Bulletin UASVM Horticulture, 66(1)/2009

Print ISSN 1843-5254; Electronic ISSN 1843-5394

In Vitro Multiplication, Conservation and Ex Vitro Acclimation

of Drosera rotundifolia

Doina CLAPA1), Alexandru FIRA1), Ioan PACURAR2)

1)
Fruit Research Station Cluj, 5 Horticultorilor St., Cluj-Napoca 400457,
Romania; www.scdpcluj.ro, doinaclapa@yahoo.com
2)
University of Agricultural Sciences and Veterinary Medicine,
3-5 Manastur St., Cluj-Napoca 400372, Romania

Abstract. At the Fruit Research Station of Cluj experiments were carried out for the
optimization of in vitro proliferation of Drosera rotundifolia as well as for the in vitro conservation, in
vitro rooting and ex-vitro acclimation of this species. For proliferation, modified Murashige & Skoog
(MS) media containing either 200 ml/l coconut water or 5 mg/l Kinetin proved to be optimal. BAP in
the media proved not to be suitable. For in vitro conservation a double layer hormone-free MS
medium was used, which ensured the long-term storage and in vitro proliferation of Drosera rosettes.
For in vitro rooting, two variants of MS medium were used, one with macroelements diluted to ¼ and
the other with macroelements diluted to ½. Both variants proved to be suitable for in vitro rooting and
increasing plantlet size; they provided 100 % rooting percentage and there were no significant
differences between the two variants regarding the biomass of the resulting plantlets. Ex-vitro
acclimation was done most efficiently by hydroculture, in uncovered trays containing water with
neutral pH.

Keywords: sundew, micropropagation, hydroculture, conservation

INTRODUCTION

Drosera rotundifolia - round leafed sundew is one of the few species of carnivorous
plants that can be found also in Romania especially in acidic peat bogs in the mountains. It is
protected by law, most of its areal is part of natural reservations.
It has weak roots and the leaves are grouped in a basal rosette. The petiole is long and
the lamina is covered with hairs. The hairs, also termed tentacles, secrete sticky substances
that gather in the form of some sparkling droplets, from which the name of the plant
originates. An insect that sits on a leaf of this plant gets stuck in the sticky secretion and, by
trying to free itself it gets stuck to more hairs. The hairs entangle the insect and secrete an
abundant sticky juice that suffocates the insect. Then the insect is digested by certain
proteolytic enzymes in a few days, so that only the skeleton remains.
One plant consumes approximately 50 insects/year. In late spring and early summer
the pentamere, pinkish-white or violet flowers appear, forming a scorpioid inflorescence.
Recently some Drosera species became important also for pharmaceutical purposes as
they contain plumbagin; and for molecular biology, as they contain genes for important
enzymes.
Plant tissue culture can provide a means for the conservation, and propagation of this
species and the ex-vitro acclimated plantlets could be used for repopulating its natural
habitats. Plant tissue culture could also provide the means for obtaining large amounts of

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biomass for plumbagin production by using bioreactors.
Research regarding tissue culture of several Drosera species was done by a few
researchers, such as: Bekesiova et al., (1999), Cachiţă-Cosma et al.,(2008), Ichiishi et
al.,(1999), Jayaram et al.,(2009), Sauerwein et al., (1994), Turcuş, (2009).
Having an unusual, interesting shape and beautiful colours, Drosera rotundifolia can
also be used as an ornamental. At the Fruit Research Station of Cluj a protocol was optimized
for the in vitro propagation and ex-vitro acclimation of Drosera rotundifolia.

