Lucrări Științifice vol. 57 (1) 2014, seria Agronomie [601318]

Lucrări Științifice – vol. 57 (1) 2014, seria Agronomie
183

THE USE OF BIOTEC HNOLOGY FOR SUPPLYING
OF PLANT MATERIAL FO R TRADITIONAL CULTUR E OF MEDICINAL,
RARE SPECIES Arnica montana L.

Iuliana PANCIU1, Irina HOLOBIUC2, Rodica CĂTANĂ2

e-mail: [anonimizat]

Abstract

Taking into account the importa nce of Arnica montana , the attempts to improve the culture technologies are justified.
Our study had the aim to optimize in vitro plant multiplication and growth as a source of plants for traditiona l culture
in this species . Aseptic germinated seedlings were used as explants, apical meristem being the origin of the direct
morphogenesis process . For induction of regeneration, to promote plant growth and rooting, we used some combination
of growth factors and supplements as ascorbic acid, glutamine , PVP and active charcoal added in culture media based
on MS formula. We improv ed the efficiency of micropropagation , the best values were recorded on variant
supplemented with PVP –.7 regenerants/explant in the first 4 weeks and increasing at 17/ initial explant ( mean 14.62)
after 8 weeks. Concerning the germination capacity of the seeds scored after 2 weeks in sterile condition, the rat e was
47.76 and in non -sterile conditions, the rate varied depending of the substrate used. Comparing to the plants obtained
through traditional seeds germination, in vitro plants grew faster and were more vigourously. The micropropagation
protocol in Arnica montana L. allowed us to regenerate healthy, developed and rooted plants in the second subculture
cycle. This in vitro methodology can provide plant material for initiation of a conventional culture after acclimatization
of the obtained vitroplants.

Key words : Arnica montana , vitroplants, improved regeneration rate.

1 University of Agronomic Scie nces and Veterinary Medicine, Bucharest
2 Institute of Biology, Romanian Academy, Bucharest A. montana is a vulnerable taxon, valuable
as medicin al plant in traditional medecine. The
habitats fragmentation, grazing, overexploitation
through excesive harvesting conducted to the
diminshig of the natural population s.
This taxon is introduced in Annex D of EU
Council Regulation No.338/97 and Annex V ( b)
Habitats Directive (92/43 EC), being included in
the red lists (Boscaiu N. et al., 1994, Oltean M. et
al., 1994, Oprea A., 2005).
Arnica montana is a valuable medicinal
plants, having an anti -inflam matory, antiseptic
(antibacterial and antifungal) and reparatory
effects, owing to its active compounds as
sesquiterpene lactones. As plant organs used in
phytotherapy and cosmetics are collected
inflorescence and rhyzomes .
The species is cul tivated in different
countries t o supply plant material for
pharma ceutical purpose, meanwhile reducing the
collecting pressure in the natural populations.

In Romania, there are some cultures of this
species but on relatively reduced areas. Taking into account the importance of
Arnica montana , several studies concernin g its
culture, seed s germination and in vitro culture were
reported (Chonchou O. et al., 1992; Malarz J. et
al., 1993; Nichterlein K., 1995; Lê C., 1998 ;
Weremczuk -Jezyna I. & Wysokinska H., 2000;
Zăpârțan M. & Deliu C., 2001; Butiuc -Keul A. &
Deliu C., 2001; Butiuc –Keul A. et al., 2002;
Trejgell A. et al., 2009; Ștefanache C.P. et al.,
2010; Du ță M. et al, 2010; Petrova M. al., 2011 ;
Nikolova M. et al., 2013 ).
The rate of regeneration is not so high in this
taxon in comparison to other related species.
Our study has the aim to improve the in
vitro plant multiplication and growth and to
compar e with traditional seeds germination as
source to provide plant material for the
establishment of a field culture in this taxon.
We tested appropriate growth factors
combination to improve the regeneration, growth
of vitroplants and rooting.

