Studii şi Cercetări Martie 2018 Biologie 27/1 104-109 Universitatea”Vasile Alecsandri” din Bacău

THE YIELD OF ARNICA MONTANA L. PLANTLETS BY MEANS OF

BIOTECHNOLOGIES IN VIEW OF REPOPULATING THE SPECIES’ NATIVE
HABITAT

Diana-Elena Maftei, Loredana - Iuliana Petrea

Key words: Arnica montana, in vitro, habitat preservation

INTRODUCTION

This present scientific study is a sequel of our

previous research on this valuable medicinal species
[5, 6, 7], aiming to provide as many arnica plants by
means of in vitro cultures in view of repopulating
some native habitats of this species in Eastern
Romania. Arnica montana (Photo 1) is a well-known
and valued species; its inflorescences are mainly used
(Arnicae flos) in the pharmaceutical industry. Arnica
comprises: anti-inflammatory sesquiterpenic lactons
(among which the most valuable for medicine is the
helenaline), flavonoids (responsible for the tonifying
action on the blood vessels, and for the local anti-
inflammatory property as well), polyphenols and
polyacetilens (antimicrobial and antifungical).
The vegetal product contains 0.2 – 0.35% Photo 1. Arnica montana L. (in bloom)
volatile oil (coloured in orange), and of a semisolid (http://www.google.ro)
consistency. Arnica serves mainly for external use. It
may be administered internally with much causion MATERIAL AND METHODS
(as an infusion, tincture, oily extract) in
hyperpressure, respiratory stimulant. Our research started with the in vitro culture
Its most important properties are: antiseptic, initiation on Arnica montana L. from seeds brought
wound healing, antitheumatic, analgesic, being from the Botanical Garden in Oberholz, Germany.
cicatrisant, antirheumatic, analgesic; it is The most effective method to provide the seedlings
recommended in the treatment of sprains, was by germination of previously disinfested seeds
dislocations, bruises, haematoma, edema associated directly on sterile solid culture medium variants. The
with fractures, superficial phlebitis, inflammation germination capacity for this species was previously
caused by insect stings, inflammation of the mucous tested [5]. It was intriguing and rather difficult to
membrane. Arnica is part of hundreds of industrial provide new plantlets from germinating seeds. The
products (e.g. tinctures, ointments, gels, infusions, references mention a 80% yield for arnica
gargles, cataplasms)[3, 6, 11, 12]. germination. The source of seeds was diverse: we
The species bears many folk names in various tested a genotype from the Botanical Garden in
countries: in England - mountain tabacco, wolfbane, Oberholz (Germany), a genotype from a population
leopard’s bane, mountain arnica; in Denmark: in Teleorman (Romania), and one from Gârda de Sus
gouldlomme, bjerg volverlei; in France: arnica, (Gheţari village). Some of our lab tests were not
amique panaceea des chutes, tabac des vosges, souci successfull. The best germination yield was achieved
des alpes, amique des montagnes; in Italy: arnica; in using the populations from Gârda de Sus and from
Germany: arnika, kraftwurz, fallkraut; in Russia: Oberholz.
arnica gornaia, amika, barannik; in Hungary: A part of the new plantlets did not survive in
mariafii, arnika, arnyekfii, amgylitoefii [8, 10, 11, lab conditions, as the species is an alpine one, with
12]. Matthaeus Sylvaticus named the species arnich, requirements specific for its native habitat. Seeds of
a word of Arab origin. [8]. all the tested genotypes were placed in Petri dishes,
as well, either on moist filter paper, or on moist
cotton wrapped in sterile gause. Despite the applied
pre-treatments, the seeds did not start to germinate.

