Botanical Studies (2006) 47: 259-266.
*
Corresponding author: Email: hstsay@cyut.edu.tw; Tel:
+886-4-2330-4920; Fax: +886-4-2330-4921.
INTRODUCTION
Scrophularia yoshimurae Yamazaki (Family -
Scrophulariaceae) is a perennial herb, native to Taiwan.
In traditional Chinese medicine in Taiwan, it is called
"Xuanshen", a substitute for Scrophularia ningpoensis
(Chiu and Chang, 1998). The species is used for treatment
of inflammation, laryngitis, tonsillitis, abscesses of
carbuncles (Reid, 1996), and constipation. It can lower
blood pressure and blood sugar levels and also has
antibacterial and anti-oxidant properties. Small doses
of it have been reported to be cardiotonic (Reid, 1996).
Populations of S. yoshimurae are distributed in the
central mountain range of Taiwan, and they are adapted
to a narrow set of environmental conditions (Liu, 1998).
Hence, it is very difficult to locate plants in the wild.
In Taiwan, the processed roots of S. ningpoensis are
imported from China and used as a crude drug since S.
yoshimurae is not cultivated on a commercial scale, and
roots collected from natural habitat are insufficient to meet
the local demand. Hence it became imperative to search
for alternative propagation methods. In vitro culture
techniques have been used successfully for propagation
of many medicinally important plant species (Tsay, 1992;
1999; Nalawade et al., 2003; Mulabagal et al., 2004).
To maintain the sterility of in vitro cultures, it is
essential to cover culture vessels with ventilation closures
(sealing). Different types of ventilation closures are
commonly used. They sometimes cause restriction of
gaseous exchange between the vessel atmosphere and
outside environment (Buddendorf-Joosten and Woltering,
1994) and result in poor aeration and hyperhydric culture
conditions. Hyperhydricity is a morphological, anatomical,
and physiological abnormality very often observed in
micro-propagated plants (Kevers et al., 1984; Debergh
et al., 1992). Anatomical features of the leaf surfaces in
tobacco and cauliflower have been reported to be affected
PHYSIOLOGY
Influence of ventilation closures on plant growth
parameters, acclimation and anatomy of leaf surface in
Scrophularia yoshimurae Yamazaki - a medicinal plant
native to Taiwan
Uei-Chern CHEN
1
,
Chi-Ni HSIA
1
, Dinesh Chandra AGRAWAL
2
, and Hsin-Sheng TSAY
2,
*
1
Agronomy Division, Agricultural Research Institute, Wufong, Taichung 413, TAIWAN
2
Graduate Institute of Biotechnology, Chaoyang University of Technology, Wufong, Taichung 413, TAIWAN
(Received November 16, 2005; Accepted February 16, 2006)
ABSTRACT.
Different ventilation closures, including aluminum foil (AF) and a varying number of dispense
papers (DP) had different effects on leaf growth parameters, in vitro rooting, survival rate and the anatomical
features of the leaf surface of in vitro and ex vitro acclimated plants of Scrophularia yoshimurae¡Xan
important medicinal plant. The lowest plant growth parameters and ex vitro acclimation rate (<7.0%) were
obtained using AF as ventilation closure. A scanning electron microscopy (SEM) study of leaf surfaces of
plants derived from different ventilation closure treatments showed that parameters¡Xincluding density and
size of epidermal cell and stomata, size of guard cells, and stomata aperture-differed significantly among
various treatments, and this in turn affected plant survival rate. Leaves derived from AF treatment had
higher epidermal cell (15094 cells/mm
2
) and stomata (38/mm
2
) densities than DP treatments. Well-ventilated
container closures, such as with DP, improved the morphological characteristics of leaves and in turn
enhanced the survival rate during ex vitro acclimation (maximum rate being 66.7%). The present study not
only provides an improved micropropagation method of S. yoshimurae but also gives scientific reasons for the
different acclimation rates obtained with various container closures.
Keywords: Hyperhydricity; In vitro culture; Scanning electron microscopy; Scrophularia yoshimurae;
Stomata; Ventilation closure.
