IN VITRO REGENERATION OF ENDANGERED
ALPINE FLOWERS
Kremen R., Lopes M. S., da Câmara Machado A., Katinger H. and Laimer da Câmara Machado M.; 1995
Institute of Applied Microbiology, University of Agriculture, Vienna/AUSTRIA
URL: http://www.boku.ac.at/iam iam@mail.boku.ac.at
Nussdorfer Lände 11, A-1190 Vienna, AUSTRIA (+43 1 3692924 - 402 FAX: 400 )
ORCHIS MACULATA, LILIUM BULBIFERUM
ORCHIDS : ORCHIS MACULATA, LISTERA OVATA
INTRODUCTION
The generative propagation of terrestrical orchids (Fig. 1) is in some cases different from that of other seed plants. Orchids produce very small seeds (0.5 - 2 mm long with a diameter up to 0.5 mm (Fig. 2 and 3)) which consist only of an embryo and the testa and they have no endosperm (Thompson, 1977; Williams, 1979). To compensate the lack of stored energy, on the one hand orchids produce up to 1 million seeds per plant and on the other hand most species require the infection with a particular species of fungi. This symbiosis is a very delicate balance between the protocorm and the fungi. The long hairy cells of this tissue facilitate the infiltration of the fungi, which provides the young orchid with energy, salts and vitamines. Therefore the medium for the asymbiontic germination of orchids has to provide all this substances to the embryo.
Fig. 1: Orchis sp.
Fig. 2: Opend capsule of a terrestrical orchid (total length 18 mm)
Fig. 3: Seed of a terrestrical orchid; inside the embryo, surrounded by the testa (total length 1.5 mm)
MATERIAL AND METHODS
Listera ovata and Orchis maculata capsules (Fig. 2) were sterilised for 20 min. with 12.5 %(v/v) Natriumhypochlorit. To prevent infection it is advisable to use only closed capsules. The seeds were spread on the medium (Murashige, 1962) and to test the influence of light on the germination one half was cultivated in light (daylight, 16 hours per day) and the other half was incubated in the dark at 23 °C.
RESULTS AND DISCUSSION
The development of the protocorm (Fig. 4) in vitro took 6 - 7 month in the darkness and one month longer in the light. Approximately 30 % of the seeds germinated. During the cultivation the media had to be optimised, concerning the pH, the addition of activated charcoal and complex components (Tab. 1).
Fig. 4: Germination of the protocorms in vitro in the darkness
(Listera ovata) diameter 1 - 2 mm
NH4NO3 0,825 g/l
KNO3 0,475 g/l
CaCl2 . 2H2O 0,44 g/l
MgSO4 . 7H2O 0,74 g/l
KH2PO4 0,17 g/l
Na2EDTA 0,0373 g/l
FeSO4 . 7H2O 0,0278 g/l
Microelements 0,50 x MS (Murashige 1962)
further compounds:
20 g/l Saccharose
8 g/l Agar
1 g/l Activated charcoal
5 g/l Banana - pulp
adjust pH to 6,1 15 min after addingactivated charcoal
Tab. 1: Example for a used medium for cultivation of Listera ovata
(Fig.: 6)
Fig. 6: Lilium bulbiferum ssp. bulbiferum;
6a:plant established by the use of bulbs
6b: regenerateded out of a seed
Eight month after germination the slow growing plantlets reached the appropriate size for acclimatisation to the greenhouse (Fig. 5 and 7).
This model for asymbiontic orchid-germination seems to be an effective way for the propagation of terrestrical orchids. To verify this system further experiments with other species should be done in the future.
Fig. 5: Listera ovata in vitro
Fig. 7: In vitro culture of Orchis maculata and Listera ovata
Thompson P. A., Orchids from Seed, Royal botanic gardens kew, 1977
Murashige T. & Skoog F. 1962; A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15: 473 - 497;
Williams, John G., Orchideen Europas, BLV Verlagsgesellschaft (1979), ISBN 3-405-11901-4
LILIUM BULBIFERUM SSP. BULBIFERUM
INTRODUCTION
Lilium bulbiferum is a member of the plant familiy Liliaceae and occurs in two subspecies: ssp. bulbiferum and ssp. croceum (Fig. 8). The natural occurrence of this plants are the mountain ranges from spain to eastern europe (Roger et. al., 1989). This species has two strategies of propagation: a.) seeds and b.) bulbs, located at the nodes.
The multiplication capability of the bulbs (Fig. 9) was investigated in tissue culture as well as in the greenhouse in order to find out the best way of propagation.
Fig. 8: Lilium bulbiferum ssp. croceum
Fig. 9: Bulb of Lilium bulbiferum ssp. bulbiferum used for the establishing of the culture
MATERIAL AND METHODS
Bulbs and immature seeds were sterilised and transfered onto medium, based on Murashige (1962) with 1.12 mg BAP and 0.20 mg NAA per liter. The regenerated plants were subcultured every four weeks.
For rooting the plantlets were cultivated either on medium containing 0,2 mg/l IBA or on hormon free medium (Novak, 1981).
RESULTS AND DISCUSSION
The in vitro culture of the bulbs with the chosen medium allowed to triple the culture volume every month.
In comparison with in vivo and in vitro propagation the tissue culture method turned out to be much more advantageous due to the higher multiplication rate of the bulbs. In addition the bulbs in the in vivo culture demanded a cold storage for one month to start growing.
The application of IBA accelerated the root development and allowed the acclimatisation of the plants one month after this treatment, compared to a 1 - 2 month delay when the plants were rooted on hormon free medium.
Bulbs, Roger Phillips and Martyn Rix, 1989, Pan Books Ltd London, ISBN 0 330 30253 1
Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant 15: 473 - 497;
Novák, F. J. and Petru, E., 1981. Tissue culture propagation of Lilium hybrids. Scientia Hortic., 14: 191 - 199;
ABBREVIATIONS
BAP 6 - Benzylaminopurine
NAA Naphtylacetic - acid
IBA Indolbutyric acid
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04/11/95 by IAM
