ISSN 1408-3671 
UDK 57(497.4) 
izdajate! j/pu bi isher 
Društvo biologov Slovenije 
ISSN 1408-3671 
UDK 57(497.4) 
8l0LOSKA KNJ/tN/Cil 
F C ' A 1• O T I I J 
10110 LJUB l J A NA 
~ACTA 
~ BIOLOGICA 
SLOVENICA 
VOL. 46 ŠT. 1 LJUBLJANA 2003 
prej/formerly BIOLOŠKI VESTNIK 
izdajatelj/publisher 
Društvo biologov Slovenije 
Acta Biologica Slovenica 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 1- 56 
Glasilo Društva biologov Slovenije - Journal of Biological Society of Slovenia 
Izdaja - Published by 
Društvo biologov Slovenije - Biological Society of Slovenia 
Glavni in odgovorni urednik - Editor in Chief 
Mihael Jožef Toman, e-mail: mihael.toman@uni-lj.si 
Tehnični urednik - Managing Editor 
Branko Vreš, e-mail: branevr@zrc-sazu.si 
Uredniški odbor - Editorial Board 
Matija Gogala (SI), Nada Gogala (SI) Peter Maček (SI), Alenka Malej (SI), Andrej Martinčič (SI), 
Harald Niklfeld (A), Livio Poldini (I), Boris Sket (SI), Robert Zorec (SI), Mitja Zupančič (SI), 
Thomas F. J. Martin (USA), Mark Tester (UK), Gerhard Thiel (D) 
Naslov uredništva - Adress of Editorial Office 
Acta Biologica Slovenica, Večna pot 111, SI-1001 Ljubljana, Slovenija 
http://bijh.zrc-sazu.si/abs/ 
Oblikovanje - Design 
Žare Vrezec 
ISSN 1408-3671 UDK 57(497.4) 
Natisnjeno - Printed on: 2003 
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Publikacijo je sofinanciralo Ministrstvo za šolstvo, znanost in šport Republike Slovenije. 
Acta Biologica Slovenica je indeksirana v - is indexed in: Biological Abstracts, 
Zoological records. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 3 - 7 
Sprejeto (accepted): 2003-10-30 
Use of direct somatic organogenesis for Agrobacterium-mediated 
transformation of onion 
Uporaba neposredne somatske organogeneze za transformacijo čebule s pomočjo 
A. tumefaciens 
Manja-Tina BASTAR, Zlata LUTHAR & Borut BOHANEC 
Centre for Plant Biotechnology and Breeding, Biotechnical Faculty, Jamnikarjeva 101, 
SI-1111 Ljubljana, Slovenia; E-mail: tina.bastar@bf.uni-lj.si 
Abstract. An Agrobacterium tumefaciens mediated DNA delivery is being 
developed for onion (Allium cepa L.) using organogenic structures formed on the 
ovaries as target tissue. An indirect transformation procedure using A. tumefaciens 
strains LBA4404 and EHA105 in combination with four plasmids was used for 
establishing an efficient transformation protocol. Studies were focused on different 
protocols for co-cultivation, selection and detection of optimal reporter and selection 
genes in onion. A histochemical GUS test and visual detection of the GFP protein 
were used for optimization of transformation treatments. Several pretreatment and co-
cultivation protocols achieving transient expression were studied, and the optimal was 
a combination of sonication (10 s) and vacuum treatment (5 min, 35 nuriHg). Among 
several selection media studied, optimal results were obtained using phosphinotricin 
(2.5 mg/1) as selection agent and timentin (150 mg/1) for good suppression ofbacterial 
growth. The transgenic nature of individual regenerants after six months on selection 
media was confirmed by polymerase chain reaction using primers for detection of 
uidA and bar genes. Using this test, three regenerants were positive for both bar and 
uidA genes and three regenerants were positive for the bar gene only. 
Keywords: Tissue culture, genetic transformation, onion, Allium cepa L., direct 
somatic organogenesis, Agrobacterium, uidA, gfp, bar, PCR 
Izvleček. Organogene strukture čebule (Allium cepa L.) nastale z direktno 
somatsko organogenezo smo uporabili za razvoj uspešne metode genske 
transformacije pri čebuli. Organogene strukture smo okuževali s sevoma LBA4404 
in EHAl 05 bakterije Agrobacterium tumefaciens in štirimi plazmidi. Proučevali smo 
različne pogoje kokultivacije, selekcije in izbirali primerne testne gene. Na podlagi 
GUS testa in z opazovanjem zelene flourescence GFP proteina z mikroskopom smo 
lahko optimizirali postopke predtretiranja in kokultivacije. Postopki predtretiranja 
so vključevali uporabo ultrazvoka (optimalno 10 s) in dveh nivojev vakuuma 
(optimalen 5 min pri 35 mmHg). Med mnogimi preizkušenimi selekcijskimi gojišči 
smo najboljše rezultate dobili pri kombinaciji selekcijskega agensa fosfinotricina 
(2,5 mg/1) ter antibiotika timentina (150 mg/1) za učinkovito odstranitev bakterije. 
4 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
Vključitev plazmidne DNA v genom čebule smo preverili s polimerazno verižno 
reakcijo za gena uidA in bar. S tem testom smo pri treh regenerantih čebule po šestih 
mesecih na selekcijskem gojišču dokazali prisotnost uidA in bar gena, pri drugih 
treh regenerantih čebule pa je prišlo do vključitve samo slednjega. 
Ključne besede: Tkivne kulture, genska transformacija, čebula, Allium cepa L., 
direktna somatska organogeneza, Agrobacterium, uidA, gfp, bar, PCR 
Onions and shallots (A. cepa L.), garlic (A. sativum L.) and leek (A. porrum L.) are important 
vegetable crops which have considerable health benefits. By manipulating agronomk and quality 
trades in this crops it may be possible to reduce the requirement for pesticide applications as it has 
been achieved in other crops (PHIPPs & PARK 2002) and improve their sustainable production and 
nutritional status. One possible approach to this is by genetic modification. An A. tumefaciens DNA 
delivery has been developed for onion using organogenic structures formed on ovaries according to an 
established somatic regeneration protocol (LUTHAR & BoHANEC 1999). The method differs from published 
protocols (EADY & al. 2000, ZHENG 2000) which have used embryo-derived callus tissue as target cells. 
Our method for direct organogenesis in oni on resulting in the formation of multiple shoot structures of 
cultured flower buds or ovaries was used as starting tissue. A. tumefaciens strains LBA4404 and EHAl 05 
in combination with four plasmids were tested. Stucties focused on different protocols for co-cultivation, 
selection and detection of optimal reporter genes for anion. A histochemical GUS test and visual 
detection of the GFP protein were used to optimize transformation treatments. 
Material and methods 
Immature flower buds were collected from five different cultivars (Belokranjka and · US inbred 
lines B1828A, B1828B, MSU2399B and B2371 x B2923B) grown in the greenhouse. A. tumefaciens 
strains LBA4404 and EHA105 containing vectors pBIN m-gfp5-ER and pCAMBIA 1301, 1303 and 
3301 were grown to log phase on liquid LB media. Ovaries with embryogenic structures were transferred 
into 15 ml of Agrobacterium suspension and exposed to sonication treatment and/or a vacuum-assisted 
transformation procedure. The embryogenic structures were strained on sterile filter paper and put on 
mectia containing 100 mM acetosyringone. After three days of co-cultivation, explants were transferred 
to BDS media containing agents for selection of transformants and prevention of Agrobacterium growth. 
The efficiency of the antibiotics, geneticin (30-50 mg/!), kanarnycin (50 mg/!), hygromycin (25 mg/!) 
and the herbicide phosphinotricin (2.5 mg/!) as selective agents in anion tissue culture was investigated. 
Certain antibiotics, cefotaxim, vankomycin (250 mg/!) and timentin (150 mg/!) were used to eliminate 
Agrobacterium from plant tissue without severely inhibiting plant growth. Tissue cultures were 
subcultured on fresh media every three weeks. Histochemical GUS assays were performed 3-6 days 
after transfer of explants to the media containing selection antibiotics following the protocol of RUEB 
& HANSGENS (1989). For GFP expression, tissues were exarnined by observation under a fluorescence 
microscope using 460-500 nm excitation and 515-560 nm barrier filters. 
Results and discussion 
The average formation of organogenic structures on ovaries of the five cultivars on BDSi media 
was 34.8%. The efficiency of the protocol for the induction of direct somatic embryogenesis on ovaries 
M. Bastar, Z. Luthar, B. Bohanec: Use of direct somatic organogenesis for Agrobacterium ... 5 
is comparable to the results obtained by LUTHAR & BoHANEC (1999). Transient expression of GUS 
product was present in in vitro onion cultures following an A. turnejaciens-mediated transformation 
procedure. Blue histochemical staining was visible on the surface area of explants. The dark blue spots 
on the forming shoots were particularly promising. After three days of co-cultivation, single cells 
expressing m-gfp5-ER could be observed, but the expression was not mainly localized on the 
embryogenic cells. Several pretreatment and co-cultivation protocols achieving transient expression 
were studied (Tab. 1), the optimal one was a combination of sonication (10 s) and vacuum treatment (5 
min, 35 mmHg) using the A. turnejaciens LBA4404 (pCAMBIA 3301). The mechanical treatment of 
ultrasound causing loosening of celi walls in the organogenic structures formed on ovaries enhanced 
the efficiency of Agrobacteriurn infection. SANTAREM & al. (1998) established that sonication included 
in the co-cultivation treatment has a major impact on the transformation efficiency of young soybean 
leaves. EADY & LrsTER (1998) reported a beneficial effect of vacuum infiltration subsided by fine 
stirring of oni on embryos in Agrobacteriurn suspension. Strong inhibition of Agrobacteriurn in cultured 
tissues was obtained with timentin at 150 mg/1. Selection media containing geneticin at 30 mg/1 had a 
deleterious effect on regenerating tissues, hygromicin at 25 mg/1 was lethal, causing excessive tissue 
damage after 6 weeks of exposure. Nine point eight percent of regenerants of Belokranjka sustained 
selection on phosphinotricin (2.5 mg/1) after six months of cultivation. 
Table l: Summary of different transformation experiments with A. turnejaciens LBA4404 and 
EHA105 (pCAMBIA1303, 1301, 3301) 
Preglednica 1: Povzetek različnih poskusov transformacij s pomočjo A. turnejaciens LBA4404 in 
EHA105 (pCAMBIA1303, 1301, 3301) 
NO. OF ORGANOGENIC OVARIES TREATMENT 
EXPERIMENT / PLASMID A. turnej aciens A. turnej aciens SONICATION VAKUUM 
LBA4404 EHA105 
3240 780 5s / 
l. / pCAMBIA 1303 270 120 / 30 min 
150 150 5 s 5 min 
120 120 5 s 30 min 
350 270 5 s / 
2. / pCAMBIA 1301 210 / 6 s 5 min 
200 / 15 s 5 min 
200 / 30 s 5 min 
420 210 5 s / 
200 / 5 s 5 min 
240 / 6s 5 min 
240 / 10 s 5 min 
3. / pCAMBIA 3301 440 / 15 s 5 min 
240 / 20 s 5 min 
200 / 30 s 5 min 
120 / 50 s 5 min 
/ 150 / 30 min 
ORGANOGENIC OVARIES/EXP. 6840 1800 
6 Acta Biologica Slovenica, 46 (1), 2003 
The transgenic nature of individual plants was confirmed by polymerase chain reaction using 
primers for uidA and bar genes. PCR on three regenerants transformed withA. tumefaciens LBA4404 
(pCAMBIA 3301) confirmed integration of uidA and bar genes into the onion genome when a 
combination of sonication and vacuum subsided transformation was used (Fig. 1). Using this analysis, 
three regenerants were positive for the bar gene only (Fig. 2). The survival of three regenerants on 
the selection media and the positive results of analysis performed by PCR show integration of only 
a fragment of pCAMBIA 3301 plasmid into the onion genome. Similar results were reported in an 
apple cultivar by YAo & al. (1995) and in rice by Hrnr & al. (1997). Studies of the transformation 
efficiency in Solanum muricatum (ATKINSON & GARDNER 1991) revealed that up to 50% of transformed 
plants have only partial incorporation of T-DNA in the plant genome. 
Figure 1: PCR amplification of genomic DNA from transformants with (A) uidA and (B) bar 
gene primers. Lanes 1, 4 and 5: untransformed regenerants, 2, 3 and 6: individual transgenic 
plants, 7: untreated plant, 8: pCAMBIA 3301, 9: water and 10: size leader no. 6 
Slika 1: Namnoženi fragmenti DNA s parom začetnih oligonukleotidov za (A) uidA in (B) bar gen 
pri regenerantih čebule po okužbi z A. tumefaciens. 1, 4 in 5: netransformirani regeneranti, 2, 3 in 6: 
transformirani regeneranti, 7: kontrola - neokuženi regeneranti, 8: pCAMBIA 3301, 9: slepi vzorec 
in 1 O: velikostni standard št. 6 
Figure 2: PCR amplification of genomic DNA from transformants with bar selection gene primers. 
Lanes 1, 3, 5 -12 and 14: untransformed regenerants, 2, 4 and 13: individual transgenics positive for 
the bar gene, 15: untreated plant, 16: pCAMBIA3301, 17: water and lane 18: size leader no. 6 
Slika 2: Namnoženi fragmenti DNA s parom začetnih oligonukleotidov za selekcijski bar gen. 1, 3, 
5 -12 in 14: netransformirani regeneranti, 2, 4 in 13: transformirani regeneranti, 15: kontrola -
neokuženi regeneranti, 16: pCAMBIA 3301, 17: slepi vzorec in 18: velikostni standard št. 6 
M. Bastar, Z. Luthar, B. Bohanec: Use of direct somatic organogenesis for Agrobacterium ... 7 
Conclusions 
On the basis of results obtained it was concluded that our protocol used for the transformation of 
onion with A. tumefaciens is suitable. The transgenic nature of six individual regenerants after six 
months on selection media was confirmed by PCR. Further analyses of their progeny are underway. 
U sing this test, three regenerants were positive for both bar and uidA genes and three regenerants were 
positive for the bar gene only. Transformation efficiency rnight be further enhanced by the application 
of more virulent strains of A. tumefaciens, specialized for monocotyledonous species (LBA4404, 
EHA105), a combination with different constructs could enhance the incorporation of transgenes into 
the target tissue, and the combination of sonication treatment and a vacuum-subsided transformation 
procedure could also contribute to the more frequent and stable incorporation of the desired genes. 
Povzetek 
Organogene strukture čebule nastale z direktno somatsko organogenezo smo uporabili za razvoj 
uspešne metode genske transformacije pri čebuli. Vključitev plazmidne DNA v genom čebule smo 
preverili s polimerazno verižno reakcijo za gena uidA in bar. S tem testom smo dokazali prisotnost 
obeh genov v treh regenerantih in samo bar gena v drugih treh regenerantih čebule. Učinkovitost 
transformacije bi lahko v prihodnje optimizirali z uporabo virulentnejših sevov A. tumefaciens 
primernih za enokaličnice (LBA4404, EHA105) ter ustreznih kombinacij sevov in plazrnidov. Za 
nadaljno optimizacijo učinkovitosti stabilne vključitve željenih genov v genom čebule bi lahko med 
ko-kultivacijo bakterijskega in rastlinskega materiala uporabili kombinacijo ultrazvoka in vakuuma. 
References 
ATKINSON R. G. & R. C. GARDNER 1991: Agrobacterium-mediated transformation of pepino and 
regeneration of transgenic plants. Plant Cell Rep 10: 208-212. 
EADY C. C. & C. E. LrsTER 1998: A comparison of four selective agents for use with Allium cepa L. 
immature embryos and immature embryo derived callus. Plant Celi Rep 18: 117-121. 
EADY C. C., R. J. WELD, C. E. LISTER 2000: Agrobacterium tumefaciens-mediated transformation and 
transgenic-plant regeneration of onion (Allium cepa L.). Plant Celi Rep 19: 376--381. 
HlEr Y., T. KOMAR!, T. Kuso 1997: Transformation of rice mediated by Agrobacteruim tumefaciens. 
Plant Mol Biol 35: 205-218. 
LUTHAR Z. & B. BoHANEC 1999: Induction of direct somatic organogenesis in onion (Allium cepa L.) 
using a two step flower or ovary culture. Plant Cell Rep 18: 797-802. 
PHIPPS R. H. & J. R. PARK 2002: Environmental benefits of genetically modified crops: Globa! and 
European perspectives on their ability to reduce pesticide use. J. Animal Feed Sci 11: 1-18. 