Abbreviations: A F, aluminum foil; BA, benzyladenine; D P, dispense paper; MS, Murashige & Skoog¡¦s
medium; NAA, £\-naphthaleneacetic acid; SEM, scanning electron microscopy.
pg_0002
260
Botanical Studies, Vol. 47, 2006
by the type of ventilation closure used and were linked to
hyperhydricity (Zobayed et al., 1999a, 1999b, 2001).
A method of de novo regeneration of S. yoshimurae has
been established in our laboratory (Sagare et al., 2001);
however, the high frequency of hyperhydric shoots was a
major problem. Hence, the aim of the present study was
to evaluate the influence of different ventilation closures
on the growth parameters of leaf, root, and plant survival
rate and also to carry out a scanning electron microscopy
(SEM) study of the leaf surfaces of S. yoshimurae plants
derived from these different ventilation closures. The
results in the present study should be of immense help
in understanding the underlying scientific principles
behind different rates of acclimation achieved with
different container closures and also boost the commercial
production and conservation of Scrophularia yoshimurae.
MATERIALS AND METHODS
In vitro culture establishment and culture
conditions
In vitro shoot multiplication in S. yoshimurae was
achieved by culturing shoot-tip explants on MS basal
medium (Murashige and Skoog, 1962) supplemented with
BA (1.0 mg l
-1
) + NAA (0.2 mg l
-1
) in a 500-ml glass flask
(TAIWANGLASS, Taiwan) containing 100 ml medium
as reported earlier (Sagare et al., 2001). The pH of all
media was adjusted to 5.7 ¡Ó 0.1 with 1 N NaOH before
autoclaving at 121¢XC, 105 kPa for 15 min. Culture vessels
were capped with two layers of aluminum foil (AF)
(Reynolds Consumer Products, Alcoa Inc., Richmond,
Virginia, USA) before autoclaving. After inoculation,
culture vessels were capped with two layers of aluminum
foil (hereafter referred as AF) or 2, 3, or 4 layers of
dispense paper [9.5 ¡Ñ 9.5 cm, 0.046 mm thick, gas flow
0.5 mls
-1
, made from soft- and hard-wood fiber (50:50),
Cheng Long Corporation, Taiwan] as ventilation closure
(hereafter referred as DP2, DP3, and DP4, respectively).
All cultures were incubated for 8 weeks at 25 ¡Ó 1¢XC under
cool white fluorescent light at 38 £gmol m
-2
s
-1
(Philips,
Holland) with a 16-h photoperiod per day.
Influence of ventilation closure on growth
parameters of leaf and root
For rooting, in vitro shoots derived from DP3 treatment
were cultured on MS basal medium devoid of growth
regulators (GR). Culture vessels were capped either with
two layers of aluminum foil or 2, 3, or 4 layers of dispense
papers as described in the previous section and incubated
for 8 weeks. Each treatment had eight explants with four
replicates.
Influence of ventilation closure on ex vitro
acclimation rate
In vitro plants after pre-trimming (about 4-cm-long
shoot and 3-cm-long root) were immersed in 1000X dilute
50% Benlate (Dupon, USA) solution for 1 h. After that,
they were transplanted into a seedling pot (56 ¡Ñ 34 ¡Ñ 4 cm,
60 wells, each well of 5 cm in diameter ¡Ñ 4 cm in depth)
containing an autoclaved mixture of BioMix:vermiculite:
perlite (1 : 1 : 1) ratio. The seedling pot was placed inside
a transparent polycarbonate (PC) box (56 ¡Ñ 34 ¡Ñ 9 cm)
with a cover (56 ¡Ñ 34 ¡Ñ 9 cm) to maintain high relative
humidity. The PC box was kept inside a growth chamber
(Hotech, Model 624 HD, Taiwan) with a light intensity
of 100 £gmol m
-2
s
-1
and a 14-h photoperiod at a 22/18¢XC
day/night temperature for 4 weeks. After that, the PC box
was taken from the growth chamber and kept outside at
an ambient room temperature for 4 weeks. Each treatment
had six rooted plantlets in three replicates.
