ArticlePDF Available

Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation


Abstract and Figures

The quality of micropropagated plants relies on the acclimatization stage. This research intends to develop an efficient protocol to obtain the acclimatization of Phalaenopsis and Cattleya. Plants of Phalaenopsis obtained from protocorms were selected. They came from flowering stalks grown at modified Murashige and Skoog (MS) (1962) medium and classified 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 results were obtained: Phalanopsis: a) Survival: 44% in R0 and 100% in R I and R II of the. b) Number of leaves: R I gave on average 1 more leaf than the range 0; c) Roots number and length: R I and R II gave on average 2 more roots than R0, and there were no significant differences in length. d) Height: R II presented greater growth than R I and Ro. Cattleya: a) The survival in MCP was 0%, MP 16 %, ACP 32% and AP 80%. b) The height in MP was significantly 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 substrate in Phalaenopsis and moss-perlite in Cattleya.
Content may be subject to copyright.
Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 27
Acclimatization of Phalaenopsis and Cattleya obtained
by micropropagation
Aclimatización de Phalaenopsis y Cattleya obtenidas
por micropropagación
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 efcient 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 modied
Murashige and Skoog (MS) (1962) medium and classied 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 signicant 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 signicantly 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.
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
BIOTECNOLOGIA XII-2 DIC 2010.indd 27 16/12/2010 23:29:10
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 eciente para la aclimatización de Phalaenopsis y Cattleya. Se seleccionaron
plantas de Phalaenopsis, obtenidas de protocormos provenientes de estacas orales cultivadas en Murashige
y Skoog modicado (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 signicativas 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
signicativamente 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 modied 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
as well.
BIOTECNOLOGIA XII-2 DIC 2010.indd 28 16/12/2010 23:29:10
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
BIOTECNOLOGIA XII-2 DIC 2010.indd 29 16/12/2010 23:29:10
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 quantied 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-
BIOTECNOLOGIA XII-2 DIC 2010.indd 30 16/12/2010 23:29:10
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 difcult, 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
potting mixtures.
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
BIOTECNOLOGIA XII-2 DIC 2010.indd 31 16/12/2010 23:29:10
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 indenite 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
(year 2007).
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 signicantly 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% condence intervals after 90 days
from implantation on MAP (year 2007).
BIOTECNOLOGIA XII-2 DIC 2010.indd 33 16/12/2010 23:29:12
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 conrms 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
22 months.
Table 2. Tukey`s test for Phalaenopsis leaf
Rank Leaf average number
RI3,6 A
RII 3,2 AB
R0 2,6 B
Treatments with the same letter do not differ significantly
(α= 0.05)
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.
BIOTECNOLOGIA XII-2 DIC 2010.indd 34 16/12/2010 23:29:12
Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 35
The gure 6 shows signicant 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% condence 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)
The authors thank the invaluable support
of Dr. Salvador Chaila and Ing. Agr. Alicia M.
Nasif on the completion of this work.
Abad, M. 1989. Los sustratos en horticultura ornamental.
Revista Agrícola Vegetal 3: 146-152.
Agramonte Peñalver, D., Jiménez Terry, F., Dita Rodríguez,
A. 1998. Aclimatización. In: Pérez Ponce, J. N. (Ed.).
Propagación y mejora genética de plantas por biotecnolo-
gía. Sta. Clara, Villa Clara, Cuba: Instituto de Biotec-
nología de las Plantas, pp. 193-206.
Arditti, J., Ernst, R. 1993. Micropropagation of Orchids.
New York: Wiley and Sons, pp. 1-25.
Avila-Díaz, I., Oyama, K., Gómez-Alonso, C., Salgado-
Garciglia, R. 2009. In vitro propagation of the en-
BIOTECNOLOGIA XII-2 DIC 2010.indd 35 16/12/2010 23:29:13
36 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
dangered orchid Laelia speciosa. Plant Cell Tiss Organ
Cult 99: 335-343.
Cha-um, S., Puthea, O., Kirdmanee, C. 2009. An eective
in-vitro acclimatization using uniconazole treatments
an ex-vitro adaptation of Phalaenopsis orchid. Scientia
Horticulturae 121: 468-473.
Colombo, L., Tadeu de Faria, R., Marinho de Assis, A.,
Batista Fonseca, I. 2005. Cattleya hybrid acclima-
tization in vegetal substrates under two irrigation sys-
tems. Acta Scientiarum Agronomy 27 (1): 145-150.
Denng, R., Donnelly, D. 1993. In vitro hardening of red
raspberry through CO2 enrichment and relative hu-
midity reduction on sugar-free medium. Can J Plan
Sci 73: 1105-1113.
