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Plant Biotechnology Reports

ISSN 1863-5466

Plant Biotechnol Rep
DOI 10.1007/s11816-013-0273-4
Improved furanocoumarin production in
Ruta graveolens L. regenerated via in vitro
stem internode cultures
Sagarika Bohidar, Suchismita Pattanaik
& Manikkannan Thiruvanoukkarasu
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ORI GI NAL ARTI CLE
Improved furanocoumarin production in Ruta graveolens L.
regenerated via in vitro stem internode cultures
Sagarika Bohidar

Suchismita Pattanaik

Manikkannan Thiruvanoukkarasu
Received: 6 November 2012 / Accepted: 10 January 2013
Korean Society for Plant Biotechnology and Springer Japan 2013
Abstract A rapid and efcient in vitro propagation pro-
tocol by enhanced multiple shoot proliferation from inter-
node cultures of Ruta graveolens was established. Mean
shoot number was maximum (55.83) in Murashige and
Skoog (MS) basal medium fortied with 1.0 mg L
-1
benzyl amino purine and 0.25 mg L
-1
indole-3-acetic acid.
The elongated shoots rooted within 1012 days in 1/2-
strength MS medium supplemented with 2.0 mg L
-1
indole 3-butyric acid. About 80 % of the rooted plantlets
survived acclimatization and transfer to the eld. Phyto-
chemical analysis revealed that micropropagated plants
produced linear furanocoumarins, characteristic of the
species, in greater quantities as compared to the in vivo-
grown plants. The results will facilitate the conservation of
this valuable medicinal plant and to obtain plants with
improved phytochemical constituents.
Keywords Auxins In vitro propagation Linear
furanocoumarins Ruta graveolens Stem internode
Introduction
Ruta graveolens L. (Family: Rutaceae) has been reported
to be a rich source of linear furanocoumarins (FCs), i.e.
bergapten (5-methoxypsoralen), and xanthotoxin (8-meth-
oxypsoralen), which have many therapeutic properties.
Many pharmacological tests have revealed that couma-
rins have antitumor, antioxidative, antimicrobial, anti-
inammatory and antimutagenic effects, as well as inu-
encing insect hormonal activities (Khatune et al. 2004;
Bapat et al. 2005; Xiao et al. 2010; Jan et al. 2012). Owing
to the high cost of chemical synthesis of bergapten, phar-
maceutical industries use it as a by-product of the essential
oil (bergamot oil) of Citrus bergamia. However, in recent
times, there has been a sharp decline in the source plants due
to over-exploitation and lack of simultaneous cultivation,
thereby posing a threat to the pharmaceutical industries and
compelling the search for new sources for bioproduction of
furanocoumarins. R. graveolens was reported to be one
of the most promising candidates (Poutaraud et al. 2000;
Diwan and Malpathak 2008), as it contains high quantities
of four linear, commercially important, FCs: psoralen,
bergapten, xanthotoxin, and isopimpinellin.
In spite of its well-known potential as a valuable
medicinal plant, R. graveolens is not available in abun-
dance in the wild and its cultivation is restricted to a few
pockets in Orissa. The species is generally propagated
through conventional vegetative methods. Propagation
through seeds is an intricate task due to the physical dor-
mancy caused by an impermeable seed coat resulting in
poor germination (Diwan and Malpathak 2008). Moreover,
the conventional propagation methods of this species are
not only inadequate to cater to the needs but it is also time
consuming. Therefore, development of a rapid mass
propagation protocol of this medicinally important plant
has become essential in order to reduce the existing pres-
sure on the wild population as well as to bring about a cost
reduction for commercial exploitation, with emphasis on
the use of in vitro cultures. Earlier reports (Massot et al.
2000; Ekiert et al. 2001), described the use of in vitro
germinated seedlings for raising shoot cultures. However,
as Ruta is a cross-pollinated plant, shoots thus obtained
would not be genetically identical to the parent plant and
S. Bohidar S. Pattanaik M. Thiruvanoukkarasu (&)
Bioresources Engineering Department, CSIR-Institute
of Minerals and Materials Technology,
Bhubaneswar 751013, Odisha, India
e-mail: mtarasu@yahoo.com; arasu@immt.res.in
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Plant Biotechnol Rep
DOI 10.1007/s11816-013-0273-4
Author's personal copy
the genetic makeup may vary with individual shoots. This
may lead to variations in FCs production. Therefore, direct
organogenesis from vegetative parts is preferred as there
are fewer chances of somaclonal variation and the plants
obtained are true to type (Diwan and Malpathak 2008).
