RESEARCHES CONCERNING IN VITRO CONSERVATION OF THE RARE

PLANT SPECIES DIANTHUS NARDIFORMIS JANKA

I.Holobiuc 1, R.Blindu 1, V.Cristea 2

1
Institute of Biology, Romanian Academy, Bucharest,
2
Cluj-Napoca Botanical Garden
irina.holobiuc@ibiol.ro
Correspondence to: Irina Holobiuc
E-mail: irina.holobiuc@ibiol.ro

ABSTRACT
In our work, we studied the possibility to use in vitro methods for the preservation of the rare species from the Red List of
Romanian Flora-Dianthus nardiformis Janka.
The plant material (whole plants and seeds) was collected from 3 populations from Natura 2000 sites.
The in vitro cultures starting from single node stem fragments had to face with high contamination degree with fungi.
Despite this fact, using different methods of disinfection, culture media and combination of growth factors, aseptic tissues
cultures and micropropagation were successfully performed. The seeds were less infected and are most suitable for in vitro
tissues cultures initiation.
The rate of regeneration was evaluated after 2 months and the tissues cultures were used for the plant production and in
the future for medium-term and long- term preservation approaches.

Keywords: Dianthus nardiformis, in vitro preservation,
Natura 2000.
Introduction
Generally, Conservation strategies rely on the management of
wild populations, but ex situ methods are complementary,
helping the plant preservation and in some cases being the
only alternative.
Ex situ conservation importance have gained the international
recognition through their including in the ninth Article of
CBD (Convention on Biological Diversity) (8). In vitro
methods have a benneficial and definite role comparing to
classical methods of conservation (7, 6, 1, 15, 16).
In vitro plant micropropagation and conservation may also
signifficantly contribute to the maintenance of the natural
populations through the reintroduction of preserved material in
the origin habitat. On the other hand, the missing of universal
valable methods for in vitro preservation of the rare taxa and
the scarcity of plant material for initiation involved extensive
testing. Meantime, the exogenous or endogen contaminations
can be an important obstacle for in vitro conservation.
The species taken in study, Dianthus nardiformis Janka,
is a rare and vulnerable, subendemic plant species, growing
only in Romania and Bulgaria, being included in Romanian
Red lists (2, 5, 12, 17, 13). Dianthus nardiformis Janka
(fig.1a) is a perennial, xerophyle species, with reduced height
and red-violet flowers, blossoming in june-august, growing
on calcareous substrates (14).
The aim of our study was the ex situ conservation in
Dianthus nardiformis using in vitro methods involving: the
collecting of plant material from 3 Natura 2000 Sites, aseptic
tissues cultures initiation, the testing of reactivity and
elaboration of in vitro protocols for conservative purpose.
Materials and methods
Three distinct Natura 2000 sites were chosen to collect plant
material for ex situ conservation: Allah Bair Hill (Constanta
District)-, Consul Hill (Tulcea District), Macin Mountain
(Tulcea District).The plants location was detected using GPS
and for every individuals was performed a field observations
sheet including locations, morphological characterization and
plant associations.
Part of viable plants were transferred and acclimatized in
the Botanical Garden Cluj- Napoca for ex situ field collection
of rare Dianthus species establishment.
In the first step, whole viable plants were collected from

