In Vitro Cell. Dev. Biol.—Plant 41:838–843, November– December 2005 DOI: 10.

1079/IVP2005698
q 2005 Society for In Vitro Biology
1054-5476/05 $18.00+0.00

EVALUATION OF THE EFFECT OF THREE GROWTH REGULATORS IN THE GERMINATION

OF COMPARETTIA FALCATA SEEDS UNDER IN VITRO CONDITIONS

JAIME PEDROZA-MANRIQUE*, CHRISTIAN FERNANDEZ-LIZARAZO, AND ANGELICA SUAREZ-SILVA

Facultad de Ciencias y Educación, Universidad Distrital Francisco José de Caldas, Bogotá D.C. Colombia

(Received 12 August 2004; accepted 27 June 2005; editor K. Dixon)

Summary
The effect of 3-indoleacetic acid (IAA), 6-furfurylaminopurine (kinetin), and gibberellic acid (GA3) on germination of
the orchid Comparettia falcata was evaluated in a factorial experiment (4 £ 4 £ 4) with Murashige and Skoog (1962) basal
medium. It was established that seeds of this orchid could be maintained under aseptic conditions as long as the necessary
nutrients and appropriate concentrations of growth regulators were provided. Of the three growth regulators used, IAA
significantly decreased seed germination of Comparettia falcata. There was a synergistic effect in the kinetin:GA3
combination that produced a positive response in both percentage seed germination and development of seedlings. This
study describes a single medium-based protocol able to achieve more than 160 000 seedlings within 21 wk, starting from a
single capsule, sufficient for both large-scale propagation and in vitro conservation of this threatened orchid.
Key words: Comparettia; orchids; in vitro; germination; kinetin; gibberellic acid; 3-indoleacetic acid.

Introduction
Colombia is host to the world’s largest orchid biodiversity with an
estimated 3500 species. Comparettia falcata is a rare epiphytic orchid
characterized by a very short life span (Arend, 1994). Seed germination
using tissue culture methods is a simple way to safeguard this endemic,
endangered orchid from extinction. In fact, several methods have been
reported on the micropropagation of orchids (Shimasaki and Uemoto,
1991; Arditti and Ernst, 1993; Seeni and Latha, 1994; Nayak et al.,
1998; Datta et al., 1999; Chan and Chang, 2000; Murthy and Pyati,
2001; Chen et al., 2002; Park et al., 2002; Pyati et al., 2002). This study
reports on preliminary attempts to germinate seeds of C. falcata in vitro.
Thus, it will be possible to maintain the natural variability in the
Andean region through the conservation of C. falcata germplasm and
remove this species from the red list of endangered species (Instituto
Alexander von Humbolt, 1997).
sucrose and gelled with 0.6% (w/v) agar. The pH of the medium was adjusted
to 5.8 with NaOH or HCl prior to autoclaving at a pressure of 1.06 kg cm22
for 20 min. The seed cultures were incubated in darkness at 25 ^ 28C for
16 wk to develop into small round protocorms, then transferred to
illuminated conditions for 5 wk at 25 –30 mmol m22 s21 (daylight fluorescent
tube FL-20D/18, 20W; China Electric Co., Taipei).
Seed viability was estimated by a 2,3,5-triphenyltetrazolium chloride
germination test (Singh, 1981), and seed germination was estimated
by recording the percentage of seeds with round or ovoid hyaline embryos
(viable embryos) with 1–3 rhizoids, for each growth regulator level.
Statistical analysis. Experiments were performed in a one-way design
consisting of a 4 £ 4 £ 4 factorial arrangement, with concentrations of 0, 5,
10, and 15 mM for IAA, GA3, and kinetin. The four concentrations of each
growth regulator were tested against the four concentrations of each of the
other plant growth regulators (PGRs), for a total of 64 treatments with 260
seeds in each treatment. Each treatment was replicated 10 times.
Observations were made after 6 wk and thereafter at 3 wk intervals up to
21 wk. The data were statistically analyzed and means were compared using
orthogonal polynomials (Steel and Torrie, 1985).

