Plant Science 160 2000 139 – 147
Cytokinin-induced somatic embryogenesis and plant regeneration in Corydalis yanhusuo Fumariaceae — a medicinal plant
A.P. Sagare
a
, Y.L. Lee
a
, T.C. Lin
b
, C.C. Chen
b
, H.S. Tsay
a,
a
Department of Agronomy, Taiwan Agricultural Research Institute,
189
, Chung-Cheng Road, Wufeng, Taichung
41301
, Taiwan
b
Institute of Chinese Pharmaceutical Science, China Medical College, Taichung
40421
, Taiwan Received 30 May 2000; received in revised form 7 August 2000; accepted 4 September 2000
Abstract
An efficient method has been developed for regeneration of complete plants via somatic embryogenesis in Corydalis yanhusuo Fumariaceae, an important medicinal plant, using tuber-derived callus. Primary callus was induced by culturing mature tuber
pieces on Murashige and Skoog’s MS medium supplemented with 2.0 mg l
− 1
N
6
-benzyladenine BA and 0.5 mg l
− 1
a -naphthaleneacetic acid NAA in darkness. Somatic embryos were induced by subculturing the primary callus on MS medium
supplemented with 0.5 – 4.0 mg l
− 1
BA, kinetin, or zeatin, within 2 weeks of culture in light. Embryos with well-developed cotyledonary leaves were transferred in half-strength liquid MS medium supplemented with 1.0 mg l
− 1
zeatin riboside for the development of roots. Converted somatic embryos were cultured on half-strength MS medium supplemented with 6 sucrose, and
with 0.5 – 10.0 mg l
− 1
abscisic acid ABA, paclobutrazol, or ancymidol, 0.5 – 5.0 mg l
− 1
GA
3
and 15 – 100 mg l
− 1
polyethylene glycol PEG 4000 for further development of plantlets and in vitro tuber formation. The development of somatic embryos over
the surface of tuber andor cotyledonary leaf base region of the converted primary somatic embryo was observed. Before ex vitro establishment of somatic embryo-derived plants, plants with well-developed tubers were cultured on half-strength MS medium
with 2 sucrose and 0.1 mg l
− 1
GA
3
for 3 weeks. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
ABA; Corydalis yanhusuo; Growth retardant; Medicinal plant; Papaveraceae; PEG-4000; Somatic embryogenesis www.elsevier.comlocateplantsci
1. Introduction
The genus Corydalis Fumariaceae or Papaver- aceae of about 320 species is widely distributed in
the Northern hemisphere and 70 species have been used in traditional herbal remedies in China,
Japan, and Korea [1]. Corydalis yanhusuo syn. Corydalis turtschanino6ii f. yanhusuo, a perennial
herb up to 20 cm tall [2], is one of the medicinally important species of Corydalis [1]. The dried and
pulverized tubers of C. yanhusuo, also called as Rhizoma Corydalis or yan-hu-suo, [3], are used in
traditional Chinese medicine in the treatment of gastric and duodenal ulcer, cardiac arrhythmia
disease [1], rheumatism and dysmenorrhea [3]. The tuber contains several alkaloids; some of the im-
portant alkaloids are
DL
-tetrahydropalmatine,
D
- corydaline, and corydalis H, I, J, K, and L [4]. The
alkaloids exhibited analgesic [5], anti-arrhythmic [6], anti-thrombic, [7], anti-inflammatory [8], anti-
cataract [9], anti-hypertensive [10] and anti-allergic [11] activity.
C. yanhusuo is cultivated mainly in Zhejiang, Jiangxi, Anhui and Hubei provinces of China as
an annual crop using tubers and the tubers are exported to other countries. C. yanhusuo is suscep-
tible to fungal diseases [2], especially downey
Abbre6iations
:
ABA, abscisic acid; Ancymidol or Anc, a-cyclo- propyl-a-4-methoxy-phenyl-5-pyrimidine methanol; BA, N
6
-benzy- ladenine; GA
3
, gibberellic acid; kinetin, 6-furfuryl amino purine; MS, Murashige and Skoog 1962; NAA, a-naphthaleneacetic acid; Pa-
clobutrazol or Pac, 2RS, 3RS-1-4-chlorophenyl-4,4-dimethyl- 21,2,4-triazol-1-ylpentan-3-ol; PEG-4000, polyethylene glycol-4000;
zeatin, 6-4-hydroxy-3-methyl-2-butenylamino purine. Corresponding author. Tel.: + 886-4-3302301, ext. 108; fax: +
886-4-3338162. E-mail address
:
hstsaywufeng.tari.gov.tw H.S. Tsay. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 7 7 - 0
mildew caused by Peronospora corydalis de Bary, which infect tubers and is responsible for 30 –
50 loss in yield when conditions are conducive for fungal growth [12]. To improve the produc-
tivity, homogeneity and quality of the tubers, it is necessary to have pathogen-free planting mate-
rial. Pathogen-free plants of C. yanhusuo could be obtained using seeds; however, the seeds have
a low percentage of germination [13] and take a long time to germinate due to extremely imma-
ture state of zygotic embryo at the time of dis- persal.
Both warm
and cold
stratification treatments are required to induce germination
from seeds [13]. Furthermore, the growth of the seedlings is very slow during the first year of
growth forming only a small, immature tuber. Mature tubers could be obtained only in the suc-
ceeding year of growth after a period of dor- mancy [13]. Plant regeneration via in vitro
culture of C. yanhusuo would be useful for mass propagation of this important medicinal plant in
a short time.
Somatic embryogenesis is the most common mode of regeneration for species in the Papaver-
aceae [14 – 23]. The genus Corydalis has shown morphogenetic potential [24,25]; to date, how-
ever, there has been no report on the complete plant regeneration of C. yanhusuo via somatic
embryogenesis. We report here a method of plant production via somatic embryogenesis from
tuber-derived callus of C. yanhusuo.
2. Materials and methods