RuEB S. & L. A. M. HANSGENS 1989: Improved histochemical staining for ~-d-glucoronidase activity 
in monocotiledonous plants. Rice Genet Newsl 6 (2): 168-169. 
SANTAREM E. R., H. N. TrucK, J. S. EssIG, J. J. FrNER 1998: Sonication-assisted Agrobacterium-
mediated transformation of soybean immature cotyledons: optimization of transient 
expression. Plant Cell Rep 19: 752-759. 
Y AO J. L., D. CoHEN, R. ATKINS, K. RrcHARDSON, B. MoRRrs 1995: Regeneration of transgenic plants 
from the commercial apple cultivar Royal Gala. Plant Cell Rep 14: 407-412. 
ZHENG S. J. 2000: Towards onion and shallots (Allium cepa L.) resistant to beet armyworm (Spodoptera 
exigua Hiibner), Ph. D. Thesis. Wageningen University, Wageningen, The Netherlands, 145pp. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 9 - 16 
Sprejeto (accepted): 2003-10-30 
Mycorrhizal potential of two forest research plots with respect to reduction 
of the emissions from the Thermal Power Plant Šoštanj 
Mikorizni potencial dveh gozdnih raziskovalnih ploskev po zmanjšanju emisij iz 
Termoelektrarne Šoštanj 
Samar AL SAYEGH PETKOVŠEK & 'Rojka KRAIGHER 
ERICo Velenje, Koroška 58, 3320 Velenje, Slovenia; 
E-mail: samar.petkovsek@erico.si 
'Slovenian Forestry Institute, Večna pot 2, 1000 Ljubljana; Slovenia; 
E-mail: hojka.kraigher@gozdis.si 
Abstract. The mycorrhizal potential of two differently polluted forest research 
plots was determined in the emission area of the Thermal Power Plant Šoštanj. 
Zavodnje is the polluted, while Mislinja is the less polluted plot. Mycorrhizal potential 
of the soils from the two sites was estimated in a pot experiment. Types of 
ectomycorrhizae were identified in the soil cores and on short roots of N orway spruce 
seedlings. The fresh weight of needles and stems, number of short roots and the 
percentage of mycorrhizal short roots on seedlings from Zavodnje were significantly 
lower in comparison with Mislinja. The results indicate that the mycorrhizal potential 
of the more polluted site is lower. Mycorrhizal potential is discussed with respect to 
the results from our earlier studies. 
Key words: ectomycorrhizae, mycorrhizal potential, Norway spruce seedlings, 
forest research plots 
Izvleček. Določili smo mikorizni potencial dveh različno onesnaženih gozdnih 
ploskev v imisijskem območju Termoelektrarne Šoštanj. Zavodnje predstavlja 
onesnaženo, Mislinja pa manj onesnaženo raziskovalno ploskev. Mikorizni potencial 
obeh območij smo določili z lončnim poskusom. Tipe ektomikorize smo identificirali 
v talnih vzorcih raziskovalnih ploskev in na kratkih koreninah semenk smreke. Sveže 
teže iglic in stebel semenk (nadzemni del), število kratkih korenin in število 
nemikoriznih kratkih korenin semenk, ki so rastle na substratu iz Zavodenj, je 
statistično značilno manjše v primerjavi Mislinjo. Rezultati kažejo, da je mikorizni 
potencial bolj onesnaženega območja nižji. Mikorizni potencial smo primerjali z 
rezultati naših prejšnjih raziskav. 
Ključne besede: ektomikoriza, mikorizni potencial, semenke smreke, gozdna 
raziskovalna ploskev 
10 Acta Biologica Slovenica, 46 (1), 2003 
Introduction 
Ectomycorrhiza is the site of exchange of nutrients between the plant and the fungus. Fungal 
hyphae exploit the soil for mobilisation and absorption of water and nutrients (BRUNNER 2001). 
They are integral, functional parts of plant roots, in which the fungi involved provide a direct link 
between the soil and the roots (LEYVAL & al. 1997). Furthermore the mycelia of ectomycorrhizal 
fungi act as temporal and spatial connections between different species of trees in the forest ecosystem 
(AMARANTHUS & PERRY 1994). 
In the last decades damages of forest trees and ecosystems have been monitored in North America 
and Europe. These can be connected with the disturbances in the ectomycorrhizal symbiosis. The 
deposition of pollutants into the forest ecosystems leads to the acidification and/or eutrophication 
of the forest soil and can consequently affect the health and vitality of forest trees (BRUNNER 
2001). The impact of pollution on forest soils can be estimated by determination of mycorrhizal 
potential of forest soil of differently polluted areas. Pollution can influence the below - ground 
diversity of ectomycorrhizal fungi since some types can better survive different stress factors than 
others (GIANINAZZI-PEARSON 1984, VODNIK & al. 1995, TAYLOR 1995). 
In Slovenia the mycobioindication method for determination of pollution stress has been used 
(KRAIGHER & al. 1996) and in spruce forest the reduction of biodiversity of types of 
ectomycorrhizae due to pollution was established (KRAIGHER 1999). On the other hand biodiversity 
indexes were high in all beech forest research plots, therefore the impacts of pollution on beech 
ectomycorrhizae was not stated (AL SA YEGH PETKOVŠEK & KRAIGHER 2000). In studies of 
oak decline KOVACS reported that the ectomycorrhizal diversity decreased slowly but significantly 
in two oak stands in the north-east of Austria and the presence of some morphotypes were highly 
correlated with the crown-status of the trees (KOVACS & al. 2000). 
The objectives of the present study were to determine mycorrhizal potential of two differently 
polluted forest research plots and to identify the types of ectomycorrhizae on short roots of mature 
trees and seedlings according to the concept of mycorrhizal succession (DIGHTON & MASON 
1985, LAST & al. 1987) and biodiversity studies (KRAIGHER 1999, KOVACS & al. 2000, FERRIS 
& al. 2000). 
Material And Methods 
The mycorrhizal potential of forest soil is defined as the capability of propagules of naturally 
occurring fungi in forest soils to colonize roots of spruce seedlings. It is expressed as the percentage 
of mycorrhizal short roots of the total number of short roots in the sample (KROPAČEK & al. 
1989). A modified method of a pot experiment for determination of the mycorrhizal potential was 
used (AL SAYEGH PETKOVŠEK 1997). Differently polluted research plots are situated in the 
emission area of the Thermal Power Plant Šoštanj (TPP) where the negative impact of pollution is 
well demonstrated in all parts of environment (SVETINA 1999, POKORNY 2000, KUGONIČ & 
STROPNIK 2001, RIBARIČ LASNIK & al. 2001, POKORNY & RIBARIČ LASNIK 2002). The 
two plots (850 m a.s.l., distric cambisol, Luzulo-Fagetum, predominant tree species Picea abies), 
were as similar regarding site characteristics as it was possible to select, but polluted differently by 
the emissions from the TPP, as indicated by the lichenological studies (BATIČ & KRALJ 1990) and 
by the total S% and Pb content in soil (Tab. 1, sampling done in August 2001). Soils from both plots 
were dug from the upper 20 cm, sieved (2 mm sieve) and used as planting substrates. At the same 
tirne types of ectomycorrhizae were identified in soil cores (275 ml volume, O - 18 cm deep) from 
both research plots. 5 seedlings (at 3 weeks of age) per pot and 5 pots per soil source were grown for 
six months in the gerrnination cabinet, where the growth conditions were the same for ali seedlings. 
After six months the seedlings were weighed, short roots were counted and the percentage of 
S. Al Sayegb Petkovšek, H. Kraiger: Mycorrbizal potential of two forest researcb plots ... 11 
mycorrhizal short roots and the types of ectomycorrhizae were determined. The determination of 
types of ectomycorrhizae followed the procedure from the "Colour Atlas of Ectomycorrhizae" 
(AGERER 1987-1999) and other primary sources of identification. 
Data from the study in 1993 and 2002 were compared. The difference was in the reduction of 
the emissions from TPP: the reduction of the emissions from the Thermal Power Plant Šoštanj was 
from 86.147 t SO
2 
in the year 1993 to 22.871 t in the year 2002. Statisticafor Windows 5.5 has been 
used for statistical analyses. The results represented in Graph 1 are the average values for seedlings 
from two different locations, the significance of the difference was evaluated by non-parametric 
statistical analyses (Man-Whitney U test). 
Table 1: Sieved soil analysis <lata for two forest research plots (P93-polluted plot in 1993; P02-
polluted plot in 2002; U93-unpolluted plot in 1993, U02-unpolluted plot in 2002) 
pH pH C% N% C/N K p S% Pb Cd Hg As 
CaCl2 Hp Ekv Ekv mg/kg mg/kg mg/kg mg/kg 
P 3,76 4,3 6,5 0,30 22 0,14 Traces 0.07 - - - -
93 
P 3.44 4,0 16,8 0,59 29 0,10 Traces 0,09 115 0,57 0,26 6,15 
02 
U 3,36 3.8 17,0 0,82 21 0,50 Traces 0.06 - - - -
93 
U 3.99 4,6 7,47 0,34 21 0,10 Traces 0,06 53,4 0,26 0,29 6,15 
02 
Results and Discussion 
The percentage of mycorrhizal short roots of seedlings from Zavodnje was significantly lower 
(p<0.05) in comparison with Mislinja (Fig. 1), consequently it could be concluded that mycorrhizal 
potential of the more polluted area is lower. Also other growth parameters of the seedlings were 
different: the fresh weights of stems and needles were higher in Mislinja, while the number of 
nonmycorrhizal roots were lower in Mislinja in comparison to Zavodnje. The average fresh weight 
of roots did not differ significantly. This is comparable also to the results of the study done eight 
years ago (Fig. 2, data on 1993 calculated from AL SAYEGH PETKOVŠEK 1997). 
12 
NUMBER OF SHOAT ROOTS 
P=0,0003 
500 
460 g <!O 1 360 
1 340 ··•· 
o 
i 300 g 
260 
220 
MISLINJA ZAVODNJE 
locaHon 
AVERAOE FRESHWEIGHT OF SHORTROOTS 
P=0.07' 
0,30 
0,28 g §0,26 ,, ~ 0,2' o o i o,22 ~ ~ ~0,20 ! 
0,18 _L. 
0,16 
lil lSllNJA ZAVODNJE 
location 
I t1.96'Std. Err. 
Otl.ll0'S!d.Err. 
a Mean 
I t1.96' S1d.Err. 
Q tUlO'Sld. Err. 
o Mean 
Acta Biologica Slovenica, 46 (1), 2003 
NUMBER OF NONMYCORAHIZAL SHORT ROOTS 
p:0,013 
60 ~ --- --- ------~ 
50 
0, 17 
0,16 
0,15 
0, 14 
§ 
i o,13 
"! 0,1 2 
[ 0,11 
~ 
0,10 
0,09 
0,08 
MISLINJA ZAVODNJE 
localion 
AVERAGEfRESHWEIGHTOFSTEMS ANDHEEOtES 
p:0,0003 
g 
---r-g 
MISLINJA ZAVODNJE 
locations 
:Cil .96'Std.Err. 
O j:t00'Sld. Err. 
• Mean 
:I_1I.H"S\J(H 
• t1m'S1t[n 
O VII~ 
Figure 1: Comparison between different parameters of Norway spruce seedlings grown in soil 
substrates Mislinja and Zavodnje. 
Our recent and previous study (AL SAYEGH PETKOVŠEK & al. 1995, AL SAYEGH 
PETKOVŠEK 1997, AL SAYEGH PETKOVŠEK & KRAIGHER 1998) on the same topic strongly 
indicate that pollution influences the mycorrhizal potential of forest soils. Negative impact is still 
present in spite of the reduction of the emissions. However the decrease in pollution seems to result 
in higher percentage of mycorrhizal short roots in current study as compared to former research. 
This increase in mycorrhization is characteristic for both plots. 
S. Al Sayegh Petkovšek, H. Kraiger: Mycorrhizal potential of two forest research plots ... 13 
Mislinja, 2002 Zavodnje, 2002 
• mycorrb.i7.al roots C nonmycorrhiz.al l'OQls • mycorrhi7.al roots Cnonmycorrhi:zal roots 
Mislinja, 1993 Zavodnje, 1993 
7% 
93% 
• mycorrltizal roots C nonmycorrb.i;,.al roots • myconttizal roots C nonmycorrhizal roots 
Figure 2: Mycorrhization of short roots of Norway spruce seedlings in pot experiments in the year 
2002 and 1993. 
Different types and different number of ectomycorrhizae were determined on short roots of 
mature trees in differently polluted forest research plots. 13 types of ectomycorrhizae in the soils 
from Mislinja and 8 types of ectomycorrhizae in the sois from Zavodnje (Tab.2) were identified. 
Three types of ectomycorrhizae were identical for both plots: Cenoccocum geophilum Fr., Lactarius 
pallidus Pers. ex Fr. and Suillus sp. These types belong to the group of types of ectomycorrhizae 
named "late stage fungi" (LAST & al. 1987). Among the type described, Scleroderma citrinum 
Pers. has been regarded as un-sensitive to pollution (ARNO LDS 1991 ). Also in our study S. citrinum 
was frequent in the soil samples from the polluted area (Zavodnje). 
14 
Table 2: Types of ectomycorrhizae identified in the soil cores. 
TYPES OF ECTOMYCORRHIZAE 
SLO 801 - SAl Xerocomus badius (Fr.) Kiihn.: Gilbert 
SLO 802 - SA2 Piceirhiza obscura 
SLO 803 - SA3 Cenoccocum geophilum Fr. 
SLO 811 - SAll Tylosporafibrillosa (Burt) Donk 
SLO 813 - SA13 Russula acrifolia Romagn. 
SLO 836 - SA24 Dermocybe sp. 
SLO 863 - SA63 Thelephora terrestris Pers. 
SLO 875 - SA75 Amphinema byssoides (Pers.) J. Erikss. 
SLO 888 - SA70 Xerocomus sp. 
SLO 897 - SA97 Russula sp. 
SLO 899 - SA99 Tuber rufum * 
SLO 890 - SA52 Lactarius pallidus Pers. ex Fr. * 
SLO 901 - SA101 Tuber borchii Vitt.* 
SLO 902 - SA102 Boletus edulis Bull.: Fr.* 
SLO 903 - SA103 Suillus sp. 
SLO 904 - SA104 Inocybe sp. 
SLO 906 - SA106 Lactarius subdulcis Bull.: Fr.* 
SLO 907 - SA107 Scleroderma citrinum Pers.* 
Acta Biologica Slovenica, 46 (1), 2003 
FOREST RESEARCH PLOT 
Mislinja 
Mislinja 
Mislinja, Zavodnje 
Zavodnje 
Mislinja 
Mislinja 
Mislinja 
Zavodnje 
Zavodnje 
Mislinja 
Mislinja 
Mislinja, Zavodnje 
Mislinja 
Mislinja 
Mislinja, Zavodnje 
Mislinja 
Zavodnje 
Zavodnje 
Legend: Types marked with * are sirnilar (not identical or described ona different plant sp.) to the 
types presented in the table. 
Two types of ectomycorrhizae were determined on six months old Norway spruce seedlings: 
Hebeloma mesophaeum Quel. and Cenoccocum geophilum. The first type is very common on young 
trees and could be considered as an "early stage fungus" (INGLEBY & al. 1990, DIGHTON & 
MASON 1985), while the C. geophilum has a world-wide distribution on a wide range of plants 
(SMITH & READ 1997) and it has been found on seedlings and mature trees (INGLEBY & al. 
1990). Since black types of ectomycorrhizae named as C. geophilum were not well differentiated 
after anatomical characteristics in the past (therefore it was also considered asa "group species" as 
in AL SAYEGH PETKOVŠEK & KRAIGHER 1999) its different ecology might possibly been due 
to an involvement of severa! species with similar characteristics in different physiological and 
ecological references. 
Conclusions 
(i) Mycorrhizal potential as a bioassay for soil substrate pollution (with S and heavy metals) is an 
acceptable mycobioindication method. 
(ii) The differences between the two sites were statistically significant, although the pollution 
effects are not highly destructive with respect to mycorrhizal soil inoculum potential. 
(iii) Eventhough the emissions from the TPP were reduced in the last decade, the revitalization of 
soil substrates is a slow process, therefore it rnight take severa! more decades before the 
mycorrhizal potential of the polluted and the unpolluted site reach the same level. 
S. Al Sayegh Petkovšek, H. Kraiger: Mycorrhizal potential of two forest research plots ... 15 
(iv) Types of ectomycorrhizae present in the soils from the two sites represent all-stage as well as 
late-stage fungi, reflecting that soil substrates originate from old-growth forests and closed 
Norway spruce stands. 
