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

ESTABLISHMENT OF IN VITRO CULTURE COLLECTIONOF ENDANGERED EUROPEAN ORCHIDS

Joanna ZNANIECKA, Ewa ¸OJKOWSKA

Intercollegiate Faculty of Biotechnology, University of Gdaƒsk and Medical University of Gdaƒsk, Department of Plant Protection and Biotechnology, K∏adki 24, 80-822 Gdaƒsk,

e-mail: lojkowsk@biotech.univ.gda.pl

SUMMARY

In order to establish in vitro culture collectionof five endangered European orchid species –Cypripedium calceolus L., Dactylorhizamajalis (Rchb.) Hunt et Summerh., Epipactisatrorubens (Hoffm.ex Bernh.) Besser, Epipactispalustris (Will.) Cr. and Orchis morio L., asym-biotic seed germination was performed. To esti-mate the method yielding the highest percentageof germination, mature and immature seeds wereused (green pod technique).

INTRODUCTION

Orchids belong to endangered species threat-ened with extinction all over the world. Thepresence of 46 species of orchids belonging to22 genera was indicated in Poland. Out of thatnumber two are already extinct in Poland:Anacamptis pyramidalis and Orchis tridentata(Szlachetko 2001). If we consider only theregion of Western Pomerania, then 11 speciesare already extinct and 16 are in direct dangerof extinction (Szlachetko 1995). Therefore allspecies of Orchidaceae family come under fullpreservation in Poland (Dz. U. Nr 27, poz. 134)(˚ukowski and Jackowiak 1995).

Despite their legal protection orchids are stilldecreasing in number. In natural conditions thelife cycle of orchids is very long, it takes themapproximately 5–10 years to bloom and pro-duce seeds. Such low proliferation rate makes itvery difficult for wild orchids to re-establishtheir position in natural habitats. That is the rea-son why a more efficient approach to conserva-tion of orchids is needed. Biotechnology andmodern technologies such as asymbiotic germi-nation and micropropagation give the opportu-nity for increasing reproduction rate and rein-troduction of orchids to natural habitats. The

conditions of asymbiotic germination of someEuropean species of orchids have already beenstudied in several research centers, in Poland atthe Botanical Garden of the University ofWroc∏aw (Arczewska 1993, 1998, Arczewskaand Kuku∏czanka 1991). Spectacular resultsconcerning asymbiotic germination and reintro-duction of rare and endangered orchids wereobtained at the Royal Botanic Gardens, Kew(Ramsay and Stewart 1998). The aim of thisproject was to compare the efficiency of asym-biotic germination of immature and matureseeds.

MATERIALS AND METHODS

Plant material – Experiments were carriedout on the following species: Cypripedium cal-ceolus L., Dactylorhiza majalis (Rchb.) Huntet Summerh., Epipactis atrorubens (Hoffm.exBernh.) Besser, Epipactis palustris (Will.) Cr.and Orchis morio L. The seeds were excisedfrom capsules produced by natural pollination.The capsules were collected from plants grow-ing in the botanical garden in Go∏ubie(Pomerania) and from natural habitats inMierzeja WiÊlana. Asymbiotic germination ofimmature and mature seeds was performed inorder to estimate the best conditions for seedgermination and to investigate the most suitablemoment in seed development to obtain thehighest percentage of their germination. Themethod based on sowing immature seeds iscalled “green pod technique”. Immature seedswere collected at different time intervals postpollination – from approximately 20 days fromflowering in the case of E. atrorubens to 50, 60and 80 days in the case of C. calceolus.

Sterilization – unripe, intact capsules weresurface-sterilized in 5% calcium hypochloritefor 15 min. Afterwards they were dipped in

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Joanna Znaniecka, Ewa ¸ojkowska

2,4-D, KIN, NAA (data not shown), asymbiot-ic germination of E. atrorubens was unsuccess-ful.

DISCUSSION

From the data presented, one can concludethat optimal conditions for asymbiotic germina-tion (maturity of seeds, methods of sterilizationand medium for germination) have to be devel-oped for each species of terrestrial orchids. Inmost cases, when seeds form as a result of nat-ural pollination and are harvested from naturalhabitants, it is very difficult to precisely deter-mine the date of pollination. Because of this theperiod after flowering was used for the descrip-tion of the stage of seed development.

The highest level of D. majalis germinationwas observed with the seeds harvested about 50days from flowering (Fig. 2). However, accord-ing to embryo colour, some of the seed capsulescontained seeds of slightly different stages ofmaturity. The majority of seeds in each capsulecould be grouped into one of the following cat-egories: 1) seeds with white to yellowish

embryos that could be wiped off with a scalpelwithout cutting into the placenta (Fig. 2A), 2) seeds with yellowish to light brown embryoswith loose contact with the placenta (Fig. 2B).The seeds from the second category did not ger-minate at all, most probably due to the fact thatthey were already entering the period of dor-mancy. This may explain the results ofArczewska (1993) who obtained low or even nogermination when mature seeds of C. calceolusand E. palustris were used for asymbiotic ger-mination. The data of Ramsay and Stewart(1998) indicate that the period of time whenimmature seeds germinate efficiently in axenicconditions is short, approximately 7–10 daysand should be experimentally estimated foreach species. Such results make it clear thatprecise evaluation of maturity of the seeds iscrucial and to ensure it, further study employinghand-pollinated seeds are planned for the nextsummer.

The media tested in the study differed wide-ly in the concentrations of inorganic nitrogen(and other ions) and varied in substances usedto supplement organic nitrogen. A wide range

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

Establishment of in vitro culture collections of endangered European orchids

of complex additives has been supplied in vari-ous media: casein hydrolysate, coconut water,peptone and yeast extract. The most efficientmedia were those supplemented with peptone,yeast extract and casein hydrolysate.

Many of the substrate recipes for seed ger-mination of terrestrial orchids have been devel-oped. The main trend has been towards reduc-ing the concentrations of mineral salts andincreasing the amount of organic compounds,for example Wetsteyn’s modification of MSmedium is based on reduction in macroele-ments to 1/5 of the original concentration andsupplementation with yeast extract. Suchattempts aim at simulating the assumed contri-bution of the mycorrhizal fungus. The soils oftypical orchid habitats are exceedingly poor ininorganic nitrogen. In a number of Europeanspecies it has been observed that the germina-tion percentage improved as the concentrationof ammonium and nitrate salts decreased. Theresults obtained confirm the earlier data ofRasmussen (1995), which show that asymbiot-ic germination is often stimulated by exogenousorganic nitrogen.

The results indicated that light proved tohave an inhibitory effect on germination. Thesefindings are completely in line with the resultsobtained by Arczewska and Kuku∏czanka(1991) and Arczewska (1993).

The failure with E. atrorubens asymbioticgermination is most probably due to the factthat seeds were harvested either too early andtherefore were not capable of germination orhad already achieved maturity and in order togerminate needed breaking seed dormancy.

The results obtained in this paper indicatethat problems with low germination of someseeds may be overcome by precise estimationof time-interval post-pollination and by con-ducting asymbiotic germination on mediaadjusted to the specific requirements of certainorchid species.

ACKNOWLEDGEMENTS

We would like to express our gratitude toProf. Dariusz Szlachetko for supplying seeds ofOrchis morio and all his help. The support for this research was provided by State Com-mittee for Scientific Research project No.3PO4C 099 25.

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http://www.orchidseed.comhttp://www.phytotechlab.com

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