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
3
.
1
. Callus induction Primary callus cultures of C. yanhusuo were
initiated using mature tuber pieces as explants. When mature tuber pieces were placed on MS
medium supplemented with 2.0 mg l
− 1
BA and 0.5
A .P
. Sagare
et al
. Plant
Science
160 2000
139 –
147
Table 2 Effect of different treatments on somatic embryos of Corydalis yanhusuo after 1 month of culture on MS basal salts medium supplemented with 6 sucrose either alone or
in combination with ABA, paclobutrazol, ancymidol, GA
3
or PEG-4000
a
Number of Treatment mg l
− 1
Length of Length of
Number of Tuber formation
Number of somatic embryos per primary roots
embryo
c
roots mm shoots
shoots mm Diameter
b
mm 26.7
bcd
3.4
cde
100 1.8
bc
0.4
c
6.7 10.8
de
1.5
bcde
None, 6 sucrose
1.1
defghi
9.3
ef
3.4
cde
100 1.6
abc
40 1.7
bc
ABA 14.1
bcd
0.5 3.3
de
80 1.9
abc
60 1.0 1.4
c
10.9
de
0.9
efghi
8.3
ef
3.4
bcde
66.7 1.7
abc
40 10.1
ef
2.0 0.9
efghi
14.9
bcd
1.3
c
6.0
f
5.0 3.2
de
80 3.7
a
73.3 1.3
c
10.2
de
0.7
hi
11.4
ef
10.0 2.8
e
13.3 1.7
abc
40 1.3
c
10.8
de
0.9
efghi
5.1
a
100 1.7
abc
73.3 9.1
fghi
0.9
fghi
Paclobutrazol 13.4
bcd
1.7
bc
0.5 0.6
i
6.0
f
4.6
ab
100 1.9
abc
66.7 1.0 1.7
bc
12.4
cde
3.8
bcde
100 1.9
abc
46.7 5.5
f
2.0 0.6
i
7.7
e
1.2
c
7.9
f
5.0 4.6
ab
60 3.0
ab
33.3 1.4
c
10.1
de
0.8
ghi
1.3
bcdefg
8.2
ef
3.8
bcde
66.7 1.4
bc
33.3 1.2
c
10.0 13.1
bcde
3.4
bcde
100 1.0
bc
26.7 17.2
def
1.3
bcdefg
11.7
cde
Ancymidol 0.5 1.7
bc
12.7
ef
1.0 3.8
bcde
100 0.6
c
20 1.3
c
12.2
cde
1.2
bcdefg
11.6
ef
2.0 4.1
abcd
100 0.0
c
1.5
bc
10.8
de
1.4
bcdef
0.0
f
0.0
c
10.4
ef
1.0
efghi
11.9
cde
1.5
bc
5.0 0.9
efghi
8.6
ef
0.0
f
0.0
c
10.0 1.2
c
10.2
de
4.3
abcd
53.3 0.4
c
6.7 35.3
ab
GA
3
0.5 1.7
abc
18.6
ab
8.2
a
42.0
a
1.0 3.7
bcde
46.7 0.4
c
6.7 7.7
a
16.7
bc
1.5
bcde
1.8
ab
42.9
a
3.9
bcde
66.7 0.4
c
6.7 7.6
a
18.1
ab
2.0 3.6
bcde
60 0.4
c
6.7 35.4
ab
22.7
a
8.6
a
1.6
abcd
5.0 19.9
cde
15 4.5
abc
100 0.5
c
13.3 3.1
b
13.9
bcd
1.4
bcdef
PEG 2.1
a
31.4
abc
3.7
bcde
100 0.9
bc
26.7 25
2.3
bc
11.8
cde
1.5
bcde
26.2
bcd
3.5
bcde
100 0.5
c
26.7 1.7
bc
10.6
de
50 3.9
bcde
87.7 0.3
c
20 28.6
bcd
1.7
bc
100 9.9
de
1.7
abc 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.
b
Data in the parentheses within a column shows percentage of converted somatic embryos forming tuber.
c
Data in parentheses within a column shows percentage of the converted primary somatic embryos showing development of somatic embryos directly over the surface of tuber andor cotyledonary leaf base region.
mg l
− 1
NAA, callus initiation occurred. When the callus was separated from the parent tissue
and transferred to fresh medium, it grew vigor- ously into yellow friable calli and could then be
regularly subcultured at 20-day intervals.
