Download ACTA BIOLOGICA CRACOVIENSIA SERIES BOTANICA Vol. 51

ACTA BIOLOGICA
CRACOVIENSIA
SERIES
BOTANICA
Vol. 51 suppl. 1
ABSTRACTS
12th National Conference
‘In vitro Cultures, Poznań 2009’
September 9–11, 2009
Poznań, Poland
Polish Academy of Sciences – Cracow Branch
Jagiellonian University
Polish Academy of Arts and Sciences
2009
ACTA BIOLOGICA CRACOVIENSIA
Series Botanica
The Official Publication
of the Biological Commission of the Polish Academy of Sciences – Cracow Branch
and the Jagiellonian University
published jointly with the Polish Academy of Arts and Sciences
DEVOTED TO PLANT ANATOMY, MORPHOLOGY, CYTOLOGY, GENETICS, KARYOLOGY,
EMBRYOLOGY, TISSUE CULTURE, PHYSIOLOGY AND BIOSYSTEMATICS
ESTABLISHED 1958
© Polish Academy of Sciences and the Jagiellonian University, Cracow 2009
The edition of this supplement is financed by the Polish Academy of Arts and Sciences.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica is published twice a year by the Polish
Academy of Sciences – Cracow Branch, ul. św. Jana 28, 31-018 Cracow, Poland
and the Jagiellonian University in Cracow, ul. Gołębia 24, 31-007 Cracow, Poland
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ACTA BIOLOGICA CRACOVIENSIA
Series Botanica
Editor
ELŻBIETA KUTA
Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52
31-044 Cracow, Poland, Tel./Fax: +48-12-422 81 07, e-mail: [email protected]
Editorial Secretary
MONIKA TULEJA
Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52
31-044 Cracow, Poland, Tel./Fax: +48-12-422 81 07, e-mail: [email protected]
Editorial Board
HARVEY E BALLARD, JR. Department of Environmental and Plant
Biology, Ohio University, Porter Hall, Athens, Ohio 45701, USA,
e-mail: [email protected]
Molecular approaches in plant systematics, ecology and evolution
TATYANA B. BATYGINA. Komarov Botanical Institute, Department of
Embryology and Reproductive Biology, Prof. Popov St. 2,
197376 St. Petersburg, Russia, e-mail: [email protected]
Plant embryology
JÓZEF BEDNARA. Department of Plant Anatomy and Cytology, Maria
Curie-Skłodowska University, ul. Akademicka 19, 20-033 Lublin, Poland,
e-mail:[email protected]
Plant embryology
BORUT BOHANEC. Biotechnical Faculty, University of Ljubljana,
Jamnikarjeva 101, 1000 Ljubljana, Slovenia,
e-mail: [email protected]
Plant biotechnology
MAURO CRESTI. Dipartimento di Biologia Ambientale, Sezione Botanica,
Universita di Siena, Via P. A. Mattioli 4, I-53100 Siena, Italy,
e-mail: [email protected]
Sexual plant reproduction; pollen biology; pollen tube; pollen-stigmastyle-ovule interaction; cytoskeleton
MARIA CHARZYŃSKA. Department of Plant Anatomy and Cytology,
Warsaw University, ul. Miecznikowa 1, 02-096 Warsaw, Poland,
e-mail: [email protected]
Cytoembryology of flowering plants; anther and pollen development
(structural and molecular aspects)
MARTA DOLEŻAL. Academy of Physical Education, Chair of Hygiene
and Health Protection, Al. Jana Pawła II 78, 81-571 Cracow, Poland,
Fax: +48-12-648 17 07
General and medical mycology; health promotion; medical microbiology
FRANCISZEK DUBERT. Department of Plant Physiology, Polish
Academy of Sciences, ul. Podłużna 3, 31-239 Cracow, Poland,
e-mail: [email protected]
Physiology of plant growth and development
OL'GA ERDELSKÁ. Institute of Botany, Slovak Academy of Sciences,
Dúbravská 14, 84223 Bratislava, Slovak Republic
Plant embryology; developmental biology
JOHANN GREILHUBER. University of Vienna, Institute of Botany,
Rennweg 14, 1030 Vienna, Austria, e-mail: [email protected]
Plant karyology
JOHN M. HERR, Jr. University of South Carolina, Department of
Biological Sciences, Columbia, South Carolina 29208, U.S.A.,
e-mail: [email protected]
Plant morphology; anatomy; embryology
ANDRZEJ JOACHIMIAK. Department of Plant Cytology and Embryology,
Jagiellonian University, ul. Grodzka 52, 31-044 Cracow, Poland,
Tel./Fax: +48-12-422 81 07, e-mail: [email protected]
Plant genetics and cytogenetics; molecular phylogeny; sex determination;
somaclonal variation
BENGT E. JONSELL. Bergius Botanic Garden, Box 50017,
S-104 05 Stockholm, Sweden, e-mail: [email protected]
Plant taxonomy; biosystematics; plant geography
ANNA KOLTUNOW. CSIRO Plant Industry, PO Box 350, Glen Osmond,
SA 5064, Australia, e-mail: [email protected]
Plant reproduction, developmental biology – particularly seed and fruit
(cellular and molecular aspects)
JOLANTA MAŁUSZYŃSKA. Department of Plant Anatomy and Cytology,
Silesian University, ul. Jagiellońska 28, 40-032 Katowice, Poland,
e-mail: [email protected]
Plany cytology; cytogenetics
ELISABETH MATTHYS-ROCHON. RDP, ENS Lyon, 46 Allée d'Italie,
69364 Lyon Cedex 07, France, e- mail: [email protected]
Plant gametes; pollination; cellular and molecular aspects of fertilization;
in vitro development
MARIA OLSZEWSKA. Department of Cytogenetics and Plant Molecular
Biology, University of Łódź, ul. Banacha 12/16, 90-237 Łódź, Poland,
e-mail: [email protected]
Plant cytochemistry and cytogenetics
MARIA PAJĄK. Department of Plant Cytology and Embryology,
Jagiellonian University, ul. Grodzka 52, 31-044 Cracow, Poland,
Tel./Fax: +48-12-422 81 07, e-mail: [email protected]
Plant embryology; apomixis
JAN J. RYBCZYŃSKI. Botanical Garden – Center for Biological
Diversity Conservation of the Polish Academy of Sciences,
ul. Prawdziwka 2, 02-973 Warsaw, Poland,
e-mail: [email protected]
Plant tissue and organ culture; biotechnology; cryopreservation
BARBARA SKUCIŃSKA. Department of Plant Breeding and Seed
Science, The Agricultural University of Cracow,
ul. Łobzowska 24, 31-140 Cracow, Poland
Plant tissue and organ culture
MICHIEL T. M. WILLEMSE. Laboratory of Plant Cell Biology,
Wageningen Agricultural University, Arboretumlaan 4,
6703 BD Wageningen, The Netherlands,
e-mail: [email protected]
Sexual plant reproduction; biology of lower plants
HONG-YUAN YANG. Key Laboratory of MOE for Plant Developmental
Biology, Wuhan University, Wuhan 430072, China,
e-mail: [email protected]
Experimental manipulation of sexual plant cells; cell and developmental
biology; fertilization and embryogenesis
MACIEJ ZENKTELER. Laboratory of General Botany,
Institute of Experimental Biology, Adam Mickiewicz University,
ul. Umultowska 89, 61-614 Poznań, Poland,
e-mail: [email protected]
Experimental embryology; plant tissue and organ culture
12th National Conference
'In vitro Cultures, Poznań 2009'
Integrating biotechnology, molecular
biology and agronomic practice
through in vitro cultures
September 9–11, 2009, Poznań, Poland
Patronage:
Rector of the Adam Mickiewicz University of Poznań
Conference organized by:
Department of General Botany, Adam Mickiewicz University, Poznań, Poland
Faculty of Biology, Adam Mickiewicz University, Poznań, Poland
Tissue Culture Section of Polish Botanical Society
Polish Academy of Arts and Sciences – Commission of Morphology and Embryology,
Cracow, Poland
Honorary Committee:
Bronisław Marciniak
Rector of Adam Mickiewicz University, Poznań, Poland
Bogdan Jackowiak
Dean of the Faculty of Biology, Adam Mickiewicz
University, Poznań, Poland
Secretary General of the Polish Academy of Arts and
Sciences, Cracow, Poland
Pro-Rector of the Jagiellonian University and Chairman
of the Commission of Morphology and Embryology of
Polish Academy of Arts and Sciences, Cracow, Poland
Jerzy Wyrozumski
Szczepan Biliński
Scientific Committee:
Anna Bach
Krystyna Bojarczuk
Teresa Cegielska-Taras
Franciszek Dubert
Ewa Łojkowska
Agricultural University of Cracow,
Department of Ornamental Plants, Cracow Poland
Polish Academy of Sciences,
Institute of Dendrology, Kórnik, Poland
Institute of Plant Breeding and Acclimatization,
Poznań, Poland
Polish Academy of Sciences, Institute of Plant
Physiology, Cracow, Poland
University of Gdansk, Department of Biotechnology
and Medical University of Gdańsk, Gdańsk, Poland
Jan Kępczyński
Elżbieta Kuta
Stefan Malepszy
Jolanta Małuszyńska
Teresa Orlikowska
Jan J. Rybczyński
Aurelia Ślusarkiewicz-Jarzina
Jan Szopa
Tomasz Twardowski
Przemysław Wojtaszek
Adam Woźny
Maciej Zenkteler
University of Szczecin, Department of Plant Physiology,
Szczecin, Poland
Jagiellonian University, Department of Plant Cytology
and Embryology, Cracow, Poland
University of Life Sciences, Department of Plant
Genetics, Breeding and Biotechnology, Warsaw, Poland
Silesian University, Katowice, Poland
Research Institute of Pomology and Floriculture,
Skierniewice, Poland
Polish Academy of Sciences, Botanical Garden,
Warsaw, Poland
Polish Academy of Sciences, Institute of Plant Genetics,
Poznań, Poland
University of Wrocław, Department of Genetic
Biochemistry, Wrocław, Poland
Polish Academy of Sciences, Institute of Bioorganic
Chemistry, Poznań, Poland
Adam Mickiewicz University, Department of Molecular
and Cellular Biology, Poznań, Poland
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Organizing Committee:
Maciej Zenkteler
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Jan J. Rybczyński
Polish Academy of Sciences, Botanical Garden,
Warsaw, Poland
Ewa Kępczyńska
University of Szczecin,
Department of Plant Physiology, Szczecin, Poland
Elżbieta Zenkteler
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Maria Katarzyna Wojciechowicz
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Agnieszka Bagniewska-Zadworna Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Rafał Mól
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Tomasz Wyka
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Barbara Stefaniak
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Łukasz Zarychta
Adam Mickiewicz University,
Department of General Botany, Poznań, Poland
Language Editor:
Technical Editor:
Tomasz P. Wyka
Agnieszka Bagniewska-Zadworna
ACTA BIOLOGICA CRACOVIENSIA
Series Botanica
CONTENTS
Volume 51, suppl. 1, 2009
Lectures
S. Malepszy – Plant biotechnology and in vitro culture – the fulfilled hopes?
T. Orlikowska, P. Sobiczewski, E. Zenkteler – What's new in the control of bacterial contamination
in plant tissue cultures?
K. Skórkowska-Telichowska, M. Żuk, A. Kulma, A. Bugajska- Prusak, K. Ratajczak and J. Szopa –
FlaxAid, a new wound dressing based on transgenic flax products
T. Twardowski – GMO – A.D. 2009
P. Wojtaszek, E. Rodakowska, A. Kasprowicz, A. Szuba, D. Kierzkowski, P. Zawadzki,
M. Maruniewicz, M. Wierzchowiecka, A. Kapczyńska, I. Kosicka, M. Michalak – Biology of cell
wall-plasma membrane-cytoskeleton continuum in plants
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Oral presentations
T. Cegielska-Taras, I. Bartkowiak-Broda – Integration of in vitro culture, biotechnology and
molecular biology in modern breeding of oilseed rape
A. Czubacka, T. Doroszewska – Obtaining tobacco double haploids containing different sources
of resistance to PVY
M. Gliwicka, S. Balazadeh, C. Caldana, B. Mueller-Roeber, M. D. Gaj – The use of multi-qPCR
platform and tan1 mutant in identification of TF genes involved in somatic embryogenesis
in Arabidopsis
K. Głowacka, S. Jeżowski – In vitro culture of Miscanthus sinensis anthers
K. L. Janczur, A. Obarska, P. Kachlicki, B. Tomaszewska – In vitro selection of varieties and
lines of Brassica napus L. resistant to multiple heavy metals
R. Konieczny, M. Libik, E. Surówka, A. Nosal, Z. Miszalski – Enzymatic markers of rhizogenic
competence in in vitro culture of common ice plant
K. Kromer, A. Raj, B. Wojtuń, D. Poturała – Use of biotechnology for conservation of a critically
endangered population of alpine saxifrage (Saxifraga nivalis L.)
E. Kuta, J. Rojek, A. Pawełek-Skoczylas, B. Ślązak, J. Bohdanowicz – Autonomous endosperm
induction in cultured unpollinated ovaries is strongly species dependent
A. Mikuła, J. J. Rybczyński – Liquid nitrogen efficacy in protection of genetic stability in plant
material
J. Nowakowska – Application of genetically modified trees in forest plantations
M. Pilarska, R. Konieczny, M. Tuleja, E. Kuta – Efficient plant regeneration of Trifolium nigrescens
via somatic embryogenesis
T. Pniewski, M. Bogusiewicz, K. Wyrwa, M. Czyż – HBV core antigen (HBcAg) as a plant-based
prototype of therapeutic hepatitis B vaccine and a carrier for HBV preS epitopes
P. M. Pukacki, M. Jarząbek, W. Jóźwiak, C. Lütz – Cryoprotective activity of thermal hysteresis
protein in evergreen plants
D. Raj, A. Kokotkiewicz , M. Łuczkiewicz – Influence of different culture strategies on growth and
indolizidine alkaloid accumulation in Securinega suffruticosa shoot cultures
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P. Sobiczewski – Bacteria in plant environment
K. Szczygieł – Somatic embryogenesis of selected coniferous tree species
W. Szypuła, J. Budziszewska, D. Delbani – In vitro culture of endophytic fungi as a potential
source of a novel biologically active secondary metabolites
K. Tomiczak, J. J. Rybczyński – Cytologial, cytometric and molecular characterization of
gentian somatic hybrids
A. Trojak-Goluch, A. Depta, M. Agacka – Induction of tetraploids in hop (Humulus lupulus L.)
using in vitro cultures
A. Wiszniewska, A. Pindel – Is that really a step forward in protoplast culture of lupins?
M. K. Wojciechowicz – Anti-mitotic agents in Salix viminalis polyploid plant induction
A. Wojciechowski, J. Janowicz, M. Stanisławska – The effect of genotype, explant type and
medium on the regeneration of linseed (Linum usitatissimum L.) in in vitro culture
A. Wójcik, J. J. Rybczyński – Agrobacterium – mediated genetic transformation
of Gentiana tibetica King
M. Zalewska, N. Miler – Topophysis in adventitious shoots regeneration in vitro
in Chrysanthemum
M. Zawadzka, T. Orlikowska, P. Sobiczewski, A. Mikiciński, M. Sulikowska, E.Zenkteler –
The control of bacterial contaminations during in vitro shoot multiplication
E. Zenkteler, P. Sobiczewski, T. Orlikowska, K. Langer, J. Langer – Application of nanomaterials
to fight and prevent bacteria in vitro
M. Zenkteler – Some remarks on the application of in vitro techniques to manipulation of sexual
reproduction of plants
J. Zimny, S. Oleszczuk, A. Z. Czaplicki, H. Woś, J. Kozdój, S. Sowa, P. Bednarek – Application
of androgenesis in basic research and breeding of cereals
M. Żuk, J. Szopa – Increase of bioactive compounds in flax plants overexpressing enzymes
of flavonoid synthesis pathway
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Posters
E. Andrzejewska-Golec, J. Makowczyńska – Micropropagation of Plantago coronopus L.
M. Arendt, R. Galek, E. Sawicka-Sienkiewicz – In vitro cultures of embryonic axes
of Lupinus mutabilis and L. albus
A. Bach, B. Pawłowska, K. Hura – The effect of the exogenous phenolic compound, caffeic acid
on organogenesis in Galanthus elwesii Hook. cultured in vitro
A. Boba, A. Kulma, K. Kostyn, J. Szopa – Changes in terpenoid level after Fusarium treatment in flax
M. Bogusiewicz, T. Pniewski – Expression of HBV core antigen (HBcAg) in transgenic tobacco
and lettuce for purposes of therapeutic vaccine against hepatitis B
A. Budzianowska, J. Budzianowski – Phenolic compounds in shoot and callus cultures
of Plantago ovata Forssk.
M. Bultrowicz, R. Mól – The use of pollen tube in vitro cultures for determination of nuclei positions
in angiosperm pollen tubes
A. Chuda, A. Adamus – Molecular analysis of interspecific Allium cepa × A. roylei hybrids
J. Ciszewska-Marciniak, M. Jędryczka, J. Przyborowski, E. Zenkteler – Two year survey
on health status of Salix × Populus hybrids in Warmia region
A. Z. Czaplicki, S. Oleszczuk, J. Zimny – Albino regenerants from isolated microspores of wheat
D. Czarnecka, A. Czubacka, T. Doroszewska – Optimization of the regeneration method of
Miscanthus × giganteus in in vitro culture
T. Czuj, M. Żuk, J. Szopa – Changes in sulfur amino acid contents in flax caused by overexpression
of yeast Met25 gene lead to an increase in antioxidant capacity and Fusarium resistance
J. Durok, E. Grzebelus, M. Szklarczyk – Phytosulfokine stimulates development of sugar beet
(Beta vulgaris L.) mesophyll protoplasts
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K. Floryanowicz-Czekalska, J. J. Rybczyński – Gentiana cachemirica Decne in in vitro culture
E. Gabryszewska, L. Kawa-Miszczak – Rooting in vitro and acclimation to the greenhouse
of herbaceous peony plantlets
M. Gliwicka, M. D. Gaj – Expression pattern of ABH1, CBP20 and HYL1 genes during somatic
embryogenesis in Arabidopsis
K. Górecka, D. Krzyżanowska, U. Kowalska, W. Kiszczak, R. Górecki – Obtaining plants from gametic
embryos of red beet – preliminary investigations
K. Górecka, D. Krzyżanowska, U. Kowalska, W. Kiszczak, R. Górecki – Yacon – in vitro propagation
trials
E. Grzebelus, M. Grabka – The effect of cefotaxime and carbenicillin on carrot protoplast cultures
E. Grzebelus, M. Gładysz, E. Widlarz, D. Grzebelus – Cytological and molecular variation of carrot
regenerants obtained through in vitro selection of carrot protoplasts against Alternaria radicina
I. Grzegorczyk, H. Wysokińska – Micropropagation of Scutellaria altissima L.
T. Hazubska-Przybył, P. Chmielarz, M. Michalak, K. Bojarczuk – Induction and proliferation of
embryogenic tissues of Serbian spruce (Picea omorika) and their maintenance in liquid nitrogen
A. Jagielska – Identification of European and Japanese larches and their hybrids based on genetic
markers
A. Kasprowicz, A. Szpitter, A. Maciejewska, M. Kamiński, E. Łojkowska, A. Królicka – In vitro
cultures of Drosera binata as a source of compounds with antimicrobial activity
A. Kawiński, J. Znaniecka, E. Łojkowska – Genetic diversity within population of endangered species
Cypripedium calceolus
A. Kiełkowska, A. Adamus – Morphological evaluation of interspecific F1 (Allium galanthum × A. cepa)
hybrids
M. Kikowska, B. Thiem – Characteristic of the cell suspension culture of Eryngium planum L.
A. Kisiała, A. Niklas-Nowak, M. Orlińska, P. Nowaczyk – In vitro androgenic response of some
pepper (Capsicum) genotypes to different levels of kinetin
M. Klimek, R. Barański – Influence of the ploidy level on growth and organogenesis of sugar beet
(Beta vulgaris L.) in vitro
J. Klocek, G. Costa, H. Mioduszewska – A study of the influence of humic substances on
in vitro potato growth and tuberization
K. Kostyn, M. Czemplik, A. Boba, A. Kulma, J. Szopa – Genes involved in flax pathogenesis
K. Kozak, R. Galek, E. Sawicka-Sienkiewicz – Induced androgenesis in anther culture of
Lupinus angustifolius
A. Krajewska-Patan, M. Dreger, M. Górska-Paukszta, A. Mścisz, S. Mielcarek, M. Baraniak,
W. Buchwald, M. Furmanowa, P. M. Mrozikiewicz – Enhancing of salidroside and rosavin
production in Rhodiola kirilowii callus cultures
H. Kruczkowska, H. Pawłowska, B. Skucińska – Comparison of micropropagation methods of
Polish cultivars of barley and oat
K. Kużdowicz, M. Gośka – Shoot regeneration of perennial wild beet species in vitro
I. Kwiecień, K. Bień, H. Ekiert – Biotransformation of p-hydroxybenzoic acid in in vitro cultures
of Ruta graveolens ssp. divaricata
I. Kwiecień, S. Zubek, H. Ekiert – Studies of the biotransformation of p-hydroxybenzoic acid
in in vitro cultures of Ruta graveolens
A. Luwanska, G. Mankowska, K. Wielgus – Artificial seeds of Linum usitatissimum L.
and Morus alba L. in long-term storage under slow growth conditions
M. Ładyżyński, J. J. Rybczyński – Regeneration of Gentiana cruciata from in vitro shoot tip culture
D. Makowski, A. Mikuła, J. J. Rybczyński – Cryobank of gametophytes of tropical woody fern species
M. Malik, B. Stawiarz – Somatic embryo formation in callus cultures of Narcissus L. 'Carlton'
multiplied in liquid media
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G. Mańkowska, A. Luwańska, K. Wielgus – The analysis of cell ploidy level in the explant tissue
culture of hemp (Cannabis sativa L.) and flax (Linum usitatissimum L.)
I. Marcińska, E. Skrzypek, I. Czyczyło-Mysza, A. Stawicka, M. Pilipowicz – Does a relationship
between the frequency of androgenesis and vernalization requirement exist in wheat?
B. Muszyńska, K. Sułkowska-Ziaja – Analysis of indole compounds in Calocera viscosa mycelium
cultured on liquid medium and in its fruiting bodies
P. Nowaczyk, L. Nowaczyk – Conversion – the critical point of induced embryogenesis
in Capsicum spp.
A. Obarska, K. L. Janczur, B. Tomaszewska – Shoot induction potential of nine Brassica napus
varieties and two types of explants
T. Orlikowska, D. Kucharska, K. Klamkowski, M. Horbowicz, L. Lahuta – Is it possible to select
drought-resistant Rubus genotypes from in vitro populations of seedlings or adventitious shoots?
M. Ożarowski, B. Thiem – The effect of cytokinins on in vitro morphogenesis of Passiflora
caerulea L.
B. Pawłowska, A. Bach – Cryopreservation of in vitro grown shoot buds of rose 'New Dawn' using
encapsulation-dehydration method
A. Płażek, F. Dubert – Optimization of medium for callus induction and plant regeneration
of Miscanthus × giganteus
B. Płoszaj, J. Przyborowski – Initiation of an in vitro culture of elder (Sambucus nigra L.) meristems
with the use of various sterilizers
B. Płoszaj, J. Przyborowski – The effect of kinetin content in the culture medium on micropropagation
of elder (Sambucus nigra L.)
M. Podwyszyńska, A. Kuras, M. Korbin – ISSR analysis of genetic stability of Polish tulip cultivars
propagated in vitro
M. Podwyszyńska, E. Gabryszewska, M. Korbin, A. Jasiński – Evaluation of somaclonal variation
in micropropagated Hemerocallis sp. plants using phenotype and ISSR markers
A. Ponitka, A. Ślusarkiewicz-Jarzina, J. Woźna, H. Pudelska – Frequencies of spontaneous doubled
haploids of winter triticale plants obtained by anther culture
M. Popielarska-Konieczna, M. Braś, K. Rzenno, M. Łojewski, I. Marcińska – Isolation and in vitro
culture of endosperm tissues in selected monocot and dicot species
A. Ptak, J. Gądek – Micropropagation of Leucojum aestivum in a temporary immersion bioreactor
system (RITA)
D. Raj, A. Kokotkiewicz, A. Skorys, M. Łuczkiewicz – Preliminary analysis of polyphenolic fraction
from intact plant and in vitro cultures of Securinega suffruticosa
M. Rojek, M. Dydak, J. Kwasniewska, K. Walas, E. Wolny, K. Nowak, J. Małuszynska –
Agrobacterium rhizogenes serves cytogenetics
M. Rokicki, A. Wojciechowski – Use of wide crossing and in vitro culture for the induction of haploid
embryogenesis in three cereal species
E. Skrzypczak-Pietraszek, J. Pietraszek – Phenolic acids in in vitro cultures of Exacum affine
Balf. f.
E. Skrzypek, I. Marcińska, A. Stawicka, I. Czyczyło-Mysza, M.Pilipowicz – Induction of androgenesis
in oat (Avena sativa L.) depending on kind of culture medium
D. Sochacki – Evaluation of phenotypic trueness-to-type for selected cultivars of narcissus propagated
by in vitro cultures
A. Stojakowska, J. Malarz – Methyl jasmonate and fosmidomycin affect mono- and sesquiterpenoid
production in root cultures of Inula royleana DC. and Inula macrocephala
Boiss. & Kotschy ex Boiss.
K. Sułkowska-Ziaja, B. Muszyńska – Mycelial cultures of some Aphyllophorales (Basidiomycetes):
optimalization of in vitro culture conditions
L. Szała, A. Olejnik, T. Cegielska-Taras – Molecular characterization of resynthesized oilseed rape
(Brassica napus L.)
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M. Sztajnert, K. Ratajczak, A. Kulma, J. Szopa – Anti-inflammatory properties of transgenic flax
fiber extract
M. Szurman, M. Gliwicka, M. Gaj – Effect of 5-azacitidine on DNA methylation pattern and somatic
embryogenesis in Arabidopsis
W. Szypuła, J. Budziszewska, D. Delbani – Identification of culturable and non-culturable
endophytic fungi isolated from shoots of Huperzia selago (L.) Bernh. ex Schrank & Mart.
(Lycopodium selago L.)
H. Ślesak, G. Góralski, P. Mizia, D. Kwolek, A. Joachimiak – Plant regeneration from liquid root
culture of Rumex acetosa L. Analysis of genetic variability: preliminary studies
J. Tarwacka, A. Szczerbakowa, B. Wielgat – Cytogenetic characteristics of the interspecific somatic
hybrids of Solanum villosum (+) S. tuberosum
B. Thiem, M. Kikowska, I. Paluch – Bioactive secondary metabolites in in vitro cultures of
Eryngium alpinum L.
M. Tomaszewska-Sowa – The regeneration of sugar beet (Beta vulgaris L.) plants from unfertilized
ovules cultured in vitro
A. Trejgell, A. Tretyn – The effect of cytokinins on morphogenetic responses of Polemonium caeruleum
seedling explants
A. Trojanowska, M. D. Gaj – Capacity for shoot organogenesis in cultures of Arabidopsis
hormone-response mutants
A. Turczynowska, Z. Broda – Evaluation of regeneration efficiency of selected double haploid lines
of rape (Brassica napus ssp. oleifera)
M. Wajand, A. Ledwon, M. D. Gaj – Efficient shoot organogenesis induced in the culture of immature
zygotic embryos of Arabidopsis
M. Warchoł, F. Dubert, T. Kusibab – In vitro culture of Cordyline australis (G. Forst) Endl.
I. Weremczuk-Jeżyna, H. Wysokińska – In vitro culture of Dracocephalum moldavica
A. Wojtania, E. Gabryszewska – Influence of growth regulators and environmental factors on shoot
multiplication of Camellia japonica in vitro
W. Wojtasik, K. Kostyn, A. Kulma, J. Szopa – Pectin metabolism in Fusarium-infected flax seedlings
T. P. Wyka, K. Ludwiczak – Regeneration of shoots from root explants in Mammillaria carmenae
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Castaneda (Cactaceae)
Ł. Zarychta, M. Zenkteler, A. Karmowska, E. Zenkteler – Intergeneric hybridization between
Salix fragilis and Populus spp. in vivo and in vitro
M. Zawadzka, T. Orlikowska – The influence of iron source in red raspberry cultures on
the chlorophyll contents and histology of the leaves
P. Zelazko, K. Kromer – In vitro reproduction of Drosera rotundifolia and conservation plant
genetic resources of this species
A. Źróbek-Sokolnik, C. Hołdyński, K. Górska-Koplińska – Propagation of Chamaedaphne calyculata
(L.) Moench via indirect somatic embryogenesis
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LECTURES
LECTURES
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Plant biotechnology and in vitro culture – the fulfilled hopes?
Stefan Malepszy
Department of Plant Genetics, Breeding and Biotechnology, Warsaw University of Life Sciences – SGGW,
Nowoursynowska 159, 02-776 Warszawa, e-mail: [email protected]
The in vitro culture is an intrinsic component of a single
area of plant biotechnology, the one dealing with cloning.
The in vitro industry maintains a strong position in the
economy and is developing well. Cloning is also a stage
required for recovery of genetically modified plants but
in this regard it is highly imperfect. There are also fields
of biotechnology in which prospects of utilizing solutions
offered by in vitro cultures once appeared very promising but have so far not been fulfilled, and it seems that
things will remain that way. What are the reasons of this
failure? Did we mis-estimate the potential of in vitro cultures? Is it rather the result of a more general trend, or
do the causes lay entirely somewhere else?
The lecture will attempt to address these questions
by analyzing specific circumstances and experimental
data. It will also try to highlight key problems within the
field of in vitro cultures, that, if solved, could likely benefit particular subdisciplines of biotechnology.
What's new in the control of bacterial contamination in plant tissue cultures?
Teresa Orlikowska1, Piotr Sobiczewski1, Elżbieta Zenkteler2
1
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice,
e-mail: [email protected]
2
Department of General Botany, Institute of Experimental Biology, Faculty of Biology, A. Mickiewicz University,
Umultowska 89, 61-614 Poznań
There are three main issues in the control of bacterial
contaminations in plant tissue cultures: 1. preventing
the introduction of bacteria with the initial plant material, 2. preventing the introduction of bacteria from the
environment during subculturing, 3. reducing bacterial
contamination in the cultures at the stage of shoot multiplication and rooting.
Problems with bacterial contamination in in vitro
cultures will probably never be fully resolved due to the
unlimited quantity and diversity of bacteria in the environment. At present, the lack of satisfactory characterization of bacteria often makes their identification and
control impossible. Furthermore, bactericidal compounds are often damaging to plant tissue cultures.
The most effective way of preventing bacterial contamination in vitro is elimination of bacteria from the
initial plant explants that are introduced into the cultures. This is not easy, especially with endogenous
bacteria, which are perfectly adapted to their hosts.
The most promising methods involve the use of
precultures of donor plants under a strict sanitary
regime and efficient sterilization of the initial
explants, reduction of the size of the initial explants
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just to meristematic tips, and early detection of bacteria in the isolated tissue.
In plant tissue cultures propagated for a long time
or in cultures stored over long periods, it is possible
for bacteria from the laboratory and human environment to accumulate. Such microorganisms can occupy
their own niche in the culture vessels or on/in explants.
Occasionally, they can also be sources of human
pathogens. In such cases, the most effective solution is
careful and frequent inspection of the cultures and the
maintenance of a reserve of clean cultures under strictly protected conditions.
Sometimes, bacteria emerge in great quantities at
the stage of intensive shoot multiplication or rooting,
affecting a large number of microplants. In this case,
the obligatory procedure is to eliminate cultures containing the deleterious bacteria (including pathogenic
species) and to add bactericidal compounds, that are
not detrimental to the explants but that can diminish
the bacterial population, to the media of the other cultures.
In the lecture we will present illustrations from the
most recent reports on this aspect of plant culture.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
LECTURES
September 9–11, 2009, Poznań, Poland
FlaxAid, a new wound dressing based on transgenic flax products
Katarzyna Skórkowska-Telichowska1,2, Magdalena Żuk1, Anna Kulma1, Ada Bugajska- Prusak1,2,
Katarzyna Ratajczak1 and Jan Szopa1
1
Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63, 51-147 Wroclaw, Poland
Department of Endocrinology and Department of Dermatology, IVth Clinical Military Hospital, Weigla 5,
50-981 Wroclaw, Poland
2
Recent reports have indicated that oxidative stress
could be an important mechanism aggravating the
chronic wound progression. It has been suggested that
reactive oxygen species are responsible for chronic
wound pathogenesis and anti-healing processes
because of the reduction in proliferating capacity of
wound cells exposed to oxidative stress. Oxidative
stress causes damage to cellular macromolecules,
down-regulates key proteins involved in DNA replication, de-regulates the cell cycle, increases the cellular
resistance to oxidative stress and promotes apoptosis
in wound fibroblasts. Multidirectional analysis has also
revealed that chronic wound fibroblasts have a
decreased ability to withstand oxidative stress. In
agreement with this is the report providing evidence
that antioxidative compounds, such as catechin, promote the healing of chronic gastric ulcers.
In the present study we propose a new material and
method of treatment of trophy lesions based on products from transgenic flax plants. The transgenic plants
overproduced antioxidative compounds of different
types. Characteristic features of those plants were an
increase in phenolic contents in fibres and unsaturated
fatty acids in seeds, and a strong increase in lignan in
seedcake. It was expected that a coordinated use of
fibres, oil emulsion and seedcake extract from transgenic plants will promote the healing of chronic skin
ulceration. Therefore the primary goal of this study
was to investigate the clinical improvement in chronic
trophy lesions by investigating changes in wound exudation and the proportion between fibrin level and
granulation level. Another goal of this study was to test
the effect of FlaxAid wound dressing on wound size and
the pain usually accompanying chronic ulceration.
FlaxAid wound dressing is a three-component product based on fibres, oil emulsion and seedcake extract
from genetically engineered flax plants. All these materials are derived from two types of transgenic flax
plants which were obtained by plant transformation
using three genes controlling the synthesis of antioxidative compounds from phenylpropanoid pathway and
genes responsible for hydrophobic polymer synthesis.
Simultaneous transformation of flax explants with three
genes coding for chalcone synthase (CHS), chalcone isomerase (CHI) and dihydroflavonol reductase (DFR)
resulted in accumulation of phenolic acids in fibres,
polyunsaturated fatty acids in oil and lignans in seedcake. Simultaneous expression of three bacterial genes
in flax vascular bundles resulted in synthesis of cellulose-polyhydroxybutyrate composite fibres. Those three
products (fibres, oil, and seedcake) of transgenic flax
that contained a broad spectrum of antioxidative compounds were tested for cytotoxicity and were sequentially used for chronic wound healing. None of these products showed negative effect on growth and morphology
of Balb/3T3 cells in the cytotoxic assay.
In this report we present the effects of linen dressing treatment alone and in combination with oil emulsion and seedcake extract on chronic wound healing in
patients. A twelve week application of FlaxAid wound
dressing gave a faster healing and, specifically, a reduction in wound exudates and wound size. In several
cases wound healing was completed during the period
of investigation. Interestingly and importantly, FlaxAid
wound dressing diminished the pain accompanying
chronic venous ulceration as reported by patients. The
molecular mechanism of this effect was based on the
action of cannabinoid (CBD), respective CB2 receptor
activation and suppression of pro-inflammatory
cytokine.
The beneficial effect of FlaxAid wound dressing on
wound healing is reported here for the first time.
GMO – A.D. 2009
Tomasz Twardowski
Institute of Biorganic Chemistry PAS, Noskowskiego 12, 61-704 Poznań
Modern biotechnology plays a critical role in the knowledge based bio-economy. For further development of
bio-economy we need science and transfer of innovation from academia to industry as well as supportive
legislation and public acceptance. Majority of members
Vol. 51, suppl. 1, 2009
of the Polish society are against genetic engineering and
majority of experts are "pro" innovative technology. The
Government is in favor of "GMO free zones" in contrast
to the EU Commission's support for GMO.
Quo vadis Polish biotechnology?
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LECTURES
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Biology of cell wall-plasma membrane-cytoskeleton continuum in plants
Przemysław Wojtaszek1,2, Ewelina Rodakowska1, Anna Kasprowicz1, Agnieszka Szuba2,
Daniel Kierzkowski1, Paweł Zawadzki1, Michalina Maruniewicz1, Magdalena Wierzchowiecka1,
Anna Kapczyńska1, Iga Kosicka1, Michał Michalak1
1
Department of Molecular and Cellular Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89,
61-614 Poznań, email: [email protected]
2
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Poznań
Cell wall-plasma membrane-ccytoskeleton (WMC)
continuum in plants is one of the major integrators of
the structure and function of plant cells. Although
spread throughout the cell, it also smoothly amalgamates into higher level structures, such as the
apoplast, and thus constitutes an important determinant of plant's organismal features. Owing to the cell
walls, it constitutes a major barrier between protoplasts and their surroundings, but it also is a dynamic entity participating in sensing of and responses to internal
and external signals.
