Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 27
ARTÍCULO DE INVESTIGACIÓN
Acclimatization of Phalaenopsis and Cattleya obtained
Aclimatización de Phalaenopsis y Cattleya obtenidas
Lucía Primitiva Díaz1, Jorge Julio Namur2, Sebastián Agustín Bollati3,
Osvaldo Ernesto Antonio Arce4
The quality of micropropagated plants relies on the acclimatization stage. This research intends to
develop an efcient protocol to obtain the acclimatization of Phalaenopsis and Cattleya. Plants of Pha-
laenopsis obtained from protocorms were selected. They came from owering stalks grown at modied
Murashige and Skoog (MS) (1962) medium and classied by growth ranks and put into moss, mesquite
wood shaving and perlite (1:1:1), into a humidity chamber. The protocorms were multiplied at MS from
Cattleya sown in Knudson C (1946) medium; regenerated plants of 1-2 cm were selected, and implanted
in humidity chamber on: moss, coal and perlite (1:1:1) MCP; mesquite wood shavings, coal and perlite
(1:1:1) ACP; moss and perlite (1:1) MP; mesquite wood shaving and perlite (1:1) AP. The following re-
sults were obtained: Phalanopsis: a) Survival: 44% in R0 and 100% in RI and RII. b) Number of leaves: RI
gave on average 1 more leaf than the range 0; c) Roots number and length: RI and RII gave on average 2
more roots than R0, and there were no signicant differences in length. d) Height: RII presented greater
growth than RI and Ro. Cattleya: a) The survival in MCP was 0%, MP 16 %, ACP 32% and AP 80%. b)
The height in MP was signicantly superior to the ones in ACP and AP. Plants from both genera need to
achieve a 2 to 4 cm growth rank in vitro to endure the greenhouse conditions. MAP was the best subs-
trate in Phalaenopsis and moss-perlite in Cattleya.
Key words: Orchidaceae, substrates, in vitro culture.
1 Doctora en Agronomía. Profesora Asociada, Facultad Agronomía y Zootecnia, UN Tucumán,Argentina.
2 Ingeniero Agrónomo. Ayudante graduado, Facultad Agronomía y Zootecnia, UN Tucumán, Argentina. jorgenamur@hotmail.
3 Becario alumno. Facultad Agronomía y Zootecnia, UN Tucumán, Argentina. firstname.lastname@example.org
4 Magíster en Estadística Aplicada. Profesor Adjunto, Facultad Agronomía y Zootecnia, UN Tucumán, Argentina. ova.arce@
Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40 27
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28 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
La calidad nal de las plantas producidas por micropropagación depende de la etapa de aclimatización. Se
intenta desarrollar un protocolo eciente para la aclimatización de Phalaenopsis y Cattleya. Se seleccionaron
plantas de Phalaenopsis, obtenidas de protocormos provenientes de estacas orales cultivadas en Murashige
y Skoog modicado (MS) (1962), por rangos de crecimiento e implantadas en musgo, viruta de algarrobo y
perlita (1:1:1), en cámara húmeda. De siembras de Cattleya en medio de Knudson C (1951) se multiplicaron
protocormos en MS; se seleccionaron plantas regeneradas de 1-2 cm, e implantadas en cámara húmeda en
los sustratos: musgo, carbón y perlita (1:1:1) MCP; viruta de algarrobo, carbón y perlita (1:1:1) ACP; musgo y
perlita (1:1) MP; viruta de algarrobo y perlita (1:1) AP. Se obtuvieron los siguientes resultados: en Phalaenopsis:
a) Supervivencia: para R0 de 44% y RI y RII del 100%; b) número de hojas: RI generó en promedio 1 hoja
más que el rango 0; c) número y longitud de raíces: RI y RII generaron en promedio dos raíces más que R0,
no detectándose diferencias signicativas en longitud; d) altura: RII presentó mayor crecimiento que RI y R0
En Cattleya: a) La supervivencia en MCP fue 0%, MP 16%, ACP 32% y AP 80%; b) La altura en MP resultó
signicativamente superior que en ACP y AP. Ambos géneros necesitan alcanzar un crecimiento de 2 a 4 cm
in vitro para tolerar las condiciones de invernáculo. El mejor sustrato fue MAP en Phalaenopsis, y la mezcla
musgo-perlita en Cattleya.