(v) Types of ectomycorrhizae are of primary importance for the functioning of the forest ecosystem, 
however the simplified situation in the pot studies of mycorrhizal potential supports the primary 
importance of the % of mycorrhizal short roots while identification of types of ectomycorrhizae 
is of a secondary importance for application in bioindication methods. 
(vi) Due to the still noticeable impact of pollution on mycorrhizal potential of forest soils further 
analyses of mycorrhizal potential are recommended in order to continuously monitor the changes 
in ectomycorrhizal communities after the reductions of the emissions from the Thermal Power 
Plant Šoštanj. 
Acknowledgements 
The study was financed by the Themal Power Plant Šoštanj and the Ministry of Education, 
Science and Sport through the research programme: Forest Biology, Ecology and Technology of the 
Slovenian Forestry Institute. 
References 
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semenke smreke. Zbornik gozdarstve in lesarstva 57: 93-110. 
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213-217. 
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AMARANTHUS M. P. & PERRY D. A. 1994: The functioning of ectomycorrhizal fungi in the field: 
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ARNOLDS E. 1991: Decline of ectomycorrhizal fungi in Europe. Agriculture, Ecosystems and 
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GIANINAZZI-PEARSON V. 1984: Host- fungus Specifity, Recognition and Compatibility in Mycorrhizae. 
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Phyton (Austria) Special issue: "Root-soil interaction" 40 (4): 109-116. 
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39: 199-202. 
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potencial of forest soils exposed to different pollutions stress. Agriculture, Ecosystems 
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Šaleški dolini. Letno poročilo, ERICo Velenje DP-24/02/01, 183 s. 
LAST F. T., DIGHTON J. & MASON P. A. 1987: Succesion of Sheating Mycorrhizal Fungi. Trees 2 (6): 
157-161. 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 17- 25 
Sprejeto (accepted): 2003-10-30 
The morphological and genetical characterisation of a native Norway 
spruce (Picea abies (L.) Karst.) population in the area of Pokljuka mire 
Morfološke in genetske značilnosti naravne populacije smreke 
(Picea abies (L.) Karst.) na območju pokljuškega barja 
Gregor BOŽIČ & Mihej URBANČIČ 
Slovenian Forestry Institute, Večna pot 2, SI-1000 Ljubljana; 
E-mail: gre gor. bozic@gozdis.si, mihej. urbancic@gozdis.si. 
Abstract. Morphometrical and genetical characteristics of two morphologically 
different sub-populations of spruce trees grown in a frost area of the mire Šijec 
(altitude 1170 m) on the Pokljuka plateau were studied. The tree height & diameter, 
needle length & needle volume of 70, approximately 120 to 200 years old trees, 
were measured. The genetic structure of the two spruce sub-populations were 
estimated by isozyme analysis on starch gel at 17 polymorphic gene loci. Soil 
conditions on the research plots examined with a gauge sound and then mapped and 
investigated in details by lab analyses of soil samples from representative soil profiles. 
The analysed spruce trees grown on the more productive site belonging to the 
association Rhytidiadelpho lorei - Piceetum in edge of the mire with mainly 
automorphic soils were in average 3,8 times taller and thicker than the trees from the 
extreme site belonging to the association Sphagno girgensohnii - Piceetum. The 
needle length of these trees was 25,7 % longer and their needJe volume 68 % bigger 
than of the trees from the mire plot on the hydromorphic peat soils. The comparison 
of the genetic structures has shown distinct differences in frequencies of some alleles 
and genotypes between the analysed spruce sub-populations. 
Key words: Picea abies (L,) Karst., sub-population, soil conditions, 
morphological variability, genetic structure, Julian Alps, Slovenia 
Izvleček. Na Pokljuški planoti smo na mraziščnem področju barja Šijec (na n. v. 
1170 m) proučevali morfološke in genetske značilnosti dveh različnih smrekovih 
subpopulacij. Okoli 120 do 200 let starim smrekam smo izmerili drevesne višine, 
premere debel v prsni višini ter dolžine in volumne iglic. Genetsko strukturo obeh 
subpopulacij smo proučili z elektroforetsko analizo 17 polimorfnih izoencimskih 
genskih lokusov v škrobnem gelu. Analizirane smreke z roba barja, ki rastejo na 
rastišču asociacije Rhytidiadelpho lorei - Piceetum, na katerem prevladujejo trdinska 
tla in je rodovitnejše, so imele v povprečju 3,8 krat večjo drevesno višino in premer 
ter za 25,7 % daljše iglice in za 68 % večjo prostornino iglic od smrek barjanske 
ploskve, ki poraščajo ekstremno rastišče asociacije Sphagno girgensohnii - Piceetum 
18 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
na hidromorfnih šotnih tleh. Primerjava genetskih struktur je pokazala izrazite razlike 
v frekvencah nakaterih alelov in genotipov med analiziranima smrekovima 
subpopuacijama. 
Ključne besede: Picea abies (L.) Karst., subpopulacija, talne razmere, 
morfološka variabilnost, genetska struktura, Julijske Alpe, Slovenija 
Forests cover about 20 km2 of the Pokljuka plateau is calcareous and mixed moraines. Natura! 
forests of beech (Fagus sylvatica L.) and fir (Abies alba Mil.) and secondary forests of Norway 
spruce (Picea abies (L.) Karst.) predominate, both with a high wood productivity. At the altitude of 
about 1200 m a.s.l. primary and supposedly autochthonous Norway spruce forests are still present 
and form two plant associations. 
The spruce plant association Rhytidiadelpho lorei - Piceetum (ZUPANČIČ 1981 emend.) covers 
automorphic soils developed on moraines, spruce growth of these stands is relatively good. The 
spruce association Sphagno girgensohnii - Piceetum (W. KUOCH 1954 corr. ZUPANČIČ 1982) 
var. geogr. Carex brizoides (ZUPANČIČ 1982 corr.) grows on hydromorphic soils of mires. This 
spruce-mire plant community on peat soils lives in extremely poor and severe site conditions, spruces 
there grow slowly and dwarfly. 
The aim of this study was to evaluate the influence of different site conditions on spruce growth 
characteristics and to determine the genetic structure of this species in two primary Norway spruce 
associations. 
Materials and methods 
Two research plots were established in the natura! growing sites of spruce in the frosty area on 
the Pokljuka plateau. The research plot "Mire" lies in the south-eastem part of the mire Šijec in the 
bushy and gappy spruce forest (association Sphagno girgensohnii-Piceetum) between dwarf-pine 
community (Sphagno-Pinetum mughi) and high spruce forest on the mire's edge. The research plot 
"Edge" is located inside a one-hectare large permanent plot "Šijec" of the Slovenian Forestry Institute 
where sites of the spruce association Rhytidiadelpho lorei-Piceetum predominate (Figure 1). The 
plots are located at 1170 m altitude, the distance between them is 500 m. 
Figure 1: Norway spruce population of the mire's Šijec area on the Pokljuka plateau (left) with 
locations of the research plots "Mire" (in the middle) and "Edge" (right). Photo: G. Božič 
G. Božič, M. Urbančič: The morphological and genetical characterisation ... 19 
Trees from every plot are considered as a sub-population from the whole spruce population of 
the mire's Šijec area. On each plot soil conditions were studied and 35 vi tal randomly chosen dominant 
spruce trees were dendrometrically analysed and their genetic structure determined. 
Soil conditions and morphologic properties of soil in the research plots (see also BOŽIČ & 
URBANČIČ 2001) were examined with a semicircular sound, which reaches down to 110 cm deep. 
According to soil heterogeneity of plots the locations for representative soil profiles were chosen. 
After the description of profiles soil samples were tak.en and analysed in the lab. For each soil 
sample pH in de-ionised water (Hp) and in 0.01 M calcium chloride (CaClz), content of: carbonates, 
organic carbon, organic matter, total nitrogen and C/N ratio were determined. Contents of 
exchangeable cations (K+, Ca2+, Mg2+, AP+, Fe3+, Mn2+ and H+), cation exchange capacities, base 
saturation and texture classes were determined for samples from mineral part of soil. 
The morphological characteristics of the sub-populations were determined by analysis of 
morphometric measurements of needle lengths and volumes and by dendrometric analyses of heights 
and diameters at the height of 1,3 m of dominant, approximately 120 to 200 years old spruce trees. 
Studied samples were tak.en from the 2 years old shoots exposed to sun in the southem upper third 
of the tree crown of 70 randomly chosen trees. Each tree sample consisted of 100 randomly chosen 
needles. All samples were taken during the second week in November and stored in the laboratory 
at -20°C until further use. Morphometric analysis of fully developed Picea abies needles were done 
by using a computer - aided image system (Optimas 5.0 programme) in the laboratory of Applied 
Botany and Physiology, Agronomy Department of Biotechnical Faculty, University of Ljubljana, 
under the supervision of MSc. T. Sinkovič. For calculation of needle volume (V) the equation of 
RIEDERER et. al. (1988) was used: V (mm3) = 0,208 x (projected needle area) 1•353 • Thy age of trees 
was estimated by measurments of tree rings from the cores taken at 0,4 m using a dendrocronological 
table LINTAB with 1/100 mm accuracy (BOŽIČ & LEVANIČ 1998). 
To determine the genetic structure of the sampled trees, endosperms from six seeds per tree 
were analysed. In the mire's spruce sub-population there was no fructification during analysis, so 
dormant winter buds were used for analysis. The genetic variability of the two spruce sub-populations 
was analysed by means of isozyme gene markers using starch electrophoresis as the separation 
method. Enzyme extraction from buds and seeds (endosperm) was performed according to RHODES 
(1977). Electrophoresis conditions, staining, and genotyping followed KONNERT & MAURER 
(1995). The following isozyme gene loci were investigated: Aco-A, Gdh-A, Got-A, Got-B, Got-C, 
ldh-A, ldh-B, Lap-B, Mdh-A, Mdh-B, Mdh-C, Mnr-A, Mnr-C, Pgi-A, Pgi-B, Pgm-A, Skdh-A, 6-
Pgdh-A, 6-Pgdh-B, 6-Pgdh-C. The results of isozyme electrophoretic analyses were evaluated by 
relative allele and genotype frequencies calculated on different gene loci (recalculated from BOŽIČ 
1997). The genetic structure was described for all loci for which polymorphisms were found in at 
least one spruce sub-population and compared with respect to the average actual heterozygosity Ha 
calculated as the arithmetic mean of single locus values (NEI 1973). The genetic differentiation 
between the sub-populations was studied with chi-square tests of homogeneity among allele 
frequencies for particular gene loci at the level a = 0,05 and estimated with allelic and genotipic 
genetic distances (d
0
) proposed by GREGORIUS (1974). 
Results and discussion 
On the bog site plot "Mire" all 35 studied spruce trees grow on hydromorphic organic soils 
which have the peat horizon lying over wet, softy and gelatinous lake sediments (gyttia). Thickness 
of the peat layer is from about 60 cm to over one meter. The reaction of its peat is very acidic 
20 Acta Biologica Slovenica, 46 (!), 2003 
(measured values of pH(Caq) are between 2,88 and 3,18). Soil is classified as ombrotrophic form 
of the middle deep to deep subtype of peat acrohistosol and according to FAO (1989) soil classification 
it belongs to soil unit of Fibric Histosols. On this site spruces have extremely bad growth conditions. 
The ages (after BOŽIČ & LEVANIČ 1998) of spruces on the plot "Mire" at the height of 0,4 m 
varied between 65 and 142 (in average 95) years, at breast height they had diameters from 6 to 19 (in 
average 12) centimetres and tree heights from 4 to 13 (in average 8) metres. Analysed needles had 
iengths from 7,2 to 14,3 (in average 10,5) milimeters and volumes from 0,8 to 3,9 (in average 1,9) 
mm3. 
On the plot "Edge" soils have developed on mixed moraine iying over lake chalk. Mixed moraine 
is composed by unconsolidated material of limestone, dolomite, mar!, cherts, shaies and sandstones. 
On this parent material heterogenous, mostly dystric soils have developed. They are covered with 
mainly acidophilic vegetation. Soil sounding discovered that 14 sampled trees grow on dystric 
cambisois, 16 on podzols and 5 on gieysois. Properties ofthese FAO (1989) soil units were examined 
closely by three representative soil profiles. Podzols and dystric cambisois have in upper half meter 
depth very acid reaction (pH 3,02--4,37) and very low base saturation levels (BS 3,15 %-34,17 %). 
Gleysols are under the influence of groundwater and lake ckalk and are less dystric (pH 3,57--4,9; 
BS 43,9 %-53,8 %). These soils are rather deep, have suitable loamy texture and moisture regime 
and are rather fertile for spruce. The age of studied spruces at the height of 0,4 m in the plot "Edge" 
varied between 87 and 147 (in average 116) years, they had diameters at breast height from 31 to 60 
(in average 46) centimetres and tree heights from 27 to 36 (in average 31) metres. Analysed needles 
had iengths from 9,9 to 17,5 (in average 13,2) milirneters and volumes from 1,0 to 6,3 (in average 
3,2) mm3 (modified after BOŽIČ 2000 and BOŽIČ & LEVANIČ 1998). 
Also according to (BOŽIČ & LEVANIČ 1998) true ages of seiected trees on the plot "Mire" 
were in average about 40 to 50 years greater and on the plot "Edge" from 30 to 35 years greater as 
in the sampling height 0,4 m. The age of the oldest trees is about 200 years, which means that trees 
in the two research plots started to grow at least 50 years before the first huge clearcuts and artificial 
regeneration with planting was started on the Pokljuka plateau in the years 1848-1859 (SMOLEJ 
1984). 
Between two sampled groups of trees on the research plots "Mire" and "Edge" remarkable 
differences in aileie and genotype frequencies were noted. The most distinct differences in frequency 
of alleles and genotypes was observed at iocus Mnr-A for alleles Mnr-A
2
, Mnr-A
4
, and genotypes 
Mnr-A
22 
and Mnr-A
24
• The homozygote type was more common in sampled trees from the plot 
"Edge" whiie the hetrozygote type was more common in sampled trees from the plot "Mire". The 
heterozygote types Mnr-A24 and Mnr-A 34 reached much higher values in sampled group of trees on 
the mire site than on the edge site. The reverse situation was occured at homozygote type Mnr-A
22 
which was much more frequent in group of trees "Edge" than in "Mire" group of trees (45,7 % vs. 
11,4 %, respectively). In average, a higher heterozygosity (H.) on the plot »Mire« then on the plot 
»Edge« was observed. If the most differentiated iocus in heterozygosity Mnr-A is taken in 
consideration, trees sampled on the »Mire« had much higher heterozygosity as sampied group of 
trees on the »Edge« plot (77,1 % and 40,0 %, respectively). Results of the contingency x2 tests for 
homogeneity of genetic structure between sub-populations has shown that ailele frequencies 
significantiy differed at the leve! a = 0,05 in two gene loci (chi-square = 8,13; 3 d.f. for Lap-B) and 
(chi-square = 5,27; 1 d.f. for Mnr-A) . At this two gene ioci, also the genetic distances between 
»Edge« and »Mire« group of trees were very high with ailelic distances of 17,2 % for Lap-Band 21 
% for Mnr-A and genotypic distances of 25,8 % for Lap-B and 37,2 % for Mnr-A. Remarkable 
differences of allele and genotype frequencies were also noted for ioci Aco-A, Got-C Lap-B, Mnr-A 
G. Božič, M. Urbančič: The morphological and genetical characterisation ... 21 
and 6-Pgdh-C. Only for gene locus Gdh-A, the genetic structures of the two sub-populations were 
identical and the genetic distances equal with zero. The single-locus values mean value of Gregorius 
allelic and genotypic genetic distance (5,9 % and 10,7 %, respectively), further confirms that the 
genetic differences between the sampled spruce sub-populations on the "Mire" and "Edge" plots 
are comparatively high. For the population genetic structure of Norway spruce a relative high levels 
of differentiation between the sub-populations were observed. 
Genetic differentiation between the sub-populations from trne different forest site conditions 
may be a consequence of different selection processes to which spruce in this location has been 
exposed. As spruces from the mire site with organic (peat) soils were 3,8 times lower and thinner as 
spruces in similar age from the site with mineral soils, is there a great opportunity for selection to 
act. Since temperature and oxygen availability in the soil are closely associated with soil moisture, 
further experiments are required to understand the environmental factors which are most directly 
associated with the selection factors which acting on the forest sites with mainly automorphic (out 
of undergroud water reach) and mineral or hydromorphic (strong influence of undergroud water) 
peat soils. 