Recent years have brought many interesting observations demonstrating the importance of WMC continuum in plants. Here, several major aspects will be presented and discussed. First, the continuum is deeply
involved in signalling processes, among them those
that affect the fate of individual cells as well as those
that influence the growth and development of the whole
plant. Moreover, more and more signalling molecules
are identified that originate from the walls. Second, the
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cell walls should no longer be considered as an invariant structure around the protoplast. They undergo
recycling and remodelling, enabling a precise regulation of mechanical and chemical properties of the various wall domains. These processes are under a tight
control by the cytoskeleton, and involve also intensive
vesicle trafficking. Third, the continuum seems to constitute the major sensor and transducer of mechanical
stimuli. This enables the control of the cell shape and,
to some extent, function which finally results in modulations of cellular fate. All these aspects will be illustrated with data coming from the work in our lab.
We would like to acknowledge the funding by the
Ministry of Science and Higher Education grants PBZMNiI-2/1/2005, N302 028 32/2243, PBZ-MNiSW2/3/2006, N303 294434, N301 164435, N302 430034,
N301 157035 and N303 360735. AK, IK, and MM participated in this work while being the students of
Biotechnology at AMU Faculty of Biology.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Integration of in vitro culture, biotechnology and molecular biology in modern
breeding of oilseed rape
Teresa Cegielska-Taras, Iwona Bartkowiak-Broda
Plant Breeding and Acclimatization Institute, Department of Genetics and Breeding of Oilseed Crops, Strzeszynska 36,
60-479 Poznan, Poland, e-mail: [email protected]
During the past 30 years, oilseed rape (Brassica napus L.)
has become an important world crop. In Poland and in the
European Union winter oilseed rape is the leading source of
edible vegetable oil and high-protein meal. The increase of
oilseed rape production during the last decades has been
due to the considerable improvement of seed quality as well
as yielding ability. The progress in the development of high
yielding and good quality varieties has been achieved owing
to the essential progress in the cellular and molecular biology of Brassica napus in the last years.
The production of haploids and doubled haploids
using microspore culture has accelerated the obtainment
of homozygous lines in oilseed rape breeding. The use of
molecular markers in marker assisted selection and
genetic transformation technique for introduction of desirable traits using doubled haploids have been improved.
Oilseed rape reveals a high level of heterosis, which is
the reason that breeders are interested in the development
of F1 hybrid varieties. Commercial production of F1 seeds
requires an effective cross pollination system. Molecular
selection of suitable genotypes, also applied to doubled
haploid lines, accelerates the production of genetically
uniform lines, especially parental lines needed for the
hybridization systems.
Genomics has been recently added to the wide range of
techniques used for development and evaluation of new
cultivars.
Brassica napus lacks many desirable traits, especially resistance to biotic and abiotic stresses, which causes
considerable economic losses. In this case, asymmetric
somatic hybridization has been carried out to achieve a
succesful introgression of desired genes. The offspring of
resynthesized oilseed rape originating from interspecific
hybridization between appropriate forms of Brassica
rapa and Brassica oleracea (in combination with embryo
rescue culture) represents a potentially important
resource to expand the genetic diversity of the narrow
gene pool of Brassica napus.
These problems will be discussed using examples of
investigations conducted at the Department of Genetics
and Breeding of Oilseed Crops of the Plant Breeding and
Acclimatization Institute in Poznań.
Obtaining tobacco double haploids containing different sources of resistance to PVY
Anna Czubacka, Teresa Doroszewska
Department of Plant Breeding and Biotechnology, Institute of Soil Science and Plant Cultivation – National Research
Institute, Czartoryskich 8, 24-100 Puławy; e-mail: [email protected]
Potato virus Y (PVY), especially its necrotic strains, is a
dangerous pathogen of tobacco. It causes necrosis of
nerves and leaf laminas leading to a significant yield
reduction. Brown vein necrosis of tobacco caused by PVY
is an important problem because new isolates able to
break resistance are still occurring. One of resistance
sources is a wild species Nicotiana africana that shows
resistance to all tested PVY isolates. Breeding BPA lines
derived from crossing N. tabacum cv. BP-210 ×
N. africana are tolerant of many PVY isolates. Another
source of resistance are transgenic tobacco lines – MN944
LMV (cultivar Mac Nair containing gene of lettuce mosaic
virus coat protein) and AC Gayed ROKY2 (cultivar AC
Gayed containing gene of PVY replicase). To increase the
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level of resistance by combining these sources of resistance, hybrids coming from crosses between transgenic
line MN 944 LMV and line BPA as well as MN 944 LMV
and AC Gayed ROKY2 were obtained. The most resistant
hybrids were used for producing haploids via androgenesis. Immature anthers were put on Nitch & Nitch medium.
Regenerated plants were analyzed with respect to the presence of transgenes and ploidy level. They were also inoculated with PVY. Most of the haploids showed to be resistant to the virus. Some of them were tolerant and only a
few were susceptible. Doubled haploids were obtained by
regeneration from stem fragments of resistant haploids.
The ploidy levels of regenerated plants were estimated by
flow cytometry.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
September 9–11, 2009, Poznań, Poland
The use of multi-qPCR platform and tan1 mutant in identification of TF genes
involved in somatic embryogenesis in Arabidopsis
Marta Gliwicka1, Salma Balazadeh2,3, Camila Caldana3, Bernd Mueller-Roeber2,3 and Małgorzata D. Gaj1
1
Department of Genetics, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland
Institute for Biochemistry and Biology, University of Potsdam, 24-25 Karl-Liebknecht Str., Haus 20,
14476 Potsdam-Golm, Germany
3
Max Planck Institute of Molecular Plant Physiology, 1 Am Muehlenberg , 14476 Golm, Germany
[email protected]; [email protected]; [email protected]; [email protected];
[email protected]
2
Somatic embryogenesis (SE) induced in vitro provides a
valuable method for plant regeneration and is widely used
in micropropagation and genetic transformation of plants.
Thus, determination of genetic factors involved in triggering, in somatic cells, of the new, embryogenic mode of
development is of practical value. The change in cell development requires a global rearrangement of the genetic
program. Regulatory genes, especially transcription factors (TF), are believed to play a key role in this process.
Here, we present the results of an experiment, in which
the multiplex qPCR platform for Arabidopsis TFs was
used to identify genes involved in the induction phase of
SE. Two Arabidopsis genotypes differing in capacity for
SE under in vitro culture were used: the tanmei/emb2757
embryonal mutant unable to undergo SE and its parental
ecotype Col-0 with a high capacity for somatic embryo formation. RNAs were isolated from freshly isolated explants
and explant-derived cultures on 5th and 10th days of in
vitro culture and the transcriptoms of the analyzed genotypes were compared in three biological replicates. The
relative expression levels of 1920 known Arabidopsis TF
genes were monitored with the use of qPCR reactions and
UBQ10 gene as standard control. It was found that only
less than 6% of the analyzed TF genes were not expressed
at any of the analyzed culture samples and over 78% of the
TFs showed differences in activity between the compared
genotype/culture time point combinations.
We found that transcriptoms of the Col-0 and mutant
explants differed significantly and over 26% of the analyzed TFs displayed at least 5-fold up- or down-regulation
in the compared tissues. The analysis indicated that 86 of
all analyzed TFs were significantly up- or down-regulated
(at least 5 fold) at different time points of embryogenic
cultures derived from the compared genotypes. Among
the differentially regulated genes, bHLH, MYB and MADS
TF families were found to be the most frequently represented. Further experiments are conducted, in which
nine of the selected genes (At1g24625, At1g27740,
At1g31040, At1g79580, At2g26320, At3g04030,
At3g56970, At5g24330, At5g65050) are analyzed in
terms of their potential functions in SE.
In vitro culture of Miscanthus sinensis anthers
Katarzyna Głowacka, Stanisław Jeżowski
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, Poland,
e-mail: [email protected]
Species of Miscanthus are perennial, rhizomatous grasses
originating from Asia, that have the potential for very high
rates of growth. Because of perenniality and C4 photosynthetic pathway, species from the genus Miscanthus possess several characteristics that make them favourable
crops for efficient biomass production. The focus of this
study was the induction of androgenesis in culture of
Miscanthus sinensis anthers. Genetic (genotype) and nongenetic (cold pre-treatment, induction medium, date of
beginning of anther culture) factors influencing androgenesis were studied. We were successful in inducting androgenesis in M. sinensis anther culture. Examination of acetocarmine squashes prepared from anthers from a sixweek-old culture showed up to 15 nonsynchronously
developing pollen-derived polycellular structures. The calVol. 51, suppl. 1, 2009
lus yield was strongly affected by the genotype. A beneficial
influence of cold pre-treatment of spikes on the induction
of androgenesis was observed. The results suggest that the
high callus yield might be caused by the late initiation of
culture. Initiating anther cultures at the end of the flowering season caused a significant increase in callus yield in
comparison to cultures initiated in the height of flowering
season. It is likely, however, that the improved efficiency
of androgenesis induction in the case of M. sinensis
anther culture initiated in autumn could be related to a
positive influence of growing donor plants under the conditions of cooler and shorter days, i.e. 11-h days with temperature around 11°C and 13-h nights with temperature
around 5°C.
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ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
In vitro selection of varieties and lines of Brassica napus L. resistant
to multiple heavy metals
Katarzyna L. Janczur1, Agata Obarska1, Piotr Kachlicki2, Barbara Tomaszewska1
1Department of Biochemistry, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland,
e-mail: [email protected]
2
Laboratory of Resistance Genetics, Institute of Plant Genetics, Polish Academy of Science, Strzeszyńska 34,
60-479 Poznań, Poland, e-mail: [email protected]
Heavy metal contamination presents a serious problem
worldwide. The existing methods of decontamination are
usually not only expensive but also invasive to the environment. Phytoremediation – a group of methods of pollutant
removal based on living plants grows in significance as a
valuable alternative. Among other methods phytoextraction,
the technology of extracting the contaminating agents from
soils through the plant root system is the most applicable
as far as heavy metal contamination is concerned.
Particular traits such as: high heavy metal accumulation
and translocation to aboveground parts, high biomass
production and well known agricultural techniques are
required for the plant to be useful for such purpose and
only a limited number of plant species can be used in phytoextraction processes (Vera-Estrella et al., 2009).
Therefore there is an increasing need to understand the
defense mechanisms that make a plant tolerant to heavy
metals as well as to directly know which plants are particularly useful. This requires testing of different species and
varieties for heavy metals resistance (Nehnevajova et al.
2007). In vitro cultures of plants constitute a powerful tool
to achieve such aim. One of the plant species known for its
relatively high tolerance and accumulation is rapeseed
(Brassica napus). In our research we tested nine Brassica
napus cultivars towards resistance to Zn, Pb, Cu and Cd
applied in combination. From each cultivar, two types of
explants – hypocotyl and petiole were cultured on MS
medium supplemented with hormones (0.15 mg/l NAA and
3 mg/l BAP), AgNO3 (2.5 mg/l) and ZnSO4, PbNO3, CuSO4
and CdSO4 at the concentrations of 30 μM, 60 μM, 7 μM
and 7 μM, respectively in order to obtain multi-heavy metal
– resistant lines from regenerated plants. By measuring the
shoot induction frequency and effectiveness parameters we
showed that the potential to regenerate on medium supplemented with heavy metals differs significantly among the
different B. napus cultivars.
REFERENCES
VERA-ESTRELLA R, MIRANDA-VERGARA M, BARKLA BJ. 2009. Zinc tolerance and accumulation in stable cell suspension cultures
and in vitro regenerated plants of the emerging model plant
Arabidopisi halleri (Brassiceae). Planta 229: 977–986.
NEHNEVAJOVA E, HERZIGR, ERISMANN KH, SCHWITHGUEBEL JP. 2007.
In vitro breeding of Brassica juncea L. to enehance metal
accumulation and extraction properties. Plant Cell Reports
26: 429–437.
Enzymatic markers of rhizogenic competence in in vitro culture of common ice plant
Robert Konieczny1, Marta Libik2, Ewa Surówka2, Agnieszka Nosal1, Zbigniew Miszalski2,3
1Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Cracow, Poland,
e-mail: [email protected]
2
Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland
3
Institute of Biology, Pedagogical University, Podbrzezie 3, 31-054 Cracow
Hypocotyl explants of Mesembryantheum crystallinum L.
were maintained on root induction medium for 1, 3, 5, 7,
10 and 14 days followed by subculture onto shoot induction
medium for 14 days. Using tissue transfer experiments,
rhizogenic determination was found to occur on the 5 day
after explantation. Histological studies revealed the presence of clumps of meristematic cells at the time of determination whilst roots with root caps were observed after 7
days of culture. The endogenous concentration of H2O2
changed throughout the culture, reaching a peak on day 3,
followed by a gradual decrease till day 14. The activity of
catalase (CAT) decreased after day 1 and became constant
till day 5. Next, a significant increase in its activity was
observed, followed by a slight decrease on days 10 and 14.
An increase in CAT activity was concomitant with the occurrence of its additional form CATII on the 5th day of culture.
The pattern of activity of superoxide dismutases (SODs)
was similar for CuZnSOD, FeSOD and MnSODI forms
throughout the culture, being generally slightly higher (days
3, 5, 7) or similar (days 10 and 14) to control. On the 5th
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day of culture, that is at the time of induction of rhizogenic
determination, an additional band of activity of MnSODII
occurred. Its intensity remained constant until day 14.
Fumarase activity and general respiration intensity reminded constant, with the exception of day 14 when a conspicuous increase was noted, while cytochrome c oxidase activity altered throughout the culture and corresponded with
the photosynthetic activity measured as chlorophyll a fluorescence. A decrease in cytochrome c oxidase activity as
well as in fluorescence parameter of chlorophyll a correlated with a slight increase of SOD activity on the 3rd day and
CAT activity on 5th day suggesting alterations in ROS levels
that preceded rhizogenesis. In summary, the occurrence of
specific forms CATII and MnSODII did not seem to be
directly related to metabolic changes accompanying regeneration but rather corresponded with the tight regulation of
endogenous H2O2 level, itself possibly directly involved in
the regenerative process.
Acknowledgements: Financial support of MNiSW (Project No
303356935) is acknowledged.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
September 9–11, 2009, Poznań, Poland
Use of biotechnology for conservation of a critically endangered population
of alpine saxifrage (Saxifraga nivalis L.)
Krystyna Kromer1, Andrzej Raj2, Bronisław Wojtuń3, Dorota Poturała1
1
Botanic Garden, University of Wrocław, Sienkiewicza 23, 50-335 Wrocław, Poland, [email protected]
Karkonoski National Park, Chałbińskiego 23, 58-570 Jelenia Góra, Poland, [email protected]
3
Department of Botany and Plant Ecology, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 24a,
50-363 Wrocław, Poland, [email protected]
2
Saxifraga nivalis is an arctic-alpine species that inhabits
rocks in the upper sub-alpine zone. In central Europe it is
known from only one location in the Karkonosze Mountains,
where it grows in crevices and windings of basalt rocks in
the glacial circus of Mały Śnieżny Kocioł. The population is
endangered because of the restricted cross pollination, small
production of viable seeds and poor vegetative reproduction
(Fabiszewski, 2001). Considering the threats to the existing
population, our efforts focus on successful maintenance of
this population and creation of supplementary habitats to
preserve the genetic pool of Polish representatives of the
genus Saxifraga. Sterilized seeds were sown on 1/2MS
medium and the cultures were kept at 25°C. After 3
months, seedlings reached 1.5 cm and their further
growth was examined under various temperature regimes
(4–25°C) and light intensities (20–80 μmol quanta m-2 s-1).
The impact of mineral nutrition and phytohormones on
growth and propagation was also evaluated. The number
of stimulated axillary buds fluctuated from 5 to 10 per
rosette on the basal medium under controlled growth conditions. Comparison of plant growth at supplementary
locations, i.e. in the Wrocław Botanical Garden (120 m
a.s.l.) and at the Jagniątków nursery of Karkonoski
National Park (650 m a.s.l.) showed that plants performed
perfectly well at the higher altitude.
Autonomous endosperm induction in cultured unpollinated ovaries is strongly
species dependent
Elżbieta Kuta1, Joanna Rojek2, Agnieszka Pawełek-Skoczylas1, Błażej Ślązak1, Jerzy Bohdanowicz2
1
Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Cracow, Poland,
e-mail: [email protected]
2
Department of Genetics and Cytology, University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland,
e-mail: [email protected]
In sexually reproducing angiosperms embryo and
endosperm development is initiated by double fertilization
and a ratio of 2 maternal genomes to 1 paternal genome
(2m:1p) is essential for endosperm formation. However,
there is a group of taxa (autonomous apomicts) representing fertilization-independent embryo and endosperm
development. Autonomous endosperm (AE) can be
induced experimentally with the use of irradiated pollen
and also through in vitro culture of unfertilized ovules or
ovaries. These results could be very helpful in understanding the mechanisms leading to endosperm development
with no involvement of paternal genome and therefore how
genomic imprinting can be overcome in sexually reproducing flowering plants.
Unpollinated pistils, ovaries, ovules of several wild (Viola
tricolor, Salix viminalis, Arabidopsis thaliana var. Columbia
and Landsberg, Alisma pantago-aquatica, Sedum acre,
Gagea lutea, Anemone nemorosa, A. ranunculoides
Capsella bursa-pastoris), cultivated (Lycopersicon esculentum cv. Adelajda, Cunero, Havana, Maeva, Shirley; Brassica
napus cv. Topas, var. Feliks, Synapis alba) and ornamental
taxa (Viola xWittrockiana, V. cornuta, Calendula officinalis,
Rudbeckia bicolor), representing annual, perennial and
woody life forms were cultured on hormone-free MS medium
(with different sucrose concentrations) as well as on media
supplemented with plant growth regulators: 2,4-D, NAA,
BAP, KIN, IAA, GA3, homoBl (homobrassinolide) in different
concentrations and combinations.
Vol. 51, suppl. 1, 2009
Partial AE development (or endosperm like structure)
was induced in unpollinated ovules on hormone-free MS
medium as well as on media supplemented with growth
regulators but the frequency was species not medium
dependent. Lack of induction was noted in V. tricolor,
V. xWittrockiana, V. cornuta, Alisma pantago-aquatica,
Sedum acre, Gagea lutea, Anemone nemorosa, Brassica
napus var. Feliks, Synapis alba; sporadically was
observed in Lycopersicon, Rudbeckia, Calendula, Salix
Capsella bursa-pastoris and Anemone ranunculoides.
The highest frequency of AE formation was found in
Arabidopis thaliana (45% of ovaries have ovules with AE)
but neither cellularization nor differentiation on specific
regions typical for endosperm of wild-type Arabidopsis,
resulting from fertilization were observed.
REFERENCES
ROJEK J, KUTA E, PRZYWARA L. 2002. Autonomous endosperm
development in unpollinated ovaries of Brassica napus L. cv.
Topas cultured in vitro. Acta Biologica Cracoviensia Series
Botanica 44: 195-202.
ROJEK J, KUTA E, BOHDANOWICZ J. 2005. In vitro culture promotes
partial autonomous endosperm development in unfertilized
ovule of wild-type Arabidopsis thaliana var. Columbia.
Sexual Plant Reproduction 18: 29-36.
KAPUSTA M, ROJEK J, BOHDANOWICZ J. 2007. Induction of
autonomous endosperm development in ovules of unpollinated pistils of Arabidopsis thaliana var. Landsberg cultured in vitro. Acta Biologica Cracoviensia Series Botanica
49/2: 53-59.
19
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Liquid nitrogen efficacy in protection of genetic stability in plant material
Anna Mikuła, Jan J. Rybczyński
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, e-mail: [email protected]
Cryopreservation is important for long-term conservation of plant genetic resources, especially for species producing recalcitrant seeds and crops that are propagated
clonally through grafting or through vegetative cuttings,
suckers, roots, tubers and bulbs. Up to now, it has been
successfully used for over 100 plant species. Despite the
fact that in the temperature of liquid nitrogen the cell
division and metabolism are arrested, the factors associated with cryotreatment, cryostorage or plant recovery
could become sources of somaclonal variability. The lecture will attempt of assess the state of knowledge concerning the influence of cryopreservation on the morphogenic capacity of tissues and genetic integrity of
plants.
Assessment of genetic and epigenetic stability of the
recovered plants derived from cryopreserved plant material is an important step to success of any storage protocol. Determination of the plant's "trueness-to-type" after
cryopreservation can be carried out at the phenotypic, histological, cytological, biochemical and molecular levels.
Several molecular markers based on different techniques
(RFLP, RAPD, AFLP, MSAP, SSR) have been applied. The
results suggest that the processes of cryoprotection and
cryostorage have an impact on DNA methylation status
and could lead to alterations in chromatin structure and
changes in gene expression. However, majority of studies
have reported insignificant or null influence of cryopreservation on the plant material.
Application of genetically modified trees in forest plantations
Justyna Nowakowska
Department of Genetics and Forest Tree Physiology, Forest Research Institute, Sękocin Stary, Braci Leśnej 3,
05-090 Raszyn, Poland, e-mail: [email protected]
Wide use of forest-tree products and progressive deterioration of natural forests make the development of new
sources of wood supply necessary. Human activities and
global climate change make forest ecosystems more prone
to biotic and abiotic agents. Some of the measures taken
to control them rely on achievements of biotechnology.
For several years, genetic engineering has been applied to
create modified trees in order to enhance their growth,
quality of wood, susceptibility to pathogens and pest
resistance. Efforts have concentrated mostly on Populus
sp. and Eucalyptus sp. whose genomes are considered to
be easily transformed via the traditional Agrobacterium
20
tumefaciens mediated method or by particle bombardment. Other tree species, i.e. spruce, pine and birch have
also been studied in the field of molecular engineering,
with main focus on the introduction of cry and aro genes,
conferring insect and herbicide resistance in forest-tree
plantations.
The paper discusses some problems in GMO foresttree breeding and summarizes recent achievements in the
transformation of tree species, in the context of possible
applications for improving and introducing novel traits
into forest plantations.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
September 9–11, 2009, Poznań, Poland
Efficient plant regeneration of Trifolium nigrescens via somatic embryogenesis
Maria Pilarska, Robert Konieczny, Monika Tuleja, Elżbieta Kuta
Department of Plant Cytology and Embryology, Jagiellonian University, 52 Grodzka str., 31-044 Cracow, Poland,
e-mail: [email protected]
This study presents an efficient protocol for regeneration
of Trifolium nigrescens Viv. plants via somatic embryogenesis (SE). The immature zygotic embryos at torpedo
(TsE) and cotyledonary (CsE) stage were cultured on
EC6 (TsE) and MS (CsE) solid media supplemented with
auxins and cytokinins. The type of explant and the combination and concentration of plant growth regulators
were the key factors for the induction of somatic
embryos. SE was observed directly during culture of TsE
on media supplemented with 2, 4 or 8 mg/l 2,4-D.
Cytokinin (kinetin or 2iP) and auxin (2,4-D or NAA)
alone did not induce somatic embryos from CsE. The
combination of 2 mg/l 2iP or kinetin along with 2,4-D or
NAA had beneficial effect for somatic embryo induction
from CsE, but the addition of cytokinin was inhibitory
for SE induction from TsE. The highest frequency of SE
(up to 100%) was obtained from CsE cultured on medium supplemented with 4 mg/l 2,4-D and with 2.0 mg/l
2iP. The appearance of somatic embryos was concomitant with the inhibition of shoot and root growth of the
original embryo. After 3–5 days the explants became
swollen. The first somatic embryos appeared directly
from upper part of the hypocotyl after 7 days of culture
(TsE and CsE) and margin of cotyledons (TsE). After 10
days the explants started to produce embryogenic callus.
Somatic embryos induced from TsE became necrotic after
10–18 days of culture. Most of the embryoids induced
from CsE and maintained on media containing 2,4-D and
kinetin or 2iP displayed several morphological abnormalities such as: fusion of embryonic axes, elongated
hypocotyls, fused cotyledons or additional cotyledons.
Subculturing somatic embryos on hormone-free medium
resulted in their conversion into plants with an average
frequency of 25%. These relatively low conversion rates
clearly seemed to be related to the various morphological
abnormalities induced under in vitro conditions. Somatic
embryos obtained from CsE on media containing NAA and
kinetin or 2iP had mostly normal morphology (clearly distinguishable root and shoot poles) and after transferring
onto hormone-free medium regenerated into flowering
plants with 64–100% frequency (depending on auxin concentration in the induction medium).
HBV core antigen (HBcAg) as a plant-based prototype of therapeutic hepatitis B
vaccine and a carrier for HBV preS epitopes
Tomasz Pniewski, Maria Bogusiewicz, Katarzyna Wyrwa, Marcin Czyż
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznań, Poland, e-mail: [email protected]
Chronic hepatitis B afflicts 400 mln people worldwide and is
the main factor inducing hepatocellular carcinoma and the
cirrhosis. The inefficiency of current anti-viral drugs encourages research on new methods of hepatitis B therapy using
durable and highly immunogenic Capsid-Like Particles
(CLPs) formed by HBV core antigen HBcAg (Lau et al.,
2002). The preS epitopes are used mainly for HBV prophylaxis but they are also considered as alternative for hepatitis
B therapy (Couillin et al., 1999). However preS epitopes
require a carrier, e.g. HBcAg, for stabilization and immunogenic display (Murray and Shiau, 1999; Chen et al., 2004).
PreS1 and preS2 epitopes were inserted into immunodominant 'c' epitope of HBcAg, displayed on the surface
of CLPs. HBcAg and HBcAg-preS1 and HBcAg-preS2 coding sequences were placed under control of CaMV 35S
promoter and the transgenes were transferred into
genomes of tobacco and lettuce via Agrobacterium-mediated transformation. Regenerated plants expressed HBcAg
at a level of up to 400 μg/g FW but HBcAg-preS only at a
level reaching 30–40 μg/g FW. HBcAg and HBcAg-preS
proteins were stably produced in plants during plant
development and in consecutive generations, as confirmed
Vol. 51, suppl. 1, 2009
by ELISA assay and western blot analysis. Selected plant
lines, verified for CLPs assembly and immunogenicity, can
be potentially exploited as an initial material to produce
therapeutic vaccine for chronic HBV carriers, orally
administered when produced in lettuce, and injective,
when produced in tobacco.
REFERENCES
CHEN X, LI M, LE X, MA W, ZHOU B. 2004. Recombinant hepatitis
B core antigen carrying preS1 epitopes induce immune
response against chronic HBV infection. Vaccine 22:
439–446.
COUILLIN I, POL S, MANCINI M, DRISS F, BRECHOT C, TIOLLAIS P, and
MICHEL ML. 1999. Specific vaccine therapy in chronic hepatitis B: induction of T cell proliferative responses specific for
envelope antigens. The Journal of Infectious Diseases 180:
15–26.
LAU GK, SURI D, LIANG R, RIGOPOULOU EI, THOMAS MG, MULLEROVA
I, NANJI A, YUEN ST, WILLIAMS R, and NAOUMOV NV. 2002.
Resolution of chronic hepatitis B and anti-HBs seroconversion in humans by adoptive transfer of immunity to hepatitis B core antigen. Gastroenterology 122: 614–624.
MURRAY K, and SHIAU AL. 1999. The core antigen of hepatitis B
virus as a carrier for immunogenic peptides. Biol Chem 380:
277–283.
21
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Cryoprotective activity of thermal hysteresis protein in evergreen plants
Paweł M. Pukacki1, Magdalena Jarząbek1, Weronika Jóźwiak1, Cornelius Lütz2
1
Physiology of Abiotic Stress Laboratory, Institute of Dendrology, Polish Academy of Sciences, PL 62-035 Kórnik, Poland,
e-mail:[email protected]
2
Department Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck,
A-6020 Innsbruck, Austria
Thermal hysteresis proteins (THPs) inhibit the growth of
ice by binding to the surface of ice crystals, preventing the
addition of water molecules to cause a local depression of
the freezing point. Recent findings demonstrate that
apoplastic THPs from Norway spruce show anti-ice nucleation activity (Jarząbek et al., 2009), and also embryonic
axes from Acer, Fagus and Quercus seeds possess specific cryoprotective activity proteins. Here, we have focused
on the most active THPs from five frost-hardy conifers of
North American and Eurasian boreal forest: Abies grandis (Douglas ex D.Don)Lindl., Picea pungens Engelm.,
Pinus nigra J.F.Arnold, Pinus sylvestris L., and Tsuga
canadensis (L.)Carriere, and additionally from one alpine
(2100 m a.s.l.) evergreen shrub Loiseleuria procumbens L.
The objective of this study was to determine whether the
thermal hysteresis proteins of frost-hardy plants influence
survival at subzero temperatures by modifying the freezing process and/or by acting as cryoprotectants.
Apoplastic extracts were obtained by vacuum infiltration
of leaves with 5 mM ascorbic acid, and the extracts were
concentrated by using a 10 kDa cut-off Ultrafree centrifugal filter device (Millipore). Proteins were analysed by
SDS-polyacrylamide gel electrophoresis. Cryoprotective
activity of apoplastic proteins was determined with the use
of the freeze/thaw inactivation, by four cycles in liquid
nitrogen (–196°C) and room temperature while the assay
of lactate dehydrogenase (LDH) was performed as
described by Wisniewski et al. (1999). Antifreeze activity
was determined by using the droplet freezing assay (Vali,
1971).
Acknowledgement: This work was supported by the Polish
Ministry of Sciences and Higher Education grant to P.M.P.
REFERENCES
JARZąBEK M, PUKACKI PM, NUC K. 2009. Cold-regulated proteins
with potent antifreeze and cryoprotective activities in
spruces (Picea spp.). Cryobiology 58: 268–274.
VALI G. 1971. Quantitative evaluation of experimental results on
the heterogeneous freezing nucleation of supercooled liquids. Journal of Atmospheric Sciences 28: 402–409.
WISNIEWSKI M, WEBB R, BALSAMO R, CLOSE TJ, YU XM, GRIFFITH M.
1999. Purification, immunolocalization, cryoprotective and
antifreeze activity of PCA60: A dehydrin from peach (Prunus
persica). Physiologia Plantarum 105: 600–608.
Influence of different culture strategies on growth and indolizidine alkaloid
accumulation in Securinega suffruticosa shoot cultures
Danuta Raj1, Adam Kokotkiewicz2 , Maria Łuczkiewicz2
1
Chair and Department of Pharmacognosy, Wroclaw Medical University, pl. Nankiera 1, 50-140 Wroclaw ,
e-mail:[email protected]
2
Chair and Department of Pharmacognosy, Medical University of Gdansk, ul. Hallera 107, 80-416 Gdańsk
Securinine is one of the indolizidine alkaloids from
Securinega suffruticosa, Phyllanthaceae. It is a CNS stimulant, and GABA receptor antagonist. Moreover, this compound may be helpful in prevention and treatment of
Alzheimer's disease as neuroprotector against neurotoxicity induced by β-amyloid (25–35) and can improve the
quality of life in people suffering from amyotrophic lateral
sclerosis (ALS) (Raj and Łuczkiewicz, 2008).
In vitro cultures allow production of biologically active
compounds in a controlled manner, independent of climatic conditions and season. Considering the special
properties of S. suffruticosa and the fact that the plant is
not native to the European climate, it seemed pertinent to
investigate in vitro cell cultures of this plant.
The aim of this work was to test different culture
strategies in order to determine the influence of investigated factors on the accumulation of indolizidine alkaloids
by S. suffruticosa shoot biomass and to create an in vitro
plant system suitable for the production of securinine.
22
Moreover, attempts were made to scale up the experimental culture of S. suffruticosa shoots to the bioreactor scale.
Within the experiment, culture type (stationary or shaken), growth conditions (light access) and production scale
(bioreactors) were modified to achieve the purpose.
Moreover, the cultures were fed with precursor of biosynthesis and growth complexes (casein hydrolysate and
coconut water) to change the production potential of the
shoots. Murashige for Lilium (ML) medium was used as
the basis. Phytochemical screening of all in vitro biomasses was carried out simultaneously. The TLC technique
with densitometric detection was used for all phytochemical analyses (Raj et al., 2009).
REFERENCES
RAJ D, ŁUCZKIEWICZ M. 2008. Securinega suffruticosa, Fitoterapia
79: 419–427.
RAJ D, KOKOTKIEWICZ A, ŁUCZKIEWICZ M. 2009. HPTLC-densitometric determination of indolizidine alkaloids in the herb and
in-vitro cultures of Securinega suffruticosa, JPC accepted,
not published.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
September 9–11, 2009, Poznań, Poland
Bacteria in plant environment
Piotr Sobiczewski
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, e-mail: [email protected]
Plant environment contains diverse bacteria whose populations are determined, both in quantitative and qualitative terms, by conditions present in a particular
biotope. Those conditions play an important role in
mutual relationships between plant and bacteria as well
as between bacteria and other organisms. Most of bacteria colonizing the rhizosphere, rhizoplane and phylloplane are commensals that do not have detectable influence on plant growth or physiology. Among mutualistic
bacteria, the microsymbionts of leguminous plants and
other bacteria promoting plant growth and yield may be
distinguished. Their activity leads to an increase in
bioavailability of nutrients, production of substances
improving plant growth (e.g. phytohormones) and/or biological reduction of pathogens and other deleterious
organisms. Endophytic bacteria colonizing healthy plant
tissue offer an enormous potential for increased agronomic performance of plants. The influence of bacteria
on plant vigor and health status may also involve their
participation in bioremediation of soil pollutants,
decomposition of organic matter and improvement of
soil structure. Although bacteria with the ability to
degrade toxic substances represent a small fraction of
soil microorganism populations present in plant habitats,
they can play an important role in plant productivity.
Of more than 5000 species of bacteria recognized, over
100 are the causal agents of plant diseases. They constitute a very important factor limiting growth and yield of
cultivated plants. Some diseases, such as fire blight of
apple and pear (Erwinia amylovora) or bacterial canker
of tomato (Clavibacter michiganensis subsp. michiganensis) can devastate the plants over considerable areas
making their production unprofitable for many years.
Pathogenic bacteria are divided into two major groups:
eubacteria possessing the cell wall and ability to grow on
artificial media and bacteria without cell wall but surrounded by cell membrane only. The latter group, called
mollicutes, formerly known as mycoplasma-like organisms, includes phytoplasmas and spiroplasmas.
In recent years the development of modern molecular
techniques has enhanced interest in the genomics and
proteomics of plant-associated bacteria. The conventional
methods of their identification have been supplemented
and often replaced by DNA-based analysis allowing the
improvement of diagnostics and the study of bacterial
phylogeny.
Somatic embryogenesis of selected coniferous tree species
Krystyna Szczygieł
Department of Genetics and Forest Tree Physiology, Forest Research Institute, Braci Leśnej Street, No 3, Sękocin Stary,
05-090 Raszyn, Poland, e-mail: [email protected]
Somatic embryogenesis in combination with cryopreservation is very effective in preservation of selected genotypes and mass clonal propagation. Experiments on
somatic embryogenesis in selected spruce (Picea abies
(L.) Karst.), silver fir (Abies alba Mill.), and European
larch (Larix decidua Mill.) were conducted to determine if
this method of micropropagation enables forest nurseries
to produce quality seedling. Mature and immature zygotic
embryos or megagametophytes with immature embryos
were used for embryogenic tissue initiation in spruce, silver fir and European larch. Experiments were also carried
out on embryogenic callus of hybrid larch (Larix×leptoeuropaea) (INRA, Orleans). High frequencies of embryogenic callus formation were achieved in spruce (23-31%)
and fir (29%) when mature zygotic embryo explants were
used, whereas immature embryos were required in larches (36% frequency). Initiation of somatic embryogenesis
from somatic embryos resulted in second and third generation of embryogenic calli. Maturation of spruce somatic embryos was achieved on BM (Gupta and Durzan,
1986) medium containing 20–40 μM ABA combined with
1 μM IBA when tissue was cultured under low intensity
Vol. 51, suppl. 1, 2009
light for 5 weeks. For larch, the MSG (Becwar et al., 1990)
medium supplemented with 20–60 μM ABA and 1 μM IBA
and also cultivation of tissue under low light intensity for
3–4 weeks was the best. Somatic embryos of silver fir
required 8–10 weeks of cultivation in darkness on modified MCM medium (Bornman and Jansson, 1981 after
Hristoforoglu et al., 1995) supplemented with 20 μM ABA
and 1 μM IBA for maturation. The present studies demonstrated the possibility of applying somatic embryogenesis
for efficient propagation of coniferous trees.
