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ORIGINAL PAPER
Callus-mediated organogenesis and effect of growth regulatorson production of different valepotriates in Indian valerian(Valeriana jatamansi Jones.)
Jayashankar Das • Ashiho A. Mao •
Pratap J. Handique
Received: 11 February 2012 / Revised: 29 May 2012 / Accepted: 19 June 2012 / Published online: 3 July 2012
� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2012
Abstract A reproducible and efficient callus-mediated
shoot regeneration system was developed for the large-scale
production of Valeriana jatamansi Jones., a highly medic-
inal plant species of global pharmaceutical importance.
Effect of Murashige and Skoog (MS) medium supple-
mented with different concentrations of 2,4-dichlorophe-
noxyacetic acid (2,4-D), a-naphthaleneacetic acid (NAA)
and indole-3-butyric acid (IBA) on callus induction and
production of valepotriates accumulation was studied by
using different explants. In V. jatamansi, the degree of
callus induction varied significantly depending on explants
type and the growth regulators used. Among different
explants used, rhizomes have the highest callus induction
potential followed by leaf. The callus induction frequency
was found to be optimum in rhizome explants on media
supplemented with 0.5 mg/l 2,4-D. The regenerative ability
of proliferated compact calli was studied by the application
of cytokinins alone and in combination with auxin. MS
medium fortified with 0.75 mg/l thidiazuron in combination
with 0.5 mg/l NAA showed the highest regeneration
frequency (88.6 %) and produced the maximum number of
shoot buds (15.20 ± 0.20) capable of growing into single
plants. Vigorous callus obtained from MS medium sup-
plemented with different concentrations of 2,4-D, NAA and
IBA were used for industrially important valepotriates
[acevaltrate (ACE), valtrate (VAL) and didrovaltrate
(DID)] analysis. High performance liquid chromatography
analysis of callus revealed that medium with 2,4-D (1 mg/l)
was found responsible for increasing ACE and DID yield,
whereas VAL production was higher in case of medium
supplemented with NAA (1 mg/l). However, the accumu-
lation of valepotriates in callus decreased in logarithmic
phase after 8 weeks. IBA was not beneficial for the va-
lepotriate synthesis, as it helped to accumulate significantly
lower concentration of ACE, VAL and DID. Micropropa-
gated plantlets with well-developed root system were suc-
cessfully acclimatized in greenhouse condition, in root
trainers containing garden soil with a survival frequency of
100 %. As Indian valerian is a highly traded medicinal plant
due to extensive use of its industrially important secondary
metabolites, the present system can be utilized to obtain
mass multiplication of the species as well as for the stable
biomass and continuous valepotriate production for the
pharmaceutical industries.
Keywords Indian valerian � Callus � Regeneration �Valepotriates
Introduction
Valeriana jatamansi Jones. syn. V. wallichi (Fam: Valeri-
anaceae) commonly known as Indian valerian is a peren-
nial herb, distributed in Himalayas from Kashmir to Bhutan
and Khasi hills of Northeast India at an altitude
Communicated by J. Van Huylenbroeck.
J. Das � A. A. Mao
Plant Tissue Culture Laboratory, Botanical Survey of India,
ERC, Shillong 793 003, India
J. Das � P. J. Handique
Department of Biotechnology, Gauhati University,
Guwahati 781084, India
Present Address:J. Das (&)
Plant Bioresources Division, Regional Centre of IBSD,
Tadong, Gangtok 737102, India
e-mail: biotechjay@yahoo.co.in
123
Acta Physiol Plant (2013) 35:55–63
DOI 10.1007/s11738-012-1047-2
1,800–3,500 m (Bos et al. 1997). V. jatamansi has been
used in Ayurvedic, Unani and modern system of medicine
due to its high medicinal and aromatic values. The essential
oil components of Indian valerian make it one of the most
demanded plant in the drug industry. Major constituents of
its volatile oil are patchouli alcohol and bornyl isovaltrate
(Bos et al. 1997). Essential oil from rhizomes exhibited
antifungal and antibacterial activities (Girgune et al. 1980).
