, Šlechtitelů 11, Olomouc – Holice 783 71, Czech Republic.

Received February 3, 2005; accepted April 11, 2005

The single-gene male-sterile 7B-1 mutant in tomato (Lycopersicon esculentum Mill.) shows reduced de-etiolation of hypocotyl growth, has an elevated level of endogenous abscisic acid (ABA) and reduced amounts of growth-active gibberellins (GAs), is supersensitive to exogenous ABA, and is resistant to abiotic stresses in light but not in the dark. The existence of crosstalk between light signaling and plant hormones, and the interaction of ABA and GA biosynthetic pathways with ethylene, led us to investigate the possible role of ethylene in the 7B-1 mutant. In the dark, 7B-1 seedlings exhibited the normal triple response to 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene precursor, similar to the wild type. In light, however, hypocotyl growth of mutant seedlings was more sensitive to ACC but less sensitive to the inhibitors of ethylene’s action, such as silver thiosulfate, than the wild type. The 7B-1 mutant seedlings produced reduced levels of ethylene, which could account for the increased sensitivity to exogenous ACC. The mutant leaf discs also contained higher chlorophyll content and showed greater chlorophyll degradation in response to ACC than the wild type, and these could also be related to low ethylene production by the mutant. Fluridone, an inhibitor of ABA biosynthesis, countered the ACC-induced inhibition of hypocotyl elongation, and it also restored the wild-type phenotype in 7B-1 plants. The results suggest that the reduced de-etiolation of hypocotyl growth and the increased sensitivity of the mutant to ethylene in light are due to reduced ethylene production, which in turn may be related to high endogenous ABA. The data presented support our earlier findings that the 7B-1 mutant has a defect in light perception which affects both hormonal sensitivity and endogenous levels, thereby affecting hypocotyl and shoot growth. 

Key words: ABA, ACC, crosstalk, ethylene, growth, light, tomato mutant.

Abbreviations: ABA - abscisic acid; ACC - 1-aminocyclopropane-1-carboxylic acid; BM - basal medium; DW - dry weight; FLU (fluridone) - 1-methyl-3-phenyl-5-[3-trifluoromethyl-(phenyl)]-4(1H)-pyridinone; FW - fresh weight; Gas - gibberellins; STS - silver thiosulfate (Ag2S2O3); WT - wild type.Abbreviations: ABA: abscisic acid; ACC: 1-aminocyclopropane-1-carboxylic acid; BM: basal medium; DW: dry weight; FLU: (fluridone) = 1-methyl-3-phenyl-5-[3-trifluoromethyl-(phenyl)]-4(1H)-pyridinone; FW: fresh weight; GAs: gibberellins; STS: silver thiosulfate (Ag2S2O3); WT: wild type.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 213-217, 2005

Reproductive Biology of Ruscus hypoglossum L. in SlovakiaReproductive Biology of Ruscus hypoglossum L. in Slovakia

Ľuboš Halada1* and Oľga Erdelská2** 
Ľuboš Halada1* and Oľga Erdelská2** 
1Institute of Landscape Ecology SAS, Branch Nitra, Akademická 2,
P.O.Box 23B, SK-949 01 Nitra, Slovakia
2Institute of Botany SAS, Dúbravská 14, SK-842 23 Bratislava, Slovakia

*e-mail: lubos.halada@savba.sk*e-mail: lubos.halada@savba.sk
**e-mail: erdelski@stonline.sk**e-mail: erdelski@stonline.sk

Received January 19, 2005; revision accepted May 24, 2005

Ruscus hypoglossum L. (Asparagaceae), a rare and endangered species of the Malé Karpaty region in Slovakia, is an evergreen geophyte having rigid leaves with special bracts developed on them. The flowers are unisexual, and the plant is dioecious. Well-developed stamens and rudimentary pistils in different stages of development occur in male flowers. Remnants of degenerated anthers persist in female flowers, which have two ovules in the ovary. Male plants prevail in local populations; the average ratio of male to female plants is 5:1. Reproduction in Ruscus hypoglossum is generative and vegetative. Fruit (red berry) development lasts from May to October. Te seeds are round, 6–8 mm in diameter, and albuminous. The endosperm is helobial, with a haustorial chalazal chamber. The endosperm cell walls in mature seeds are thickened and are composed of reserve cellulose. The mature embryo consists of one elongated cotyledon, a plumule, short hypocotyl and radicle. Seed dormancy lasts one year. Vegetative propagation is accomplished by rhizome fragmentation. The populations should be protected above all against direct destruction, shoot collecting, and grazing by animals, especially deer.

Key words: Ruscus hypoglossum, seed, embryo, reproduction.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 219-226, 2005

Morphological and Histological Aspects of 2,4-D Effects
on Rape Explants (Brassica napus L. cv. Kana) Cultured In vitro

Halina Ślesak, Marzena Popielarska*, and Grzegorz Góralski

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

* e-mail: m.popielarska@iphils.uj.edu.pl

Received January 22, 2005; accepted April 12, 2005

The effect of different durations of exposure to 2,4-D on hypocotyls and cotyledons cultured in vitro was studied in Brassica napus L., cv. Kana. Organogenesis or callogenesis depended on the duration of explant exposure to MS medium supplemented with 2 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid). Short treatment (1 and 3 days) with 2,4-D resulted in rhizogenesis on hypocotyls (100% and 98% of explants, respectively) and cotyledons (80% and 54% of explants, respectively). Adventitious shoots formed sporadically, with the highest frequency (14% of explants) on hypocotyls cultured 3 days on MS supplemented with 2,4-D and then transferred to hormone-free medium. Histological analysis clearly indicated that the basal part of hypocotyls is involved in root formation and callus production, and the apical part for shoots. Meristematic sites originated from groups of cells in the cortex layer (including cells of the endoderm), but the procambium, phloem and pericycle also showed meristematic activity. The present study indicated that the response of explants cultured on media containing 2,4-D at constant concentration depends on the duration of explant exposure to growth regulator.