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The pre-treatments were: a seed batch from all the (temperature of about 22o C, light 2500 lux,
tested genotypes were treated with hydrogen photoperiod 16/8 hours).
peroxide (H2O2) and subsequently immersed in a
fertilizer (Atonik); half of the seeds were grown in RESULTS AND DISCUSSIONS
soil pots afterwards, and half on Petri dishes to
induce germination. This present scientific study is a sequel of our
Other pre-treatments applied to the seeds previous research on this valuable medicinal species
consisted of their maintainance in a solution of [5, 6, 7], thoroughly studying many references on
giberellic acid for 6 hours at 20°C, subsequently arnica [3, 4, 9, 13], in order to provide as many
placed in still water in a refrigerator for 24 hours. arnica plants by mean of in vitro cultures in view of
The seeds were inoculated on the sterile culture repopulating some native habitats of this species in
medium variants MS, BA1, and BGA. Eastern Romania.
The most efficient method to provide Our preliminary studies reavealed the
seedlings was by seed germination (previously following : the morphogenetic response on the A2
sterilized in ethylic alcohol and HgCl2) directly on medium variant was extremely favourable, the
sterile culture medium (Oberholz genotype) and by multiple shoots grew intensely on this variant. This
seed germination directly on soil (the genotype from medium stimulated the root growth (the root length
Gârda de Sus)(photo 6, 8). was of about 3 cm, the merystematic root tips could
The seeds from Gârda de Sus were be well observed). On the basal hormone free MS,
characterized by a good germination on the soil pots the shoots were characterised by a better length
surface (in our laboratory). The soil was an acidic growth compared to the previous culture medium (3-
one, favourable for alpine species (pH = 3). 7 cm in height). The multiple shooting was absent.
The disinfestation of seeds followed some The roots are very long, extremely thin, without
steps: 1. A former sterilization in absolute ethyl secondary ramifications.
alcohol 10”; 2. Disinfestation 5-10” in HgCl2 10/00, The BD medium variant provided friable
followed by 5 rinses in sterile distilled water. The average proliferative callus, cream greenish colour.
medium formuli used were Murashige-Skoog (1962) 50 % of the explants provided callus as well, and
(tab.1) and other MS variants, as well (A2, BB2, shoots via callus (indirect caulogenesis). The shoots
BD), various combinations and concentrations of had small thick dark green leaves. It was observed
growth regulators (auxins and cytokinins). that the rhyzogenesis was absent on this medium
The charbon source was saccharose (30 g/l); variant. The best morphogenetic response was
the agar-agar (8,5 g/l) solidified the medium culture. evinced on the BB2 medium variant, consisting of:
After culture mediums preparation pH fixing with multiple vigorous shoots, and a rapid shoot growth.
NaOH (5.5), they were sterilized in an autoclave at The caulogentic response was very appropriate on the
121°C for 20 minutes, the pressure being 1 atm. newly tested medium variants ZB and BK. The
The inoculated vials comprising many variants rhyzogenesis was well represented on this medium
of the culture medium (table 1) were incubated in a variant (the roots were more developed and greener
room with half-climatised conditions within the compared to the rest of the tested variants). The main
Genetics and Biotechnologies Laboratory of the root’s length reaches 10 cm, and it is endowed with
"Vasile Alecsandri" University of Bacău secondary ramifications.

Table 1. The culture medium variants used within the experiments

Amount of growth regulators (mg/l)

Medium Basic
Species
variant medium
IAA IBA BAP KIN 2.4-D ZEA NAA GA3

MS MS - - - - - - - -
Half MS MS - - - - - - - -
A2 MS 2.0 - - - - - - -
BD MS - - 0.5-1.0 - 0.5 - - -
BB2 MS - 1.0 2.0 - - - - -
Arnica montana L.
BB1 MS - 0.5 1.0 - - - - -
BA1 MS 0.5 - 1.0 - - - - -
BK MS - - 1.0 0.5 - - - -
BN MS - - 0.5 - - - 0.5 -
ZB MS - 0.5 - - - 2.0 - -
BGA MS 0.5 - 0.5 - - - - 0.5
IAA = indole acetic acid; NAA = naphtyl acetic acid; BAP = benzylaminopurine; KIN = kinetine;
IBA = indole butiric acid; Zea – zeatyn ; 2.4-D = diclorfenoxiacetic acid

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 The plantlets grown from seeds germinated on
CONCLUSIONS sterile medium variants (photo 8) are an important
explant source in view of micropropagation.
Arnica montana is a rare and vulnerable Regarding the morphogenetic response, there was
species, and it is on the Red List of IUCN [2]. The ascertained that: the best caulogenetic response was
advantages of micropropagation are: a better genetic provided on the BB2 medium variant, followed by:
control on the plant species, several successive BK, ZB, A2, MS, BD. The best rhyzogenesis was
harvests in a shorter period of time as if in obtained on BB2 medium variant, as well; the
conventional cultures, regardless of the seasons. The rhyzogenesis was absent on the BD variant, that
septic and the asptic germination of the arnica seeds, provided callus, and shoots via callus (photos 9-13).
as well as the in vitro seedlings’ growth are time- The in vitro plants were acclimatized, and they will
consuming, thorough processes. The plant growth be further tested phytochemically. The shoots
(either in soil pots, or on sterile medium culture provided by means of micropropagation are meant to
variants) is rather slow for all the studied arnica repopulate some of this species’ native habitats.
genotypes.

