Environmental and Experimental Botany 100 (2014) 26–33

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Physiological and biochemical changes during acclimatization in a

Doritaenopsis hybrid cultivated in different microenvironments
in vitro
Kong-Sik Shin a , So-Young Park b,∗ , Kee-Yoeup Paek c,∗∗
a
Biosafety Division, National Academy of Agricultural Science, RDA, Suwon 441-707, Republic of Korea
b
Department of Horticultural Science, Chungbuk National University, Cheongju 361-763, Republic of Korea
c
Research Center for the Development of Advanced Horticultural Technology, Chungbuk National University, Cheongju 361-763, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Photoautotrophic culture has advantages over conventional micropropagation methods, and its poten-
Received 18 September 2013 tial benefits appear to be promising. Nevertheless, little is known regarding the effects of such culture
Received in revised form conditions on plant physiology during ex vitro acclimation. In this study, Doritaenopsis plantlets were
29 November 2013
grown under three different microenvironmental conditions: (1) in vitro under photoautotrophic condi-
Accepted 3 December 2013
tions (high photosynthetic photon flux density [PPFD], high CO2 concentrations and an increased number
of air exchanges), (2) photomixotrophic conditions (photoautotrophic conditions, with 30 g L−1 sucrose
Keywords:
added to the culture medium) and (3) heterotrophic conditions (conventional culture conditions; low
Ex vitro acclimatization
Heterotrophic PPFD, low CO2 concentration and no air exchange). The plantlets were subsequently transferred into ex
Photoautotrophic vitro conditions, and the physiological and biochemical changes were investigated during the acclimation
Photomixotrophic process. Photoautotrophic and photomixotrophic in vitro cultivation markedly stimulated the growth of
Doritaenopsis plantlets during ex vitro acclimatization. The development of photosynthetic function (PEP-
carboxylase activities and carbohydrate metabolism) appeared to be fully achieved in ex vitro plantlets
that had been cultured in photoautotrophic and aerated photomixotrophic conditions in vitro. The strong
enhancement of growth by CO2 enrichment, along with the exposure of photoautotrophic plantlets to
high PPFD, may explain why the plants adjust more effectively (exhibiting higher rates of photosynthesis
under high-light conditions) during ex vitro acclimatization. The results of this study suggest that the
growth and survivability of Doritaenopsis during the acclimatization process can be enhanced by CO2
supplementation and increased light levels during in vitro culture.
© 2013 Elsevier B.V. All rights reserved.

1. Introduction
In the micropropagation system of plant, ex vitro acclimatiza-
tion, or hardening, is one of the main processes performed in the
production of healthy plantlets before their transplantation to field
conditions. The conventional in vitro environment is characterized
by high relative humidity levels, constant temperatures, low pho-
tosynthetic photon flux density (PPFD), large diurnal fluctuations in
CO2 concentration, high concentrations of sugar, salts and growth-
regulating substances in the medium, gradual accumulation of toxic
substances and the absence of microorganisms (Xiao et al., 2011;
Zobayd et al., 2004). These conditions often lead to low rates of
transpiration, photosynthesis, water and nutrient uptake and CO2
∗ Corresponding author. Tel.: +82 43 261 2531; fax: +82 43 271 0414.
∗∗ Co-corresponding author. Tel.: +82 43 261 3445; fax: +82 43 271 0414.
E-mail addresses: soypark7@chungbuk.ac.kr (S.-Y. Park),
paekky@chungbuk.ac.kr (K.-Y. Paek).
uptake but a high rate of dark respiration, all of which result in poor
growth in vitro and, subsequently, decrease adaptability during the
acclimatization period (Xiao et al., 2011). Photoautotrophic culture
with enriched CO2 levels during in vitro culture of plantlets using
environmental controls has successfully been applied to improve
the survival percentage of many species of plants under ex vitro
conditions (Kozai, 1991; Hazarika, 2006; Kozai et al., 2005; Lian
et al., 2002).
Doritaenopsis is a monopodial epiphytic CAM (Crassulacean
acid metabolism)-type orchid with succulent leaves, which has
high economic value because of its worldwide popularity as a cut
flower and as a potted plant (Park et al., 2003). The leaves of Dori-
taenopsis are produced alternately, with younger leaves exposed
to complete irradiance and mature leaves gradually becoming
shaded by the formation of the juvenile leaves above them. This
phenomenon strongly inhibits the absorbed photon flux density
(PFD) of the leaves situated on the lower half of the stem and
seriously hampers the process of carbon assimilation. Recently,
Yoon et al. (2009) demonstrated that CO2 enrichment and sugar