Materials and Methods Results

Plant material and sterilization. A limited number of mature
capsules of C. falcata were washed with soapy solution, followed by
treatment with 70% alcohol for 5 min. The capsules were then surface-
sterilized with a 5% sodium hypochlorite solution for 5 min and rinsed four
times with sterile distilled water.
Culture medium and conditions. Capsules were cut open, the seeds
scooped out and placed in a beaker with 100 ml of sterile distilled water, and
stirred with a magnetic bar at 700 rpm. Then, 75 ml aliquots were taken
and placed inside culture bottles (15 ml capacity) with 5 ml of MS
(Murashige and Skoog, 1962) culture medium. Each aliquot contained
approximately 26 orchid seeds. The medium was supplemented with
3-indoleacetic acid (IAA; 0, 5, 10, 15 mM), gibberellic acid (GA3; 0, 5, 10,
15 mM), 6-furfurylaminopurine (kinetin; 0, 5, 10, 15 mM), and 3% (w/v)
*Author to whom correspondence should be addressed: Email jpedroza@
udistrital.edu.co
According to the germination test, C. falcata seeds have a viability
of 98%. All treatments presented significant statistical differences
throughout the six observation periods. It is important to point out that
there was an increase in the percentage of germinated seeds in
the sixth week (Table 1). During the final periods of observation
(weeks 9 –21), only slight change was observed with no further
progressing of seed germination. New germinants were rarely
observed during the final periods of observation in this study. Despite
seed germination exhibiting a negative correlation with the age of the
culture, germination occurred with all treatments. The medium with
all growth regulators tested (treatment 64) resulted in a lower
percentage of seed germination (19%).
IAA effect. Treatments without IAA (1– 16) are superior in
germination response compared to 5, 10, and 15 mM IAA. In fact,

838
 EFFECT OF PGRS ON C. FALCATA GERMINATION 839

TABLE 1

EFFECT OF IAA, KINETIN, AND GA3 ON THE PERCENTAGE C. FALCATA SEED GERMINATION

% Germination at each observation interval (wk)

