In Vitro Cell.Dev.Biol.—Plant (2012) 48:609–612 DOI 10.
1007/s11627-012-9468-6
MICROPROPAGATION
Thidiazuron enhances shoot organogenesis from leaf explants
of Saussurea involucrata Kar. et Kir
Bin Guo & A
manda R. Stiles & C
hun-Zhao Liu
Received: 21 November 2011 /Accepted: 20 September 2012 /Published online: 24 October 2012 / Editor: Praveen Saxena © The
Society for In Vitro Biology 2012
Abstract An efficient protocol for the in vitro micrpropa- gation of Saussurea involucrata Kar. et Kir, an endangered
Chinese medicinal plant, was developed. Shoot organogen- esis was obtained following culture of leaf explants on
Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ). After 28 d of culture, 15.6±1.4 shoots
were regenerated per leaf explant on MS medium containing 0.5 μM TDZ. After transfer of shoots to a medium
contain- ing 5.0 μM indole-3-acetic acid, approximately 80% of the regenerated shoots formed roots and whole
plantlets. After transfer of rooted shoots to the greenhouse, 83% of the regenerated plantlets survived and grew
vigorously. The regeneration protocol developed in this study provides a basis for germplasm conservation and for
the production of plant material necessary to study the medicinally active components of S. involucrata.
. . .
Keywords Saussurea involucrata Shoot organogenesis Thidiazuron Germplasm conservation
Introduction
Saussurea involucrata Kar. et Kir., one of the most well- known Chinese medicinal plants, is commonly used for
treating rheumatoid arthritis, gynopathy, and high-altitude diseases (Li and Zhao 1989). S. involucrata extracts also
show anti-inflammatory, anti-tumor, and analgesic activities (Liu et al. 1985; Jia et al. 2005). The overexploitation of
native plants for commercial purposes has resulted in the near extinction of S. involucrata i n China, and the species
is listed as a nationally protected wild plant (Fu 1992).
As demand has increased for S. involucrata, there is an urgent need to develop methods for the efficient propagation
and conservation of this plant. Conventional propagation methods using seeds are ineffective due to the high
mortality rate of the seedlings in the early stages of growth, while the use of rhizomes for vegetative propagation
may destroy the already endangered mother plants. In vitro propagation tech- niques provide useful systems for the
mass multiplication and germplasm conservation of many threatened plant spe-
. .
B. Guo A. R. Stiles C.-Z. Liu National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese
Academy of Sciences, Beijing 100080, People’s Republic of China
cies (Liu et al. 2004a, b) and offer great potential for the propagation of plant species such as S. involucrata.
In plant tissue culture systems, the balance between auxin and cytokinin plays an important role in determin- ing the
morphogenetic development of an explant (Skoog A. R. Stiles Department of Plant and Microbial Biology University of
California, Berkeley, CA 94720, USA
and Miller 1957; Gaspar et al. 1996). A high cytokinin to auxin ratio generally favors the formation of shoots, while
a low cytokinin to auxin ratio induces root forma- tion. A balance between the two growth regulators pro- C.-Z. Liu (*)
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190,
People’s Republic of China e-mail: czliu@home.ipe.ac.cn
motes callus formation. Manipulation of the composition and ratio of these plant growth regulators (PGRs) is often
the primary empirical approach used for optimization of in vitro m icropropagation methods (Shukla et al. 2012).
Thidiazuron (N- phenyl-N-(1, 2, 3-thidiazol-5-yl) urea;2003). The objectives of the current study were to test the
TDZ), a phenylurea derivative with cytokinin-like activity, iseffectiveness of TDZ for the induction of S. involucrata s hoot
effective in a wide variety of plant species for the induction oforganogenesis, compare the TDZ-induced morphogenesis with
both somatic embryogenesis (Malik and Saxena 1992; Murthythe response stimulated by balanced auxin/cytokinin treat-
et al. 1998; Akasaka et al. 2000; Jones et al. 2007) and shootments, and develop an effective protocol for the regeneration of
organogenesis (Li et al. 2000; Murch et al. 2000; Liu et al.S. involucrata from leaf explants.
explant was compact, green, and readily formed shoots, while
callus from stems and root explants was less compact, white–
Materials and Methods green, and shoot formation was less reliable. Therefore, leaf
explants were used throughout the study. Leaf explants (ap-
ar. et Kir. seeds were obtained from Tianshan proximately 0.5×0.5 cm in size) were sectioned from the 30-d
S. involucrata K
mountain, Xinjiang, China. Seeds were surface sterilized by old seedlings (approximately 4.0 cm height) and incubated on
placement in 70% ethanol for 30 s, followed by immersion in MS medium supplemented with 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0,
5.4% sodium hypochlorite for 20 min, and then rinsing three 10, 15, or 20 μM TDZ. The effect of exposure time to TDZ
times with sterile distilled water. Surface-sterilized seeds werewas evaluated by cultivating the leaf explants on MS medium
germinated and maintained on Murashige and Skoog (1962;with 0.5 μM TDZ (identified as the optimal concentration in
MS) solid medium for 30 d in a growth chamber, with a 16- hthe previous experiment) for a specific period (0, 7, 14, 21, 28,
photoperiod under cool-white light (30–40 μmolm−2s−1) at35, 42, and 49 d) followed by subculturing onto fresh TDZ-free
25°C. Preliminary results comparing callus induced from leaf,medium. The frequency of shoot regeneration and the number
stem, and root material indicated that the callus from leafof
610 GUO ET AL.
Figure 1. TDZ-induced plant
regeneration from S. involucrata
leaf explants. (A) An intact S.