0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.envexpbot.2013.12.004
 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33
deprivation in vitro significantly increase the survival rates
of Phalaenopsis ex vitro. Hahn and Paek (2001) also reported
that the growth and net photosynthetic rate of in vitro-grown
orchid plantlets are influenced by the culture environment,
including aspects such as PPFD, CO2 concentration, relative
humidity and the number of air exchanges. The optimiza-
tion of these parameters resulted in the development of a
more efficient micropropagation system for these orchid plants.
Recently, we reported the effect of microenvironment con-
ditions on growth and sugar metabolism in in vitro-grown
Doritaenopsis plantlets (Shin et al., 2013). We found that pyru-
vate, produced by the decarboxylation of malate, is required
for optimal photoautotrophy under high PPFD. We also found
that growth was greatest in plantlets grown under CO2 -enriched
photoautotrophic and photomixotrophic conditions with a high
PPFD. A transition from C3 to CAM was observed in in vitro-
grown Doritaenopsis plants prior to acclimatization (Shin et al.,
2013).
During ex vitro acclimatization, many changes can occur in mor-
phological and physiological metabolism as well as photosynthesis
due to differences in environmental conditions, but studies on
this aspect of acclimatization are still limited. Healthy, acclima-
tized plantlets have often been identified using plant morphological
characteristics and survival rates only. Therefore, the aim of this
study was to determine how elevated CO2 concentrations and PPFD
during in vitro culture affect the characteristics of photosynthesis
and sugar metabolism in Doritaenopsis, a representative CAM plant,
during subsequent acclimation under ex vitro conditions.
2. Materials and methods
2.1. Plant material
In vitro-grown seedlings of a Doritaenopsis hybrid of Doritaenop-
sis ‘New Candy’ and Dtps. ‘Mary Anes × Ever Spring’ were used
during the present investigation. Dtps plantlets (2.5 cm in shoot
length and 0.8 g in weight) were cultured on Hyponex medium
(6.5N: 6P: 19K, 1 g + 20N: 20P: 20K, 1 g L−1 ) containing 2 g L−1
peptone, 0.5 g L−1 activated charcoal and 8 g L−1 agar (without the
addition of hormones) in 500 mL culture vessels (Phytohealth, SPL,
Korea). The medium was sterilized by autoclaving at 121 ◦ C at 15 psi
of pressure. Ten plantlets per vessel and 20 vessels per condition
(Table 1) were cultured as previously reported (Shin et al., 2013),
and after ten weeks of in vitro culture, the plantlets were harvested
for the acclimatization experiment.
For ex vitro acclimatization, Doritaenopsis plantlets were trans-
planted to culture boxes (55 cm × 45 cm × 25 cm) containing pine
bark (average particle size >5 mm) as a potting medium. During
the acclimatization period, the photosynthetic photon flux in the
daytime was adjusted to 100 ± 20 ␮mol m−2 s−1 on average, and
the temperature during the day and at night was maintained at
25 ± 2/22 ± 2 ◦ C (70 ± 20% RH) in greenhouse. During this time, the
plants were irrigated every 3 days, treated with foliage dressing
every week using Hyponex (6.5N-4.5P-19K 1 g L−1 ), and acclima-
tized for 40 days under the conditions described above.
2.2. Growth parameters
The growth of the plants was measured, including the leaf
length, leaf width, leaf area, fresh weight and dry weight. The leaf
area was measured using a leaf area meter (Skye, Skye Instruments
Ltd., Powys, UK). The dry weights of the aerial and subterranean
parts of the plants were measured separately after drying the mate-
rial for 2 days in a dryer (FO-600M, Jeio Tech Co., Korea) set at 70 ◦ C