A large biomass of herbal raw material with fewer
efcacies will not fulll the quality control requirement of
the international market and will also not fetch high eco-
nomic values for a sufcient time in the domestic market.
In this scenario, a detailed comparative phytochemical
evaluation of micropropagated and the mother plants
becomes indispensable to select the right quality plant
material for better quality of drugs. A plant may grow well
in different situations but may fail to produce the same
constituents, because active constituents present in the
plant system are inuenced by the physiological and
environmental factors. The advantage of developing plants
through tissue culture is that they are grown under
controlled environmental conditions, and hence, their
phytochemical constituents are believed to be maintained.
The overall objective of the current study was to develop
an in vitro system for rapid propagation of R. graveolens
plantlet cultures that could contain high levels of
furanocoumarins.
Materials and methods
Source material and explants preparation
Healthy shoots, with 45 nodes, were collected from
6-month-old plants of R. graveolens maintained in the
experimental garden of the CSIR-Institute of Minerals and
Materials Technology (IMMT), Bhubaneswar (2017
0
45
00
N,
8549
0
15
00
E; altitude 58 m), India. After trimming the
leaves, the shoots were cut into pieces (5.07.0 cm long),
washed thoroughly under running tap water, then disinfec-
ted with 0.1 % HgCl
2
solution for 34 min, and nally
washed with sterile double-distilled water under sterile
condition. Stem internode segments measuring 2.53.0 cm
were prepared from the sterilized shoots and used for
in vitro culture.
Culture medium and culture condition
Murashige and Skoog (1962) basal media containing
30 g L
-1
sucrose and 7 g L
-1
agar with benzyl amino
purine (BAP) either alone or in combination with auxins
indole-3-acetic acid (IAA) or a-naphthalene acetic acid
(NAA) was employed in the present study. Previous
experiments on multiple induction in nodal segments cul-
ture (Bohidar et al. 2008) and petiole cultures (unpublished)
revealed that synthetic cytokinin BAP at 1.0 mg L
-1
con-
centration exerted the highest shoot formation. Hence,
throughout the study, BAP 1.0 mg L
-1
concentration
was xed while auxin was used in varied concentrations
(0.25, 0.5, 1.0, 2.0, and 3.0 mg L
-1
). For root induction,
half-strength MS medium supplemented with different
auxins, indole-3-butyric acid (IBA), NAA, and IAA, in
concentrations ranging from 0.25 to 4 mg L
-1
were used.
The pH of the media was adjusted at 5.8 prior to autoclaving
at 121 C and 108 kPa for 20 min. All chemicals used in the
present work were of analytical grade (Sigma Chemical,
USA; Merck; Hi-Media; and Qualigens India). All the
cultures were maintained at 25 2 C and 60 5 % rel-
ative humidity in a culture room under a 16-h photoperiod
of 40 lmol m
2
s
-1
light intensity provided by cool white
uorescent tubes.
Shoot induction, in vitro rooting and acclimatization
Disinfected stem internode segments were inoculated onto
the various shoot induction media compositions (Table 1)
and maintained for 4 weeks and sub-cultured onto the same
media compositions following expiry of a period of
4 weeks. When the shoots were more than 3.0 cm long,
they were transferred to rooting media (Table 2) and cul-
tured for 5 weeks. Regenerated plants with healthy root
systems were washed (especially the root portions) under
running tap water for about 15 min and the plantlets were
then transferred to root trainers containing pre-soaked
sterilized vermiculite medium and kept inside the mist
chamber. The timer was set to 1 min on and 30 min off,
and the relative humidity was set to 90 %. After 4 weeks,
they were transferred to poly-bags containing soil ?
sand ? farmyard manure (1:2:1) and shifted to a shade-net
house for 6 weeks and subsequently transferred to the eld.
The acclimatized plants were successfully transferred and
established in the eld, and these plants are termed as ex
vitro plants. The survival percentage was recorded after
4 weeks of transfer to the eld.
Estimation of furanocoumarins
Healthy, 5-month-old ex vitro plants of R. graveolens were
evaluated for their furanocoumarin contents by HPLC and
the values were compared with that of in vivo-grown plants.