Scanning Electron Microscopy (SEM) study
of leaf surfaces of in vitro plants and ex vitro
acclimated plants
For SEM study, 8-week-old (4 weeks in growth
chamber and 4 weeks under room condition), ex vitro
acclimated plants and 4-week-old in vitro plants derived
from different ventilation closures treatments were used.
Leaves from the same position in the plant (2
nd
or 3
rd
pair from apex) of the same approximately size were
sampled and frozen in liquid nitrogen immediately. SEM
(JEOL-JSM-6330F, Japan) examination of both abaxial
and adaxial surface of leaves was carried out. For each
treatment, 30 epidermal cells and ten stomata in four
replicates were recorded.
Statistical analysis
Quantitative data were first analyzed by ANOVA (SAS
R
Inc., 2001). For those treatments where ANOVA showed
significance, (P<0.05), the least significant difference
(LSD) test was used to compare means.
RESULTS
Influence of ventilation closure on growth
parameters of leaf and root
In vitro plants grown under ventilation closure
treatments DP2 and DP3 had larger leaves than plants
derived from AF and DP4 treatments. Plants in AF
treatment developed the smallest leaves (Table 1).
In vitro shoots of S. yoshimurae cultured on MS basal
medium without growth regulators induced 100% rooting
in all ventilation closure treatments (data not shown).
However, number and length of roots varied among
the treatments. The maximum number of roots (2.93
per shoot) and the shortest root length (6.81 cm) were
observed in AF treatment (Table 1). Among the three DP
treatments, number and length of roots showed marginal
differences.
Influence of ventilation closure on ex vitro
acclimation rate
In vitro plants derived from different ventilation closure
pg_0003
CHEN et al. ¡X
A medicinal plant native to Taiwan
261
treatments showed varying survival percentages at 4 and 8
weeks of observation (Figure 1). The highest survival rate
(77.8%) was obtained with DP3 treatment after 4 weeks
of acclimation in the growth chamber. This percentage
decreased to 66.7% when plants were shifted to room
conditions. AF resulted in the lowest survival (27.8%)
which further decreased to < 7% on shifting plants to
ambient conditions. Among the three DP treatments, DP4
showed the poorest survival rate, but it still exceeded AF
(Figure 1).
Figure 2A shows the in vitro plantlets of S. yoshimurae
under the DP3 treatment after 8 weeks of incubation.
Plants derived from DP3 and DP4 treatments had better
growth than AF and DP2 treated ones (Figure 2B). In
vitro plants transferred to the PC box became established
(Figure 2C) when PC box was kept inside a growth
chamber for 4 weeks and on its subsequent transfer to
9-inch pot under outdoor conditions (Figure 2D).
Scanning Electron Microscopy (SEM) study
of leaf surfaces of in vitro plants and ex vitro
acclimated plants
AF as ventilation closure affected the maximum density
of epidermal cells (15094 cells/mm
2
) on the adaxial
surface of leaves (Table 2). However, cell dimensions in
AF were the lowest of all the DP treatments. We observed
a direct correlation between ventilation closure and
epidermal cell density. Higher ventilation resulted in lower
density. Among the four ventilation closure treatments, the
minimum cell density (640 cells/mm
2
) was observed with
DP2. However, a reverse trend was observed with respect
to cell dimensions. Among all the ventilation closure
treatments, the maximum cell dimensions were obtained
with DP2 (Table 2).
As with epidermal cells, a more or less identical
trend was observed for stomata density and dimensions
(Table 2). All four ventilation closure treatments and ex
vitro acclimated plants showed a marked difference in
the number of open and closed stomata. The maximum
amount of open stomata (33/mm
2
) was recorded under AF
treatment. Again, the reverse was true for the number of
closed stomata. The highest density of closed stomata (21/
mm
2
) was observed in leaves of ex vitro acclimated plants.
The sizes of guard cells and stomatal apertures were also
influenced by the type of ventilation closure treatment in
leaves of S. yoshimurae. The largest guard cells (0.27 £gm)
and maximum aperture (0.08 £gm ) were recorded in the AF
treatment (Table 2). The SEM examination of leaf surface
of S. yoshimurae plants derived from different ventilation
closure treatments is shown in Figure 3A-F. Abaxial
surface of leaves derived from DP as ventilation closure
showed both open and closed stomata (Figure 3C-3E)
while all the stomata were open on both the adaxial
(Figure 3A) and abaxial (Figure 3B) surfaces of leaves
under AF treatment. In leaves from ex vitro acclimated
plants, all the stomata were found closed (Figure 3F).