to completely remove the water.
27
2.3. Chlorophyll fluorescence
Chlorophyll fluorescence variables were measured on the abax-
ial sides of freshly detached leaf discs. The plants were incubated for
30 min in the dark prior to measurement. Modulated fluorescence
was measured using a PAM chlorophyll fluorometer (PAM-200;
Heinz Walz, Effeltrich, Germany) connected to the leaves with leaf
clip holders (2030-B, Walz), and the DA-200 (Walz) program was
used to collect the data.
Minimal fluorescence (Fo) was measured in 30 min dark-
adapted leaves using a light level of <250 ␮mol m−2 s−1 , and
maximal fluorescence (Fm) was measured in the same leaves after
a 3 s saturating pulse (>2500 ␮mol m−2 s−1 ) of light. Maximum
variable fluorescence (Fv = Fm − Fo) and photochemical efficiency
of PSII (Fv/Fm) were calculated for dark-adapted leaves. In light-
adapted leaves, the steady state fluorescence yield (Fs), maximal
fluorescence (Fm) after a 3 s saturating pulse (>2500 ␮mol m−2 s−1 )
and minimal fluorescence (Fo) values were measured when the
actinic light was turned off. The fluorescence values measured
in the light-adapted leaves were used to calculate the following
parameters: variable fluorescence (Fv = Fm − Fo), photochemi-
cal quenching [qp = (Fm − Fs)/(Fm − Fo)], and non-photochemical
quenching [qN = 1 − (Fm − Fo )/(Fm − Fo)].
2.4. Net photosynthesis rate
The CO2 level was measured with a gas chromatograph (HP 6890
series, Hewlett Packard Co., Wilmington, DE, USA) equipped with a
Poraplot Q capillary column (25 m × 0.53 mm, Hewlett Packard Co.),
with the flow of He (as the transportation gas) set at 13 mL min−1 ,
an oven temperature of 90 ◦ C and an injector temperature of 200 ◦ C,
using a TCD detector. The net photosynthesis rate (NPR) of the
plants was measured according to the Kozai and Iwanami method
(1988) using the CO2 concentration measured by the method
described above.
2.5. Carbohydrate extraction and analysis
Frozen samples were ground in liquid nitrogen using a mor-
tar and pestle and then extracted in three volumes of 80% ethanol
to separate the ethanol-soluble fraction containing sugars from the
ethanol-insoluble starch. The ethanol-soluble supernatant was col-
lected after centrifugation for 15 min at 8000 × g. The supernatant
was passed sequentially through a C18 Sep-Pak cartridge (Waters,
Milford, MA, USA) and a 0.2 ␮m membrane filter. The samples
were injected into a Waters HPLC system (Waters 626 pump and
600 s controller) using a Rheodyne 7725I injector equipped with a
20 ␮l sample loop. The isocratic system was operated at 1 mL min−1
using degassed 80% acetonitrile as the mobile phase. Soluble
sugar separation was achieved using a carbohydrate WATO44355
(250 × 4:6 mm2 ) stainless steel column (Waters). Soluble sugars
were measured using a refractive index detector (Waters 410). For
starch determination, the precipitate was washed three times with
2–3 volumes of 40% ethanol to remove the soluble sugar residues.
The starch was solubilized using an HCl–DMSO pretreatment and
hydrolyzed by incubation with amyloglucosidase (50 units/mL in
0.1 M Na-acetate buffer, pH 4.2) for 48 h at 55 ◦ C, with occasional
agitation. The starch was then determined (as glucose) using a dini-
trosalicylic acid assay (Chaplin, 1986).
2.6. Carbohydrate and related enzyme assay
The same leaf regions (the centers of two leaves per plant)
were used for various enzyme analyses. A gram of each sam-
ple was triturated in a mortar using liquid nitrogen, and 3 mL of
enzyme extraction buffer solution (50 mM HEPES-NaOH, pH 7.5,
28 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33

