LANKESTERIANA


of the culture media has remained elusive to the
scientific community for commercial reasons. Most
research on this and related genera is limited to studies
on systematics and evolution (e.g., Cox et al. 1997,
1998). Investigations on phenology in this genus,
describing characteristics of seed capsules and their
development, are scarce (Arditti and Ghani 2000).
 When proper conditions for germination are to
be assessed, it is important to know the viability of
the seeds under study. This allows distinguishing
the proportion of seeds that do not germinate as a
consequence of unfavorable germination conditions
from those seeds that are not capable of germinating at
all due to lack in viability. The most common method
used to evaluate seed viability is the tetrazolium
(C19H,1C1N4) stain. Dehydrogenases, which are active
in living tissues, reduce the colorless tetrazolium
chloride to a red compound, coloring viable seeds. The
intensity of the tint could vary from pink to dark red
(Singh 1981). This technique has been successfully
used to test viability of orchid seeds (Lauzer et al.
1994, Vujanovic et al. 2000).

 The aims of this study were to describe the
development of P. humboldtii, P. longifolium, and P.
pearcei capsules, to evaluate the seed viability and to
establish a method for in vitro germination of mature
seeds and for growing and acclimatizing plantlets
of these three Phragmipedium species aiming at
conservation.

 Materials and methods

Pollination of flowers and capsule development.
 Manual pollination of flowers of Phragmipedium
humboldtii, P. longifolium, and P. pearcei, growing at
Lankester Botanical Garden, Universidad de Costa
Rica, was conducted using pollen from a different
plant. Capsule length, diameter and color were
evaluated weekly, until full maturity. The time taken
for each capsule to open was also recorded.

Seed viability. The percentage of viable seeds was
determined in each ripe capsule using the method of
tetrazolium stain (Singh 1981). For that purpose, four
subsamples of each of the two P. humboldtii, one P.
longifolium, and four P. pearcei capsules were placed
in tetrazolium chloride (1%, pH 6-7), in a water bath

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


(300C) and in dark conditions for 24 h. Subsequently,
they were transferred to Petri dishes and the percentage
of viable (stained) seeds was determined with aid of
a stereomicroscope (model 222279, Nikon). Seeds
from the corresponding capsules were used in the
germination studies.

In vitro germination. Seeds from open capsules
were sterilized in sodium hypochlorite (NaOC1,
0.6% w/v) and Tween 20 (1 drop/100 ml) for 10
min. They were subsequently screened through
sterile Albet  filter paper (quantitative quality), in
the laminar hood. They were then rinsed three times
with sterile distilled water, while adhered to the filter
paper and, after decanting the water of the last rinse,
distributed evenly, with a scalpel (blade No. 22) on
20 ml of semisolid culture medium contained in 90
mm-Petri dishes.
 Knudson C (Knudson 1946) and half-strength
Murashige and Skoog (1962) mineral salts, both
supplemented with 1 mg 1-1 thiamine, nicotinic acid
and pyridoxine, together with 20 g 1-1 sucrose, were
compared for seed germination. pH was adjusted to
5.7 and media gelled with agar (0.8%). Media were
autoclaved at 1.05 kg cm2 for 25 min.
 Additionally, two light regimes were evaluated
during germination: dark conditions and photoperiod
of 12 hours (10.9 imol m2 s-1, Sylvania Supersaver
Cool White, 32 W, F48%12/CW/SS). Cultures were
grown at 25+10C.
 To determine the percentage of germination, three
squares, of 1 cm2 each, were drawn in each Petri dish.
Areas of the Petri dishes in which individual seeds
could be clearly observed using a stereomicroscope
were selected to draw the squares. Total number of
seeds in each section was counted at the first day
of culture. Afterwards, the number of germinating
seeds (those in which rupture of the testa occurred
by the enlarging embryo) was assessed weekly. Three
Petri dishes were evaluated for every combination
of culture medium, light condition and species.
Considering seed viability, germination rate was
corrected and expressed as the rate of viable seeds
that germinated in each treatment. For example, if
50% of the seeds of a capsule with a 50% germination
rate, according to the tetrazolium test, germinated on
culture medium, it was considered that 100% of the