MuNOZ & JIMENEZ Capsule development and germination in Phragmipedium


 Knowing the time the capsule of a particular
orchid species requires until full maturity is very
useful to determine proper harvesting time for in
vitro germination. Ripe capsules usually have larger
amounts of viable seeds than unripe ones. Moreover,
collecting seeds after capsule opening usually
reduces success during in vitro establishment due to
contamination problems and damage caused by the
sterilization process, and should be avoided when
possible.
 Seed viability measured in Phragmipedium (Fig. 3)
is similar to that observed in other terrestrial orchids.
For example, the seed viability of Cypripedium acaule
varied from 20% to 40% in the capsules studied by
Lauzer et al. (1994), and the viability of Calopogon
tuberosus was 35% in the study conducted by Kauth
et al. i2 *.. ,. High values, as measured inP. pearcei in
this study, have been observed in mature capsules of
Ophrys (Kitsaki et al. 2004). Given that seed viability
can vary significantly among capsules within the
same species (up to 60%, as observed in P. pearcei),
it is important to evaluate viability in each capsule.
This is necessary to assess the real effect of particular
conditions on seed germination based on the actual
amount of viable seeds per capsule.
 The seeds of P. pearcei and P. longifolium
germinated faster (2-3 weeks) (Fig. 4) than those
of several terrestrial orchids. Henrich et al. (1981)
showed that 7-12 weeks were necessary for the
beginning of germination in 17 terrestrial orchid
species. In the study of Shiau et al. i2 ', the seeds
of Anoectochilusformosanus germinated eight weeks
after sowing. On the other hand, there are other
terrestrial orchids that germinate faster, as in the case
of C. tuberosus, whose seeds germinated one week
after sowing and reached the maximum germination
in 4-6 weeks (Kauth et al. 2006).
 The percentage of germination in terrestrial
orchids varies among species. Henrich et al. (1981)
found 100% germination in Orchis fucssi and
Epipactis gigantea, 75% in Goodyera oblongifolia
and Spiranthes nrolton/offinima. 50% in Platanthera
stricta and Orchis macula, and 25% in other seven
species, Platanthera dilatata, Liparis laeselii and
Cypripedium reginae among them. These authors
also measured very low germination rates (1%) in


 Cypripedium calceolus, C. candidum, Plantanthera
 hiperboria and P. flava. However, they did not
 evaluate the viability of the seeds used; therefore the
 real percentage of germination of viable seeds could
 not be determined.
 The seeds of P. humboldtii had very low
 germination rate (2.9%) in spite of their relatively
 high viability (34.3%). However, the large quantity of
 seeds in each capsule (data not shown) and the low rate
 of dead protocorms (Fig. 3) allowed the regeneration
 of enough plants using the protocol developed in
 this work for conservation and reintroduction of this
 species.
 It has been considered that both germination and
 embryo staining (e.g., with tetrazolium chloride)
 are comparable viability tests. However, although
 the latter test has been successfully employed with
 epiphytic tropical orchids and several European
 and North American terrestrial orchids, there are
 some reports on inconsistencies in several species,
 probably attributed to variation in the permeability
 of the seed coat (reviewed by Vujanovic et al.
 2000). In one of these cases, Lauzer et al. (1994)
 did not find any correlation between the percentage
 of germination and the percentage of seeds stained
 with tetrazolium chloride in Cypripedium acaule.
 They attributed these differences to a prolonged
 pretreatment with NaOC1 (40 min with 0.6% w/v
 NaOC1, as compared to 10 min with the same
 concentration in our study), a compound that can
 promote dormancy release in terrestrial orchid
 seeds (St-Arnaud et al. 1992).
 Numerous orchid species grow better in the
 dark, mainly during the first phases of development.
 Activated carbon has also been used to darken the
 culture medium and, in this way, improve germination
 (Arditti and Ernst 1993). In some species, the role of
 light during germination is not clear; for example,
 some authors recommend germination of C.
 tuberosus in the dark and others in light conditions
 (reviewed by Kauth et al. 2006). The light or dark
 treatments did not have any effect over germination
 in the Phragmipedium species studied. However,
 larger protocorms in the three species were obtained
 in dark conditions. The protocorms that germinated
 in the dark were white because they do not produce

LANKESTERIANA8(2), August 2008. 0 Umversidad de Costa Rica, 2008.