Table 1
Description of the treatments used for the in vitro culture.

Treatment codea CO2 concentration outside the vessel (␮mol mol−1 ) PPFD (␮mol m−2 s−1 ) Sucrose (g L−1 ) Gas exchange (times/h)

Photoautotrophic
AL 350 ± 20b 40 ± 2 0 2.83
AH 350 ± 20 120 ± 5 0 2.83
ALC 1500 ± 100 40 ± 2 0 2.83
AHC 1500 ± 100 120 ± 5 0 2.83

Photomixotrophic
ML 350 ± 20 40 ± 2 30 2.83
MH 350 ± 20 120 ± 5 30 2.83
MLC 1500 ± 100 40 ± 2 30 2.83
MHC 1500 ± 100 120 ± 5 30 2.83

Heterotrophic
HL 350 ± 20 40 ± 2 30 0.25
HH 350 ± 20 120 ± 5 30 0.25
a
A: autotrophic, M: mixotrophic, H: heterotrophic, L: low light, H: high light, C: CO2 enrichment.
b
Numbers followed by ± are standard errors.

20 mM MgCl2 , 10 mM iso-ascorbic acid, 1 mM EDTA) was added
to the sample. Insoluble polyvinylpyrrolidone (PVP) was added
to absorb/remove phenol. The sample was shaken 2–3 times and
centrifuged at 12,000 rpm. After centrifugation, removal of the
precipitate and filtration through a 0.45 ␮m membrane filter, the
supernatant was utilized as a coenzyme liquid (Van Huylenbroeck
et al., 1998). The temperature was maintained at 4 ◦ C at each step in
the tests, and the process was repeated three times for each group.
The protein content of the liquid enzyme was measured using the
Lowry method (Lowery et al., 1951).
The activated enzyme reaction of PEP-carboxylase was mea-
sured with 950 ␮L of reaction buffer solution (25 mM Tris-HCl pH
8.0, 5 mM MgCl2 , 2 mM NaHCO3 , 5 mM glucose-6-p, 5 mM PEP,
0.2 mM NADH, and 2 units of L-MDH mL−1 ) and 50 ␮L of coenzyme
liquid. The reaction began with the addition of glucose-6-P. The
activation of the enzyme was measured by determining the amount
of NADH that was reduced by the oxidation of oxaloacetate for
10 min at 340 nm (Uvikon 930, Kontron Instruments Co., Zurich,
Switzerland) following Kumar et al.’s method (1988).
3. Results and discussion
3.1. Plantlet growth
Acclimation to ex vitro conditions and the subsequent growth
of Doritaenopsis plants were markedly better under elevated CO2
concentrations with higher light intensity than under standard
conditions, which favored adaptation of the plantlets to the ex
vitro environment (Fig. 1). Doritaenopsis plantlets grown under
1500 ± 100 ␮mol mol−1 CO2 concentration, when in vitro cultured,
exhibited longer leaf length and width, a greater leaf area, as
well as higher leaf fresh/dry weight and root fresh/dry weight
ratios, compared with those grown under control CO2 levels
(350 ± 20 ␮mol mol−1 ), irrespective of the light intensity provided
(Fig. 1).
In vitro proliferated plants have various physiological abnormal-
ities due to the stress that they experience from sudden changes in
the environment during the ex vitro acclimatization stages. Their
survival rate is also influenced by environmental conditions (Xiao
et al., 2011). To solve these problems, the in vitro culture envi-
ronment must be adjusted to minimize the loss of plants during
cultivation (Kozai et al., 1997). However, very limited information