IAA mM Kinetin mM GA3 mM Treatment 6 9 12 15 18 21 Total percentage

0 0 0 1 40.6 13.1 0.0 0.0 0.0 0.0 53.7

5 2 53.8 1.7 6.4 0.0 0.0 0.0 62.0
10 3 57.5 5.3 1.0 2.5 0.0 0.0 66.3
15 4 83.2 3.2 0.0 0.0 4.9 0.0 91.4
5 0 5 45.7 11.1 0.0 0.0 4.2 1.2 62.2
5 6 52.1 3.0 14.3 0.0 2.0 0.0 71.4
10 7 53.4 3.5 10.4 1.5 4.7 2.0 75.4
15 8 63.7 0.0 2.7 0.0 18.5 0.0 84.9
10 0 9 50.9 7.9 0.0 0.0 7.9 0.5 67.2
5 10 45.7 3.0 6.9 4.5 10.1 0.0 70.2
10 11 41.7 9.6 6.2 8.4 5.7 2.5 74.1
15 12 47.2 11.4 1.2 10.6 9.7 0.0 80.1
15 0 13 56.5 3.2 0.0 0.0 4.2 0.0 64.0
5 14 55.1 2.7 0.0 0.0 5.7 0.0 63.5
10 15 61.2 5.2 2.0 0.0 3.5 3.2 75.1
15 16 77.0 1.4 9.6 0.0 10.3 1.7 100.0
5 0 0 17 31.2 0.0 2.2 1.5 0.0 0.0 34.9
5 18 31.1 1.7 2.5 0.5 0.5 0.0 36.3
10 19 32.6 1.60 2.9 0.0 8.6 0.0 45.7
15 20 36.0 5.9 3.7 4.4 0.0 0.0 50.1
5 0 21 24.6 9.0 0.0 0.0 1.2 0.0 34.8
5 22 36.5 3.4 6.9 0.0 0.0 1.5 48.3
10 23 36.0 4.8 4.0 0.0 0.7 5.9 51.5
15 24 37.9 15.5 5.7 0.2 0.0 0.0 59.3
10 0 25 44.2 4.8 0.8 0.0 5.0 0.0 54.8
5 26 38.8 19.8 0.0 0.0 1.2 0.0 59.7
10 27 37.2 16.0 7.9 0.0 4.0 0.0 65.1
15 28 39.4 6.7 8.2 3.2 1.4 2.8 61.6
15 0 29 37.5 0.0 2.2 0.0 3.2 0.0 42.9
5 30 46.0 0.0 2.0 0.0 4.2 3.7 55.9
10 31 47.3 10.6 0.0 5.4 0.0 0.0 63.3
15 32 49.6 8.6 0.0 0.0 0.0 5.4 63.7
10 0 0 33 13.6 2.1 0.0 1.9 0.7 3.7 22.1
5 34 15.6 2.9 1.2 0.0 1.7 2.2 23.5
10 35 20.6 4.7 0.0 0.0 4.4 0.0 29.7
15 36 17.5 15.1 0.0 0.0 0.0 0.0 32.6
5 0 37 19.9 7.7 0.0 1.0 3.0 0.0 31.5
5 38 13.6 7.9 4.9 0.0 4.9 4.2 35.6
10 39 25.3 16.3 0.0 3.5 0.0 0.0 45.1
15 40 41.2 14.1 0.0 4.7 0.0 0.0 60.0
10 0 41 11.9 17.3 2.2 0.0 0.0 0.0 31.4
5 42 28.6 0.0 9.4 0.0 5.9 0.0 43.9
10 43 29.5 8.1 5.9 0.0 0.0 0.0 43.6
15 44 40.9 7.4 2.2 0.0 0.0 0.0 50.5
15 0 45 18.8 18.8 0.0 0.0 0.7 0.0 38.3
5 46 24.3 5.9 9.6 0.0 0.0 6.7 46.5
10 47 24.3 0.5 0.0 4.2 1.0 2.0 32.0
15 48 34.0 8.1 0.0 11.9 0.0 0.0 54.0
15 0 0 49 12.2 0.0 6.4 0.0 3.5 0.0 22.1
5 50 12.6 0.0 8.1 0.0 3.0 0.0 23.7
10 51 19.5 5.9 1.0 0.0 4.2 0.0 30.6
15 52 26.3 3.70 0.0 0.0 3.5 0.0 33.5
5 0 53 6.7 8.9 0.0 4.2 0.0 5.9 25.7
5 54 7.3 17.5 0.0 1.5 3.2 0.0 29.5
10 55 19.8 15.3 0.0 0.0 0.0 0.0 35.1
15 56 18.3 13.3 0.0 2.7 0.0 0.0 34.3
10 0 57 13.7 4.2 2.6 0.0 11.2 0.0 31.8
5 58 6.3 13.8 8.9 0.0 1.7 1.5 32.2
10 59 19.1 0.0 7.9 0.0 0.0 0.0 27.0
15 60 19.6 4.7 0.0 2.2 0.0 7.4 33.9
840 PEDROZA-MANRIQUE ET AL.
TABLE 1 – continued
% Germination at each observation interval (wk)
IAA mM Kinetin mM GA3 mM Treatment 6 9
15 0 61 13.6
5 62 15.4
10 63 11.1
15 64 10.9
Data analyzed by an orthogonal polynomial test (Steel and Torrie, 1985) using 10 replicates per treatment for a total n ¼ 2600 seeds per treatment, showed
P , 0.001 for C. falcata seed germination and a standard deviation of 7.07. Percentages in columns 5–10 are new germinants at each observation interval.
Column 11 shows the total cumulative percentage germination after 21 wk.
C. falcata seed germination on medium without IAA exhibited the
highest percentage germination (53.7– 100%). IAA alone at
5– 15 mM reduces the percentage seed germination to below 50%,
indicating that its presence does not optimize in vitro germination
and development. After 12 wk in the same medium without
subculture, poor germination occurred in IAA supplemented media.
IAA was also not able to improve seed germination in combination
with kinetin and/or GA3.
GA3 effect. C. falcata seed germination showed a positive
correlation with GA3 concentration. There was an increase in
percentage germinated seeds in the sixth week (Table 1). High
germination percentages were obtained when seeds were incubated
in the presence of GA3 (5 – 15 mM), indicating that this growth
regulator does have an optimizing effect on in vitro germination
and development. The highest percentages of germination were
obtained at 15 mM GA3 (Table 1).
Kn effects. There is a significant increase in the percentage of
germinated seeds from 6 to 9 wk in response to kinetin treatments 5,
9 and 13 (5, 10, 15 mM kinetin only). Meanwhile, in the absence of
kinetin, the increase shown in the 6 wk is very slight. Furthermore,
there is an evident decrease in the percentage of embryos in the
subsequent observations. In contrast, high kinetin (15 mM)
increased seed germination, although 10 mM kinetin was also