Palabras clave: Orchidaceae, sustratos, cultivo in vitro.
Recibido: junio 16 de 2010 Aprobado: noviembre 23 de 2010
Pospišilová (1999) stated that in vitro
acclimatization is one of the key factors in
producing healthy plantlets before they are
transplanted to ex vitro conditions. According
to Preece and Sutter (1991) acclimatization will
allow the plant to reach a state of autotrophic
growing (Teixeira da Silva et al., 2005) in en-
vironments of lesser relative humidity, more
light and septic substrates. Transferring plants
from an almost ideal situation to a greenhouse
or ex vitro situation presents a challenge for sur-
vival. Plants will move to a heterotrophic state
to an autotrophic one, undergoing physiolo-
gical and morphological changes as well as a
greater exposition to the action of plagues and
diseases. The phenotype is one of the characte-
ristics being modied under in vitro conditions,
i.e; stems are thinner, with lesser wax quanti-
ties, reduction of support mechanical tissues,
greater content of cell water and heterotro-
phic growing (Denng and Donnelly, 1993). All
these alterations make necessary to include an
acclimatization stage within the micropropa-
gation protocols for the plant to recover their
morphological and physiological characteris-
Micropropagation is a massal culture sys-
tem developed under conditions of asepsis, high
humidity and controlled lightness and tempe-
rature. Acclimatization is a critical stage in mi-
cropropagation. During this period the higher
percentages of plant losses occur due to several
reasons (Kozai, 1991; van Huylendroeck et al.,
1998). This is the reason why is necessary to ob-
tain quality plantlets under in vitro conditions to
ensure a high survival percentage and an appro-
priate growth under greenhouse conditions. In
order to survive ex vitro a plantlet must achieve
a growth stage with an appropriate sprout num-
ber, foliage area and radicular system, conside-
ring roots number and length.
Another aspect to be considered is that
many plantlets do not survive the acclimatiza-
tion stage when transferred to a septic subs-
trate because of media contamination. The
effects of temperature, humidity and lightness
in the greenhouse and also the plantlets nutri-
tional conditions should be taken into account
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Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 29
tics. The conditions during this stage involve
increased lightning, reduced humidity, termic
variations, septicity, the right selection of subs-
trate and an optimal growth stage in order to
obtain an adequate survival percentage.
Cha-um et al.(2009) state that environ-
mental conditions for ex vitro growth are quite
different from those used for in vitro cultiva-
tion Plant growth retardants, i.e., uniconazo-
le (UCZ), paclobutrazol (PBZ) triapenthenol
(TPN), triadimefon (TDM) and hexaconazole
(HCZ) have been reported as effective agents
in reducing the size of plants, but retaining
dark-green leaves and thick roots, which de-
ne them as healthy plantlets, and aiding anti-
wilting, leading to better survival and growth in
ex vitro condition
The selection of a proper substrate with
low septicity, high aeration, permeability and a
correct acidity grade is a requisite to guaran-
tee conditions of initiation and autotrophic
growth. It is also necessary that the substrates
keep these conditions for a long period without
deteriorating to avoid compaction and lack of
aeration and permeability.
A substrate is considered to be a solid and
porous, natural or syntethic material, which
combined or not, permits and adequate plants
growth under controlled environment condi-
tions (Abad, 1989). The substrate function is
to provide mechanical support and to improve
air and water absorption by the roots (Tortosa,
1990). The substrate may be related or not to
the mineral nutrition management. Sanitation
is a major issue so that the substrate should be
obtained from inert or easy to disinfect material
like earthworm humus, compost (Agramonte
Peñalver et al., 1998; Díaz et al., 2004), tezont-
le, tezontle –vermiculite, tezontle-dicalite, pine
bark and dark lava rock chippings ( Avila-Díaz
et al., 2009).