6-Methylapigenin and hesperidin isolated from rhizome of
Indian valerian showed anxiolytic and sedative activities
(Wasowski et al. 2002). The sedative and tranquilizing
properties of the plant are also due to the presence of
nonglycosidic iridoid esters known as valepotriates. The
major valepotriates are valtrate (VAL), acevaltrate (ACE)
and didrovaltrate (DID) due to which V. jatamansi pos-
sesses antispasmodic, anticonvulsive and antidepressant
properties (Gupta et al. 1986). DID, VAL and their
degraded product baldrinal were found to be cytotoxic in
rat hepatoma cells (Bounthanh et al. 1981). In addition, the
antitumor activity of DID was demonstrated in vivo on
female mice KREBS II ascetic tumors (Marder et al. 2003).
Due to extensive use in the modern pharma and perfumery
industries, V. jatamansi is listed as one of the most
exploited plant of Himalayan range. However, it is not yet
cultivated anywhere in India for the large-scale production
and all demands for its domestic and foreign trade are met
from its wild population (Gupta et al. 2006). Over the years
its indiscriminate collection has led to its large-scale
depletion in the wild and has necessitated its replenishment
and cultivation.
During the last several years, there is an increase
demand of in vitro culture techniques which offer a feasible
tool for rapid clonal propagation and germplasm conser-
vation of rare, endangered and threatened medicinal and
aromatic plants (Abraham et al. 2010). Cultured plant cells
synthesize, accumulate and sometimes exude many classes
of secondary metabolites. Numerous alkaloids, saponins,
cardenolides, anthraquinones, polyphenols and terpenes
have been reported from in vitro cultures (Verpoorte et al.
2002; Vanisree and Tsay 2004). In dedifferentiated cells,
some biosynthetic potential typical for the developed
organs from which they were initiated can be conserved. In
Pueraria lobata callus cultures, the bioactive isoflavonoid
content depended on the source organ, reflecting relations
in the mature plant (Matkowski 2004). A range of envi-
ronmental and nutritional factors are known to influence
the biosynthetic pathways of secondary metabolites
(Stintzing and Carle 2004). Plant growth regulators have
been widely used in promoting the biosynthesis of both
inducible and constitutive secondary metabolites, including
medicinal compounds such as anticancer alkaloids
(Vanisree and Tsay 2004; Verpoorte et al. 2002).
The continuous monitoring of a chosen metabolite is a
prerequisite for the successful development of production
technology. Also, the super-efficient clones of cultured cell
or tissues can be selected by monitoring the level of the
compound of interest, or can be complemented by a
selecting agent facilitating the process.
Though an earlier fragmentary report was published on
micropropagation of V. jatamansi (Kaur et al. 1999), the
result was scanty towards standardization of the protocol
for the commercial scale. The present research work is
based on twofold objectives, viz., to develop an efficient
and rapid propagation protocol of V. jatamansi for the
fulfillment of market demand and to quantify the indus-
trially important valepotriates (VAL, ACE and DID)
accumulated in the calli of different ages. This is a first
attempt to study the effect of different growth regulators on
in vitro accumulation of valepotriates in the callus of
V. jatamansi.
Materials and methods
Callus induction
Leaf (ca. 1 9 1 cm), petiole (ca. 0.5–0.7 cm) and rhizomes
(ca. 1 9 1 cm) explants were taken from a 12-month-old
single genotype of V. jatamansi maintained in the green-
house, BSI, ERC, Shillong, for callus induction studies. The
explant cuttings of 2.0–2.5 cm long were rinsed in running
tap water three times and washed in a 2 % (v/v) Tween 20
detergent solution for 15 min. Then the plant materials were
surface-sterilized in a solution of 10 % (v/v) sodium
hypochlorite for 5 min followed by 0.1 % (w/v) mercuric
chloride for 1 min. Finally, the explants were rinsed 3 times
with sterilized distilled water. The explants were estab-
lished in Murashige and Skoog’s (1962) basal medium
supplemented with 3 % (w/v) sucrose and 0.8 % (w/v)
agar-agar (Hi-media, Mumbai, India). The pH of media was
adjusted to 5.8 before autoclaving at 15 psi and 121 �C for
20 min. All the explants were cultured on MS medium
supplemented with different concentrations of 2,4-D, NAA
and IBA (0.25, 0.5, 1.0, 2.0, 3.0 mg/l). Callus cultures were
subcultured at 4-week intervals on respective media.