Table 1. Influence of container closure type on leaf growth and in vitro rooting in S. yoshimurae
x
.
Container closures Layers
Leaf dimension (cm)
y
Rooting
y
length
width
Number
Length (cm)
Aluminum foil
2
1.52 ¡Ó 0.15
c
0.92 ¡Ó 0.10
b
2.93 ¡Ó 0.41
a
6.81 ¡Ó 2.67
c
Dispense paper
2
1.92 ¡Ó 0.09
a
1.36 ¡Ó 0.07
a
2.67 ¡Ó 0.18
ab
9.46 ¡Ó 0.15
a
Dispense paper
3
1.90 ¡Ó 0.17
a
1.30 ¡Ó 0.10
a
2.07 ¡Ó 0.47
c
8.62 ¡Ó 0.02
b
Dispense paper
4
1.78 ¡Ó 0.09
b
1.10 ¡Ó 0.05
b
2.47 ¡Ó 0.13
b
8.84 ¡Ó 0.48
b
Means ¡Ó standard error followed by the same letter/s are not significantly different at 5% level by LSD test.
x
In vitro shoots cultured on MS basal medium with BA 1 (mg/l) + NAA (0.2 mg/l) for 8 weeks.
y
Each value is the mean for 15 leaves or 15 roots.
Figure 1. Influence of container closure type on s urvival
rate of ex vitro acclimated plants of S. yos himurae. In vitro
rooted plantlets of S. yoshimurae derived from various closure
treatments were kept in growth chamber for 4 weeks (¡¼) and
there after under room condition for another 4 weeks i.e total 8
weeks (¡½). The same letter/s above columns with same symbol
is not significantly different from each other at the 5% level by
LSD test. Each treatment consisted of 18 plants.
pg_0004
262
Botanical Studies, Vol. 47, 2006
DISCUSSION
Growth rate and many other physiological and
morphological characteristics of plants developed under
in vitro conditions have been reported to be influenced by
the physical and chemical micro-environment of culture
vessels (Walker et al., 1988). In the present study, in vitro
plants grown under higher ventilation closure treatments
had larger leaves than plants derived from lower
ventilation closures. These observations are consistent
with earlier reports on different plant species (Sallanon
and Maziere, 1992; Lai et al., 1998; Zobayed et al., 1999a,
1999b, 2001), which showed that plants under a diffused
ventilation conditions had larger leaf area.
Though different ventilation closure treatments had
no effect on the rooting response of in vitro shoots of
S. yoshimurae cultured on MS basal medium without
growth regulators, growth parameters in terms of number
and length of roots varied among the treatments. This
difference was marginal among the DP treatments. We
have observed similar results with carnation (Chen et al.,
1998) and B. kaoi (Chen et al., 2004a; 2006).
Our results on survival percentages of plants
demonstrate that ventilation closure has a direct bearing on
success in the hardening process and further acclimation
of in vitro plants of S. yoshimurae. In general, DP as a
container closure improved ex vitro acclimation of plants.
Similar observations in other crops have been reported
earlier (Whish et al., 1992; Majada et al., 1998; Chen et
al., 2004b; 2005). In our previous research on carnation,
DP as a container closure resulted in better plant growth
and non-hyperhydric shoots (Chen et al., 1998), B. kaoi
(Chen et al., 2004a, 2006) and S. miltiorriza (Chen et al.,
2005).
SEM studies demonstrate that the type of ventilation
closure affected the anatomical features of leaf surfaces,
Figure 2. A: In vitro rooted shoots of S. yoshimurae under DP2 treatment (Bar=1 cm); B: rooted plantlets under different container
closure treatments (Bar=2 cm); C: plantlets inside transparent PC plastic box with cover, kept inside a growth chamber for 4 weeks
followed by exposure to room conditions for 4 weeks (Bar=5 cm); D: Plant established in pot kept in outdoor conditions for 2 months.