The substrate choice is conditioned by
the plant species, just as the case of the epi-
phytic orchids genera studied in this paper. The
substrate requirements are: acidity, aeration
and permeability. It is also necessary to know
the appropriate environmental conditions for
growing in greenhouse (Iriarte et al., 2002).
According to Northen (1990) Phalaenopsis de-
mands temperatures between 15 ºC and 35 ºC,
10% solar light and 70% relative humidity (RH)
while Cattleya needs temperatures between 10
ºC and 35 ºC, 30% solar light and 50% RH.
In Argentina both genera are economically im-
portant because of their ornamental value as
owers and interior plants. A basic need to ful-
ll the requirements of orchid producers is to
develop protocols allowing a high quality mas-
sal propagation and plants uniformity.
This research intends to develop an ef-
cient protocol to obtain the acclimatization of
Phalaenopsis and Cattleya and the objectives were:
I) Achieving the adaptability of Phalaenopsis
from in vitro to ex vitro conditions using a mix-
ture of substrates and evaluating the effects of
different growth ranks on survival and growth.
II) Achieving the adaptability of Cattleya from
in vitro to ex vitro conditions by using substrate
combinations from different origins.
Materials and methods
Four substrates were used as cultivation
media: A) Perlite (P): it is a substrate widely
used in the preparation of compost from di-
fferent cultures. It is ground inert volcanic lava
expanded at 800ºC, thus facilitating sustained
aeration and permeability conditions in any
mixture. Their inherent properties of porosi-
ty and sanity are important when used at the
acclimatization stage. B) Moss (Meteoropsis onus-
tum) (M): vegetal substrate being able to absor-
be water up to 20 times their own weight. It is
acid (pH 5-6), creating optimal conditions for
epiphytic orchids cultivation. The disadvantage
is that after 2 to 4 months this characteristic va-
nishes so it has to be replaced after that period.
C) Vegetal charcoal (C): it is an organic product
obtained by burning wood from different plant
species. It is stable in cultivation media but it
is hydrophobic so it has to be submerged in
water for at least 24 hours in order to eliminate
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30 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
the air and gets to absorbed water. D) Mesqui-
te wood shaving (A): it is a product obtained
by manual or mechanical wood planing, widely
used in carpentry. Their physical characteristics
persist for a period of over a year since mes-
quite provides a hard wood. It has to be sifted
before being used in order to separate the big
shavings. An intermediate size is required to fa-
cilitate small plants cultivation.
The vegetal material comes from the fo-
llowing in vitro cultivated genera:
I) Phalaenopsis: the hybrids were cultiva-
ted from oral nodes (Arditti and Ernst, 1993)
(Photo 1) incubated in Murashige and Skoog
(MS) (1962) medium with salts and vitamins
(50%); 6 benzyl-amino-purine, BAP, (10 mg/l);
1-naphtalen acetic acid, ANA, (1 mg/l); sucro-
se (3%), activated charcoal (0,2%) and agar
(0,5%). Protocorms and plantlets were regene-
rated (Photo 2). The material was selected by
growth ranks: R0: 1 to less than 2 cm height and
two 1-4 cm long roots; RI: 2 to less than 3 cm
height and three 1-6 cm long roots; RII: 3 up to
4 cm height and three 1-8 cm long roots. The
substrate composition was a mixture of moss,
mesquite wood shavings and perlite (MAP) in
1:1:1 proportion (Photo 3). 75 plantlets (25 in
each rank category) were implanted in three 40
x 60 cm plastic collective trays, at the end of
springtime, and kept in humid chamber during
the rst two weeks. After that period intermit-
tent ultra low volume (fog) irrigation system,
10% luminosity and 30% solar light (using half
shadow or saran) was applied. The greenhouse
conditions were: 10 °C (night) and 25 °C (day)
during the winter; 20 °C (night) and 35 °C (day)
during the summer.