effective in C. falcata seed germination (Table 1). Akin to GA3,
kinetin enhanced the percentage seed germination within 6 wk.
IAA:kinetin interaction effect. In general terms, there was a
remarkable antagonism between IAA and kinetin in the first three
observation periods. Combinations of kinetin with IAA were less
stimulative than media supplemented with kinetin alone (Table 1). On
media with combinations of kinetin with IAA, most seeds turned
yellowish green and became compact in appearance, which gave rise
to a lower percentage seed germination (10.9%) in the sixth week.
IAA:GA3 interaction. The effect of the IAA:GA3 interaction was
negative when the two regulators were present. Seed germination
was high (53– 100%) on media containing GA3 and without IAA
(treatments 2 – 16). In contrast, seed germination was poor on media
with the highest concentration of IAA in the presence of GA3
(treatments 49 – 64).
Kinetin:GA3 interaction. There was a positive effect in the
kinetin:GA3 interaction, particularly in the first three observations.
In the present study both kinetin and GA3 were essential for
C. falcata seed germination even at high concentrations (15 mM).
The optimum growth regulator concentrations for germination of
C. falcata seeds was found to be 15 mM kinetin and 15 mM GA3,
where the highest percentage seed germination (100%) was
12 15 18 21 Total percentage
8.3 2.0 2.0 1.7 1.0 28.4
13.7 0.0 0.0 0.0 1.7 30.8
5.1 2.2 0.0 6.2 0.0 24.6
5.3 0.5 0.0 0.2 2.1 19.0
observed. Moreover, the superiority of the kinetin:GA3 interaction
in promoting seed germination was observed in the absence of IAA
(Table 1). Using 15 mM kinetin and 15 mM GA3 we estimate that
approximately 160 000 seedlings could be obtained from one
capsule after 21 wk of culture (Fig. 1).
IAA:Kinetin:GA3 interaction. This interaction showed positive
and negative effects. Good response is stimulated by the kinetin and
GA3 interaction. The percentage seed germination was greater
(100%) on medium containing 0 mM IAA, 15 mM kinetin, and
15 mM GA3, as compared with other combinations of IAA, kinetin,
and GA3. The lowest germinative response (19%) resulted at 15 mM
IAA, 15 mM kinetin, and 15 mM GA3. Medium supplemented with
0 mM IAA, 15 mM kinetin, and 15 mM GA3 appears to be the best
medium to use as germination medium for C. falcata seeds. Of the
three regulators used, kinetin and GA3 significantly promoted
C. falcata seed germination. When GA3 interacts with IAA in the
absence of kinetin, it produces low results. Moreover, when GA3
interacts with kinetin in the absence of IAA, or when it is alone in
high concentrations, the germinative response increases.
Discussion
According to Hartman et al. (1997), specific endogenous growth
promoting and inhibiting compounds are involved directly in the
control of seed development, dormancy, and germination. Evidence
for hormone involvement comes from correlations of hormone
concentration with specific developmental stages, effects of applied
hormones, and the relationship of hormones to metabolic activities.
The activation of the metabolic machinery of the embryo leading to
the emergence of a new seedling plant is known as germination. For
germination to be initiated, three conditions must be fulfilled: first,
the seed must be viable – that is, the embryo must be alive and
capable of germination; second, the seed must be subjected to the
appropriate environmental conditions; third, any primary dormancy
condition present within the seed must be overcome. These
conditions make it reasonable to hypothesize that exogenous
hormonal requirements are needed to induce in vitro morphogenesis
in rare orchids such as C. falcata. It is therefore essential to
research the need for exogenous PGRs (Pierik, 1990).
IAA effect. It is possible to assume that in the first stages of
germination the embryos did not develop a large amount of
hormonal receptors because they needed very little or no IAA. This
could explain the germination inhibition at the tested concen-
trations (5, 10, and 15 mM) of IAA. Inhibition might have occurred
because of the intoxication process or because of competition with