The very high allelic and genotypic distances observed in loci Lap-B and Mnr-A indicated that 
specific site conditions could have an impact to these two loci. The response to site conditions could 
affect the observed locus directly, or any other loci tightly linked to them, as well as any coadapted 
gene complexes that they mark (HAMRICK 1989). As the isoenzyme variants (allozymes) may be 
both, selectively equivalent (i.e. neutral) and adaptive, depending on the respective environmental 
conditions under which the particular population is living (BERGMANN 1991), present results would 
allow to hypothesise that decrease in frequency of allele MNR-A
2 
from 65,7 % observed in forest site 
with mainly automorphic and mineral soils to 44,3 % on mire site, may have an adaptive origin and 
could be connected with increased leve! of heterozigosity of individuals existing in environmental site 
conditions with hydromorphic soils because only heterotzigotic individuals can be better adapted to 
exteme site conditions. 
Althought the obtained results are incomplete, also because of possible sampling mistakes on 
account of the large size of analysed samples, and do not allow us to infer any certain conclusions 
on the possible adaptive role of the enzyme systems under analysis, they verify the existence of 
morphological and genetical differentiation between spruce sub-populations within single population 
associated with variation of environmental conditions resulting from differences in site conditions. 
Conclusions 
The research plots were established on Pokljuka in sites within the area of natura! distribution of 
Norway spruce. On both plots dominant from about 120 to 200 years old spruces were chosen. The 
oldest trees were growing at least 50 years before the first huge clearcuts and artificial regeneration 
with spruce seedlings on Pokljuka plateau in the middle of 19th century began so the analysed spruce 
sub-populations in the Šijec area can be regarded as autochthonous. 
Striking differences in soil and other site conditions between the research plots have been reflected 
in the spruce's growth. Spruces from the plot "Edge" were in average 3,8 times taller and thicker as 
trees from the plot "Mire". Needle length and needle volume of spruces from the site with better 
growing conditions were 26 % longer and 68 % bigger than of the trees from the peat bog. Different 
site conditions resulting from soil conditions were attributed particularly to the tree growth and its 
morphological attributes. 
22 Acta Biologica Slovenica, 46 (1), 2003 
Environmental conditions resulting from differences in site conditions could have an impact to 
the genetic structures of sub-populations growing in such habitats. The obtained results would also 
allow to hypothesise an adaptive response of the allele Mnr-A
2 
to different environmental conditions 
resulting from differences in site conditions. 
Aknoweledgement 
The authors acknoweledge the generous support of Dr. Monika Konnert and MSc. Tomaž Sinkovič 
for their guidance in laboratory works. Thanks are also due to. Dr. Rojka Kraigher for helpful 
comments on the manuscript. 
Povzetek 
Raziskovalni ploskvi sta bili osnovani v mraziščnem delu Pokljuke, v okolici visokega barja 
Šijec, v dveh smrekovih subpopulacijah, na njenih naravnih rastiščih. Na vsaki ploskvi smo za 
raziskavo izbrali 35 vitalnih, nadraslih oziroma soraslih smrek različnih starosti (od 120 do 200 let). 
Najstarejša drevesa so rastla vsaj 50 let pred prvimi velikopovršinskimi goloseki in sadnjami 
smrekovih sadik sredi 19 stoletja, zato menimo, da so analizirane smreke še avtohtone. 
Ploskev »Mire«, ki jo porašča asociacija barjanskega smrekovja (Sphagno - Piceetum s. lat. R. 
KUOCH 1954 emend. ZUPANČIČ 1981), leži na šotnem barju. Njena srednje globoka do globoka 
šotna tla po FAO (1989) razvrstitvi spadajo v talno enoto fibrični histosoli. Na njej vladajo za smreko 
skrajno neugodne rastiščne razmere. 
Ploskev »Edge« smo osnovali na robu barja. Njeno rastišče porašča rastlinska združba smreke 
in smrečnega resnika (Rhytidiadelpho lorei - Piceetum (M. WRABER 1953 n.nud.; ZUPANČIČ 
(1976), 1981 em. 1999). Na njem so se na mešani moreni, ki prekriva jezersko kredo, razvili podzoli, 
distrična rjava tla in hipogleji. Po mednarodni FAO (1989) klasifikaciji jih uvrščamo v talne enote: 
podzoli, distrični kambisoli in glejsoli. Ta tla so distrična (imajo zelo kisle reakcije in zelo nizke 
stopnje nasičenosti z izmenljivimi bazami) in so vsaj pol metra globoka. Imajo ugodno ilovnato 
teksturo in vodni režim, tako da so za smreko dokaj rodovitna. 
Analizirane smreke z roba barja so bile v povprečju 3,8- krat višje in debelejše ter so imele v 
povprečju za 25,7 % daljše in za 68,4 % volumensko večje iglice od smrek s šotnega barja. Te 
razlike v rasti in v morfoloških lastnostih smrek so po našem prepričanju odraz velikih razlik v 
talnih in drugih rastiščnih razmerah med ploskvama. 
Te razlike v okoljskih razmerah pa bi lahko vplivale tudi na genetski strukturi analiziranih 
smrekovih subpopuacij. Izidi naših analiz genetske variabilnosti subpopulacij smreke z metodo 
izoencimske gelske elektroforeze, ki je zajemala uporabo škrobnega gela in ekstrakcijo izoencimov 
iz popkov in endosperma semen, podpirajo to domnevo. Razlika v heterozigotnosti med 
subpopulacijama na genskem lokusu Mnr-A nakazuje možni učinek preživetvene selekcije. 
G. Božič, M. Urbančič: The morphological and genetical characterisation ... 23 
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Variation in European Populations of Forest Trees. - J. D. Sauerlander's Verlag: 67-78, 
Frankfurt am Main. 
Božič, G. 1997: Genetska variabilnost dveh subpopulacij domnevno avtohtone smreke (Picea abies 
(L.) Karst.) na Pokljuki. Magistrsko delo. Univerza v Ljubljani, Biotehniška fakulteta, 
Oddelek za agronomijo, Ljubljana, 83 p. 
Božič, G., 2000: Dendrokronološka, morfološka in populacijskogenetska struktura smreke (Picea 
abies (L.) Karst.) na stalni raziskovalni ploskvi Šijec na Pokljuki. (In: H. Kraigher (ed.), I. 
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fiziološke parametre gozdnega drevja v izbranih gozdnih ekosistemih, sestojnih tipih in 
razvojnih fazah gozda. - Strokovna in znanstvena dela, Gozdarski inštitut Slovenije, 118: 
34--46, Ljubljana. 
Božič, G., LEVANIČ, T. 1998: Starost in morfološke značilnosti domnevno avtohtone smreke (Picea 
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gozd, Zbornik referatov Gorski gozd, Biotehniška fakulteta, Oddelek za gozdarstvo in 
obnovljive gozdne vire: 243-254, Ljubljana. 
Božič, G., URBNČIČ, M. 2001: Influences of the soils on the morphological characteristics of an 
autochthonous Norway spruce on the Pokljuka plateau. Glasnik za šumske pokuse, 38: 
137-147, Zagreb. 
FAo, 1989: FAO-Unesco Soil Map of the World.- FAO, Unicef, Intemational soil reference and 
information centre, Wageningen, 138 p. 
GREGORIUS, H. R. 1974: Genetischer Abstand zwischen Populationen. I. Zur Konzeption der 
genetischen Abstandsmessung. Silvae Genetica 23: 22-27. 
HAMRICK, J. L., 1989: Isozymes and the Analysis of Genetic Structure in Plant Populations. (In: D. 
E. Soltis (editor), P. S. Soltis (editor) Isozymes in Plant Biology. -Advance in Plant Series, 
Washington State University, 4: 87-105. Washington. 
KoNNERT, M., MAURER, W. 1995: Isozymic Investigations on Norway Spruce (Picea abies (L.) Karst.) 
and European Silver Fir (Abies alba Mill.). A Practical Guide to Separation Methods and 
Zymogram Evaluation, 79 p. 
NEI, M. 1973: Analiysis of gene diversity in subdivided populations. Proc. Nat. Acad. Sci. USA, 70, 
pp. 3321-3323. 
RHODES, M. J. C., 1977: The extraction and purification of enzymes from plant tissues. Proceedings 
of the Biochemical Society, 14, pp. 254-248. 
RIEDERER, M., KuRBASIK, K., STEINBRECHER, R., Voss, A. 1988: Surface areas, lenghts and volumens 
of Picea abies (L.) Karst. needles: detrermination, biological variability and effect of 
environmental factors. Trees, 2: 165-172. 
SMOLEJ, I. 1984: Prispevek k zgodovini blejskih gozdov. Kronika, Iz zgodovine Bleda, 32, pp. 145-154. 
ZUPANČIČ, M. 1999: Smrekovi gozdovi Slovenije. Dela, 4. r., SAZU, 36, Ljubljana: 222 p. 
24 Acta Biologica Slovenica, 46 (1), 2003 
Appendices 
Appendix 1: The tree age on the height of 0,4 m, diameter at 1,3 m, tree height, needle length and 
needle volume of sampled spruce trees on the research plots "Mire" and "Edge" 
Parameter Plot "Mire" Plot "Edge" 
Age Min. (years) 65 87 
Max. (years) 142 147 
Mean (years) 95 116 
Diameter Min. (cm) 6 31 
Max. (cm) 19 60 
Mean (cm) 12 46 
Height Min. (m) 4 27 
Max. (m) 13 36 
Mean (m) 8 31 
Needle length Min. (mm) 7,2 9,9 
Max. (mm) 14,3 17,5 
Mean (mm) 10,5 13,2 
Needle volume Min. (mm3) 0,8 1,0 
Max. (mm3) 3,9 6,3 
Mean (mm3)· 1,9 3,2 
Appendix 2: The distribution of needle length and needle volume values of sampled spruce needles 
per each research plot 
20 ..------------------, 
E 1s t-- ---+-------- ----t 
.s 
f-
:i: 
(9 
z 
Lij 
Lij 
o 
14 ----·-·,-----''-----,_ 
tlf 10 t-----~---i 
z '----~ 
f 5 
.s 
~ 4 
:, 
g 3 
Lij 
o 
UJ 2 
UJ 
z 
8 -----
Edge 
Edge 
Mire 
PLOT 
Mire 
PLOT 
I Min.-Maks 
D 25%-75% 
• Mediana 
I Min.-Maks. 
D 25%-75% 
• Mediana 
G. Božič, M. Urbančič: The morphological and genetical characterisation ... 25 
Appendix 3: Genotype frequencies at the 20 analysed enzyme gene loci of sampled spruce trees 
on the research plots "Mire" and "Edge" 
Locus Genotype Plot Plot Locus Genotype Plot Plot 
"Mire" "Edge" ''Mire'' "Edge" 
ACO-A 11 0,086 0,086 MDH-C 24 0,086 0,000 
12 0,200 0,343 44 0,914 1,000 
22 0,714 0,571 
MNR-A 12 0,057 0,000 
GDH-A 12 0,029 0,029 22 0,114 0,457 
22 0,971 0,971 24 0,600 0,400 
34 0,114 0,000 
GOT-A 12 0,000 0,029 44 0,114 0,143 
22 1,000 0,971 
MNR-C 12 0,057 0,000 
GOT-B 12 0,000 0,029 22 0,943 1,000 
22 1,000 0,971 
PGI-A 22 1,000 1,000 
GOT-C 22 0,171 0,086 
24 0,514 0,429 PGI-B 22 0,000 0,086 
25 0,029 0,029 23 0,600 0,514 
44 0,257 0,429 33 0,400 0,400 
45 0,029 0,029 
PGM - A 22 0,829 0,857 
IDH-A 23 0,029 0,114 23 0,143 0,114 
33 0,971 0,886 33 0,029 0,029 
IDH-B 22 1,000 1,000 SKDH-A 13 0,029 0,057 
22 0,143 0,086 
LAP-B 11 0,000 0,029 33 0,829 0,800 
13 0,000 0,029 35 0,000 0,029 
14 0,029 0,086 36 0,029 0,029 
33 0,000 0,029 
34 0,114 0,200 6PGDH-A 12 0,000 0,029 
36 0,000 0,029 22 0,971 0,971 
44 0,571 0,429 23 0,029 0,000 
46 0,229 0,143 
47 0,029 0,000 6PGDH-B 22 0,400 0,486 
66 0,029 0,029 25 0,486 0,343 
35 0,000 0,029 
MDH-A 22 1,000 1,000 55 0,114 0,143 
MDH-B 12 0,029 0,000 6PGDH- C 15 0,000 0,029 
22 0,914 0,971 22 0,457 0,314 
23 0,057 0,029 25 0,257 0,400 
26 0,029 0,000 
55 0,257 0,257 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 27 - 33 
Sprejeto (accepted): 2003-10-30 
Impact of air pollution on the mitotic activity in meristematic cells in shallot 
(Allium cepa L. var. ascalonicum) 
Vpliv onesnaženega zraka na mitotsko aktivnost v meristemskih celicah šalotke 
(Allium cepa L. var. ascalonicum) 
Erika GLASENČNIK1, Dieter GRil.V, Maria MULLER3, Cvetka RIBARIČ-LASNIK1 & Franc BATIČ2 
11 ERICo Velenje; Environmental Research & Industrial Co-operation Institute, Koroška 58, 3320 
Velenje, Slovenia, E-mail: Erika.Glasencnik@erico.si 
21 Biotechnical Faculty, Agronomy Department, Applied Botany, Ecology and Plant Physiology, 
Jamnikarjeva 101, 1000 Ljubljana, Slovenia. 
31 Institut fiir Pflanzenphysiologie, Universitat Graz, SchubertstraBe 51, A-8010 Graz, Austria. 
Abstract. Test shallot plants Allium cepa L. var. ascalonicum were exposed to 
field conditions in research plots in the most polluted areas in Slovenia in the 
vegetation season in 1999. The intention of this research was to evaluate the influence 
of air pollution on mitotic activity in meristematic tissues of root tips of bioindication 
plants. At each sampling site the mitotic activity was determined under field conditions 
and in a pot experiment. The maturated bulbs were collected from the field and after 
winter dormancies cytogenetic analyses were run on them in a lab. Root tips were 
fixed in Clark's fixative and afterwards stained with Schiff's reagent. 
Significant differences in the mitotic activity in different sampling plots in pot 
experiments were found. The results showed the presence of cytotoxic substances at 
chosen sampling sites which caused the decrease of mitotic cell division. 
Key words: Allium cepa L. var. ascalonicum, cytotoxicity, mitotic index, 
environmental pollution. 
Izvleček. Izbrane testne rastline šalotke (Allium cepa L. var. ascalonicum) so 
bile v času vegetacijske dobe izpostavljene naravnim razmeram na vzorčna mesta v 
bližini virov onesnaženja na ozemlju Slovenije. Namen raziskave je bil oceniti vpliv 
onesnaženega zraka na mitotsko aktivnost v meristemskih celicah koreninskih 
vršičkov bioindikatorskih rastlin. Vpliv onesnaženega okolja smo analizirali pri 
rastlinah izpostavljenih v lončnem in poljskem poskusu. 
Posušene in očiščene čebulice so bile po zimski dormanci izpostavljene 
v laboratoriju, koreninski vršički so bili fiksirani v Clarkoven fiksativu in obarvani s 
Schiffovim reagentom. 
Ugotovljeno je bilo, da se pojavljajo statistično značilne razlike med posameznimi 
lokacijami pri rastlinah izpostavljenih v lončnem poskusu. Rezultati kažejo, da so 
na izbranih lokacijah citotoksične substance, ki vplivajo na mitotsko aktivnost celic 
izpostavljenih rastlin. 
28 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
Ključne besede: Allium cepa L. var. ascalonicum, citotoksičnost, mitotski indeks, 
onesnaženost okolja. 
Besides the analysis of chromosomal aberrations to evaluate genotoxicity a mitotic index is 
most frequently used as a parameter to evaluate cytotoxicity to show effect of chosen substances in 
cytogenetic bioindication. Mitotic activity decreases while the influence of environmental stress is 
stronger (FlSKESJ0 1994, PARADIŽ 1996, 1998, PARADIŽ & al. 1996, SMAKA-K.!NCL & al. 1996, BAVCON 
& al. 1999, FREISZMUTH & al. 2000, Mi.iLLER & al. 2000, PAVLICA & al. 2000, PARADIŽ & DRUŠKOVIČ, 
2001). In the Alliaceae family shallot is not commonly used for bioindication (PAVLICA & al. 1997, 
2000, ZOLDOŠ & al. 1997). 
Test shallot plants (Allium cepa L. var. ascalonicum) were exposed to field conditions at research 
plots of differently polluted areas of Slovenia in 1999: in the Šalek Valley the most polluted sites are 
either those which are very close to the thermal power plant (Veliki Vrh, ash dump of the Šoštanj 
Thermal Power Plant) or those which are highly polluted due to climatic conditions and topography 
but more distant from the thermal power plant (Zavodnje) or sites with relatively clean air in relation 
to the wind direction from the thermal power plant (Arnače, Škale); in the Zasavje region (Kovk) 
and in the Upper Meža Valley (Žerjav) two very polluted sites were taken for one vegetation period. 