The plant materials were oven dried at 60 C for 8 h. The
dried plant materials were powdered using a mixer grinder.
Dried plant material weighing 200 mg was subjected to
cold extraction with ethyl acetate overnight. The macera-
tion process was repeated thrice and, at the end of the third
day, the ethyl acetate extracts were pooled together. The
solvent was removed under vacuum at temperature below
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50 C in a rotary evaporator and the extracts were stored in
a refrigerator at 4 C until further use.
One milligram of each of the plant extracts was weighed
separately and dissolved in methanol. The quantitative
analysis of two FCs, i.e. xanthotoxin (xanthotoxin) and
bergapten (5-MOP), was carried out by HPLC methods
using Shimadzu Japan/LC-10Avp apparatus coupled to a
photo diode array (DAD) on Xtimate
TM
C-18, 4.6 9
250 mm, 5 lm column. The isocratic solvent system was
methanol: water 1:1.2 v/v. The ow rate was 1 mL min
-1
and detection was performed at 310 nm. Identication and
quantication of the secondary metabolites was carried out
by comparing the retention time of the relevant peaks with
their reference standards.
Experimental design and statistical analysis
At the end of the 6 weeks of the in vitro multiplication
cycle, the following parameters were determined: the per-
centage of explant response, number of shoots longer than
8.0 mm, and the length of the shoots produced by each
explant. Rooting experiments were evaluated by deter-
mining rooting percentage, root number, and root length.
Length of shoots and roots were intended as a cumulative
length of each organ on one plant. Each treatment consisted
of 10 explants and all the experiments were repeated thrice.
The data were analyzed statistically using analysis of var-
iance ANOVA, and the means were compared by Duncans
multiple range test at a 5 % probability level according to
Gomez and Gomez (1984).
Table 1 Shoot regeneration from stem internode segments of Ruta graveolens cultured in MS ? cytokinin (BA 1.0 mg L
-1
) along with auxins
(IAA and NAA) supplements
Cytokinin ? auxin (A)
Conc. mg L
-1
(B)
% Response Mean shoot numbers Mean shoot length (cm)
BAP ? IAA BAP ? NAA BAP ? IAA BAP ? NAA BAP ? IAA BAP ? NAA
0.25 96.66 76.66 55.83
a
8.73
f
3.29
a
3.20
a
0.5 90.00 70.00 43.00
b
7.50
f
3.43
a
2.17
b
1.0 83.33 66.66 33.50
c
6.50
f
3.62
a
1.70
c
2.0 83.33 76.66 27.00
d
5.67
f
2.44
b
1.67
c
3.0 93.33 76.66 21.57
e
4.37
f
2.04
b
1.27
d
Mean shoot numbers Mean shoot length (cm)
A B A B
F value 759.74* 40.16* 194.29* 66.35*
SEM 0.760 1.202 0.049 0.077
CD at 5 % 2.116 3.346 0.136 0.215
Means followed by the same letter are not signicantly different at P = 0.05 of Duncans multiple range test
Observations were made 6 weeks after culture (n = 30)
SEM standard error of mean, CD critical difference
* P = 0.05
Table 2 Effect of auxins on rooting of in vitro shoots of R. graveo-
lens in half-strength MS medium submitted with auxins
Auxin concentrations
(mg L
-1
)
Rooting
rate (%)
Mean root
number/shoot
Mean root
length/shoot (cm)
IBA
0.25 63.33 4.57
k
6.82
a
0.5 76.66 5.63
j
5.15
b
1.0 86.66 5.73
j
4.95
b
2.0 73.33 8.40
g
4.85
b
3.0 73.33 7.57
h
4.14
c
4.0 73.33 6.30
i
3.97
c
NAA
0.25 80.00 24.47
c
2.40
f
0.5 80.00 25.23
b
2.35
f
1.0 83.33 26.73
a
1.76
g
2.0 83.33 17.87
d
2.24
f
3.0 80.00 14.40
e
1.44
h
4.0 83.33 10.70
f
0.81
i
IAA
0.25 80.00 4.03
l
3.41
d
0.5 86.66 6.30
i
3.00
e
1.0 83.33 7.67
h
4.52
c
2.0 70.00 8.20
g
4.34
c
3.0 73.33 6.40
i
4.03
4.0 70.00 5.03
j
3.96
c
Observations were made after 4 weeks of culture
Means followed by the same letter are not signicantly different at
P = 0.05 of Duncans multiple range test
Means obtained from 30 observations
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Results and discussion
In vitro culture
Variation in shooting response was observed due to the
auxin concentration in the medium (Table 1). Initiation of
multiple shoots in the stem internode explants observed on
the both ends within 710 days of inoculation (Fig. 1a).