is available on the physiological changes that occur during the tran-
sition of micropropagated plantlets from in vitro conditions to the
acclimatization phase.
In this study, we determined the elevated CO2 concentrations
and PPFD in vitro affect the characteristics of photosynthesis and
sugar metabolism in Doritaenopsis, a CAM plant, during acclimation
to ex vitro conditions. Superior plantlet growth under photoau-
totrophic culture conditions has been attributed to the notion that
these conditions provide the proper environment for the photo-
synthesis of plantlets, as recently reviewed by Xiao et al. (2011).
Under natural in vitro conditions, the CO2 concentration inside the
culture vessels increased during the dark periods and decreased
during the light periods because of plantlet respiration and photo-
synthesis. Under CO2 -enriched conditions in the vessels, the CO2
levels were never lower than in plantlets performing photosyn-
thesis under light-saturated conditions. It has been reported that
the increased growth of plantlets is a consequence of the increased
photosynthetic rate of the plantlet due to the control of environ-
mental conditions during in vitro culture, which also affects plantlet
growth and survival during ex vitro acclimatization (Solarova and
Pospisilova, 1997; Hahn and Paek, 2001; Xiao et al., 2011).
Several studies have indicated that photoautotrophic growth in
cultured plantlets can be significantly promoted by increasing the
CO2 concentration and the light intensity as well as by removing
growth retardant gases, such as ethylene from the culture vessel
(Lian et al., 2002; Xiao et al., 2011; Yoon et al., 2009). Furthermore,
in our previous report (Shin et al., 2013), the growth of Doritaenopsis
was greatest in plantlets grown under CO2 -enriched photoau-
totrophic and photomixotrophic conditions with high PPFD. In that
observation, the starch, reducing sugar and non-reducing sugar
levels were higher in Doritaenopsis plantlets grown under photoau-
totrophic or photomixotrophic conditions versus heterotrophic
conditions (Shin et al., 2013). The net photosynthetic rate was also
higher under the former two conditions, and the plants exhibited
better growth as well.
3.2. Photosynthesis characteristics
After 20 and 40 days of ex vitro transfer, plantlets from the
MHC and AHC groups exhibited higher photosynthetic rates than
those of the other treatment groups. Moreover, plantlets grown
under photoautotrophic or photomixotrophic conditions without
CO2 enrichment recovered their photosynthetic ability within 20
days after ex vitro transfer, whereas no such recovery was observed
in plantlets grown under heterotrophic conditions (Table 2). These
results indicate that during in vitro culture, the former plantlets
never lost their photosynthetic ability, and, thus, their growth rate
was higher during acclimatization than that of plantlets grown
under heterotrophic conditions. During heterotrophic growth, the
 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33 29

