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.