REFERENCES
BECWAR MR, NAGMANI R, WANN SR. 1990. Initiation of embryogenic
cultures and somatic embryo development in loblolly pine
(Pinus taeda). Canadian Journal of Forest Research 20:
810–817.
GUPTA PK, DURZAN DJ. 1986. Plantlet regeneration via somatic
embryogenesis from subcultured callus of mature embryos
of Picea abies (Norway spruce). In Vitro Cellular &
Developmental Biology – Plant 22: 685–688.
HRISTOFOROGLU K, SCHMIDT J, BOLHAR-NORDENKAMPF H. 1995.
Development and germination of Abies alba somatic
embryos. Plant Cell, Tissue and Organ Culure 40: 277–284.
23
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
In vitro culture of endophytic fungi as a potential source of novel biologically
active secondary metabolites
Wojciech Szypuła1, Julia Budziszewska2, Daria Delbani1
1
Department of Biology and Pharmaceutical Botany, The Medical University of Warsaw, Banacha 1, 02-097 Warsaw,
Poland, e-mail: [email protected]
2
Department of Systematics and Plant Geography, University of Warsaw, Al. Ujazdowskie 4, 00-478 Warsaw, Poland,
e-mail: [email protected]
An endophyte is a bacterial (including actinomycetes) or fungal microorganism, which spends the whole or part of its life
cycle colonizing inter- and/or intra-cellular locations inside
healthy tissues of the host plant, typically causing no apparent symptoms of disease (Hallmann et al., 1997; Tan and
Zou, 2001). The endophytic population of a given species
varies from several to a few hundred of bacterial and fungal
strains. Endophytes can be isolated from mildly surface disinfected plant tissues and cultivated on nutrient agar (l.c).
The relationship between the endophyte and its host plant
may range from latent phytopathogenesis to mutualistic
symbiosis. Almost all vascular plant species examined to
date were found to harbor endophytic bacteria and/or fungi.
Endophytes colonizing plant tissues usually get nutrition
and protection from the host plant. In return, they profoundly enhance fitness of the host plants by producing certain functional metabolites. Primarily, endophytes can produce bioactive secondary compounds, such as alkaloids,
steroids, terpenoids, sesquiterpenes, isocumarin derivatives, phenylpropanoids, lignans and others (Tan and Zou,
2001). Moreover, endophytes can influence host's interactions with other species, altering plant community composition (Tan and Zou, 2001; Rudgers et al., 2004; Clay et al.,
2005). As a poorly investigated store of microorganisms
'hidden' within the host plants, endophytes are obviously a
rich and reliable source of bioactive and chemically novel
compounds with huge medicinal and agricultural potential.
For example, the advantage of production of some important
phytochemicals such as paclitaxel and huperzine A in endophytes lies in the fact that it provides an alternative strategy
for easing the impact of the growing human population on
plants which are needed for the preservation of biodiversity
and the ecosystem (Tan and Zou 2001; Szypuła et al., 2005,
2006). The aim of our investigation was to establish the culture of Huperzia selago endophytes that had been isolated
from shoots and to perform their phytochemical analysis.
After surface disinfection, shoot fragments were cut into 2
mm pieces and plated onto 4% potato-dextrose agar (PDA).
Plates were incubated at room temperature for 10 weeks or
until fungal growth was observed. The isolated fungi were
transferred onto solid medium and incubated in the culture
room at 25°C for 5 days. In order to determine the presence
of secondary metabolites in the mycelium, HPLC-UV and
HPLC-MS/MS-ESI analyses were performed in the positive
ion mode.
REFERENCES
CLAY K, HOLAH J, RUDGERS JA. 2005. Herbivores cause a rapid
increase in hereditary symbiosis and alter plant community
composition. PNAS 102(35): 12465–12470.
HALLMANN J, QUANDT-HALLMANN A, MAHAFFEE WF, KLOEPPER JW.
1997. Bacterial endophytes in agricultural crops. Canadian
Journal of Microbiology 43: 895–914.
RUDGERS JA, KOSLOW JM, CLAY K. 2004. Endophytic fungi alter
relationships between diveristy and ecosystem properties.
Ecology Letters 7: 42–51.
SZYPUŁA W, PIETROSIUK A, SUCHOCKI P, OLSZOWSKA O, FURMANOWA M,
KAZIMIERSKA O. 2005. Somatic embryogenesis and in vitro
culture of Huperzia selago shoots as a potential source of
huperzine A. Plant Science 168: 1443–1452.
SZYPUŁA W, OLSZOWSKA O, FURMANOWA M. 2006. In vitro culture of
Lycopodiaceae (club mosses). Botanical Guidebooks
29:163–175.
TAN RX, ZOU WX. 2001. Endophytes: a rich source of functional
metabolites. Natural Product Report 18: 448–459.
Cytologial, cytometric and molecular characterization of gentian somatic hybrids
Karolina Tomiczak, Jan J. Rybczyński
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, e-mail: [email protected]
The aim of the presentation is to show and summarize the
results obtained with the help of various methods, of somatic hybridization between selected species of Gentiana.
In a breeding program, somatic hybridization can overcome sexual incompatibility through the somatic cell fusion.
In contrast to sexual hybridization, after protoplast fusion all
nuclear and cytoplasmic DNA from both parents are united
in one individual. However, regeneration of plants from
fused protoplasts is often accompanied by polyploidization
and/or the elimination of genome parts of one or both fusion
parents. Thus, detailed investigations are needed not only to
confirm the hybrid status of regenerates, but also to exactly
characterize their genome size and composition.
24
In interspecific somatic hybridization program involving
Gentiana, numerous calli and plants were produced by electrofusion between G. kurroo Royle and G. cruciata L. as well
as between G. cruciata and G. tibetica King protoplasts.
Several methods, including flow cytometry, chromosome
counting, stomatal characteristics and AFLP were applied
for identification and description of the recovered somatic
hybrids. Results of cytological and cytometric analyses
revealed higher genome sizes and chromosome numbers of
regenerates in comparison to parental species. Some evidence of polyploidy and mixoploidy as well as partial
genome elimination was found. According to molecular data,
hybrids showed differential levels of symmetry.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
ORAL PRESENTATIONS
September 9–11, 2009, Poznań, Poland
Induction of tetraploids in hop (Humulus lupulus L.) using in vitro cultures
Anna Trojak-Goluch, Anna Depta, Monika Agacka
Department of Plant Breeding and Biotechnology, Institute of Soil Science and Plant Cultivation, State Research
Institute, Czartoryskich 8, 24-100 Puławy, Poland, e-mail: [email protected]
Hop (Humulus lupulus L.) is a diploid, perennial plant
whose secondary metabolites are used for brewing as well
as in pharmaceutical industry. The Polish hop breeding
programme centres around the generation of triploid cultivars which are superior to diploids because they produce more acid resins and essential oils. In the process of
breeding triploids, diploid cultivars need to be raised to
the tetraploid level (Koutoulis et al., 2005).
In this study tetraploids were induced through culturing apical buds of Polish diploid cultivar Sybilla on liquid
MS media containing three colchicine concentrations:
0.01%; 0.05%; 0.1%. Buds were incubated on an orbital
shaker for 24h or 48h and then transferred to shoot multiplication medium. Flow cytometric analysis of ploidy
level revealed that the highest induction of tetraploids was
achieved with the exposure to 0.05% colchicine for 48h
while the lowest with 0.01% colchicine for 48h. Out of 116
plants, 8.7% were found to be tetraploids whereas others
were diploids (63.3%) and mixoploids (30.7%). Shoot
internodes of mixoploids were selected and calli derived
from shoots were obtained. Subculture of these calli on
different regeneration media supplemented with
indoleacetic acid (IAA) and several concentrations of
cytokinins promoted shoot regeneration. The best caulogenic potential was achieved on medium with 0.5 mg/l IAA
and 5 mg/l zeatin riboside. The regeneration rate was
42.9%. Plantlets were successfully rooted and transferred
to the greenhouse. Individual plants were classified as
mixoploids or tetraploids after screening by flow cytometry.
This study provided a population of tetraploids that
will be used in further hop breeding program.
REFERENCE
KOUTOULIS A, ROY AT, PRICES A, SHERIFF L, LEGGETT G. 2005. DNA
ploidy level of colchicine- treated hops (Humulus lupulus).
Scientia Horticuturae 105: 263–268
Is that really a step forward in protoplast culture of lupins?
Alina Wiszniewska, Anna Pindel
Department of Botany, Faculty of Horticulture, Agricultural University of Cracow, Al. 29 Listopada 54, 31-425 Cracow,
Poland, email: [email protected]
Considering the agricultural importance of lupins, studies
aiming at an expansion of the gene pool effectively contribute to the improvement of breeding programmes in the
genus. Lupin species, both those commercially grown and
their wild relatives, are considered to be recalcitrant to in
vitro culture. On the other hand, some valuable characters
may be successfully introduced into the plant genomes by
means of such manipulations. Protoplast and single-cell
culture could be suitable methods for overcoming interspecific barriers and for increasing the genetic variability
of lupins.
Comprehensive studies were undertaken to determine
optimal conditions for yellow lupin (Lupinus luteus L.)
protoplast culture. The adapted procedure of isolation
resulted in a high yield of protoplasts obtained from various explants. Cultivar 'Parys' proved to be the most promising material for manipulations. Various media and cul-
Vol. 51, suppl. 1, 2009
ture techniques were evaluated in terms of their usefulness in promoting survivability and morphogenetic
responses of protoplasts. Medium solidification enhanced
the development of cultures initiated from hypocotyls and
cotyledons by significantly increasing the mitotic division
rate. However, an unfavourable phenomenon appeared in
both liquid and solid media: after first regular division
daughter cells did not undergo consecutive mitoses. An
important breakthrough in the development of cultures
occurred in media supplemented with 0.1% activated
charcoal. Overcoming the suppression of mitosis led to
the formation of small aggregates from hypocotyl protoplasts. The noteworthy progress achieved here indicates
that in spite of yellow lupin recalcitrance it is possible to
obtain responsive cultures with a higher morphogenetic
potential and thus provide new materials for exploitation
in modern and sustainable agriculture.
25
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Anti-mitotic agents in Salix viminalis polyploid plant induction
Maria Katarzyna Wojciechowicz
Department of General Botany, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland,
e-mail: [email protected]
The genus Salix contains many economically important
species used for energy, chemicals and fibers. In vitro
polyploidization system of the species can be an essential step to generate variation for selection of desirable
traits. In the present study, the potential of microtubule
depolymerising agents trifluralin, pronamide (KERB),
oryzalin amiprophos-methyl (AMP) and colchicine for in
vitro genome doubling in S. viminalis clones was investigated. The mitotic inhibitors were dissolved in 2%
dimethyl sulfoxide and added to MS medium with double
concentration of Fe-EDTA. Embryos, shoots apices and
axillary buds of the basket willow were exposed to various concentrations of the inhibitors for 1, 2, 3 and 6
days. The control test was performed with explants cul-
tured on MS medium supplemented with 2% dimethyl
sulfoxide. All cultures were kept under continuous light
(cool white fluorescence lamps; 35 μmol quanta m-2 s-1)
at 22±2°C. Flow cytometry was applied to estimate ploidy
level. Artificially induced tetraploid plants from S. viminalis embryos were obtained on media supplemented
with 0.025 and 0.05% colchicine, 0.1% trifluralin or 0.3%
KERB when the treatment lasted two and three days. The
highest efficiency of doubling chromosome numbers was
achieved on medium supplemented with 7 % trifluralin.
In cultures of shoots apices and axillary buds no
tetraploids were detected. Oryzalin was very toxic for tested explants of S. viminalis even at a very low concentration (0.001%).
The effect of genotype, explant type and medium on the regeneration of linseed
(Linum usitatissimum L.) in in vitro culture
Andrzej Wojciechowski, Jowita Janowicz, Marzena Stanisławska
Department of Genetics and Plant Breeding, Poznan University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznań,
e-mail: [email protected]
Linseed (Linum) belongs to the family Linaceae that comprises about 200 species. Most of them are wild and only
a few, such as L. usitatissimum L., are cultivated. This
work reports the results of studies on the regeneration
capacities in in vitro cultures of two Linum usitatissimum
cultivars: Modran and Selena. Cotyledon (3 different fragments), hypocotyl and hypocotyl-cotyledon explants of
tested cultivars were obtained from 6-, 7- and 8-day old
seedlings. They were cultured on MS basic and MS medium supplemented with 1mg l-1 2,4-D, NAA and BAP.
The genotype, type of media and explant significantly
affected the ability and direction of regeneration.
Irrespective of the genotype and age of explants, the best
formation of shoots was achieved from hypocotyl and
hypocotyl-cotyledon explants cultured on MS control and
MS+BAP media. The largest number of regenerated
plants in the soil was obtained from 6-day old hypocotyl
explants incubated on MS medium with BAP. There were
no plants transferred to the soil from 8-day old hypocotyl
26
explants of both tested linseed cultivars. The addition of
2,4-D did not stimulate regeneration from most explants
except for 6-day old hypocotyl-cotyledon explants of
Modran cultivar which formed shoots. The callus formation and later the roots were the best on MS medium containing NAA irrespective of age of seedlings from which
they were collected. Endogenous ethylene that accumulated in the culture vessels was likely responsible for stimulation or inhibition of regeneration. With respect to cotyledon and hypocotyl-cotyledon explants it caused them to
turn yellow and consequently to die. However, it stimulated callus formation on hypocotyl explants. Explants from
6- and 7-day old seedlings showed a higher regeneration
efficiency than 8-day old explants. Hypocotyl-cotyledon
explants showed a better regeneration if they were harvested from 7-day old seedlings compared to 6 day old
seedlings and as a result, a higher number of plants were
transfered to the soil.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
September 9–11, 2009, Poznań, Poland
ORAL PRESENTATIONS
Agrobacterium – mediated genetic transformation of Gentiana tibetica King
Anna Wójcik and Jan J. Rybczyński
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, e-mail: [email protected], [email protected]
The discovery of natural ability of bacteria from genus
Agrobacterium to infect plants and to integrate bacterial
genetic material into the plant genome opened a new chapter in contemporary plant experimental biology. For over
30 years Agrobacterium has been used in biotechnological laboratories all over the world (Vain, 2007) and
Agrobacterium – mediated genetic transformation has
became an indispensable genetic engineering tool.
Species from genus Gentiana are valued as ornamental plants and material for isolation of pharmacologically active compounds (Skrzypczak et al., 1993). Their
aesthetic and medicinal quality could be increased by a
genetic transformation. The aim of our work was to
develop an Agrobacterium – mediated transformation
technique of Gentiana tibetica and to regenerate genetically modified plants. C58C1 Agrobacterium tumefa-
ciens strain containing pDraGON-G:GFP vector was
used (Wróblewski et al., 2005).
On the basis of the experiments it was shown that
genetic transformation of G. tibetica leaf explant cells took
place only in the presence of L-glutamine in the medium.
Transformation efficiency increased with its concentration. The most efficient regeneration of transgenic plants
was observed on the WPM medium without any plant
growth regulators.
The transgenic character of tissues was determined on
the basis of β-glucuronidase histochemical reaction. GUS
activity was examined in transformed leaf explants, callus
tissues and regenerated plants. PCR analysis of regenerants
confirmed the transgene integration into plant genome while
Agrobacterium contamination was excluded. Randomly
selected plants were checked by Southern hybridization.
Topophysis in adventitious shoot regeneration in vitro in Chrysanthemum
Małgorzata Zalewska, Natalia Miler
Department of Ornamental Plants and Vegetable Crops, Laboratory of Biotechnology, University of Technology and Life
Sciences, Beranrdyńska 6, 85-029 Bydgoszcz, Poland, e-mail: [email protected]
Adventitious shoots originate from a single cell or several
cells and therefore the adventitous shoots technique is
widely applied in plant breeding e.g. for induction of mutagenesis, genetic transformation and separation of components of chimeras. The success of breeding programmes
often depends on the regeneration efficiency. The aim of
this study was to investigate the influence of topophysical
position of explants on the efficiency of adventitious shoot
regeneration.
Uniform single shoots of chrysanthemum 'Satinbleu'
propagated in vitro and consisting of 12 nodes were divided equally into three topophysical zones: distal, central
and proximal. Two leaves and two internodes were isolated from each zone. The explants were isolated from the
distal end of the distal zone, the central portion of the central zone, and the proximal end of the proximal zone.
Explants were cultured for 12 weeks on MS medium supplemented with 0.6 mg l-1 BAP and 2.0 mg l-1 IAA. The
number of adventitious shoots and regenerating explants
were recorded. Regeneration curves were plotted as a
function of time.
Vol. 51, suppl. 1, 2009
Regeneration proceeded differently in leaves and
internodes. Internodes produced almost four times more
shoots than did leaves. Internodes excised from the distal
and proximal zones were more efficient in shoot regeneration, while explants taken from the central zone produced the lowest number of adventitious shoots. Leaves
isolated from the central zone produced eight times fewer
shoots than did leaves taken from the distal zone, whereas leaves excised from the distal zone did not regenerate
at all. The earliest formation of adventitious shoots was
observed on internodes isolated from the distal zone,
where it started in the third week and continued until the
seventh week of culture and then declined. The regeneration curves for proximal internodes as well as for distal
leaves were shifted by two weeks towards the regeneration
curve for distal internodes.
Due to the highest adventitious shoot regeneration
capacity and the earliest formation of shoots, the best
explants for chrysanthemum breeding programmes are
internodes excised from distal parts of in vitro plantlets.
27
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
The control of bacterial contaminations during in vitro shoot multiplication
Marta Zawadzka1, Teresa Orlikowska1, Piotr Sobiczewski1, Artur Mikiciński1, Monika Sulikowska1,
Elżbieta Zenkteler2
1
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice,
e-mail: [email protected]
2
Department of General Botany, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań
Multiannual tissue cultures can be contaminated by different bacteria that do not reveal themselves via symptoms in the medium or on the shoot explants. Such contaminations are often transferred to subsequent subcultures. During a two-year period we obtained a number of
isolates belonging to 24 species of bacteria from cultures of
hosta, rose, raspberry, elder, blackberry, anthurium, narcissus, apple rootstock and orchid. Their identification was
based on phenotypic and molecular analyses. The most frequently isolated bacteria were Pseudomonas putida,
Bacillus sp., Staphylococcus pasteuri, Methylobacterium
lusitanum and Serratia marcescens. The following compounds or commercial products known to have bactericidal
activity: dichloroisocyanuric acid (Aldrich), thymol oil
(Riedel-deHaën), Triclosan (Irgasan, Fluka), PPM (Plant Cell
Technology, Inc), Biosept 33SL (Cintamani), Biochicol 020
PC (Poly-Gumitex Farm), silver nitrate (Sigma), benzoic
acid (Chempur), menthol oil (Fluka), Decaben C (Jan
Dekker), acetylsalicylic acid (Duchefa) and Proclin 300
(Supelco) were added to the media on which selected isolates were inoculated.
The growth of bacterial colonies was inhibited by
Biosept, thymol oil, Triclosan and PPM. A small degree of
inhibition or lack of growth was observed on media with
silver nitrate, Biochicol, menthol oil, benzoic acid, acetylsalicylic acid, Decabenen C and Proclin 300.
Concentrations of 0.5 and 1% were used to evaluate
the phytotoxicity of the compounds. After 2–3 days on the
media containing thymol oil or Triclosan, explants from
all of the tested cultures died. Phytotoxic effects of
Decaben C were observed for cultures of apple rootstocks,
strawberry, raspberry and hosta, but it did not affect the
growth and development of gerbera explants. No phytotoxicity symptoms were observed after the application of
PPM and Biosept at concentrations of 0.5 and 1%.
The next step was the use of bactericides in shoot cultures artificially contaminated with the studied bacteria.
Silver nitrate, Biosept, Decaben C, PPM and Proclin 300
were added to multiplication cultures of strawberry, hosta
and gerbera.
The most effective treatment against all tested bacteria
in gerbera cultures was Proclin 300 at 0.05%. In cultures
infected with Bacillus sp. or Methylobacterium lusitanum, the growth of bacteria was inhibited by 0.3% PPM,
0.05% Decaben C, 1% AgNO3 and 0.5% Biosept. In hosta
cultures, only Decaben C was not effective when cultures
were contaminated with Pseudomonas putida. No signs of
bacterial presence were recorded in cultures of strawberry supplemented with Proclin 300 (0.05%), AgNO3
(0.5–1%) and Biosept (0.1–0.5%). In cultures contaminated with Methylobacterium lusitanum, only PPM at 0.3%
was effective. The bacteriostatic effect lasted for 4 weeks.
Application of nanomaterials to fight and prevent bacteria in vitro
Elżbieta Zenkteler1, Piotr Sobiczewski2, Teresa Orlikowska2, Krzysztof Langer3, Jerzy Langer3
1Department of General Botany, Institute of Experimental Biology, Faculty of Biology,
A. Mickiewicz University, Umultowska 89, 61-614 Poznań
2
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice
3
Laboratory for Materials Physicochemistry and Nanotechnology, Faculty of Chemistry, A. Mickiewicz University,
Grunwaldzka 10, 63-100 Śrem
Nowadays, among the most promising nanomaterials
that have revealed antibacterial properties are the nanosized metal particles and nanotubes of polypyrrole (Ppy)
or protoneated polyaniline (PAni) (Xia et al., 2004;
Srivastava et al., 2007). In the present study we report
the effectiveness of nanosilver (NS) particles sized 5–10
nm against Microbacterium strains during micropropagation of Alocasia sanderiana. Contaminated explants
were dipped in 50, 100 and 200 mg l-1 solutions of NS.
Results of the treatment recorded after one month of culture on modified MS medium showed a high number of
decontaminated explants without any adverse effect on
their growth characteristics. We also investigated the
activity of polyaniline fixed directly onto filter paper by
28
electrochemical oxidative polymerization against
Microbacterium. We showed that A. sanderiana cultured
in liquid MS medium on bridges of filter paper coated with
the conducting polymers were decontaminated and developed without any visible symptoms of bacteria in comparison to the control.
REFERENCES
SRIVASTAVA S, BERA T, ROY A, SINGH G, RAMACHANDRARO P, DASH D.
2007. Characterization of enhanced antibacterial effects
of novel silver nanoparticles. Nanotechnology 8:
424081–424090.
XIA H, CHAN H, XIAO CH, CHENG D. 2004. Self-assembled oriented
conducting polianiline nanotubes. Nanotechnology 15:
1807–1811.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
September 9–11, 2009, Poznań, Poland
ORAL PRESENTATIONS
Some remarks on the application of in vitro techniques to manipulation
of sexual reproduction of plants
Maciej Zenkteler
Department of General Botany, Institute of Experimental Biology, Faculty of Biology, A. Mickiewicz University,
Umultowska 89, 61-614 Poznań,
The in vitro culture conditions enable researchers to control and modify the processes of pollination, fertilization
and the course of embryogenesis in seed plants.
Technological development and the improvement of various procedures make it possible not only to obtain a deeper knowledge about sexual reproduction but, in particular, to provide strategies to transfer this knowledge into
crop improvement. Sophisticated techniques are now
available to manipulate the female and male organs, thus
allowing to introduce new genetic material into breeding
lines.
The presented lecture will be based on investigations
carried out in our laboratory. It will mainly summarize
those achievements that concern wide hybridization
among various species.
Application of androgenesis in basic research and breeding of cereals
Janusz Zimny1, Sylwia Oleszczuk1, Andrzej Z. Czaplicki1, Henryk Woś2, Janusz Kozdój1,
Sławomir Sowa1, Piotr Bednarek1
1
2
Plant Breeding and Acclimatization Institute, Radzików, Poland, e-mail: [email protected]
HR Strzelce Sp. z o.o. Grupa IHAR
The techniques for obtaining double haploids opened up
an opportunity for practical breeding to obtain, in a short
time, homozygotic lines possessing alleles of genes in the
recessive form. The first varieties of wheat that were
obtained by anther culture appeared on the markets in
China and France in the 80-ties. Later, the more sophisticated method of isolated microspore cultures has been
applied. Microspore culture allows for examination of
large numbers of cells in a small area and in a short period of time as well as the selection of cell lines with the
desired characteristics.
On the basis of many laboratories' reports and the
subsequent publications on the issue, there has emerged
a hierarchy among cereal crops in terms of their morphogenetic capacity in anther cultures. In this respect, rye and
oat have always been considered the most difficult to
regenerate, while wheat was found to be less recalcitrant
Vol. 51, suppl. 1, 2009
and barley and triticale the least. One can observe a growing interest in homozygotic lines among scientists and
breeders. They can be used in basic research as a "clean"
material and in experimental plant breeding to obtain
lines whose offspring does not segregate.
On the other hand, double haploid lines are a source
of variation because each microspore, which is a source of
regenerated homozygotic line has a certain set of gene alleles that can appear important in the breeding process. For
this reason, breeding of cereals in many countries in
recent years has been based mainly on double haploids.
Polish breeding companies slowly start to follow in the
steps of foreign breeding companies, who have been using
that method. Based on our experience we may even risk
the statement that in vitro induced androgenesis is the
most efficient biotechnological method, used in practical
breeding.
29
ORAL PRESENTATIONS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Increase of bioactive compounds in flax plants overexpressing enzymes of
flavonoid synthesis pathway
Magdalena Żuk, Jan Szopa
Faculty of Biotechnology, University of Wrocław, Poland, e-mail: [email protected]
Flavonoids are a group of secondary plant metabolites
important for plant growth and development. They are
also well known as very effective antioxidants with a major
significance for human health. We obtained flavonoid-rich
plants using two strategies. The first strategy involved a
simultaneous overexpression of three genes of flavonoid
synthesis (chalcone synthase, chalcone isomerase and
dihydroflavonol reductase) and the second strategy consisted in overexpression of the broad substrate specificity
enzyme glucose transferase.
Both groups of plants had increased levels of phenylopropanoid compounds important for the antioxidant
potential, such as kaempferol, quercetin, phenolic acids,
poli-unsaturated fatty acids and lignans. Because the constitutive promoter (CaMV 35S) was used for modification,
the positive changes were observed in the entire plant
body – green parts and seeds.
Flax seeds are a very good source of the plant lignan
precursor secoisolariciresinol diglucoside (SDG), which is
metabolised by bacteria in the colon to produce the mammalian lignans enterolacton and enterodiol. Lignans
inhibit cell proliferation and growth, which makes these
30
compounds potential anticancer agents (in hormon-sensitive cancers). In our transgenic plants a significant
increase of SDG level (up to 30-fold) was noticed. A dramatic increase of ferulic acid level (about 40 fold) was also
observed. The high level of lignans was also found in seedcakes – the residual material left after pressing of oil from
linseed of transgenic plants. For this reason, seedcake
obtained from flavonoid-rich plants should be use as
a very good source of bioactive compounds.
A significantly higher level of antioxidants was also
detected in oil from transgenic plants. Remarkably, such
oil is rich of poli-unsaturated fatty acids and the ratio of
ω6/ω3 was close to that recommended by FAO/WHO for
optimal human diet. The presence of antioxidants in this
oil also resulted in increased unsaturated acid stability.
Flax fibers obtained from transgenic plants on a semitechnical scale have increased contents of many bioactive
phenylopropanoids such as a phenolic acids and lignans.
In fibers we also identified canabidiol – a compound that
can reduce pain. For this reason fabrics produced from
these fibers may be recommended as wound dressing.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Micropropagation of Plantago coronopus L.
Emilia Andrzejewska-Golec, Joanna Makowczyńska
Department of Biology and Pharmaceutical Botany, Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland,
e-mail: [email protected]
Buckhorn
plantain
Plantago
coronopus
L.
(Plantaginaceae) is a vanishing species in Poland, belonging to the "critically endangered" category in Polish Red
Data Book of Plants, and subject to an active protection
(Ministry of Environment's Directive, 9 July 2004). To our
knowledge there is no information in literature about in
vitro culture of this taxon. However, some other species in
the genus Plantago were studied in in vitro culture
(Andrzejewska-Golec and Makowczyńska, 2008;
Budzianowska et al., 2004; Makowczyńska and
Andrzejewska-Golec, 2003; 2009).
We propagated Plantago coronopus from different
explants of 4-week-old seedlings. Murashige and Skoog
basal medium, supplemented with indole-3-acetic acid
with various concentrations of cytokinin 6-benzyladenine
(0.2; 1; 2 or 5 mg/dm3) was used. After 6 weeks of culture,
micropropagation rates were calculated. The plants
obtained as a result of micropropagation were not phenotypically changed.
Our study proved that Plantago coronopus was
amenable to propagation in vitro. This method may have
significance for protection of this plantain.
REFERENCES
ANDRZEJEWSKA-GOLEC E, MAKOWCZYŃSKA J. 2008. Micropropagation
of Plantago camtschatica Link. Acta Societatis
Botanicorum Poloniae 77: 269–273.
BUDZIANOWSKA A, SKRZYPCZAK L, BUDZIANOWSKI J. 2004.
Phenylethanoid glucosides from in vitro propagated plants
and callus cultures of Plantago lanceolata. Planta Medica
70: 834–840.
MAKOWCZYŃSKA J, ANDRZEJEWSKA-GOLEC E. 2003. Micropropagation
of Plantago asiatica L. through culture of shoot-tips. Acta
Societatis Botanicorum Poloniae 72: 191–194.
MAKOWCZYŃSKA J, ANDRZEJEWSKA-GOLEC E. 2009. Micropropagation
of Plantago maritima L. – a vanishing species in Poland.
Acta Societatis Botanicorum Poloniae 78: 13–18.
In vitro cultures of embryonic axes of Lupinus mutabilis and L. albus
Małgorzata Arendt, Renata Galek, Ewa Sawicka-Sienkiewicz
Department of Genetics, Plant Breeding and Seed Production, Wrocław University of Environmental and Life Sciences;
Plac Grunwaldzki 24 A, Poland, e-mail: [email protected]
Lupine plants are difficult to cultivate under in vitro conditions. The aim of this study was to evaluate the regeneration ability of embryonic axes of two L. mutabilis genotypes (KW 7/01/05 and KW 10/01/05) and one L. albus
cultivar Butan. Seeds were surface sterilized in a commercial bleach (Domestos). Having removed the seed coat,
embryonic axes with cotyledons were additionally disinfected in Citrosept solution. Explants were divided into
two groups: embryonic axes with cotyledon fragments and
axes without cotyledons. All explants were placed on B5
medium supplemented with different types and combinations of growth regulators. The following concentrations
32
and types of growth regulators were used: 3.0 mg/L BA,
3.0 mg/L 2iP, 3.0 mg/L BA + 0.2 mg/L NAA and 3.0 mg/L
2iP + 0.2 mg/L IAA.
After six weeks of culture, length of roots, hypocotyls
and stem height of regenerated plantlets were measured.
The number of leaves was also counted. The highest mean
stem length (5.01 cm) was observed in Butan on B5 medium containing 3.0 mg/L 2iP + 0.2 mg/L IAA. The largest
leaf number was found in KW 10/01/05 (6.25) on B5 medium containing 3.0 mg/L BA + 0.2 mg/L NAA. The above
results were recorded on explants with cotyledon fragments.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
The effect of the exogenous phenolic compound, caffeic acid on organogenesis
in Galanthus elwesii Hook. cultured in vitro
Anna Bach1, Bożena Pawłowska1, Katarzyna Hura2
1
Department of Ornamental Plants, Agricultural University of Cracow, Al. 29 Listopada 54, 31-425 Cracow,
e-mail: [email protected]
2
Department of Plant Physiology, Agricultural University of Cracow, Podłużna 3, 30-239 Cracow
Phenolic compounds belong to secondary metabolites
whose function in plants is unknown. Caffeic acid is a simple phenolic compound, a phenylpropene derivative, with
two -OH groups, that inhibits IAA oxidase and, therefore,
is considered to be an auxin cofactor. The aim of the present experiments was to determine the effect of exogenous
caffeic acid on organogenesis (formation of roots, bulbs,
leaves) in giant snowdrop in in vitro cultures. Bulb
explants obtained from plants multiplied by adventitious
buds were placed on MS (1962) media containing 3%
sucrose, growth regulators BA and IAA (0.1–1.0 μM) and
caffeic acid at 0, 100 and 500 mg/dm3. The cultures were
maintained in the dark at 23/25°C. Observation of the
regeneration process revealed that exogenous caffeic acid
at the optimal concentration (500 mg/dm3) completely
inhibited shoot regeneration and leaf and roof formation
by significantly decreasing the number of the organs
formed and their browning. It also reduced bulb fresh
mass growth. A five-fold lower dose of exogenous caffeic
acid in the medium inhibited only adventitious root elongation and caused explant browning. Analysis of the content of phenolic compounds in tissues and organs of giant
snowdrop showed that the contents were diverse and
depended on developmental stage and reproduction conditions. It was demonstrated that exogenous caffeic acid
reduced the total content of phenolic compounds in regenerating plants from in vitro cultures, independently of
cytokinin and auxin concentration in the medium. The
highest content of phenolic compounds was observed in
the control bulbs originated from plants growing in the
open air, in basal plate (1723 μg/g fresh weight). Bulbs
formed in vitro on control media were characterized by a
similar content of phenolic compounds in inner and middle scales (247 μg/g fresh weight) though it was lower than
in bulbs grown in the open air. The level of phenolic compounds was significantly lower in bulbs formed in the
presence of 100 and 500 mg/dm3 caffeic acid (80 and 30 μg/g
fresh weight, respectively). It is suggested that the content
of phenolic compounds can be a biological quality marker
in cultures of plants reproduced by in vitro techniques.
REFERENCES
TILLY-MANDY A, JAMBOR-BENCZUR E, SZABO J. 2006. Results with the
micropropagation of Galanthus elwesii and Galanthus
nivalis 'Flore Pleno'. Acta Horticulturae 725: V International
Symposium on in vitro Culture and Horitcultural Breeding:
439–444.
SINGLETON VS, ROSSI JA JR. 1965. Colorimetry of total phenolics
with phosphomolybdic-phosphotungstic acid reagent.
American Journal of Enology and Viticulture 16: 144–157.
Changes in terpenoid level after Fusarium treatment in flax
Aleksandra Boba, Anna Kulma, Kamil Kostyn, Jan Szopa
Department of Genetic Biochemistry, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/77,
51-148 Wrocław, e-mail: [email protected]
Terpenoids are a group of both primary and secondary
plant metabolites playing important roles in plant growth
and survival (gibberellic acid, chlorophyll, carotenoids).
They are involved in responses to stress situations such as
pathogen infection (tocopherol). But these compounds are
also very important in the human diet because they can
protect against many civilization-related diseases, such as
cancer (lycopen, squalene). The understanding of synthesis pathway is important for genetic manipulation of levels
Vol. 51, suppl. 1, 2009
of terpenoids in plants. Even though the pathway is well
known in model plants, in Linum those genes have not
been identified. Using degenerated primers we found 16
sequences of major terpenoid synthesis genes in flax
(Linum usitatissimum var. Nike). We used those
sequences to verify the level of expression of terpenoid
genes after a Fusarium treatment. This knowledge will be
used for creation of plants with terpenoid levels beneficial
for human health.
33
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Expression of HBV core antigen (HBcAg) in transgenic tobacco and lettuce for
purposes of therapeutic vaccine against hepatitis B
Maria Bogusiewicz, Tomasz Pniewski
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska 34, 60-479 Poznań, Poland,
e -mail: [email protected], [email protected]
Hepatitis B is the most common infectious liver disease in
the world, with it's prevalence at 2 billion people infected
worldwide and 400 million chronically infected. Hepatitis
B is the main factor inducing hepatocellular carcinoma
and one of the most important factors causing the cirrhosis. For the reasons of inefficiency and difficulty of today's
expensive anti-viral therapies using INF-α or antiviral
drugs such as Lamivudin, Adefovir, etc., many scientists
work on new therapies against Hepatitis B Virus (HBV),
such as therapeutic vaccines, consisting of HBcAg
(Hepatitis B core Antigen).
The aim of the study was to analyze the expression of
HBcAg in transgenic plants of tobacco and lettuce for purposes of defining appropriate conditions for efficient
expression and selecting plants with high HBcAg level for
further work on therapeutic vaccine for chronic HBV car-
riers, and also finding the best method of isolation of
HBcAg from plant tissue.
The transgenic plants revealed 1–5 copies of T-DNA
integrated into genomic DNA, which were stably inherited.
Expression of native HBcAg, probably assembled into
highly immunogenic Capsid-Like Particles (CLPs), was
confirmed by ELISA assay and western-blot. Observed
level of HBcAg – up to 400 μg/g FW, displayed a relative
stability during plant development and in sequential generations. Selected plants can be used for further research
on HBcAg assembly into CLPs in plant cells and animal
immunization studies with plant-associated HBcAg. In the
outlook, the plants obtained by us can be exploited as an
initial material to produce therapeutic vaccine for chronic
HBV carriers, orally administrated when produced in lettuce, and injective, when produced in tobacco.
