Bulletin of Botanical Gardens, 13: 69–73, 2004

ESTABLISHMENT OF IN VITRO CULTURE COLLECTION OF ENDANGERED EUROPEAN ORCHIDS

Joanna ZNANIECKA, Ewa ¸OJKOWSKA

Intercollegiate Faculty of Biotechnology, University of Gdaƒsk and Medical University of Gdaƒsk, Department of Protection and Biotechnology, K∏adki 24, 80-822 Gdaƒsk, e-mail: [email protected]

SUMMARY conditions of asymbiotic germination of some European species of orchids have already been In order to establish in vitro culture collection studied in several research centers, in Poland at of five endangered European orchid species – the Botanical Garden of the University of L., Dactylorhiza Wroc∏aw (Arczewska 1993, 1998, Arczewska majalis (Rchb.) Hunt et Summerh., and Kuku∏czanka 1991). Spectacular results atrorubens (Hoffm.ex Bernh.) Besser, Epipactis concerning asymbiotic germination and reintro- palustris (Will.) Cr. and Orchis morio L., asym- duction of rare and endangered orchids were biotic seed germination was performed. To esti- obtained at the Royal Botanic Gardens, Kew mate the method yielding the highest percentage (Ramsay and Stewart 1998). The aim of this of germination, mature and immature seeds were project was to compare the efficiency of asym- used (green pod technique). biotic germination of immature and mature seeds. INTRODUCTION MATERIALS AND METHODS Orchids belong to endangered species threat- ened with extinction all over the world. The Plant material – Experiments were carried presence of 46 species of orchids belonging to out on the following species: Cypripedium cal- 22 genera was indicated in Poland. Out of that ceolus L., (Rchb.) Hunt number two are already extinct in Poland: et Summerh., Epipactis atrorubens (Hoffm.ex and Orchis tridentata Bernh.) Besser, Epipactis palustris (Will.) Cr. (Szlachetko 2001). If we consider only the and Orchis morio L. The seeds were excised region of Western Pomerania, then 11 species from capsules produced by natural pollination. are already extinct and 16 are in direct danger The capsules were collected from grow- of extinction (Szlachetko 1995). Therefore all ing in the botanical garden in Go∏ubie species of family come under full (Pomerania) and from natural habitats in preservation in Poland (Dz. U. Nr 27, poz. 134) Mierzeja WiÊlana. Asymbiotic germination of (˚ukowski and Jackowiak 1995). immature and mature seeds was performed in Despite their legal protection orchids are still order to estimate the best conditions for seed decreasing in number. In natural conditions the germination and to investigate the most suitable life cycle of orchids is very long, it takes them moment in seed development to obtain the approximately 5–10 years to bloom and pro- highest percentage of their germination. The duce seeds. Such low proliferation rate makes it method based on sowing immature seeds is very difficult for wild orchids to re-establish called “green pod technique”. Immature seeds their position in natural habitats. That is the rea- were collected at different time intervals post son why a more efficient approach to conserva- pollination – from approximately 20 days from tion of orchids is needed. Biotechnology and flowering in the case of E. atrorubens to 50, 60 modern technologies such as asymbiotic germi- and 80 days in the case of C. calceolus. nation and micropropagation give the opportu- Sterilization – unripe, intact capsules were nity for increasing reproduction rate and rein- surface-sterilized in 5% calcium hypochlorite troduction of orchids to natural habitats. The for 15 min. Afterwards they were dipped in

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Joanna Znaniecka, Ewa ¸ojkowska

Fot. 2. Dactylorhiza majalis plants after 2 years of culture on medium Fast supplemented with activated charcoal a) 0.02% and b) 0.2%.

2,4-D, KIN, NAA (data not shown), asymbiot- embryos that could be wiped off with a scalpel ic germination of E. atrorubens was unsuccess- without cutting into the placenta (Fig. 2A), ful. 2) seeds with yellowish to light brown embryos with loose contact with the placenta (Fig. 2B). DISCUSSION The seeds from the second category did not ger- minate at all, most probably due to the fact that From the data presented, one can conclude they were already entering the period of dor- that optimal conditions for asymbiotic germina- mancy. This may explain the results of tion (maturity of seeds, methods of sterilization Arczewska (1993) who obtained low or even no and medium for germination) have to be devel- germination when mature seeds of C. calceolus oped for each species of terrestrial orchids. In and E. palustris were used for asymbiotic ger- most cases, when seeds form as a result of nat- mination. The data of Ramsay and Stewart ural pollination and are harvested from natural (1998) indicate that the period of time when habitants, it is very difficult to precisely deter- immature seeds germinate efficiently in axenic mine the date of pollination. Because of this the conditions is short, approximately 7–10 days period after flowering was used for the descrip- and should be experimentally estimated for tion of the stage of seed development. each species. Such results make it clear that The highest level of D. majalis germination precise evaluation of maturity of the seeds is was observed with the seeds harvested about 50 crucial and to ensure it, further study employing days from flowering (Fig. 2). However, accord- hand-pollinated seeds are planned for the next ing to embryo colour, some of the seed capsules summer. contained seeds of slightly different stages of The media tested in the study differed wide- maturity. The majority of seeds in each capsule ly in the concentrations of inorganic nitrogen could be grouped into one of the following cat- (and other ions) and varied in substances used egories: 1) seeds with white to yellowish to supplement organic nitrogen. A wide range