Regeneration of multiple shoot bud from callus
Randomly selected compact calli were transformed to
growth regulator-free MS basal medium to overcome the
carryover effect of auxins. To evaluate the effect of growth
regulators on the callus potential for shoot regeneration,
calli were excised, divided into small pieces (0.5 9
0.5 cm) transferred to the regeneration medium for shoot
induction. MS medium supplemented with different con-
centrations of TDZ (0.5, 0.75 mg/l) and kinetin (Kn; 2.0,
56 Acta Physiol Plant (2013) 35:55–63
123
3.0 mg/l) alone and in combination with NAA (0.5 mg/l)
was used for shoot proliferations from callus in 150-ml
culture flasks (Borosil, India). Callus along with the initi-
ated multiple shoot buds were subcultured on respective
regeneration medium after 4 weeks interval to obtain
healthy shoots.
Culture conditions
The cultures were maintained at 24 ± 2 �C and relative
humidity (RH) of 50 ± 5 % under 16 h photoperiod with
30 lmol m-2s-1 photosynthetic photon flux density
(PPFD) provided by cool-white fluorescent light tubes
(Philips, India).
Extraction and HPLC analysis
Vigorous callus obtained from rhizome explants supple-
mented with 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l)
and IBA (1 mg/l) were used for valepotriates (ACE, VAL
and DID) analysis. The calli were dried and subjected to
dichloromethane extraction 3 times at room temperature
(25 ± 2 �C). The extract was dissolved in methanol to
obtain a concentration of 1 mg/ml, filtered through a
membrane filter (0.22 lm pore size, Merck), analyzed by
HPLC and compared with the reference compounds (Sigma
Aldrich, USA).
HPLC analysis of valepotriates was performed in a
Shimadzu LC-10A gradient HPLC coupled with 2 LC-
10AD pumps, 10A UV detector and manual injector with a
20 ll sample loop. The separation conditions were: Waters
Nova pack C18 column (25 9 4.6 mm i.d.); mobile phase
acetonitrile: water; 80:20 (v/v); flow rate 1 ml/min;
detector wavelength 254 nm and sensitivity 0.04 Aufs.
Rooting and acclimatization
Healthy shoots of 4–5 cm were excised and cultured for
rooting on MS medium supplemented with different con-
centrations of auxins viz. indole-3-acetic acid (IAA), NAA
and IBA (0.05, 0.10, 0.20 mg/l) in 150-ml culture flasks
(Borosil, India). MS medium without growth regulators
was used as control. Plantlets with well-developed roots
were removed from the culture medium, washed gently
under running tap water, and transferred to root trainers
containing garden soil and acclimatized under greenhouse
condition (24 ± 2 �C temp. and 80 ± 5 % RH) without
use of any organic fertilizers.
Data analysis
The callus induction, shoot regeneration and rooting
experiments were repeated three times with minimum 24
explants each. All the valepotriates were analyzed with 3
replicates. To analyze the effect of various treatments, the
data were subjected to analysis of variance (ANOVA) and
statistical significance between mean values was assessed by
Duncan’s multiple range test at P \ 0.05 by using statistical
software SPSS ver. 15 (SPSS Inc., Chicago, USA).
Results and discussion
Callus induction
The disinfection treatment used was efficient for in vitro
establishment with approximately 94 % of the explants
remaining aseptic. In V. jatamansi callus induction varied
significantly depending on the explant type. Among dif-
ferent explants used, rhizomes have the highest callus
induction potential followed by leaf. Rhizome explants
started swelling within 6–8 days, whereas leaf and petiole
materials started within 10–12 days of inoculation. MS
basal medium without growth regulators exhibited no cal-
lus proliferation. All the calli in the growth regulators-
supplemented media were observed to be initiated from the
cutting edge of the explants. The induced calli were fast
growing, yellowish green and compact (Fig. 1a, b). The
callus induction frequency was found optimum in rhizome
explants (Table 1) on media supplemented with 0.5 mg/l
2,4-D (86 %) followed by 0.5 mg/l NAA (75.8 %). In our
study, 0.5 mg/l 2,4-D was the best auxin responsible for
obtaining the vigorous and compact callus from rhizome
(0.23 ± 0.06 g), leaf (0.17 ± 0.06 g) and petiole
(0.13 ± 0.03 g) explants. Effect of different explants and
growth regulators on callus induction of Allium chinense
was observed by Yan et al. (2009). Different tissues may
have different levels of endogenous hormones, and there-
fore, the type of explants source would have a critical
impact on the callus induction and its regeneration success.