3
.
2
. Induction and de6elopment of somatic embryos
Various concentrations
of cytokinins
BA, kinetin or zeatin were tested for their potential
to induce either caulogenesis or somatic embryo- genesis in C. yanhusuo using tuber-derived pri-
mary callus. The response is summarized in Table 1. All the three cytokinins stimulated so-
matic embryo formation including the basal medium consisting of MS inorganic salts and vi-
tamins, 100 mg l
− 1
inositol and 3 sucrose, but the efficiency varied. Somatic embryo induction
usually involves the use of auxins [27], mostly 2,4-D, with or without a low level of cytokinin;
however, initiation
of somatic
embryos on
medium containing cytokinin as the sole growth regulator has also been reported in few species
such as Trifolium [28], Coffea [29], Gladiolus [30], Helianthus [31], Spinacia [32] and Medicago [33].
The development of somatic embryos occurred on the surface of the tuber-derived primary cal-
lus. The highest number of somatic embryos was obtained on medium supplemented with either
4.0 mg l
− 1
kinetin, 0.5 – 1.0 mg l
− 1
BA, or 0.5 mg l
− 1
zeatin, however, the embryos induced on BA and kinetin reverted to callus. The emergence
of somatic embryos Fig. 1A, arrows occurred after 15 days of culture on 1.0 mg l
− 1
zeatin containing medium. The embryos progressed
through the globular, late-globular, heart, early cotyledonary and cotyledonary stages Fig. 1B.
Cotyledonary-stage somatic
embryos were
formed after 21 days of culture of primary calli on 1.0 mg l
− 1
zeatin containing medium Fig. 1C. After 5 weeks of culture, somatic embryos
showed development of cotyledonary leaves Fig. 1D, however, root development was arrested.
3
.
3
. Con6ersion of somatic embryos Roots developed readily after transfer of em-
bryos along with calli to half-strength liquid MS medium supplemented with 1.0 mg l
− 1
zeatin ri- boside. In liquid medium, the embryos could eas-
ily be separated without any damage, from the surrounding callus tissue. Somatic embryos 96
converted to form well-developed roots and shoots Fig. 1E. Over 90 of the converted so-
matic embryos showed development of only one cotyledon into a cotyledonary leaf. A few species
of the genus Corydalis, although being a dicot, showed a single cotyledon in the mature seeds
[34]. The presence of a single cotyledon in seed- derived plantlets of C. yanhusuo has been re-
ported recently [13].
3
.
4
. Effects of ABA, paclobutrazol, ancymidol, GA
3
and PEG-
4000
on de6elopment of con6erted somatic embryos
In a preliminary experiment, none of the 30 converted somatic embryos survived when trans-
planted to sand:peat moss, immediately after conversion in liquid medium containing 1.0 mg
l
− 1
zeatin riboside. To promote development of roots, shoots and in vitro tuberization, converted
somatic embryos were cultured on medium sup- plemented with 6 sucrose and different levels of
either ABA, growth retardants paclobutrazol, ancymidol, PEG-4000, or GA
3
Table 2. An 8 sucrose concentration [35] and plant growth re-
tardants [36,37] have been used for in vitro mi- crotuber formation in potato. ABA and PEG as
an osmoticum promoted accumulation of storage reserves in somatic embryos of white spruce [38].