Universitatea de Științe Agric ole și Medicină Veterinară Iași
184 MATERIAL AND METHOD

The plant material was represented by seeds
purchassed from Germany, which were sterilized
through washing in runnig tap water for 2 hours,
short immersion 1 minute in 70 ș ethylic alcohol,
sterilization in 0.1% mercuric chloride, three washing
in sterile di stilate water. The sterilized seeds were
cultivated for one week in sterile distilled water
supplenmented with 5 mg/l gibberelic acid and than
transferred on MS( Murashige T. and Skoog F. , 1962)
medium free of growth factors for plant development.
Despite the first seeds geminated after 7 days
of culture, the germination capacity (% of germinated
seeds) were recorded after 2 weeks.
Different media variants based on MS formula
of micro and macroelements, added with Gamborg
vitamins ( Gamborg O.L. et al., 1968 ), 30 g/l sucrose,
7 g/l Agar Duchefa Biochimie, adjusted at pH 5.8 and
supplemented with different compounds were tested
for direct morphogenesis ( table 1 ). The explants were
represented b y aseptic germinated seedlings of ~
1cm height.
The subcultures wer e made at every 4 weeks.
For each variant were cultured 2 explants/ petri
dish in 4 repetitions in the first stage and 4 -5
explants/ Duchefa polypropylene autoclavable box of
9x10X10 cm. All the cultures were maintained in the
growth chamber at 3000 lux il lumination and 16/8
photoperiod and 250 temperature regime.
In the second stage of multiplication, we used
media variants added with active charcoal A C (0.5g/l)
to improve plant vigourosity and to help rooting. A C
also prevents phenolic compounds accumulat ion in
the culture medium.
The cultures were evaluated using 2
parameters: the mean number of regenerants/ initial
explant scored after two time intervals (4 weeks, 8
weeks, respectively), and the maximum length of the
developed plants (in cm).
Also the maximum height of plants were
compared in vivo and in vitro .
Non-sterilized seeds originated from the same
source were treated for 2 days with distilled water and
5 mg/l GA 3 and susequently were sowed on different
substrates as V1(mixture of soil substate 50%+
perlite 50 % ), V2 ( mixture of soil substate
50%+peat 25% +perlite 25 %),V3( mixture of soil
substate 50%+ sand 50 %.
The seeds were sowed ~ 100 for each
treatment in 3 -5 repetitions and were maintained at
250C temperature and normal day/night re gime.
The germination of seeds after 2 weeks and
the growth of in vivo seedlings after 8 weeks were
recorded.
Graphic values are expressed as mean values
±SD. One-way analysis of variance (ANOVA) was
applied to calculate the statistical significance at
p<0.05.

RESULTS AND DISCUSSIONS

The in vitro developmental way in A.
montana , similarly to other Asteraceae taxa is
direct and indirect morphogenesis, but bot h for conservative purpose and also for multiplication
the direct way is preferred because the
regeneration is better and plants are more
genetically stable.
Our work involves the establisment of an
efficient in vitro regeneration protocol through
direct morphogenesis, which was compared with
traditional seeds germination.
In our study, w e tested usual growth factors
as citokinine benzyl -amino purine BAP, Kinetin or
more expensive Zeatin associated with alfa –
naphthyl acet ic acid (more stable as IAA ). For
induction of higher regeneration rate, to promote
plant growth and rooting, we used some
combi nation of growth factors and active charcoal
(0.5 g/l). 2, 4-D used as auxin instead of NAA, did
not significantly improve the regeneration.
Gibberelic acid presence in the culture media
help shoots to elongate.
Zeatin adding in M2 variant also did not
improve s significantly the regeneration comparing
to other variants tested.
The use of classical culture media based on
MS formula and supplemented with citokinin s and
auxin s in 10/1 ratio , gibberelic acid (0,25 mg/l) and
some other compounds as ascorbic aci d, glutamine
or polyvinyl pir rolydone, conducted to impoved
regeneration ( figure 1), good growth and vigour of
plants. The best values were recorded on variant
M7 supplemented with PVP -7 regenerants/explant
in the first 4 weeks ( figure 3) and increasing at 17/
initial explant ( mean 14.62) after 8 weeks (figure
4). Rooting also occu red on the same media
variants ( figure 2) without to be necessary the
transfer of shoo ts on rooting medium –in phase III.
The ex vitro acclimatization of the rooted
plants was made starting with 9 -10 weeks after
initiation phase on V1 variant , with 75% survival
rate in the recorded after 3 weeks.
Our results were satisfactory when are
compared to previously reported works.
Conchou O. et al.,(1992) reported 7.7
regenerants/explants on medium MS and 9
regenerants on B5 medium suplimented with BA
(1 mg /l) and NAA (0,1 mg/l), after 6 weeks of
culture.
Weremczuk -Jezyna & Wysokinska (2000)
obtained plants through direct shooting on MS
mediu m (Murashige & Skoog, 1962) added with
IAA (beta indole acetic acid) 0,5 M /l and zeatin
(0,05 M/l) with a medium rate of 6 shoots/ explant.
In Romania, Butiuc -Keul A. &Deliu C.
(2001) obtained direct shoot formation on MS
medium added with N6 -[2-izopen thenyil] adenine
(2-iP), zeatin and alfa -naftilacetic acid (NAA)
with a maximum regeneration of 3,2 neoplantuls
per explant. Butiuc –Keul A. et al., 2002, reported