involucrata seedling germinated in
MS medium, (B) Callus formation
from a leaf explant (B1; bar 1.0 cm)
and shoot primordia on the surface
of the callus after 14 d on MS
medium containing 0.5 μM TDZ
(B2; bar 5 .0 mm). (C) Regenerated
shoots cultivated for 49 d on MS
medium containing 0.5 μM TDZ.
(D) Proliferation and elongation of
regenerated shoots cultivated on 0.5
μM TDZ-supplemented medium for
28 d followed by subculture on MS
medium without PGRs for 21 d. (E)
Rooting of regener- ated shoots on
half-strength MS medium
supplemented with 5 μM IAA after
28 d. (F) Micropropagated plants
trans- planted to soil after 60 d.
shoots per leaf explant were recorded after 49 d of culture (starting from the initial day of inoculation).
a
To induce root organogenesis, green regenerated shoots larger than 30 mm were excised from the explant tissue and
cultured on half-strength MS medium supplemented with 1, 5,
t nalpxef aelr eps toohsd etarenegerf or ebmu 20 1612
N Duration of TDZ exposure (day)
Figure 3. Effect of the duration of TDZ exposure on shoot regenera- tion from leaf explants of S. involucrata. Columns w ith different
letters a re significantly different at P<0.05 according to Tukey’s multiple range test. Bars r epresent SE values.
Table 1. Effect of IAA on in vitro rooting of S. involucrata r egen- erated shoots after 28 d. Values represent the mean±SE
IAA (μM)
b
b
or 10μM indole-3-acetic acid (IAA). All media were adjusted to pH5.8 and supplemented with 0.6% agar
(Bacterialogical
8
grade, Sanland International Inc, Shanghai, China) and 3% sucrose before autoclaving at 121°C for 18 min. The
b bc
450 ml plastic culture boxes (Gentel Co. Ltd., Beijing, China) were
4
cd sealed with Parafilm® and incubated for 28 d under a 16/8 h (light/dark) photoperiod with a light intensity of
30–40 μmol
0 −2 −1
d 0 7 14 21 28 35 42 49 m s provided by cool-white fluorescent lamps.
Rooted plantlets were removed from the media, rinsed in water, and transferred to a potting soil mixture containing
south nutrition soil/perlite/vermiculite (3:1:1, v/ v/ v; Hebei, China) in the greenhouse. Each plantlet was covered with
a polyethylene bag in order to maintain a high humidity (~90%). After 21 d, the polyethylene covers were removed
and the plants were gradually exposed to ambient green- house conditions. Supplemental lighting was not supplied
and the average light level on the benches over the course of the experiment was 244 μmolm−2s−1.
All experiments were conducted using a completely ran- domized design and each experiment consisted of five
explants per culture dish and 10 replicate dishes per treatment. Each experiment was repeated twice. All data are
presented as the mean±standard error. The data were subjected to a one- way analysis of variance and the Tukey’s
honest significant difference multiple range test was used to calculate significant differences. SPSS for windows
(SPSS Inc., version 7.5.1, Chicago, USA) was used for all statistical analyses and a value of P< 0.05 was considered
significant.
Results and Discussion
Leaf explants from S. involucrata seedlings (Fig. 1A) were incubated on solid MS medium supplemented with
varying levels of TDZ for the induction of shoot regeneration. After 14 d culture, compact, light green calli
developed from the cut margins of the leaf explants, and after 21 d culture, regenerated shoots appeared (Fig 1B) .
After 49 d, signifi- cantly more adventitious shoots were observed on leaf explants cultured on media containing 0.5
μM TDZ com- pared to the other TDZ levels, with an average of 8.5±0.6 shoots per leaf explant and a frequency of
shoot regenera- tion of 69.0±2.0% (Figs. 1C a nd 2). The number of regen- erated shoots decreased at TDZ
concentrations higher than 0.5 μM.
The duration of exposure to TDZ also affected the shoot t nalpxef aelr eps toohsd etarenegerf or ebmuN20 1612840Number
regeneration from the leaf explants of S. involucrata. The
of regenerated shoots per leaf explant
maximum average number of shoots per leaf explant (15.6±
Percentage of responding leaf explant
1.4) was produced in cultures grown on 0.5 μM TDZ- supplemented medium for 28 d followed by subculturing on
MS medium without PGRs for 21 d (Fig. 1D). Exposure times longer or shorter than 28 d resulted in significantly
fewer shoots per explant (Fig. 3).