At each sampling plot plants were growing under field conditions and pot experiment (see Material 
and methods for a short description of growing conditions). Mitotic activity was later determined in 
a laboratory. 
In this study we attempted to determine the influence of air pollutants on plants (pot) and indirect, 
accumulated effects in soil exposed to environmental pollution on the cytogenetic material of the 
shallot. The aims of the study were to make a comparison among the sampling plots regarding the 
mitotic activity to define cytotoxic effect arising from environmental pollution and to confirm the 
applicability of chosen method for relatively fast, simple and cost-effective determination of 
cytotoxicity of polluted air. In comparison with the pots experiments (where plants were exposed 
primarily to air pollution) we expected decrease of mitotic activity in plants in field conditions, 
since these shallots were exposed to stress due to air and soil pollution. 
Materials and methods 
For every sampling site we used approximately equal-sized shallot bulbs (Allium cepa L. var 
ascalonicum Escalote de Jersey' , 2n=l6) . 
The choice of sampling sites and sampling procedure was carried out as described by GLAsENČNIK 
& al. (2002) and the method for cytogenetic analyses was performed as described by FrsKEsJo (1994) 
and detailed by GLASENČNIK & al. (2002). 
In the pot experiment plants were exposed in 15-litre self-watering pots (five per site, three 
bulbs per pot) and for experiment under field conditions plants were put clirectly in field soil (six 
bulbs per site) near the pots in the end of May 1999. After maturation all plant material was removed 
from sampling plots and bulbs were stored under dry conditions (4°C) for two months. 
The soil from pots and field soil were analysed. The analysis of soils was carried out after 
homogenisation (ISO 11464) and digestionation (ISO 11466) using the methods for spectroscopy 
(ICP-MS, ET-AAS, CV-AAS). 
Cytogenetic analyses were run after winter dormancies. Root tips were treated with using Feulgen 
"squash" technic. A mitotic activity was determined in five bulbs per sampling site, one slide per 
bulb and 2000 cells per slide. A mitotic index (MI) is a percentage of dividing cells scored. 
E. Glasenčnik, D. Grili, M. Milller, C. Ribarič-Lasnik, F. Batič: Irnpact of air pollution on the ... 29 
All statistical treatments were performed using the Statistica for Windows 5.5 software package 
(STATS0Ff 1999). Differences among sampling sites were tested using Kruskal-Wallis ANOV A. Cross 
comparisons between means were performed using the Mann-Whitney U test. 
Results 
Significant differences were found in MI among the sampling sites for shallots grown in pots 
(Kruskal-Wallis ANOVA: H <6, 47l = 19,49, p = 0,003; Fig. lb), while for plants grown in field soils no 
significant differences could be found (H <5•41> = 7,68, p = 0,17; Fig. la) . Detailed statistics of pair-
wise comparisons using Mann-Whitney U test are presented in Table 1. 
The analysed plants material in pot experiment showed that the MI was between 3 and 14 %. 
The lowest value of MI was determined in Arnače near Velenje (3,7±2,5 %), a very low value was 
also at Veliki Vrh and at Kovk (6,1±2,6 % and 6,1±1,6 %), the highest was at Žerjav (10,93±3,5 %). 
The results of shallot grown under field conditions showed that the value of MI was between 6 
and 10 %, the lowest was determined at Veliki Vrh (6,4±3,7 %), the highest at the sampling site of 
Žerjav (10,93±3,5 %). 
A:Soll 
22 
20 
18 
16 
14 
~ 12 
~ @~~~® :ii 10 
4 
Arnače Ash dump Velik Vrh Zerjav • Mean 
Kovk Vnajnarje Zavodnje Skale D±SE 
Sampllng site 
I±1,96'SE 
B: Pots 
22 
20 
~ 
18 
16 
14 
-- ~ ~ 12 :ii 10 1® 
,@~ ~ 
~ 
Arnače A5h dump Velik Vrh Zerjav c Mean 
Kovk Vnajnarje Zavodnje Skale D ±SE 
Sampllng site 
::C ±1 .96'SE 
Figure la, b: Mitotic activity (%) in root tip cells of shallot, exposed to field conditions in soils (A) 
and in pots (B) at eight different localities in Slovenia in the vegetation season in 1999. 
30 Acta Biologica Slovenica, 46 (1), 2003 
Table 1: Significance of differences in mitotic activity between sampling sites (Mann-Whitney 
U-test). 
Soil 
1999 ARNAČE KOVK ASH ŠKALE VNAJNARJE VELIKI ZAVODNJE ŽERJAV 
MI DUMP VRH 
ARNACE / / / / / / / 
KOVK * / NS NS NS NS NS 
ASHDUMP NS NS / / I I I 
SKALE ** ** NS NS * NS NS 
VNAJNARJE * * NS NS NS / NS 
VELIKI VRH NS NS NS * * NS NS 
ZAVODNJE I / / / I I NS 
ZERJAV * I NS NS NS NS * 
***: p<0.001; **: p<0.01 , *: p<0.05; NS: not significant. 
Discussion 
The inhibition of mitotic activity was often used for tracing cytotoxic substances. Cytotoxicity 
was defined as a decrease in the mitotic index and as an increase in the frequencies of chromosomal 
aberrations (FrsKESJb 1994). The analysis of chromosomal aberrations for evaluation of genotoxicity 
was already done (for results see Gt.ASENČNIK & al. 2002). In our study a decrease in the mitotic 
index of sballot root meristems was found depending on the chosen location in pot experiment with 
probability p<0,01. We used the same soil for all pots and therefore plants grown in pots were 
exposed only to air pollutants. Analysis of chromosomal aberrations showed no significant differences, 
but the frequency of chromosomal aberrations was at least two-fold higher in comparison to published 
<lata for a control environment (3%, e.g. VIDAKOVIČ & al. 1993). Therefore air pollution had a strong 
influence on mitotic activity and also on the frequency of chromosomal aberrations. These results 
are in accordance with the results of fumigation experiments of a research group in Graz: ozone 
treatments consistently increased the rate of chromosomal aberrations, the Ml remained unchanged, 
with fumigation with SO
2 
and ~S the MI was lower and the chromosomal aberrations were also 
consistently increasing (MDLLER & al. 2000). 
Arnače sampling plot showed, against expectation, a very small mitotic activity, though the 
finding was in accordance with the recent results from other research fields (SVETINA 1999, KuooNIČ 
& STROPNIK 2001 ), which revealed elevated levels of Co, Cr and Ni in meadow soil and higber Jevels 
of heavy metals in vegetables and forage were found in comparison with other sampling sites in the 
bottom of the Šalek Valley. The highest frequency of chromosomal aberrations (for details see 
Gt.ASENČNIK & al. 2002) in our pot experiment confirmed the presence of stress inducing factor at 
this sampling site. 
Since the cleaning device was built on the Šoštanj Power Plant in 1995, the emissions have been 
drastically reduced, which has reflected in an improvement of ecosystem conditions (RIBARIČ-LASNIK 
& al. 1999b). This is nota case at some sites, which are stili polluted due to their position or due to 
accumulation of pollutants in the past. At the sampling site of Veliki Vrh just facing power plant 
chimneys and the prevailing wind direction a high concentration of air pollutants is directly brought 
from chimneys to this highly exposed site. This was confirmed by severa! studies (e.g. high levels of 
Cd in soil and vegetables KuooNIČ & STROPNIK 2001, high levels of Cd, As and Hg in fungi AL 
SAYEGH-PETKOVŠEK & al. 2002, the most acid precipitation (pH<5,6) SEVŠEK & al. 2000). The low 
value of MI deterrnined and the highest frequences of chromosomal aberrations (see Gt.ASENČNIK & 
al. 2002) at that sampling site were in agrement with the above mentioned studies. 
E. Glasenčnik, D. Grili, M. Miiller, C. Ribarič-Lasnik, F. Batič: Impact of air pollution on the ... 31 
Considering the extreme pollution with heavy metals in the Upper Meža Valley (see RIBARIČ­
LASNIK & al. 1999a and references therein) the high value of MI in both experimented plots in 
žerjav were surprising because we expected a Iow value of MI at this site due to extreme value of 
heavy metals in the surroundings (e.g. 4,99±6,92 mgkg·1Cd, 1496±1071,02 mgkg·1 Pb, 498,22±546,0 
mgkg·' Zn KuooNIČ & STROPNIK 2001). More investigations are needed at this research plot to verify 
the results. · 
Contrary to our expectation no significant differences in the mitotic activity could be found 
among Iocalities in the experiment under field conditions. We found significant differences in the 
frequency of chromosomal aberrations in different sampling sites conceming shallot grown in the 
field (see GLASENČNIK & al. 2002). The results are in accordance with the statement of FlsKESJ• 
(1997) saying that when chromosomal aberrations occur, there are almost always some growth 
restrictions. These results indicated that the influenece of chronic exposure (soil) is far more genotoxic 
while the acute exposure (pots) is more cytotoxic. Results obtained with our investigation were 
confirmed also with the results of an assessment of cytogenetic hazard for plants caused by highway 
traffic: the mitotic activity decrease and the frequency of chromosomal aberrations increase with 
the duration oftraffic influence in the onion (Allium cepa L.) exposed in short-lasting in situ exposure 
near highways in Slovenia (PARADIŽ & DRUŠKOVIČ 2001 ). In spruce and other perennial plants growing 
spontaneously on experimental sites near the highways as bioindicators of long-lasting exposure, 
cytogenetic damages increased in correlation with the duration of traffic, but only at sites with more 
than 20 years' traffic influence (ibid). 
Nevertheless, we also found out that the values of MI in the field conditions and in the pot 
experiment were lower than the average values from the literature for the control (10-15 %; e. g. 
Oud & RICKARDS 1999; 5-10%; e. g. PARADIŽ 1996) and these results showed a decrease in mitotic 
activity and a cytotoxic effect of chosen sites. 
Considering the present results we can conclude that the polluted air does have a cytotoxic 
effect on mitotic activity with the decrease of MI and that more polluted sites have a higher effect on 
mitotic activity. We can also say that the polluted air and soil together probably had a stronger effect 
at the majority of investigated plots. Therefore we conclude that the mitotic activity is the appropriate 
testing system for determination of MI in shallot root tip meristematic cells for quick, easy and 
useful estimation of cytotoxic effect of environmental pollution. 
Acknowledgements 
This work was financially supported by the Šoštanj Therrnal Power Plant and by the Ministry of 
Education, Science and Sport of the Republic of Slovenia. We owe a deep debt of gratitude to ali 
colleagues who helped us with many very useful comments and suggestions or assisted us at field 
work: Karin Savinek, Melita Stropnik, Meta Zaluberšek, Leopolda Pačnik, Andreja Bienelli, Stane 
Vanovšek, Nataša Kopušar, Boštjan Pokomy for statistical advice (ERICo Velenje), Gabrijel Leskovec 
and Tomaž Sinkovič (Biotechnical Faculty, Department for Applicative Botany). Colleagues from 
the Institute for Plant Physiology, Graz, introduced us to the Allium test. Finally, we are grateful to 
Marta Pačnik, who helped us linguistically, and to my family for support. 
32 Acta Biologica Slovenica, 46 (1), 2003 
References 
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BAVCON J., DRUŠKOVIČ B. & N. GOGALA 1999: Effects of UV-B radiation on growth and Initotic activity 
in the norway spruce (Picea abies (L.) Karst.) Acta Biologica Slovenica 42 (2): 9-16. 
FlsKESJ0 G. 1994: Technical Methods Section -Allium Test II. Environmental Toxicology and Water 
Quality 9: 235-241. 
F1sKEs10 G. 1997: Allium test for screening chemicals; evaluation of cytological parameters. In: 
Wang W., J. W. Gorsuch & J. S. Hughes (ed.): Plants for Environmental studies. CRC 
Press LLC, New York, pp. 307-333. 
FREISZMUTH D., M0LLER M. & H. GuTTENBERGER 2000: The mitotic index in the female gametophyte 
of Picea abies. In: Guttenberger H., Borzan Ž., Schlarbaum S. E ., Hartman T. P. V. (ed.): 
Cytogenetic studies of forest trees and scrubs - review, present status and outlook on the 
future. IUFRO Cytogenetics Working Party, Graz, pp. 51-56. 
GLASENČNIK E. , C. RIBARIČ-LASNIK, D. GRILL, M. M0LLER & F. BATIČ 2002: Impact of air pollution on 
genetic material of the shallot (Allium cepa L. var ascalonicum), exposed in the vicinity of 
major Slovene local emission sources in 1999. Phyton (Hom) 42 (2): 237-250. 
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Šalek Valley. ERICo Velenje, DP-513/1999, Velenje, pp. 183. (unpublished). 
MDLLER M., M . TAusz, A. WoNISCH, H. GUTTENBERGER & D. GRILL 2000: Evaluation of chromosomal 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 35 - 39 
Sprejeto (accepted): 2003-10-30 
Growth and root respiration of C4 plants under CO2 enrichment 
Rast in dihanje korenin C4 rastlin pri povečani koncentraciji CO
2 
Irena MAČEK, 1 Hardy PFANZ,2 Dominik VODNIK1 
1University of Ljubljana, BF, Agronomy Dep., Jamnikarjeva 101, SI-1001 Ljubljana, Slovenia; 
E-mail: irena.macek@bf.uni-lj.si 
2Institut fiir Angewandte Botanik, Universitat Essen, D-45117 Essen, FRG 
Abstract. Respiratory measurements of apical root parts of severa! C4 plant 
species (Echinochloa crus-galli var. crus-galli, Setaria pumila and Zea mays DK 
312 (Dekalb, USA) subjected to an elevated CO
2 
regime during growth in clirnatic 
chambers or at natura! CO
2 
springs were performed. 
Biomass production, root respiratory potential and root respiration of Echinochloa 
was not significantly changed by high atmospheric CO
2 
treatment in the climatic 
chambers, compared to ambient CO
2 
treatment. 
Root respiratory potential of C4 weeds (Echinochloa crus-galli and Setaria 
pumila) growing in natura! CO
2 
spring area was not significantly affected by extremely 
high CO
2 
in the rhizosphere. Yet, respiratory potential of one and a half month old 
sown maize seedlings was significantly lower in the roots exposed to naturally elevated 
CO
2 
concentrations. 
Key words: root respiration, ETS activity, respiratory potential, C4 plants, 
Echinochloa crus-galli, Zea mays, Setaria pumila, elevated CO
2
, natural CO2 springs, 
co2 mofette 
Izvleček. V pričujoči raziskavi smo merili dihalno aktivnost apikalnih delov 
korenin nekaterih C4 vrst (navadne kostrebe Echinochloa crus-galli var. crus-galli, 
sivozelenega muhviča Setaria pumila in koruze Zea mays DK 312 (Dekalb, ZDA), 
izpostavljenih povečani koncentraciji CO
2 
med rastjo v klimatskih komorah ali ob 
naravnih izvirih CO
2
• 
Pri navadni kostrebi povečana koncentracija CO
2 
v klimatskih komorah ni 
značilno vplivala na produkcijo biomase, dihalni potencial in dihanje korenin. 
Ekstremno povečana koncentracija CO
2 
v rizosferi rastlin (navadne kostrebe in 
sivozelenega muhviča) ob naravnih izvirih CO
2 
ni značilno vplivala na dihalni 
potencial v koreninah. Taje bil značilno manjši le pri mesec in pol stari sejani koruzi 
rastoči na področju naravnih izvirov CO
2
• 
Ključne besede: dihanje korenin, aktivnost ETS, dihalni potencial, C4 rastline, 
Echinochloa crus-galli , Zea mays, Setaria pumila, povečana koncentracija CO
2
, 
naravni izviri co2, co2 mofeta 
36 Acta Biologica Slovenica, 46 (1), 2003 
Introduction 
An elevated atmospheric CO
2 
concentration can have a significant effect on growth and carbon 
metabolism of many plant species. On a daily basis, more than 50% of the photosynthates produced, 
may be simultaneously respired by roots (LAMBERS et al. 2002). Compared to the number of 
papers on inhibition of aboveground shoot respiration by elevated CO
2 
( e.g. AMTHOR 1991, DRAKE 
et al. 1997, TJOELKER et al. 2001), very little information on the effects of elevated CO
2 
on root 
respiration has been published so far. Furthermore, the reported CO2 effects on root respiration are 
rather heterogeneous, lirnited to only few plant species and differently discussed among authors 
(e.g. BURTON et al. 1997, YODER et al. 2000, LAMBERS et al. 2002). LAMBERS et. al. (1996) 
report that there is insufficient evidence to state that root respiration is inhibited by the [CO
2
] around 
roots in a sirnilar manner as leaf respiration is inhibited by elevated CO
2 
in the atmosphere. Moreover, 
there is no convincing evidence for a direct effect of elevated atmospheric CO
2 
on the specific rate 
of root respiration or the fraction of carbon required for root respiration. However, there are probable 
indirect effects of elevated CO
2 
on the carbon requirement of plants in natura! systems. 