Díaz, L., Medina, L., Latife, J., Digonzelli, P., Sosa, S. 2004.
Aclimatación de plantas micropropagadas de caña
de azúcar utilizando el humus de lombriz. RIA 33
Font uer, P. 1965. Diccionario de Botánica. Barcelona:
Labor, Labor.
Goh, H., Ng, W-Y., Huang, S-Y., Tan, I., Zhang, X-S., an,
F-Y., Lim-Ho, C-L. 2003. Anatomical study on the
development of in vitro orchid roots infected with
mycorrhiza. Acta Hort. (ISHS) 616:475-480. htttp:// [veried
November/21st/ 2010]
Iriarte, A., Saravia, L., Matias, C. 2002. Acondicionamiento
térmico con energía solar de un invernadero rusticade-
ro para la producción de plantas. Congreso regional de
ciencia y tecnología NOA. Secretaría de Ciencia y Tec-
nología, UNCa.
Jeon, M., Mohammad, Babar, A, Eun-Joo, H., Kee-Yoeup, P.
2005. Eects of photon ux density on the morphol-
ogy, photosynthesis and growth of a CAM orchid,
Doritaenopsis during post-micropropagation acclima-
tization. Plant Growth Regulation 45: 139-147.
Knudson, L. 1951. Nutrient solution for orchids. Botan
Gaz 112: 528-532.
Kozai, T. 1991. Autotrophic micropropagation. In: Ba-
jaj (ed.) Biotechnology in agriculture and forestry
17: High-tech and Micropropagation I. New York:
Springer-Verlag. Pp. 313-343.
Lee, Y., Yeung, E., Lee, N., Chung, M. 2008. Embryology
of Phalaenopsis amabilis var. formosa: embryo develop-
ment. Botanical Studies 49: 139-146.
Maene, L., Debergh, P. 1983. Rooting of tissue cultured plants
under in vivo conditions. Acta Hort 212: 335-448.
Murashige, T., Skoog, F. 1962. A revised medium for rapid
growth and bioassays with tobacco tissue cultures.
Physiol Plantarum 15: 473-497.
Northen, T. 1990. Home Orchid Growing, New York: Pren-
tice Hall.
Pierik, R. 1990. Cultivo in vitro de las plantas superiores.
Madrid: Mundi-Prensa.
Pospišilová, J., Tichá, I., Kadleček, P., Haisel, D., Plázkova,
Š. 1999. Acclimatization of micropropagated plants to
ex vitro conditions. Biologia Plantarum 42 (4): 481-
Preece, J., Sutter, E. 1991. Acclimatization of micropropa-
gated plants to the greenhouse and eld. In: Debergh,
P.; Zimmerman, R . H. (eds.). Micropropagation: tech-
nology and application. e Netherlands: Kluwer Aca-
demic Publishers.
R Development Core Team. 2010. R: A language and en-
vironment for statistical computing. R Foundation
for Statistical Computing, Vienna, Austria. ISBN
3-900051-07-0. [veri-
ed November/21st/2010].
Shushan, S. 1959. Developmental Anatomy of an Orchid,
Cattleya xTrimos. In: Carl Withner (ed.) e Orchids.
A Scientic Survey. New York: e Ronald.
Shiau, Y., Sagare, A., Chen, U., Yang, S., Tsay, H. 2002.
Conservation of Anoectochulis formosanus Hayata by
articial cross-pollination and in vitro culture of seeds.
Bot Bull Acad Sin 43: 123-130.
Teixeira Da Silva, J., Giang, D., Tanaka, M. 2005. In Vitro
Acclimatization of banana and Cymbidium. Interna-
tional Journal of Botany 1 (1): 41-49.
Torres, J., Laskowski, L., Sanabria, M. 2006. Efecto del am-
biente de desarrollo sobre la anatomía de la epidermis
foliar de Cattleya jenmanii Rolfe. Bioagro 18 (2): 93-
Tortosa, J. 1990 La turba, su caracterización física y quí-
mica, evaluación para cultivos en contenedor. Revista
Agrícola Vergel 106: 777-783.
van Huylenbroeck, J., Piqueras, A., Debergh, P. 1998. Pho-
tosynthesis and carbon metabolism in leaves formed
prior and during ex vitro acclimatization of micro-
propagated plants. Plant Science 134 (1): 21-30.
BIOTECNOLOGIA XII-2 DIC 2010.indd 36 16/12/2010 23:29:13
Acclimatization of Phalaenopsis and Cattleya obtained by micropropagation 37
Photo 1. Phalaenopsis protocorms regenerated from oral nodes.
Photo 2. Phalaenopsis in vitro plantlets.