In our studies with C. yanhusuo, converted so- matic embryos cultured on 0.5 – 5.0 mg l
− 1
ABA, 0.5 – 10.0 mg l
− 1
paclobutrazol, 0.5 – 2.0 mg l
− 1
ancymidol and 15 – 100 mg l
− 1
PEG-4000 con- taining medium showed tuber formation Table
2. However, embryos cultured on ABA, ancymi- dol, paclobutrazol and PEG-4000 did not show
development of new shoots and the primary shoot wilted. Medium with 6 sucrose alone
promoted tuber, root, and shoot development Fig. 1F. The tubers were well developed on
medium containing 0.5 mg l
− 1
ABA and 0.5 mg l
− 1
paclobutrazol and differed in their morphol- ogy Fig. 1G and H. Embryos cultured on 0.5 –
5.0 mg l
− 1
GA
3
containing medium produced around eight new shoots and thin, long roots.
Fig. 1I shows a converted somatic embryo after 1 month of culture on medium with 5.0 mg l
− 1
GA
3
.
Fig. 1.
3
.
5
. De6elopment of somatic embryos o6er the surface of tuber andor cotyledonary leaf base
region of the con6erted primary somatic embryo Somatic embryos developed directly over the
surface of tuber andor cotyledonary leaf base region of the primary somatic embryo after 1
month in culture on different treatments Table 2. The maximum number 3.7 of somatic embryos
per primary embryo was obtained on medium with 5.0 mg l
− 1
ABA. Somatic embryos developed on the surface of tuber showed non-synchronous de-
velopment Fig. 1J and embryos were observed at globular g, heart h and cotyledonary stage c
of development. In contrast, somatic embryo de- velopment on the basal region of the cotyledonary
leaf was more synchronous Fig. 1K. Primary embryos cultured on either 0.5 – 5.0 mg l
− 1
GA
3
,
2 – 10 mg l
− 1
ancymidol, or growth regulator-free medium did not support development of somatic
embryos on either tuber or cotyledonary leaf base region. Induction of somatic embryos on the
cotyledons andor hypocotyl region of previously differentiated somatic embryos has been reported
earlier in other plant species [39] and references cited therein. The somatic embryos developed
over the surface of tuber and cotyledonary leaf base region of the primary somatic embryo devel-
oped normally and could be converted into plantlets data not shown.
3
.
6
. Ex 6itro establishment of plantlets To prevent dormancy and promote development
of new shoots, plantlets with well-developed tu- bers, formed after culture on 6 sucrose contain-
ing medium without growth regulators control treatment, Table 2, were cultured on a medium
supplemented with 2 sucrose and 0.1 mg l
− 1
GA
3
. After 2 weeks of culture, the plantlets showed development of new shoots and roots. Fig.
1L shows somatic embryo derived plantlet with well-developed roots r, tuber t and shoot apical
meristem sm. Plants with well-developed roots, shoots and tubers were transplanted to a sand:peat
moss mixture. The acclimatization rate of plants derived from somatic embryos was 80 after 2
months of culture in a growth chamber. Fig. 1L shows a plant that survived 2 months after trans-
fer to sand:peat moss.
The optimal procedure for plant production via somatic embryos in C. yanhusuo is summarized as:
i primary callus cultures were established in 3 months by culturing mature tuber pieces on MS
basal medium supplemented with 2.0 mg l
− 1
BA and 0.5 mg l
− 1
NAA in darkness; ii somatic embryos were induced in 5 weeks by culturing the
tuber-derived primary callus on MS basal medium with 0.1 mg l
− 1
zeatin in light; iii ninety six percent somatic embryos converted in 2 weeks by
culturing in half-strength MS liquid medium sup- plemented with 1.0 mg l
− 1
zeatin riboside; iv converted somatic embryos showed good shoot,
root and tuber development when cultured on half-strength MS medium supplemented with 6
sucrose for 1 month; v before transplanting to soil:peat moss, the plants were cultured on half-
strength MS medium with 2 sucrose and 0.1 mg l
− 1
GA
3
for 3 weeks; vi eighty percent plants survived 2 months after transfer to sand:peat moss
and incubation in a growth chamber; vii the entire protocol starting from establishment of the
tuber-derived primary callus to well-developed plants in soil takes around 8 months. However,
this period could be reduced to around 5 months once the primary callus cultures are established.
Around 25 plants could be obtained from 1 g of tuber-derived primary callus after acclimatization.
4. Conclusions