II) Cattleya: the Cattleya maxima x nobilior
hybrid was used, originated from seeds cultiva-
ted in vitro in Knudson C (1951) medium and
multiplied from small and medium size proto-
corms in MS (1962) with salts and vitamins MS
(50%), BAP (0,5 mg/l), ANA (0,1 mg/l), sucro-
se (3%) and agar (0,5%) (Photo 4). 1-2 cm long
regenerated plants were selected and implanted,
in humid chamber, on the following substrates:
MCP: moss, charcoal and perlite (1:1:1) (Photo
5); ACP: mesquite wood shavings, charcoal and
perlite (1:1:1) (Photo 6); MP: moss and perlite
(1:1) (Photo 7); AP: mesquite wood shavings
and perlite (1:1:1) (Photo 8). 100 plantlets were
implanted in four 40 x 60 cm plastic collective
trays, each one with a different substrate (25
plantlets per subtrate). The implanted plant-
lets had between 1 and 2 cm length with two
roots, 1-4 cm long, corresponding to rank 0.
No enough material corresponding to ranks
I and II was achieved in order to carried out
and experiment similar to the one in Phalaenop-
sis. Experimental environment conditions were
the same as in Phalaenopis mentioned before.
In both genera, supplementary lighting
was provided during the fall, winter and spring
initiating from 6 AM to 11 PM i.e. a 17-hours
photoperiod. The plants were fertilized every
fteen days by using a mixture of N:P:K
(7:3,1:7,3) and ANA (40 mg/l). After three wee-
ks of implantation the plantlets were watered
with intermittent ultra low volume (fog) irri-
gation system. Water was biologically stabilized
or chlorine free.
The following evaluations were carried
out: I) Phalaepnosis: survival on four evaluation
dates (25, 40, 55 and 85 days after implanta-
tion), and roots height, number and length after
60 and 90 days from implantation. II) Cattleya:
survival was quantied at 35, 50, 75 and 105
days after implantation. Plant length was mea-
sured after 90 days from implantation.
The following substrate measurements
were evaluated: pH, electric conductivity (EC)
(1:10 dilution) and ashes by calcination (%)
(Table 1). Analyses were conducted at the labo-
ratory of Cátedra Edafología. FAZ-UNT.
The perlite chemical composition was
also determined (%): Si O2:74 -79; Al2O3:
13-17; K2O: 0.5- 5; Na2O: 2-5; CaO: 0,4-0,6;
Fe2O3: 0,3-0,9 and MgO: 0,04-0,15.
The following statistical techniques were
used: a) Survival: percentage of survived plants
was calculated in relation to the initial im-
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Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 31
planted number, on every evaluation date; b)
Treatments effect on plants height, root num-
ber and length: analysis of variance and Tukey’s
pairwise comparison test.
Graphics and analysis were run on R (R
Development Core Team, 2010).
Results and discussion
Phalaenopsis and Cattleya adaptation is
slow and difcult, however, no references were
found concerning in vitro adaptation by using
different origin substrates for these species.
Doritaenopsis post micropropagation acclimati-
zation CAM orchids, was studied by Jeon et al.
(2005) who evaluated the effect of light ow
density on morphology, photosynthesis and
growth. Teixeira da Silva et al., (2005) worked
on banana and Cymbidium in vitro acclimatiza-
tion and they included the evaluation of growth
parameters in ex vitro conditions. In the present
paper the effect of light on acclimatization was
not evaluated. A gradual plantlets adaptation to
light was accomplished by using half-shadow
as well as controlled watering and nutrition.
The plantlets were fertilized every fteen days
by using a mixture of N:P:K (7:3,1:7,3) and
ANA (40 mg/l).
Colombo et al. (2005) worked on the accli-
matization of a Cattleya hybrid by using several
vegetal substrates and two irrigation systems.