The selection of proper donor plants and organs should
already be considered when starting the culture, unless it
can be overcome by a suitable treatment. However, in most
circumstances the dedifferentiation apparently also
involves some of the biochemical properties of the cells.
The modification of relative biosynthesis to degradation
ratios of a desired product can also influence the final levels
of a desired compound in the culture (Stintzing and Carle
2004).
The degree of callus formation was varied within all the
explants as well as PGRs used. Within different concen-
trations of NAA and IBA, 0.5 mg/l NAA produced the
richest callus from rhizomes (0.21 ± 0.07 g) with callus
induction frequency 75.8 % followed by 1.0 mg/l NAA
(0.16 ± 0.01 g) with 68.2 %. No somatic embryos or
adventitious roots formed from any callus. 2,4-D was
Acta Physiol Plant (2013) 35:55–63 57
123
considered as one of the most effective auxins for the callus
induction and found effective in case of Pennisetum
glaucum (Jha et al. 2009), Pinus caribaea (Akaneme and
Ene-Obong 2005), Tylophora indica (Thomas 2009) and
Juncus effusus (Xu et al. 2009). Medium fortified with
different concentrations of IBA produced significantly less
biomass than the other auxins used. However, the rate of
callus proliferation was inversely proportional to the con-
centrations of auxins in V. jatamansi (Table 1). Compact
calli are important in the in vitro cultures as they have
Fig. 1 Callus induction and regeneration of plantlets from callus in
V. jatamansi Jones. a 8-week-old vigorous callus (bar 10 mm)
induction from leaf explant on MS medium with 0.5 mg/l 2,4-D. b 8-
week-old vigorous callus induction (bar 10 mm) from rhizome
explant on MS medium with 0.5 mg/l 2,4-D. c, d Regenerated
plantlets (bar 15 mm) from callus on MS medium with 0.75 mg/l
TDZ and 0.5 mg/l NAA. e 8-week-old in vitro-raised plantlet with a
healthy root system. f Acclimatized plants in the greenhouse
condition and g 1-year-old established plants
58 Acta Physiol Plant (2013) 35:55–63
123
ability for organogenesis. They are more efficient to
develop chlorophyll than friable calli from the same
explants; this might be due to the chloroplast development
and integrity favored by cell aggregation (George and
Sherrington 1984).
Shoot regeneration from callus
The regenerative ability of proliferated compact calli was
studied by the application of cytokinins alone and in
combination with auxin. After 2 weeks of culture, most of
the calli turned green in order to give response towards
regeneration of plantlets. Shoot primordia appeared after
2 weeks in the regeneration medium. MS medium sup-
plemented with 0.75 mg/l TDZ in combination with
0.5 mg/l NAA showed the highest regeneration frequency
(88.6 %) and produced the highest number of shoot buds
(15.20 ± 0.20) capable of growing into single plants
(Table 2). Medium fortified with equal concentration
(0.5 mg/l) of TDZ and NAA produced 12.30 ± 0.21 shoots
having 80.4 % regeneration potential. Addition of NAA at
low concentration with TDZ-supplemented medium
Table 1 Effect of different concentrations of auxins on callus induction from different explants of V. jatamansi
Plant growth regulators (mg/l) Callus induction frequency (%) Mean weight of the callus (gm)
2,4-D NAA IBA Rhizome Petiole Leaf Rhizome Petiole Leaf
0.25 0.0 0.0 54.6 48.2 51.0 0.19 ± 0.09c 0.12 ± 0.02b 0.14 ± 0.07b