MATERIALS AND METHODS

In our study the experiments were started from Drosera rotundifolia cultures already
available in vitro.
For the in vitro multiplication experiments Magenta vessels a modified MS medium
was used (Table 1, variant 0), to which various growth regulators were added (Table 2).
The coconut water used in the experiments resulted from ripe fruit.
All the components were added to the media before autoclavation. The media were
poured into Magenta GA7 vessels (cca 50 ml/vessel) and autoclaved at 121 ˚C for 20 minutes.
For the proliferation experiments, 5 rosettes ca 2 cm in diameter were inoculated into
each vessel. For the rooting experiments, 9 rosettes of the same size were inoculated into each
vessel. The cultures were kept at cca 300 lux and 16-hour photoperiod, at temperatures of 23-
26 ˚C in the growth chamber. Both for in vitro multiplication and rooting the cultures were
kept for 2 months.
For the rooting experiments, 2 variants of MS medium were used (Table 1, Variants 1
and 2) as well as liquid MS medium with perlite as solid support. In the latter case, cca 10 g
of perlite+100 ml MS medium were used/Magenta vessel
For conservation, liquid MS medium (cca 100 ml/vessel) was poured onto Drosera
rotundifolia cultured in 2 Magenta vessels in solid, hormone-free MS medium).
Tab. 1
The media used for Drosera maintenance and rooting

Component Variant 0 Variant 1 Variant 2

MS macroelements Full concentration Diluted to 1/4 Diluted to la 1/2
MS microelements Full concentration Full concentration Full concentration
FeNaEDTA 36.7 mg/l 36.7 mg/l 36.7 mg/l
Myo-inositol 100 mg/l 100 mg/l 100 mg/l
Vitamin B1 1 mg/l 1 mg/l 1 mg/l
Vitamin B6 0.5 mg/l 0.5 mg/l 0.5 mg/l
Nicotinic Acid 0.5 mg/l 0.5 mg/l 0.5 mg/l
Sugar 30 g/l 30 g/l 30 g/l
Plant Agar (Duchefa) 6 g/l 6 g/l 6 g/l
PH adjusted to 5.8

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 Tab. 2
The experimental variants usedfor proliferation and the resulting multiplication rate

Variant Mediul nutritiv Vessel No. of rosettes Rosettes Multiplication rate

no. resulted/vessel resulted/ (rosettes
vessel resulted/inoculm
1 Hormone-free MS 1 169 154.5 30.9

2 140

2 Hormone-free MS 1 107 110.5 22.1

without agar
2 114

3 MS+ 100 ml/l 1 219 231 46.2

coconut water
2 243

4 MS+ 200 ml/l 1 271 240 48

coconut water
2 209

5 MS+ 2 mg/l kinetin 1 168 168 33.6

6 MS+ 5 mg/l kinetin 1 256 256 51.2
7 MS+ 10 mg/l kinetin 1 154 154 30.8
8 MS+ 0.5 mg/l BAP 1 183 171.5 34.3
2 160
9 MS+3 mg/l BAP+0.2 1 No viable plant
mg/l kinetin

For ex-vitro acclimation, several variants were tested: plastic trays and pots containing
peat, perlite, peat+perlite in 1:1 volume to volume ratio, peat+soil, perlite+soil.
Acclimation in hydroculture was also tested; for this purpose plastic trays and glass
jars containing a layer of 2-3 cm of tap water from Cluj-Napoca with the pH=7 were used.
The plant material used on the solid substrates consisted in Drosera plantlets cultured
on full-strength MS medium (Tab. 1, Variant 0). In hydroculture, plantlets cultured on all 3
variants were tested.

RESULTS AND DISCUSSION

In the proliferation stage, the highest multiplication rate was provided by variant 4
(MS+ 200 ml/l coconut water), but without great differences from variant 3 (MS+ 100 ml/l
coconut water). MS+ 5 mg/l kinetin also provided a high proliferation rate. BAP did not
increase multiplication rate very significantly and the resulting rosettes were rather deformed.
Hormone-free MS gelled with agar provided a higher proliferation rate and better developed
plantlets in comparison with liquid MS (Tab. 2 )
The rooting percentage was of 100 % on full-strength hormone-free MS medium
(Table 1, variant 0) as well as the variants tested for rooting (Variants 1 and 2). On Variant 0
the plantlets proliferated and rosette sizes were much smaller than those on Variants 1 and 2,
where there was weak proliferation and the plantlets grew larger, with robust leaves covered

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with well-developed tentacles, each bearing a red droplet of digestive secretion. The green
biomass of the Drosera plantlets grown on the rooting media was remarkably high (Fig. 1)
and the two rooting variants gave remarkably similar results.
The Drosera plantlets also rooted on liquid hormone-free MS medium containing
perlite as substrate; rooting percentage was also 100 %, but the plantlets were less robust than
in media gelled with agar (Fig. 2).

a. b.