Fig. 1. Doritaenopsis hybrid plantlets (A) and fresh and dry weights of leaves and roots of plants grown in a greenhouse for 40 days. Plantlets were transferred ex vitro after
10 weeks of culture under photoautotrophic, photomixotrophic and heterotrophic conditions. L: low PPFD (40 ± 2 ␮mol m−2 s−1 ), H: high PPFD (120 ± 5 ␮mol m−2 s−1 ), LC:
low PPFD + CO2 enrichment (1500 ± 100 ␮mol mol−1 ), HC: high PPFD + CO2 enrichment (1500 ± 100 ␮mol mol−1 ).

plantlets were developed in the culture vessels under low levels poor growth and mortality of the plantlets (Xiao et al., 2011). Fur-
of light and the medium containing ample sugar and nutrients, thermore, Chakrabarty et al. (2005) suggested that the substantial
but during their transfer to ex vitro conditions, these plantlets had reduction in photosynthesis that occurs under heterotrophic con-
to adjust from heterotrophic to autotrophic growth, resulting in ditions largely results from a severe disruption of the photosystem

Table 2
CO2 uptake of Doritaenopsis hybrid plantlets in a greenhouse, as affected by three different culture conditions previously applied in vitro.

Days after transplanting (days) CO2 uptake (␮mol CO2 m−2 s−1 ) LSD (p < 0.05)

Photoautotrophic Photomixotrophic Heterotrophic

AL AH ALC AHC ML MH MLC MHC HL HH

20 1.25 0.83 3.23 2.23 1.04 1.24 2.86 2.60 0.78 1.24 0.43
40 2.07 2.47 2.73 2.53 1.90 2.27 2.63 2.58 1.73 1.89 0.40

Net CO2 exchange rate was measured at 1–3 AM during 40 days of growth in a greenhouse. Plantlets were transferred to a greenhouse after 10 weeks of in vitro culture under
the following conditions: L: low PPF (40 ± 2 ␮mol m−2 s−1 ), H: high PPF (120 ± 5 ␮mol m−2 s−1 ), LC: low PPF + CO2 enrichment (1500 ± 100 ␮mol mol−1 ), HC: high PPF + CO2
enrichment (1500 ± 100 ␮mol mol−1 ).
30 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33
Fig. 2. Changes in Fv/Fm, F0 and Fm values during acclimatization of Doritaenopsis hybrid plantlets during 40 days of growth in a greenhouse. Plantlets were transferred to
a greenhouse after 10 weeks of culture. The vertical bars represent the mean ± standard error of three replicate vessels.
(or at least of PSII) and is most likely not the result of any major
effects on other aspects of photosynthesis. Therefore, any treat-
ment before ex vitro transfer (in this case, CO2 enrichment and
high PPFD) that increases the photosynthetic capacity of plants may
further improve plant establishment (Kozai and Iwanami, 1988;
Kitaya et al., 1995; Van Huylenbroeck et al., 2000; Hahn and Paek,
2001).
Chlorophyll fluorescence (Fv/Fm) reflects the maximal effi-
ciency of excitation energy captured by open PSII reaction centers;
a decrease in this parameter indicates down-regulation of pho-
tosynthesis or photoinhibition (Maxwell and Johnson, 2000). In
the current study, the value of Fv/Fm was not critically different
between the photoautotrophic and photomixotrophic condition
groups (Fig. 2). AHC and MHC displayed only small changes
in this value and maintained steady photosynthetic efficiency
within the 0.77–0.8 range. Similarly, no significant changes were
observed in other chlorophyll fluorescence parameters, including
the PS II photochemical reaction (ФPS II), photochemical quench-
ing (qP ) and non-photochemical quenching (qN ), after 40 days
of ex vitro acclimatization (Fig. 3). This indicates that the pho-
tosynthetic efficiency and apparatus were not damaged in any
treatment group. The results demonstrate that in vitro-developed
leaves of Doritaenopsis are photosynthetically competent and dis-
played photosynthetic activity during the acclimatization phase.
However, the steady rate of photosynthetic efficiency observed
from the beginning of ex vitro acclimatization in AHC and MHC
again support the notion that changing the in vitro environ-
mental conditions improves the ex vitro survival and growth of
plantlets.
3.3. Carbohydrates and enzymes
The sugar concentration in leaves and roots during ex vitro
acclimatization through day 40 varied with the in vitro cultural
treatments. The starch and sugar contents were the highest in
plantlets grown under AHC and MHC conditions (Figs. 4 and 5). In
general, the starch and total sugar contents were high in photoau-
totrophically and photomixotrophically grown plants compared
with heterotrophically grown plants (Figs. 4 and 5). These vari-
ations were obvious because the NPR rates were very high in
the photoautotrophically and photomixotrophically grown plants.
Under photoautotrophic conditions, the carbon requirement was
fulfilled by photosynthesis, and high PPFD and CO2 enrichment may
have helped the plants by promoting transient sucrose storage in
the roots. In Spathiphyllum ‘petite’ plantlets during acclimatization,
Van Huylenbroeck and Riek (1995) detected strong increases in all
carbohydrate pools at the end of acclimatization; these increases
were related to enzymatic sucrose metabolism. However, in the
current study, we found that plantlets grown under heterotrophic
conditions in vitro had decreased pools of all carbohydrates except
starch after 40 days of ex vitro acclimatization. The increased con-
tent of starch observed at the later stage of the acclimatization
process indicates that the photosynthetic efficiency of these plants
started to recover approximately 30 days after ex vitro transfer
(Fig. 4). This observation can also be correlated with the observed
recovery in PS II system efficiency (Fv/Fm) 30 days after ex vitro
transfer (Fig. 2).
We examined the time course of PEP-carboxylase activity
in different culture treatment groups during the acclimatization
 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33 31

Fig. 3. Changes in photochemical quenching parameters ФPSII, qN and qP during acclimatization of Doritaenopsis hybrid plantlets during 40 days of growth in a greenhouse.
Plantlets were transferred to a greenhouse after 10 weeks of culture. The vertical bars represent the mean ± standard error of three replicate vessels.