Lucrări Științifice – vol. 57 (1) 2014, seria Agronomie
185 later an improved regeneration of 3,6
shoots/explant on semisolide medium added with
yeast extract, BAP 1mg/l and IBA 1mg/l.
Trejgell A. et al. (2009) used aseptic
germinated seedlings as explants source, just apical
meristems showed morphogenic response, the best
was recorded in presence of 3,0 mg /l BAP (2,5
shoots/ explant). The shoots were rooted on auxin
free media.
Ștefanache C.P. et al., (2010) also used
aseptic germinated seedlings collected from natural
populations or inflorescences but the regeneration
was quite reduce d after 4 weeks , just 3 -4
regenerants/ explants w ere obtained on MS
medium + BAP 1mg/l or 2 mg/l associated with
NAA 0,3 mg.l . Ștefanache C.P. et al., (2011) also
made a comparative analysis between in vivo and
in vitro obtained plant material concerning active
compounds content
Duță M. et al, 2010 , used a ratio of 0.02
auxin s:4 citokinin s, 40g/l dext rose and active charcoal (0,3 g/l) to regenerate 5 plants/initial
explant in the II stage of multiplication.
Petrova M. et al., 2011 , reported indirect
morphogenes at very low rate(0.86)
regenerants/explant and direct morphogenesis
(16,3 generants/explan t) on MS + 1 mg /l BA + ,1
mg /l IAA (beta indole acetic acid) .The shoots were
rooted after 4weeks on MS1/2 medium added with
0.5 g/l Indole buthylic acid (IBA).
Concerning the germination capacity (%) of
the seeds scored after 2 weeks in sterile condi tion,
the medium rate was 47.76 and in non -sterile
conditions, the rate varied depending of the
substrate used, better values were recorded on V1
and V3 (table 2 ).
Vârban D.I. et al., 2012 have also made a
study concerning germinative energy and capacity
of seeds from 3 different origins and used 4
different substrates, the best response being
obtained on V1 variant consisted in 50% peat, 25%
terra rosa, 25% sand.

Table 1
Media compositi on used for the induction of direct morphogenesis in Arnica montana L.
Components M1 M2 M3 M4 M5 M6 M7
Macroelements MS MS MS MS MS MS MS
Microelements MS MS MS MS MS MS MS
Vitamins B5 B5 B5 B5 B5 B5 B5
Growth factors
(mg/l) BAP 1 – 1 1 1 1 1
Kin – – 1 1 1 1 1
Zea – 1 – – – – –
NAA 0.1 0.1 – 0.2 0.2 0.2 0.2
2,4-D – – 0.2 – –
GA 3 0.25 0.25 0.25 0.25 0.25 0.25 0.25
Other
compounds
(mg/l) A a – – – 20 – –
Glut – – – – 200 –
PVP – – – – – 10.000
Legend: MS – Murashige & Skoog medium ; B5 Gamborg – vitamins; BAP – benzyl aminopuryne; Kin – kinetin; Zea – zeatin, NAA – alfa-
naphtyl acetic acid, 2.4-D-dichlor phenoxy acetic acid, GA3 – gibberelic acid,A a Ascorbic acid, Glut – glutamine, PVP -polyvinyl pyrrolidone.