BIOTECNOLOGIA XII-2 DIC 2010.indd 37 16/12/2010 23:29:14
38 Rev. Colomb. Biotecnol. Vol. XII No. 2 Diciembre 2010 27-40
Photo 3. Phalaenopsis plantlets on MAP potting mixture.
Photo 4. In vitro Cattleya plantlets.
BIOTECNOLOGIA XII-2 DIC 2010.indd 38 16/12/2010 23:29:15
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.
BIOTECNOLOGIA XII-2 DIC 2010.indd 39 16/12/2010 23:29:17
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.
BIOTECNOLOGIA XII-2 DIC 2010.indd 40 16/12/2010 23:29:18
... One of the difficult stages is a high percentage of dying plants after transferal to ex vitro conditions; therefore, it is very important to obtain good quality plantlets with properly developed roots [3]. A rooting system is necessary to allow water and nutrient intake by plants and to maintain the plants stably in soil [4]. These problems can usually be successfully solved by the use of appropriate plant hormones, mainly auxins [5]. ...
... During the rooting stage, limitations of water and nutrient transfer might cause a decrease in relative water content in orchids tissues [27]. According to Diaz et al. [4], leaves formed during in vitro growth are not able to further grow in ex vitro conditions and they are replaced by new ones. ...
Full-text available
To obtain healthy and good quality plants from in vitro cultivation, it is necessary to produce plantlets with well-developed rooting systems because they must undergo acclimatization, a final and a very difficult stage of micropropagation. In the present research, the effect of auxins NAA, IAA and IBA in concentrations of 0.5; 1; 2.5 and 5 mg·dm−3 on the Paphiopediluminsigne in vitro rooting was studied, and it was noted that 1 mg·dm−3 of IAA or IBA enabled the obtaining of a lot of rooted and good quality plantlets. The subsequent influence of the two most advantageous auxins on the acclimatization of plantlets in different substrates (sphagnum moss, sphagnum moss + substrate for orchids, substrate for orchids, substrate for orchids + acid peat) was tested, in the means of morphological features of plants and their physiological parameters, i.e., chlorophyll fluorescence (FV, Fm, Fv/Fm), stress enzyme activity (catalase, ascorbate peroxidase), and water balance. Considering all the tested features, it might be stated that the best results were obtained when explants were rooted in vitro in the presence of 1 mg·dm−3 of IAA and then planted ex vitro in substrate for orchids.
... The ability of seedlings to tolerate stress during transplantation often determines the success or failure of the clonal micropropagation process in in vitro culture [39]. The choice of a suitable substrate with high aeration, permeability, and an appropriate acidity grade is a prerequisite for ensuring autotrophic growth [40]. The most used substrates for the adaptation of orchids consist of bricks or charcoal, clay tiles, bark, coconut fibers, sawdust, perlite, vermiculite, peat, or sphagnum moss in different ratios [41,42,43]. ...
Full-text available
Cymbidium erythrostylum Rolfe is one of the most beautiful species of the genus cymbidium which is used as a cut flower or indoor plant. However, it was registered as a rare species even in its original habitat. This study was carried out to develop a protocol for in vitro propagation of C. erythrostilum. We used protosomes obtained by the asymbiotic germination of seed on 1/2 of Murashige and Skoog nutrient medium (MS) supplemented with 1 mg/l 2-Isopentenyl adenine (2iP) as objects of study. During the multiplication stage, the number of formed protosomes on a culture medium containing 0.8 mg/L 6-Benzylaminopurine (6-BAP) was higher (3.8 ± 0.2 protosomes). During the subsequent cultivation on ½ MS medium supplemented with 2 mg/l 6-BAP and 0.5 mg/l a-naphthaleneacetic acid (NAA), the highest numbers of shoots (4.00 ± 0.19 shoots/plantlet) and leaves (4.50 ± 0.14 leaves/plantlet) were obtained. At the rooting stage under in vitro conditions, the most effective was the use of ½ MS nutrient medium with the addition of 0.5 mg/l of indolyl-3-butyric acid IBA, 1 g/l charcoal, and 50 g/l banana puree. The obtained plants were successfully adapted to a substrate consisting of bark, perlite, and peat in a ratio of 1:1:1.
... 18 | Page among others, in the ability to maintain the humidity of the media. Media that can be used for acclimatization of orchid species are fern, peat moss, kadaka root, cocopeat, charcoal, and tile fragments 15,14,13,17,16 . The effects of the growing media characteristics at different shade levels on the growth and vigor of tiger plantlets has not been widely studied. ...