Key words: Brassica napus L., 2,4-D, regeneration, callus, histology.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 227-236, 2005

Induction, Maintenance and Preservation of Embryogenic Competence of Gentiana cruciata L. Cultures

Anna Mikuła, Agnieszka Fiuk, and Jan J. Rybczyński

Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2, 02-976 Warsaw, Poland

e-mail: amikula@ob.neostrada.pl

Received January 27, 2005; revision accepted April 19, 2005

The paper describes structural and ultrastructural changes in primary explants, induction of embryogenesis, somatic embryo development, and four protocols for cryopreservation of cell suspensions. The changes during tissue culture of hypocotyl and cotyledon explants from 10-day-old seedlings and fragments of leaf explant of Gentiana cruciata L. were studied. Seedling explants were cultured on MS medium supplemented with 1.0 mg/l dicamba + 0.1 mg/l NAA + 2.00 mg/l BAP + 80.0 mg/l adenine sulphate. The hypocotyl callus tissue was initiated by cell divisions of the vascular cylinder, but in cotyledons only parenchyma cells took a part in callus formation. The leaf blade explants usually responded only by proliferation of the wounded surface. The effect of auxins (2,4-D, NAA, DIC) and cytokinins (kinetin, zeatin, BAP) in various concentration and combinations on leaf explant response was examined. Generally, embryos were formed sporadically on media containing NAA (1.64% responding explants) or 2,4-D (0.38%), but were not produced in the presence of dicamba. 
	Production of somatic embryos was more effective from suspension culture than from agar medium. Liquid culture made it possible to maintain the cell suspension’s embryogenic competence for 5 years. For preservation of proembryogenic masses, four protocols of cryopreservation were studied: direct cooling, sorbitol/DMSO treatment, vitrification, and encapsulation. Direct cooling and sorbitol/DMSO treatment was unsuccessful. Vitrified tissue required a minimum 3 weeks culture on solid medium for cell proliferation to reach the proper fresh weight for manipulation. Alginate beads with PEMs were transferred directly to liquid medium for post-freezing culture. Vitrification and encapsulation maintained high viability of post-freezing PEM, but encapsulation ensured faster restoration of G. cruciata cell suspension.

Key words: Gentiana cruciata L., hypocotyl, leaf explants, dedifferentiation, suspension culture, cryopreservation.

Abbreviations: AS – adenine sulphate; BAP – 6-benzylaminopurine; GA3 - gibberellic acid; Kin – kinetin - furfurylamonopurine; NAA – á-naphtaleneacetic acid; Dic - dicamba- 3,6-dichloro-o-anisic acid; LN - liquid nitrogen; DMSO - dimethylsulfoxide; PEM – proembryogenic mass; Zeat – zeatin - 6-(4-hydroxy-3-methyl-2-enylamino)purine; PGR – plant growth regulator; G.cruc/C – suspension derived from cotyledon callus; G.cruc/H - suspension derived from hypocotyls callus.

ACTA BIOLOGICA CRACOVIENSIA Series Botanica 47/1: 237-245, 2005

Is the Blue Zinc Violet (Viola guestphalica Nauenb.) a Taxon of Hybrid Origin? Evidence from Embryology

Aneta Siuta1, Monika Bożek1, Monika Jędrzejczyk2, Adam Rostański2, 
and Elżbieta Kuta1*

1Department of Plant Cytology and Embryology, Jagiellonian University,
 ul. Grodzka 52, 31-044 Cracow, Poland
2Department of Plant Systematics, University of Silesia, 
ul. Jagiellońska 28, 40-032 Katowice, Poland

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

Received March 29, 2005; revision accepted May 10, 2005

Investigations of reproductive processes of the blue zinc violet (Viola guestphalica Nauenb.) from a natural location in D-Blankenrode and from two sites of introduction in Poland (Wełnowiec zinc spoil and a private garden in Sosnowiec-Ostrowy Górnicze) showed significant disturbances resulting in reduced plant fertility. Pollen viability estimated by acetocarmine staining was relatively low, with 54% viable pollen grains in plants from Wełnowiec and 62% from the garden. Specimens from Blankenrode had 80% viable pollen but the pollen grains differed in size conspicuously. Giant abnormal pollen grains accompanied very small ones. 
	Necrosis affected anthers and pistils, including degeneration of whole anthers and ovules, whole embryo sacs or embryo sac elements, and abortion of embryos. The pathway of female gametophyte and embryo development was normal in 61% of the ovules. Necrosis of somatic tissues and generative cells at different developmental stages was found in 28% of the ovules, and irregular development in 11% of them. As a consequence, embryo viability was reduced to 33%. Embryological data indicated that V. guestphalica is not a well-stabilized genotype. The possible origin of the blue zinc violet is discussed.

Key words: Viola L., zinc violets, pollen, megasporogenesis, female gametophyte, embryo, heavy metals.