 EFFECT OF PGRS ON C. FALCATA GERMINATION
FIG . 1. C. falcata seed germination on MS medium with 15 mM Kn and 15 mM GA3 after: A, 6 wk (100 £ ); B, 9 wk (400 £ ); C, 12 wk
(100 £ ); D, 15 wk (100 £ ); E, 18 wk (bar ¼ 5 mm); F, 21 wk (bar ¼ 5 mm).
the other growth regulators. In vitro seed germination using
techniques similar to those used with epiphytic orchids has been
successful both with and without fungal assistance (Hartman et al.,
1997). It is evident that the mycorrhizal fungi used symbiotically by
the C. falcata seeds to germinate possibly supply them with a low
amount of auxin in the first stages of their germinative development
(Gil-Martı́nez et al., 1995). In relation to the results obtained in this
study, the situation can partly be the cause of a strong hormonal
toxicity of the already IAA-germinated C. falcata seeds, because
free IAA is high during development, but is reduced in mature
seeds (Hartman et al., 1997). When light was introduced at week
16, germination increased at weeks 18 and 21 at IAA 15 mM,
probably because IAA can be oxidized nonenzymatically when
exposed to light and its photodestruction may be promoted by plant
pigments (Taiz and Zeiger, 1998). Moreover, synthetic auxins are
not destroyed by IAA oxidases, so those auxins persist in plants
much longer than does IAA (Salisbury and Ross, 2000). The
interaction of exogenous hormones with light is complex. Treatments
with hormones can offset the light effect (Hartman et al., 1997).
GA3 effect. According to Jones and Stoddart (1980), GA3 has the
potential to regulate germination in several ways, notably through
the elaboration of the endo-membrane system and the synthesis of
new mRNA types that can be used to regulate the protein synthesis
required in germination. On the other hand, the changes in the
characteristics of the cellular membrane and the cell wall can
regulate the materials required for growth during their cultures.
This is why, in natural conditions, the C. falcata seeds might need
GA3 provided by a mycorrhizal fungus in order to initiate the
841
germinative process successfully. Mycorrhizal fungi may vary the
level of gibberellins in the seeds with which they form symbiosis
(Gil-Martı́nez et al., 1995), and this is possibly the reason why GA3
has been important at all levels (5, 10, and 15 mM) in seed
germination of C. falcata. According to Hartman et al. (1997),
active forms of GA decline at seed maturity and are replaced by
biologically active conjugated forms of gibberellins that are utilized
during germination. Gibberellins occur at relatively high concen-
trations in developing seeds, but usually drop to a lower level in
immature seeds.
Kinetin Effect. Kinetin has been shown to have a morphogenetic
response in processes associated to micropropagation of orchid spp.
(Martin, 2003). However, in some cases, it is a growth inhibitor of
callus tissue (Tokuhara and Mii, 2003). Cytokinins are good
inductors of protocorm-like bodies from leaf explants (Murthy and
Pyati, 2001; Lee and Lee, 2003). The highest concentration of
kinetin (15 mM) developed a maximum percentage seed germination
in combination both with or without IAA and GA3 (Table 1),
indicating an overall positive effect on C. falcata seed germination.
IAA:kinetin interaction effect. In some orchids, a kinetin – auxin
combination is not as stimulating as a medium that has only kinetin
(Martin, 2003). The hormonal control of germination involves a
balance between the stimulating and inhibitory components of the
seed (Thomas, 1980; Kinderen, 1990). However, seed germination
increased progressively in the first and second observations, when
kinetin is positively assimilated by the cells. This is in part due to
the two regulators, but especially due to IAA, which diminished the
percentage germinated seeds. The tendency toward a decrease in
842 PEDROZA-MANRIQUE ET AL.
germinative response appeared to be influenced by IAA,
presumably due to the fact that at high concentrations, auxins
may increase the cytokinin-oxidase activity (Palni et al., 1993).
Furthermore, the efficacy of kinetin has also been reported in
Phalaenopsis spp. (Tanaka, 1992).
IAA:GA3 interaction. The embryos do not seem to need