(Bar=2 cm).
pg_0005
CHEN et al. ¡X
A medicinal plant native to Taiwan
263
which in turn influenced the survival rate of in vitro
raised plants. AF or an increased number of DP layers
reduced gaseous exchange between the culture vessel
and the outside environment. Similar observations in
B. kaoi (Chen et al., 2006), cauliflower and tobacco
(Zobayed et al., 2001), D. caryophyllus (Majada et al.,
2000), Delphinium spp. (Santamaria et al., 1993), and
rose (Sallanon et al., 1993) have been reported. Higher
ventilation was shown to decrease stomatal density,
resulting in more functional stomata. The hyperhydric
and/or in vitro plants derived from airtight culture vessels
having a higher number of improperly functioning stomata
have been reported. These disorders result in excessive
water loss and poor photosynthesis during ex vitro
acclimation, which lead to a lower survival rate (Preece
and Sutter, 1991; Ziv, 1991; Sallanon et al., 1993; Jeong
et al., 1995; Majada et al., 1998; Zobayed et al., 1999a,
1999b, 2001; Chen et al., 2006). Our results confirm
earlier findings that normal functioning of stomata in in
vitro plants grown under high relative humidity conditions
(AF as ventilation closure in the present study) is disrupted
(Sallanon et al., 1993; Santamaria et al., 1993; Cassells
and Walsh, 1994; Majada et al., 1998; 2000) and adversely
affects survival rate.
Culture vessels can be considered tiny greenhouses.
Micro-environmental conditions in culture vessels can be
controlled by techniques similar to greenhouse control
such as enhancing natural/forced ventilation. Controlled
micropropagation systems, especially under forced
ventilation have been shown to produce morphologically
superior as well as physiologically normal plants (Zobayed
et al., 1999a, 1999b, 2001). The head space of vessels
with low ventilation accumulates components including
ethylene, CO
2
, acetaldehyde, and ethanol (Zobayed et al.,
1999a, 1999b, 2001). These micro-environment changes
in the vessels are a result of altered anatomical features
in leaves as evident from the present study. Under a low
ventilation closure like AF in our previous study and
under the higher number of DP in the present study, an
accumulation of ethylene and CO
2
was demonstrated (Lai
et al., 2005). The poor growth and low survival percentage
of plants under AF can be attributed to the inhibitory effect
of ethylene as well as to the non-utilization of CO
2
due
to low density and the high number of non-functioning
stomata (Lai et al., 2005). Further, it could be understood
that improved growth parameters under high ventilation
closures are a function of a balanced CO
2
supply enabling
the plants to benefit from net photosynthetic assimilate
production (Zobayed et al., 1999a, 1999b).
CONCLUSION
Results obtained in the present study clearly
demonstrate that ventilation closure of culture vessels
has a definite influence on plant growth parameters,
anatomical features of leaf and in turn survival rate
of plants raised under in vitro culture conditions. Use
of dispense papers as ventilation closure instead of
pg_0006
264
Botanical Studies, Vol. 47, 2006
commonly used aluminum foil improved not only
plant growth parameters but also the morphological
characteristics of leaves, ex vitro acclimation and survival
of plants.
Acknowledgements. The authors thank Dr. W.Y. Su
Figure 3. SEM examination of adaxial (A) and abaxial (B-F) surfaces of leaves of S. yoshimurae. A, B: In vitro leaves derived from
containers having two layers of aluminum foil (AF); C: two layers of dispense paper (DP); D: three layers of DP; E: four layers of DP;
F: leaf derived from ex vitro acclimated plant. (Bar, A=100 £gm; B-F=10 £gm).
(Department of Pesticide Application, Taiwan Agricultural
Chemicals and Toxic Substances Research Institute,
Taichung, Taiwan) for carrying out SEM, and the Council
of Agriculture (Project No. 94 Agri.-5.2.1-Sci-al.) and
the National Science Council (NSC 94-2313-B324-001)
Taiwan for their financial support.
pg_0007
CHEN et al. ¡X
A medicinal plant native to Taiwan
265
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