Fig. 1. Green biomass of Drosera rotundifolia on the rooting media: a) on MS with macroelements diluted to ¼;
b) on MS with macroelements diluted to ½;

a b c d

Fig 2. Drosera in vitro: a) and b) on modified MS (Table 1, Variant 1); c) on modified liquid MS + perlite; d) on
full-strength liquid MS

For conservation (Fig. 3), liquid MS medium (cca 100 ml/vessel) was poured onto
Drosera rotundifolia cultured in Magenta vessels in solid, hormone-free MS medium)for 2
months. After another 9 months in culture the plantlets were still alive, resulting a mass of
abundantly proliferated plantlets. The results regarding the number of rosettes harvested from
the 2 vessels and plantlet biomass are presented in Table 3.
Drosera acclimation (Fig. 4) failed in all the experiments using solid substrate for
acclimation, except for the plastic pots containing perlite, where the plantlets were covered
with glass jars. In the latter case, the plantlets were acclimated in 3 months, with a survival
rate of cca 1/3.

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 Tab. 3.
Number and biomass of plantlets during conservation

Vessel no. No. of Total Biomass/plantlet (g)

resulting plantlets biomass (g)
1 625 65.48 0.1047
2 942 79.88 0.0847
Average 783.5 72.68 0.0947

Fig. 3. Drosera conservation on double layer medium

Drosera plantlets cultured on full-strength MS medium could not be acclimated in

hydroculture; mortality rate was of 100 %.
Acclimation in hydroculture was entirely successful in the case of Drosera plantlets
cultured on modified MS media with low amounts of macroelements (Table 1, Variants 1 and
2), where survival rate was of 100 % during a 2-month culture period in all types of vessels
used for acclimation (plastic trays, glass jars), the plantlets grew during the acclimation phase,
they flowered and slightly proliferated. The leaves became visibly more robust and the
number and size of the tentacles containing droplets of digestive secretion increased.

Fig. 4. Acclimated Drosera plantlets

CONCLUSIONS

Drosera rotundifolia is a rare, endangered and interesting species. It is an indicator of

soil acidity and moisture.
For the in vitro propagation of this species, several media proved to be suitable.
Among the variants that we tested, modified MS medium containing coconut water proved to
be suitable, but high multiplication rates were obtained on MS medium containing 5 mg/l
Kinetin as well as on hormone-free MS gelled with agar.
In vitro storage was achieved on double-layer MS medium, on which massive
proliferation of the rosettes took place during the storage period.
In vitro rooting was successful on all the variants of hormone-free MS medium and ex-

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vitro acclimation was achieved by using the technique of hydroculture, in the case of plantlets
rooted on MS media with reduced amounts of macroelements.

REFERENCES

1. Bekesiova, I., J. P. Nap and L. Mlynarova (1999). Isolation of High Quality DNA and RNA from
Leaves of the Carnivorous Plant Drosera rotundifolia, Plant Molecular Biology Reporter 17: 269–277.
2. Cachiţă-Cosma, D., V. Turcuş, A. Petruş-Vancea, L. Barbutudoran and C. Crăciun (2008). Drosera
Rotundifolia L. Vitroculture Associated with a Saprophyte Fungus, Sudia Universitatis, pp.103-105.
3. Ichiishi S., T. Nagamitsu, Y. Kondo, T. Iwashina, K. Kondo and N. Tagashira (1999). Effects of
Macro-components and Sucrose in the Medium on in vitro Red-color Pigmentation in Dionaea muscipula Ellis.
and Drosera spathulata Labill., Plant Biotechnology, 16 (3), 235-238.
4. Jayaram, K., and M. N. V. Prasad (2005). Rapidly in vitro multiplied Drosera as reliable source for
plumbagin bioprospection, Current Science, Vol. 89, No. 3, pp 407-408.
5. Sauerwein M., S. Schmidt, J. Reichling and M. Wink (1994). Naphtoquinone production in in vitro
cultures of Drosera communis St. Hil., BIOForum Extra, Prague, pp. 26-27.
6. Turcuş, V. (2009). Studii Morfo – Studii anatomice si citologice efectuate la nivelul vitroplantulelor
de Drosera Rotundifolia L., Teza de doctorat.

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