INTRODUCTION
Ramie (Boehmeria nivea Gaud), commonly known
as China grass, is a perennial fiber plant in Urticaceae,
which has been grown in China for thousands of years and
was used for Chinese burial shrouds over 2,000 years. It
is used for the production of textiles and ropes because
it is extremely absorbent, dries quickly, dyes fairly easy,
resists shrinkage, and is unusually tolerant with bacteria,
mildew and insect attacks. Ramie is frequently used in
fabric blends due to its poor elasticity and brittleness.
Consequently, when added to cotton, ramie can increases
strength, color and luster without compromising the
flexibility of the fabric.
Improvement of ramie mainly focused on fiber quality,
yield and resistance. However, traditional breeding of
ramie is limited owing to its complex genetic background.
Comparatively, in vitro techniques can provide alternative
means for cultivar improvement via Agrobacterium-
mediated transformation. Successful application of in vitro
methods is largely dependent on a reliable regeneration
Botanical Studies (2007) 48: 173-180.
*
Corresponding author: E-mail: dxpeng@mail.hzau.edu.
cn; Tel: +86-27-87280550; Fax: +86-27-87281311; Dr. Bo
WANG: E-mail: wangbo@webmail.hzau.edu.cn.
system, which, nevertheless, has not been established in
ramie. Since the first publication on tissue culture of ramie
was reported over two decades ago (Zhou et al., 1980),
efforts have been made to regenerate ramie plantlets
from different explants such as cotyledons (without
regeneration data) (Huang et al., 1980), stem segments
(Pan et al., 1995), hypocotyls (Huang et al., 1981) and
leaves (Guo et al., 1998; Pan et al., 1995). It is noted that
in all of the above studies plants were regenerated via
morphogenesis from callus induced from the explants. The
callus was always subcultured for several cycles before
plant regeneration. One disadvantages of such process
is the recalcitrance of the callus to differentiation (Pan
et al., 1995), which negatively affected the regeneration
efficiency. Furthermore, a low rate of regeneration (not
more than 50%) in these studies limited the application of
previous protocols in genetic transformation, which, so far,
has been performed in work (Dusi et al., 1993). Therefore,
it is necessary to establish a reliable and efficient
regeneration system of ramie, which can be employed for
genetic transformation in the future.
Cotyledon, which has been widely used for many other
plants (Colijin-Hooymans et al., 1994; Yang et al., 2001;
Han et al., 2004; Sul III-Whan et al., 2004; Zhang et al.,
2005), was scarcely used in ramie tissue culture. Thidi-
azuron (TDZ), a substitute urea with both cytokinin activ-
phySIOlOgy
An efficient adventitious shoot regeneration system for
ramie (Boehmeria nivea
gaud) using thidiazuron
Bo WANG, Dingxiang PENG*
,
Lijun
LIU, Zhenxia SUN, Na ZHANG, and Shimei GAO
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, P.R. China
(Received May 22, 2006; Accepted October 25, 2006)
ABSTRACT.
An efficient culture system for direct adventitious shoot regeneration of plantlet from cotyledon
of ramie (Boehmeria nivea Gaud) was established and factors affecting shoot regeneration efficiencies were
evaluated. Cotyledons excised from 4-day-old seedlings showed the highest regeneration capacity. Various
concentrations of thidiazuron (TDZ) and indoleacetic acid (IAA) gave rise to adventitious shoots with differ-
ent efficiencies. The cultures of cotyledons from 4-day-old ramie seedlings on MS medium supplemented with
2.27 £gM TDZ and 0.057 £gM IAA were shown to have the highest efficiency (83.6%) of shoot regeneration
among the four auxin supplements (2,4-D; IAA; IBA; NAA). Regeneration of shoot in six cultivars was geno-
type-dependent, ¡¥5041-3¡¦ being the most responsive one. The regenerated shoots were transferred to hormone-
free medium for shoot elongation, after successfully rooted on half-strength MS medium supplemented with
0.27 £gM NAA. The rooted plantlets with 4-5 leaves were transplanted to greenhouse for further growth. To
our knowledge, this is the first report on efficient regeneration of plantlet from cotyledon of ramie, which will
be of value for genetic improvement in the near future.