Phenolic compounds in shoot and callus cultures of Plantago ovata Forssk.
Anna Budzianowska, Jaromir Budzianowski
Department of Pharmaceutical Botany and Plant Biotechnology, Poznań University of Medical Sciences, Św. Marii
Mgdaleny 14, Poznań, Poland, e-mail: [email protected]
Plantago ovata Forssk. (Plantaginaceae) is native to
flora of S.E. Spain, N. Africa, S.W. Asia. It is cultivated for
seeds and seed husks, which are commercially important
laxative remedies due to the high content of mucilage. The
species is propagated from seeds and does not have the
ability for vegetative propagation.
In vitro regeneration of P. ovata through induction of
axillary shoots, indirect organogenesis and somatic
embryogenesis have been described (Fons et al., 2008). So
far, only seeds of P. ovata have been studied with respect
to phenolic metabolites demonstrating the presence of two
flavonoids and two phenethyl glycosides (Nishibe et al.,
2001).
The aim of the present work was to establish shoot
and callus cultures of P. ovata and to investigate their
phenolic compound pattern.
Micropropagation from shoot-tips of seedlings was
carried out on MS medium supplemented with NAA/KIN
or IAA/KIN and resulted in only 2–3 axillary shoots per
explant. No shoots developed through direct organogenesis on leaf and root fragments, in contrast to Plantago
lanceolata. Callus initiation was readily achieved on
roots, leaves and hypocotyls on MS medium supplemented with 2,4-D/Kin or NAA/BAP, however, growth of callus
cultures was poor. Therefore, a reduction of NH4NO3 con-
34
tent in the medium was tested, which appeared useful in
the case of callus cultures of P. lanceolata (Budzianowska
et al., 2004).
Extracts from shoot and callus cultures and from
seeds of P.ovata were prepared and analysed by 1D-TLC
and 2D-TLC. The shoots and callus exhibited the presence
of phenethyl glycosides (similarly to seeds), while shoots
additionally contained flavonoids different from those of
the seeds and leaves of P. lanceolata (Budzianowski and
Budzianowska, 2008).
REFERENCES
BUDZIANOWSKA A, SKRZYPCZAK L, BUDZIANOWSKI J. 2004.
Phenylethanoid glucosides from in vitro propagated plants
and callus cultures of Plantago lanceolata L. Planta Medica
70: 834–840.
BUDZIANOWSKI J, BUDZIANOWSKA A. 2008. Improved analytical and
preparative separation of Plantago lanceolata L. flavonoid
glucuronides using mobile phases with ionic modifiers. 6th
Int. Symp. Chromatogr. Nat. Prod., Lublin (Poland), June
15–18. Abstracts. p. 51.
FONS F, GARGADENNEC A, RAPIOR S. 2008. Culture of Plantago
species as bioactive components resources: a 20-year review
and recent applications. Acta Biologica Gallica 155:
277–300.
NISHIBE S, KODAMA A, NOGUCHI Y, HAN Y. 2001. Phenolic compounds of Plantago ovata and P. psyllium. Natural
Medicines 55: 258–261.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
The use of pollen tube in vitro cultures for determination of nuclei positions
in angiosperm pollen tubes
Marta Bultrowicz, Rafał Mól
Department of General Botany, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University in
Poznań, Umultowska 89, 61-614 Poznań, Poland, e-mail: [email protected]
Appropriate sperm cell delivery to the embryo sac is one
of the crucial step of fertilization in flowering plants, and
it determines reproductive success of a plant.
Mechanisms of sperm cell transport during pollen tube
growth are not fully understood. The concept of male
germ unit (MGU) is a very attractive hypothesis on sperm
cell transport within the pollen tube (Mogensen, 1992).
However, MGU has been described for sperm cells or generative cells and the vegetative nucleus only for ten or so
angiosperms, and in some cases the spatial relationship
between sperms or generative cell and the vegetative
nucleus were temporal only and found either in pollen
grain or pollen tube. Here we claimed to determine the
position of the vegetative and generative nuclei in the
pollen tubes of lily, hyacinth, and common snowdrop.
Pollen grains germinated on Brewbaker-Kwack media
optimized for each species and pollen tubes were growing
in vitro for 24 or 48 hours. The vegetative and generative
nuclei were visualized by various dyes, and the ethidium
bromide staining of fixed pollen tubes was chosen for
major analyses. Nuclear position related to the pollen tube
tip showed great variability in three species studied. No
general model on vegetative nucleus guidance could be
established for each species, and only in one culture variant per species the vegetative nucleus was ahead of the
generative nucleus in the pollen tube (statistically significant). Also the distance between both nuclei in pollen
tubes was variable within each species. This suggests that
MGU could only be temporal in the pollen tubes growing
in vitro or culture conditions affected the MGU formation
in many pollen tubes. Based on nuclear position analyses,
the approximate region of nuclei appearance was approx.
50% or 35% length of 48-hour-old pollen tubes. In vitro
cultures of pollen tubes can be used for MGU investigation
in plants, however, the results should be compared to the
in planta situation.
REFERENCES
MOGENSEN HL. 1992. The male germ unit:concept, composition,
and significance. Inernational Review of Cytology 140:
129–143.
Molecular analysis of interspecific Allium cepa × A. roylei hybrids
Alicja Chuda, Adela Adamus
Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Cracow,
Al. 29 Listopada 54, 31-425 Cracow, Poland, e-mail: [email protected]
To achieve introgression of resistance to downy mildew
(Peronospora destructor) into the bulb onion, interspecific crosses between Allium cepa and Allium roylei were
conducted. F1 A. cepa × A. roylei plants were obtained via
embryo rescue technique (Chuda and Adamus, 2005).
Verification of the hybrid character of the F1 regenerants
was based on three different molecular markers. SIRF/SIR-R and ACS-F/ACS-R primers were based on
AY753557 and AY585678 onion cDNA clones, respectively, while A-F/A-R primers were designed on the basis on
Allium cepa alliinase gene according to van Heusden et al.
(2000). Analysis of the electrophoretic profiles of the
Vol. 51, suppl. 1, 2009
examined accessions revealed that 97.6% of the tested F1
plants were interspecific Allium cepa × Allium roylei
hybrids.
REFERENCES
CHUDA A, ADAMUS A. 2005. Allium cepa x Allium roylei hybrids –
production and identification. Allium
Improvement
Newsletter 15: 49–51.
VAN HEUSDEN AW, VAN OOIJEN JW, VRIELINK-VAN GINKEL R, VERBEEK
WHJ, WIETSMA WA, KIK C. 2000. A genetic map of an interspecific cross in Allium based on amplified fragment length
polymorphism (AFLPTM) markers. Theoretical and Applied
Genetics 100: 118–126.
35
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Two year survey of health status of Salix × Populus hybrids in the Warmia
region
Joanna Ciszewska-Marciniak1, Małgorzata Jędryczka1, Jerzy Przyborowski2, Elżbieta Zenkteler3
1
Institute of Plant Genetics, Polish Academy of Sciences, Poznań
Department of Plant Breeding and Seed Production, University of Varmia and Mazury, Olsztyn
3
Department of Botany, Faculty of Biology, Adam Mickiewicz University of Poznań
2
Recently willows have become increasingly popular as
crop plants used for renewable energy (providing an alternative to fossil fuels). However, they require abundant
water supply during the whole growth period. To avoid
this problem, hybrids between willows and poplars have
been produced at the Department of Botany, Faculty of
Biology of Adam Mickiewicz University in Poznań.
Leaf rust is the most common and serious disease of
both willows and poplars. The disease is caused by several species of the genus Melampsora. Willows and poplars
are infected by different ranges of Melampsora species
and also by various special forms and pathotypes, each
capable of infecting a certain range of species and geno-
types. The aim of this study was to investigate the resistance of hybrids obtained from the crossings between Salix
viminalis and Populus tremula.
Field experiment was located in Bałdy, near Olsztyn in
Warmia region (north-east Poland). Observations were
conducted over a two year period (2008–2009). Each year,
the first observation took place in July/August and the second in September/October. All studied hybrids (six genotypes) were infested with willow rust, similarly to parental
plants. Identification of Melampsora spp. isolates
obtained from the infected plants is under way using pathogenicity tests and molecular tools.
Albino regenerants from isolated microspores of wheat
Andrzej Z. Czaplicki, Sylwia Oleszczuk, Janusz Zimny
Plant Breeding and Acclimatization Institute, Radzików, 05-870 Błonie, Poland, e-mail: [email protected]
Significant progress has been observed in recent years in
the development of culture techniques of isolated cereal
microspores. One can expect a considerable improvement
in the efficiency of haploid plant regeneration due to the
highly embryogenic nature of microspores and a short
path of the culture.
The purposes of the experiments were: to induce the
androgenic development of microspores isolated using a
blender, to produce embryos and to regenerate plants.
Microspores of wheat spring variety Pitic 62 were isolated using the method described by Zheng (Zheng et al.,
2001, 2002) and then grown in the dark at 26°C. Isolated
ovaries of spring wheat variety Orofen were used as a
36
nurse culture. Microspore divisions and further embryo
development were observed. As a result of the experiment,
five albino plants have been regenerated so far.
REFERENCES
ZHENG MY, LIU W, WENG Y, POLLE E, KONZAK CF. 2001. Culture of
freshly isolated wheat (Triticum aestivum L.) microspores
treated with inducer chemicals. Plant Cell Reports 20:
685–690.
ZHENG MY, WENG Y, LIU W, KONZAK CF. 2002. The effect of
ovaryconditioned medium on microspore embryogenesis in
common wheat (Triticum aestivum L.). Plant Cell Reports
20: 802–807.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Optimization of the regeneration method of Miscanthus × giganteus in in vitro
culture
Diana Czarnecka, Anna Czubacka, Teresa Doroszewska
Institute of Soil Science and Plant Cultivation – State Research Institute, Czartoryskich 8, Puławy,
e-mail: [email protected]
Miscanthus × giganteus Greef & Deuter is a triploid
hybrid species belonging to the family Poaceae. This
species is characterized by a fast biomass increase,
because its shoots can growth up to 3 meters in height in
the second year of cultivation. Therefore miscanthus can
be used as a source of biomass for the energy industry, in
phytoremediation and also in the paper industry. Under
our climatic conditions Miscanthus x giganteus does not
set seeds and the best methods for its propagation are
multiple division of the rhizome and propagation in vitro.
The objective of the present experiment was to develop
a method of obtaining regeneration of Miscanthus ×
giganteus plants under in vitro conditions. Inflorescence
explants (0.5–2.5 cm in length), internodes, fragments of
leaves and rhizomes were the research material. They
were sterilized using 10% hydrogen peroxide. Three basic
media were used: MG1 (LS medium containing 5 mg l-1
2,4 D and 0.1 mg l-1 BAP), MG2 (LS medium with 2.5 mg l-1
2,4 D and 0.5 mg l-1 BAP) and MG3 (WPM medium
enriched with 2.5 mg l-1 2,4 D and 0.5 mg l-1 BAP). The
experiment was conducted both in the light and in the
dark. Regenerated plants were transferred to LS medium
supplemented with 0.2 mg l-1 IAA and 0.2 mg l-1 NAA.
Callus was not observed on explants maintained in the
dark or on fragments of leaves and rhizomes kept in the
light. The most suitable explants for callus induction and
plant regeneration were fragments of inflorescences. Less
abundant callus tissue was observed on internodes, from
which only a few plants regenerated. On the remaining
explants neither callus formation nor organogenesis were
noticed. The largest numbers of regenerated plants were
obtained on MG1 and MG3 media. They were rooted and
acclimated to greenhouse conditions.
REFERENCES
LINSMAYER EM, SKOOG F. 1965. Organic growth factor requirements of tobacco tissue cultures. Physiologia Plantarum 18:
100–127. (LS medium)
LLOYD G, MCCOWN B. 1980. Commercially feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip
culture. Comb. Proc. Int. Plant Prop. Soc. 30: 421–427.
(WPM medium)
Changes in sulfur amino acid contents in flax caused by overexpression of yeast
Met25 gene lead to an increase in antioxidant capacity and Fusarium resistance
Tadeusz Czuj, Magdalena Żuk, Jan Szopa
Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63–77, 51-148 Wrocław, Poland,
e-mail: [email protected]
Cysteine synthesis in plants is the crucial point in sulfate
assimilation. It introduces inorganic sulfide into the carbon skeleton. In plants and bacteria, this reaction is catalyzed by O-acetyloserine sulfhydrylase (OASTL) with free
sulfide and O-acetyloserine (OAS) as the substrates. In the
present study we use Sacharomyces cerevisiae gene
Met25 encoding O-acetyloseine-O-acetylohomoserine-sulphydrylase, yeasts analogue of plant OASTL protein in
flax.
The yeast enzyme utilizes both O-acetylhomoserine
and, as discovered in in vitro experiments, O-acetylserine
as substrates, while the plant enzyme uses only Oacetylserine as its substrate in cysteine biosynthesis
(Matityahu et al., 2006). An important difference between
the plant OASTL and its yeast analogue is that the plant
enzyme has an additional activity: it is able to degrade cysteine to ammonia, H2S and pyruvate (Riemenschneider et
al., 2005). The yeast enzyme does not seem to be able to
do this (Matityahu et al., 2006; Harms et al., 2000; Sirko
et al., 2004), thus we expect to have an increased production of cysteine and it's derivatives in flax. Overexpressing
the yeast Met25 in flax resulted in a significant increase in
Vol. 51, suppl. 1, 2009
cysteine and methionine biosynthesis. This overproduction of sulfur amino acids also increases the synthesis of
glutathione, a tripeptide containing cysteine. The increase
in glutathione content in the transgenic plants increases
their antioxidative potential. We also observed improvements in plants' protection against Fusarium infection.
REFERENCES
HARMS K, BALLMOOS, PV, BRUNOLD C, HÖFGEN R, HESSE H. 2000.
Expression of a bacterial serine acetyltransferase in transgenic potato plants leads to increased levels of cysteine and
glutathione. The Plant Journal 22: 335–343.
MATITYAHU I, KACHAN L, BAR ILAN I, AMIR R. 2006. Transgenic tobacco plants overexpressing the Met25 gene of Saccharomyces
cerevisiae exhibit enhanced levels of cysteine and glutathione
and increased tolerance to oxidative stress. Amino Acids 30:
185–194.
RIEMENSCHNEIDER A, RIEDEL K, HOEFGEN R, PAPENBROCK J, HESSE H.
2005. Impact of reduced O-acetylserine(thiol)lyase isoform
contents on potato plant metabolism. Plant Physiology 137:
892–900.
SIRKO A, BLASZCZYK A, LISZEWSKA F. 2004. Overproduction of SAT
and/or OASTL in transgenic plants: a survey of effects.
Journal of Experimental Botany 55: 1881–1888.
37
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Phytosulfokine stimulates development of sugar beet (Beta vulgaris L.)
mesophyll protoplasts
Joanna Durok, Ewa Grzebelus, Marek Szklarczyk
Department of Genetics, Plant Breeding and Seed Science, Faculty of Horticulture, Agricultural University of Cracow,
Al. 29-Listopada 54, 31-425 Cracow, e-mail: [email protected]
Phytosulfokine (PSK) is a peptide plant growth factor that
was originally isolated from a conditioned medium
derived from asparagus mesophyll cell culture
(Matsubayashi and Sakagami, 1996). In some plant
species PSK was found to stimulate cell proliferation
(Matsubayashi, 2003). On the other hand, so called "recalcitrant species" are known, that are difficult either to
regenerate using tissue culture methods and/or to transform with foreign DNA. Sugar beet is such a recalcitrant
crop, particularly with respect to protoplast-based techniques. The aim of this work was to determine phytosulfokine influence on the division activity of sugar beet protoplasts. We tested 3 clones of sugar beet male-sterile line
growing in 2 different culture media (CPP with 8 mg/l
putrescine and 2.12 mg/l propyl gallate; CPP with 8 mg/l
putrescine, 2.12 mg/l propyl gallate, Kao and Michayluk
vitamins and 2 % coconut water) with 2 concentrations of
phytosulfokine (10-7 and 10-8 M). Viability of protoplasts
was determined by fluorescein diacetate (FDA) staining on
the day of isolation as well as 24 and 48 hours after isolation. Cell proliferation activity was evaluated on 7th, 14th
and 21st days of culture. There were no differences in viability of protoplasts between media and PSK concentrations. Cell division activity was dependent on both medium composition and PSK concentration. The highest division frequency was observed in CPP containing 8 mg/l
putrescine, 2.12 mg/l propyl gallate and 10-7 M PSK. It
varied from 13.9 to 29.1% on 21st day of culture whereas
in the same medium containing 10-8 M PSK, proliferation
efficiency reached approximately 6%. In no-PSK control,
proliferation efficiency varied between 0.1 and 2.6%. This
result indicates that PSK strongly stimulates the division
of sugar beet mesophyll protoplasts under cell culture
conditions.
REFERENCES
MATSUBAYASHI Y. 2003. Ligand-receptor pairs in plant peptide signaling. Journal of Cell Science 116: 3863–3870.
MATSUBAYASHI Y, SAKAGAMI Y. 1996. Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of
Asparagus officinalis L. Proceedings National Academy of
Sciences USA 93: 7623–7627.
Gentiana cachemirica Decne in in vitro culture
Katarzyna Floryanowicz-Czekalska1,2, Jan J. Rybczyński1
1
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, email: [email protected], [email protected]
2
Department of Molecular Carcinogenesis, Medical University of Łódź, Mazowiecka 6/8, 92-215 Łódź
Most species from genus Gentiana described so far are
protected by law in Poland. They are valued because of
important secondary metabolites and attractive flowers.
They are also well known for possessing a high morphogenetic potential in vitro. The above characteristics have
made this genus very attractive for breeding.
Micropropagation through somatic embryogenesis is an
alternative pathway to the conventional breeding methods
for the propagation of gentians. Up to now it has been
described for only a few species of the genus.
In the present study, extensive experiments with
G. cachemirica, a new in vitro Gentiana species are
38
reported. Callus tissue derived from seedling explants was
used as a source for the cell suspension. The morphological potential of the culture expressed by its embryogenic
capacity was assessed by counting the number of somatic
embryos on agar regeneration medium and by their ability to become converted into plants.
Long-term culture brings about the risk of somaclonal
variation. Two techniques were evaluated to perform cytological analysis of the regenerants. Chromosome counting
was carried out on well spread metaphase plates. DNA
content was determined using a flow cytometer.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Rooting in vitro and acclimation to the greenhouse of herbaceous peony
plantlets
Eleonora Gabryszewska1, Ludwika Kawa-Miszczak2
1
Department of Physiology and Morphogenesis of Ornamental Plants, Research Institute of Pomology and Floriculture,
Pomologiczna 18, 96-100 Skierniewice, Poland, e-mail: [email protected]
2
Food Safety Laboratory, Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice,
Poland, e-mail: [email protected]
The influence of sucrose concentration (10 and 30 g l-1),
low level of nitrogen salts (1/8 KNO3, 1/8 NH3NO4), auxins
(1 mg l-1 IBA + 1 mg l-1 IAA + 0.01 mg l-1 NAA) and temperature (15°C, 20°C) on the rooting in vitro and acclimation to the greenhouse conditions of two cultivars
('Jadwiga' and 'Profesor Wójcicki') of Paeonia lactiflora
was investigated. The level of endogenous carbohydrates
in the peony shoots and roots during the rooting and acclimation phases were analysed.
Rooting and acclimation success rates were higher for
cultivar 'Profesor Wójcicki' than for cultivar 'Jadwiga'. Also
more roots were produced by shoots of cultivar 'Profesor
Wójcicki'. The presence of auxins in the medium and the
higher concentration of sucrose (30 g l-1) stimulated a
greater number of roots/shoot. On the other hand, a higher rooting rate was found on the auxin-free medium in the
presence of high level of sucrose. The shoots of 'Profesor
Wojcicki' rooted best when cultured at 20°C, but shoots of
'Jadwiga' rooted best at the lower temperature (15°C).
During the rooting stage, the major sugars detected in the
peony microplants were sucrose, glucose and fructose.
After four weeks of acclimation, plantlets accumulated
starch and showed a strong inhibition of shoot growth,
and dormant buds were developed.
Expression pattern of ABH1, CBP20 and HYL1 genes during somatic
embryogenesis in Arabidopsis
Marta Gliwicka, Małgorzata D. Gaj
Department of Genetics, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland
[email protected]; [email protected]
Genetic dissection of somatic embryogenesis (SE) is of
great interest since the process is commonly applied for
plant regeneration in transgenesis and micropropagation.
Identification of genes involved in embryogenic transition
of somatic cells is of interest for the improvement of the
existing protocols on plant regeneration, including those
for recalcitrant plants. Arabidopsis mutants impaired in
SE provide valuable material in the search for SE-specific
genes (Gaj et al., 2006). Among such SE-defective genotypes, the ABA-response (abh1, cbp20 and hyl1) and
auxin-response (axr4-1) mutants were found, implicating
the involvement of the mutated genes in the process of
somatic embryo formation. The ABH1 and CBP20 genes
encode the subunits of the cap binding complex (CBC)
involved in mRNA metabolism while the protein product
of HYL1 gene takes part in miRNA biogenesis.
To provide insight into the role of the ABH1, CBP20
and HYL1 genes in SE, Real-time qPCR analysis was
applied to monitor the expression level of these genes at
various time points of embryogenic cultures derived from
Vol. 51, suppl. 1, 2009
the highly embryogenic (Col-0) and the SE defective
mutant (axr4-1 and cbp20) genotypes. The level of ABH1,
CBP20 and HYL1 activity was evaluated between 0 and
30d of embryogenic cultures. It was observed that the
genes were expressed at the same level during the whole
monitored period of embryogenic cultures, implicating
their basic role in cell growth and differentiation, rather
than SE specificity. Moreover, the expression pattern of
the ABH1, CBP20 and HYL1 genes found in cultures
derived from the axr4-1 and cbp20 mutants were found to
be similar to control culture (Col-0), implicating that the
defects in embryogenic response observed in these
mutants did not result from changes in expression of the
analysed genes.
REFERENCES
GAJ MD, TROJANOWSKA A, UJCZAK A, MĘDREK M, KOZIOł A and
GARBACIAK B. 2006. Hormone-response mutants of
Arabidopsis thaliana (L.) Heynh. impaired in somatic
embryogenesis. Plant Growth Regulation 49: 183–197.
39
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Obtaining plants from gametic embryos of red beet-preliminary investigations
Krystyna Górecka, Dorota Krzyżanowska, Urszula Kowalska, Waldemar Kiszczak, Ryszard Górecki
Research Institute of Vegetable Crops, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland,
e-mail: [email protected]
Red beet ranks second (after carrots) in statistics of root
vegetable consumption in Poland. Red beet hybrids characterised by high yields and size uniformity are becoming
more and more important. Using traditional methods to
obtain homozygotic parental forms to create hybrids is
difficult and time-consuming. Making use of biotechnological methods (androgenesis and gynogenesis in vitro)
could shorten the time needed to obtain F1 cultivars.
In experiments conducted at the Research Institute of
Vegetable Crops in Skierniewice success was achieved in
obtaining the first embryos by gynogenesis and calluses in
anther cultures of red beet cv. 'Opolski'. Attempts at plant
regeneration from the obtained structures were made on
the media used by Barański (1996). Multiplication of
shoots was obtained on MS regeneration medium
(Muraschige and Skoog, 1962) containing cytokinin and a
lower level of sucrose. The next step involved rooting of
the obtained rosettes. They were transferred onto the
same medium supplemented with IAA, and after about 6
weeks rooted on a medium containing 1/2 of the MS
macroelements and IBA.
REFERENCES
BARAŃSKI R. 1996. Efektywność procesu gynogenezy u buraka
ćwikłowego. Praca doktorska wykonana w Katedrze
Genetyki Hodowli i Nasiennictwa Wydziału Ogrodniczego
Akademii Rolniczej im. H. Kołłątaja w Krakowie.
MURASHIGE T, SKOOG F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum 15: 473–497.
Yacon – in vitro propagation trials
Krystyna Górecka, Dorota Krzyżanowska, Urszula Kowalska, Waldemar Kiszczak, Ryszard Górecki
Research Institute of Vegetable Crops, Konstytucji 3 Maja 1/3, 96-100 Skierniewice, Poland,
e-mail: [email protected]
The yacon (Polymnia sonchifolia) is a native Andean
plant, cultivated mainly for its tubers, which have a particularly high fructan content. The whole plant is edible.
The leaves are used in folk medicine. Extracts from yacon
leaves demonstrate antioxidant activity. The plant can be
cultivated in the European climate. Natural methods of
propagation through tubers or cuttings are not very efficient because of the transmission of bacterial and viral
pathogens (Zardini, 1990). Micropropagation in vitro is
the best alternative for the propagation of this species
(Mogor et al., 2003).
The aim of our work was to carry out trials on in vitro
propagation of yacon: multiplication of shoots, their rooting and plant weaning.
In our preliminary experiments, we obtained the best
shoot multiplication on MS medium (Muraschige and
40
Skoog, 1962) supplemented with BAP and 3% sucrose. A
medium containing 1/2 MS macroelements, IBA and 3%
sucrose proved to be the best for rooting of shoots. Up to
90% of rooted plants became adapted to ex vitro conditions.
REFERENCES
MOGOR G, MOGOR AF, LIMA GPP. 2003. Bud source, asepsis and
benzylaminopurine (BAP) effect on Yacon (Polimnia sonchifolia) micropropagation. ISHS Acta Horticulturae 597:
311–313.
MURASHIGE T, SKOOG F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiologia Plantarum 15: 473–497.
ZARDINI E. 1991. Ethnobotanical notes on "yacon" Polymnia
sonchifolia (Asteraceae). Economic Botany 45: 72–85.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
The effect of cefotaxime and carbenicillin on carrot protoplast cultures
Ewa Grzebelus, Mikołaj Grabka
Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Cracow, Al. 29 Listopada 54,
31-425 Cracow, Poland, e-mail: [email protected]
In vitro culture techniques, especially protoplast cultures,
are expensive, time consuming, and laborious. Therefore
exogenous (accidental) and endogenous microbial contamination is one of the most critical points in the
research relying on in vitro cultures. Application of antibiotics may help to solve this problem. The ideal antibiotic
should efficiently affect a broad spectrum of bacteria and
be non-toxic to plant cells.
In the present study we examined the toxicity of two
antibiotics, i.e. cefotaxime and carbenicillin, to carrot protoplasts. The antibiotics belong to the betalactam and
cephalosporin group, respectively. Protoplasts were isolated from the mesophyll tissue of cv. 'Dolanka' according
to Dirks et al. (1996) protocol with modifications and cultured at five concentrations (0.4, 0.8, 1.2, 1.6, 2.0 mg ml-1)
of cefotaxime or carbenicillin. Protoplast viability, their
mitotic activity and regeneration capacity were analyzed to
characterize the toxic effect of the antibiotics on carrot
cells. Reduction of protoplast viability was observed after
24 hours of culture for carbenicillin and after 48 hours for
cefotaxime. The inhibitory effect of antibiotics on cell divisions was visible after 10 days of the culture. The concentrations of cefotaxime and carbenicillin equal or higher
than 1.2 mg ml-1 in culture medium decreased the division frequency of protoplasts. However, carbenicillin
showed a more dramatic negative effect on carrot cells,
lowering their mitotic activity two- to ten-fold, as compared to cefotaxime. Despite the different reactions of cells
at the beginning of the culture, callus tissue and somatic
embryos were obtained later, both in the system with cefotaxime and carbenicillin. Owing to the fact that a higher
level of plant regeneration was observed in the presence of
cefotaxime, this antibiotic should be recommended to protect carrot cell or tissue cultures from bacterial contamination.
REFERENCES
DIRKS R, SIDOROV V and TULMANS C. 1996. A new protoplast culture
system in Daucus carota L. and its applications for mutant
selection and transformation. Theoretical and Applied
Genetics 93: 809–815.
Cytological and molecular variation of carrot regenerants obtained through
in vitro selection of carrot protoplasts against Alternaria radicina
Ewa Grzebelus, Mirosława Gładysz, Estera Widlarz, Dariusz Grzebelus
Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Cracow, Al. 29 Listopada 54,
31-425 Cracow, Poland, e-mail: [email protected]
A population of carrot regenerants was produced from
protoplasts isolated from three accessions (cvs. 'Dolanka',
'Amsterdamska', and a breeding line 9304B). The protoplasts were cultured in the presence of different concentrations of crude culture filtrate from Alternaria radicina,
the causal agent of black rot.
We examined the ploidy level of regenerants by flow
cytometry and chromosome counting, the level of
somaclonal variation by RAPD-PCR, and pollen viability.
Cytometric analysis was performed on ca. 500 plants
regenerated after in vitro selection and on ca. 260 control
plants. 18% regenerants obtained under selection stress
and 9% control plants were tetraploids, the rest of regenerants being diploids. Chromosomes were counted for ca.
110 plants, of which 72 represented plants regenerated in
Vol. 51, suppl. 1, 2009
the presence of the A. radicina culture filtrate. The results
of chromosome counting were consistent with the cytometric data, indicating that no aneuploids were produced
in the course of protoplast cultures. Also, no major differences were generally observed on the RAPD-PCR profiles.
Only sporadically additional amplicons were present in
the profiles of individual regenerants. Pollen viability was
the most variable trait, both for the regenerants from in
vitro selection and for the control plants. Individuals
could be divided into three groups of pollen viability: (1)
up to 20%, (2) from 40 to 70%, and (3) from 80 to 90%.
The results suggest that the level of variability induced in
the process of in vitro selection was only slightly higher
than that observed for control plants.
41
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Micropropagation of Scutellaria altissima L.
Izabela Grzegorczyk, Halina Wysokińska
Department of Biology and Pharmaceutical Botany, Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź,
e-mail: [email protected]
Genus Scutellaria includes over 300 species of perennial
plants which show multiple pharmacological activities.
Their main secondary metabolites are flavonoids
(baicalin, wogonin and scutelarein derivatives) and ditepenoids (clerodane and neoclerodane derivatives). So far
only one species Scutellaria baicalensis, listed in Chinese
and Japanese Pharmacopeias, has been investigated using
in vitro methods, but other Scutellaria species, such as
Scutellaria altissima could also become sources of compounds with pharmacological properties.
The aim of the present study was to establish optimal
conditions for micropropagation of Scutellaria altissima.
Shoot tips and stem fragments with single node from fiveweek-old shoots grown in in vitro culture were used as
explants. The influence of various cytokinins (6-benzylaminopurine-BAP, zeatin, thidiazuron-TDZ, kinetin,
6-dimethylallylaminopurine-2iP) on shoot proliferation was investigated. The best results were obtained when
BAP and zeatin at a high concentration were used. In our
work we obtained also the valuable shoots from callus tissue. The callus tissue was obtained from sterile seedlings
(fragment of hypocotyls, roots and cotyledons). This investigation is concerned with selection of the most suitable
medium for organogenic callus formation. The best conditions for differentiated callus tissue development were on
MS medium with a low concentration of thidiazuron.
Higher TDZ concentration caused hyperhydricity syndrome and shoot deformation.
Induction and proliferation of embryogenic tissues of Serbian spruce (Picea
omorika) and their maintenance in liquid nitrogen
Teresa Hazubska-Przybył1, Paweł Chmielarz2, Marcin Michalak2, Krystyna Bojarczuk1
1
Laboratory of Vegetative Reproduction, Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik,
Poland, e-mail: [email protected]
2
Laboratory of Seed Biology, Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik, Poland,
e-mail: [email protected]
Picea omorika is an endemic species threatened with
extinction in the wild. Besides, it is a valuable species for
nursery production. Hence it is necessary to protect its
genetic resources ex situ.
Somatic embryogenesis is a very beneficial micropropagation method, because of the potentially high regeneration rate and possibilities to store the embryogenic tissue
and somatic embryos in liquid nitrogen (LN). The aim of
this study was to induce embryogenic culture of P. omorika and develop a protocol for cryopreservation of embryogenic tissues.
Embryogenic tissues of Serbian spruce were induced
from mature zygotic embryos. They were dissected from
seeds of P. omorika originating from the Kórnik
Arboretum (Poland). Explants were cultured in two
media: BM-3 (Gupta and Durzan, 1986) and 1/2 LM
(Litvay et al., 1985), supplemented with 9 μM auxin (2,4-D,
NAA or Picloram) and 4.5 μM cytokinin (BA).
Embryogenic tissues were proliferated in 1/2 LM medium
with the addition of 9 μM 2,4-D or NAA or Picloram and
4.5 μM BA.
Embryogenic tissue of P. omorika was cryopreserved
using two methods (E-LN and E+LN). In the first method
(E-LN), microfuge tubes containing only embryogenic tis-
42
sue were immersed in LN for 24 h and then rapidly
thawed in warm water (42°C) for 2 min. In the second
method (E+LN), microfuge tubes containing LN and
embryogenic tissue were immersed in LN for 24 h and
then embryogenic tissues were taken out of the tubes and
rapidly thawed in a warm 1.2 M sucrose solution (42°C)
for 2 min.
Induction rate of embryogenic tissue of P. omorika was
the highest (23.75%) on 1/2 LM medium supplemented
with 9 μM Picloram and 4.5 μM BA. The growth of the
embryogenic tissue in vitro was best on the same medium.
Cryopreservation in microfuge tubes containing only
embryogenic tissue (thawed in water inside the tubes) was
more appropriate than in microtubes containing LN and
embryogenic tissue (thawed directly in sucrose solution).
REFERENCES
GUPTA PK, DURZAN DJ 1986. Plantlet regeneration via somatic
embryogenesis from subcultured callus of mature embryos
of Picea abies (Norway spruce). In vitro Cellular and
Developmental Biology – Plant 22: 685–688.
LITVAY JD, VERMA DC, JOHNSON MA. 1985. Influence of loblolly pine
(Pinus taeda L.) culture medium and its components on
growth and somatic embryogenesis of wild carrot (Daucus
carota L.). Plant Cell Reports 4: 325–328.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Identification of European and Japanese larches and their hybrids based
on genetic markers
Anna Jagielska
Department of Genetics and Forest Tree Physiology, Forest Research Institute,
Braci Leśnej 3, Sękocin Stary, 05-090 Raszyn, Poland, e-mail: [email protected]
Larix decidua Mill. is a native species, and Larix
kaempferi Sarg. is the most common of foreign larch
species. Morphological differences between pure Larix
decidua and Larix kaempferi and their interspecific
hybrid (Larix x eurolepis) are difficult to assess. Hybrids
between European and Japanese larches combine the
properties of both parental species (Scheepers, 2000).
The interspecific hybrids combine traits of both
parental species and contribute to their morphological
variability. Molecular markers are needed to identify larch
hybrids and their parental species.
The identification strategy involved a combination of
maternally inherited markers from the mitochondrial
genome (mtDNA) and paternally inherited markers from
the chloroplast genome (cpDNA). Hybrids were identified
by the presence of a mitochondrial sequence inherited
from one parental species and a chloroplast sequence
inherited from the other parental species.
For the identification of diagnostic markers 96 larch
individuals were sampled. Sequences of cpDNA and
mtDNA were amplified by polymerase chain reaction
using specific primers (Acheré, 2004). The chloroplast (llTaq I) and mitochondrial (f13) diagnostic markers were
used to identify pure species of larches and their hybrids.
Marker ll-Taq I revealed an interspecific polymorphism. This marker discriminated between all individuals
of L. decidua and L. kaempferi. All L. kaempferi individuals possessed a specific 601 bp fragment. This polymorphism is due to a single nucleotide substitution in the
restriction site: TCGA recognised by Taq I in L. decidua
and TCAA in L. kaempferi.
Marker f13 of approximately 1,300 bp, was amplified
only in L. kaempferi. The test based on the f13 and ll-TaqI
markers is easy to perform and can constitute a very effective instrument for identification of European and
Japanese larches and their hybrids.
REFERENCES
ACHERÉ V, FAIVRE RAMPANT P, PÂQUES LE, PRAT D. 2004. Chloroplast
and mitochondrial molecular tests identify European ×
Japanese larch hybrids. Theoretical and Applied Genetics
108:1643–1649.
SCHEEPERS D, ELOY MC, BRIQUET M. 2000. Identification of larch
species (Larix decidua, Larix kaempferi and Larix ×
eurolepis) and estimation of hybrid fraction in seed lots by
RAPD fingerprints. Theoretical and Applied Genetics:
1071–1074.