Full-text available
Background: Tiger orchid (Grammatophyllum speciosum. Blume) is designated as a protected species based on Government Regulation of the Republic of Indonesia, number 7 of 1999. Efforts to preserve the tiger orchid can be carried out by plant propagation using tissue culture techniques. This study aims to analyze the effects of growing media and shade levels on the growth and vigor of acclimatized plantlets. The research was conducted at the Permanent Nursery Greenhouse of BPDAS Barito, Guntung Manggis, Banjarbaru City, South Kalimantan. Materials and Methods: The experimental method is employed as the research method with an experimental design using a split plot design, the main plot is the shade levels consisting of 3 levels (paranet 50%, paranet 70%, and paranet 90%), and the growing media consisting of 4 levels (peat moss, fern, husk charcoal, and cocopeat) with 3 replications. The observed parameters included plant height, leaf length, leaf growth, survival percentage, and plantlet vigor. Results: The results showed that the interaction of growing media and shade level treatments had significant effects on plantlet growth, except for the parameters of number of leaves (plant height p < 0.01, leaf length p 0.04, leaf growth p 1.344, and survival percentage p 0.015). The main plot treatment had significant effects on the parameters (height p 0.002, leaf length p 0.035, number of leaves p 0.014, and survival percentage p 0.011). Sub-plot treatments had significant effects on plant height p < 0.01, leaf length p < 0.01, and survival percentage p 0.001, but had no significant effect on the parameter of number of leaves p 0.517. Conclusion: The best treatment combination was paranet 50%+ cocopeat. The vigor of plantlets was classified into some categories, namely very low (3 plantlets), low (7 plantlets), medium (13 plantlets), good (12 plantlets), and very good (1 plantlet).
... During acclimatization, plants must have a well-developed root system [35], enabling the plant to have properly assimilated water and nutrients, in addition to being well fixed to the substrate [36]. Accordingly, findings of this study indicate that plants grown in the 1 + 4 g humic acid substrate exhibited, in general, better morphological development. ...
Full-text available
In strawberry micropropagation, several challenges must be overcome to obtain quality plants and achieve high survival rate to ex vitro acclimatization. In this study, therefore, a set of protocols were evaluated to optimize explant (meristem) disinfection, in vitro growth (multiplication and rooting), and ex vitro acclimatization of strawberry. The results showed that explants treated with 1.0% NaClO for 5 min had a lower percentage of contamination, and achieved a higher percentage of viability, height, and number of leaves. In vitro growth was favored by the use of 1 mg L−1 zeatin, since it allowed greater seedling growth (number of shoots, seedling height, number of leaves, number of roots and root length), and a higher pre-acclimation rate (100%). In the acclimatization phase, plants grown in a substrate composed of compost + peat combined with 4 g of humic acid achieved better response in morphological and physiological variables. In fact, the results of this study could be used to cultivate strawberry plants of the ‘Aroma’ variety with high commercial quality.
... The study revealed that different combination of potting mixture plays a vital role on survivability of in vitro raised plants (Table 4). The main characteristics of appropriate potting mixture are water holding capacity, helps in aeration and draining out of excess water for proper establishment of plantlets (Diaz et al. 2010;Kang et al. 2020). Among the different combination used for hardening, maximum survival rate (91.66%) of plants was observed in potting medium comprising small brick chips (̴ ̴ ̴ 5À7 mm in size), charcoal pieces (̴ 5À7 mm in size) and coco peat (1:1:1) as it can provide higher water retention capacity and good aeration to transplanted orchids. ...
Bulbophyllum odoratissimum (Sm.) Lindl. ex Wall. is widely used as traditional medicine for the treatment of tuberculosis, chronic inflammation and is listed as an endangered orchid due to over-exploitation and habitat destruction. The present study was carried out to establish a genetically stable in vitro regeneration protocol for mass multiplication using nodal segments and to determine phytochemical constituents, the antioxidant capacity of different parts (leaf, pseudobulb and root) of mother plants and acclimatized in vitro raised plants of B. odoratissimum. The synergistic effect of BA (4.0 mgl⁻¹) and IBA (0.5 mgl⁻¹) was found to be optimum for producing maximum shoot proliferation. IBA was found to be more effective for root proliferation than IAA, NAA and an average (3.08 roots/shoot) was obtained in 0.5 mgl⁻¹ IBA. The well rooted plantlets were successfully acclimatized (more than 90% survival rate) in greenhouse, exhibiting normal development. The ISSR banding profiles confirmed high degree of genetic homogeneity among the clones. Extraction of all plant material from both mother plant and in vitro raised plant possess relevant TPC, TFC, radical scavenging activity (DPPH and ABTS) as well as total reducing power ability. HPLC chromatogram exhibited that phytochemical compounds detected in regenerated plants were similar to those present in mother plant and there was no remarkable difference. High regeneration frequency along with its genetic stability and secondary metabolite production validate the efficiency of this micropropagation protocol of B. odoratissimum for species recovery program as well as a potential source of commercial application.