Fig. 4. Changes in starch contents of leaves (A, B, C) and roots (D, E, F) of Doritaenopsis hybrid plantlets during acclimatization during 40 days of growth in a greenhouse.
Plantlets were transferred to a greenhouse after in vitro culture. The vertical bars represent the mean ± standard error of three replicate vessels.
32 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33

Fig. 5. Changes in total sugar contents of leaves (A, B, C) and roots (D, E, F) in Doritaenopsis hybrid plantlets during acclimatization during 40 days of growth in a greenhouse.
The vertical bars represent mean ± standard error of three replicate vessels.

process. The activation of PEP-carboxylase decreased during the
early phases of acclimatization under photoautotrophic and pho-
tomixotrophic conditions, and it increased gradually after 15 days
of acclimatization (Fig. 6). The early decrease in PEPC activity
observed in these groups indicates that enriched CO2 with high
PPFD during in vitro culture produces enough reserved carbohy-
drate (as shown in Figs. 4 and 5) to supplement plant growth during
the acclimatization period. In addition, the high net CO2 uptake (AH
and MH after 20 days), coupled with high PEPC activity, suggest that
PEPC mediates CO2 uptake in plants grown under elevated levels

Fig. 6. Changes in ADGP pyrophosphorylase and PEP-carboxylase activities during acclimatization of Doritaenopsis hybrid plantlets during 40 days of growth in a greenhouse.
Plantlets were transferred to a greenhouse after 10 weeks of culture. The vertical bars represent the mean ± standard error of three replicate vessels.
 K.-S. Shin et al. / Environmental and Experimental Botany 100 (2014) 26–33
of CO2 and sugar-deprived conditions, as suggested by Yoon et al.
(2009). In contrast, the PEPC activity did not increase after 15 days of
acclimatization in heterotrophically treated plantlets. It is possible
that the decrease in photosynthetic rate caused by environmental
stress (which is quite common during the normal ex vitro acclima-
tization process) during the early phases of acclimatization also
caused the decrease in activation (Xiao et al., 2011).
A recovery of SPS activity was observed during the later stages of
acclimatization under all conditions, whereas SS activity declined
in plantlets that were grown under heterotrophic conditions in vitro
(Fig. 6). The sucrose content decreased in these plantlets in a similar
manner during the later stages of acclimatization. Sucrose synthe-
sis in higher plants occurs through the action of SPS and SS. All of
the growth parameters in plantlets grown under heterotrophic con-
ditions were low under subsequent ex vitro conditions, indicating
that these plants had low metabolic activity during acclimatiza-
tion. Thus, low SS activity may be due to the slower growth of
these plants, resulting in low carbohydrate content in their leaves,
whereas the SPS activity may recover after transfer to ex vitro condi-
tions, which was the same for all plantlets. SS is rapidly degraded as
the photosynthetic machinery develops (Nguyen-Quoc et al., 1990).
In heterotrophically grown plants, after a certain period of acclima-
tization, plants convert from heterotrophy to autotrophy, which
may involve changes in the photosynthetic machinery.
4. Conclusions
The results of this study demonstrate that in vitro condi-
tions with CO2 enrichment and high PPFD can improve the
acclimatization of in vitro-grown Doritaenopsis plantlets to ex vitro
conditions. Photoautotrophically and photomixotrophically plants
grown in vitro exhibited the best growth in the ex vitro environ-
ment, which may be a consequence of the presence of higher
sugar reserves in these plants, as well the early recovery of PEP-
carboxylase activity.
Acknowledgment
This research was supported by grants from the National
Academy of Agricultural Science (PJ00863404), Rural Development
Administration, Republic of Korea.
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