In vitro cultures of Drosera binata as a source of compounds with antimicrobial
activity
Aleksandra Kasprowicz1, Anna Szpitter1, Anita Maciejewska2, Marian Kamiński2, Ewa Łojkowska1,
Aleksandra Królicka1
1
Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and Medical University of
Gdańsk, Kladki 24, 80-822 Gdańsk, Poland, e-mail: [email protected]
2
Gdańsk University of Technology, Department of Analytical Chemistry, Gabriela Narutowicza 11/12, 80-952 Gdańsk,
Poland, e-mail: [email protected]
Extracts of plants Drosera binata (Droseraceae) is of
potential medicinal use due to the content of naphthoquinones and flavonoids. The aim of this work was to
establish the most efficient method for the production of
secondary metabolites in the tissue of D. binata grown on
1/2 MS medium, 2% sucrose, pH 5.6 in order to study
their antibacterial activity and ability to inhibit bacterial
quorum-sensing (QS). For increasing the accumulation of
secondary metabolites a series of elicitors has been used:
autoclaved overnight suspension of Agrobacterium rhizogenes, jasmonic acid (JA), additional nitrogen salts and
nitrogen deprivation in culture medium. Quantitative and
qualitative determination of naphthoquinones and
flavonoids in chloroform (CHCl3) and methanol extracts
from D. binata was performed using NP – HPLC/UV-DAD.
JA occurred to be the most efficient elicitor, which
increased the accumulation of plumbagin in D. binata tissue about 1.4 fold in comparison to control. Bactericidal
Vol. 51, suppl. 1, 2009
activity of extracts was tested against 4 species of human
pathogens: Escherichia coli, Enterococcus faecalis,
Klebsiella pneumoniae and Staphylococcus aureus. To
measure antibacterial activity minimal bactericidal concentrations (MBC) of extracts were determined. S. aureus
strains occurred to be the most sensitive among human
pathogens tested (MBC for CHCl3 extract was 70 μg of dry
weight of extract/ml). Indicator strains of Agrobacterium
tumefaciens and E. coli expressing β-galactosidase or lux
genes in the presence of N-acyl homserine lactones
(AHLs) were used to study QS inhibition. It was shown
that extract from D. binata significantly inhibited AHLinduced response of bioassay strains with IC50 values
ranging from 14 to 27 μg/ml depending on the indicator
strain used.
Acknowledgements: This research was supported by Foundation
for Polish Science & by the Grant B051–5–0293–9.
43
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Genetic diversity within population of the endangered species Cypripedium
calceolus
Adam Kawiński, Joanna Znaniecka, Ewa Łojkowska
Laboratory of Plant Protection and Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdańsk and
Medical University of Gdańsk, Kładki 24, 80-822 Gdańsk, Poland, e-mail: [email protected]
Cypripedium calceolus (L.) (Lady's Slipper) is a 15–60 cm
tall terrestrial herb. It grows best in partially shady areas
under hazel, oak, conifer or ash. Flowering period
depends on geographical location; in Northern Poland, it
occurs between May and July. According to the Polish Red
List of Threatened Plants this species belongs to the vulnerable group. The aim of the study was to check the suitability of RAPD-PCR to determine the genetic diversity of
Lady's Slipper population growing in the nature reserve
'Ostrzycki las'. The results should provide information on
Lady's Slipper's manner of reproduction. Fresh leaves of
the Lady`s Slipper taxa were collected from the hereby
listed sites in the nature reserve and deep (–80°C) frozen.
Four DNA isolation methods were tested: PVP isolation
described in Pirtilla et al. (2001), CTAB isolation
described in Bekesiova et al. (1999), DNA isolation by
Fermentas Genomic DNA Purification Kit and A&A
Biotechnology Genomic mini AX Plant Kit. RAPD-PCR
reaction was carried out in 25 μl volume containing reaction buffer, MgCl2, dNTP`s, primers [10 pmol/μl], all prepared as 'master mix', then aliquoted into reaction tubes.
Template DNA (5 ng) was added to the reaction mixture
after first adding 1 unit of Taq DNA polymerase (all from
Fermentas). Amplification products were separated electrophoretically on a 2% agarose gel and afterwards stained
with ethidium bromide. After examining yield and quality
of the DNA, the method of DNA isolation described by
Bekesiova et al. (1999) with slight modifications was chosen. Approximately 0.05–0.15 g of leaf material were used
for DNA extraction experiment. 28 arbitrary 10-mer
primers were tested, and seven that gave polymorphic and
scorable amplified products, ranging between 100–1200
bp, were used for RAPD profiling. Preliminary experiments proved that RAPD-PCR can be used to determine
genetic diversity of Lady`s Slipper.
REFERENCES
BEKESIOVA I, NAO JP and MLYNAROWA L. 1999: Isolation of high
quality DNA and RNA from leaves of the carnivorous plant
Drosera rotundifolia. Plant Molecular Biology Reporter 17:
269–277.
PIRTILLA AM, HIRSIKORPI M, KAMARAINEN T, JAAKOLA L and HOHTOLA A.
2001. DNA isolation methods for medicinal and aromatic
plants. Plant Molecular Biology Reporter 19: 273.
Morphological evaluation of interspecific F1 (Allium galanthum × A. cepa)
hybrids
Agnieszka Kiełkowska, Adela Adamus
Department of Genetics, Pant Breeding and Seed Science, Agricultural University of Cracow,
29 Listopada 54, 31-425 Cracow, Poland, e-mail: [email protected]
The interspecific F1 hybrids (Allium galanthum × A. cepa)
were obtained by natural seed forming process and via
embryo rescue technique. For hybrids and parental plants
(A.galanthum and A.cepa) evaluation of the morphological traits in vegetative and generative phases was conducted. In the vegetative stage the number of bulbs, the number, length and diameter of leaves, length and diameter of
the stem were evaluated. In the generative phase the number and length of the flower stems, and the length and
diameter of the inflorescence were evaluated. In tested
44
populations, the majority of the investigated features of
vegetative as well as generative organs showed an intermediate (V% = 21–50%) or lower variability.
The hybrids were similar to the maternal form with
regard to only one feature (number of bulbils), while leaf
and stem characters were similar to the paternal form.
The remaining morphological features of interspecific
hybrids were intermediate in relation to maternal and
paternal plants.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Characteristic of the cell suspension culture of Eryngium planum L.
Małgorzata Kikowska, Barbara Thiem
Department of Pharmaceutical Botany and Plant Biotechnology, Poznań University of Medical Sciences,
Św. Marii Magdaleny 14, 61-861 Poznań, Poland, e-mail: [email protected]
Eryngium planum L. (Apiaceae) is used in folk medicine
in Europe. The presence of active constituents found in
majority of Sea Holly species, including triterpene
saponins, flavonoids, phenolic acids, essential oils and
coumarins, determines their multidirectional pharmacological activity: diuretic, expectorant and antimycotic
(Duke J.A.et al.,2002).
Numerous studies have been carried out using plant
cell suspension cultures as potential sources of valuable
constituents. The main problem is that the desired compound usually occurs at a high concentration during a
short period of culture growth. For that reason, the shakeflask suspensions have been analyzed in terms of biomass
concentration suitable for elicitor addition over the course
of a typical growth cycle.
This work reports the initiation, maintenance, measurement of cell culture growth and the influence of MeJA
on secondary metabolites in the liquid culture of Flat Sea
Holly. Methyl jasmonate (100 μM) was employed to
enhance the accumulation of the secondary metabolites –
triterpene saponins, flavonoids and phenolic acids.
Liquid suspension cultures of petiole-derived, stabilized callus of E. planum were conducted by shaking at
110 rpm in MS medium supplemented with 0.5 mg/l 2,4D in the darkness. Subculturing was conducted periodi-
cally (every 8–10 days) by inoculating 5 ml of old culture
into 50 ml of fresh medium to maintain cell vitality. Cell
suspension growth was measured by cell fresh and dry
weights. In order to quantify growth, a stabilized suspension culture was used. From this suspension aliquots of
5 ml were inoculated into 9 flasks. The experiment was
set up in a completely randomized design with 9 treatments. Evaluations were made at 3-day intervals up to the
24th day.
Suspension culture followed a typical growth curve
with the complete cycle showing a lag phase, followed by
exponential, linear and stationary phases.
The ethanolic extracts of suspension cultures were
phytochemically analyzed by TLC for presence of complex
triterpene saponins, flavonoids and phenolic acids. The
data demonstrated that the addition of MeJA on the 12th
day of culture caused an increased secondary metabolite
accumulation.
Acknowledgements: This work was supported by the Ministry of
Science and Higher Education, Warsaw, Poland (grant no NN 05
065334)
REFERENCES
DUKE JA. et al., 2002. Handbook of Medicinal Herbs, CRC PRESS,
277–278
In vitro androgenic response of some pepper (Capsicum) genotypes to different
levels of kinetin
Anna Kisiała, Aleksandra Niklas-Nowak, Marta Orlińska, Paweł Nowaczyk
Department of Genetics and Plant Breeding, University of Technology and Life Sciences, Kaliskiego 7,
85-789 Bydgoszcz, Poland, e-mail: [email protected]
In the reported experiment, the effect of culture media
composition on the effectiveness of androgenesis in anther
culture of selected pepper (Capsicum) breeding lines and
hybrids was tested. Anther culture was conducted according to Dumas de Vaulx et al. (1981), with slight modifications concerning the level of kinetin in R1 medium (0.1,
0.2, 0.3, 0.5 mg·dm-3 KIN). The efficiency of gametophytic
embryogenesis varied significantly and depended both on
the genotype and on the kinetin level tested. In case of
annual pepper hybrids, the effectiveness of androgenesis
was relatively high and ranged from 2.6% to 15.3%. The
highest effectiveness of androgenic embryo development
was observed for (ATZ1 CDT) F1 form, in the medium
supplemented with 0.1 mg·dm-3 KIN. The effectiveness of
Vol. 51, suppl. 1, 2009
anther culture of the lines originated from interspecific
hybrid C. annuum L. × C. frutescens L. was considerably
lower and very rarely exceeded 2%. However, increase of
kinetin concentration in R1 medium visibly improved the
androgenic response of most of these lines. According to
the cytometric analyses, there were haploid, diploid and
some mixoploid plants among the androgenic pepper
regenerants.
REFERENCES
DUMAS DE VAULX R, CHAMBONNET D, POCHARD E. 1981. Culture in
vitro d'anthéres de piment (Capsicum annuum L.) amélioration des taux d'obtention de plantes chez différents génotypes par des traitements á +35°C. Agronomie 1: 859–864.
45
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Influence of the ploidy level on growth and organogenesis of sugar beet
(Beta vulgaris L.) in vitro
Magdalena Klimek, Rafał Barański
Department of Genetics, Plant Breeding and Seed Science, Agricultural University of Cracow, Al. 29 Listopada 54,
31-425 Crakow, Poland, e-mail: [email protected]
Tissue culture techniques are utilized in breeding of new
sugar beet (Beta vulgaris L.) cultivars. They allow the production of homozygous clones after diploidization of haploid plants developed from unfertilized ovules. However,
after haploidization and treatment with antimitotic agents,
material with various ploidy level may develop.
The aim of the present work was to evaluate the influence of the ploidy level on the susceptibility of sugar beet
tissues to culture in vitro and their ability for shoot development.
Biological material used in this study was derived from
24 clones of haploids obtained from developing unfertilized ovules in vitro, which were later treated with
colchicine. In consequence, groups of plants of common
origin were obtained; each group comprised plants not
responding to colchicine (1n) and plants with a multiple
number of chromosomes (2n and 4n). These materials
were assessed for shoot morphology, necrotic changes
during the culture and the ability for shoot development.
Considerable variation in shoot and leaf morphology
was observed among the used genotypes. Usually, 4n
plants had shortened shoots, hardened petioles and much
wider leaf blades in comparison to 1n and 2n plants.
Additionally, they produced fewer shoots during micropropagation. Regeneration ability of leaf explants placed
on MS medium supplemented with 1 mg/l BAP was
assessed after 3 and 6 weeks of the culture. Preliminary
observations indicate that the reaction of explants strongly depended on the genotype of donor plant and, additionally, on the ploidy level. Usually, 1n plants exhibited a
higher potential for organogenesis than did 2n and 4n
plants. However, there was no difference between explants
of different ploidy levels in their survival rates.
A study of the influence of humic substances on in vitro potato growth and
tuberization
Józef Klocek1, Guy Costa2, Halina Mioduszewska1
1
Department of Plant Physiology and Genetics, University of Podlasie, Prusa 12, 08-110 Siedlce, Poland,
e-mail [email protected]
2
Laboratoire de Chimie des Substances Naturelles (LCSN EA 1069) Faculté des Sciences & Techniques,
Universitie de Limoges,123 Avenue Albert Thomas, 87060 Limoges, e-mail [email protected]
It has long been recognized that humic substances (HS)
have many beneficial effects both on the soil and on plant
growth. These heterogeneous and complex molecules,
ubiquitous in the environment, may produce various morphological, physiological and biochemical effects on higher plants (Chen and Aviad, 1990). The influence of the
humic material on plant growth have been investigated
with various measurement methods and numerous studies have shown that humic substances not only enhance
the root, leaf and shoot growth but also stimulate germination of various crop species (Piccolo et al., 1993). These
positive effects are explained by the direct interaction of
HS with the physiological and metabolic processes (Nardi
et al., 2002). When added to the soil or medium, the HS
increase nutrient uptake and cell permeability and they
modify the mechanisms involved in plant growth stimulation.
We examined the influence of three different HS concentrations (1%, 2% and 3%) on growth of potato plants.
These substances were added to the MS medium on which
single node cuttings of potato plants were grown in order
46
to observe what influence these substances might exert on
the length of the shoots and the number of leaves and
roots as well as on the tuberization process. When added
to the medium, the HS stopped the growth of the shoots
and reduced the number of the leaves and roots in comparison to the control sample. However, the tuberization
process began earlier in case of the plants grown on the
medium with the HA. This paper discusses the influence
of HS on the above mentioned processes.
REFERENCES
CHEN Y, AVAID T. 1990. Effects of humic substances on plant
growth. In: American Society of Agronomy and Soil Science
Society of America [eds.], 161–186. Humic substances in
soil and crop science; Selected Readings. American Society
of Agronomy, Madison, WI.
NARDI S, PIZZEGHELLO D, MUSCOLO A, VIANELLO A. 2002.
Physiological effects of humic substances in higher plants.
Soil Biology and Biochemistry 34: 1527–1537.
PICCOLO A, CELANO G, PIETRAMELLARA G. 1993. Effects of fractions
of coal-derived humic substances on seed germination and
growth of seedlings (Lactuga sativa and Lycopersicum
esculentum). Biology and Fertility of Soils 16: 11–15.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Genes involved in flax pathogenesis
Kamil Kostyn, Magdalena Czemplik, Aleksandra Boba, Anna Kulma, Jan Szopa
Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63, 51-148 Wrocław, Poland;
e-mail: [email protected]
Fusarium is the most common flax pathogen causing serious plant diseases and in most cases leading to plant
death. It is estimated that over 20% of damages in flax cultivation is caused by Fusarium infection. Plant response
to pathogen attack is a complex mechanism, and involves
multiple defense strategies and different biochemical
pathways (Dixon and Harrison, 1990). The identification
of the plant genes that respond to infection, and generation of suitable transgenic plants is the approach that has
been used in our study. Fifty four flax genes have been
identified by means of cDNAs subtraction method as those
that respond to pathogen infection. Subtracted genes were
classified into several classes, and the prevalence of the
genes involved in the broad spectrum of antioxidant
biosynthesis has been noticed. Compounds known for
their antioxidative properties, like terpenoids, phenylpropanoids, catecholamines, play a well established role
in the plant pathogen response. Phenylpropanoids are of
special interest among them, as they are connected with
cell wall biosynthesis. The phenolic alcohols are constituents of lignins and lignans, which reinforce the cell
wall, the first barrier against pathogens. In addition, phenolic acids form amides with catecholamines and settle in
the cell wall, making it more resistant to pathogens
(Zakarés et al., 2007). By means of semi-quantitative PCR,
the involvement of subtracted genes controlling phenylpropanoid pathway in flax upon infection was positively
verified. Moreover, the metabolite profiling obtained from
the GCMS analysis constitutes a supplementary verification. The data from analysis of the transgenic flax plants
overexpressing genes of the phenylpropanoid pathway
showed and confirmed the protective effect of phenolic
compounds against Fusarium infection.
REFERENCES
DIXON RA, HARRISON M. 1990. Activation, structure, and organization of genes involved in microbial defense in plants.
Advances in Genetics 28: 165–234.
ZACARÉS L, LÓPEZ-GRESA MP, FAYOS J, PRIMO J, BELLÉS JM,
CONEJERO V. 2007. Induction of p-Coumaroyldopamine and
Feruloyldopamine, two novel metabolites, in tomato by the
bacterial pathogen Pseudomonas syringae. Molecular
Plant-Microbe Interactions 20: 1439–1448.
Induced androgenesis in anther culture of Lupinus angustifolius
Kamila Kozak, Renata Galek, Ewa Sawicka-Sienkiewicz
Department of Genetics, Plant Breeding and Seed Production, The Wrocław University of Environmental and Life
Science, Pl. Grunwaldzki 24 A, 50-363 Wrocław, Poland, e-mail: [email protected]
The aim of this work was to evaluate the reaction of seven
blue lupine (Lupinus angustifolius) genotypes – cvs.
'Graf', 'Emir', breeding line 'LAE-1' and four hybrids originated from crossing 'LAE-1 × Graf' (204, 205) and 'Emir
× LAE-1' (207, 209) – after inducing androgenesis in tissue culture. Some effort was made to increase the efficiency of androgenesis through optimization of conditions, such as: developmental stage of microspores,
method of pretreatment and medium composition. A
cytological analysis of developmental stage of
Vol. 51, suppl. 1, 2009
microspores was performed on anthers in vivo and on
explants sampled during the in vitro culture. Microscopic
observations of all materials revealed the occurrence of
multicellular microspores, which indicate the induction
of divisions. The tendencies of the analyzed genotypes to
produce multicellular structures resembling embryos at
the globular stage and to induce callus formation were
observed. To confirm the microspore origin of the developed embryos, chromosome counts of root tip meristematic cells were also made.
47
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Enhancing of salidroside and rosavin production in Rhodiola kirilowii callus
cultures
Anna Krajewska-Patan1, Mariola Dreger1, Małgorzata Górska-Paukszta1, Alina Mścisz1,
Sebastian Mielcarek1, Marek Baraniak1, Waldemar Buchwald1, Mirosława Furmanowa2,
Przemysław M. Mrozikiewicz1
1
The Branch of Medicinal Plants of the Institute of Natural Fibres and Medicinal Plants, Libelta 27, 61-707 Poznań,
e-mail: [email protected]
2
Department of Biology and Pharmaceutical Botany of Medical University in Warsaw, Banacha 1, 02-097 Warsaw
Rhodiola kirilowii (Regl.) Maxim (Crassulaceae) is a traditional medicinal plant used in North Asia and China.
Animal studies have shown a protective effect of the root
extract in cardiopulmonary disorders in the hypoxic conditions induced by high altitude. Callus tissues of R. kirilowii synthesize only trace amounts of salidroside and
rosavins – the supposed biologically active compounds of
R. rosea, a related medicinal plant from genus Rhodiola.
The aim of this study was to enhance the production of
salidroside and rosavins in R. kirilowii callus tissues by
exogenous addition of precursors to the medium. Callus
tissue (line derived from cotyledons) cultured on solid MS
medium was used in the experiments. The supplementation with p-tyrosol (at 3–5 mM/L) resulted in a significant
increase of salidroside content – up to 1106 mg/100g dry
weight (110-fold increase compared to control). The addition of cinnamyl alcohol (at 2.5 mM/L) induced rosavins
and especially rosin production (1193 mg/100g d.w.) in
the treated tissues. The supplemented callus tissues synthesized also: p-tyrosol, epigallatecatechine gallate, tannins and polyphenolic acids. These results demonstrated
that supplemented R. kirilowii callus was able to synthesize high amounts of salidroside and rosavins (in contrast
to intact plants where traces amounts of rosavins have
been found).
Acknowledgment: The project was supported by a grant of the
Ministry of Science and Higher Education, project No: N 405 025
32/1687.
Comparison of micropropagation methods of Polish cultivars of barley and oat
Hanna Kruczkowska, Helena Pawłowska, Barbara Skucińska
Department of Plant Breeding and Seed Science, Agricultural University of Cracow, Łobzowska 24, 31-140 Cracow,
Poland, e-mail: [email protected]
Obtaining transgenic plants in cereal cultivars of agronomic value requires an efficient method of micropropagation which does not cause an additional variation.
Recently suggested protocols consist in inducing regeneration of multiple shoots from apical meristems of
seedlings derived in vitro from dry mature seeds. We compared the efficiency of three protocols: after Zhang et al.
(1996), Sharma et al. (2004) and Geneshan et al. (2006)
in several Polish cultivars of barley and oat. In a preliminary experiment, two procedures, after Zhang et al. and
Sharma et al., were compared for oat cv. Chwat. We
showed that numbers of shoots obtained from one explant
in the same period of culture did not differ significantly.
The procedure after Zhang et al. was found to be more
laborious (dehusking of grains, cutting of shoots), so in
the next experiment, involving two oat and two barley cultivars, we compared protocols by Sharma et al. and
Geneshan et al. In the examined cultivars, the protocol
after Geneshan et al. proved to be more efficient, less
laborious and faster. After 10–11 weeks of culture we
obtained, on average, in barley cv. Basza – 6.3, cv. Stratus
48
– 17.1 shoots per plated explant; in oat cv. Bohun – 28.1,
cv. Szakal – 54.2 shoots per plated explant. The lesser efficiency of the protocol after Sharma et al. in the same period of culture resulted mainly from a number of nonproliferating explants in barley and from slower development
and elongation of shoots.
REFERENCES
GENESHAN S, CHODAPARAMBIL SV, BAGA M, FOWLER DB, HUCL P,
ROSSNAGEL BG, and CHIBBAR RN. 2006. In vitro regeneration
of cereals based on multiple shoot indiction from mature
embryos in response to thidiazuron. Plant Cell Tissue and
Organ Culture 85: 63–73.
SHARMA VK, HÄNSCH R, MENDEL RR, and SCHULZE J. 2004. A highly
efficient plant regeneration system through multiple shoot
differentiation from commercial cultivars of barley
(Hordeum vulgare L.) using meristematic shoot segments
excised from germinated mature embryos. Plant Cell
Reports 23: 9–16.
ZHANG S, ZHONG H, and STICKLEN MB. 1996. Production of multiple shoots from shoot apical meristems of oat (Avena sativa
L.). Journal of Plant Physiology 148: 667–671.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Shoot regeneration of perennial wild beet species in vitro
Kamilla Kużdowicz, Maria Gośka
Plant Breeding and Acclimatization Institute, Powstańców Wlkp. 10, 85-090 Bydgoszcz, Poland,
e-mail: [email protected]; [email protected]
Perennial wild beet species are characterized by the high
level of resistance to main beet diseases and tolerance to
environmental stresses such as low temperature and
drought (Asher et al., 2001). In vitro multiplication ability of
most wild beet species is not sufficiently known. The purpose of this investigation was to examine the possibility of
in vitro shoot regeneration of perennial wild beet species.
Seeds without hard coat of B. macrorhiza, B. trigyna,
B. lomatogona, B. corolliflora and B. vulgaris as a control
were used in this study. For surface sterilisation, seeds
were treated with 70% ethanol for 30 s, 5% sodium
hypochlorite for 25 minutes and washed four times in a
sterile distilled water. Sterilized seeds were incubated in
darkness for 4–8 days at 25°C on sterile moist blottingpaper in glass Petri dishes to obtain germination which
was observed three days later. For each species, 36 shoot
tips from 7 or 9 day old seedlings with 5 mm hypocotyls
were cultured at 25°C on MS basal medium (Murashige
and Skoog, 1962) containing different concentrations and
combinations of growth regulators (BAP, TDZ, KIN, 2iP)
under artificial daylight conditions. Explants were trans-
ferred to fresh medium every four weeks. Aseptic leaf and
petiole segments were also cultured.
After 10 days, 16–55% of explants from seedlings of
perennial species and 99% of explants of B. vulgaris started
to regenerate shoots or shoot-like structures. Leaf and petiole
explants showed no morphogenetic responses. MS medium
emriched with 1 mg l-1 BAP was the best for an efficient plant
regeneration of perennial wild beet species. Shoot regeneration ability was the best for seedlings incubated in darkness
for 8 days. Some of regenerated plants of perennial wild beet
species occasionally developed root-like structures but
majority of them did not form roots on the basal medium.
Acknowledgement: This work was financed by the research project No. 2 PO6A 021 30.
REFERENCES
ASHER MJC, LUTERBACHER MC, FRESE L. 2001. Wild Beta species as
a source of resistance to sugar-beet pests and diseases.
International Sugar Journal 103: 447–456.
MURASIGE T, SKOOG F. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiologia
Plantarum 15: 473–497.
Biotransformation of p-hydroxybenzoic acid in in vitro cultures of Ruta
graveolens ssp. divaricata
Inga Kwiecień, Katarzyna Bień, Halina Ekiert
Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, Medyczna 9,
30-688 Cracow, Poland, e-mail: [email protected]
The cells of Ruta graveolens ssp. divaricata* cultured in
vitro have been shown to transform the exogenous precursor, hydroquinone into its β-D-glucoside, arbutin
(Skrzypczak et al., 2005). The maximal obtained contents
of this product (12–13% d.w.) were very interesting from
practical point of view (Zubek et al., 2009).
p-Hydroxybenzoic acid has been reported to be a good
precursor of arbutin in in vitro cultures of many different
plant species (Dušková et al., 1999). The aim of the present studies was to test the usefulness of this compound as
a precursor of arbutin.
The agitated cultures of R. g. ssp. divaricata were
maintained using Linsmaier-Skoog medium (Linsmaier
and Skoog, 1965), supplemented with 2 mg/l NAA and 2
mg/l BAP. Different concentrations of the p-hydroxybenzoic acid (96–384 mg/l of medium) were administered into
the culture flasks in one, two or three portions. The products of the biotransformation were qualitatively determined in methanol extracts from dry biomass and
lyophilized media by HPLC method.
The main product of biotransformation in the extracts
from biomass had retention time (tR= 12.20 min) different from arbutin. Unknown compound was found also in
the media when the higher dose of the precursor was
administered. Identification of this compound is under
way. Additionally, in the biomass and the media extracts
we detected hydroquinone, which is the evidence of decarboxylation of the p-hydroxybenzoic acid.
REFERENCES
DUŠKOVÁ J, DUŠEK J, JAHODÁR L. 1999. Zur Biotransformation von
Hydrochinon zu Arbutin in den In vitro-Kulturen. Herba
Polonica 45: 23–26.
LINSMAIER EM, SKOOG F. 1965. Organic growth factor requirements
of tabacco tissue cultures. Physiologia Plantarum 18:
100–127.
SKRZYPCZAK-PIETRASZEK E, SZEWCZYK A, PIEKOSZEWSKA A, EKIERT H.
2005. Biotransformation of hydroquinone to arbutin in plant
in vitro cultures – preliminary results. Acta Physiologiae
Plantarum 27: 79–87.
ZUBEK S, JANAS E, EKIERT H. 2009. 5th German-Polish Symposium
"New challenges for pharmaceutical sciences", Poznań,
Abstracts, p.126.
* The cultures were obtained thanks to cooperation of our Department with Institute of Biosciences, Würzburg University (Germany) –
Prof. F. Ch. Czygan, Dr. A. Abou-Mandour
Vol. 51, suppl. 1, 2009
49
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Studies of the biotransformation of p-hydroxybenzoic acid in in vitro cultures of
Ruta graveolens
Inga Kwiecień, Szymon Zubek, Halina Ekiert
Chair and Department of Pharmaceutical Botany, Jagiellonian University, Collegium Medicum, Medyczna 9,
30-688 Cracow, Poland, e-mail: [email protected]
Arbutin, β-D-glucoside of hydroquinone, is a well-known
plant metabolite possessing both therapeutic and cosmetic values. Shoot cultures of Ruta graveolens maintained
at the Chair of Pharmaceutical Botany JU CM are a good
source of this compound.
The cells of the Ruta graveolens shoots cultured in
vitro successfully transformed the exogenous hydroquinone into arbutin. The maximal content of this product, obtained so far is 7.8 % d.w. (Zubek et al., 2009).
In order to obtain higher biotransformation efficiency,
the usefulness of p-hydroxybenzoic acid as a potential
arbutin precursor was tested. p-Hydroxybenzoic acid has
been shown to be a good precursor of arbutin in in vitro cultures of many different plant species (Dušková et al., 1999).
The agitated cultures of R. graveolens were maintained using Linsmaier-Skoog medium (Linsmaier and
Skoog, 1965), supplemented with 2 mg/l NAA and 2 mg/l
BAP. Different concentrations of the p-hydroxybenzoic
acid (96 – 384 mg/l of medium) were administered into the
culture flasks in one, two or three portions. The products
of the biotransformation were qualitatively determined in
methanol extracts from dry biomass and in lyophilized
media by HPLC method.
The main product of biotransformation in the extracts
from biomass had retention time (tR= 12.20 min) different from arbutin. Identification of this compound is under
way. This unknown compound was detected also in the
media when the higher dose of the precursor was administered. Additionally, in the biomass extracts we detected
hydroquinone, which is the evidence of decarboxylation of
the p-hydroxybenzoic acid.
REFERENCES
DUŠKOVÁ J, DUŠEK J, JAHODÁR L. 1999. Zur Biotransformation von
Hydrochinon zu Arbutin in den In vitro-Kulturen. Herba
Polonica 45: 23–26.
LINSMAIER EM, SKOOG F. 1965. Physiologiae Plantarum 18:
100–127.
ZUBEK S, PIEKOSZEWSKA A, EKIERT H. 2009. Arbutin production in
Ruta graveolens L. and Hypericum perforatum L. in vitro
cultures. Acta Physiologiae Plantarum. accepted.
Artificial seeds of Linum usitatissimum L. and Morus alba L. in long-term
storage under slow growth conditions
Aleksandra Luwanska1, Grazyna Mankowska1, Karolina Wielgus1
1
Department of Biotechnology, Institute of Natural Fibres & Medicinal Plants, Wojska Polskiego 71b, 60-630 Poznań,
Poland; e-mail: [email protected]
Techniques of in vitro culture are used not only for micropropagation but also long-term storage of plant material in
gene banks. They are an effective tool for maintaining gene
resources of crop plants, rare, extinct and elite plants, collections of botanic gardens, as well as plants incapable of
generative propagation or plants with modified features.
Fragments of tissues or entire organs of such plants can
be stored under slow growth conditions and in the form of
50
artificial seeds. The aim of this study was to create artificial seeds of flax (Linum usitatissimum L.) as an example
of a crop plant and white mulberry (Morus alba L. cv.
Żólwińska Wielkolistna) as a genotype of a rare tree. The
vitality of plants was assessed after creating artificial
seeds and after three-month storage. The results proved
the effectiveness of storing plant material in the form of
artificial seeds on medium that conditions slow growth.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Regeneration of Gentiana cruciata from in vitro shoot tip culture
Maciej Ładyżyński and Jan J. Rybczyński
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, e-mail: [email protected]
Different explants and types of culture have been used for
studies of somatic embryogenesis and organogenesis in
genus Gentiana. Shoot tips and liquid medium have been
used relatively rarely, and not at all in G. cruciata. Such a
combination should be conducive to a low variation and
an increased regeneration efficiency.
The study aimed to achieve an efficient regeneration
protocol for the legally protected G. cruciata. Explants,
i.e. shoot tips (< 1 mm) containing 2–4 leaf primordia,
were dissected from 1–1.5-month-old seedlings growing
under in vitro conditions. Single explants were placed in
100 ml Erlenmeyer flasks containing 20 ml of MS medium (half-strength macroelements) supplemented with different levels of growth regulators: cytokinin BAP and
auxin NAA. Two ways of medium mixing were applied.
Cultures were maintained on an orbital shaker at 100 rpm
and on a rotary device at 12 rpm. Temperature was 25°C
and photoperiod was 16/8 h (day/night).
Among hormone combinations and concentrations
studied, the best results were obtained on media supplemented with 0.2 mg/L NAA, or with 2.0 mg/L NAA and 0.2
mg/L BAP. After 9–10 weeks of culture, more or less developed regenerants (from green points on tissue aggregates
to 2–4-leaf shoots) were the main part of the biomass. It
was possible to obtain 86 shoots, each with several
leaflets, from one shoot tip after 3 months of culture. The
type of equipment used (rotary or shaker device) did not
influence the development of explants and the results were
similar. Multiplied shoots transferred onto agar media
with or without growth regulators were easily rooted at a
high rate.
Acknowledgement: Supported by project no 39/N-COST/2007/0 of
the Polish Ministry of Science and Higher Education
Cryobank of gametophytes of tropical woody fern species
Damian Makowski, Anna Mikuła, Jan J. Rybczyński
Botanical Garden – Center for Biological Diversity Conservation, Polish Academy of Sciences, Prawdziwka 2,
02-973 Warsaw, Poland, e-mail: [email protected]
Maintaining representative collections of biological diversity of woody ferns outside of their natural habitats is very
difficult, because of their large size. Mainly vegetative
propagation of tropical ferns leads to constraining of their
gene pool. Ferns need special procedures to introduce
them into gene banks. Now, it is possible to develop new
cryopreservation methods.
The aims of this study were: 1) to establish the most
efficient cryopreservation conditions for gametophytes of
seven woody fern species and 2) to create their gene bank
in liquid nitrogen (LN). Experiments were carried out on
the following fern gametophyte cultures: Cyathea australis, Cyathea dealbata, Cyathea smithii, Cyathea
schan-chin, Cibotium schiedei, Cibotium glaucum,
Dicksonia fibrosa. Encapsulation-dehydration method
was applied. The influence of time of preculture, ABA
Vol. 51, suppl. 1, 2009
addition and type of preculture 1/2 MS medium (solid/liquid) were tested.
Encapsulated gametophytes of D. fibrosa and
C. schan-chin survived at 100% rate after the NL treatment, independently of preculture type and time.
Gametophytes of other five species survived at 60–80%
rates. Application of two-week preculture with ABA gave
better results than preculture without ABA for these
species.
Cryopreservation by encapsulation-dehydration allows
to create a cryobank of tropical woody fern gametophytes
with the possibility of a high level viability conservation.
Acknowledgement: The research was supported by project no
39/N-COST/2007/0 of the Polish Ministry of Science and Higher
Education.
51
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Somatic embryo formation in callus cultures of Narcissus L. 'Carlton' multiplied
in liquid media
Małgorzata Malik, Barbara Stawiarz
Department of Ornamental Plants, Agricultural University of Cracow, 29 Listopada 54, 31-425 Cracow, Poland,
e-mail: [email protected]
Until now only a few studies have reported propagation of
Narcissus L. based on the process of somatic embryogenesis in liquid and liquid/solid culture systems (Malik,
2008; Malik and Molenda, 2008; Sage and Schroeder,
2002). These studies imply that cultivation in liquid media
has a positive effect on somatic embryogenesis. The objective of this study was to investigate the effects of an 8-week
exposure of callus to the liquid medium as well as that of
growth regulator contents in the media on biomass growth
and somatic embryo formation.
Seven lines (LC1-7) of embryogenic calluses of different origins (initial explant, initial medium, culture system)
were used for the experiment. Calluses were cultured for
8 weeks in liquid proliferation media with Picloram or
2,4-D (10 or 25 μM) and BA (1 or 5 μM) and then for 24
weeks on solid proliferation or regeneration medium containing 0.5 μM NAA and 5 μM BA. The control cultures
were maintained for 32 weeks on solid media.
The highest biomass growth was observed in the LC7
callus line culture obtained on ovary explants under the
influence of 25 μM Picloram and 5 μM BA. The highest number of somatic embryos was noted in the LC5 callus line culture initiated from ovary explants and cultured on medium
containing 25 μM 2,4-D and 5 μM BA in the liquid/solid culture system (Malik, 2008). The 8-week liquid medium treatment did not result in a biomass increase but promoted
somatic embryo formation. Proliferation media stimulated
the process of callus culture multiplication. In turn, the
regeneration medium promoted somatic embryo formation.
Acknowledgement: The research were supported by Ministry of
Education and Science (No. 2P06R12129).
REFERENCES
MALIK M. 2008. Comparison of different liquid/solid culture systems in the production of somatic embryos from Narcissus
L. ovary explants. Plant Cell, Tissue and Organ Culture 94:
337–345
MALIK M, MOLENDA M. 2008. Formowanie zarodków somatycznych
narcyza (Narcissus L.) z tkanki kalusowej w systemie okresowego zalewania pożywką RITA® oraz na pożywce stałej.
Zeszyty Problemowe Postępów Nauk Rolniczych 525:
237–243.