Photo 2. Seeds of Arnica montana L.( Gârda de Sus Photo 3. Seeds of Arnica montana L. (Oberholz,
population) Germany)

Photo 4. Germinating seeds on moist filter paper in Photo 5. Germinating seeds on moist cotton in Petri
Petri dishes. dishes.

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 Photo 6. Seedlings provided by germinated seeds on Photo 7. Arnica montana L. seeds with pappus.
soil pots (Gârda de Sus population)

Photo 8. Sterile arnica seeds on solid culture medium in Photo 9. Arnica montana shoots on the BB2 medium
the in vitro growth chamber

Photo 10. Arnica seedlings on the A2 medium variant Photo 11. Friable cream and green callus BD

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 Photo 12. Vials with arnica shoots in the in vitro Photo 13. Arnica montana in vitro (several madium
growth chamber culture variants)

3. BUTIUC-KEUL, DELIU C., 2001 - Clonal

ABSTRACT propagation of Arnica montana L., a medicinal
plant, In vitro Cell Dev. Biol.-Plant, 37, p. 581-
Arnica montana L. is one of the most rare and 585;
valuable medicinal species, one of the best-known 4. BUTIUC-KEUL, 2002 - The role of nature
homeopathic remedies. It is an alpine plant, growing extracts on the in vitro multiplication of Arnica
in nutrient-poor soil. It can potentially reach a height montana L. Contribuţii Botanice vol. 37 p. 183-
of 60 cm, but this is unusual given the harsh 189;
conditions at high altitudes. It grows in meadows up 5. MAFTEI DIANA - ELENA, MAFTEI DANIEL
to 3,000 metres above sea level, where it is exposed -IOAN, VÂRBAN DAN -IOAN, 2015 -
to strong sunlight. The higher the altitude, the more Preliminary research on Arnica montana L.
aromatic the plant will become. It is found provided in vitro. Studii şi Cercetări, Seria
throughout Europe. Biologie. Biologie Vegetală, Universitatea
Arnica (mountain arnica) is included in the „Vasile Alecsandri” din Bacău, vol. 24, nr.1, p.
Compositae family, about 30cm tall, with yellow 34-37;
flowers that bloom in June-July. The inflorescences 6. MAFTEI DIANA-ELENA, 2013 – Culturi in
are used (Arnicae flos) for treatment and they vitro la plante aromatice, Editura Alma Mater,
comprise volatile oil (0.05 - 0.15%). Used externally Bacău, ISBN 978-606-527-284-2, p.240;
as an anti-inflammatory, and in external bruises, 7. MAFTEI DIANA-ELENA, NICUŢĂ
sprains, arthritis, phlebitis and thrombophlebitis, DANIELA, 2013 – Biotehnologii vegetale. Ghid
venotonic, varicose veins, etc. pentru lucrări practice. Ed. Alma Mater, Bacău,
Used internally, it causes gastrointestinal ISBN 978-606-527-284-2, p.182;
disorders and hypertension. The study aims to 8. PÂRVU C., 1997 - Universul Plantelor, Editura
provide seedlings and plantlets of mountain arnica by Enciclopedică, Bucureşti, p. 54;
conventional culture and by means of plant 9. SAND SAVA CAMELIA, 2015 - Arnica
biotechnologies, in view of repopulating some native montana L. as a medicinal crop species.
habitats of this species in Eastern Romania. Further Scientific Papers Series-Management, Economic
phytochemical and biometrical comparative studies Engineering in Agriculture and Rural 6
will be accomplished on several genotypes of Arnica Development, 15 (4):303-307;
montana L. 10. SĂVULESCU T., 1964 - Flora Republicii
Populare Române. Editura Academiei Republicii
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13. VÂRBAN D.I., VÂRBAN E., ODAGIU A., AUTHORS’ ADDRESS
2012 – Testing Germination capacity on Seeds of
Arnica montana L. Species, ProEnviroment 5, MAFTEI DIANA ELENA – „Vasile
p.251-255. Alecsandri” University of Bacău, Faculty of Science,
e-mail: diana.maftei@ub.ro;
PETREA LOREDANA – IULIANA – „Vasile
Alecsandri” University of Bacău, Faculty of Science,
e-mail lollyana.petrea@yahoo.com.

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