Keywords: Boehmeria nivea Gaud; Cotyledon; Organogenesis; Ramie; Thidiazuron.
Abbreviations: BA, Benzyladenine; FCM, Flow cytometry; 2, 4-D, 2, 4-Dichlorophenoxyacetic acid;
IAA, Indoleacetic acid; IBA, Indolebutyric acid; MS medium, MS medium basal medium; NAA,
£\-Naphthaleneacetic acid; TDZ, thidiazuron (N-phenyl-N¡¦-1,2,3-thidiazol-5-ylurea).
pg_0002
174
Botanical Studies, Vol. 48, 2007
ity and auxin activity (Mok et al., 1982; Visser et al., 1992;
Murthy et al., 1998), has been widely used in plant tissue
culture as powerful cytokinin (Huettman et al., 1993; Lu
et al., 1993; Colijin-Hooymans et al., 1994; Ahroni et al.,
1997; Kim et al., 1997; Gless et al., 1998; Li et al., 2000;
Gu et al., 2005). But it has never been reported in ramie
tissue culture. Therefore, in the present study, attempts
were made to establish an efficient regeneration system
based on culture of ramie cotyledon explants and use of
TDZ as an important hormone, which is expected to hold
potential for genetic transformation in the future.
MATERIAl AND METhODS
plant material
Mature seeds of six cultivars of ramie, ¡¥Luzhuqing¡¦,
¡¥5041-3¡¦, ¡¥1690¡¦, ¡¥Dama No. 1¡¦, ¡¥Nianhong¡¦ and ¡¥Huang-
pigun¡¦, were collected from Ramie Germplasm Resources
Garden located at Huazhong Agricultural University, Chi-
na. The seeds were first soaked in 70% (v/v) ethanol for
30 seconds and then treated with 10% (w/v) sodium hypo-
chlorite (NaOCl) for 15 min, followed by 3-4 rinses with
sterile water for surface sterilization. The sterilized seeds
were germinated on half-strength MS medium (Murashige
and Skoog, 1962) without growth regulators to produce
cotyledons. When cotyledons were observed and fully
developed, they were carefully excised from each seedling
for culture without any other wounding.
Culture of cotyledons
The cotyledons from the cultivar ¡¥Luzhuqing¡¦ were col-
lected at 2-day intervals from the seedlings 2 to 12 days
after germination, which were cultured on MS medium
supplemented with 2.27 £gM TDZ and 0.057 £gM IAA. Each
cotyledon was placed with its abaxial side contacting the
medium. Since 4-day-old seedlings were proven to be the
optimum donor of cotyledons in the experiment described
above, they were employed to study effect of various plant
hormone combinations on shoot regeneration. Cotyledons
excised from 4-day-old seedlings were cultured on MS
medium supplemented with 2.27 £gM TDZ and either of
IAA (0.0285, 0.057, 0.285, 1.43 or 2.85 £gM), IBA (0.0246,
0.049, 0.246, 1.23 or 2.46 £gM), NAA (0.0268, 0.054,
0.268, 1.34 or 2.68 £gM) or 2,4-D (0.0226, 0.045, 0.226,
1.13 or 2.26 £gM). The effects of different concentrations
of TDZ and IAA on shoot regeneration were further tested
using cotyledons excised from 4-day-old seedlings that
were cultured on MS medium supplemented with 0.045,
0.23, 1.14, 2.27 or 4.54 £gM TDZ in combination with 0,
0.028, 0.057, 0.285 or 2.85 £gM IAA, respectively. Finally,
in order to test the selected regeneration systems for dif-
ferent genotypes, cotyledons excised from 4-day-old seed-
lings of six ramie cultivars, ¡¥5041-3¡¦, ¡¥Luzhuqing¡¦, ¡¥1690¡¦,
¡¥Dama No. 1¡¦, ¡¥Nianhong¡¦ and ¡¥Huangpigun¡¦, were cul-
tured on MS medium supplemented with 2.27 £gM TDZ
and 0.057 £gM IAA or with 2.27 £gM TDZ and 0.054 £gM
NAA.