exogenous IAA. In fact, the presence of this regulator in all
interactions does not improve seed germination in any observation.
This suggests that IAA is the responsible agent for the negative
effect of the IAA:GA3 interaction, which is consistent with previous
reports (Garcidueñas and Ramı́rez, 1993). They state that the level
of GA3 rises depending on the embryo’s development, and then
decreases. This means that there was an antagonistic effect in the
IAA:GA3 interaction that produces a negative response in both
percentage seed germination and development of seedlings. The
reason for this is that germination requires sequential activation of
different genetic programs (Jann and Amen, 1980). Moreover, not
all genotypes of one particular species respond in the same way
under the same culture conditions (Park et al. 2002; Lee and Lee,
2003). Furthermore, gibberellins appear to play a role in two
different stages of germination. The first occurs in the initial enzyme
induction and the second is in the activation of reserve food-
mobilizing systems (Hartman et al., 1997).
Kinetin:GA3 interaction. According to Khan (1980), the action
of cytokinins in the germinative process is interpreted as a
contribution to the physiological activity of the gibberellins, acting
as ‘permissive’ agents of germination, especially counteracting the
actions of the inhibitors like abscisic acid (ABA). The germination
of some orchids is influenced by the ABA concentrations of the
seeds; if the ABA concentration is high, poor germination will
result. On the other hand, there are treatments that lack kinetin, but
have a high GA3 concentration with a high germinative response. It
can then be deduced that at these concentrations, GA3 manages to
neutralize the action of a possible inhibitor and/or generates an
adequate hormonal balance. Furthermore, it is important to point
out that orchids rely on mycotrophy to obtain the required energy in
their life cycle (Rasmussen, 1995), and it is then that fungal
colonization is especially needed to stimulate gluconeogenesis in
order to mobilize reserves and provide a nutritional support to non-
photosynthetic seedlings (Smith and Read, 1997). The in vitro
germination of land orchids has been tested with fungi recovered
from mature plants (Dixon, 1987; Takahashi et al., 2000; Zettler
et al., 2001; Bowles et al., 2002; Sharma et al., 2003). Presumably,
mycorrhizal fungi, which assist with germination of C. falcata seeds,
initiate symbiosis and regulate gibberellin content at the beginning
of germination, and supply low auxin concentrations at a specific
stage of the germinative process.
IAA:kinetin:GA3 interaction. Treatments with auxins diminish
the percentage of germinated embryos, while IAA appears to provoke
a negative behavior in PGR interaction, presumably because there
must be a sequential action of these hormones: first the gibberellin,
then the cytokinin, and finally the auxin (Van Overbeek et al., 1993).
One of IAA, GA3, or kinetin may be a restrictive hormone in some
species but not in others. As a matter of fact, there are cultures that
may not need any regulators, other cultures that may need one of
them and there may be other cultures that need the interaction of
them all. This suggests the need for growth regulators added
exogenously, which also depends on the plant species. In summary,
our results underscore the importance of both kinetin and GA3 in
improving C. falcata seed germination, which may produce up to
160 000 seedlings within 21 wk. Moreover, this system serves as a
low-cost, high-volume propagation system.
In conclusion, a reliable protocol via seed germination for C. falcata
was established. The best seed germination was obtained from seeds
incubated on basal medium containing 15 mM kinetin and 15 mM
GA3, while IAA is inhibitory. Under optimal conditions, 160 000
seedlings could be obtained from one capsule after 21 wk of culture.
Acknowledgments
We thank Margarita Perea-Dallos, Universidad Nacional de Colombia,
and Julio C. Calvo, Universidad Distrital Francisco José de Caldas, for
reading and commenting on this article.
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