LANKESTERIANA8(2) 23-31 2008


 CAPSULE DEVELOPMENT, IN VITRO GERMINATION

 AND PLANTLET ACCLIMATIZATION

IN PHRAGMIPEDIUMHUMBOLDTII, P LONGIFOLIUMAND P PEARCEI

 MELANIA MUNOZ' & VICTOR M. JIMENEZ2

 CIGRAS, Universidad de Costa Rica, 2060 San Pedro, Costa Rica
 Jardin Botanico Lankester, Universidad de Costa Rica, P.O. Box 1031, 7050 Cartago, Costa Rica
 imelaniamunozg @yahoo.com; 2victor.jimenez @ucr.ac.cr


 ABSTRACT. Capsule development from pollination to full ripeness was evaluated inPhragmipedium longifolium,
 P. pearcei and P. humboldtii. Besides, seed viability, analyzed in each capsule by means of the tetrazolium
 chloride staining, was determined. Considering seed viability, germination rate was corrected and expressed as
 the rate of viable seeds that germinated in the presence and absence of light, on Knudson C and on half-strength
 Murashige and Skoog culture media. Capsule length remained constant during the evaluation period, while the
 diameter increased during the first 6-8 weeks and then stagnated. Capsule opening occurred 16 weeks after
 pollination in P. longifolium, after 9.8 weeks in P. pearcei and after 32 weeks in P. humboldtii. Seed viability
 averaged 44.7% in P. longifolium, 82.3% in P. pearcei and 34.3% in P. humboldtii. No significant effect of
 light conditions was evident in any of the species. However, a higher proportion of seeds of P. longifolium and
 P. pearcei germinated earlier on half-strength Murashige and Skoog medium than on Knudson C. Only 2.9%
 of the viable seeds of P. humboldtii germinated, while approximately 40% germination occurred in the other
 two species. Initial growth of the embryos was better in the dark on Knudson C medium, compared to the
 other treatments studied. Further growth of the seedlings took place under light conditions. Developed plants
 formed roots and were successfully acclimatized in the greenhouse.

 KEY WORDS: capsule development, in vitro seed germination, pollination, Phragmipedium, terrestrial orchids,
 tetrazolium chloride, tropical orchids


 Introduction
 Slipper orchids belonging to the genus
Phragmipedium (Subfamily Cypripedioideae Lindl.)
are distributed in Meso and South America (Cox et al.
1998, Dressler 2003). They are seriously threatened
because of alteration and destruction of their habitat
and over collection from their natural environment
(Arditti 1992, Salazar 1996).
 Use of in vitro protocols has been foreseen as a
successful approach for ex-situ conservation and
reintroduction of endangered orchids (Stenberg
and Kane 1998, Decruse et al. 2003, Sarasan et al.
2006). Plants regenerated from seeds have a broader
genetic background than those developed by clonal
propagation methods. Therefore, the former meet
the goals of a reintroduction program better, in the
sense of warranting sufficient genetic resources in


the reintroduced population to undergo adaptive
evolutionary change (Guerrant and Kaye 2007).
 This strategy has been successfully employed for
the reintroduction of the orchid species Bletia urbana
(Rubluo et al. 1989), Ipsea malabarica (Gangaprasad
et al. 1999) and Spiranthes brevilabris (Steward et al.
2003). An additional advantage of mass-propagating
orchids for conservation purposes is that increasing
availability of plants from preferred species with
adequate phytosanitary standards and at affordable
prices would reduce illegal collection from the wild
populations (Ramsay and Dixon 2003, Salazar and
Mata 2003).
 Propagation of Phragmipedium through seeds
does not seem to be extremely difficult, because
commercial formulations for asymbiotic germination
in this genus are available. However, the composition