... An improved method of acclimatization of micropropagated. Cattleya was reported [45] . Da Silva et al. [46] studied the effect of Kefir, Knudson medium and sucrose on in vitro growth of Cattleya walkeriana. ...
Full-text available
Cattelya is one of the most popular orchid genera. The exquisite beauty of its flowers accounts for its high demand the global floriculture trade. The present review provides an overview of Cattelya orchid literature. It aims to sensitize the readers about general botanical information and commercial importance of cattelya orchids that will encourage the readers to take up cattelya cultivation. Conventional and biotechnology based methods of Cattelya propagation have been discussed that will be extremely useful to Cattelya growers. Considering the fact that ex vitro maintenance of orchid is a challenge, special emphasis has been given to summarize the methods for Cattelya growth in pots and maintenance of conducive physical environment for optimal growth and development of Cattelya orchids. The review also aims to highlight the status of Cattelya cultivation in the country as compared to other nations. While countries like Thailand, Singapore, Malaysia lead the world production of orchids, despite vast orchid diversity; India's orchid cultivation enterprise is still in its prime. Overall, the study only expands the use of Cattelya for propagation by the growers, it also presses upon the need and significance of taking up Cattelya cultivation on a commercial scale in the country.
... A pré-aclimatização em casa de vegetação é uma prática comum entre produtores comerciais de orquídeas e melhora diversas características das plantas, propiciando maior sobrevivência durante a aclimatização e reduzindo custos de produção (Cardoso et al. 2013;Silva et al. 2017). Muitos experimentos avaliam a influência de substratos no processo de aclimatização (Díaz et al. 2010;Kunakhonnuruk et al. 2018;Juras et al. 2019), até mesmo com plântulas produzidas simbioticamente (Guimarães et al. 2013). Outros fatores abióticos que interferem na aclimatização, como a qualidade da luz durante o crescimento in vitro (Massaro et al. 2018), também são encontrados na literatura. ...
Conference Paper
Full-text available
Introdução A interação das plantas com microrganismos é regra. A maioria deles colonizam externamente as esferas de seus órgãos e/ ou internamente os seus tecidos, sem que se observe efeitos negativos ou sintomas de patogenia. Ao contrário, eles contribuem de forma positiva ao desenvolvimento da planta, em interações classificadas mutualísticas (Sylvia et al. 2004, Cardoso e Andreote 2016). As orquídeas abrigam em suas raízes uma larga gama de microrganismo mutualistas. A interação com fungos endofíticos micorrízicos (EM), em um mutualismo simbiótico especializado, é indispensável ao ciclo natural das orquídeas (Dearnaley et al. 2017). Outros fungos endofíticos, aqui tratados como não micorrízicos (ENM), são destacados por contribuirem nutricionalmente e fisiologicamente ao crescimento das orquídeas (Ma et al. 2015). Bactérias associativas também colonizam externamente e/ ou internamente (bactérias endofíticas BEs) as raízes das orquídeas (Bayman e Otero 2006). Muitas das BEs atuam diretamente na promoção do crescimento das orquídeas, fixando nitrogênio, solubilizando nutrientes e produzindo fitohormônios (e/ ou seus os precursores), em contrapartida elas recebem abrigo e fotoassimilados (Galdino et al 2011; Faria et al. 2013). Os benefícios dessas associações devem ser considerados para o desenvolvimento de estratégias que garantam melhores resultados durante a produção comercial e conservacionista de mudas (Pereira e Valadares 2017, Fay 2018). Dessa forma, este trabalho busca descrever os endofíticos e seus benefícios ao desenvolvimento das orquídeas, considerando os aspectos da germinação e da aclimatização da mudas. Os Endofíticos de Orquídeas A diversidade de endofíticos tem sido estudada de duas formas: isolamento e identificação dos simbiontes e identificação pela amplificação de genes conservados utilizando Sequenciamento de Nova Geração (Next-Generation Sequencing NGS) (Bayman e Otero 2006, Pereira et al. 2014, Waud et al. 2016). A comunidade microbiana varia consideravelmente entre as diferentes fases do ciclo de vida da orquídea (Rasmussen et al. 2015). Mesmo que os Ems se associem às orquídeas na fase de semente e possam perdurar por toda a vida da planta, a colonização por outros microrganismos ocorre durante o desenvolvimento do protocormo, da plântula, e na fase adulta (Dearnaley et. 2017). A interação com EMs é caracterizada pela presença de enovelados de hifas fúngicas em células parenquimáticas do embrião, do protocormo e em células do córtex radicular (Peterson et al. 2004). Os enovelados, denominado pelotons, permitem a classificar a interação como simbiose mutualística, uma vez que permitem a proximidade entre os parceiros para que as trocas sejam efetivadas (Yeh et al. 2019). A identificação dos EMs por muito tempo se baseou em caracteres morfológicos, quando se priorizava o grupo artificial dos fungos rizoctonióides (Currah 1992). Esse grupo abrangia alguns gêneros do filo Basidiomycota considerados: Ceratobasidium, Tanathephorus, Tulasnella, Sebacina e Serendipita (Dearlaney et al. 2012). Dearlaney et al. (2012) apresentam uma classificação mais ampla dos fungos micorrízicos de orquídeas. Nela, o filo Basidiomycota se destaca por abrigar o maior número de táxons de fungos micorrízicos, sendo 20 táxons identificados em nível de gênero. Por outro lado, Ascomycota é representada por 3 gêneros da classe Pezizomycetes. Diante o papel dos EMs em sustentar a germinação das sementes e auxiliar as raízes das plantas adultas na absorção de nutrientes (Arditti 1992, Peterson et al. 2004), a maioria dos estudos se voltam para eles, os quais são considerados um fator essencial a dispersão e manutenção das orquídeas em seus habitats (Dearnaley et al. 2017). No Brasil, cinco gêneros recebem mais (Pereira e Valadares 2012). Sebacina e Tulasnella são os gêneros mais relatados. Eles atuam na germinação de grande parte das orquídeas estudadas, podendo ser detectados nas raízes