TDZ concentration (uM)
100 80604020) %(t nalpxef aelg nidnopserf oe gatnecre .1
P0 0 0Figure 2. Effect of TDZ on shoot regeneration from leaf explants of S.
involucrata. Columns w
ith different letters are significantly different at P 0.05 according to Tukey’s multiple range test. Bars represent
<
SE values.
a
c
c
a
ab
a
b
b
b
bbb
bc
bc
c
c
cc d
.25
0
.5 .0 .5 .0 0.0 5.0 0.0
0 1 2 5 1 1 2 Rooting
Number of roots per
Root length percentage
regenerated shoot
(mm)
d
0 15.0±1.0 d 1.3±0.1 d 0.9±0.0 d 1 53.0±3.0 c 3.1±0.2 cd 9.3±1.0 b 5 81.0±7.0 a 9.2±0.5 a 12.0±0.8 a 10 66.0±6.0 b 7.5±0.6 b 5.5±0.8 c
Values followed by the same letter are not significantly different at P
< 0.05 according to Duncan’s multiple range test
THIDIAZURON ENHANCES SHOOT ORGANOGENESIS FROM LEAF EXPLANTS 611
The number of regenerated shoots per leaf explantculture (Table 1). Increasing the concentration of IAA above 5
using TDZ obtained here higher than that was obtained inμM decreased the rooting percentage, the number of roots per
previ- ous studies where benzylaminopurine (BAP) andregenerated shoot, and the average length of the roots. The
naphtha- leneacetic acid (NAA) were used (Guo et al. 2007). Inplantlets were trans- ferred to soil after 45 d and cultivated to
the previous study, the most effective combination (10 μMmaturity with a survival rate of 83.0% (Fig. 1F) .
BAP and 2.5 μM NAA) produced an average of 5.2±0.4 shoots TDZ was surprisingly effective for shoot regeneration
per leaf explant and a shoot regeneration frequency of 66.0±from leaf explants of S. involucrata. At the optimum exposure
9.2%. Although the shoot regeneration frequency was sim- ilartime of 28 d, a relatively low level of TDZ (0.5 μM) induced
using 0.5 μM TDZ (69.0±2.0%), exposing the shoot explants tomore than 15 regenerated shoots per leaf explant, representing a
0.5 μM TDZ increased the average number of regeneratedthreefold increase over the optimized BAP–NAA combination
shoots per explant. previously reported (Guo et al. 2007). Similar results were
Regenerated shoots larger than 30 mm were separatedrecorded for other medicinal plants, including Scutellaria
and used for rooting media evaluation. All of the media-baicalensis a nd Artemisia judaica, in which large numbers of
induced rooting, including media without PGRs, however, inde novo s hoots were regenerated in response to TDZ ex- posure
the absence of PGRs, fewer roots were induced, and those that(Liu et al. 2003; Li et al. 2000).
were produced were much shorter compared to those induced This study has resulted in a protocol which can be uti-
using IAA treatments (Table 1). The optimal root- ing waslized for the regeneration and mass propagation of S. invo-
observed on the medium containing 5 μM IAA (Fig. 1E). Thelucrata. This method may also be used to select and clone
frequency of root formation on this medium was 81.0±7.0%,superior individual genotypes which could be further im-
the regenerated shoots developed an aver- age of 9.2±0.5 rootsproved using genetic engineering approaches. In addition, mass
per shoot and an average root length of 12±0.8 mm after 28 d
propagation may be used to produce the large quanti- ties of systems. Plant Cell Rep 26:13–19 Li GH, Zhao RC (1989) Studies on
plant tissue needed for the biochemical characteriza- tion of the pharmacological actions of Saus-
medicinally active constituents of S. involucrata. Lastly, this surea involucrata Kar. et Kir. Acta Pharm Sin 15:368–369 Li
H, Murch SJ, Saxena PK (2000) Thidiazuron-induced de novo shoot
protocol may be used for the generation of the large number of
organogenesis on seedlings, etiolated hypocotyls and stem seg- ments of
viable plantlets needed to meet commercial demand and
Huang-qin. Plant Cell Tiss Org Cult 62:169–1 73 Liu CZ, Murch SJ,
potentially replenish natural populations. EL-Demerdash M, Saxena PK (2003) Regeneration of the Egyptian
medicinal plant Artemisia judaica L. Plant Cell Rep 21:525–530 Liu CZ,
Acknowledgments This work was funded by the National Natural Science Murch SJ, El-Demerdash M, Saxena PK (2004b) Artemisia judaica L.:
Foundation of China (no. 21150110459), the Knowledge mass propagation and antioxidant potential. J Biotech- nol 110:63–71 Liu
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