Control treatments involving no plant growth regulators
produced no shoots at all. Taking the data on shoot pro-
duction from stem internode explants into account,
increasing concentrations of auxin caused a dramatic
decrease in terms of both the mean number of shoots per
explant and the percentage of explants producing shoots.
Between the two auxins IAA and NAA tested for efcacy in
shoot induction, IAA showed superior to NAA. This is in
line with the result obtained in Solanum nigrum in vitro
culture (Sridhar and Naidu 2011). The highest number of
shoots (55.83 shoots per explant at 96.6 % frequency) was
obtained when 1.0 mg L
-1
BAP was combined with
0.25 mg L
-1
IAA (Table 1). In such combinations, shoots
regenerated directly without the intervention of callus for-
mation. Multiple shoot buds occurred as mass clusters from
both cut ends. After a period of 4 weeks, a cluster of
regenerating shoot buds covered the explant. Hence, the
explants with shoot clumps were dissected into two
pieces and sub-cultured on the same media composition
(Fig. 1b, c) for further shoot elongation. The rate of mul-
tiplication increased as the number of subcultures increased
(every subculture was made at 4-week intervals). This was
probably due to adaptation of the explants to in vitro con-
ditions. Similar observations have been reported for Pic-
rorhiza kurroa (Upadhyay et al. 1989), Plumbago zeylanica
(Rout et al. 1999) and Clitoria ternatea (Rout 2004).
Many authors report that cytokinin is required in optimal
quantity for shoot proliferation in many genotypes, but that
inclusion of a low concentration of auxin along with
cytokinin increases the rate of shoot multiplication (Shas-
any et al. 1998; Rout et al. 2000; Abdullah et al. 2003;
Thirunavoukkarasu et al. 2006; Nayak et al. 2007). MS
medium fortied with 1.0 mg L
-1
BAP in combination
with NAA (0.253.0 mg L
-1
) were less effective than IAA
comprising media for multiple shoot induction (Table 1).
Contrary to the present nding, Faisal et al. (2005)
observed better shoot formation in the presence of NAA
rather than IAA in nodal segment culture of R. graveolens.
This contradiction may be attributed to the explant type
and its source. Shoot inducing capacity was better when
Fig. 1 Plantlet regeneration
from stem internode culture of
Ruta graveolens L. a Initiation
of multiple shoots in the stem
internode explants on the both
ends, cultured in
MS ? 1.0 mg L
-1
BA ? 0.25 mg L
-1
IAA
(10 days old). b, c Shoot clumps
developed on the stem internode
were dissected and sub-cultured
on the same medium for further
shoot elongation (4 weeks old).
d A single shoot rooted in 1/2-
MS ? 2.0 mg L
-1
IBA
(4 weeks old). e Acclimatized
plants showing normal growth
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explants were inoculated in BAP 1.0 mg L
-1
? NAA
0.25 mg L
-1
. With a further increase in NAA concentra-
tion, there was decline in the percentage of cultures with
multiple shoots and the mean number of shoots per culture.
A higher concentration (3.0 mg L
-1
) favored callus growth
immediately after a week of culture.
Auxin type and concentration used in combination with
BAP profoundly affected shoot growth of the in vitro-
proliferated shootlets. Plants gained higher overall shoot
length in BAP ? IAA-fortied medium as compared to
BAP ? NAA comprising the media. MS medium supple-
mented with BAP 1.0 mg L
-1
? IAA 1.0 mg L
-1
pro-
duced shoots with highest shoot length (3.62 cm);
however, a further increase in the IAA concentration had
an antagonistic effect on shoot length. Likewise, in the case
of BAP ? NAA-fortied media, maximum shoot length
(3.20 cm) was obtained at 0.25 mg L
-1
concentration
which further diminished with increasing NAA concen-
tration. Consequently, differential responses of explants to
the auxin concentrations may be attributed to endogenous
hormone levels of explants. Wernicke et al. (1986) opined
that increased auxin concentrations prevented meristematic
cell divisions, resulting in decreased reactions.