SAGE DO, SCHROEDER MB. 2002. Growth and development of nodular callus of Narcissus pseudonarcissus cvs in temporary
immersion 'Rita' bioreactors. In: Abstracts of the 1st international symposium on liquid systems for in vitro mass
propagation of plants, Norway, May 29 – June 2 2002.
The analysis of cell ploidy level in the explant tissue culture of hemp (Cannabis
sativa L.) and flax (Linum usitatissimum L.)
Grażyna Mańkowska, Aleksandra Luwańska, Karolina Wielgus
Department of Biotechnology, Institute of Natural Fibres and Medicinal Plants, 60-630 Poznań, Poland,
e-mail: [email protected]
The influence of ploidy of hemp and flax explants on their
capacity for regeneration during in vitro culture makes the
determination of the cell ploidy necessary at the beginning
of breeding and genetic studies.
The aim of the study was to evaluate the suitability of
different hemp and flax explants for organogenesis by
monitoring the ploidy level by the means of flow cytometry. Stems, leaves, roots and cotyledons of hemp (Beniko)
and flax (Nike, Alba, Aleksim) were used. The analysis was
performed for the explants at different stages of plant
growth: cotyledons of swollen seeds, young seedlings (five
days after germination), two week and five week old
plants. Additionally, at the beginning of flowering, flax
anthers containing microspores with one nucleus were
isolated from flowers. The determination of ploidy level
was conducted using flow cytometer PARTEC CA II. The
samples were fragmented directly in analysis buffer and
then isolated nuclei were filtered through polyamide filter
(50 μm). The released nuclei were stained with 4',6diamidino-2-phenylindole (DAPI). The fluorescence of
52
individual nuclei was evaluated. The experiments were
carried out in three replicates and 3500–6000 nuclei per
sample were analysed. Histograms from different samples
of DNA were compared with standard with known DNA
content (erythrocytes of trout).
The results with respect to hemp demonstrated that
young seedling and their cotyledons were mixoploid
(2n+4n) which makes them unsuitable for in vitro culture. A clear decrease of DNA content was observed in
leaves and in upper part of shoots from five week old
plants. Two week old hemp was found to be the best
source of explants due to their stable 2n DNA content.
The experiment conducted on flax revealed that explants
obtained from five day and two week old plants were the
most suitable for in vitro culture. In the leaves of five
week old flax there was a decrease in DNA content. Based
on the results of this research, it may be concluded that
the best explants for regeneration purposes originated
from two week old hemp, as well as five and fourteen day
old flax.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Does a relationship between the frequency of androgenesis and vernalization
requirement exist in wheat?
Izabela Marcińska, Edyta Skrzypek, Ilona Czyczyło-Mysza, Agata Stawicka, Marta Pilipowicz
The F. Górski Institute of Plant Physiology, Polish Academy of Sciences, 30-239 Cracow, Niezapominajek 21, Poland,
e-mail: [email protected]
Androgenesis was achieved in Polish spring and winter
wheat cultivars, which were all derived from F1 hybrids.
Anthers were incubated on C17 medium and embryo-like
structures on 190-2 regenerating medium. The relationship between anther culture traits and selected phenotypic traits of donor plants were evaluated by correlation,
regression and variance analysis. It was found that the frequency of production of embryo-like structures and the
number of regenerated green and albino plants were significantly different for winter and spring genotypes. The
effectiveness of androgenesis was as much as two times
higher in winter cultivars compared to spring cultivars. Is
it possible that vernalization stimulated this process? The
exposure of germinating seeds to a prolonged period of
low temperature promoted flowering in adult plants of
wheat (Chaudhary et al., 2003). It is possible that genes
involved in vernalization, can also be involved in the
molecular mechanism of androgenesis, including the role
of DNA methylation. Androgenesis was also related to
some morphological traits of the main shoot and yield
structure independently of the kind of genotype within the
winter and spring cultivars. Such relationships may be
due to the fact that these traits are influenced by the genetic basis of hybrid parental lines (Dodig et al., 2008). These
results suggest that it is possible to screen genotypes with
good anther culture traits directly by morphological markers, which are practical and simple to use for breeders, to
select highly productive wheat lines in anther culture. The
advantages of double haploid technology for accelerating
breeding programs include reduction of costs of cultivar
development, via the greater efficiency, with which the
homozygous double haploid lines can be evaluated for
selected desired traits.
REFERENCES
CHAUDHARY HK, DHALIWAL I, SINGH S, SETHI GS. 2003. Genetics of
androgenesis in winter and spring wheat genotypes.
Euphytica 132: 311–319.
DODIG D, ZORIC M, MITIC N, NIKOLIC R, SURLAN-MOMIROVIC G. 2008.
Tissue culture and agronomic traits relationship in wheat.
Plant Cell, Tissue and Organ Culture 95: 107–114.
Analysis of indole compounds in Calocera viscosa mycelium cultured on liquid
medium and in its fruiting bodies
Bożena Muszyńska, Katarzyna Sułkowska-Ziaja
Chair and Department of Parmaceutical Botany, Jagiellonian University Collegium Medicum, Medyczna 9,
301-688 Cracow, Poland
In Poland Calocera viscosa (Pers.; Fr.) Fr.
(Macromycetes) is a widespread species of mushroom.
The aim of this study was to investigate the contents of
indole compounds in fruiting bodies of this species and in
its mycelium cultured in vitro.
Fruiting bodies were collected in mixed forests in
southern Poland. In vitro cultures was initialized from
fruiting bodies taken from the natural state. The optimal
medium composition for a submerged culture was determined. Fresh material: fruiting bodies (61.5 g) and mycelium (170 g) was frozen and then dried by lyophilization.
The crushed dry biomass was extracted with petroleum
ether to remove the oil fraction which was discarded. The
remaining biomass was extracted with methanol.
Vol. 51, suppl. 1, 2009
Analysis of indole compounds was performed in
methanol extracts using chromatographic methods: TLC,
PTLC and HPLC.
The HPLC method allowed to estimate the contents of
the following metabolites (mg/100g d.w.): tryptophan
(0.92), skatol (0.37), 5-methoxytryptamine (0.14), melatonin (0.79) in fuiting bodies and tryptophan (0.89), skatol (0.31), 5-hydroxytryptophan (0.23) and melatonin
(0.45) in mycelium from in vitro cultures.
It was demonstrated that in vitro cultures of Calocera
viscosa and its fruiting bodies contain comparable
amounts of indole derivatives. In vitro cultures will make
a good model for studies of accumulation of this group of
metabolites.
53
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Conversion – the critical point of induced embryogenesis in Capsicum spp.
Paweł Nowaczyk, Lubosława Nowaczyk
Department of Genetics and Plant Breeding, University of Technology and Life Sciences, Bernardynska 6,
85-069 Bydgoszcz, e-mail: [email protected]
The creation of new, useful genetic variation is the main
aim of in vitro gametic embryogenesis, while the somatic embryogenesis provide an opportunity for multiplication of heterozygotic genotypes. In both these processes,
the conversion of the embryos into plantlets is the limiting
factor for their practical exploitation. From this point of
view the effectiveness of induced embryogenesis should be
presented as the relationship of obtained plantlets to
explant number. The success rate of converting haploid
embryos to plants in Capsicum annuum L. ranged
between zero to over 50%, according to genotype and conditions of anther culture. In the study on individual reaction of Capsicum F2 plants (Nowaczyk et al., 2009), interspecific hybrids showed a higher conversion coefficient
(84%) than did C. annuum L. (52%). In both of these genotype groups the share of haploids was lower than that of
diploid plants. Therefore, spontaneous diploidization of
haploid embryos derived from anther cultures seems to be
a very interesting phenomenon. The lines obtained as a
result of distant hybrid progeny selection showed that the
conversion efficiency depends mainly on the genotype
characteristics. Similar effectiveness has been observed
when the microspores were used as explants (Supena et
al., 2006). In some of hot pepper genotypes 15–33% of
embryos converted to complete plants. This information
is not good for pepper breeders because conversion is the
limiting factor in the rapid use of the new genes and character arrangements in hybrid recombinants. In addition,
research on somatic embryogenesis has not supplied satisfactory results concerning this way of propagation, however the maximum conversion effectiveness currently
described for one cultivar has reached 80% (Khan et al.,
2006).
REFERENCES
KHAN H, SIDDIQUE I, ANIS M. 2006. Thidiazuron induced somatic
embryogenesis and plant regeneration in Capsicum annuum. Biologia Plantarum 50 (4): 789–792.
NOWACZYK P, OLSZEWSKA D, KISIAłA A. 2009. Individual reaction of
Capsicum F2 hybrid genotypes in anther cultures. Euphytica
DOI:10.1007/s10681–009–9909–4
SUPENA EDJ, SUHARSONO S, JACOBSEN E, CUSTERS JBM. 2005.
Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper
(Capsicum annuum L.). Plant Cell Reports 25: 1–10.
Shoot induction potential of nine Brassica napus varieties and two types of
explants
Agata Obarska, Katarzyna L. Janczur, Barbara Tomaszewska
Department of Biochemistry, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland,
e-mail: [email protected]
Rapeseed is recognized as the most important source of
vegetable oil and protein-rich meal worldwide. It is well
known that the improvement of plant breeding methods is
slow, time consuming and labor-intensive. Non-conventional genetic improvement programs based on tissue culture and molecular genetics are essential as a complement
to standard breeding. Efficient micropropagation and
transformation methods require a reliable and efficient
callus induction and plantlet regeneration protocols therefore B. napus has become an object of extensive tissue culture studies (Moghaieb et al., 2005). A wide range of target explants of B. napus have been studied, including
hypocotyls, petioles, thin cell layers, stem segments and
protoplasts. The effects of culture media and genotypes on
shoot regeneration in oilseed Brassica species have also
been examined (Tang et al., 2003). Due to the highly variable and genotype specific regeneration in B. napus, it is
especially important to determine the varieties and types
of explants with particularly high potential for tissue culture micropropagation among Polish accessions. In our
research we tested nine rapeseed varieties registered in
54
Poland and two types of explants to detect the variation in
frequency of callus induction, shoot induction and shoot
induction effectiveness. We cultured hypocotyl and petiole
explants of each variety on MS medium supplemented
with hormones (0.15 mg/l NAA and 3 mg/l BAP) and
AgNO3 (2.5 mg/l). By measuring the shoot induction frequency and effectiveness parameters we showed that the
differences in shoot regeneration ability of various
explants were significant within the same variety as well as
in different genotypes. Thus, the shoot regeneration from
rapeseed explants highly depended on the genotype and
kind of explant.
REFERENCES
MOGHAIEB REA, EL-AWADY MA, EL MERGAWY RG, YOUSSEF SS, ELSHARKAWY AM 2006. A reproducible protocole for regeneration and transformation in canola (Brassica napus L.).
African Journal of Biotechnology 5: 143–148.
TANG GX, ZHOU WJ, LI HZ, MAO BZ, HE ZH, YONEYAMA K. 2003.
Medium, explant and genotype factors influecning shoot
regeneration in oliseed Brassica ssp. Journal of Agronomy
and Crop Science 189: 351–358.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Is it possible to select drought-resistant Rubus genotypes from in vitro
populations of seedlings or adventitious shoots?
Teresa Orlikowska1, Danuta Kucharska1, Krzysztof Klamkowski1, Marcin Horbowicz2,
Lesław Lahuta3
1
Research Institute of Pomology and Floriculture, Skierniewice, Poland, e-mail: Teresa.Orlikowska.insad.pl
University of Podlasie, Siedlce, Poland
3
University of Warmia and Mazury, Olsztyn, Poland
2
Raspberry is an important fruit in the Polish horticulture,
fresh produce and food industries. Irrigation during
spring and summer is essential in some years and on
some plantations, but this increases the production costs.
Therefore, drought stress tolerance should be one of the
breeding goals. We would like to develop a reliable system
enabling the preliminary screening of seedlings and
adventitious regenerants at the in vitro stage.
We compared the reactions of 6 raspberry and 2
blackberry cultivars propagated in vitro to drought stress
simulated by the addition of polyethylene glycol 6000
(PEG) to the media. We measured the contents of osmoprotectants (proline and soluble carbohydrates) and of the
percentage dry mass at different PEG concentrations and
after different culture times. We also observed the reaction
to water deficit of one-year old plants grown in pots in the
greenhouse using the same genotypes, by measuring: leaf
water potential, the intensity of gas exchange and chlorophyll fluorescence.
A metabolic reaction in vitro was already observable
after one week, but the patterns of reactions differed
between genotypes depending on the stressor concentration and the length of the stress period. Differences in the
osmoprotectant contents were also found in the control
shoots (non-stressed). The water deficit in the soil limited
the intensity of gas exchange and the water potential in the
leaves, and decreased the ETR coefficient, but differences
between genotypes were also observed. All the parameters, whether in vitro or in vivo, indicated cv. Latham as
the most resistant to water stress.
The effect of cytokinins on in vitro morphogenesis of Passiflora caerulea L.
Marcin Ożarowski, Barbara Thiem
Department of Pharmaceutical Botany and Plant Biotechnology, Poznań University of Medical Sciences,
Św. Marii Magdaleny 14, 61-861 Poznań, Poland, e-mail: [email protected]
The development of an effective method of in vitro regeneration of Passiflora caerulea L. (blue passion flower) is essential for the improvement of cultivation and rapid micropropagation of the plant, whose expected medicinal properties include anxiolytic, sedative, analgesic, antiepileptic activities and utility as a remedy for opiate withdrawal.
The aim of the research was to estimate the effect of
cytokinins on the production of morphogenic callus and
shoot induction from different explants of P. caerulea.
Several concentrations of 6-benzylaminopurine (BAP),
alone or in combination with indole-3-acetic acid (IAA) or
gibberelic acid (GA3), were used in vitro to induce indirect
morphogenesis and to achieve micropropagation. Three
types of explants obtained from in vitro seed germination:
leaf discs, root segments and shoot tips (30-day seedling)
were used in experiments.
The frequency of callus and shoot formation was
dependent on the origin of the explants, concentration of
Vol. 51, suppl. 1, 2009
cytokinin and medium supplementation. The culture
medium containing 0.5 mg/l BAP was the most suitable for
all the explants. The highest rate of induction of indirect
morphogenesis was observed in leaf discs. Direct morphogenesis was observed on root segments. The best
results for shoot regeneration, elongation and propagation
from node explants were obtained on media containing
1.0 mg/l BAP or 1.0 mg/l BAP and 1.0 mg/l IAA, without a
statistical difference.
REFERENCES
DHAWAN K, DHAWAN S, SHARMA A. 2004. Passiflora: a review update.
Journal of Ethnopharmacology 94: 1–23.
OżAROWSKI M, THIEM B. 2008. Kultury in vitro Passiflora quadrangularis L. i P. caerulea L. – wstępne badania nad
indukcją kalusa i organogenezą. Zeszyt abstraktów. VIII
Ogólnopolska Konferencja "Kultury in vitro w fizjologii
roślin". Polska Akademia Nauk, Kraków, 4–5 grudnia
2008 r.
55
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Cryopreservation of in vitro grown shoot buds of rose 'New Dawn' using
encapsulation-dehydration method
Bożena Pawłowska, Anna Bach
Department of Ornamental Plants, Agricultural University of Cracow, Al. 29 Listopada 54, 31-425 Cracow,
e-mail: [email protected]
In the present study, a protocol for preservation in liquid
nitrogen by encapsulation-dehydration method of shoot
meristems of the park rose 'New Dawn' has been developed. Shoot apical and axillary meristems about 2 mm in
diameter were collected from plants propagated in vitro
on the mineral medium according to Quoirin et al. (1977)
supplemented with 5 μM BA, 0.3 μM GA3, 0.5 μM NAA and
0.06 M sucrose. Moreover, some explants for cryopreservation were pre-cultured on the medium containing a
higher sucrose level (0.25 M) for 8 weeks. Before encapsulation some explants were cultivated on medium supplemented with 2.5 g dm-3 activated charcoal for 3 days.
After encapsulation plant material was dehydrated by the
quick method (capsules were placed in liquid medium
containing 0.75 M sucrose for 18 h) or by the gradual
method (capsules were transferred to liquid solutions of
media with sucrose concentrations increasing from 0.3 M
to 1 M for 7 consecutive days).
Results indicate that the survival rate of plant tissue
after freezing in liquid nitrogen depended on plant mate-
rial: apical meristems of roses regenerated callus and
shoots but axillary meristems did not survive the freezing.
Cutting capsules after freezing did not influence the regeneration of the cryopreserved plant explants. It was shown
that the slow dehydration method used in preparation of
plant material for cryopreservation allowed to obtain
viable tissues of Rosa 'New Dawn'. After cryopreservation,
34% of explants incubated on the pre-culture medium
supplemented with a high level of sucrose (0.25M) and
activated charcoal developed shoots.
REFERENCES
QUOIRIN M, LEPOIVRE P And BOXUS P. 1977. Un premier bilan de 10
annees de recherches sur les cultures de meristemes st la
multiplication in vitro de fruitiers ligneus. In: C.R.Rech.
1976–1977 et repports de Synthese, Stat. des Cult. Fruit.
Et Maraich. Glemblous: 93–117
WANG QC and LAAMANEN J. 2005. Cryopreservation of in vitrogrown shoot tips of raspberry (Rubus idaeus L.) by encapsulation-vitrification and encapsulation-dehydratation. Plant
Cell Reports 24: 280–288.
Optimization of medium for callus induction and plant regeneration
of Miscanthus × giganteus
Agnieszka Płażek1, Franciszek Dubert2
1
Department of Plant Physiology, Agricultural University of Cracow, Podłużna 3, 30-239 Cracow, Poland
e-mail: [email protected]
2
Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland
e-mail: [email protected]
Miscanthus × giganteus is a sterile triploid, which reproduces only vegetatively from rootstocks (Lewandowski,
2006). The result of vegetative reproduction is a small
range of genetic variability within this species. The in vitro
culture can be used as a method to increase its genetic
variability via somaclonal embryogenesis. Miscanthus
produces a lot of phenolics, which, when oxidized and
secreted into the medium, show toxicity to tissue development. The aim of this work was to optimize composition
of medium for callus induction and plant regeneration.
The medium inducing callus was supplemented with compounds that inhibit oxidization of phenolics.
Callus was induced from immature inflorescences on
MS (Murashige and Skoog, 1962) medium containing 6.5
mg dm-3 2.4 D, 0.25 mg dm-3 BAP, 500 mg dm-3 of casein
hydrolysate and 30 g dm-3 of sucrose as a control and on
the same medium supplemented with 1) 200 mg dm-3 of
chitosane; 2) 65 g dm-3 of banana pulp; 3) 100 mg dm-3 of
cysteine; 4) 30 g dm-3 of honey instead of sucrose. Callus
was induced at 25°C in the dark. After 2 months, callus
was transferred onto regeneration MS medium supple-
56
mented with 1) 0.2 mg dm-3 of BAP or 2) 0.2 mg dm-3 of
kinetin. Regeneration was performed at 20°C in the light
(PPFD=300 mol m-2 s-1).
The best initiation of callus with numerous somatic
embryos was observed on MS medium containing honey
(84% of all explants) and banana pulp (79%), while on control medium such callus tissue was obtained with 54% frequency. On the regeneration medium with 0.2 mg of BAP no
plants were obtained, while on medium containing 0.2 mg
dm-3 of kinetin, 52 plants were obtained but only from calli
induced on medium supplemented with banana pulp.
REFERENCES
LEWANDOWSKI I. 2006. Miscanthus – a multifunctional biomass
crop for the future. In: Jeżowski S, Wojciechowicz MK,
Zenkteler E [eds.], Alternative plants for sustainable agriculture. PAGEN, Centre of Excellence in Plant Agrobiology
and Molecular Genetics, Institute of Plant Genetics, Polish
Academy of Sciences, Poznań, vol. 5: 83–90.
MURASHIGE T, SKOOG F. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiologia
Plantarum 15: 473–497.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Initiation of an in vitro culture of elder (Sambucus nigra L.) meristems
with the use of various sterilizers
Beata Płoszaj1, Jerzy Przyborowski2
1
Department of Horticulture, University of Warmia and Mazury in Olsztyn, Prawocheńskiego 21, 10-719 Olsztyn,
e-mail: [email protected]
2
Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Plac Łódzki 3,
10-719 Olsztyn, e-mail: [email protected]
Elder (Sambucus nigra L.) is a shrub from the family
Caprifoliaceae. It is used in phytotherapy, food processing industry and as a decorative plant. The objective of
this study was to develop an effective method for surface
sterilization of initial material for in vitro cultures of elder
var. Aurea. The initial material consisted of axillary and
apical buds sampled from the shoots of shrubs grown at
the Experimental Station of the University of Warmia and
Mazury in Olsztyn. Shoot samples were collected at the
beginning of the growing season in 2007 and 2008. Buds
were isolated from the shoots, rinsed under running water
for 1 hour and then immersed in water with the addition
of a detergent for 5 minutes. Prior to disinfection, buds
were immersed in 70% ethanol for 30 seconds. The material was disinfected with various combinations of disinfectants applied over different periods of time. Treatment 1
involved the use of 0.1% mercury chloride (HgCl2) for 60,
120 and 240 seconds. In treatment 2, buds were disinfected with 3% sodium hypochlorite (NaOCl) for 5, 10, 15
and 30 minutes. In treatment 3, the samples were disin-
fected with 0.1% mercury chloride (HgCl2) for 60, 120 and
240 seconds, after which the incisions were immersed in
silver nitrate (AgNO3). In the last treatment, plant material was immersed in a calcium hypochlorite [Ca(OCl)2]
solution for 5, 10 and 15 minutes. After sterilization, buds
were rinsed three times in sterile distilled water. Explants
of tissue fragments containing the meristem were isolated
from the disinfected material. Every explant was placed in
a 10 ml test tube containing 1.5 ml MS medium
(Murashige and Skoog, 1962) whose macronutrient and
micronutrient contents were reduced by half. Culture
media were additionally enriched with 8 g·dm-3 agar and
30 g·dm-3 saccharose. The medium pH was set at 5.5
before autoclaving. Every tratment was represented by 20
explants. Incubation was carried out in a phytotron at
23°C with 16 h of light exposure. The highest percentage
share of sterile plants occurred in cultures where the initial material was disinfected with calcium hypochlorite. In
the remaining treatments infections were more prevalent
and led to the death of explants within a short time.
The effect of kinetin content in the culture medium on micropropagation
of elder (Sambucus nigra L.)
Beata Płoszaj1, Jerzy Przyborowski2
1
Department of Horticulture, University of Warmia and Mazury in Olsztyn, Prawocheńskiego 21, 10-719 Olsztyn,
e-mail: [email protected]
2
Department of Plant Breeding and Seed Production, University of Warmia and Mazury in Olsztyn, Plac Łódzki 3,
10-719 Olsztyn, e-mail: [email protected]
Elder var. Aurea is an attractive decorative shrub with
golden yellow leaves that maintain their color throughout
the growing period. Elder shrubs of this variety reach 2
meters in height, and they create a highly attractive combination when set against plants with dark foliage. As an
additional advantage, this elder variety has low soil
requirements. This study investigated the effect of the
kinetin content in the culture medium on in vitro micropropagation of elder var. Aurea. The experiment was carried out on a medium (Murashige and Skoog, 1962)
whose micronutrient and macronutrient contents were
reduced by half, enriched with 8 g·dm-3 agar and 30 g·dm3
saccharose. The medium pH was adjusted to 5.5 before
autoclaving. Plant material was obtained from a sterile
and stable culture. Explants consisting of two nodes were
placed individually in 10 ml test tubes containing 1.5 ml
Vol. 51, suppl. 1, 2009
medium each. The kinetin (KIN) concentrations used in
the experiment were: 2, 4 and 6 mg·dm-3. Each treatment
was represented by 40 explants. After an 18-week propagation cycle (3 subcultures), the propagation ratio was
computed for every treatment, and the morphological
characters of the plants were assessed.
The highest propagation ratio occurred in the treatment with 4 mg·dm-3 kinetin. Most plants growing in this
medium were not affected by any developmental anomalies. Lower propagation ratios were noted in culture
media with the lower kinetin content, while higher concentrations of this phytohormone resulted in similar or
lower propagation ratios. The resulting plants were characterized by short internodes, fewer and smaller leaves
and lower root mass.
57
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
ISSR analysis of genetic stability of Polish tulip cultivars propagated in vitro
Małgorzata Podwyszyńska, Anita Kuras, Małgorzata Korbin
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland,
e-mail: [email protected]
The aim of the study was an early detection of somaclonal
variation (SV) which could occur within the micropropagated plant material. Shoot cultures of nine Polish cultivars
were used. They were propagated in vitro for one or two
years. Plant material was propagated in vitro by the means
of adventitious shoot regeneration in the presence of thidiazuron (Podwyszyńska and Marasek, 2003). The SV was
detected with the inter-simple sequence repeat (ISSR) markers. For ISSR analysis, leaf samples were taken from in vitro
propagated plant material and from conventionally propagated true-to-type plants which served as standards. Ten
ISSR primers were used (TIBMOLBIOL, Poznań and IBB,
Warszawa): 824, 843, 844, 845, 846, 852, 853, 857, 973
and 895. The DNA extraction was performed using the DNA
purification kits Genomic Mini AX PLANT (A&A
Biotechnology). The molecular markers were first used for
cultivar identification and then for the detection of SV. The
degree of genetic similarity between the micropropagated
plants and the standard of each cultivar was calculated
based on the number of monomorphic and polymorphic
bands. The Cafe-Jaccard coefficient was used to plot a den-
drogram using the UPGMA method. The statistical analyses
were performed in XLSTAT software environment
(Addinsoft, 2006). The UPGMA dendrogram of genetic similarity showed nine clusters grouping cultivars, their standards and micropropagated plants. For the six studied genotypes, the ISSR analysis, performed with ten primers, did
not reveal polymorphisms between the standard and the
micropropagated plants. Analysis of the other three cultivars
showed that some of the plants, micropropagated for either
one or two years, differed slightly from standards (in cultivar
A, three of the five analyzed plants; in cultivar B, two of the
four plants); whereas in cultivar C, all plant showed a high
(96%) similarity. These results indicate that the changes in
DNA structure may occur during the first year of the plant
micropropagating process.
REFERENCE
PODWYSZYŃSKA M, MARASEK A. 2003. Effect of thidiazuron and
paclobutrazol on regeneration potential of tulip flower stalk
explants in vitro and subsequent shoot multiplication. Acta
Societatis Botanicorum Poloniae 72:181–190.
Evaluation of somaclonal variation in micropropagated Hemerocallis sp. plants
using phenotype and ISSR markers
Małgorzata Podwyszyńska1, Eleonora Gabryszewska1, Małgorzata Korbin1, Artur Jasiński2
1
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland,
e-mail: [email protected]
2
Horticulture, Wierzbowa 1, 05-820 Piastów, Poland
The genetic fidelity of micropropagated daylily plants was
evaluated by phenotypic observation and inter-simple
sequence repeat (ISSR) techniques. Plants of nine daylily
cultivars were used in this study. The plants which were
propagated in vitro for the period of one to five years, were
then grown outdoors in the years 2002 to 2006. The observations comprised 1600 plants and concerned the number
and quality of flowering plants. The colour, the size and the
shape of flowers were evaluated and compared to the published cultivar descriptions in accordance with American
Hemerocallis Society (AHS). The nine selected somaclones
were analized with ISSR markers in order to determine the
character of variation. The first micropropagated plants
flowered sporadically in the first growing season. In the second season, the highest percentage of flowering plants was
found for the cultivar 'Moonlit Masquerade' (84%) and the
lowest for 'Pink Debutante' (20%). The latter cultivar had the
flowering rate of 70–100% in the third season. In general, the
length of flower stems of all studied cultivars was similar to
the lengths given in the AHS cultivar descriptions.
Somaclonal variants occurred within the micropropagated plants of the five genotypes. The somaclones differed
from the true-to-type plants in terms of the colour and the
58
shape of flowers. The frequency of SV ranged from 1.4%
to 100%. In 'Moonlit Masquerade', 62.6% of plants developed flowers with narrower petals and a smaller brown
eye compared to the true-to-type. In 2006, for 'Ruby
Moon', the colour of the eye was changed from ruby-red to
a lighter colour in all of the plants, while in 2007 this was
observed only for 25% of the plants. For 'Pink Debutante',
only one plant was found with a malformed flower in the
first growing season, while in the second season, the SV
frequency was 11.1%. The observation performed during
2004–2006 revealed that for 'Amethyst Jewel' the colour of
all the flowers changed from light amethyst-violet to light
grey pink. However, in 2007, all plants of this cultivar had
flowers with colour similar to the type. Four cultivars
appeared to be genetically stable.
The ISSR analyses of the selected somaclones
revealed that the phenotypic changes observed had the
characteristics of a mutation.
To summarize: 1) the SV frequency depends on the
genotype, 2) a reliable information on the SV, introduced
during the in vitro propagation, can be obtain when more
than 70% of the plants have flowered; this can occur as
early as in the second or third growing season.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Frequencies of spontaneous doubled haploids of winter triticale plants obtained
by anther culture
Aleksandra Ponitka, Aurelia Ślusarkiewicz-Jarzina, Jolanta Woźna, Hanna Pudelska
Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznań, e-mail: [email protected]
The haploids and spontaneous doubled haploids are
obtained with varying efficiency in the process of cereal
androgenesis. The aim of this study was to evaluate the
production frequency of spontaneous doubled haploids in
winter triticale plants derived from anther culture.
Anthers from ten F1 hybrids of winter triticale 5953
were plated on a C17 medium containing 0.5 mg/l KIN +
2.0 mg/l 2,4-D + 90 g/l maltose. 5412 (average of 91.2%)
embryo-like structures were obtained (29.7 to 150.4%,
depending on the genotype), from which 463 green plants
were regenerated (average of 7.8% in relation to anthers
plated and from 2.2 to 26.7% depending on the genotype)
on a 190-2 medium supplemented with 0.5 mg/l KIN + 0.5
mg/l NAA + 30g/l sucrose.
The ploidy levels of green plants were determined by
flow cytometry of the DNA content of DAPI – stained nuclei
from leaves. Among the 463 green regenerants analyzed,
239 (average of 51.6%) were spontaneous double haploid,
with frequency varying from 32.1 to 85.7%, depending on
the genotypes. The identified haploid plants were subjected to colchicine treatment (0.1% aqueous solution of
colchicine was used together with 4% DMSO and 25 mg/l
GA3 for 6 hours in the light at 25°C). All plants were potted and vernalized for 8 weeks at 4°C, and then moved to
the greenhouse where they were grown to maturity. The
efficiency of chromosome doubling was determined based
on the fertility of spontaneous doubled haploids as well as
individuals subjected to colchicine treatment.
Isolation and in vitro culture of endosperm tissues in selected monocot
and dicot species
Marzena Popielarska-Konieczna1, Marta Braś1, Krystian Rzenno1, Maciej Łojewski1, Izabela
Marcińska2
1
Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Cracow, Poland,
e-mail: [email protected]
2
Institute of Plant Physiology of Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland
The competence of isolated endosperms of selected
important crop plants for proliferation and differentiation
in vitro was assessed on culture media supplemented with
the plant growth regulators. Mature endosperm tissues
were excised from seeds of kiwifruit (Actinidia deliciosa
cv. Hayward). Ovules of eight cultivars of winter, spring
and durum wheat (Triticum aestivum and T. durum) and
two lines of T. monococcum were used as sources of
immature endosperm.
The basal medium consisting of MS salts and vitamins
was supplemented with 3% (w/v) sucrose, auxin (2,4-D),
cytokinins (kinetine, thidiazuron – TDZ) and 5-azacytidine
(5-azaC). Culture were incubated in the dark or exposed to
a 16 h photoperiod provided by cool-white fluorescent
tubes (60–90 μmol photons m-2 s-1). The material for sectioning (freshly isolated and cultured endosperm) was prepared by embedding tissues in Technovit 7100 (Heraeus
Kulzer) and stained with toluidine blue or auramine O.
In kiwifruit the long-term culture of endospermderived callus, organogenesis induction and histological
events have been described previously (Popielarska et al.,
2006; Popielarska-Konieczna et al., 2008). In the present
work localization of cutine on callus and meristematic
protuberances were examined.
Vol. 51, suppl. 1, 2009
Studies on proliferation and histological analysis of
isolated endosperm of heksaploid plants T. aestivum,
tetraploid T. durum and diploid T. monococcum cultured
in vitro have been described recently (PopielarskaKonieczna et al., in press). The present experiments were
conducted on immature endosperm tissues isolated from
6–8 DAP-old grains of T. aestivum. Flowers and/or isolated endosperm were treated with 5-azaC. The aim of the
experiment was to induce hypomethylation and expression of silencing genes.
REFERENCES
POPIELARSKA M, ŚLESAK H, GÓRALSKI G. 2006. Histological and SEM
studies on organogenesis in endosperm-derived callus of
kiwifruit (Actinidia deliciosa cv. Hayward). Acta Biologica
Cracoviensia Series Botanica 48: 97–104.
POPIELARSKA-KONIECZNA M, KOZIERADZKA-KISZKURNO M, ŚWIERCZYŃSKA J,
GÓRALSKI G, ŚLESAK H, BOHDANOWICZ J. 2008. Ultrastructure
and histochemical analysis of extracellular matrix surface
network in kiwifruit endosperm-derived callus culture. Plant
Cell Reports 27: 1137–1145.
POPIELARSKA-KONIECZNA M, MARCIŃSKA I, NOWAKOWSKI P. Preliminary
studies on isolated endosperm of wheat (Triticum) species
cultured in vitro. Advances of Agricultural Sciences
Problem Issues (in press).
59
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Micropropagation of Leucojum aestivum in a temporary immersion bioreactor
system (RITA)
Agata Ptak, Joanna Gądek
Department of Plant Breeding and Seed Science, Agricultural University of Cracow, Łobzowska 24, 31-140 Cracow,
Poland, e-mail: [email protected]
Leucojum aestivum belongs to the family Amaryllidaceae,
the alkaloids of which are known to exhibit a wide range
of biological activities. Galanthamine, an acetylocholinesterase inhibitor, is used for symptomatic treatment of Alzheimer's disease (Heinrih and Lee Teoh, 2004).
Our previously published results have shown that in vitro
cultures of L. aestivum are a source of galanthamine and
other Amaryllidaceae alkaloids (Ptak et al., 2009). In
order to optimize somatic embryogenesis of L. aestivum
we used RITA temporary immersion bioreactor systems
(TIBS). Embryogenic callus obtained by the method
described previously was transferred to solid and liquid
MS media (Ptak et al., 2009). Liquid cultures were carried
out in RITA vessels, and every 2 hours 5 or 15-minute
flushing with the medium was applied. The medium was
supplemented with: picloram (2, 5, 10, 25 μM) and BAP
(0.5 μM). Callus growth on the solid medium enriched
with 2 or 5 μM of picloram and 0.5 μM of BAP was characterized by a higher multiplication index. However, the
greatest number of somatic embryos was observed on the
callus multiplied on the liquid medium containing 2 μM of
picloram and 0.5 μM of BAP, while using 5-minute flush-
ing with the medium every 2 hours. Globular embryos
developed into torpedo-stage embryos under the influence
of NAA (0.5 μM) and zeatin (5 μM). In our experiment, the
TIBS also promoted the development of L. aestivum
somatic embryos. Torpedo embryos derived from RITA
vessels were characterized by a greater increase in fresh
weight compared with the embryos cultivated on the solid
medium. The RITA derived embryos converted into normal plantlets on the medium enriched with NAA (0.5 μM)
and zeatin (5 μM). The analysis of the ploidy level of the
regenerated L. aestivum plantlets from the solid medium
and the RITA TIBS did not reveal any changes.
REFERENCES
HEINRICH M, LEE TEOH H. 2004. Galanthamine from snowdropthe development of a modern drug against Alzheimer's disease from local Caucasian knowledge. Journal of
Ethnopharmacology 92(2–3): 147–62.
PTAK A, TAHCHY AE, DUPIRE F, BOISBRUN M, HENRY M, CHAPLEUR Y,
MOŚ M, LAURAIN-MATTAR D. 2009. LCMS and GCMS for the
screening of alkaloids in natural and in vitro extracts of
Leucojum aestivum. Journal of Natural Prododucts 72 (1):
142–147.
Preliminary analysis of polyphenolic fraction from intact plant and in vitro
cultures of Securinega suffruticosa
Danuta Raj1, Adam Kokotkiewicz2 , Agnieszka Skorys1 , Maria Łuczkiewicz2
1
Chair and Department of Pharmacognosy, Wrocław Medical University, pl. Nankiera 1, 50-140 Wroclaw,
e-mail: [email protected]
2
Chair and Department of Pharmacognosy, Medical University of Gdańsk, Hallera 107, 80-416 Gdańsk
Securinega suffruticosa, Phyllanthaceae, is one of the basic
Chinese folk medicines used in the treatment of various diseases, e.g. rheumatoid disease or quadriplegia (Yuan et al.,
2005). Many studies have been conducted on the alkaloid
fraction of the plant, however there are very few reports on
other types of compounds, especially on the polyphenolic
constituents (Lee, 1994; 1996). To better understand the
complex mode of action of plant medicines, we need to
detect all the physiologically active compounds. Considering
the well known health promoting properties of polyphenolics, it seemed advisable to analyze flavonoids, tannins and
phenolic acids from S. suffruticosa.