The partial pressure of CO2 in soil may differ from the CO2 partial pressure above ground and it 
seems to be more variable. The concentration of CO
2 
in soil rapidly increases after rainfall because 
its diffusion through the gas phase is restricted by the water saturation of the soil. Thus, plant roots 
are frequently exposed to relatively high CO
2 
concentrations. Water flooding can also present a 
longer exposure of roots to elevated CO
2 
and consequently hypoxic environment. Plants growing 
near CO
2 
springs are exposed to extreme CO
2 
regimes that can also lead to hypoxic conditions in the 
rhizosphere. Vegetation at CO
2 
springs has been exposed to such conditions for long periods, giving 
tirne for acclimation, and perhaps also genetic adaptation of plants (VODNIK et al. 2002). Thus, 
natural CO
2 
springs may represent a good model ecosystem to study effects of elevated CO
2 
on 
plants (RASCHI et al. 1997, BADIANI et al. 1999). 
Our work was performed on three C4 plant species Echinochloa crus-galli var. crus-galli, Setaria 
pumila and Zea mays DK 312 (Dekalb, USA) subjected to an e!evated CO
2 
regime during growth in 
climatic chambers as well as in a CO
2 
spring-area Stavešinci in NE Slovenia. 
Material and methods 
Seedlings of Echinochloa, the offsprings of plants growing near the CO
2 
springs, were grown in 
climatic chambers under ambient (368.4 ± 15.6 µmol CO
2 
moJ·1) and elevated (1906.2 ± 195.0 µmol 
CO
2 
mo[·1) CO
2 
for seven weeks at the temperature 25 °C during the light (14 h) and 16 °C during 
the dark period (1 Oh) of the day. The fumigation started two weeks after seed germination. After the 
growth period plant roots were sampled for respiratory measurements. 
In the July 2002, root samples were also collected for natura! growing Echinochloa, Setaria and 
one and a half month old plants of sown maize (Zea mays DK 312, Dekalb, USA) growing in the 
natura! CO
2 
spring area Stavešinci in NE Slovenia (for detailed information on the site characteristics 
and other details see VODNIK et al. in press). The plants were chosen according to their height and 
the preliminary measured soil CO
2 
concentration in the rooting zone by a gas analyzer GA 2000 
(Ansyco, FRG). A significant correlation between plant height and plant exposition to elevated CO
2 
from CO
2 
springs is known from our previous studies (VODNIK et al. 2002•-b, TURK et al. 2002). 
I. Maček, H. Pfanz, D. Vodnik: Growth and root respiration of C4 ... 37 
Root respiration rates were measured as oxygen consumption on root tip segments (1 cm length) 
using Clark-type oxygen electrodes (Hansatech, Norfolk, UK). Measurements were performed in 
50 mM MES (morpholinoethanesulfonic acid) buffer solution pH 6 at 20 °C. 
The root respiratory potential - electron transport system (ETS) activity was determined on root 
tip segments (1 cm length) using the iodonitrotetrazolium salt (INT) method described by KENNER 
& AHMED (1975). 
Statistical analyses were performed by Statgraphic Plus 4.0 (Statistical Graphics Corp.). 
Results and discussion 
In the fumigation experiment no significant impact of high atmospheric CO
2 
concentration on 
biomass production of Echinochloa was found (Tab. 1). In general, the growth response of C4 
plants to elevated [CO
2
] is inconsistent and depends on various environmental factors (GHANNOUM 
et al. 2000). For Echinochloa the increase of plant biomass under elevated CO
2 
concentration 
(690 µmol moI·1) was documented by ZISKA et al. (1997). 
Measurements of root oxygen consumption revealed no significant differences in root respiration 
of elevated CO
2 
chamber-grown Echinochloa plants. There was also no significant difference in 
root respiratory potential (Tab. 1). 
Table 1: Biomass production, root respiration and root respiratory potential of E. crus-galli var. 
crus-galli subjected to elevated atmospheric CO2 in climatic chambers, (avg ±SD). 
Dry weight of shoot (g) (ll 
Dry weight of root (g) (ll 
Root respiration<2l 
ETS roots<3l 
Ambient CO
2 
(368.4 ± 15.6 µmol CO, mol·1) 
0.95 ± 0.26 
0.83±0.16 
1.5 ± 0.6 
1.0 ± 0.2 
Elevated CO
2 
(1906.2 ± 195.0 µmol CO, moI·1) 
1.15 ± 0.26 
0.89 ± 0.33 
1.8 ± 0.5 
1.0 ± 0.2 
<1>By AN0VA, n = 24. <2>Given as nmol 0
2 
g· ' fresh wt s·', by AN0VA, n = 15. <31Given as µg 0
2 
g·' fresh wt 
h·', by AN0VA, n = 15 
In addition to these findings, measurements of Echinochloa exposed to different CO
2 
regimes at 
the natura! CO
2 
spring Stavešinci showed no significant effects ofhigh rhizospheric CO
2 
concentration 
on root respiratory potential of the root-tip segments. The same was true for another C4 plant Setaria 
pumila (Tab. 2). It is to conclude that both species are relatively insensitive to high CO
2
• A high 
tolerance of E. crus-galli to hypoxia is known from different studies and is especially well documented 
for its variety E. crus-galli var. oryzicola (BUCHANAN & al. 2000). Germination of E. crus-galli 
could be stimulated by elevated CO
2 
as it was shown by YOSHIOKA & al. (1998). In this study 
germination was stimulated by exposure to 30 mmol mor-1 CO
2 
and it was concluded that soil CO
2 
is 
responsible for causing intermittent flushes of seed germination of this species after heavy rainfall. 
This could also explain the presence of germinating and growing Echinochloa plants at the sites 
38 Acta Biologica Slovenica, 46 (1), 2003 
with extreme C0
2 
concentrations in the natural C0
2 
spring Stavešinci as reported by KALIGARIČ 
(2001). No similar reports have been published on Setaria purnila. 
Table 2: Shoot height and root respiratory potential of E. crus-galli var. crus-galli, S. purnila and Z. 
rnays subjected to elevated soil and atmospheric C02 at a natural C02 spring (avg ± SD). 
Plant species CO exposureni Mean height (cm)\1l ETStl1 
Echinochloa crus-galli Low (0.4%) 62.3 ± 11.7 1.52 ± 0.19 
High (26%) 15.9 ± 1.8 1.55 ± 0.18 
Setaria purnila Low (0.4%) 51.0 ± 6.8 0.34 ± 0.05 
High (26%) 28.0 ± 5.6 0.32 ± 0.06 
Zea rnays Low (0.1-0.4%) 239.0 ± 21.0 1.12 ± 0.13 
High ( over 10%) 114.2 ± 7.9 0.95 ± 0.13 
<1>Measured as soil C0
2 
concentration (25 cm depth) by a gas analyzer GA 2000 (Ansyco, FRG). <2lBy ANOVA, 
n = 10. <31Given as µg 0
2 
g·' fresh wt h·', by ANOVA, n = 12. 
Results on Echinochloa and Setaria, could indicate a general low sensitivity of root respiration 
to a high C0
2 
concentration, which is suggested by LAMBERS et al. (1996, 2002). Yet, root 
respiratory potential in root tips of Zea mays measured in our study was significantly lower in the 
roots exposed to high soil C0
2 
than in those growing in the low C02 environment (Tab. 2). Different 
results obtained for native species (Echinochloa , Setaria) and sown maize suggest that plants growing 
as a part of natural vegetation could be adapted to extreme conditions. This however, has to be 
confirmed in the future work. 
Conclusions 
At natural C0
2 
springs, the growth of Echinochloa and Setaria can be decreased by high C0
2 
concentrations and physiological processes in shoots can be severely affected. Despite this, no 
significant impact of C0
2 
exposure on root respiratory potential of the root-tip segments was found 
for the same species. More detailed physiological studies on root respiration are needed, regarding 
to the different parts of the root system, different ontogenetic development and measurements on 
different plant species. Further research is also needed in connection to different environmental 
factors affecting root respiration. 
Acknowledgements 
Research was granted and performed in the frame of COST 627 program, granted by Forschungs-
Pool 2001 (09112000) of the University of Essen and by grants 14-2186 and 24-3196 from Ministry 
of Science, Education and Sport of Slovenia. 
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; a 
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concentration 
on sugar maple root respiration. Tree Physiol. 17: 421-427. 
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rising atmospheric CO
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GHANN0UM O., VoN CAEMMERER S., ZISKA L. H. & CoNROY J. P. 2000: The growth response of C4 
plants to rising atmospheric CO2 partial pressure: a reassessment. Plant, Cell and Environ 
23: 931-942. 
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functioning. In: Waisel Y., ESHEL A. & KAFKAFI U. (ed.): Plant Roots The Hidden 
Half, 3rd ed. Marcel Dekker, Inc., New York, pp. 521-552. 
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by elevated CO
2 
is minor in 12 grassland species. New Phytol. 150: 419-424. 
TURK B., PFANZ H., VoDNIH D., BERNIK R., WrTTMANN C., SINKOVIČ T. & BATIČ F. 2002: The effects of 
elevated CO
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on bog rush (Juncus ejfusus L.) growing near natural CO
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on shoot anatomy. Phyton (Hom - Austria) 42: 13-23. 
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ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 41 - 45 
Sprejeto (accepted): 2003-10-30 
Influence of in vitro propagation on the economically important traits of 
strawberry cv. Marmolada 
Vpliv in vitro razmnoževanja na ekonomsko pomembne lastnosti 
jagod cv. Marmolada 
Mojca VIRŠČEK MARN\ Jasna BERLJAK2, Darinka KORON1 
1Agricultural Institute of Slovenia, Hacquetova 17, 1001 Ljubljana, Slovenia; 
Fax: +386 1 2805 255, E-mail: mojcavm@kis.si 
2Semenama Ljubljana d.d., Dolenjska cesta 242, 1000 Ljubljana, Slovenia; 
Fax: +386 1 4273 538, E-mail: jasna.berljak@semenama.si 
Abstract. In vitro propagation of strawberries is a promising alternative to 
traditional propagation, since it provides better sanitary status of plants. The 
possibility of somaclonal variation presents one of the drawbacks of this method, 
but this phenomena can be minimised by the choice of optimal tissue culture 
procedure. 
To evaluate the influence of in vitro growing on economically important traits of 
strawberry cv. Marmolada, rooted plants were produced in vitro from a long term 
and from a newly established culture. These plants were used as mother plants for 
short multiplication in the field. Vegetative and generative traits of their runner plants 
were compared with conventionally produced cold storage plants and plants derived 
directly from tissue culture. Statistically significant differences were observed among 
different plant types in the number of runners per plant, number of flowers per plant, 
number of fruits per plant and yield per plant. The most pronounced differences 
were observed in the number of fruits per plant and in the yield per plant. Plants 
obtained directly from tissue culture were almost twice as productive as 
conventionally produced cold storage plants. Conventionally produced cold storage 
plants had statistically significantly higher yields and fruit number per plant than 
runner plants derived from micropropagated mother plants. High yields of plants 
obtained directly from in vitro were mainly the results of their significantly prolonged 
ripening. 
Keywords: strawberry, micropropagation, yield, quality, somaclonal variation 
Izvleček. In vitro razmnoževanje jagod predstavlja zanimivo alternativo 
klasičnemu razmnoževanju, saj zagotavlja boljše zdravstveno stanje sadik. 
Somaklonska variabilnost je lahko omejujoč dejavnik pri uveljavljanju 
mikrorazmnoževanja jagod, vendar lahko z izbiro optimalne in vitro tehnike ta pojav 
znižamo na minimum. 
42 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
Za ugotavljanje vpliva in vitro gojenja na ekonomsko pomembne lastnosti jagod 
cv. Marmolada smo vzgojili ukoreninjene rastlinice iz dolgotrajne in novo inicirane 
kulture te sorte in jih posadili v matični nasad. Vegetativne in generativne lastnosti 
njihovih hčerinskih rastlin smo primerjali z vegetativnimi in generativnimi lastnostmi 
standardno vzgojenih hlajenih sadik in rastlin, posajenih v poskus neposredno iz 
tldvne kulture. Med različnimi tipi sadik smo ugotovili statistično značilne razlike v 
številu živic na rastlino, številu cvetov na grm, številu plodov na grm in pridelku na 
grm. Največje razlike smo opazili v številu plodov na grm in pridelku na grm. Rastline, 
izvirajoče neposredno iz tkivne kulture, so imele skoraj enkrat večji pridelek v 
primerjavi s standardno vzgojenimi hlajenimi sadikami. Te so v številu plodov na 
grm in v pridelku na grm statistično značilno presegle hčerinske rastline matičnih 
rastlin, vzgojenih in vitro. Visoki pridelki rastlin, posajenih neposredno iz in vitro 
pogojev, so bili predvsem posledica njihovega močno podaljšanega zorenja. 
Ključne besede: jagode, mikrorazmnoževanje, pridelek, kakovost, somaklonska 
variabilnost 
Strawberries can only be multiplied by vegetative metbods, which explains the great dissemination 
of parasites. Most of strawberry producing countries thus have drawn up a program of certification 
to provide healthy planting material, which requires a minimum delay of 3 years before a healthy 
cultivar is at producer's disposal (Boxus & al. 1977). In vitro propagation of strawberries can speed 
up the propagation process. The possibility of somaclonal variation presents one of the drawbacks 
of this method. Mainly epigenetic changes such as hyperflowering, smaller fruit size and lower 
yields have been reported to occtir. The quality of the material obtained in vitro and its behaviour in 
the field depends on genotype and tissue culture procedure (L6PEZ-ARANDA & al. 1994). 
To determine the influence of tissue cul ture on field performance of cv. Marmolada, traditionally 
produced cold storage plants were compared with plants either derived directly from tissue culture 
or from micropropagated plants further propagated for one cycle in the nursery. 
Materials and methods 
Plant propagation 
Meristems from terminal buds of dormant plants of cv. Marmolada, one of the main varieties 
cultivated in Slovenia, were isolated in December 1999 and grown in vitro. In the beginning of February 
2000, newly developed shoots were multiplied according to the procedure described by Boxus & al. 
(1977). The same was done with shoots of cv. Marmolada grown in vitro over 3 years. Multiplied 
shoots originating from both newly established and old culture were transferred to a rooting medium 
for strawberries (Boxus & al. 1977). On 19 th of April 2000 rooted shoots were transferred to soil. After 
14 days of acclimatisation plants were transferred in larger pots and maintained in nethouse until 
beginning of August, when they were planted in the nursery. In spring 2001 runner plants were separated 
from mother plants and stored at -1 ° C until planting in the experimental field. 
On 20th of June 2000 a new in vitro culture of cv. Marmolada was established from meristems 
excised from runner tips of field grown plants. Shoots were multiplied and rooted according to 
Boxus & al. (1977). On 1st of March 2001 rooted shoots were transferred to soil. After 14 days of 
M. Viršček Mam, J. Berljak, D. Koron: Influence of in vitro propagation on the economically ... 43 
acclimatisation plants were transferred to larger pots and grown in the greenhouse until planting in 
the experimental field. 
Field tria! 
On 22nd of May 2001 the field trial was planted on the Experimental Orchard of Agricultural 
Institute of Slovenia at Brdo near Lukovica. Four different plant types of strawberry cv. Marmolada 
were tested: 
A. micropropagated plants originating from the in vitro culture established in June 2000, 
B. runner plants from micropropagated mother plants, originating from the in vitro culture established 
in December 1999, 
C. runner plants from micropropagated mother plants, originating from a culture maintained in vitro 
over 3 years, 
D. conventionally produced cold storage plants of A quality. 
A randomised block design with 4 treatments (plant types), 4 replications and 10 plants per plot 
was used. Following data were recorded: number of runners per plant, number of flowers per plant, 
number of fruits per plant and yield per plot. The STATGRAFICS Plus version 3.1 computer 
programme was used for statistical analyses. The analysis of variance was performed for the number 
of runners, number of flowers on 26th of July 2001, number of fruits and yield. 
Results and discussion 
Sanitary status of all plants included in the trial was very good. As expected, mutations were not 
observed, since propagation in vitro through axillary branching is generally considered to produce 
genetically stable material. Nevertheless, yellow leaf variants were reported to occur among 
micropropagated plants of strawberry cv. Redcoat by NEHRA & al. (1994). 