72 Establishment of in vitro culture collections of endangered European orchids of complex additives has been supplied in vari- REFERENCES ous media: casein hydrolysate, coconut water, peptone and yeast extract. The most efficient Arczewska A. 1993. Wst´pne stadia rozwojowe media were those supplemented with peptone, storczyków europejskich w kulturze zacho- yeast extract and casein hydrolysate. wawczej in vitro. Acta Universitatis Wratisla- Many of the substrate recipes for seed ger- viensis 1515. Prace botaniczne LVII: 215-221. mination of terrestrial orchids have been devel- Arczewska A. 1998. Badania nad wzrostem i roz- oped. The main trend has been towards reduc- wojem europejskich storczyków w kulturach in ing the concentrations of mineral salts and vitro. Acta Universitatis Wratislaviensis 2038. increasing the amount of organic compounds, Prace botaniczne LXXVI: 15-51. for example Wetsteyn’s modification of MS Arczewska A., Kuku∏czanka K. 1991. Asymbio- medium is based on reduction in macroele- tyczne rozmna˝anie europejskich storczyków. ments to 1/5 of the original concentration and Prace Ogrodu Botanicznego PAN, Zesz. 1, 69-73. supplementation with yeast extract. Such Fast G. 1981. Orchideen Kultur. Stuttgart, 220. attempts aim at simulating the assumed contri- Kuku∏czanka K., Paluch B. 1971. Zastosowanie bution of the mycorrhizal fungus. The soils of peptonu Peptobak-Bacutil w hodowli merys- typical orchid habitats are exceedingly poor in tematycznej tkanki Cymbidium Sw. Acta inorganic nitrogen. In a number of European Agrobotanica XXIV, 1: 52-62. species it has been observed that the germina- Kuku∏czanka K., Sarosiek J. 1971. Merystema- tion percentage improved as the concentration tyczne kultury storczyków. WiadomoÊci Bota- of ammonium and nitrate salts decreased. The niczne XV, 1: 29-40. results obtained confirm the earlier data of Murashige T., Skoog F. 1962. A revised medium Rasmussen (1995), which show that asymbiot- for rapid growth and bioassays with tobacco ic germination is often stimulated by exogenous tissue cultures. Physiol. Plant 15: 473-497. organic nitrogen. Ramsay M.M., Stewart J. 1998. Re-establish- ment of the lady’s slipper orchid (Cypripedium The results indicated that light proved to calceolus L.) in Britain. Botanical Journal of have an inhibitory effect on germination. These the Linnean Society 126: 173-181. findings are completely in line with the results Rasmussen H.N. 1995. Terrestrial orchids from obtained by Arczewska and Kuku∏czanka seed to mycotrophic plant. Cambridge Univer- (1991) and Arczewska (1993). sity Press, 67-70. The failure with E. atrorubens asymbiotic Reinert R.A., Mohr H.C. 1967. Propagation of germination is most probably due to the fact Cattleya by tissue cultures of lateral bud meri- that seeds were harvested either too early and stems. Proc. Am. Soc. Hortic. Sci. 91: 664-71. therefore were not capable of germination or Szlachetko D. 1995. Zagro˝enia gatunków z ro- had already achieved maturity and in order to dziny Orchidaceae na Pomorzu Zachodnim germinate needed breaking seed dormancy. [W:] ˚ukowski W., Jackowiak B. (red.), Ginà- The results obtained in this paper indicate ce i zagro˝one roÊliny naczyniowe Pomorza that problems with low germination of some Zachodniego i Wielkopolski. Bogucki Wydaw- seeds may be overcome by precise estimation nictwo Naukowe. Poznaƒ, 123-126. of time-interval post-pollination and by con- Szlachetko D.L. 2001. Flora Polski, Storczyki. Mul- ducting asymbiotic germination on media tico Oficyna wydawnicza Sp. z o. o. Warszawa. adjusted to the specific requirements of certain Vacin E.F., Went F.W. 1949. Some pH changes orchid species. in nutrient solutions. Bot. Gaz. 110: 605-613. Van Waes J.M., Deberg P.C. 1986. In vitro ger- ACKNOWLEDGEMENTS mination of some Western European orchids. Physiol. Plant. 67: 253-261. We would like to express our gratitude to ˚ukowski W., Jackowiak B. 1995. Ginàce i za- Prof. Dariusz Szlachetko for supplying seeds of gro˝one roÊliny naczyniowe Pomorza Zachod- Orchis morio and all his help. The support niego i Wielkopolski. Bogucki Wydawnictwo for this research was provided by State Com- Naukowe. Poznaƒ. mittee for Scientific Research project No. http://www.orchidseed.com 3PO4C 099 25. http://www.phytotechlab.com

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