Shoot elongation and rooting
After culture for 8 weeks, the adventitious shoots
regenerated from cotyledon explants were transferred to
hormone-free MS medium for shoot elongation. When the
shoots reached 2 cm in height, they were transferred onto
half-strength MS medium supplemented with 0.27 £gM
NAA (Guo et al., 1998) for rooting. Young plantlets with
4-5 leaves were transplanted to plastic pots, in which soil,
vermiculite and sand (2:1:1) were blended, before they
were moved to the greenhouse for further growth.
Culture conditions
All the experiments except for plantlets acclimation
were conducted in 150 ml conical flask containing 30 ml
medium. All of the media were supplemented with 30 g/l
glucose, solidified with 8 g/l agar, with pH adjusted to 5.8
prior to autoclaving at 121¢XC for 20 min. Cultures were in-
cubated at 25¡Ó2¢XC under a 16/8-h (light/dark) photoperiod
with a light intensity of 3,000 lux.
Data collection and statistical analysis
All experiments were conducted 3 replicates with 3 150
ml conical flasks in each and 12-15 explants were cultured
in every 150 ml conical flask. Data on the percentage of
shoot formation and average number of shoots per explant
were analyzed using SAS ver 6.12 (SAS Institute, 1995,
Cary, N.C.). Analysis of variance (ANOVA) was used
to test the statistical significance, and the significance of
differences among means was carried out using Duncan¡¦s
(1955) multiple range test at P=0.05.
ploidy analysis by flow cytometry (FCM)
The ploidy level of the regenerated plantlets was
determined with flow cytometry as described by Xu et
al. (2006). 40 plantlets were selected randomly from the
regenerated plantlets described above and 1.5 cm
2
young
leave was cut into small pieces in nuclear extraction buffer
(solution A of High Resolution Kit for Plant DNA, Partec,
Germany) respectively. Then it was filtered through
a nylon sieve (Partec CellTrics, Germany) with mesh
diameter of 30 £gm, followed by adding a staining solution
containing DAPI (solution B of the kit). The samples
were then measured by a flow cytometer (PA-I, Partec).
The relative DNA content of ¡¥Luzhuqing¡¦ seedlings was
used as a ploidy standard. The ploidy of the cell lines was
calculated by comparing the relative fluorescence intensity.
RESUlTS AND DISCUSSION
Effect of donor plant age on shoot induction
Effect of cotyledons sampled at different time after ger-
mination was evaluated, which showed that cotyledon age
influenced the efficiency of adventitious shoot formation
(Table 1). Cotyledons excised from 4-day-old seedlings
gave rise to the highest percentage of shoot formation
(51.1%), whereas 12-day-old cotyledons gave rise to the
pg_0003
WANG et al. ¡X Efficient shoot regeneration system for ramie
175
lowest percentage (7.9%) and the least number of shoots
per explant (3.6). Regeneration rate was inversely related
to the age of plants within 4 days after culture. In addi-
tion, 2-day-old cotyledons produced the largest number
of shoots per explant (5.6), but there was no significant
difference between the 2-day-old and 4-day-old seedlings.
These results seemed to reveal that the regeneration poten-
tial of cotyledon explants depended on the developmental
stage of the donor plant, in agreement with that reported in
cucumber by Colijin-Hooymans et al. (1994) and in bottle
gourd by Han et al. (2004). However, it did not agree with
Pan et al. (1995) who discovered that regeneration rate
increased with the age of hypocotyls sampled from 7 to
16-day-old of ramie seedlings, which might be due to the
different explant types and/or genotypes.