Considerable differences in vigour were observed among different plant types. Plants derived 
directly from tissue culture (plant type A) showed uniform and extremely vigorous growth with 
large leaves and very large flowers. Control plants (plant type D) were also uniform in vigour and 
had large leaves and flowers. In contrast, runner plants derived from micropropagated mother plants 
(plant type B and C) exhibited weak and variable growth and had small leaves and very small 
flowers. 
Runner plants deri ved from micropropagated plants also produced the lowest number of flowers 
per plant as recorded on July 26th 2001 (Tab. 1). Plants of the type C had statistically significantly 
lower number of flowers per plant in comparison with plants derived directly from tissue culture as 
well as with traditionally propagated plants (Tab. !). The number of flowers per plant of plant type 
B differed statistically significant only from plants obtained directly from tissue culture (Tab. 1). 
Since in strawberry not all of the flowers develop fruits, the number of fruits per plant was lower 
than the number of flowers recorded for all the plant types with exception of .plants derived directly 
from tissue culture. The latter exhibited prolonged flower development. Consequently fruit ripening 
was also markedly prolonged. Fruits of plants derived directly from tissue culture were harvested 
from 4th of July until 24th of August whereas fruit ripening of other plant types ended more or less by 
2nd of August. Plants derived directly from tissue culture gave statistically significant higher number 
of fruits and higher yields per plant than all other plant types including conventionally produced 
44 Acta Biologica Slovenica, 46 (1), 2003 
cold storage plants mainly due to prolonged ripening (Tab. 1). In spite of long ripening tirne of type 
A plants there was very little variation in fruit number and fruit yield among picking dates. Fruit 
weight diminished only slightly towards the end of the ripening tirne. 
Table 1: Vegetative and generative traits of different plant types of strawberry cv. Marmolada in the 
field 
Plant Number of Number of Number of Yield per plant 
type runners per plant flowers per plant fruits per plant in g 
A 10.1 a 13.4 a 16.15 a 97.6 a 
B 7.0 C 8.1 bc 6.45 C 31.1 C 
C 9.3 ab 7.3 C 6.25 C 30.9 C 
D 7.6 bc 10.5 ab 9.0 b 54.2 b 
Mean separation within column by Duncan's multiple comparison procedure 
Values followed by a same letter do not differ significantly, P < 0.05 
In the experiment of KARHu & HAKALA (2002) micropropagated plants of cv. Senga Sengana, 
planted directly in the field, also showed more abundant flowering and gave statistically significant 
higher marketable crop than control runner plants in the first cropping year. However no differences 
in yielding were recorded between micropropagated and control runner plants in cv. Zefyr (KARHu 
& HAKALA 2002). Similarly, an increased productivity of directly used micropropagated plants that 
was not accompanied by reduced weight was observed by NEHRA & al. (1994) for cv. Redcoat, 
whereas the yielding of cv. Veestar was not affected by the propagation method. In contrast to the 
results of KARHU & HAKALA (2002), SzczYGIEL & al. (2002) recorded higher yields but diminished 
fruit quality of micropropagated plants of cvs. Senga Sengana, Kent and Dukat in comparison with 
the traditionally propagated runner plants. L6PEZ-ARANDA & al. (1994) and SzczYGIEL & al. (2002) 
quote other authors who observed a sharp decrease in average fruit weight, which was usually 
limited mainly to the plantlets coming directly from micropropagation. SzczYGIEL & al. (2002) 
therefore suggested, that micropropagated plants should at least once be reproduced by runners 
before planting in the field . In our experiment runner plants derived from mother plants originating 
from either old or newly established in vitro culture both behaved poorly. The low vigour, flowering 
and yield of these plants were probably the result of the late planting of mother plants in the nursery 
and/or unfavourable weather condition during winter. As a consequence runner plants could not 
fully develop. Earlier planting of micropropagated plants in the nursery was prevented by poor 
development of rooted plantlets after acclimatisation due to extremely high temperatures. 
Conclusions 
Plants of cv. Marmolada derived directly from tissue culture showed markedly prolonged flower 
and fruit development. The number of fruits and yield of these plants were therefore almost twice as 
high as in conventionally produced cold storage plants, which showed statistically significantly 
better productivity than runner plants deri ved from micropropagated mother plants. The low vegetative 
and generative characteristics of the latter were probably the result of the late planting of mother 
plants in the nursery and/or unfavourable weather condition during winter. 
M. Viršček Marn, J. Berljak, D. Koron: Influence of in vitro propagation on the economically ... 45 
Acknowledgement 
This research was performed in the frame of the project No. V 4-0292-99, financed by the Ministry 
of Education, Science and Sport and Ministry of Agriculture, Forestry and Food of the Republic of 
Slovenia. 
Literature 
Boxus P., M. QuOIRIN, J.M. LAJNE 1977: Large scale propagation of strawberry plants from tissue 
culture. In: Rainert J, Y.P.S. Bajaj (eds.): Applied and fundamental aspects of plant cell, 
tissue, and organ culture. Springer, Berlin Heidelberg New York, pp. 130-143. 
K.ARHU S., K. HAKALA 2002: Micropropagated strawberries in the field. Acta Horticulturae 567: 
321-324. 
L6PEZ-ARANDA J.M., F. PLIEGO-ALFARO, I. L6PEZ-NAVIDAD, M. BARCEL6-MUNOZ 1994: 
Micropropagation of strawberry (Fragaria x ananasa Dutch.). Effect of mineral salts, 
benzyladenine levels and number of subcultures on the in vitro and field behaviour of the 
obtained microplants and fruiting capacity of their progeny. Joumal of Horticultural Science 
69: 625-637. 
NEHRA N.S. , K.K. KARTHA, C. STUSHNOFF, K.L. G1LES 1994: Effect of in vitro propagation methods 
on field performance of two strawberry cultivars. Euphytica 76: 107-115. 
SzczYGJELA, K. PJERZGA, B. BoRKOVSKA 2002: Performance ofmicropropagated strawberry plantlets 
after planting in the field. Acta Horticulturae 567: 317-320. 
ACTA BIOLOGICA SLOVENICA 
LJUBLJANA 2003 Vol. 46, Št. 1 : 47 - 50 
Sprejeto (accepted): 2003-10-30 
In vitro plant regeneration from somatic tissue of strawberry 
Fragaria x ananassa Duch. 
In vitro regeneracija poganjkov v somatskem tkivu jagode 
Fragaria x ananassa Duch. 
Jasna BERLJAK1, Mojca MARN2, Darinka KORON2 
1Semenarna Ljubljana d.d., Dolenjska cesta 242, SI-1000 Ljubljana, Slovenia 
2Agricultural Institute of Slovenia, Hacquetova 17, SI-1000 Ljubljana, Slovenia 
Abstract. Successful shoot regeneration in somatic tissue is the basic requirement 
for in vitro induction of genetic variability as the new tool in plant breeding. 
Somatic tissue excised from in vitro multiplied strawberry plants were tested on 
ability for plant regeneration. Leaves, petiole and stipules were inoculated on initial 
medium with BA and 2,4-D, or on medium with BA only after 1 hour pulse treatrnent 
with 2,4-D. Callus was induced on all sliced surfaces of explants inoculated on initial 
medium with growth regulators BA and 2,4-D during first 7 days of culture. Explants 
inoculated on initial medium with BA, after pulse treatment with 2,4-D did not develop 
callus but abundantly produced fenolic compounds, and tured necrotic in the first 24 
hours. 
Spontaneous plant regeneration was noticed on leaves explants with less 
developed callus tissue on initial medi um with growth regulators during second week 
of culture. High percentage of explants with regenerated shoots were obtained after 
transfer on hormone-free medium. 
The highest plant regeneration ability was in leaf tissue, less in petiole and stipules. 
Ca!lus induced in leaf tissue showed ability for constant plant regeneration during 
three months of culture and careful 4-week interval transfer on basal MS medium 
with 4.4¼M BA and 40 g/l sucrose. 
Keywords: in vitro, somatic tissue, leaf explants, plant regeneration, strawberry, 
Fragaria x ananassa Duch., cv. Elsanta, cv. Marmolada 
Izvleček. Uspešna regeneracija poganjkov v somatskem tkivu je osnovni pogoj 
za in vitro indukcijo genetske raznolikosti kot novega pristopa v žlahtnjenju rastlin. 
Somatsko tkivo, pridobljeno iz in vitro razmnoženih rastlinic jagod, smo testirali 
glede sposobnosti za regeneracijo rastlin. Del izsečkov iz listov, pecljev in stipul 
smo prenesli neposredno na začetno gojišče z BA in 2,4-D, del pa predhodno 1 uro 
tretirali z 2,4-D in nato prenesli na gojišče s samo BA. Kalus se je induciral v prvih 
7 dneh rasti kulture na rezani površini vseh izsečkov na gojišču z rastlinskimi hormoni 
BA in 2,4-D. Izsečki, predhodno tretirani z 2,4-D in inokulirani na gojišče z BA, 
niso razvili kalusa, ampak so proizvedli obilo fenolnih sestavin ter po 24 urah propadli. 
48 
Introduction 
Acta Biologica Slovenica, 46 (1), 2003 
Tekom drugega tedna rasti kulture na začetnem gojišču z rastnimi regulatorji 
BA in 2,4-D smo opazili spontano regeneracijo poganjkov na listnih izsečkih s slabo 
razvitim kalusnim tkivom. 
Visok odstotek izsečkov z regeneriranimi poganjki smo pridobili po prenosu na 
gojišče brez rastnih regulatorjev. 
Največjo sposobnost za regeneracijo poganjkov smo ugotovili pri listnem tkivu, 
slabšo pa pri tkivu pecljev in stipul. 
Kalus, induciran v listnem tkivu, je pokazal sposobnost za stalno regeneracijo 
poganjkov tekom treh mesecev rasti kulture in po previdnem 4-tedenskem 
prestavljanju na bazalno MS gojišče z 4.4µM BA in 40 g/1 sladkorja. 
Ključne besede: in vitro, somatsko tkivo, listni izseček, regeneracija poganjkov, 
jagoda, Fragaria x ananassa Duch., cv. Elsanta, cv. Marmolada 
Development and application of reliable protocols for plant regeneration from tissue culture 
become the new tools for the improvement of strawberry (Fragaria x ananassa Duch.) cultivars. 
Strawberry regeneration bas been obtained in vitro from different somatic tissue: celi suspension 
(DAMJANO et al. 1995), leaves (NEHRA et al. 1989; NEHRA et al. 1990), petioles (JONES et al. 1988), 
stipules (RuGINI and ORLANDO 1992, 1EMMALI et al. 1992), cotyledons (MILLER and CHANDLER 1990), 
anthers (RosATI et al., 197 5). 
Because regeneration efficiency is strongly dependent on the cultivar, regeneration methods for 
strawberry shoots from nonmeristematic tissue have not been well defined yet. 
The purpose of the present work was the evaluation of ability for in vitro shoot regeneration in 
strawberry somatic tissue. 
Material and methods 
Donor strawberry plants (Fragaria x ananassa Duch.) cv. Elsanta and cv. Marmolada, were 
propagated in vitro by protocol described by Boxus (Boxus et al. 1977). 
Leaf strips, petioles and stipules were inoculated on basal MS medium with thiamine 0.4 mg/!, 
agar 8 g/1, pH 5.8. Four treatments were used: 1) BA 4.4¼M + 2,4-D 2.3¼M + 40 g/1 sucrose; 2) BA 
4.4¼M + 2,4-D 2.3¼M + 80 g/1 sucrose; 3) BA 4.4¼M + 40 g/1 sucrose; explants were under pulse 
treatrnent in MS liquid medium with 2,4-D 22.6¼M 1 hour prior inoculation; 4) is as (2) and also 
with pulse treatrnent as mentioned above. 
Hundred and twenty leaf explants, 100 petiole explants oflength 0.5 mm, and 60 stipule explants 
were inoculated for each treatment. 
Explants with regenerated shoots were cultivated on MS horrnone-free rnediurn which promoted 
shoot elongation. Shoots grew in dense clusters, but it was possible isolated well developed plantlets. 
It was successfully aclirnatized 194 plantlets of strawberry cultivar Elsanta. 
Results 
Regenerative ability was strongly influenced by genotype for three type of explants tested. Shoot 
regeneration frorn leaf, petiole and stipule explants in the strawberry cultivars Elsanta and Marmolada 
is presented in Table l. Cultivar Elsanta showed better ability for callus initiation and shoot 
J. Berljak, M. Marn, D. Koron: In vitro plant regeneration from somatic tissue of strawberry ... 49 
regeneration in both treatments used. High percentage of leaf explants developed callus in the presern;e 
of BA and 2,4-D, and sucrose 40 g/1 . Presence of 80 g/1 sucrose suppressed callus development. 
Shoots which were initiated and regenerated on leaf explants cultured on initial medium, continued 
to grow on medium with BA 4.4¼M. It was possible to harvest regenerated plantlets from leaf 
explants during three months of culture, when explants with regenerative callus were transferring 
on fresh medium MS with BA every 4th week. 
Petiole explants also developed callus and regenerated shoots, but in lower percentages than 
leaf explants. 
In this experiment was not possible to obtain callus initiation and shoot regeneration in stipule 
explants of strawberry cv. Elsanta. 
Induction of callus was very poor in leaf explants and petioles of cv. Marmolada. Stipule explants 
showed slightly better ability for shoot regeneration. 
Pulse treatment with 22.6¼M 2,4-D on all three explant types of strawberry cultivars Elsanta 
and Marmolada caused abundant production of fenolic compounds and browning of plant tissue. 
Discussion 
The results show that ability of strawberry cv. Elsanta and cv. Marmolada to regenerate in vitro 
from somatic tissue (leaf, petiole, stipule) depends on the genotype. This fact also confirmed the 
results of experiments on cv. Redcoat and cv. Honeoye carried out by NEHRA et al. (1988). ŽEBROWSKA 
et al. (2002) tested leaf and petiole of cv. Kama and clone B-302 on capacity for plant regeneraton, 
and also confirmed influence of the genotype. In their results petiole explants showed higher ability 
for regeneration, but higher number of regenerated plantlets per explant was in leaf tissue. We 
obtained better capacity for plant regeneration in leaf tissue of strawberry cv. Elsanta, and no ability 
for shoot regeneration in leaf and petiole tissue of cv. Marmolada. Only low percentage of stipule 
explants of cv. Marmolada regenerate shoots in our experiment. But MoNTICELLI et al. (1995) reported 
on very high competence to regeneration from stipules of strawberry cv. Teodora and cv. Clea. They 
claimed also importance of 2,4-D as inducing factor in initial medium for callus induction, and 
reported similar as JEMMALI et al. (1992) that BA is sufficient to induce shoot regeneration. We 
noticed that explants with callus induced on medium with BA 4.4¼M and 2,4-D 2.3¼M, continously 
regenerate shoots in leaf explants subsequently subcultured on medium with BA alone. PoPEscu et 
al. (1997) reported on the organogenetic potential of petiole-derived calli for a long period from at 
least 18 up to 29 weeks, but shoot formation was no Ionger recorded after 19 weeks. 
In our experiment leaf explants of cv. Elsanta were able to regenerate shoots during 12 weeks. 
50 Acta Biologica Slovenica, 46 (1), 2003 
Table 1: Shoot regeneration from leaf, petiole and stipule explants in two strawberry cultivars at two 
clifferent treatments. 
Cultivar/explant Tretment % callus % shoot Number of 
regeneration regeneration plantlets explani- 1 
explant-1 explant-1 
Elsanta 
Leaf 1 100 31.2 12* 
2 79.5 9.6 8* 
Petiole 1 61.3 3.5 3 
2 20.8 2.8 3 
Stipule 1 2.0 o o 
2 o o o 
Marmolada 
Leaf 1 3.5 o o 
2 1.8 o o 
Petiole 1 1.2 o o 
2 o o o 
Stipule 1 1 1 3 
2 o o o 
*shoots were harvested in vitro during 3 months 
Conclusion 
Shoot regeneration from somatic tissue (leaves, petioles, stipules) of strawberry (Fragaria x 
ananassa Duch.) is highly dependent on genotype. 
2,4-D was essential for callus induction, while BA alone was later sufficient to induce shoot 
regeneration. 
Regenerative calli induced on leaf explants of strawberry (Fragaria x ananassa Duch.) cv. Elsanta 
showed shoot regeneration ability during next three months in the presence of 4.4 ¼M BA. 