Plant tissue cultures are considered to be an interesting source of attractive secondary metabolites, allowing
the production of biologically active compounds in a controlled way, independent of climatic conditions and seasonality. Considering the potential ability of some plant
cell cultures to accumulate high amounts of polyphenolic
compounds, it seemed pertinent to investigate in vitro cell
cultures of this plant.
The aim of this work was to determine qualitatively
and quantitatively the main compounds of the polypheno-
60
lic fraction from S. suffruticosa in vitro cultures and
compare them with results from intact plant grown under
Polish conditions. To this end, callus cultures, shoot cultures and tissues of intact plants were analyzed with
respect to their polyphenolic fraction. Moreover, all plant
matrices were compared to each other to determine "the
best producer" of the analyzed compounds. Calli were
grown on the Schenk – Hildebrandt (SH) medium and SH
medium modified with plant growth regulators; shoots
were grown on the Murashige for Lilium (ML) medium.
TLC was the method of choice for the preliminary qualitative analyses. For the quantitative analyses of polyphenols
HPLC with DAD UV detection was chosen.
REFERENCES
LEE S. 1994. Chemical study on the phenolic compounds from the
leaves of Securinega suffruticosa, Sayengyak Hakhoechi
25: 105–112.
LEE S. 1996. Phenolic compounds from stems of Securinega suffruticosa, Korean. Journal of Pharmacognosy 27: 1–5.
YUAN W, LU Z, LIU Y, MENG C, CHENG K, ZHU P. 2005. Three new
podocarpane-type diterpenoids from callus of Securinega
suffruticosa, Chemical and Pharmaceutical Bulletin 53:
1610–1612.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Agrobacterium rhizogenes serves cytogenetics
Magdalena Rojek, Marta Dydak, Jolanta Kwasniewska, Karolina Walas, Elzbieta Wolny,
Krystyna Nowak, Jolanta Małuszynska
Department of Plant Anatomy and Cytology, Faculty of Biology and Environmental Protection, University of Silesia,
Jagiellonska 28, 40-032 Katowice, e-mail: [email protected], [email protected], [email protected],
[email protected]
Transformation using Agrobacterium rhizogenes has
become a very popular and widely used method in plant
biotechnology. A. rhizogenes is a Gram negative soil bacterium, a natural pathogen of many plants. In the process
of plant tissue infection, a fragment of the bacterial Ri
plasmid, the T-DNA, is transferred and integrated with
the plant genome. Expression of specific bacterial genes
from T-DNA cause the production of hairy roots. This
natural process has been used to produce hairy roots
from many different species. Hairy roots have several
characteristic features which makes them noteworthy and
easy to work with.
The hairy roots culture is a valuable source of material in different areas of studies, such as bioproduct and
secondary metabolite production or phytoremediation. In
this work we present the use of hairy roots in cytogenetics
studies. A hairy root culture is a very good tool for maintaining rare genotypes for further analysis, especially if
species are not self-fertilizing. Hairy root cultures are usually cytogenetically stable for a long time, but in some
species, such as Brassica, chromosome elimination or
chromosomal rearrangements may occur.
A large number of root tips with many dividing cells in
a hairy roots culture, makes cytogenetic analysis much
easier than using seedlings. Additionally, chemical treatment e.g. with hydroxyurea can increase the metaphase
index even to 50%. Such a high metaphase index together
with a large number of root tips in the culture create the
possibility for chromosome isolation using flow cytometer
equipped with sorter.
We discuss the use of hairy roots from long and short
term cultures in cytogenetic analysis of individual chromosomes and interphase nuclei with different numbers of
B chromosomes.
Also we have used C. capillaris hairy roots for evaluation of genotoxicity. Comparison of cytogenetic effects in
hairy roots and roots of seedlings showed a much higher
sensitivity of hairy roots, which makes them a convenient
material for monitoring the DNA damage after mutagenic
treatment.
Use of wide crossing and in vitro culture for the induction of haploid
embryogenesis in three cereal species
Michał Rokicki, Andrzej Wojciechowski
Department of Genetics and Plant Breeding, Poznań University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznań,
e-mail: [email protected]
Wide crossing is an important tool for expansion of variability and for haploid production. The bulbosum method
is one of the oldest approaches, in which crosses of cultivated barley with wild Hordeum bulbosum are made to
help produce haploid plants. In other cereal species crosses with maize are more popular. There is evidence in the
literature showing that pollination of cereals with maize
pollen results in haploids production (Lauriea and Benett,
1986; Wędzony, 1999). However, there are also doubts
concerning the actual fertilization. Therefore, the aim of
this work was to explain whether there is a complete conjunction of maize sperm with cereal egg cell or whether the
presence of maize sperm is sufficient for the induction of
embryogenesis from an unpollinated cereal egg cell.
Crosses of three cereal species (wheat, triticale and
rye) with maize were carried out in a greenhouse belonging to the Department of Genetics and Plant Breeding,
Poznań University of Natural Sciences. Spikes of these
species were castrated shortly before earing. The pollination was conducted from 2 to 6 days after castration. Next
Vol. 51, suppl. 1, 2009
day after pollination the ears were sprayed with 50 mg l-1
solutions of 2,4-D and picloram. Embryos or ovules from
wide pollinations were isolated and placed on White or MS
medium depending on the embryo developmental stage.
For the observation of pollen grain germination and pollen
tube growth, the pollinated pistils were fixed in the Carnoy
solution and stained with aniline blue (Martin, 1959).
Based on our results it can be concluded that there
were differences in the intensity of pollen tube growth and
the development of embryos between particular crosscombinations.
REFERENCES
LAURIE DA, BENNETT MD. 1986. Wheat × maize hybridization.
Canadian Journal of Genetics and Cytology 28: 313–316.
MARTIN F. 1959. Staining and observing pollen tubes by means of
fluorescence. Stain Technology 34: 125–128.
WĘDZONY M. 1999. Wpływ analogów auksyn na aktywność
otrzymywania linii podwojonych haploidów pszenżyta
i pszenicy metodą krzyżowania z kukurydzą. Monografie: 9,
Zakład Fizjologii Roślin PAN, Kraków.
61
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Phenolic acids in in vitro cultures of Exacum affine Balf. f.
Ewa Skrzypczak-Pietraszek1, Jacek Pietraszek2
1
Chair and Department of Pharmaceutical Botany, Collegium Medicum, Jagiellonian University, Medyczna 9,
30-688 Cracow, Poland, e-mail: [email protected]
2
Institute of Applied Computer Science, Cracow University of Technology, Jana Pawła II 37, 31-864 Cracow, Poland,
e-mail: [email protected]
Plant tissue cultures can be the source of many various
secondary metabolites (Malepszy, 2001). Exacum affine
Balf. f. (Gentianaceae) grows on Socotra Island (Aden
Bay). In Poland it is known as a decorative pot plant.
Secondary metabolites such as secoiridoid glucosides,
flavonoids, phenolic acids and saponins were found in
plants of the genus Exacum (Das et al., 1984). Chemical
composition of Exacum affine has been only partly
analysed (Kuwajima et al., 1996). The only known phenolic acid that has been reported from this plant is pcoumaric acid. We found no literature information on
chemical analysis of in vitro cultures of Exacum affine.
The aim of the present investigation was chemical
analysis of phenolic acids in in vitro cultures of Exacum
affine Balf. f. and their comparison with constituents of
pot plants of this species. Free and bound phenolic acids
were determined.
In vitro cultures of Exacum affine (shoot cultures)
were maintained on the MS medium supplemented with
BAP (1 mg/l), NAA (0.5 mg/l), GA3 (0.25 mg/l). Dried plant
materials (shoots from in vitro cultures and aerial parts of
pot plants) were extracted with methanol. Additional samples of plant materials were hydrolysed with 2 M HCL
before extraction. Methanolic extracts were qualitatively
and quantitatively analysed by HPLC. Compounds were
detected at 254 nm. The identification of phenolic acids
was accomplished by the comparison of their retention
times with standards and followed by the internal standard method. Some phenolic acids were identified in the
investigated extracts (protocatechic, p-hydroxybenzoic,
vanillic, syringic, p-coumaric and ferulic acid). Free phenolic acids were usually present in small or even trace
amounts. More compounds were found after hydrolysis.
REFERENCES
DAS S, SHARMA RP, BARUAH JN, KULANTHAIVEL P. 1984. Secoiridoids
from Exacum tetragonum. Phytochemistry 23: 908–909.
KUWAJIMA H, SHIBANO N, BABA T, TAKAISHHI K, INOUE K, SHINGU T.
1996. An acetophenone glycoside from Exacum affine.
Phytochemistry 41: 289–292.
MALEPSZY S. 2001. Biotechnologia roślin. PWN, Warszawa.
Induction of androgenesis in oat (Avena sativa L.) depending on kind of culture
medium
Edyta Skrzypek, Izabela Marcińska, Agata Stawicka, Ilona Czyczyło-Mysza, Marta Pilipowicz
The F. Górski Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland,
e-mail: [email protected]
The production of doubled haploids under laboratory conditions allows to obtain a high level of homozygotic DH
lines, to shorten the breeding time and to increase the
selection effectiveness of required genotypes. However
androgenic response of plants highly depends on the
species and even genotype. An extremely low efficiency of
androgenesis is observed in oat in comparison to wheat or
triticale.
The aim of the study was to compare the effect of type
and physical properties of the medium on anther vitality
and induction of embryo like structures (ELS).
Five F1 generations, nine F2 generations and two cultivars of oat were used. Donor plants were grown in the
greenhouse. Anthers were isolated after a cold pretreatment and cultured on solid media: C17 (Wang and Chen,
1983), W14 (Ouyang et al., 1989) and modified W14
(Kiviharju et al., 2005) or on a liquid and semi-solid modified W14. The analyses of results showed that anther
62
vitality and ELS formation mostly depended on oat genotype and kind of media but less on media fluidity. Forty
one ELS were obtained from eleven genotypes. The ELS
from five genotypes regenerated eight plants. Only two
plants survived colchicine treatment used for chromosome doubling.
REFERENCES
KIVIHARJU E, MOISANDER S, LAURILA J. 2005. Improved green plant
regeneration rates from oat anther culture and thr agronomic performance of some DH lines. Plant Cell Tissue and
Organ Culture 81: 1–9.
OUYANG JW, JIA SE, ZHANG C, CHEN X, FENG G. 1989. A new synthetic medium (W14) for wheat anther culture. Ann Rep Inst
Genet Aca Sinica, Beijing, China, 91–92.
WANG P, CHEN Y. 1983. Preliminary study on prediction of height
of pollen H2 generation in winter wheat grown in the field.
Acta Agronomica Sinica 9: 283–284.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Evaluation of phenotypic trueness-to-type for selected cultivars of narcissus
propagated by in vitro cultures
Dariusz Sochacki
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice, Poland,
e-mail: [email protected]
Micropropagation of 13 narcissus genotypes was carried
out in 2001. After acclimation, the plants were cultivated
in an insect-proof mesh tunnel. Single plants of two cultivars ('Tete-a-Tete' and 'Golden Ducat') produced first flowers in 2004. The following year, eight cultivars were in full
blossom after 4 years of ex vitro cultivation. All blooming
plants of 11 cultivars were evaluated according to UPOV
descriptors during the next two growing seasons – 2006
and 2007. Nineteen morphological characteristics, including the shape and colour of perianth segments, and the
type, shape and colour of corona, were observed and
measured. The observations of plants propagated in vitro
were compared with the observations of the same cultivars growing in the field and propagated traditionally, or
with the descriptions available in the literature.
Narcissi obtained via in vitro propagation, showed no
phenotypic differences in comparison with the initial genotypes. Small differences in the colour gradation of flowers
or the length of flower stems were a result of growing conditions. The lower colour intensity in the case of plants
cultivated in the mesh tunnel was probably caused by
extensive shading due to the very dense mesh. However, in
the case of one cultivar – 'Marie-Jose' from the split-corona group, significant modifications in the shape and
colour of perianth segments were observed. A considerable fraction of plants (22%) showed no creamy-white
stripes on the orange split corona. In addition, changes in
the corona (from split, via partly split, to completely connate, like in small-cupped narcissi) were noticed.
Methyl jasmonate and fosmidomycin affect mono- and sesquiterpenoid
production in root cultures of Inula royleana DC. and Inula macrocephala
Boiss. & Kotschy ex Boiss.
Anna Stojakowska, Janusz Malarz
Department of Phytochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, 31-343 Cracow,
Poland, e-mail: [email protected]
Root cultures of Inula macrocephala and Inula royleana
were obtained as described by Stojakowska et al. (2006)
and cultivated either in WP (Lloyd and McCown, 1980) or
in 1/2 GB5 (Gamborg et al., 1968) liquid media, respectively, at 25°C in the dark. The cultures produced
monoterpenoids (thymol derivatives) and sesquiterpene
lactones of eudesmane type. Methyl jasmonate or fosmidomycin solution was added to the flasks containing 20
day old cultures, to reach the final concentration of 100
μM in the nutrient medium. Roots from both treated and
control cultures were harvested 72 h after the beginning of
experiment, frozen and lyophilized. Contents of selected
mono- and sesquiterpenoids were measured by means of
analytical RP-HPLC (Stojakowska et al., 2006).
Jasmonates are elicitor signal transducers for the production of secondary metabolites in plants and plant tissue cultures. Methyl jasmonate added to I. macrocephala
and I. royleana root cultures caused an enhanced accumulation of eudesmanolides and thymol derivatives in the
roots. The eudesmanolide content in the treated cultures
was up to four fold higher than that in the controls. The
C5 unit (IPP) for isoprenoid biosynthesis in plants is gen-
Vol. 51, suppl. 1, 2009
erated either through the plastidic (GAP, DXP or MEP) or
the cytoplasmic (mevalonate) pathway. Mono- and diterpene biosynthesis is generally attributed to the plastidic
compartment of the plant cell. In contrast, the enzyme
responsible for the C15 isoprenoid chain (FPP) synthesis
seems to be bound to the cytoplasmic compartment.
Fosmidomycin, an inhibitor of the plastidic pathway of
terpenoid biosynthesis, decreased monoterpenoid production in the Inula roots, as expected. The eudesmanolide content remained unaffected.
REFERENCES
GAMBORG OL, MILLER RA, OJIMA K. 1968. Nutrient requirements of
suspension cultures of soybean root cells. Experimental Cell
Research 50: 151–158.
LLOYD G, MCCOWN B. 1980. Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia by use of shoot-tip
culture. The International Plant Propagators Society 30:
421–427
STOJAKOWSKA A, MICHALSKA K, MALARZ J. 2006. Simultaneous quantification of eudesmanolides and thymol derivatives from tissues of Inula helenium and I. royleana by reversed-phase
high-performance liquid chromatography. Phytochemical
Analysis 17: 157–161.
63
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Mycelial cultures of some Aphyllophorales (Basidiomycetes): optimization of
in vitro culture conditions
Katarzyna Sułkowska-Ziaja, Bożena Muszyńska
Chair and Department of Pharmaceutical Botany, Jagiellonian University Collegium Medicum, Medyczna 9,
301-688 Cracow, Poland
Sarcodon imbricatus (L.) P. Karst., Sparassis crispa
Wulf.: Fr. and Hydnum repandum L.: Fr. are species of
fungi belonging to Aphyllophorales (Basidiomycetes).
S. imbricatus and S. crispa are under strict legal protection in Poland.
In vitro cultures were established from fruit bodies
growing naturally in the forests of southern Poland. The
aim of this study was to optimize the conditions of submerged culture for mycelial biomass increments (MBI)
during 4 week cycles of culture. The optimal medium
composition for submerged culture was determined. To
investigate the effect of carbon and nitrogen source on
hyphal growth, the mycelium was cultivated on the medium containing various carbon (fructose, glucose, maltose,
lactose, sucrose) and nitrogen (amonium nitrate, sodium
nitrate, casein hydrolyzate of, malt extract, yeast extract)
sources. Additionally the optimum initial value of pH and
optimal temperature for mycelial increment were determined. The optimal medium composition for biomass
increment of Sarcodon imbricatus was 5% fructose
(MBI=8.0 g d.w/dm3) and 1% casein hydrolyzate
(MBI=9.6 g d.w/dm3). Maximal growth of biomass was
observed at initial pH 6.0 (MBI=4.8 g d.w/dm3) and the
optimal temperature of incubation was 30°C (MBI=9.4 g
d.w/dm3). The optimal medium composition for biomass
increments of Sparassis crispa was 5% glucose
(MBI=13.7 g d.w/dm3) and 1% casein hydrolyzate
(MBI=7.95 g d.w/dm3). Maximal growth of biomass was
observed at initial pH 6.0 (MBI=9.92 g d.w/dm3) and
optimal temperature of incubation was 30°C (MBI=7.95 g
d.w/dm3). The optimal medium composition for biomass
increments of Hydnum repandum was 5% glucose
(MBI=11.0 g d.w/dm3) and 1% casein hydrolyzate
(MBI=14.7 g d.w/dm3). Maximal growth of biomass was
observed at initial pH=6.0 (MBI=9.8g d.w/dm3) and optimal
temperature of incubation was 25°C (MBI=9.6 g d.w/dm3).
Preliminary investigation of chemical compounds from
these species showed the presence of endo- and egzogenic
amino acids, fatty acids and phenolic acids. All these
compounds were determined by chromatographic methods (TLC, HPLC).
These results show that submerged cultures of investigated fungi species could make a useful subject for future
chemical analysis.
Molecular characterization of resynthesized oilseed rape (Brassica napus L.)
Laurencja Szała, Anna Olejnik, Teresa Cegielska-Taras
Plant Breeding and Acclimatization Institute, Department of Genetic and Breeding of Oilseed Crops, Strzeszyńska 36,
60-479 Poznań, Poland, e-mail: [email protected]
Oilseed rape (Brassica napus L; genome AACC, 2n =38)
is a relatively young species that originated through a
spontaneous hybridization between turnip rape (Brassica
rapa L.; genome AA, 2n=20) and cabbage (Brassica oleracea L., genome CC, 2n=18). Today oilseed rape is one of
the most important oilseed crops in the world. However,
its limited geographical range has led to a comparatively
narrow genetic basis currently available for breeding.
Resynthesized (RS) rapeseed genotypes developed
through interspecific crosses between different B. rapa
and B. oleracea genotypes have the potential to increase
significantly the available gene pool and provide important
64
germplasm base for further improvement of seed quality
and resistance to biotic and abiotic stress.
Embryo rescue techniques considerably assist in
obtaining distant hybrids in cases when hybrid embryos
abort at early stages of development.
This work focuses on the development of interspecific
hybrids between Brassica rapa L. subsp. pekinensis and
Brassica oleracea L. var. acephala subsp. lanciniata
through embryo rescue culture in vitro. The objectives of
this research were: to develop an in vitro culture system
for hybridization and to analyze the regenerated resynthesized plants using RAPD molecular markers.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Anti-inflammatory properties of transgenic flax fiber extract
Monika Sztajnert1, Katarzyna Ratajczak1,2, Anna Kulma1 and Jan Szopa1
1
Department of Genetic Biochemistry, University of Wrocław, Przybyszewskiego 63/77, 51-148 Wrocław, Poland,
Department of Traumatology and Hand Surgery, Wrocław Medical University, Borowska 213, 50-556 Wrocław,
Poland
2
Flax (Linum usitatissimum L.) is a very important source
of natural fibers used by the textile industry and also a
source of valuable oil. Also seed cakes left after oil pressing contain many valuable components. Flax oil and seed
cake meal is widely used for its health promoting properties. Many health-promoting components have been identified to date, including lignans, phenylopropanoids and
Omega 3 fatty acids. However, most data concern compounds found in flax seeds.
We observed the anti-inflammatory and wound healing
properties of wound dressing made of transgenic flax
fibers and so we decided to identify compounds present in
flax fibers responsible for those properties. Several components of fiber extract were identified by UPLC method,
including phenolic acids and terpenoids. One of the most
interesting compounds found were cannabinoid derivatives, not reported previously from flax fibers,. Therefore
we concentrated our research on cannabinoid-like compounds know for their anti-inflammatory and analgesic
properties. The effects of flax fiber extract on the expression levels of cannabinoid receptor and several pro- and
anti -inflammatory genes were tested in mouse 3T3 Balb
cell line in which the inflammation state was mimicked by
LPS or TNFa treatment. The gene expression was estimated by means of semi-quantitative PCR. The most interesting finding was the induction of anti-inflammatory SOCS1 (suppressor of cytokine signaling) gene and a decrease
in the expression of Interleukin 6 and MCP-1 (monocyte
chemotactic protein1) genes responsible for propagation
of inflammation. Thus anti-inflammatory effects of flax
fiber extracts were confirmed and it was shown that at
least some of the effects are caused by activation of endocannabinoid signaling pathway.
Effect of 5-azacitidine on DNA methylation pattern and somatic embryogenesis
in Arabidopsis
Miriam Szurman, Marta Gliwicka, Małgorzata D. Gaj
Department of Genetics, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland,
e-mail: [email protected]
Methylation of DNA is an epigenetic feature, controlling gene
expression and maintaining proper functioning of cells. The
aim of this work was to evaluate the pattern of DNA methylation in embryogenic culture of Arabidopsis thaliana (L.)
Heynh. To evaluate DNA methylation level Methylation
Sensitive Amplified Polymorphism (M-SAP) method was
used. The method is a modification of AFLP technique taking advantage of enzymes sensitive to DNA methylation:
MspI and HpaII. Moreover, the influence of 5-azacitidine (5AzaC) on embryogenic capacity of the culture was evaluated. 5-Aza-C is an analogue of cytosine nucleoside and thus
changes the level of methylated cytosine in DNA.
Embryogenic cultures of Arabidopsis were derived
from immature zygotic embryos of Col-0, the ecotype possessing a high capacity for somatic embryogenesis (SE).
The standard conditions for induction of embryogenic culture in Arabidopsis were used (Gaj, 2001) and in addition
to the basal induction medium (E5), media supplemented
with 5-Aza-C (8 μM and 10 μM) were used. A significant
reduction in SE efficiency (64.7%) and productivity
(61.5%) was observed on E5+8 μM 5-AzaC medium while
10 μM of 5-AzaC resulted in a total inhibition of the
embryogenic response. M-SAP-based experiments
Vol. 51, suppl. 1, 2009
involved 26 primer combinations analyzed at various time
points (0, 5, 15 and 30th day) of embryogenic cultures
induced on E5 and E5+8μM 5-AzaC media. The results
confirmed that 5-AzaC influences the level of DNA
demethylation as 142 new short DNA fragments in 5-Aza-C
treated tissue were observed. This indicated that the
observed negative impact of 5-AzaC on embryogenic
capacity of cultured tissue resulted from a reduced level of
DNA methylation. Moreover, DNA methylation patterns at
various time points of SE culture were compared to identify the loci differentially methylated during the time
course of SE. In total 1108 loci were analyses but none of
them were found to be polymorphic in the standard
embryogenic culture. The analysis of culture induced on
E5+5-AzaC medium revealed one polymorphic DNA fragment which indicated a demethylation event on 5th day of
culture.
REFERENCES
GAJ MD. 2001. Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis
thaliana (L.) Heynh. Plant Cell, Tissue and Organ Culture
64: 39–46.
65
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Identification of culturable and non-culturable endophytic fungi isolated from
shoots of Huperzia selago (L.) Bernh. ex Schrank & Mart. (Lycopodium selago L.)
Wojciech Szypuła1, Julia Budziszewska2, Daria Delbani1
1
Department of Biology and Pharmaceutical Botany, The Medical University of Warsaw, Banacha 1, 02-097 Warsaw,
Poland, e-mail: [email protected]
2
Department of Systematics and Plant Geography, University of Warsaw, Al. Ujazdowskie 4, 00-478 Warsaw, Poland,
e-mail: [email protected]
Gametophytes as well as sporophytes of H. selago are colonized by endophytic fungi (Higgins et al., 2007; Winther
and Friedman 2008). The class Dothideomycetes is the
main group of fungi mentioned among the endophytes of
club-moss sporophytes (Higgins et al., 2007). Some representatives of this group are known as dark septate endophytes -DSE (Schmid and Oberwinkler, 1993).
In recent years, the pharmaceutical industry has been
becoming increasingly interested in huperzine alkaloids
found in some club-mosses (genus Huperzia). Excessive
harvesting of sporophytes as a source of huperzine has
resulted in a marked decline of these plants in some countries. Studies are now being conducted that focus on the
use of in vitro cultures for propagation of plant material
for pharmaceutical industry. In vitro cultures of club
mosses may also contribute to ex situ protection of these
plants. However fungal contamination is a serious problem that causes severe loss of in vitro grown cultures of a
number of plants. This problem becomes even more acute
if the fungal contamination is of endophytic origin. In such
cases, identification and characterization of the contaminants is essential for achieving specific control of the contaminants through selective use of antibiotic agents, especially if the routinely used contamination control methods
practiced elsewhere in tissue culture studies are ineffective (Kulkarni et al., 2007). Such is the case with the fungal contamination observed in the present study.
The aim of the present study was to detect the presence of endophytic fungi in in vitro grown shoots of
H. selago originating from different European sites
(Szypuła et al., 2005, 2006). From 420 tissue fragments,
66
132 isolates representing 13 species of endophytic fungi
were recovered in culture. The isolated endophytes
belonged to the Zygomycota (1 species) and Ascomycota
(12 species). The representatives of the Ascomycota phylum belonged to 3 classes: Dothideomycetes,
Soradriomycetes and Eurotiomycetes. The most abundant
taxon were members of Sordariomycetes. Representatives
of Dothideomycetes were detected most often in shoots
originating from mountain sites.
REFERENCES
HIGGINS LK, ARNOLD AE, MIADLIKOWSKA J, SARVATE SD, LUTZONI F.
2007. Phylogenetic relationships, host affinity, and geographic structure of boreal and arctic endophytes from three
major plant lineages. Molecular Phylogenetics and
Evolution 42: 543–555.
KULKARNI AA, KELKAR SM, WATVE MG, KRISHNAMURTHY KV. 2007.
Characterization and control of endophytic bacterial contaminants in in vitro cultures of Piper spp., Taxus baccata
subsp. wallichiana, and Withania somnifera. Canadian
Journal of Microbiology 53(1): 63–74
SCHMID E, OBERWINKLER F. 1993. Mycorrhiza – like interaction
between the achlorophyllus gametophyte of Lycopodium
clavatum L. and its fungal endophyte studied by light and
electron microscopy. New Phytologist 124: 69–81.
SZYPUŁA W, PIETROSIUK A, SUCHOCKI P, OLSZOWSKA O, FURMANOWA M,
KAZIMIERSKA O. 2005. Somatic embryogenesis and in vitro
culture of Huperzia selago shoots as a potential source of
huperzine A. Plant Science 168: 1443–1452.
SZYPUŁA W. OLSZOWSKA O, FURMANOWA M. 2006. In vitro culture of
Lycopodiaceae (club mosses). Botanical Guidebooks No.
29: 163 – 175.
WINTHER JL, FRIEDMAN WE. 2008. Arbuscular mycorrhizal associations in Lycopodiaceae. New Phytologist 177(3): 790–801.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Plant regeneration from liquid root culture of Rumex acetosa L.
Analysis of genetic variability: preliminary studies
Halina Ślesak, Grzegorz Góralski, Patryk Mizia, Dagmara Kwolek, Andrzej Joachimiak
Department of Plant Cytology and Embryology, Jagiellonian University, Grodzka 52, 31-044 Cracow, Poland,
e-mail: [email protected]
Rumex acetosa L. is one of the few dioecious species that
have sex chromosomes (2n = XX + 12 in females, 2n =
XY1Y2 + 12 in males). In vitro culture of R. acetosa cv.
Lionski adventitious roots was initiated and maintained on
a hormone – free, liquid MS medium, containing 1/2
strength macronutrients (Mosiołek et al., 2005). This long –
term root culture was genetically stable. Recently, we were
successful in obtaining a method of high efficiency plant
regeneration (via indirect organogenesis) in vitro from roots
of Rumex acetosa. Root fragments (approx. 5 mm.) derived
from liquid male root culture (RAY1 and RAY2 lines) were
placed on MS medium (solidified with agar) supplemented
with growth regulators: 2,4-D, NAA, kinetin, BAP and TDZ
at different concentrations and combinations. The highest
frequency of callus induction was found on MS + 0.5 mg/l
TDZ medium after 5–6 weeks of culture. Callus culture
maintained on MS medium with 0.5 mg/l TDZ resulted in a
high frequency (90%) of shoot formation after 6 weeks.
Regenerated shoots were isolated and inoculated onto a
rooting medium (1/2 MS + 2% sucrose + 0.5 mg/l IBA) that
had been proved to be optimal for root induction among the
tested media. Complete plants with well developed root systems (after 2 weeks of culture on the rooting medium) were
transferred to soil and acclimated to in vivo conditions in a
phytotron chamber and then in the field. In vitro culture
method of Rumex acetosa with a high frequency of plant
regeneration was used for a preliminary analysis of genetic
variation. Genomic DNA was extracted (using CTAB buffer)
from plant material representing each of the following culture steps: roots from liquid culture, callus, plants regenerated in vitro from callus and plants acclimated in vivo, and
analyzed using RAPD markers.
REFERENCES
MOSIOŁEK M, PASIERBEK P, MALARZ J, MOŚ M, JOACHIMIAK A. 2005.
Rumex acetosa Y chromosomes: constitutive or facultative
heterochromatin? Folia Histochemica et Cytobiologica 43:
161–167.
Cytogenetic characteristics of the interspecific somatic hybrids of Solanum
villosum (+) S. tuberosum
Justyna Tarwacka, Anna Szczerbakowa, Bernard Wielgat
Institute of Biochemistry and Biophysics PAS, 5A Pawińskiego St, 02-106 Warsaw, Poland
Solanum villosum, a wild tetraploid (2n=2x=48) nontuber bearing species resistant to Phytophthora infestans
could be a potential source of resistance genes for cultivated potato. Since S. villosum is genetically and therefore
reproductively isolated from S. tuberosum, the fusion of
leaf mesophyll protoplasts was used to produce interspecific somatic hybrids between S. villosum and the diploid
S. tuberosum. The objective was to transfer the late blight
resistance genes from S. villosum into the susceptible
potato. The expected ploidy level of the hybrids was 6x,
but the real ploidy of the regenerants varied from 4x+ to
Vol. 51, suppl. 1, 2009
6x. The ploidy level was estimated by the direct method of
counting chromosomes in metaphase plates of the root tip
meristem cells. Analyses showed that the most vigorously
growing hybrid plants were heksaploid, although all
hybrids were quite similar morphologically and resembled the dominating wild tetraploid species. Selected
euploid hybrids will be assessed for their resistance to
P. infestans.
Acknowledgement: The research was supported by the Polish
Ministry of Science and Higher Education as project PBZ-MNiSW2/3/2006/33.
67
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Bioactive secondary metabolites in in vitro cultures of Eryngium alpinum L.
Barbara Thiem, Małgorzata Kikowska, Izabela Paluch
Department of Pharmaceutical Botany and Plant Biotechnology, Poznań University of Medical Sciences,
Św. Marii Magdaleny 14, 61-861 Poznań, Poland, e-mail: [email protected]
Eryngium alpinum L. (Apiaceae), an endangered subalpine perennial is easily recognizable by its blue amethyst
coloured flowers heads. This taxon is protected all over
Europe and cultivated mainly for decorative purposes.
Rosmarinic acid with antioxidant activity has been detected in this species (Le Claire et al., 2005). E. alpinum in
natural populations produces very few seeds that become
dormant soon after harvest, so their germination is poor
(Gaudeul and Till-Bottraud, 2004).
Plant material regenerated from in vitro cultures offers
an opportunity to carry out phytochemical investigations
of rare or vulnerable plants without collecting them from
natural sites. Moreover, in vitro techniques could be used
to complement ex situ conservation of endangered
species.
The aim of our study was to establish in vitro cultures
of E. alpinum and to conduct a preliminary phytochemical study. Seedlings obtained from seeds that had been
isolated from fruits after their stratification and scarification, were used for initiation of in vitro cultures. Shoot
culture was developed via the induction of axillary buds
from shoot tips and established on MS medium supplemented with BAP (1.0 mgl-1) and IAA (0.1 mgl-1). The
shoots were rooted on MS medium containing IAA, IBA or
NAA (0.1 mgl-1). Root cultures were obtained from root
tips placed on MS liquid medium enriched with auxins
(0.5 mgl-1) and were grown in 300 ml flasks on a rotary
shaker, in darkness.
To detect the presence of selected secondary metabolites, ethanol extracts from shoot and root cultures were
analyzed chromatographically (TLC) on cellulose and silica gel F254 in different mobile phases. Spray specific
reagents were employed for detection of phenolics,
flavonoids and saponins. We showed that both types of in
vitro material were able to produce those bioactive compounds.
The study was also undertaken to develop some in
vitro techniques for conservation of E. alpinum.
Acknowledgements: This work was supported by the Ministry of
Science and Higher Education, Warsaw, Poland (Grant NN 405
065334)
REFERENCES
GAUDEUL M, TILL-BOTTRAUD I. 2004. Reproductive ecology of the
endangered alpine species Eryngium alpinum L. (Apiaceae):
phenology, gene dispersal and reproductive success. Annals
of Botany 93: 711–721.
LE CLAIRE E et al. 2005. Distribution of a new rosmarinic acid
derivative in Eryngium alpinum L. and other Apiaceae.
Journal of Agricultural and Food Chemistry 53:
4367–4372.
The regeneration of sugar beet (Beta vulgaris L.) plants from unfertilized ovules
cultured in vitro
Magdalena Tomaszewska-Sowa
Department of Plant Physiology, University of Technology and Life Sciences, Bernardyńska 6, 85-029 Bydgoszcz,
Poland, e-mail: [email protected]
The aim of the experiment was to improve the effectiveness
of regeneration of sugar beet (Beta vulgaris L.) plants from
unfertilized ovules cultured in vitro. A two-step method was
applied: (1) culture in liquid medium that initiated meristem differentiation, followed by (2) transfer of explants onto
Murashige and Skoog media solidified with agar or gelrite,
which stimulated shoot development and growth.
During the experiments 30 media were tested in terms
of their influence on shoot formation. The media differed
in the composition and content of growth regulators, carbohydrates and gelling compounds. The best efficiency of
organogenesis was observed when 4.4 μM BAP, 0.09 M
glucose and 0.7% agar were used. Root formation was
induced after addition of 14.8 μM IBA and 0.049 μM 2iP,
and the rooting rate ranged between 0.0 and 65.2%
68
depending on the experimental treatment. After planting
into pots and several weeks of acclimation in the greenhouse, the plants were transferred to the field, where their
morphological characteristics were observed.
The ploidy level analysis of 413 regenerants performed
by flow cytometry showed that 32.7% of plants were haploids, 45.8% diploids and 21.5% mixoploids. Haploid
plants had narrower and more extended leaves, longer
petioles and smaller roots in comparison to diploids. It
also turned out that in some plants the chromosome number doubled spontaneously.
Detailed DNA study with the RAPD method revealed
large differences among the regenerants. There were 13
polymorphic sequences identified in total, and 11 of them
typified only the diploids' DNA.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
The effect of cytokinins on morphogenetic responses of Polemonium caeruleum
seedling explants
Alina Trejgell, Andrzej Tretyn
Department of Biotechnology, Institute of General and Molecular Biology, Nicolaus Copernicus University, Gagarina 9,
87-100 Toruń, Poland, e-mail: [email protected]
The aim of this study was to estimate the morphogenetic
response of Polemonium caeruleum explants. The donor
material were 10-day-old seedlings. Surface sterilized
seeds were germinated on MS medium (Murashige and
Skoog, 1962) supplemented with GA3 (1 mg·dm-3).
Seedling explants (shoot tips, fragments of cotyledons,
hipocotyls and roots) were isolated and transferred onto
solidified MS medium supplemented with different types
of cytokinins (BA, Kn, Zea, 2iP) at concentrations of 1.0,
3.0 and 5.0 mg·dm-3 in combination with NAA (0.1
mg·dm-3).
All explant types produced callus proliferation. It was
observed that calli developed on the entire surface of
hipocotyl and root fragments. On the other hand, shoot
tips and cotyledonary petioles formed callus tissue at the
cut ends, and petioles only at abaxial ends. The growth of
calli on all explant types was strongly stimulated by Zea.