... Phalaenopsis y CattleyaConventional Pre-hardening In vitro and MS salts aplication during the first month. 90 -100 Díaz., et al.[29]; Deb., et al.[2] ...
... Appropriate media selection with low septicity, high aeration, suitable permeability, and acidity level are required to ensure initial and autotrophic growth condition. The media is also required to maintain a long-term condition with no reduction in quality to avoid compaction and lack of aeration and permeability (Diaz et al., 2010). The substrate is a solid and porous material, synthetic or natural, combined or uncombined, which allows plants to grow well under controlled environmental conditions (Abad, 1989). ...
Full-text available
This study was to evaluate the effect of plant media variation, time of fertilization, and combination of both on the growth of orchid plant Dendrobium sylvanum Rchb. f. in acclimatization phase. This study was designed using 6x4 factorial experimentation. The first factor was the variation of plant media: charcoals, coconut husk, bagasse, mixing of charcoal and coconut husk, combination of charcoal and bagasse, combination of coconut husk and bagasse. The second factor was fertilization time: no fertilization, 7 day-fertilization, 14 day-fertilization, and 20 day-fertilization for three months. The data were analysed by using Two Way MANOVA (α = 5%) and Duncan test. Based on the results, there were differences between media variation and fertilization time on the increase of leaf length and width, stem diameter, and root length of Dendrobium orchid in acclimatization phase. Combination of bagasse and coconut husk media showed the best result on media treatment. The best of fertilization time was 14 days. Moreover, the best of interaction between media and fertilization time showed by the combination of bagasse and coconut husk with 14 days-fertilization.
Produksi bibit Phalaenopsis Hibrid umumnya dilakukan melalui teknik kultur jaringan. Tahap selanjutnya adalah aklimatisasi plantlet di lingkungan ex-vitro. Penggunaan naungan dan tipe substrat berperan terhadap pertumbuhan dan kelangsungan hidup plantlet selama periode awal aklimatisasi. Penelitian ini mengkaji peran naungan dan tipe substrat yang berbeda terhadap pertumbuhan dan kelangsungan hidup plantlet Phalaenopsis Hibrid selama periode awal aklimatisasi di greenhouse. Penelitian menggunakan plantlet Phalaenopsis Hibrid dalam botol dan naungan paranet. Plantlet diberi 3 tipe substrat yaitu serabut kelapa, akar paku kadaka, sphagnum serta semua perlakuan dinaungi paranet secara bertahap. Parameter penelitian yang diukur meliputi: jumlah akar, total panjang akar, panjang daun, berat segar dan persentase kematian plantlet. Rancangan Acak Lengkap dengan satu faktor dan 3 ulangan digunakan dalam penelitian ini. Data dianalisis menggunakan ANOVA dan uji LSD pada taraf signifikansi 95% (P < 0,05). Penelitian dilakukan selama 5 minggu di greenhouse Jurusan Biologi, Fakultas Sains dan Matemetika, UNDIP. Plantlet tumbuh dan mampu bertahan hidup pada semua substrat dan naungan paranet secara bertahap. Pembentukan pori besar pada substrat serabut kelapa dan akar kadaka meningkatkan jumlah dan panjang akar. Kemampuan menjerap air tinggi pada spagnum meningkatkan berat segar plantlet. Penutupan paranet secara bertahap dan penggunaan substrat serabut kelapa, akar kadaka serta spagnum mempertahankan kelangsungan hidup plantlet selama periode awal aklimatisasi ex-vitro. Kata kunci:aklimatisasi, Phalaenopsis Hibrid, serabut kelapa, akar kadaka, sphagnum
Full-text available
Phalaenopsis amabilis var. formosa is an endemic epiphytic orchid variety native to Taitung and Lanyu of Taiwan. A ⊥-shaped, four-celled embryo is produced by two successive cell divisions of a zygote. Soon after, two of the four cells toward the micropyle enlarge and divide two more times resulting in the formation of eight tubular suspensor cells. The suspensor cells are highly vacuolated; the bottom tier of suspensor cells elongates towards the micropyle, and the upper tier elongates towards the chalazal end of the seed. During the early stages of embryo development, lipid droplets appear in the elongating suspensor cells and disappear soon afterwards, indicating the suspensor functions in nutrient uptake and as a temporary food storage site for the developing embryo. In the mature seed, a differentiated apical zone containing the relatively small cells can be seen in the embryo proper. Protein and lipid bodies are the main storage products in the embryo proper cells. The results of Nile red staining indicate that a cuticular layer is present only on the surface walls of the embryo proper, but is absent from the suspensor cell wall Cuticular material is also present in the outermost layer of the seed coat and persists through seed maturation.