Povzetek 
Pri testiranju sposobnosti regeneracije jagod cvs. Elsanta in Marmolada iz različnih somatskih 
tkiv in vitro gojenih rastlin in učinkovitosti različnih načinov tretiranja s hormoni smo ugotovili: 
* neučinkovitost tretiranja izsečkov z 2,4-D za 1 uro pred prenosom na gojišče z BA, 
* velike razlike v sposobnosti regeneracije med proučevanimi kultivarjema, 
* zelo slabo sposobnost regeneracije cv. Marmolada, pri kateri smo regeneracijo opazili samo pri 
1 % izsečkov iz stipul, 
* zelo dobro tvorbo kalusa in regeneracijo poganjkov cv. Elsanta iz izsečkov iz listov in nekoliko 
slabšo, a dobro regeneracijo iz izsečkov iz listnih pecljev 
* neučinkovitost regeneracije iz stipul na preskušanih gojiščih, 
* boljšo tvorbo kalusa oz. regeneracijo poganjkov na gojišču s 40 g/1 saharoze v primerjavi z 80 
g/1 saharoze. 
J. Berljak, M. Marn, D. Koron: In vitro plant regeneration from somatic tissue of strawberry ... 51 
References 
Boxus, PH., QuoIRIN, M., LAJNE, J .M . 1977: Large scale propagation of strawberry plants from tissue 
culture. In: Reinert J. and Bajaj Y.P.S. (ed.): Applied and fundamental aspects of plane celi. 
tissue and organ culture, Springer Verlag, New York, pp.130-143. 
DAMJANO, C., AscARELLI, A., FRATTARELLI, A ., LAURI, P. 1995: Adventitious regeneration and genetic 
variability in strawberry. Acta Hort 392:107-114. 
DAMJANO, C. , MoNTICELLI, S., CoRAZZA, L. 1997: Somaclonal variability and in vitro regeneration of 
strawberry. Acta Hort 447:87-93. 
JEMMALI, A., Boxus., PH., KINET, J.M. 1992: Are strawberry plantrets arising from adventitious stipule 
buds also true to type? Acta Hort 319:171-176. · 
MILLER, A., CHANDLER, C.K. 1990: Plant regeneration from excised cotyledons of mature strawberry 
achenes. HortSci 25:569-571. 
MoNTICELLI, S., DAMJANO, C., GALLELLI, A. 1995: Regeneration from strawberry stipules. Med. Fac. 
Landbouww., Univ. Gent 60/4a:1679-1682. 
NEHRA, N.S ., STUSHNOFF, C., KARTHA, K.K. 1989: Direct shoot regeneration from strawberry leaf 
disks. J. Amer. Soc. Hort. Sci l 114(6):1014-1018. 
NEHRA, N.S., STUSHNOFF, C., KARTHA, K.K. 1990: regeneration of plants from immature Jeaf-derived 
callus of strawberry (Fragaria x ananassa Duch.). Plant Sci 66: 119-126. 
PorEscu, A. N., IsAc, V. S., CoMAN, M. S., RADULAscu, M. S. 1997: Somaclonal variation in plants 
regenerated by organogenesis from callus culture of strawberry (Fragaria x ananassa). 
Acta Hort 439:89-96. 
RosATI, P. , DEVREUX, M., LANERI, U. 1975: Anther cul ture of strawberry (Fragaria x ananassa Duch.). 
HortSci 10:119-120. 
RuGINI, E., ORLANDO, R. 1992: High efficiency shoot regeneration from calluses of strawberry (Fragaria 
x ananassa Duch.) stipules ofin vitro shoot cultures. J. Hort. Sci 67 (4) : 577-582. 
ŽEBROWSKA, J.I., HoRTYNSKI, J. 2002: Plant regeneration from leaf explants in strawberry (Fragaria x 
ananassa Duch). Acta Hort 567:313-315. 
NAVODILA AVTORJEM 
l. Vrste prispevkov 
a) ZNANSTVENI ČLANEK je celovit opis originalne raziskave in vključuje teoretični pregled 
tematike, podrobno predstavljene rezultate z diskusijo in sklepe ter literatumi pregled: shema 
IMRAD (lntroduction, Methods, Results And Discussion). Dolžina članka, vključno s tabelami, 
grafi in slikami, ne sme presegati 15 strani; razmak med vrsticami je dvojen. Recenzirata ga dva 
recenzenta. 
b) PREGLEDNI ČLANEK objavi revija po posvetu uredniškega odbora z avtorjem. Število 
strani je lahko večje od 15. 
c) KRATKA NOTICA je originalni prispevek z različnih bioloških področij (sistematike, bio-
kemije, genetike, mikrobiologije, ekologije itd.), ki ne vsebuje podrobnega teoretičnega pregleda. 
Njen namen je seznaniti bralca s preliminarnimi ali delnimi rezultati raziskave. Dolžina na sme 
presegati 5 strani. Recenzira ga en recenzent. 
d) KONGRESNA VEST seznanja bralce z vsebinami in sklepi pomembnih kongresov in po-
svetovanj doma in v tujini. 
e) DRUŠTVENA VEST poroča o delovanju slovenskih bioloških društev. 
2. Originalnost prispevka 
Članek, objavljen v reviji Acta Biologica Slovenica, ne sme biti predhodno objavljen v drugih 
revijah ali kongresnih knjigah . 
3. Jezik 
Teksti naj bodo pisani v angleškem jeziku, izjemoma v slovenskem, če je tematika zelo lokalna. 
Kongresne in društvene vesti so praviloma v slovenskem jeziku. 
4. Naslov prispevka 
Naslov (v slovenskem in angleškem jeziku) mora biti kratek, informativen in razumljiv. Za 
naslovom sledijo imena avtorjev in njihovi polni naslovi (če je mogoče, tudi štev. faxa in e-mail). 
5. Izvleček • Abstract 
Podati mora jedrnato informacijo o namenu, uporabljenih metodah, dobljenih rezultatih in 
zaključkih. Primerna dolžina za znanstveni članek naj bo približno 250 besed, za kratko notico pa 
100 besed. 
6. Ključne besede • Keywords 
Število naj ne presega 10 besed; predstavljati morajo področje raziskave, obravnavane v članku. 
Člankom v slovenskem jeziku morajo avtorji dodati ključne besede v angleškem jeziku. 
7. Uvod 
Nanašati se mora le na tematiko, ki je predstavljena v članku ali kratki notici. 
8. Slike in tabele 
Tabele in slike (grafi, dendrogrami, risbe, fotografije idr.) naj v članku ne presegajo števila 10, 
v članku naj bo njihovo mesto nedvoumno označeno . Ves slikovni material naj bo oddan kot fizični 
original (fotografija ali slika). Tabele in legende naj bodo tipkane na posebnih listih (v tabelah naj 
bodo le vodoravne črte). Naslove tabel pišemo nad njimi, naslove slik in fotografij pod njimi. 
Naslovi tabel in slik ter legenda so v slovenskem in an-leškem jeziku. Pri - itiranju tabel in slik v 
besedilu uporabljamo okrajšave (npr. Tab. l ali Tabs. 1-2, Fig. 1 ali Figs. 1-2; Tab. 1 in Sl. 1). 
9. Zaključki 
Članek končamo s povzetkom glavnih ugotovitev, ki jih lahko zapišemo tudi po točkah. 
10. Povzetek - Summary 
Članek, ki je pisan v slovenskem jeziku, mora vsebovati še obširnejši angleški povzetek. Velja 
tudi obratno. 
11. Literatura 
Uporabljene literatume vire citiramo med tekstom. Če citiramo enega avtorja, pišemo ALLAN (1995) 
ali (ALLAN 1995), če sta dva avtorja (TRINAISTic & FRANJIC 1994), če je več avtorjev (PuLLIN & al. 
1995). Kadar navajamo citat iz večih del hkrati, pišemo (HONSIG-ERLENBURG & al. 1992, WARD 
1994a, ALLAN 1995, PuLuN & al. 1995). V primeru, če citiramo več del istega avtorja, objavljenih 
v enem letu, posamezno delo označimo s črkami a, b, c itd. (W ARD 1994a,b ). Če navajamo dobesedni 
citat, označimo dodatno še strani: TOMAN (1992: 5) ali (TOMAN 1992: 5-6). Literaturo uredimo po 
abecednem redu, začnemo s priimkom prvega avtorja, sledi leto izdaje in naslov članka, mednarodna 
kratica za revijo (časopis), volumen poudarjeno, številka v oklepaju in strani. Npr.: 
HoNSIG-ERLENBURG W., K. KRAINER, P. MILDNER & C. WrnsERR 1992: Zur Flora und Fauna des 
Webersees. Carinthia II 182/102 (1) : 159-173. 
ThINAJSTIC I. & J. FRANJIC 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (1): 10-15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10-15. 
Knjige, poglavja iz knjig, poročila, kongresne povzetke citiramo sledeče: 
ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & 
Hall, London, 388 pp. 
PuLLIN A. S., I. F. G. McLEAN & M. R. WEBB 1995: Ecology and Conservation of Lycaena dispar: 
British and European Perspectives. In: PuLLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall, London, pp. 150-164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s po-
svetovanja DZVS, Gozd Martuljek, pp. 17. 
12. Format in oblika članka 
Članek naj bo poslan v elektronski obliki v Microsoft Word formatu ( doc) ali kot obogateno besedilo 
(rtf) v pisavi "Times New Roman CE 12" z dvojnim medvrstnim razmakom in levo poravnavo ter s 
3 cm robovi na A4 formatu. Odstavki naj bodo med seboj ločeni s prazno vrstico. Naslov članka in 
poglavij naj bodo pisani krepko in v velikosti pisave 14. Vsa latinska imena morajo biti napisana 
ležeče. V besedilu navedemo uporabljene nomenklatume vire. Tabele in slike so posebej priložene 
tekstu. Glavnemu uredniku je potrebno oddati original, dve kopiji in disketni zapis v elektronski 
obliki na disketi 3,5", CD-romu ali kot priponko elektronske pošte (slednjega odda avtor po oprav-
ljenih strokovnih in jezikovnih popravkih). 
13. Recenzije 
Vsak znanstveni članek bosta recenzirala dva recenzenta (en domači in en tuji), kratko notico pa 
domači recenzent. Avtor lahko v spremnem dopisu predlaga tuje recenzente. Recenziran članek, ki 
bo sprejet v objavo, popravi avtor. Po objavi prejme 50 brezplačnih izvodov. V primeru zavrnitve 
se originalne materiale vrne avtorju skupaj z negativno odločitvijo glavnega urednika. 
INSTRUCTIONS FOR AUTHORS 
l. Types of Articles 
a) SCIENTIFIC ARTICLES are comprehensive descriptions of original research and include a 
theoretical survey of the topic, a detailed presentation of results with discussion and conclusion, 
and a bibliography according to the IMRAD outline (Introduction, Methods, Results, and 
Discussion). The length of an article including tables, graphs, and illustrations may not exceed 
fifteen (15) pages; lines must be double-spaced. Scientific articles shall be subject to peer review 
by two experts in the field. 
b) REVIEW ARTICLES will be published in the joumal after consultation between the editorial 
board and the author. Review articles may be longer than fifteen (15) pages. 
c) BRIEF NOTES are original articles from various biological fields ( systematics, biochernistry, 
genetics, rnicrobiology, ecology, etc.) that do not include a detailed theoretical discussion. Their 
aim is to acquaint readers with prelirninary or partial results of research. They should not be longer 
than five (5) pages . Brief note articles shall be subject to peer review by one expert in the field. 
d) CONGRESS NEWS acquaints readers with the content and conclusions of important 
congresses and seminars at home and abroad. 
e) ASSOCIATION NEWS reports on the work of Slovene biology associations. 
2. Originality of ArticJes 
Manuscripts submitted for publication in Acta Biologica Slovenica should not contain previously 
published material and should not be under consideration for publication elsewhere. 
3. Language 
Articles and notes should be subrnitted in English, or as an exception in Slovene if the topic is 
very local. As a rule, congress and association news will appear in Slovene. 
4. Titles of Artides 
Titles (in Slovene and English) must be short, informative, and understandable. The title should be 
followed by the name and full address of the author (and if possible, fax nurnber and e-mail address). 
5. Abstract 
The abstract must give concise information about the objective, the methods used, the results 
obtained, and the conclusions. The suitable length for scientific articles is approximately 250 words, 
and for brief note articles, 100 words. 
6. Keywords 
There should be no more than ten (10) keywords; they must reflect the field ofresearch covered 
in the article. Authors must add keywords in English to articles written in Slovene. 
7. Introduction 
The introduction must refer only to topics presented in the article or brief note. 
8. Illustrations and Tables 
Articles should not contain more than ten (10) illustrations (graphs, dendrograrns, pictures, photos 
etc.) and tables, and their positions in the article should be clearly indicated. AII illustrative material 
should be provided as physical originals (photographs or illustrations). Tables with their legends 
should be subrnitted on separate pages (only horizontal lines should be used in tables). Titles oftables 
should appear above the tables, and titles of photographs and illustrations below. Titles of tables and 
illustrations and their legends should be in both Slovene and English. Tables and illustrations should 
be cited shortly in the text (Tab. 1 or Tabs. 1-2, Fig. 1 or Figs. 1-2; Tab. 1 and Sl. 1). 
9. Conclusions 
Articles shall end with a summary of the main findings which may be written in point form. 
10. Summary 
Articles written in Slovene must contain a more extensive English summary. The reverse also applies. 
11. Literature 
References shall be cited in the text. If a reference work by one author is cited, we write ALLAN 
( 1995) or (ALLAN 1995); if a work by two authors is cited, (TRINAJSTIC & FRANJIC 1994 ); if a work 
by three or more authors is cited, (PULLIN & al. 1995); and if the reference appears in several works, 
(HONSIG-ERLENBURG & al. 1992, WARD 1994a. ALLAN 1995. PuLLIN & al. 1995). If several works by 
the same author published in the same year are cited, the individual works are indicated with the 
added letters a, b, c, etc.: (WARD 1994a,b ). If direct quotations are used, the page numbers should 
be included: TOMAN (1992: 5) or (TOMAN 1992: 5-6). 
The bibliography shall be arranged in alphabetical order beginning with the sumame of the first 
author followed by the year of publication, the title of the article, the intemational abbreviation for the 
joumal (periodical), the volume (in bold print), the number in parenthesis, and the pages. Examples: 
HoNSIG-ERLENBURG W., K. KRAINER, P. MrLDNER & C. WrESER 1992: Zur Flora und Fauna des We-
bersees. Carinthia II 182/102 (1): 159-173. 
TRINAJSTIC l. & J. FRANJIC 1994: Ass. Salicetum elaeagno-daphnoides (BR.-BL. et VOLK, 1940) 
M. MOOR 1958 (Salicion elaeagni) in the Vegetation in Croatia. Nat. Croat. 3 (2): 253-256. 
WARD J. V. 1994a: Ecology of Alpine Streams. Freshwater Biology 32 (l): 10--15. 
WARD J. V. 1994b: Ecology of Prealpine Streams. Freshwater Biology 32 (2): 10--15. 
Books, chapters from books, reports, and congress anthologies use the following forms: 
ALLAN J. D. 1995: Stream Ecology. Structure and Function of Running Waters, 1st ed. Chapman & 
Hall, London, 388 pp. 
PuLLIN A. S., l. F. G. McLEAN & M. R. WEBB 1995: Ecology and Conservation of lycaena dispar: 
British and European Perspectives. In: PuLLIN A. S. (ed.): Ecology and Conservation of 
Butterflies, 1st ed. Chapman & Hall, London, pp. 150--164. 
TOMAN M. J. 1992: Mikrobiološke značilnosti bioloških čistilnih naprav. Zbornik referatov s po-
svetovanja DZVS, Gozd Martuljek, pp. 17. 
12. Format and Form of Articles 
Articles should be send as Microsoft Word document (doc) or rich text format (rtf) using 
"Times New Roman CE 12" font with double spacing, align left and margins of 3 cm on A4 pages. 
Paragraphs should be separated with an empty line. The title and chapters should be written bold in 
fant size 14. All scientific names must be properly italicized. Used nomenclature source should be 
cited. Tables and illustrations shall accompany the texts separately. The original manuscript, two 
copies, and an electronic copy ona 3.5" computer diskette, on CD-ROM or by e-mail must be 
given to the editor-in-chief. All articles must be proofread for professional and language errors 
before submission. 
13. Peer Review 
All Scientific Articles shall be subject to peer review by two experts in the field (one Slovene 
and one foreign) and Brief Note articles by one Slovene expert in the field. Authors may nominate 
a foreign reviewer in an accompanying letter. Reviewed articles accepted for publication shall be 
corrected by the author. Authors shall receive fifty (50) free copies of the joumal upon publication. 
In the event an article is rejected, the original material shall be returned to the author together with 
the negative determination of the editor-in-chief.