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
2
.
1
. Induction of somatic embryogenesis Mature tubers of Corydalis yanhusuo W.T.
Wang syn. Corydalis turtschanino6ii Bess. f. yan- husuo Y.H. Chou et C.C. Hsu were obtained
from Shenyang, Liaoning province, China in November 1995. Tubers were cleaned under run-
ning tap water and surface-disinfected in 70 ethanol for 1 min, followed by 0.5 sodium
hypochlorite Clorox, The Clorox Co., Oakland, CA with two drops of Tween 20
®
per 100 ml Hayashi Pure Chemical Industries Ltd., Osaka,
Japan under ultrasonic vibration Branson Ul- trasonic Cleaner, Branson Cleaning Equipment
Co., Shelton, CT for 10 min and rinsed five times with sterile distilled water. After steriliza-
tion, tubers were cut into 5 × 5 × 2 mm pieces and cultured in 22 × 120 mm glass test tubes,
each containing 10 ml of medium. The medium consisted
of Murashige
and Skoog’s
MS medium [26] MS inorganic salts and vitamins +
100 mg l
− 1
myo-inositol, referred to hereafter as MS basal medium, 3 sucrose, 0.9 Difco
Bacto agar Difco Laboratories, Detroit, MI and 2.0 mg l
− 1
BA in combination with 0.5 mg l
− 1
NAA. The cultures were incubated at 25 9 1°C in darkness for 1 month. The primary callus
produced from tuber pieces was subcultured ev- ery 20 days for three times on the same medium.
The primary callus 200 mg each was trans- ferred on MS basal medium with 3 sucrose,
0.9 Difco Bacto agar and supplemented with 0, 0.5, 1.0, 2.0 and 4.0 mg l
− 1
BA, kinetin or zeatin Sigma Chemical Co., St. Louis, MO
Table 1. Zeatin stock solution was filter-steril- ized 0.22 mm Millipore and added to the
medium after autoclaving. Twenty calli were evaluated per treatment. The pH of the medium
was adjusted to 5.7 9 0.1 with 1 N NaOH or HCl before autoclaving at 121°C, 105 kPa for 15
min. The culture vessels were capped with two layers of aluminum foil before autoclaving and
sealed with three layers of Parafilm M after cul- ture. The cultures were incubated at 25 9 1°C
under
cool white
fluorescent light
at 38
Table 1 Effect of cytokinins on induction of somatic embryogenesis
from tuber-derived primary callus of Corydalis yanhusuo after 5 weeks
a
Cytokinin Average number of somatic embryos
mg l
− 1
produced per callus None
2.9
c
0.5 BA
10.0
ab
1.0 9.0
ab
2.0 4.7
bc
4.0 0.6
c
0.5 Kinetin
7.5
b
1.0 7.5
b
7.6
b
2.0 4.0
12.6
a
0.5 Zeatin
9.0
ab
1.0 6.2
bc
2.0 5.6
bc
4.0 3.8
bc a
Means within a column followed by the same letter are not significantly different from each other at the 5 level as
determined by the LSD test.
m mol·m
− 2
·s
− 1
Philips, Eindhoven, The Nether- lands with a 16-h photoperiod per day for 5
weeks.
2
.
2
. Con6ersion of somatic embryos Somatic embryos with cotyledonary leaves, dif-
ferentiated on the surface of the primary calli on zeatin 1.0 mg l
− 1
containing medium, were trans- ferred along with the undifferentiated calli in half-
strength MS liquid medium half-strength of MS inorganic salts + full complement of vitamins and
inositol supplemented with 3 sucrose and 1.0 mg l
− 1
zeatin riboside Sigma in 250-ml Erlen- meyer flasks with 20 ml medium. The pH of the
medium was adjusted to 5.2 9 0.1 prior to auto- claving. Zeatin riboside stock solution was filter-
sterilized and
added to
the medium
after autoclaving. The flasks were placed on an orbital
shaker Model SK-302A, Sun Kuan Instruments Co., Taichung, Taiwan with rotary motion of 100
rpm, and incubated for 2 weeks at 25 9 1°C under cool
white fluorescent
light at
38 m
mol·m
− 2
·s
− 1
·with a 16-h photoperiod per day.
2
.
3
. Effects of ABA, paclobutrazol, ancymidol, GA
3
, and PEG on de6elopment of con6erted somatic embryos
Converted somatic embryos with well-developed shoots and roots were transferred individually on
half-strength MS medium supplemented with 6 sucrose, 0.9 Difco Bacto agar and i 0.5, 1, 2, 5
or 10 mg l
− 1
ABA, ancymidol or paclobutrazol; ii 0.5, 1, 2 or 5 mg l
− 1
GA
3
; iii 15, 25, 50 or 100 mg l
− 1
PEG-4000 MW 4000 Table 2, in 22 × 120 mm glass test tubes, each containing 10 ml of
medium. Ancymidol Sigma, ABA Sigma, pa- clobutrazol Wako Pure Chemical Industries Ltd.,
Osaka, Japan and GA
3
Sigma stock solutions were filter-sterilized and added to the medium
after autoclaving. The cultures were incubated un- der the conditions described in Section 2.2. Five
converted somatic embryos were evaluated per treatment.
The number of shoots and roots produced per somatic embryo derived plantlet, average length of
shoots and roots, tuber diameter and number of somatic embryos developed was recorded after 1
month of culture. The experiment was repeated three times.
2
.
4
. De6elopment of plantlets Plantlets with well-developed tubers were trans-
ferred to half-strength MS medium supplemented with 2 sucrose, 0.18 Gelrite Sigma and 0.1
mg l
− 1
GA
3
in 250-ml Erlenmeyer flasks with 100 ml medium, and incubated for 3 weeks under the
conditions described in Section 2.2.
2
.
5
. Ex 6itro establishment of plantlets Sixty somatic embryo-derived plants with well-
developed roots, shoots and tubers were thor- oughly washed under tap water for 2 – 3 min to
remove traces of agar-gelled medium sticking to them. Plants were then dipped in 0.1 wv Ben-
late Du Pont De Nemours and Co. Inc., Taoyuan, Taiwan for 1 min and planted in 6-cm
plastic pots containing a mixture of autoclaved sand and peat moss 1:1 by volume. The pots
were kept in the growth chamber Model 624 HD, Hotech Instruments Corp., Taipei, Taiwan under
a light intensity of 100 mmol·m
− 2
·s
− 1
for a 16-h photoperiod per day and daynight temperatures
of 2016°C. The plants were initially covered with polypropylene bags to maintain humidity and irri-
gated once in a week with tap water. After 15 days, the polypropylene bags were removed. The
percentage survival of plants was calculated after 2 months.
2
.
6
. Statistical analysis For statistical analysis, number of somatic em-
bryos, shoot number and length, number of so- matic embryos showing tuber formation and
number of somatic embryos developed over the surface of tuber andor cotyledonary leaf base
region of the converted primary somatic embryo were recorded. Least significant difference LSD
test was used for statistical analysis.
3. Results and discussion