Full-text available
Anoectochilus formosanus is an important ethnomedicinal plant of Taiwan. We have optimized a method for mass propagation of A. formosanus by artificial cross-pollination and asymbiotic germination of seeds. The success of pollination and fruit set was found to be dependent on the developmental stage of male and female gametophytes. Fruit set of hand-pollinated flowers was 86.7%. The seeds from 7-week-old capsules were germi- nated by culturing on half-strength Murashige and Skoog's (MS) medium supplemented with 0.2% activated char- coal and 8% banana homogenate for four months. Germinated seedlings were cultured in half-strength liquid MS medium containing 2 mg/l N 6 -benzyladenine (BA) in 125-ml Erlenmeyer flasks for two months. Before ex vitro establishment of seedlings, seedlings with well-developed rhizomes and shoots were cultured on half-strength MS medium with 0.2% activated charcoal, 8% banana homogenate, 2 mg/l BA and 0.5 mg/l a-naphthaleneacetic acid (NAA) for further growth. Around 90% seed-derived plants survived two months after transfer to peat moss:ver- miculite potting mixture and incubation in the growth chamber.
Full-text available
La calidad de las plantas y la eficiencia del proceso de micropropagación dependen de la aclimatación del material vegetal. El objetivo de este trabajo fue determinar efectos del lombricompuesto como sustrato, en aclimatación de plantas micropropagadas de caña de azúcar en condiciones de invernadero. El lombricompuesto se obtuvo de deyecciones de la lombriz roja californiana, Eisenia foetida, alimentada con estiércol bovino descompuesto. Las plantas micropropagadas de NA 63-90 se obtuvieron del meristema apical con dos o tres primordios foliares, incubado en MS (1962) modificado. Los plantines se trasplantaron a bandejas de plástico de 72 celdas con los siguientes tratamientos en %: T1: 0 lombricompuesto +100 tierra; T2: 20 lombricompuesto + 80 tierra; T3: 40 lombricompuesto + 60 tierra; T4: 60 lombricompuesto + 40 tierra; T5: 80 lombricompuesto +20 tierra y T6: 100 lombricompuesto + 0 tierra. A los 60 días, 24 plantas por tratamiento se llevaron a macetas de plástico (9,5 x 10 cm). Cada 20 días se evaluó longitud de raíces, número y altura de plantas. El diseño experimental fue totalmente al azar, con 5 repeticiones. Se realizó análisis de la varianza y test de Tukey. Respecto a la longitud de raíces, 100 % lombricompuesto fue significativamente superior a 0 % lombricompuesto. El ANOVA mostró un incremento del crecimiento de raíces a través del tiempo, aunque la tendencia en él no fue lineal. En materia de altura de plantas, T1 fue significativamente superior a T6 y T2; el efecto del tiempo fue significativo y la tendencia no fue lineal. Con respecto al número de plantas, los tratamientos con lombricompuesto favorecieron el macollaje, mientras el testigo mantuvo el número inicial de plantas. El uso de lombricompuesto como sustrato, en diferentes concentraciones, favorece el crecimiento de raíces y número final de plantas, sin incrementos de crecimiento de la parte aérea de plantas micropropagadas de caña de azúcar
Full-text available
Micropropagated plantlets are fragile and often lack sufficient vigour to survive the acclimatization shock during transplantation to the soil. Effects of photosynthetic photon flux densities (PPFDs) on growth, photosynthesis and anatomy of micropropagated Doritaenopsis were studied after 4months of acclimatization in a greenhouse at 25C. The plantlets were transferred to three different PPFDs for four months, i.e. low light (175), intermediate light (270) and high light (450molm–2s–1). For most of the growth parameters measured i.e. leaf length, leaf area, leaf width, fresh weight, dry weight, chlorophyll (Chl) a/b ratio, were greater for the intermediate light levels after 4months of acclimatization. The only exception was leaf thickness, which was increased more under high light levels. Results showed that the survival of Doritaenopsis plantlets was greatest (90%) in low light and intermediate light (89%) compared with only (73%) at high light. However, at low light levels, pigment concentrations (chlorophyll a, b and total chlorophyll) were higher. Net CO2 assimilation (A), stomatal conductance (g) and