Effect of different auxins on shoot induction
Different auxins in combination with TDZ have been
used for plant regeneration in other plant species, i.e.
gypsoghila (Ahroni et al., 1997), sweetgum (Kim et al.,
1997), winter jujube (Gu et al., 2005). Herein, we also
investigated effects of auxin on shoot induction, which
demonstrated that culture of cotyledons on MS medium
added with 2.27 £gM TDZ and different auxins led to
significant differences in percentage of shoot formation
and average number of shoots per explant (Table 2). The
optimum concentration of a given auxin to induce plant
regeneration from cotyledons of ramie was different. IAA
at 0.057 £gM and NAA at 0.054 £gM were optimal for shoot
regeneration compared with IBA at 1.23 £gM and 2,4-D
at 1.13 £gM for regeneration rate and number of shoots
per explant. The regeneration rate and number of shoots
per explant were reversely related to IAA concentration
over 0.057 £gM and NAA concentration over 0.054 £gM,
whereas they increased with IBA and 2,4-D except at the
highest concentration. In addition, IAA at concentrations
higher than 0.057 £g£O, NAA at concentrations higher than
0.054 £g£O, IBA higher than 1.23 £g£O and 2,4-D higher
than 1.13 £g£O, combined with TDZ, induced more callus
Table 1. Effect of cotyledon explant age of ¡¥Luzhuqing¡¦ cul-
tivar on percentage of s hoot form ation (SF%) and average
number of shoots per explant (AS) (means ¡Ó SE) 8 weeks after
culture.
Cotyledon age
SF%
a
AS
a
2 d
37.8 ¡Ó 7.3 b
5.6 ¡Ó 1.1 a
4 d
51.1 ¡Ó 7.8 a
5.5 ¡Ó 0.4 ab
6 d
28.1 ¡Ó 4.5 c
5.1 ¡Ó 1.2 ab
8 d
18.3 ¡Ó 4.8 d
4.7 ¡Ó 1.0 ab
10 d
13.4 ¡Ó 3.0 de 4.6 ¡Ó 0.9 ab
12 d
7.9 ¡Ó 2.5 e
3.6 ¡Ó 1.7 b
a
Different letters in the same column shows that the values are
significantly different at P<0.05.
pg_0004
176
Botanical Studies, Vol. 48, 2007
from cotyledon explants than those at lower concentration
auxin (data not shown). Our results indicated that low
concentration of IAA combined with TDZ was the best
condition for inducing shoots from ramie cotyledon.
Effect of concentrations of TDZ and IAA on
shoot induction
In order to test the effect of TDZ and IAA on shoot
induction, five concentrations of TDZ in combination
with five concentrations of IAA were used, which showed
percentage of shoot formation of two ramie cultivars
(¡¥Luzhuqing¡¦ and ¡¥5041-3¡¦) increased with increase
in TDZ except the highest concentration at the same
IAA levels (Table 3). The highest percentage of shoot
formation (54.3% and 86.2%, respectively) and the largest
number of shoots per explant (4.6 and 4.4, respectively)
for both genotypes were obtained when 2.27 £gM TDZ and
0.057 £gM IAA were used. ANOVA showed significant
differences in both percentage of shoot formation and
average number of shoots per explant of ¡¥Luzhuqing¡¦
Table 3. Effect of TDZ and IAA concentration on percentage of shoot formation (SF%) and average number of shoots per explants
(AS) from cotyledons of ¡¥Luzhuqing¡¦ and ¡¥5041-3¡¦ (means ¡Ó SE) 8 weeks after culture.