They found that the coconut powder substrate
and intermittent irrigation system were the most
indicated for the acclimatization of the Cattleya
chocolate drop (C. guttata x L. tenebrosa) orchid
with a 90% survival. In this paper only the inter-
mittent irrigation system was applied. Colombo
et al. (2005) also found that moss showed the
lowest survival (72%) in Cattleya when combined
with a manual irrigation system
Torres, Laskowski and Sanabria (2006)
evaluated Cattleya jenmanii Rolfe leaf epidermis
anatomy, in vitro multiplication and acclimati-
zation in orchidarium. They determined that,
during the acclimatization stage, leaves from in
vitro plants increased the stomata size and the
thickness of anticlinal walls in typical cells in
order to favor mechanical resistance and stiff-
A few references were found on different
origin substrates usage on micro propagated
plants acclimatization (Agramonte Peñalver et
al., 1998). Díaz et al. (2004) used earthworm
humus as a substrate for sugar cane micro-
propagated plants acclimatization. Shiau et al.
(2002) worked on the establishment of Anoec-
tochilus formosanus Hayata ex vitro plantlets, which
achieved 90% survival after transferring the
material to ex vitro conditions in coconut ber
closed recipients and then incubated in peat
moss and vermiculite (1:1). Avila-Díaz et al.
(2009) studied the survival and acclimatization
of seedlings from Laelia speciosa using different
Table 1. Substrates pH, electric conductivity and ashes (%)
pH EC Ashes (%)
Mesquite 6.63 0.27 2,15
Moss 6.03 2.00 7,78
Perlite 6.83 0.00 99,2
Charcoal 7.69 0.05 2,61
Charcoal H2O 7.34 1.20 _
Laboratory Cátedra Edafología. FAZ-UNT
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32 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
In this research was intended to repro-
duce the natural conditions under which these
epiphytes grow using permeable, aerated, dura-
ble, acid or neutral substrates so as to guarantee
the in vitro material survival, establishment and
growth under greenhouse conditions (table 1).
I) Phalaenopsis: Christenson (2001) quoted
by Lee et al. (2008) states that the genus Phalae-
nopsis (Orchidaceae) comprises about 63 spe-
cies that have produced numerous attractive
hybrids and cultivars. This genus presents the
following characteristics: monopodial growth
with indenite growing apical meristem, fast
growth, owering from the second life year on
and producing up to 9 leaves.
The survival of the material implanted
on MAP was: 44% in R0 and 100% in R I and
RII. This result indicates that the plant needs to
achieve certain in vitro growth rank to endure
the external conditions in greenhouse.
Figure 1. Phalaenopsis survival percentage according to growth ranks and days after implantation (year 2007).
Figure 2. Phalaenopsis mean height (cm) according to growth rank and days after implantation on MAP
BIOTECNOLOGIA XII-2 DIC 2010.indd 32 16/12/2010 23:29:11
Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 33
As far as growth concerns (gure 2), a di-
fferential behavior among ranks was observed.
Plants in the ranks I and II achieved a greater
mean height during the same time period (sta-
tistically different at α = 0.05).
According to the resulst a critical thres-
hold was observed in order to ensure the sur-
vival and posterior growth in Phalaenopsis. This
threshold should be plantlets in RI and RII, i.e.
2 to 4 cm height and three 1-8 cm root long.
Shushan (1959) in Cattleya mossiae x C. trianae
observed that the appropriate aerial develop-
ment was 0.5 to 2 cm.
In relation to roots growth, during the rst
60 days no plants with new roots were observed.
However, in the second evaluation (90 days), the
ranks I and II generated a signicantly greater
root number (α=0.05) than rank 0. The plants
in the ranks I and II generated, on average, two
more roots than the ones in rank 0 (gure 3).
No difference was found in roots length when
comparing the three ranks (gure 4).
Figure 3. Phalaenopsis roots number according to rank and days after implantation on MAP (year 2007).
Figure 4. Phalaenopsis roots length mean and 95% condence intervals after 90 days
from implantation on MAP (year 2007).
