ASPECTS REGARDING TRADITIONAL CULTURE OBTAINED THROUGH THE IN VITRO MULTIPLICATION OF THE RARE, VULNERABLE SPECIES Arnica montana L. Iuliana-Ruxandra… [601442]

ASPECTS REGARDING TRADITIONAL CULTURE
OBTAINED THROUGH THE IN VITRO MULTIPLICATION OF THE
RARE, VULNERABLE SPECIES Arnica montana L.

Iuliana-Ruxandra PANCIU1, Gheorghe SIN2

1University of Agronomic Sciences and Veterinary Medicine of Bucharest,
59 Mărăști Blvd, District 1, 011464, Bucharest, Romania
2Academy of Agricultural and Forestry Sciences "Gheorghe Ionescu- Șișești",
61 Mărăști Blvd, District 1, 011464, Bucharest, Romania

Corresponding author email: [anonimizat]

Abstract

Taking into account the importance of Arnica Montana, we think that it is important to know as much as possible about
the multiplication and the cultivation technologies of this species.
The purpose of our study is to initiate traditional culture obtained from in vitro multiplication of this species.
The aseptic germinated seedlings were used as explants, the origin of the direct morphogenesis beeing in the apical meristem. So as for induction of regeneration, to promote plant growth and rooting, we have tested different
combinations of growth factors and other components such as the ascorbic acid, polyvinylpyrrolidone-PVP, glutamine
and active charcoal added in culture media based on Murashige&Skoog formula. The best results were recorded on variant supplemented with PVP, reaching a number of 17 regenerants per explant,
after eight weeks of cultivation.
The ex vitro transfer of the rooted plants was made after 9-10 weeks, achieving a survival rate of 75% during the first month after acclimatization.
The multiplication protocol that was used allowed us to obtain healthy, developed and well- rooted plants that made it
possible to start our first culture in a private nursery situated in the locality of Corbeni, Arges County.

Key words : Arnica montana L., multiplication in vitro, traditional culture.

INTRODUCTION

Arnica montana is classified as a rare and
vulnerable taxon, as a result of the habitats fragmentation, excessive grazing and harvesting, most of the populations being diminished and eventually disappearing. It is included in Annex D of Regulation no.
338/97 of the European Council and Annex V
of the Habitats Directive (92/43/EC-
Council Directive 92/43/EEC of 21 May 1992 on the conservation of natural habitats and of wild fauna and flora).
This species is cultivated to reduce harvesting
pressure on the natural populations and to provide the raw material for the national
industry and for exportation. In Romania, there
are cultures, but they stretch on rather small surfaces and our country continues to be one of the main providers of raw material. Given the importance of the species, there have been made a lot of studies regarding its cultivation, the in vitro multiplication and the evaluation of the content of active principles
both in vivo and at the in vitro regenerants.
Our study aimed at inducing and stimulating the in vitro seed germination and using the
aseptic germinated seedlings as explants for the in vitro multiplication at optimized levels, as
well as evaluating the growth and regeneration of the in vitro plants, so that they could be used
for the initiation of traditional cultures. After the finalization of the acclimatization process, the obtained plants were transported to the planting area.
MATERIALS AND METHODS

As raw material, there were used seeds
purchased from Germany, of the 2013 harvest,
which were sterilized through washing in running tap water for two hours, and then they were immersed in ethyl alcohol at 70
o for one
minute, followed by the treatment with 0.1% mercuric chloride and three washing in sterile distilled water.
291Scientific Papers. Series A. Agronomy, V ol. LVIII, 2015
ISSN 2285-5785; ISSN CD-ROM 2285-5793; ISSN Online 2285-5807; ISSN-L 2285-5785

The sterilized seeds were cultivated for one
week in sterile distilled water + 5mg/l of Gibberellic Acid and then transferred on MS medium, medium free of growth factors for the plant development.
The seed germination top rate was reached after
two weeks. The cultures were started on seven different culture mediums based on the MS formula of micro and macroelements, supplemented with Gamborg vitamins (Gamborg et al., 1968), 30 g/l sucrose, 7 g/l agar (Duchefa Biochemie), adjusted to pH 5.8, supplemented with various components. The in vitro cultures were started with
approximately 1 cm-high seedlings resulted from the aseptic seeds germination.

Figure 1. Aseptic germinated seedlings used as explants
in vitro culture

Each four weeks we initiated the sub-cultures, in the first stage for each variant of the 7 culture mediums there were inoculated two explants/ Petri dish, repeating the process 4 times, and afterwards 4-5 explants/ autoclavable polypropylene dish (Duchefa –
9x10x10cm). In the second stage of the multiplication, there were tested 3 culture mediums, initial mediums tested were M5, M6 and M7 from the first stage (Table 1), supplemented with 0.5 g/l
active charcoal so as to improve vigorously of
the plant and to help rooting, we registered the maximum growth of plants after 2 months (Figure 6).

Figure 2. Regenerated rooted plant obtained on M7
variant before ex vitro transplantation

RESULTS AND DISCUSSIONS

For the multiplication of the species Arnica
montana it is preferred the direct
morphogenesis, because the regeneration is more efficient and the plants are more stable from a genetic point of view, in comparison with indirect morphogenesis. Starting from aseptic germinated seedlings that were cultivated on different medium variants,
there was evaluated the efficiency of the
regeneration and growth of the new plant. We tested growth factors such as cytokinin benzyl-aminopurine, kinetin or zeatin associated with alpha-naphtyl acetic acid. In order to improve regeneration rate, to promote plant growth and rooting, we tested different combinations of growth factors and 0.5 g/l active charcoal. The Gibberellic Acid was used on all culture mediums in order to stimulate the newly-formed shoots elongation. The best results recorded after 4 and 8 weeks (Figure 5) regarding the number of regenerants on the initial inoculum, were those of variant M7 supplemented with polyvinylpyrrolidone –
PVP. 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. The ex vitro transfer of the rooted plants was
made after 9-10 weeks, achieving a survival
rate of 75% during the first month after the
acclimatization.
The planting was made at the end of August in a private nursery of the Arges County, situated at approximately 850 m altitude.
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Figure 3. Initiation of traditional culture

Figure 4. Arnica montana after 3 weeks in culture A number of 100 plants were planted directly
in the slightly acid soil (pH of 5.5-5.8), at 25 cm distance between the plants and 30 cm between the rows. No herbicides were applied on the soil, which
was manually tilled by digging and harrowing;
any kind of herbicide would have a negative impact on the development of the plant. The plant irrigation was also manually carried out, with well water. In the period before wintering, there were taken protection measures against strong air draughts by hedging. In order to avoid the plant destruction as a result of the massive snow falls, the land plot was protected with a twig hedge, providing at the same time the necessary amount of water.

Table 1. Media composition used for the induction of direct morphogenesis in Arnica montana L.
Components Average of tested variants
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.

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Figure 5. The number of regenerants/ initial explant after
4 and 8 weeks of culture (mean values + SD) Figure 6. The maximum growth of plants after 2
months

CONCLUSIONS

The ex vitro acclimatization of well-developed
plants (4-5 cm-high with roots 7-10 cm-long) was carried out on a perlite 50% – soil 50% substrates, at a temperature of 20
oC,
illumination of 2000 lux and humidity between
95 and 75%, with gradual lowering to 75%
during the third week. The in vitro multiplication protocol that we
elaborated for Arnica montana can provide in a
short period a high number of vigorous plants, with a well-developed root system. The quick obtaining of a high number of plants helps the establishment of cultures and the production of the raw material without destroying the natural environment where this plant grows, the results that were obtained following our study encouraging the establishment of traditional cultures.
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