Figure 1 Regenerated roo ted plant in the phase II of
culture on M6 variant – transplantation .

Figure 2 Regenerated rooted plant obtained in the
phase II of culture on M7 variant before ex vitro

Universitatea de Științe Agric ole și Medicină Veterinară Iași
186
2.5
2.1254 3.8753.6254.257
0123456789
1
Regeneration media testedmean number of regenerants / explantM1
M2
M3
M4
M5
M6
M7
Figure 3 The number of regenerants/explant after 4 weeks of culture (mean values+SD).

4.875
2.6255.756.25 6.1256.7514.625
024681012141618
1
Media variants testedmean number of regenerants/initial explantM1
M2
M3
M4
M5
M6
M7

Figure 4 The number of regenerants/initial explant after 8 weeks of culture (mean values +SD).

Lucrări Științifice – vol. 57 (1) 2014, seria Agronomie
187
0.731253.43753.7
2.95625
00.511.522.533.544.5
1
Variants usedthe maximum length of plants (cm)V1
V2
V3
V4

Figure 5 The maximum growth of plants cultured in different conditions (V1 – in non -sterile condition on
soil/peat/sand mixture; V2 – variant supplemented with Ascorbic acid and AC;V3 – variant with glutamine
and AC, V4 – medium added with with PVP and AC (mean values +SD) after 2 months.

Table 2
Seeds geermination
in different experimental conditions ( %)
Germination capacity of seeds
V0 sterile condition 47.76 %
Non-sterile conditions on different substrates
V1 soil 50%+perlite 50% 58.8%
V2 50 % soil+25% peat+25% perlite 20%
V3 50 % soil +50% sand 66.6 %

Our seeds showed a lower germination
capacity, the best response was recorded on V3
variant, but provided us enough material to initiate
in vitro cultures.
Concerning the growth of non -sterile
seedlings and regenerated plants, we observed that
in vitro plants grew faster and were more
developed (V2-V4) compared to those obtained
through classical germination -V1 (fig.5).

CONCLUSIONS

Our in vitro multiplication protocol for
Arnica montana can provide healthy, developed
and rooted plants in the second phase of culture ,
starting from aseptic germinated seeds as source of
explant s, through direct organogenesis originated
from apical meristem of seedlings. The regenerants rate of growth is higher comparing to plants
derived from the seeds traditionally germinated.
The protocol can ensure plant material for
the initiation of traditional cultures after
acclimatization of the regenerants .
Using affordable plant growth factors, but
combined with some supplements and active
charcoal help to improve regeneration, growth and
rooting. It is not necessary the culture of neo –
formed shoots separat ely on rooting media as in
previous reports.

REFERENCES

Boșcaiu N., Coldea G., Horeanu C., 1994, Lista Roșie
a Plantelor Vasculare dispărute, periclitate,
vulnerabile și rare din Flora României . Ocrot. Nat.
Med. Inconj. Vol 38 (1) , p 45-56.
Butiuc -Keul A., Deliu C., 2001, Clonal propagation of
Arnica montana L ., a medicinal plant. In Vitro
Cell. Dev. Biol. —Plant vol. 37, p. 581–585,
September –October 2001.
Butiuc -Keul A, Suteu A, Keul M, Deliu C. 2002 , The
role of natural extract on the in vitro multiplication
Arnica montana L , Contributii Botanice, vol vol
XXXVII, p. 183-188.
Conchou O , Nichterlein K , Vömel A ., 1992, Shoot Tip
Culture of Arnica montana for micropropagation .
Planta Med ica Feb; vol. 58(1) , p.73-6.
Directive for Conservation of Natural Habitats and
wild Fauna and Flora 92/43/EC, Annex V(b) –