transpiration (E) were higher in plantlets grown at high level PPFD than at low after 4months of acclimatization. Photosynthetic efficiency (Fv/Fm) decreased insignificantly; only at mid day for the high light treatment whereas leaf temperature and stomatal closure increased compared to low light. Scanning electron microscopic (SEM) images of leaves from acclimatized plantlets showed an increase in wax formation for the high light grown plantlets compared to those at low light. Microscopic analysis of acclimatized root sections showed highly developed multiseriate-velamen layers and higher root cell activity; while shoots had larger leaf air spaces than those of in vitro grown plantlets. These results suggest that physiological acclimation occurs at the intermediate PPFD (270molm–2s–1) in Doritaenopsis compared to treatment at the high light level.
Full-text available
Laelia speciosa is an endangered epiphytic orchid. The effects of various media components on germination of L. speciosa were evaluated. Pods were collected at 4, 7, and 9months following hand-pollination, and seeds were germinated on Murashige and Skoog (MS) media with 30gl−1 sucrose and five concentrations (0.0, 0.04, 0.22, 0.44, and 2.22μM) of benzyladenine (BA) under light and dark conditions. Gibberellic acid (GA3; 0.0, 0.29, 1.44, 2.89, 14. 43 and 28.87μM) with naphthaleneacetic acid (NAA; 0.0, 0.54, 1.34, 2.69, and 5.37μM) were evaluated for in vitro subcultivation. MS medium with 30gl−1 sucrose was effective for germination. The effects of BA and light on germination of L. speciosa seeds differed with pod maturity. All mature seeds germinated using 0.44μM BA and light. The highest frequency of germinated seedlings (60%) was obtained using mature seeds grown on MS medium without BA and under light conditions. For subculture, MS with 30gl−1 sucrose, 2.69μM NAA, and 0.29μM GA3 was effective. Plantlets of 5cm in length were transplanted to the greenhouse, and a 77.5% of survival rate was obtained. A successful protocol for micropropagation by seed germination will contribute to the development of a sustainable management program for L. speciosa.
Tissue culture techniques are now widely used to produce commercially grown orchids. This process takes about 8-12 months to produce plantlets large enough to withstand the rigours of nursery conditions. However, during the acclimatization phase, tissue-cultured plantlets undergo a lag phase before they are able to establish. In nature, orchid plants rely on their close association with mycorrhiza fungi for growth and development. Thus, we have developed a method to infect in vitro orchid roots with mycorrhiza. Stage III micropropagated orchid plantlets were artificially inoculated with mycorrhiza strain MS6A (isolated from Mokara Sunkist) in vitro. The roots of the plantlets were harvested 5, 8 and 20 weeks after inoculation to study the stages and development of mycorrhiza infection. Using biological microtechniques, the orchid roots were cut into sections of 8 μm thickness and stained with 1% Toludine Blue O. The slides were examined under light microscope and the developmental stages of mycorrhiza in the cells were examined. The aim is to study the developmental stages of the association of mycorrhiza strain MS6A in Vanda 'Tan Chay Yan' orchid plantlets in vitro. Orchid roots after 5 weeks of infection had masses of fungal hyphae at the velamen region. At week 8, intracellular fungal aggregates have spread to the cortical cells. At week 20, coils of fungal hyphae, also known as pelotons, have infected cells near the central vascular cylinder. This was similar to the infection pattern observed in plants in vivo.
The special conditions during in vitro culture result in the formation of plantlets of abnormal morphology, anatomy and physiology. After ex vitro transfer, these plantlets might easily be impaired by sudden changes in environmental conditions, and so need a period of acclimatization to correct the abnormalities. This review is focused upon contemporary information on the changes in leaf structure, water relations and photosynthesis during acclimatization of plantlets to ex vitro conditions. It also describes some ways of improving plant survival and for the speeding up of acclimatization.