TDZ (£gM)
IAA (£gM)
SF%
a
AS
a
Luzhuqing
5041-3
Luzhuqing
5041-3
0.045
0
0.0 ¡Ó 0.0 i
0.0 ¡Ó 0.0 o
0.0 ¡Ó 0. 0 i
0.0 ¡Ó 0.0 j
0.029
14.8 ¡Ó 1.7 g
7.7 ¡Ó 0.6 lmn
1.3 ¡Ó 0. 3 h
1.7 ¡Ó 0.6 hi
0.057
6.5 ¡Ó 0. 8 h
9.9 ¡Ó 3.8 klmn
1.7 ¡Ó 0. 3 fgh
2.7 ¡Ó 0.4 cdefg
0.285
8.5 ¡Ó 1. 4 h
21.5 ¡Ó 1 .5 feg
2.0 ¡Ó 0. 3 efg
2.0 ¡Ó 0.3 ghi
2.85
0.0 ¡Ó 0. 0 i
12.5 ¡Ó 0 .8 ijkl
0.0 ¡Ó 0.0 i
1.7 ¡Ó 0.3 ghi
0.23
0
0.0 ¡Ó 0.0 i
4.9 ¡Ó 4.3 no
0.0 ¡Ó 0. 0 i
1.3 ¡Ó 1.2 hi
0.029
15.8 ¡Ó 1.7 fg
16.2 ¡Ó 0.7 ghij 1.4 ¡Ó 0.3 h
1.9 ¡Ó 0.6 ghi
0.057
16.9 ¡Ó 5.4 efg 17.5 ¡Ó 0.8 fghi 3.0 ¡Ó 0.4 c
2.8 ¡Ó 0.6 cdef
0.285
17.4 ¡Ó 2.4 efg 24.3 ¡Ó 5.7 de
2.2 ¡Ó 0.3 def
2.1 ¡Ó 0.2 fghi
2.85
16.7 ¡Ó 4.1 efg 14.3 ¡Ó 1.0 hijk
1.6 ¡Ó 0.1 gh
2.0 ¡Ó 0.5 ghi
1.14
0
7.9 ¡Ó 1. 9 h
6.8 ¡Ó 0.3 mn
1.7 ¡Ó 0. 3 fgh
1.7 ¡Ó 0.6 hi
0.029
19.4 ¡Ó 2.4 cdefg 18.3 ¡Ó 4.3 fgh
2.6 ¡Ó 0.5 cd
2.3 ¡Ó 0.3 efgh
0.057
23.5 ¡Ó 4.7 bcd 32.6 ¡Ó 2.9 c
3.7 ¡Ó 0.3 b
3.0 ¡Ó 0.2 bcde
0.285
18.3 ¡Ó 1.7 defg 28.7 ¡Ó 2.1 cd
2.5 ¡Ó 0.5 cde
2.7 ¡Ó 0.4 cdefg
2.85
21.4 ¡Ó 8.1 bcdef 17.8 ¡Ó 3.8 fghi 1.9 ¡Ó 0.1 fg
2.5 ¡Ó 0.2 cdefg
2.27
0
15.7 ¡Ó 1.2 fg
22.7 ¡Ó 3.5 ef
2.2 ¡Ó 0.4 def
3.3 ¡Ó 0.5 bc
0.029
26.7 ¡Ó 1.8 b
48.3 ¡Ó 6.5 b
2.5 ¡Ó 0. 2 cde
3.1 ¡Ó 0.3 bcd
0.057
54.3 ¡Ó 4.2 a
86.2 ¡Ó 3.4 a
4.6 ¡Ó 0.4 a
4.4 ¡Ó 0.5 a
0.285
24.9 ¡Ó 1.8 bc
43.9 ¡Ó 5.4 b
3.9 ¡Ó 0.8 b
3.7 ¡Ó 0.6 ab
2.85
22.1 ¡Ó 2.6 bcde 29.8 ¡Ó 3.4 c
2.6 ¡Ó 0.4 cd
2.6 ¡Ó 0.2 cdefg
4.54
0
7.8 ¡Ó 2.7 h
13.7 ¡Ó 0 .5 hijk
1.3 ¡Ó 0.3 f
1.7 ¡Ó 0.3 hi
0.029
16.3 ¡Ó 2.7 fg
18.6 ¡Ó 3.8 fgh
1.5¡Ó 0.1 gh
2.0 ¡Ó 0.3 ghi
0.057
26.5 ¡Ó 9.2 b
44.3 ¡Ó 5 .2 b
2.5¡Ó 0 .2 cd
2.3 ¡Ó 0.3 defgh
0.285
23.2 ¡Ó 1.8 bcd 18.8 ¡Ó 1.2 fgh
2.2 ¡Ó 0.3 def
2.5 ¡Ó 0.5 cdefg
2.85
20.8 ¡Ó 2.5 cdef 12.1 ¡Ó 1.1 jklm
2.0 ¡Ó 0.0 efg
2.1 ¡Ó 0.4 fghi
Analysis of variance
SF%
AS
Source of variation
F-test
b
F-test
TDZ
86.55**
267.54**
99.80**
30.77**
IAA
86.29**
161.67**
96.11**
15.03**
TDZ¡ÑIAA
10.17**
29.27**
5.55**
1.58
a
Different letters in the same column shows that the values are significantly different at P<0.05.