Universitatea de Științe Agric ole și Medicină Veterinară Iași
188 Plant and animal species of Community interest
whose taking in the wild and exploitatio n are
likely to be subject to management measures.
http://eur -lex.europa.eu/legal -content/EN –
Duță M ., Teodorescu A ., Alexiu V ., Neblea M ., 2010,
In vitro propagation scheme of planting material
for Arnica montana L species. Scientific papers of
the RIFG Pitești, Vol 24 , p. 156-159.
Gamborg O.L., Miller R.A., Ojima K. 1968 , Nutrient
requirements of suspension cultures of soybean
root cells . Exp. Cell. Res. Vol. 50,p. 151-158.
Lê C.L., 1998, In vitro clonal multiplication of Arnica
montana L . In:Proc. Plant Biotechnology as a tool
for the Exploitation of Mountain lands, Acta
Horticulture vol.457, p.195-203.
Malarz J., Stojakovska A., Donhal B., Kisiel W., 1993,
Helenalin acetate in vitro propagated plants of
Arnica montana . Planta Med ica vol. 59, p. 51-53.
Murashige T. & Skoog F. 1962 , A revised medium for
rapid growth and bioassays with tobacco tissue
culture . Physiol ogia Plant arum Vol.15, p. 473-
497.
Nichterlein, K. ,1995, Arnica montana (mountain arnica):
in vitro culture and the production of
sesquiterpene lactones and other secondary
metabolites . Medicinal and aromatic plants VIII/
Berlin Heidelberg New-York, Ed . Springer -Verlag,
Biotechnology in Agriculture and Forestry vol.33,
p.: 47-61.
Nikolova M., Petrova M., Zayova E., Vitkova A., 2013,
Comparative study of in vitro, ex vitro and in vivo
grown plants of Arnica montana -polyphenols and
free radical scav enging activity . Acta Bot anica
Croat ica vol. 7, p.13-22. Oltean M ., Negrean G., Popescu A., Roman N.,
Dihoru Gh., Sanda V., Mihăilescu S., 1994,
Lista Rosie a Plantelor Superioare din România .
Stud. Sint. Doc. Ecol., Acad. Rom., București.
Oprea A., 2005 , Lista Critica a Plantelor Vasculare
din Romania .”A.I. Cuza” University Press, Iași,
p. 377.
Petrova M., Yazova E., Zankova E., B aldshiiev G.,
2011, Plant regeneration from callus culture of
Arnica montana . Romanian Biotechn ological
Letter vol. (16), No.1, Supplement, p. 92-97.
Ștefanache C.P., Dănila D., Necula R., Gille E., 2010 ,
Studies regarding in vitro regeneration of Arnica
montana L. from natural populations -Bistrita
Valley (Eastern Carpathians ). Analele științifice
ale Universității “Al. I. Cuza” I ași Tomul LVI, fasc.
1, s. II a. Biologie vegetală, p . 33-42
Trejgell A. , Michalska M. , Tretyn A. , 2009 , Effect of
cytokinins on the micropropagation of Arnica
montana L. Zeszyty Problemowe Postępów Nauk
Rolniczych 12/2009; vol. 534, p.289-296.
Vârban D. I., Păcurar F., Vârban R., Odagiu A., 201 2,
The study of the seed germination in some Arnica
montana L. descents, 2012 , Analele Universit ății
Oradea. Fascicula Protectia Mediului. Vol. XIX, p.
200-207.
Weremczuk -Jezyna I., Wysokinska H., 2 000,
Micropropagation of Arnica montana L. and
sesquiterpene lactone s in regenerated pla nts,
Biotechnologia vol. 4, p. 118-122.
Zăpârțan M., Deliu C., 2001 , Studies of in vitro
regeneration and multiplication of Arnica montana
L., (Asteraceae) . Contrib uții Botanice vol XXXVI ,
p. 131-135.