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the natural habitats and explants represented of single node
stem fragments were detached from them and used as
explants for in vitro cultures induction.
In the second step, from the same populations, were
collected seeds used also for tissue cultures initiation and for
Botanical Garden collection.
The sterilization procedures consisted first in the washing
of explants in running tap water for 2 hours, then the
immersion in 700ethyl alcohol (for 30 seconds), the
disinfection using combinations of different sterilizing factors
or a single agent.
In the case of single node stem fragments, owing to high
contamination degree with fungi, a single explant was
cultured in every vessel (at least 10 explants/individuals).
Several sterilization methods with various agents were
used to overcome the high contamination of plant shoots:
1.mercuric chloride 0,1 %, - for 7 minutes;2. sodium dichloro
isocyanurate 1%- 7minutes ; 3. mercuric chloride 0,1 % -2
times X 5 minutes; 4. sodium dichloro isocyanurate 1%- 10
minutes; 5. sodium dichloro isocyanurate 0,1%, 16 hours and
then sodium dichloroisocyanurate 1%, 10 minutes;
6.Domestos 2,4%- 5 minutes and then mercuric chloride,1 %
- 5 minutes. All the sterilization methods were followed by
three washing with sterile distilled water.
Different sterilization procedures were also used for seeds, first
were washed for 2 hours in running tap water and subsequently
were disinfected with: 1. mercuric chloride 0,1 % 2 times x 5
minutes; 2. sodium dichloro isocyanurate 2% for 5 minutes; 3.
Domestos 2,4% for 10 minutes; 4. hydrogen peroxide 2,5% for 16
hours followed by hydrogen peroxide 10% treatment for 15
minutes and finally, three washing in sterile distilled water.
About 10-20 seeds/ Petri dish of 10 cm diameter x 4
repetitions were inoculated.
To asses the efficiency of sterilization procedures, the rate
of contamination (no. of infected explants or seeds/total of
inoculla x100) were determined.
For in vitro regeneration, were tested many culture media
variants having macro- and microelements according
Murashige& Skoog formula (11), supplemented with B5
vitamins (8) and pH adjusted at 5.8. Into culture media
different growth factors and supplements have also been
used. As carbon source was added sucrose at 30g/l and the
media were solidified with 8 g/l Plant agar.
The media variants were: M1 ( supplemented with benzyl
amyno purine-BAP- 1 mg/l);M2(BAP 1 mg/l, α-naphthyl
acetic acid-NAA 0.1 mg/l ), M3 (Kinetin-kin-1mg/l, 2,4-
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dichloro phenoxy acetic acid-2,4-D- 0.1 mg/l), M4 (benzyl
amino purine-BAP- 1 mg/l, kin- 1 mg/l, NAA- 0.2 mg/l), M5
(BAP- 1 mg/l, kin- 1 mg/l, NAA- 0.2 mg/l, glutamine- 0,2
g/l),M6 (BAP- 1 mg/l, kin- 1 mg/l, adenine- 50 mg/l, NAA -
0.2 mg/l),M7 (BAP- 1 mg/l, kin- 1 mg/l, 2,4-D- 0.2 mg/l),
M8 (zeatin- 2 mg/l, NAA- 0.2 mg/l), M9 (adenine -100 mg/l,
NAA- 0.1 mg/l), M10 (thydiazuron- 0.05, NAA -0.01 mg/l).
The cultures with single node stem fragments were
maintained in the growth chamber, at 250C, at 2000-lux
illumination and a photoperiod of 16/8 hours. The seeds were
cultured at dark at the same temperature.
The number of the regenerants obtained per the initial
inocullum was scoredafter 2 months of cultures (minimum-
maximum/ explant).
Results and Discussion
The In a previously experiment, aseptic in vitro cultures were
started from seeds originated from Munich Botanical Garden
using hydrogen peroxide as disinfection agent and
regeneration were made on media supplemented with BAP
and NAA in1/1 and 1/10 ratio (4).
In Dianthus nardiformis, the sterilization of the explants
detached from mature plants were difficult to perform owing
to high degree of contamination with fungi (Actynomycetes)
resistant to the sterilization agents used by us. The problem of
contamination represents an important step to overcome in
the tissues culture initiation (3).
The first contamination appeared at the begining to the fifth
day, with rates between 20 -30% and afterwards varied between
47-75 % during of the first month of culture (table 1.I).
The use of complex methods of sterilization, the
extension of the duration of treatment and the use of many
explants allowed us finally to obtain viable and sterile
inoculla for the majority of individuals.
In the case of seeds, the sterilization efficiency was better
and the degree of contamination of them was lower (varying
between 0% and ~ 5%), being more appropriate to use for
aseptic cultures initiation (table 1.II).
Concerning the in vitro response of D. nardiformis, this
species showed a good reactivity- the way of regeneration
was the direct morphogenesis (fig.1.b), being appropriate for
short –term in vitro preservation and production of viable
plants. (table 2).
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 TABLE 1. supplementary of adenine as citokinine or glutamine as
The contamination rate of the single node stem fragments and nitrogen source had a positive effect concerning the
seeds in D. nardiformis, sterilised using different methods regeneration rate (table2).
I. Single node stem fragments TABLE 2.
II. Seeds sterilization
sterilization The in vitro response of D. nardiformis on different culture
Variant of Contamination Variant of Contamination media after 2 months
sterilization rate (%) sterilization rate (%)
The number of
1 64.8 1 4,25 Culture media
regenerants/inocullum
2 75 2 5,17 variants tested
(min-max)
3 47,1 3 0
M1 1-7
4 51,6 4 0
M2 7-11
5 62,9
M3 5-10
6 60,5
M4 8-10
M5 20-35
M6 14-25
M7 40-50
M8 20-50
M9 5-8
M10 5-7
Also, the zeatin use in the regeneration medium had a
positive effect, but this growth factor is more expensive that
the other combinations of growth factors.
Variant M7 of culture medium induced the best
a)
regeneration, lower cost of the growth factors used
(BAP, kin and 2,4-D) recomends this protocol for the
large scale of multiplication.
Every clone will be subsequently characterised for in
vitro behavior and also at the biochemical and molecular
level.
The preserved plant material will be introduced in a
collection during medium-term period of time and tested for
cryopreservation.

b)
Fig.1-a.In situ D.nardiformis (Macin Mountain), b. In vitro regeneration
in D. nardiformis on M7 medium after 2 months of culture
The in vitro behavior in this taxon was similarly with
those reported by us in other rare Dianthus species (10).
In D. nardiformis, the best results of regeneration rate
were registered on M5-M9 variants
The combination of BAP/K/ANA or BAP/K/2,4-D had a
benneficial effect on the regeneration process. The presence
Conclusions
1. For ex situ conservation purpose, in Dianthus
nardiformis species, three natural populations from Natura
2000 sites were chosen. Mature plants and seeds were
collected and there were used for ex situ conservation
approach: for in vitro tissues cultures and for plant cultivation
in Cluj Botanical Garden.
2. Owing to high contamination degree of fragments of
plants shoots and the need of extensive labour, in vitro
cultures are difficult to induce starting from this kind of
explants. The lower infection rate of the seeds recomened
that tissues cultures to be induced from aseptically
germinated seeds.

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 3. Using in vitro preserved plant material a collection
for ex situ conservation purpose in this rare species can be
established.
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