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34 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
The development of the root system
(root number and length) is vital to anchor the
plant and also to ensure water and nutrients
absorption. This is coincident with the fact
expressed before related to the existence of a
minimum plantlet length threshold. Maene and
Debergh (1983) found that 2.5 to 5 cm long
micro shoots of Cordilyne terminalis rooted bet-
ter while the ones shorter than 2.5 and larger
than 6 cm plantlets showed a decreased per-
centage of roots. The results in this research
showed that the best results in survival and
growth were achieved with plants between 2
to 4 cm and 3 roots. Ávila- Díaz et al. (2009)
transplanted Laelia speciosa plantlets of 5 cm in
length to the greenhouse and a survival rate of
77.5% of was obtained.
The table 2 shows that the leaves number
in RI is, on average, one more unit than in R0.
This fact conrms the results from Preece and
Sutter (1991) and Dietrich et al. (1992), quoted
by Pospisilova et al. (1999), who stated that in
many plant species leaves formed in vitro are
not capable of keep growing under ex vitro con-
ditions and that they are replaced by new ones.
These authors did not study the growth rank
effects in their research. A differential effect
was found among ranks, particularly in RI, in
this experience. Sushan (1959) observed under
greenhouse conditions in the primary hybrid
Cattleya x Trimos, the formation of 6 to 7 leaves
in 12 months and up to 9 leaves were found in
Table 2. Tukey`s test for Phalaenopsis leaf
Rank Leaf average number
RII 3,2 AB
R0 2,6 B
Treatments with the same letter do not differ significantly
II) Cattleya: is an American tropical genus
with pseudobulbs and sympodial growth (Font
Quer, 1965). Its growth is slow reaching phy-
siological maturity after seven years.
80% survival was obtained on AP subs-
trate, 90 days after implantation; MCP showed
the worst performance (0% at 90 days). The
other two media were intermediate: 16% MP
and 32% ACP (gure 5).
Figure 5. Cattleya survival percentage according to days after implantation on four substrates.
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Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 35
The gure 6 shows signicant differences
among average plants height. MP produced
plants 1 cm larger on average. Shushan (1959)
determined an aerial development of 0.5 to 2
cm in 12 months under greenhouse conditions.
This size was the right one in the sense that
the plantlet reached the proper roots number
and length and foliage area to survive under
greenhouse conditions. Ávila-Díaz et al. (2009)
determined the relationship between survival
and seedling size in different substrates and
arrived to the conclusion that seedlings of 5cm
in length had the highest frequency of survival
(77.5%) whilst sizes of 2 and 1 cm long showed
5 to 0% survival respectively.
Figure 6. 95% condence intervals for Cattleya plant height (year 2007) for three substrate mixtures.
In this research was found, for both gene-
ra, that the plantlets must achieve a 2-4 cm long
growth. By reaching this size the plantlets also
gets a proper number of sprouts, foliage area
(leaves size and number) and roots number and
length thus allowing for maximal survival un-
der ex vitro conditions.
Plants from Phalaenopsis and Cattleya need
to achieve from 2 to 4 cm growth rank in vi-
tro in order to endure the external conditions
in greenhouse. MAP cultivated Phalaenopsis got
the better growth response when length was 2
to 4 cm with three roots and 1 to 8 cm long;
these values being a critical survival threshold.
The best substrate in Cattleya was the mixture
of moss-perlite (MP)
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of Dr. Salvador Chaila and Ing. Agr. Alicia M.
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Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 37
Photo 1. Phalaenopsis protocorms regenerated from oral nodes.
Photo 2. Phalaenopsis in vitro plantlets.
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Photo 3. Phalaenopsis plantlets on MAP potting mixture.
Photo 4. In vitro Cattleya plantlets.
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Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 39
Photo 5. Cattleya plantlets on MCP potting mixture.
Photo 6. Cattleya plantlets on ACP potting mixture.
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40 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
Photo 7. Cattleya plantlets on MP potting mixture.
Photo 8. Cattleya plantlets on AP potting mixture.
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