Among the explants tested, only shoot tips exhibited shoot
organogenesis. The highest frequency of shoot organogenesis was observed if the explants were cultured on medium supplemented with 5.0 mg·dm-3 BA (100%) or
5.0 mg·dm-3 Zea (97%). The highest shoot number per
explant (8.4 on average) was obtained in the presence of
5 mg·dm-3 Zea. The presence of BA or Zea in the proliferation medium inhibited rhizogenesis and the elongation
growth of shoots. However, root organogenesis was supported by exposure to Kn.
REFERENCE
MURASHIGE T, SKOOG F. 1962. A revised medium for rapid growth
and bioassays with tobacco tissue cultures. Physiologia
Plantarum 15: 437–497.
Capacity for shoot organogenesis in cultures of Arabidopsis hormone-response
mutants
Aneta Trojanowska, Małgorzata D. Gaj
Department of Genetics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland,
e-mail: [email protected]
Plant hormones are considered to be the key factors
involved in plant morphogenesis in vitro. Mutants featuring altered hormonal responses are useful in studies of
molecular mechanisms involved in in vitro induction of
plant morphogenesis.
In Arabidopsis, adventitious shoots can be efficiently
regenerated via indirect organogenesis induced in the culture of root explants. The objective of this work was to
evaluate the shoot organogenic (ORG) capacity of root
explant cultures of 32 mutants defective in responses to
plant hormones. The studied genotypes included auxin
(7), ABA (12), cytokinin (2) and gibberellin (11) mutants
and their parental genotypes (Columbia, Landsberg erecta, Wassilewskija). Explants isolated from 14-day old sterile plants were preincubated on an auxin-rich liquid callus
induction medium (CIM), and then transferred to a
cytokinin-rich shoot solid induction medium (SIM)
according to Feldman and Marks (1986) method. The
capacity for ORG was evaluated in 4-week old cultures
and the ORG productivity (average number of shoots produced per 1 cm of explant) was evaluated.
Vol. 51, suppl. 1, 2009
The majority of the analyzed mutants (84%) displayed
ORG productivity at the level similar to their parental
genotypes. However, 5 of the analyzed mutants displayed
distinct defects in the ORG response. Three of these
mutants: aba1-3, abi4-1 (ABA) and axr4-1 (auxin)
showed a significant decrease in organogenic response,
while in two other mutants: hyl1 (ABA) and pkl1 (gibberelin) shoot regeneration was completely inhibited.
Except for the abi4-1, all mutants impaired in ORG
(aba1-3, hyl1, axr4-1 and pkl1) were previously reported
to display significant defects in their capacity for somatic
embryogenesis (Gaj et al., 2006) implicating the involvement of the mutated genes in basic cellular processes governing plant cell growth and differentiation.
REFERENCES
FELDMAN KA, MARKS MD. 1986. Rapid and efficient regeneration of
plants from explants of Arabidopsis thaliana. Plant Science
47: 63–69.
GAJ MD, TROJANOWSKA A, UJCZAK A, MĘDREK M, KOZIOł A,
G ARBACIAK B. 2006. Hormone-response mutants of
Arabidopsis thaliana (L.) Heynh. impaired in somatic
embryogenesis. Plant Growth Regulation 49: 183–197.
69
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Evaluation of regeneration efficiency of selected double haploid lines of rape
(Brassica napus ssp. oleifera)
Anna Turczynowska, Zbigniew Broda
Department of Genetics and Plant Breeding, Poznań University of Life Sciences, Wojska Polskiego 71c, 60-625 Poznań,
e-mail:[email protected]
Regeneration, determined by the genotype and environment, is the most important process leading to production
of plants under in vitro conditions. Genus Brassica
belongs to the family Brassicaceae whose representative
species regenerate in vitro with a high efficiency
(Wojciechowski, 1998).
The aim of this research was to characterize the regeneration efficiency of 14 doubled haploid lines of rape
(Brassica napus ssp. oleifera) that were expected to show
a range of different tissue response in vitro.
Plant material consisted of cotyledon and hypocotyl
explants gathered from 6-day old seedlings. Explants
were placed on basic MS medium (Murashige and Skoog
1962) with the addition of 1 mg/l or 10 mg/l BAP.
Evaluation of regeneration efficiency was conducted after
28 days of culture and was based on the number of
explants regenerating shoots in relation to the total number of explants used. The examined genotypes varied in
their regeneration ability.
REFERENCE
WOJCIECHOWSKI A. 1998. Zdolności regeneracyjne wybranych genotypów Brassica w kulturach in vitro. Roczniki Akademii
Rolniczej w Poznaniu, Rozprawy naukowe, Zeszyt 289.
Efficient shoot organogenesis induced in the culture of immature zygotic
embryos of Arabidopsis
Malgorzata Wajand, Agnieszka Ledwon, Malgorzata D. Gaj
Department of Genetics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland,
e-mail: [email protected]
To study the genetic determination of somatic embryogenesis induced in in vitro cultures, the model plant
Arabidopsis thaliana is widely used because of its
advanced genomics and the availability of numerous
mutants. In the model system for SE analysis, immature
zygotic embryos (IZEs) of Arabidopsis provide an efficient
explant source (Gaj, 2001). The aim of this study was to
establish in vitro conditions for IZE culture that would
promote adventitious shoot organogenesis as an alternative to the SE mode of plant regeneration. The IZE
explants of commonly used genotypes, Columbia (Col-0)
and Wassilewskija (Ws), were analysed and four methods
differing in media sequence and composition (ZOR1 –
ZOR4) were tested. Efficient shoot regeneration was
observed when cotyledons of IZEs were cultured on MS
medium supplemented with B5 vitamins and plant growth
regulators: 1 mg/l BAP and 0.1 mg/l NAA (ZOR3); however
the method was found to be genotype-specific. Explants of
Col-0 cultured with the use of ZOR3 media developed
shoots at 65% rate while the protocol was much less efficient for Ws culture (15% of responding explants). An
improvement of Ws explant response was achieved when
70
7 day pre-culture of the explants in CIM (0.5 mg/l 2,4-D;
0.1 mg/l kinetin) medium was employed (ZOR4).
Additionally, ZOR4 media resulted in an increase of average culture productivity and more rapid shoot formation.
The regenerants developed into fertile plants upon culture
on hormone free medium.
Moreover, the expression of embryogenesis-specific
LEC genes (LEC1, LEC2 and FUS3) was monitored with
the use of RT-PCR. The analysis was performed at various
time points of the culture induced by the ZOR4 method.
In contrast to IZE-derived embryogenic culture, in which
activity of the LEC genes was detected up to 30d of culture
(Ledwoń, 2008), in IZE culture induced for shoot ORG,
the expression of LEC1 and FUS3 genes disappeared
gradually and vanished by 7 and 21d, respectively.
REFERENCES
GAJ MD. 2001. Direct somatic embryogenesis as a rapid and efficient system for in vitro regeneration of Arabidopsis
thaliana. Plant Cell, Tissue and Organ Culture 64: 39–46.
LEDWON A. 2008. Analiza ekspresji genów LEAFY COTYLEDON
podczas somatycznej embriogenezy u Arabidopsis thaliana.
PhD Thesis, University of Silesia, Katowice, 55–104.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
In vitro culture of Cordyline australis (G. Forst) Endl.
Marzena Warchoł1, Franciszek Dubert2, Tadeusz Kusibab3
1
Department of Ornamental Plants, Agricultural University of Cracow, 29 Listopada 54, 31-425 Cracow, Poland,
e-mail: [email protected]
2
Institute of Plant Physiology, Polish Academy of Sciences, Niezapominajek 21, 30-239 Cracow, Poland
3
Gospodarstwo Ogrodnicze Tadeusz Kusibab, Zbyszka z Bogdańca 16, 31-979 Cracow, Poland
Cordyline australis "Red Star" with it's beautiful and
attractive red decorative foliage is one of the most important ornamental house plant. Since the growth of plants in
genus Cordyline is very slow, the interest in using in vitro
propagation is increasing (DeMason and Wilson, 1985).
Shoot apices of about 5 cm length used for this study
were excised from plants growing under greenhouse conditions. After removing the apical sheathing leaves,
explants were washed thoroughly and surface sterilized
for 20 min with 20% Domestos, followed by three rinses
in sterile distilled water each for 5 min and a second sterilization with 2% Domestos for 5 min. Explants (about 1
cm long) were isolated and transferred to culture vessels.
Full strength MS (1962) medium containing 3% sucrose
and 0.7% agar was used as the basal medium.
Two different cytokinins: BA (5 μM i 17 μM) and zeatin
(5 μM) were tested for shoot bud induction in combination
with 0.5 μM NAA. For the development of shoot buds from
the shoot apex, positive responses were observed only in
the culture media that were supplemented with the lower
concentration (5 μM) of BA. Shoot buds began to appear
after 12 weeks in culture and the highest frequency of
explants forming shoots was 55%. The effect of BA was
quite inhibitory when used at the higher concentration
(17 μM). Small clusters of shoots (10–15 shoots in each
cluster) were subcultured to the proliferation medium
containing four different cytokinins: BA, Zeatin, metaTopolin and 2iP. Maximum increase in shoot number was
15.80 after four weeks in culture. It occurred in media
containing a combination of 5 μM zeatin and 0.5 μM NAA.
Tiny shoots, when transferred to a fresh medium continued to produce adventitious shoots.
Individual shoots were cultured separately in the rooting medium enriched with IAA or IBA and containing
0.01% activated charcoal. Most of the plants showed normal and healthy growth after they were transferred to
pots.
REFERENCE
DEMASON DA, WILSON MA. 1985. The continuity of primary and
secondary growth in Cordyline terminalis (Agavaceae).
Canadian Journal of Botany 63: 1907–1913.
In vitro culture of Dracocephalum moldavica
Izabela Weremczuk-Jeżyna, Halina Wysokińska
Department of Biology and Pharmaceutical Botany, Medical University of Łódź, Muszyńskiego 1, 90-151 Łódź, Poland
e-mail: izabela.weremczuk-jezyna.umed.lodz.pl
Dracocephalum moldavica L. (Lamiaceae) is an annual
plant native to Central Asia and naturalized in Central and
Western Europe. D. moldavica has been reported to be
used as a food ingredient as well as tea and a herbal drug
for the treatment of stomach, kidney and liver disorder in
folk medicine. The spectrum of D. moldavica effects is
ascribable principally to the presence of an essential oil,
phenolic acids, flavonoids and tannins.
In the investigation reported here, we attempt to
induce callus and suspension cultures. Callus cultures of
D. moldavica were initiated from hypocotyls and roots of
4-week-old seedlings. The explants were incubated on
Vol. 51, suppl. 1, 2009
Murashige and Skoog agar (0.7%) medium with 2,4-D or
NAA at various concentrations (0.1, 0.5 or 1.0 mg/l). The
most abundant callus proliferation was observed on root
explants, after a 3-week culture on MS medium with 2,4-D
at 0.5 mg/l. In addition to callus proliferation, somatic
embryo formation was also observed. Suspension cultures were initiated by transfer of the callus tissues into
MS liquid medium supplemented with 0.1 or 0.5 mg/l
2,4-D and 0.1 mg/l BAP. Suspension cultures with high
biomass production were established on medium with
2,4-D at 0.5 mg/l. Cell suspension growth curves were
obtained.
71
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
Influence of growth regulators and environmental factors on shoot multiplication
of Camellia japonica in vitro
Agnieszka Wojtania, Eleonora Gabryszewska
Research Institute of Pomology and Floriculture, Waryńskiego 14, 96-100 Skierniewice,
e-mail: [email protected]
Camellia japonica is known to be relatively recalcitrant to
tissue culture. Strong apical dominance, shoot-tip necrosis, leaf browning and difficulties with rooting are the
main problems, on which this study was focused.
Shoot cultures were initiated from axillary buds and
shoot tips collected from a juvenile plant of Camellia
japonica. MS medium supplemented with 0.1 mg l-1 BAP
i 0.5 mg l-1 GA3 was used. The aim of this investigations
was to determine the influence of growth regulators
(MemT, TDZ, GA3) and environmental factors – temperature (15°C, 20°C) and sucrose (5, 10, 20, 30 g l-1) on the
growth and development of Camellia japonica shoots.
The experiments were carried out with cooled (15°C) and
non-cooled shoots (20°C). The highest multiplication rate
and high quality of shoots were noted on the WPM supplemented with MemT (0.5 mg l-1), TDZ (0.01 mg l-1), GA3
(1.0 mg l-1) and sucrose at concentrations of 10–20 g l-1.
The higher sucrose concentration stimulated elongation
and strong lignification of shoots. Low sucrose concentration, as well as the 15°C temperature regime decreased
growth of Camellia shoots. The addition of GA3 to the
medium together with cytokinins and 10–20 g-1 sucrose
activated axillary shoot development and leaf formation of
both cooled and non-cooled shoots.
Pectin metabolism in Fusarium-infected flax seedlings
Wioleta Wojtasik, Kamil Kostyn, Anna Kulma, Jan Szopa
Department of Genetic Biochemistry, Faculty of Biotechnology, University of Wrocław, Przybyszewskiego 63/77,
51-148 Wrocław, Poland, e-mail: [email protected]
Flax (Linum usitatissimum L.) is a common crop,
highly valued as a source of fibre, oil and linseed. The
greatest losses in flax crops are caused by fungal diseases. Fusarium culmorum and Fusarium oxysporum
are fungal pathogens that cause the most severe and
imminent infections of flax. Two subtractive cDNA
libraries were constructed from flax seedlings treated
with salicylic acid or infected by F. oxysporum. Further
analyses revealed two sets of gene sequences, which are
potentially involved in flax defence responses. In order to
ascertain the level of expression of these genes, 9-day old
flax seedlings infected by Fusarium culmorum and
Fusarium oxysporum were subjected to semi-quantitative
reverse transcription PCR analysis. Metabolite profiling
was performed by means of GS-MS and UPLC analysis
and revealed major changes in several metabolite groups
such as phenolic acids, amino acids and sugars. Among
genes found in the subtractive libraries, two (UDP-D-glu-
72
curonate 4-epimerase and formate dehydrogenase) are of
particular interest to us, as they are connected with cell
wall sugar polymers in plant. UDP-D-glucuronate
4-epimerase delivers the main substrate for pectin polysaccharide biosynthesis. We have determined that the
expression level of UDP-D-glucuronate 4-epimerase gene
was decreased after the Fusarium infection. However, the
content of pectins remained unchanged. Formate dehydrogenase participates in the metabolism of formic acid,
a toxic side-product of pectin demethylation. The expression level of formate dehydrogenase gene increased after
the Fusarium treatment, which can be due to formate
release from pectins under the influence of fungi. This
observation is supported by research on transgenic plants
overexpressing pectin degrading enzymes, where the level
of formic acid is elevated. Our research point to early
changes in flax plants, especially in the cell wall, occurring
after the Fusarium treatment.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
~
Regeneration of shoots from root explants in Mammillaria carmenae Castaneda
(Cactaceae)
Tomasz P. Wyka, Katarzyna Ludwiczak
Department of General Botany, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań,
Poland, e-mail: [email protected]
Micropropagation of cacti from adult tissues has been difficult because of the low organogenic potential and heavy
microbial contamination of potential explants, such as
areoles or stem segments. Another problem is the damage
to the stock specimen associated with collecting explants
from these stem-succulent plants. Two alternative sources
of explants are flowers and roots. Whereas a complete
cycle of micropropagation from flowers has been demonstrated for three Mammillaria species (Wyka et al., 2006,
2009), the only report of micropropagation from roots
concerns root explants harvested from in vitro culture of
Coryphantha elephantidens (Bhau 1999).
We attempted to use root tips of pot-grown M. carmenae rinsed with 70% ethanol followed by disinfection in
NaOCl (0.05–0.1%), CaOCl (2–5%), or HgCl2 (0.1%). In no
case was there a viable root culture obtained because of
persistent heavy contamination and the resulting mortality of explants. Irrigation of the plants with fungicide did
not improve the outcome.
In contrast, when 10 mm long root tips were harvested
from existing long-term perianth-derived shoot cultures of
M. carmenae and transferred to MS medium containing
30 g l-1 sucrose, 1 mg l-1 kinetine and 0.5 or 2.0 mg l-1
2,4-D, they became hypertrophic and produced white calli
that soon greened up. When calli were subdivided and
transferred to regeneration medium (MS containing 30 g l-1
sucrose, 0.1 mg l-1 NAA and 5 or 10 mg l-1 BAP or kinetin),
within 12 weeks shoot organogensis was recorded. At 24
weeks after transfer, the greatest efficiency of shoot formation (1.7 shoots per callus) was on medium containing
0.1 mg l-1 NAA and 5.0 mg l-1 BAP. Root-derived shoots
were then used to re-establish a proliferating shoot culture. New shoots were rooted in perlite saturated with MS
medium containing 0.1 mg l-1 NAA and later acclimated to
ex vitro conditions.
Our results confirm the earlier finding by Bhau (1999)
that roots of Cactaceae are a viable source of explants and
point to the need to develop novel disinfection methods.
REFERENCES
BHAU BS. 1999. regeneration of Coryphantha elephantides (Lem.)
Lem. (Cactaceae) from root explants. Scientia Horticulturae
81: 337–344
WYKA TP, HAMERSKA M, WRÓBLEWSKA M. 2006. Organogenesis of
vegetative shoots from in vitro cultured flower buds of
Mammillaria albicoma (Cactaceae). Plant Cell, Tissue and
Organ Culture 87: 27–32
WYKA TP, WRÓBLEWSKA M, HAMERSKA M. 2009. Use of cactus flowers and explants for micropropagation. The Journal of
Horticultural Science and Technology, in press.
Intergeneric hybridization between Salix fragilis and Populus spp.
in vivo and in vitro
Łukasz Zarychta, Maciej Zenkteler, Aneta Karmowska, Elżbieta Zenkteler
Department of General Botany, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań,
Poland, e-mail: [email protected]
As a result of experimental pollinations in vivo and in
vitro between species of Salix and Populus, embryos and
plantlets were obtained from crosses of Salix fragilis ×
Populus tremula and Salix fragilis × Populus simonii.
Fully developed stigmas were pollinated with pollen grains
of six poplar species: Populus tremula, Populus simonii,
Populus alba, Populus trichocarpa, Populus violascens
and Populus nigra. Under in vivo conditions, pollen
grains were placed on stigmas using pencil tip. For pollinations in vitro, basal catkin segments were disinfected
for 10 s in 70% ethanol, 4–5 min in chlorine water and
washed three times in sterile water. After pollination,
catkins were placed on basal MS medium. At 24 hours
after pollination pollen germination occurred and 3 DAP
Vol. 51, suppl. 1, 2009
pollen tube growth as well as style and ovule penetration
were observed. Globular embryos were formed 5–6 days
after pollination. After 18–24 days fully developed
embryos were transferred aseptically to MS-based medium modified by the addition of l mg l-1 KIN. Three week
old plantlets were transferred on MS-based medium supplemented with 0.2 mg l-1 NAA. Both media contained 3%
sucrose and were solidified with 8 mg l-1 agar and their pH
had been adjusted to 5.8 before autoclaving. Plantlets that
possessed 3–4 leaves were transferred into pots containing a mixture of sand and peatmoss. Fully developed
embryos (18–21 days after in vivo pollination) died after
2–4 weeks from transfer to MS medium.
73
POSTERS
12th National Conference • ‘In vitro Cultures, Poznań 2009’
The influence of iron source in red raspberry cultures on the chlorophyll
contents and histology of the leaves
Marta Zawadzka, Teresa Orlikowska
Research Institute of Pomology and Floriculture, Pomologiczna 18, 96-100 Skierniewice,
e-mail: [email protected]
Iron is an essential nutrient for plants and is involved in
fundamental metabolic processes: photosynthesis and
respiration, growth regulator synthesis and many enzymatic reactions. Interveinal chlorosis of leaves is the most
visible symptom of iron deficiency. Such chlorosis was
observed in raspberry shoot cultures when a typical MS
formula containing FeEDTA was used. The most chlorotic cultivars were also the most recalcitrant to adventitious
regeneration. Supplementing the medium with FeEDDHA
(i.e. a more photostable iron chelate) resulted in the elimination of chlorosis, an increase in the rate of shoot multiplication and rooting and an increase in the efficiency of
adventitious leaf regeneration.
We compared the effects of FeEDDHA and FeEDTA on
the chlorophyll contents and leaf histology of five raspberry cultivars: Beskid, Canby, Malling Seedling, Norna and
Veten. Leaf samples were obtained after 4 weeks on shoot
multiplication medium, after 4 weeks on rooting medium,
and after 2 and 4 weeks of acclimation in the greenhouse.
The chlorophyll content was measured spectrophotometrically. For histological observations leaf sections of 10μm were stained with safranin and fast green.
FeEDDHA significantly increased the chlorophyll
content at each stage of propagation (shoot multiplication, rooting and microplant acclimatization) in all cultivars, but the degree of increase was genotype dependent.
Differences in leaf histology depended on the source of
iron in the medium, and the stage of propagation. In
leaves produced on the medium containing FeEDDHA,
the parenchyma and palisade cell layers were thicker
and the cells more abundant and condensed at the multiplication stage, whereas on the medium containing
FeEDTA, such leaf structure was not observed until the
rooting stage.
In vitro reproduction of Drosera rotundifolia and conservation plant genetic
resources of this species
Przemyslaw Zelazko, Krystyna Kromer
Botanical Garden, University of Wrocław, Plant Tissue Culture Laboratory, Sienkiewicza 23, 50-335 Wrocław, Poland,
e-mail: [email protected], [email protected]
All carnivorous plants that can be found in Poland are
under legal protection. Recently, the number of their natural stands has been dramatically decreasing. The principal causes of their reduction include draining, exploiting
and eutrophication of peat bogs. All possible measures to
prevent populations of these plants from further decline
should be taken.
The objective of our study was to develop a reproduction method of Drosera rotundifolia using in vitro cultures. Results of our experiments indicate the possibility
of effective generative reproduction of Drosera rotundifolia in vitro, involving the production of small amounts of
vital seeds. Our data confirmed earlier reports that seeds
of this species require a stratification period for plants to
develop normally. Moreover, it has been confirmed that
100% of plants survive in temperature 0–2°C, however,
their growth is inhibited and also formation of winter
buds can be observed. After transfer to a fresh medium
and moving from refrigerator to a phytotron with temperature of 25°C, the plants resume growth. Storing Drosera
74
rotundifolia at a reduced temperature is useful for preservation of their germplasm, maintenance of long-term cultures and development of a gene bank of this species in
vitro. Examination of fertilization in Drosera rotundifolia
confirmed other reports of self-fertilization in this species.
The contents of phenolic compounds in water collected from natural stands of Drosera rotundifolia did not
show any relationship with the type of stand for a given
population. Moreover, the use of liquid medium indicated
that Drosera rotundifolia does not tolerate excessive
amount of water, as the plants became curled and reddening of the plants could be observed, which is characteristic of mineral deficiency.
Chemical analysis of water taken from individual
stands of populations that are developing dynamically or
disappearing, did not show any significant differences in
the contents of mineral compounds. This indicates the
possibility that there could be other critical factors, not
connected with the contents of nutrients in the water at
the given stand.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica
POSTERS
September 9–11, 2009, Poznań, Poland
Propagation of Chamaedaphne calyculata (L.) Moench via indirect somatic
embryogenesis
Anna Źróbek-Sokolnik1, Czesław Hołdyński1, Kamilla Górska-Koplińska2
1
Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Plac Łódzki 1,
10-727 Olsztyn, Poland, e-mail: [email protected]
2
Biosensors in Food Department, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences,
Tuwima 10, 10-747 Olsztyn, Poland
Leather leaf Chamaedaphne calyculata (L.) Moench is a
wintergreen dwarf shrub belonging to family Ericaceae. In
Poland it is a rare species and is under full legal protection. Because of the limited number of existing stands of
this plant (presently 9 remaining of the 13 historically
known stands), it is important to determine the possibility of Chamaedaphne calyculata propagation for the purpose of ex situ protection.
Somatic embryogenesis may be used for propagation
of non-seed producing plants, sterile male or female
plants and plants that do not respond to conventional
propagation techniques. It can also be used for
germplasm preservation of valuable genotypes or critically endangered species. According to current literature,
there are only a few reports concerning somatic embryogenesis in the Ericaceae (eg. Vejsadova and Pretova,
2003; Antony et al., 2004). We tried to find if indirect
somatic embryogenesis could be used for an effective
propagation of Chamaedaphne calyculata. This study
may have significant implications in the management of
leather leaf in restoration and conservation programs.
Leaves removed from shoots derived from in vitro cultures were used as explants. Leaves were cut transversely
and placed with the abaxial surface in contact with the
medium.
Vol. 51, suppl. 1, 2009
Embryogenic callus production and initiation of
somatic embryos was achieved using Gamborg's B5
(Gamborg et al., 1968) medium (with pH adjusted to 5.6)
and supplemented with 3% sucrose, 0.7% agar, 10 μM
TDZ and 5 μM IAA. Somatic embryos were removed from
the parent tissue and transferred to Anderson's (1980)
medium containing 2.28 μM zeatin for elongation. Root
development occurred on hormone-free Anderson's
(1980) medium. After transfer to a mixture of sphagnumpeat and perlite (3:1), approximately 65% of plantlets survived.
REFERENCES
ANDERSON WC. 1980. Tissue culture propagation of red and black
raspberries, Rubus idaeus and R. occidentalis. Acta
Horticulturae 112: 13–20.
ANTHONY JM, SENARATNA T, DIXON KW, SIVASITHAMPARAM K. 2004.
Somatic embryogenesis for mass propagation of Ericaceae –
a case study with Leucopogon verticillatus. Plant Cell,
Tissue and Organ Culture 76: 137–146.
GAMBORG OL, MILLER RA, OJIMA K. 1968. Nutrient requirements of
suspension cultures of soybean root cells. Experimental Cell
Reserach 50: 11–158.
VEJSADOVA H, PRETOVA A. 2003. Somatic embryogenesis in
Rhododendron
catawbiense
Grandiflorum.
Acta
Horticulturae 616: 467–470.
75
Index of Authors
Adamus A 35, 44
Agacka M 25
Andrzejewska-Golec E 32
Arendt M 32
Bach A 33, 56
Balazadeh S 17
Baraniak M 48
Barański R 46
Bartkowiak-Broda I 16
Bednarek P 29
Bień K 49
Boba A 33, 47
Bogusiewicz M 21, 34
Bohdanowicz J 19
Bojarczuk K 42
Braś M 59
Broda Z 70
Buchwald W 48
Budzianowska A 34
Budzianowski J 34
Budziszewska J 24, 66
Bugajska-Prusak A 13
Bultrowicz M 35
Caldana C 17
Cegielska-Taras T 16, 64
Chmielarz P 42
Chuda A 35
Ciszewska-Marciniak J 36
Costa G 46
Czaplicki AZ 29, 36
Czarnecka D 37
Czemplik M 47
Czubacka A 16, 37
Czuj T 37
Czyczyło-Mysza I 53, 62
Czyż M 21
Delbani D 24, 66
Depta A 25
Doroszewska T 16, 37
Dreger M 48
Dubert F 56, 71
Durok J 38
Dydak M 61
Ekiert H 49, 50
Floryanowicz-Czekalska K 38
Furmanowa M 48
Gabryszewska E 39, 58, 72
Gaj MD 17, 39, 65, 69, 70
Galek R 32, 47
Gądek J 60
Gliwicka M 17, 39, 65
Gładysz M 41
Głowacka K 17
Gośka M 49
Góralski G 67
Górecka K 40
Górecki R 40
Górska-Koplińska K 75
Górska-Paukszta M 48
Grabka M 41
Grzebelus D 41
Grzebelus E 38, 41
Grzegorczyk I 42
Kozdój J 29
Krajewska-Patan A 48
Kromer K 19, 74
Królicka A 43
Kruczkowska H 48
Krzyżanowska D 40
Kucharska D 55
Kulma A 13, 33, 47, 65, 72
Kuras A 58
Kusibab T 71
Kuta E 19, 21
Kużdowicz K 49
Kwasniewska J 61
Kwiecień I 49, 50
Kwolek D 67
Hazubska-Przybył T 42
Hołdyński C 75
Horbowicz M 55
Hura K 33
Lachuta L 55
Langer J 28
Langer K 28
Ledwon A 70
Libik M 18
Ludwiczak K 73
Lütz C 22
Luwanska A 50, 52
Jagielska A 43
Janczur KL 18, 54
Janowicz J 26
Jarząbek M 22
Jasiński A 58
Jeżowski S 17
Jędryczka M 36
Joachimiak A 67
Jóźwiak W 22
Kachlicki P 18
Kamiński M 43
Kapczyńska A 14
Karmowska A 73
Kasprowicz A(leksandra) 43
Kasprowicz A(nna) 14
Kawa-Miszczak L 39
Kawiński A 44
Kiełkowska A 44
Kierzkowski D 14
Kikowska M 45, 68
Kisiała A 45
Kiszczak W 40
Klamkowski K 55
Klimek M 46
Klocek J 46
Kokotkiewicz A 22, 60
Konieczny R 18, 21
Korbin M 58
Kosicka I 14
Kostyn K 33, 47, 72
Kowalska U 40
Kozak K 47
Ładyżyński M 51
Łojewski M 59
Łojkowska E 43, 44
Łuczkiewicz M 22, 60
Maciejewska A 43
Makowczyńska J 32
Makowski D 51
Malarz J 63
Malepszy S 12
Malik M 52
Małuszynska J 61
Mankowska G 50, 52
Marcińska I 53, 59, 62
Maruniewicz M 14
Michalak M(arcin) 42
Michalak M(ichał) 14
Mielcarek S 48
Mikiciński A 28
Mikuła A 20, 51
Miler N 27
Mioduszewska H 46
Miszalski Z 18
Mizia P 67
Mól R 35
Mrozikiewicz PM 48
Mścisz A 48
Mueller-Roeber B 17
Muszyńska B 53, 64
Niklas-Nowak A 45
Nosal A 18
Nowaczyk L 54
Nowaczyk P 45, 54
Nowak K 61
Nowakowska J 20
Obarska A 18, 54
Olejnik A 64
Oleszczuk S 29, 36
Orlikowska T 12, 28, 55, 74
Orlińska M 45
Ożarowski M 55
Paluch I 68
Pawełek-Skoczylas A 19
Pawłowska B 33, 56
Pawłowska H 48
Pietraszek J 62
Pilarska M 21
Pilipowicz M 53, 62
Pindel A 25
Płażek A 56
Płoszaj B 57
Pniewski T 21, 34
Podwyszyńska M 58
Ponitka A 59
Popielarska-Konieczna M 59
Poturała D 19
Przyborowski J 36, 57
Ptak A 60
Pudelska H 59
Pukacki PM 22
Raj A 19
Raj D 22, 60
Ratajczak K 13, 65
Rodakowska E 14
Rojek J 19
Rojek M 61
Rokicki M 61
Rybczyński JJ 20, 24, 27, 38, 51
Rzenno K 59
Sawicka-Sienkiewicz E 32, 47
Skorys A 60
Skórkowska-Telichowska K 13
Skrzypczak-Pietraszek E 62
Skrzypek E 53, 62
Skucińska B 48
Sobiczewski P 12, 23, 28
Sochacki D 63
Sowa S 29
Stanisławska M 26
Stawiarz B 52
Stawicka A 53, 62
Stojakowska A 63
Sulikowska M 28
Sułkowska-Ziaja K 53, 64
Surówka E 18
Szała L 64
Szczerbakowa A 67
Szczygieł K 23
Szklarczyk M 38
Szopa J 13, 30, 33, 37, 47, 65, 72
Szpitter A 43
Sztajnert M 65
Szuba A 14
Szurman M 65
Szypuła W 24, 66
Ślązak B 19
Ślesak H 67
Ślusarkiewicz-Jarzina A 59
Tarwacka J 67
Thiem B 45, 55, 68
Tomaszewska B 18, 54
Tomaszewska-Sowa M 68
Tomiczak K 24
Trejgell A 69
Tretyn A 69
Trojak-Goluch A 25
Trojanowska A 69
Tuleja M 21
Turczynowska A 70
Twardowski T 13
Wajand M 70
Walas K 61
Warchoł M 71
Weremczuk-Jeżyna I 71
Widlarz E 41
Wielgat B 67
Wielgus K 50, 52
Wierzchowiecka M 14
Wiszniewska A 25
Wojciechowicz MK 26
Wojciechowski A 26, 61
Wojtania A 72
Wojtasik W 72
Wojtaszek P 14
Wojtuń B 19
Wolny E 61
Woś H 29
Woźna J 59
Wójcik A 27
Wyka TP 73
Wyrwa K 21
Wysokińska H 42, 71
Zalewska M 27
Zarychta Ł 73
Zawadzka M 28, 74
Zawadzki P 14
Zelazko P 74
Zenkteler E 12, 28, 36, 73
Zenkteler M 29, 73
Zimny J 29, 36
Znaniecka J 44
Zubek S 50
Źróbek-Sokolnik A 75
Żuk M 13, 30, 37
INSTRUCTIONS TO AUTHORS
ACTA BIOLOGICA CRACOVIENSIA Series Botanica is an English-language journal founded in 1958, devoted to plant anatomy and morphology, cytology, genetics, karyology, embryology, tissue culture, physiology
and biosystematics. It is published twice a year.
1. ACTA BIOLOGICA CRACOVIENSIA Series Botanica publishes original papers embodying the results
of experimental or theoretical research, invited reviews, and brief communications. Manuscripts will
be considered only on the understanding that they have not been published and are not being considered for publication elsewhere. Authors are charged a fee for publication of their articles. The bill for
publication will be sent with the galley proof. The fee, which is calculated after all articles are accepted, will be not more than $20.00 USD per printed page.
2. Manuscripts including illustrations should be sent to the Editor:
Prof. dr. ELŻBIETA KUTA
Department of Plant Cytology and Embryology
The Jagiellonian University
ul. Grodzka 52, 31-044 Cracow, Poland
Tel./Fax: +48-12-422 81 07
e-mail: [email protected]
Manuscripts will be examined by two referees.
3. Manuscripts should be submitted via e-mail as an attached pdf file to the address of the Editor. To
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(1982) or (Ward, 1950; Hiroshi and Ohta, 1970). For three or more authors, use the form: Zinkowski
et al. (1991).
Examples of style for references:
a) citations of journal papers:
PALMER TP. 1962. Population structure, breeding system, interspecific hybridization and alloploidy. Heredity 17:
278–283.
CHEN BY, HENEEN WK, and SIMONSEN V. 1989. Comparative and genetic studies of isozymes in resynthesized and
cultivated Brassica napus L., Brassica campestris L., and B. alboglabra Baitey. Theoretical and Applied
Genetics 77: 673–679.
b) citations of books, congress proceedings, theses:
BERGRREN DJ. 1981. Atlas of Seeds, part 3. Swedish Museum of Natural History, Stockholm.
BING D, DOWLEY RK, and RAKOW GFW. 1991. Potential of gene transfer among oilseed Brassica and their weedy
relatives. Proceedings of the GCTRC Eighth International Rapeseed Congress, 9–11 July 1991, 1022–1027.
Saskatoon, Saskatchewan.
ROMEO JT. 1973. A chemotaxonomic study of the genus Erythrina (Leguminosae). Ph.D. disseration, University
of Texas, Austin, TX.
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PHILLIPS RL. 1981. Pollen and pollen tubes. In: Clark G [ed.], Staining Procedures, 61–366. Williams and Wilkins,
Baltimore, MD.
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Indexed in Science Citation Index, Current Contents (Agriculture, Biology & Environmental Sciences),
Biological Abstracts, BIOSIS Data, Index Copernicus, Polish Scientific Journals Contents – AGRIC. &
BIOL. SCI., AGRO-AGEN.
ACTA BIOLOGICA CRACOVIENSIA Series Botanica on the Internet
The home page of Acta Biologica Cracoviensia Series Botanica can be found at
http://www.ib.uj.edu.pl/abc/abc.htm
Inquiries should be sent to the Biological Commission of the Polish Academy of Sciences,
ul. św. Jana 28, 31-018 Cracow, Poland.
HOW TO ORDER
ACTA BIOLOGICA CRACOVIENSIA
Series Botanica
PL ISSN 0001-5296
For further information, subscriptions, publication exchange visit home page of Polish Academy of
Sciences, Cracow Branch at:
http://www.pan-krakow.pl/english/sklep.php
or contact:
Polish Academy of Sciences Publishing House in Cracow
ul. św. Jana 28, 31-018 Cracow, Poland
Tel.: +48-12-422 36 43 ext. 22 or 15
Fax: +48-12-422 27 91
Printing: Skleniarz, Kraków
Number of volumes: 250