0.5 0.0 0.0 86.0 64.1 74.2 0.23 ± 0.06a 0.13 ± 0.03a 0.17 ± 0.06a
1.0 0.0 0.0 72.5 62.7 66.0 0.21 ± 0.04b 0.12 ± 0.03b 0.14 ± 0.06b
2.0 0.0 0.0 62.6 48.2 50.4 0.17 ± 0.08d 0.12 ± 0.04b 0.13 ± 0.06c
3.0 0.0 0.0 55.2 42.8 44.4 0.14 ± 0.01f 0.11 ± 0.05c 0.11 ± 0.06e
0.0 0.25 0.0 70.5 52.6 61.4 0.17 ± 0.01d 0.12 ± 0.02b 0.13 ± 0.03c
0.0 0.5 0.0 75.8 59.0 68.9 0.21 ± 0.07b 0.11 ± 0.06c 0.14 ± 0.09b
0.0 1.0 0.0 68.2 51.6 58.5 0.16 ± 0.01e 0.11 ± 0.06c 0.13 ± 0.08c
0.0 2.0 0.0 54.0 46.2 48.7 0.12 ± 0.01h 0.11 ± 0.06c 0.11 ± 0.07e
0.0 3.0 0.0 50.6 40.5 42.8 0.10 ± 0.01j 0.08 ± 0.09ef 0.09 ± 0.03fg
0.0 0.0 0.25 44.7 38.9 42.4 0.11 ± 0.01i 0.09 ± 0.04de 0.10 ± 0.05f
0.0 0.0 0.5 64.8 50.5 56.9 0.12 ± 0.01h 0.10 ± 0.04d 0.11 ± 0.05e
0.0 0.0 1.0 70.8 56.2 62.0 0.13 ± 0.07g 0.09 ± 0.04de 0.12 ± 0.07d
0.0 0.0 2.0 42.8 40.1 42.2 0.10 ± 0.05j 0.07 ± 0.04efg 0.08 ± 0.08gh
0.0 0.0 3.0 36.5 30.5 32.6 0.09 ± 0.04jk 0.07 ± 0.04efg 0.07 ± 0.04ghi
Values represent mean ± SE of three replicates with 24 explants each. Means within a column followed by different letters differ significantly at
P B 0.05 as compared by Duncan’s multiple range test
Table 2 Influence of different plant growth regulators on regeneration of shoot buds and elongation of proliferated shoots from callus
of V. jatamansi
Plant growth regulators (mg/l) Regeneration frequency (%) Mean number of shoots/callus Mean shoot length (cm)
TDZ Kn NAA
0.5 0.0 0.0 56.3 7.20 ± 0.24d 2.20 ± 0.17de
0.75 0.0 0.0 64.2 11.90 ± 0.31c 2.50 ± 0.08c
0.5 0.0 0.5 80.4 12.30 ± 0.21b 2.80 ± 0.07b
0.75 0.0 0.5 88.6 15.20 ± 0.20a 3.60 ± 0.07a
0.0 1.0 0.0 31.0 3.50 ± 0.09g 2.10 ± 0.11cd
0.0 2.0 0.0 34.8 3.70 ± 0.15g 2.30 ± 0.12cd
0.0 3.0 0.0 52.1 5.10 ± 0.17e 2.40 ± 0.11cd
0.0 1.0 0.5 53.0 5.30 ± 0.14e 2.10 ± 0.10cd
0.0 2.0 0.5 66.2 5.40 ± 0.30e 2.40 ± 0.12cd
0.0 3.0 0.5 48.2 4.70 ± 0.21ef 2.30 ± 0.06cd
Values represent mean ± SE of three replicates with 24 explants each. Means within a column followed by different letters differ significantly at
P B 0.05 as compared by Duncan’s multiple range test
Acta Physiol Plant (2013) 35:55–63 59
123
enhanced the callus regeneration frequency with more
number of shoot response (Fig. 1c, d). The same effect was
observed in case of Kn supplemented media in combina-
tion with NAA. However, 2 mg/l Kn along with 0.5 mg/l
NAA showed 66.2 % regeneration frequency proliferating
5.40 ± 0.30 shoots (Table 2). Synergistic effect of auxin
and cytokinin on shoot regeneration from calli were
observed in Solanum tuberosum (Shirin et al. 2007),
Cynondon dactylon (Zhang et al. 2007), Ipomoea obscura
(Mungole et al. 2009), Juniperus excels (Shanjani 2003),
Aframomum corrorima (Tefera and Wannakrairoj 2006)
and Pennisetum glaucum (Jha et al. 2009). Huetteman and
Preece (1993) and Gyves et al. (2001) emphasized the
potential use of TDZ in the regulation of adventitious shoot
proliferation and hypothesized on the synergism existing
between TDZ and other endogenous and exogenous auxins.
However, significant decreased shoot regeneration found in
case of medium supplemented with cytokinin alone than in
combination with auxin. The efficiency of TDZ on shoot
regeneration in many medicinal plants was reported
(Thomas 2003, 2007; Mithila et al. 2003; Sanikhani et al.
2006). According to George and Sherrington (1984) cell
differentiation and morphogenesis may be promoted by
insufficient oxygen environment such as in compact calli.