b
F-test: *, ** Significant at P.0.05 or 0.01, respectively.
pg_0005
WANG et al. ¡X Efficient shoot regeneration system for ramie
177
among different concentrations of TDZ and IAA, and
significant interactions existed between TDZ and IAA.
Similar results were obtained in ¡¥5041-3¡¦ except that no
significant interaction between TDZ and IAA was found
in average number of shoots per explant. The results
showed that TDZ, which has been proven to be powerful
in stimulating shoot regeneration in many plant species
including gypsophila (Ahroni et al., 1997), oat (Gless et
al., 1998), cucumber (Colijin-Hooymans et al., 1994),
Huang-qin (Li et al., 2000), sweetgum (Kim et al., 1997)
and winter jujube (Gu et al., 2005), was an effective plant
growth regulator for the induction of shoot regeneration
from ramie cotyledons. However, in this study, it is worth
noting that percentage of shoot formation was inhibited
when 4.54 £gM TDZ was used compared with TDZ at
lower concentration. In addition, the shoot elongation in
this study was also retarded by 4.54 £gM TDZ, since the
regenerated shoots induced on medium supplemented
with 4.54 £gM TDZ were shorter than those at lower
concentration (below 1.14 £gM) of TDZ within 8 weeks
(data not shown). The inhibition of TDZ observed herein
was possibly due to toxicity of TDZ, as has been reported
elsewhere (Huettman et al., 1993; Lu et al., 1993; Kim et
al., 1997).
genotypic response to the shoot regeneration
system
Cotyledons of six genotypes were cultured on MS me-
dium containing 2.27 £gM TDZ and 0.057 £gM IAA or 2.27
£gM TDZ and 0.054 £gM NAA to investigate the genotypic
response to in vitro culture (Table 4). Regeneration of
shoots occurred in all of the genotypes with regeneration
rate higher than 50% in most cases. Regeneration rate of
¡¥5041-3¡¦ was the highest, whereas the least responsive
genotype was ¡¥Huangpigun¡¦. ANOVA analysis showed
significant differences in percentage of shoot formation
among the six cultivars, which indicated that shoot re-
generation was genotype-dependent, in agreement with
the findings in ramie (Pan et al., 1995), carnation (Henni
Kallak et al., 1997), melon (Ficcadenti et al., 1995), rape
(Khehra et al., 1992) and red pepper (Christopher et al.,
1996).
plant regeneration, elongation, rooting and
transplantation
Cotyledons cultured on MS medium supplemented
with TDZ and auxins began to swell 7 day later, and the
first bud formation was observed at the cutting edge 3
weeks after culture (Figure 1A, B). Some cotyledons only
produced one shoot (Figure 1C), but others produced
multiple shoots (Figure 1D). It is noted that shoot
regenerated from the cutting edge of cotyledon explant
without callus phase. It is known that regeneration from
callus that has been maintained for several cycles is always
coupled with somaclonal variation (Cassells and Curry,
2001). Therefore, regeneration from cotyledon directly
may minimize somaclonal variation (Skirvin et al., 1994;
Cassells and Curry, 2001; .u.ek et al., 2002), which seems
superior to the previous reports on ramie tissue culture
involving callus (Huang et al., 1980; Huang et al., 1981;
Pan et al., 1995; Guo et al., 1998).
