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. 
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