On the other hand, they reported that due to this factor
friability of callus is mostly associated with somatic
embryogenesis while compact callus can be readily used
for organogenesis. Also, in our case no somatic embryo-
genesis occurred during plant regeneration.
Effect of PGRs on valepotriates accumulation in callus
Callus derived from medium supplemented with different
concentrations of 2,4-D (0.5, 1.0 mg/l), NAA (0.5, 1.0 mg/l)
and IBA (1 mg/l) were subjected to HPLC analysis for
quantifying ACE, VAL and DID and the result revealed
that all treatments showed the presence of valepotriates.
Medium with 2,4-D (1 mg/l) was found to be responsible
for increasing ACE (Fig. 2a) and DID (Fig. 2c) yield,
whereas VAL (Fig. 2b) production was higher in case of
medium supplemented with NAA (1.0 mg/l). The
increased concentration of auxins like 2,4-D and NAA
could have caused a mild but chronic oxidative stress
response in undifferentiated mass of cells potentially
capable of inducing valepotriate accumulation. Reactive
oxygen species have been shown to trigger the production
of various secondary metabolites, including terpenes (Zhao
et al. 2005). 8-week-old cultures produced optimum bio-
mass and valepotriates yield (Fig. 2). The accumulation of
valepotriates in callus decreased in logarithmic phase after
8 weeks. Previously, the presence of valepotriates was
characterized in callus of Valeriana glechomifolia (Maur-
mann et al. 2009). In our study, ACE and DID were the
major valepotriates found in V. jatamansi callus.
IBA was not beneficial for the valepotriate production,
as it helped to accumulate significantly lower concentration
of ACE, VAL and DID than 2,4-D and NAA. The observed
impact of auxins on valepotriate metabolism was not nec-
essarily dependent on phytohormone-induced develop-
mental changes on callus (De Klerk et al. 1999). The
benefits of auxin exposure were apparently correlated to
auxin stability, since metabolically stable type of auxins,
such as IBA was not beneficial for valepotriate yield.
Differences in auxin stability and auxin-induced secondary
effects could also explain the concentration and auxin-type
dependence of valepotriate content responses in calli (Bello
0
0.2
0.4
0.6
0.8
1
1.2
0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBAAce
valtr
ate
cont
ents
(g%
dry
wei
ght)
Auxin type and concentration
4 wk
8wk
12wk
0
0.1
0.2
0.3
0.4
0.5
0.6
0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBA
Val
trat
e co
nten
ts (
g% d
ry w
eigh
t)
Auxin type and concentration
4 wk
8wk
12wk
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0.5 mg/l 2,4-D 1.0 mg/l 2,4-D 0.5 mg/l NAA 1.0 mg/l NAA 1.0 mg/l IBADid
rova
ltria
te c
onte
nt (
g% d
ry w
eigh
t)
Auxin type and concentration
4 wk
8wk
12wk
a
b
c
Fig. 2 Time-course effect of different plant growth regulators on
valepotriate accumulation in callus of V. jatamansi from rhizome
explants: a influence of different efficient auxins on acevaltrate accu-
mulation, b influence of different efficient auxins on valtrate
accumulation, c effect of different efficient auxins on didrovaltrate
accumulation. Bar indicates mean of 3 replicates ±SE (n = 10)
60 Acta Physiol Plant (2013) 35:55–63
123
de Carvalho et al. 2004). However, the comparative anal-
ysis of individual valepotriate accumulation within every
4-week interval showed significant variation among all the
treatments analyzed. Active secondary metabolite produc-
tion through callus culture is known to be an effective
process in pharmaceutical industries. Several industrially
important secondary metabolites, viz., anthocyanin from
Panax sikkimenesis (Mathur et al. 2010), cerpegin from
Ceropegia juncea (Nikam and Savant 2009), cynarin
from Cynara cardunculus (Silvia et al. 2006), rosmarinic
acid from Lavandula officinalis (Georgiev et al. 2006),
rutin from Hemidesmus indicus (Misra et al. 2005), crocin
from Crocus sativus (Chen et al. 2003), lithospermic acid
from Salvia miltiorrhiza (Morimoto et al. 1994) and
piceatannol from Arachis hypogaea (Ku et al. 2005) were
produced by in vitro callus culture.