The regenerated shoots were cut carefully from
cotyledon explants 8 weeks after culture and were cultured
on hormone-free MS medium for shoot elongation. The
shoots grew quickly on MS medium and reached 2 cm
in height within one month (Figure 1E), which were
then transferred to half-strength MS medium added with
0.27 £gM NAA to induce root. About 95% of the shoots
rooted successfully (Figure 1F). The rooted plantlets were
transplanted to plastic pots containing soil, vermiculite and
sand (2:1:1) and almost all of the plantlets survived in the
greenhouse (Figure 2A, B).
ploidy analysis of regenerated plantlets
FCM, a technique which has been described as a
timesaving method for ploidy analysis (Xu et al., 2006),
was used to analyze the ploidy levels of 40 plantlets
Table 4. Effect of genotype on percentage of shoot formation (SF%) and average number of shoots per explants (AS) (means ¡Ó SE)
8 weeks after culture using cotyledons explants from 4-day-old seedlings.
Genotypes
SF%
a
AS
a
TDZ/IAA
b
TDZ/NAA
c
TDZ/IAA
b
TDZ/NAA
c
5041-3
86.2 ¡Ó 3.4 a
64.5 ¡Ó 5.2 a
4.4 ¡Ó 0.6 a
5.2 ¡Ó 0.6 ab
1690
52.9 ¡Ó 5.4 cd
56.2 ¡Ó 9.9 ab
4.3 ¡Ó 0.4 a
5.5 ¡Ó 0.5 a
Luzhuqing
62.2 ¡Ó 3.9 b
47.8 ¡Ó 3.8 b
4.6 ¡Ó 0.4 a
4.8 ¡Ó 0.6 ab
Dama No. 1
59.5 ¡Ó 3.5 bc
48.2 ¡Ó 1.8 b
4.5 ¡Ó 0.4 a
5.2 ¡Ó 0.5 ab
Nianhong
55.0 ¡Ó 2.9 c
50.1 ¡Ó11.4 b
4.4 ¡Ó 0.4 a
4.6 ¡Ó 0.5 bc
Huangpigun
46.5 ¡Ó 3.1 d
33.4 ¡Ó 3.8 c
3.4 ¡Ó 0.1 b
3.8 ¡Ó 0.1 c
a
Different letters in the same column shows that the values are significantly different at P<0.05.
b
TDZ=2.27 £gM, IAA =0.057 £gM.
c
TDZ=2.27 £gM, NAA=0.054 £gM.
pg_0006
178
Botanical Studies, Vol. 48, 2007
selected randomly from the regenerated plantlets, which
has never been conducted in the previous study on ramie
tissue culture. Results showed that the regenerated
plantlets demonstrated the fluorescence intensity similar
to the control whose fluorescence intensity was preset to
50 (Figure 3A), which indicated that they were diploids
(Figure 3B).
In conclusion, we report herein for the first time a
protocol for highly efficient regeneration of plantlets from
ramie cotyledon. The established regeneration system
holds great potential for Agrobacterium tumefaciens-
mediated transformation with the efforts of cultivar
improvement for ramie, which is underway at present in
our laboratory.
Acknowledgements. This research was supported by
High-technology Project of China. The authors wish to
thank Prof. J.H. Liu and Prof. D.M. Jin for critically read-
Figure 3. FCM histograms. (A) The diploid control; (B) One of
the regenerated plantlets.
Figu re 1. Plant regeneration from the cotyledons of ramie.
(A) Shoot formation at the cutting edge of cotyledon 15 d after
culture; (B) Regeneration of adventitious shoots from the coty-
ledons cultured for 8 weeks on MS medium with 2.27 £gM TDZ
and 0.057 £gM
IAA; (C) Induction of one shoot from a cotyledon
8 weeks after culture; (D) Multiple shoots regenerated from a
cotyledon; (E) Elongated shoots on hormone-free MS medium;
(F) A well-rooted plantlet.
Figure 2. A and B, Acclimatized plants in plastic pots contain-
ing soil, vermiculite and sand (2:1:1) in the greenhouse.
160
120
90
60
30
0
0 100 200 300 400 500
1 FL stain
A
B
160
120
90
60
30
0
0 100 200 300 400 500
1 FL stain
pg_0007
WANG et al. ¡X Efficient shoot regeneration system for ramie
179
ing the manuscript. B. Wang wishes to thank Doc. M.
Zhang for his helpful work in ploidy check.
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