Rooting and acclimatization
In vitro shoots, regenerated from shoot clusters proliferated
on multiple shoot induced medium and callus regeneration
medium were separated and used for the rooting experi-
ments. Rooting initiated after 2 weeks in all cultures
including control. But, the response of rooting (%), number
and length of roots were achieved significantly higher
when cultured with auxin-supplemented media. Among
different auxins used, NAA (0.05, 0.1 mg/l) and IAA
(0.1 mg/l) were effective in producing longer and healthy
roots with 100 % response (Table 3). IBA showed signif-
icantly lower root-inducing potential both in response to
root numbers and root length. MS medium fortified with
0.1 mg/l NAA produced the highest number of roots
(17.30 ± 2.01) followed by 0.05 mg/l NAA (12.40 ±
1.20). NAA when used as low concentrations was consid-
ered as an effective rooting hormone in many plant systems
(Mao et al. 1995; Sanches-Gras and Calvo 1996; Rout et al.
2000). Also, IAA was reported to enhance the root for-
mation in case of Hedeoma multiflorum (Koroch et al.
1997) and Woodfordia frusticosa (Krishnan and Seeni
1994). However, MS basal medium without auxins pro-
duced significantly lower number of roots and were not
found healthy for hardening in greenhouse. There are many
reports on the microshoots of various medicinal plants
rooted on only MS medium without the growth regulators
(Christine and Chan 2007; Mao et al. 1995). Explants
having a functional rooting system are more likely to sur-
vive transition to greenhouse. Roots were washed thor-
oughly before being transferred to root trainers.
Micropropagated plantlets with well-developed root system
were successfully acclimatized in greenhouse condition, in
root trainers containing garden soil with a survival
frequency of 100 % (Fig. 1f). The in vitro-derived plants
were phenotypically similar to the parental stock and no
morphological abnormalities have been observed in the
micropropagated plants.
Conclusion
A highly efficient and reproducible protocol was developed
for the large-scale production of V. jatamansi. The indirect
organogenesis system of Indian valerian can provide a
mass production of disease-free and genetically uniform
plant materials throughout the year for the fulfillment of
market demand globally and conservation of the species.
The standardized protocol can offer stable biomass and
continuous valepotriate production for the pharmaceutical
industries. Furthermore, using bioreactor, callus induction
protocol can be useful for continuous callus culture and
the extraction of valepotriate on a commercial scale.
Table 3 Effect of different concentrations of auxins on rooting of in vitro-raised elongated shoots in V. jatamansi
Auxins (mg/l) Response of rooting (%) Mean number of roots/explant Mean root length (cm)
NAA IBA IAA
0.0 0.0 0.0 34.2 3.30 ± 0.61e 4.10 ± 0.21e
0.05 0.0 0.0 100 12.40 ± 1.20b 8.30 ± 0.24b
0.10 0.0 0.0 100 17.30 ± 2.01a 9.30 ± 0.39a
0.20 0.0 0.0 64.6 7.20 ± 0.82d 7.90 ± 0.29bc
0.0 0.05 0.0 38.5 6.15 ± 0.98d 7.70 ± 0.21bc
0.0 0.10 0.0 60.8 10.80 ± 1.85bc 8.10 ± 0.21bc
0.0 0.20 0.0 42.0 5.20 ± 0.61d 6.60 ± 0.20d
0.0 0.0 0.05 75.2 7.80 ± 0.38d 7.90 ± 0.32bc
0.0 0.0 0.10 100 12.10 ± 1.29b 9.20 ± 0.39a
0.0 0.0 0.20 52.5 5.80 ± 0.61d 7.60 ± 0.20bc
Values represent mean ± SE of three replicates with 24 explants each. Means within a column followed by different letters differ significantly at
P B 0.05 as compared by Duncan’s multiple range test
Acta Physiol Plant (2013) 35:55–63 61
123
However, the valepotriate accumulation mainly depends on
the growth regulators used and age of the calli. As V. jat-
amansi is a highly traded medicinal plant due to extensive
use of its industrially important secondary metabolites, the
present micropropagation system can be utilized for further
research on metabolic pathways and transgenic approach to
build up the value added products and production of quality
material.
Author contribution J. Das performed the experiment,
analyzed the data and wrote the manuscript. A.A. Mao and
P.J. Handique designed the work, edited the manuscript
and supervised the entire research.
Acknowledgments The authors are thankful to the Joint Director,
BSI, Shillong, for providing the facilities and to the Department of
Biotechnology (DBT), Government of India, New Delhi, India, for
the award of research fellowship to J.D.
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