Well-developed shoots with a length of ([3.0 cm) were
excised and transferred to half-strength MS medium sup-
plemented with different concentrations of auxins such as
NAA, IBA and IAA (0.251.0 mg L
-1
). Although the
highest number of roots per shoot was observed with
1.0 mg L
-1
NAA (26.73 roots per shoot at 83.33 % fre-
quency), the roots failed to elongate, attained mean root
length of 1.76 cm, remained fused, and exhibited poor
growth. The number of healthy roots produced per shoots
was higher when the rooting medium contained IBA,
especially at the 2.0 mg L
-1
concentration which induced
lengthy roots (Table 2; Fig. 1d). From the present ndings,
auxin IBA proved to be better than IAA and NAA in terms
of inducing healthy roots per shoot. This is in close
conformity with the results obtained in Solanum nigrum
(Sridhar and Naidu 2011) in vitro culture studies. Reports
concerning in vitro rooting studies reveal that IBA has been
widely used as a root-inducing hormone in difcult-to-root
plants both under in vitro and in vivo conditions (Minocha
1987). The stimulatory effect of IBA on root formation has
also been reported in many medicinal plants like Murraya
koenigii (Bhuyan et al. 1997), Ocimum basilicum (Sahoo
et al. 1997), and Clitoria ternatea (Barik et al., 2007). The
control treatment involving no auxins did not produce any
roots at all. Addition of any of these three root-inducing
hormones was essential for rooting of in vitro shoots.
Plantlets with well-developed root systems were sepa-
rated from the culture tubes, washed, and transferred
to polypots containing vermiculite for hardening. After
3 weeks of hardening, the plantlets were transferred to the
polybags lled with a mixture of soil, sand, and FYM
(2:1:1). The acclimatized plants showed normal growth
(Fig. 1e). Finally, the hardened plantlets were transferred
to eld conditions. The survival rate was 80 % after
1 month. Field-transferred plants were periodically
watered and after 5 months growth period in the eld, a
few plants were randomly selected and processed for fur-
anocoumarin estimation.
Furanocoumarin quantication
The results of the analysis of concentrations of the FCs
(xanthotoxin and bergapten) in root, shoot and fruit organs
of in vitro- and eld-raised plants are shown in Fig. 2. The
data revealed that the concentrations of both the FCs was
always greater in ex vitro-derived plants, i.e. plants
regenerated via in vitro culture, as compared to that of
in vivo-grown plants.
Xanthotoxin concentration in shoots of ex vitro-raised
plants was 671 lg/g of DW which was about 3.4 times
greater than that of in vivo plants (197 lg/g of DW). Roots
of micropropagated plants contained maximum amounts of
xanthotoxin (9,089 lg/g of DW) whereas the same extracts
from in vivo plants had very negligible concentrations of
the secondary metabolite, amounting to only 185 lg/g of
DW. Likewise, fruit organs of ex vitro plants accumulated
more xanthotoxin (209 lg/g of DW) than the in vivo fruit
parts (87 lg/g of DW).
Similar to xanthotoxin, bergapten concentrations were
also strikingly higher in ex vitro shoot (10,076 lg/g
of DW), root (10,656 lg/g of DW), and fruit organs
(7,193 lg/g of DW) as compared to in vivo plants. The
different levels of bergapten concentrations detected in
in vivo plant parts were: shoots (143 lg/g of DW), roots
(3,014 lg/g of DW), and fruits (3,107 lg/g of DW),
respectively.
Fig. 2 Bergapten (5-MOP) and xanthotoxin (8-MOP) production
from different organs (fruit, root and shoot) in ex vitro and in vivo
plants of R. graveolens
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Baskaran and Jayabalan (2008) reported higher psoralen
contents in ex vitro plant parts as compared to in vivo plant
parts. Similarly, Kang et al. (2004) reported enhanced
production of tropane alkaloids in ex vitro-propagated
plants than in the in vivo plants of Scopolia parviora.
Enhanced accumulation of secondary metabolites in tissue
culture cells typically occurs under specic conditions.
Mulabagal and Tsay (2004) have established that optimi-
zation of culture conditions results in increased accumu-
lation of several secondary metabolites in cultured cells as
compared to the native plants. Therefore, in the present
study, increased furanocoumarin levels may be attributed
to the in vitro stress conditions (incubation culture condi-
tion). The change in secondary metabolite patterns with the
introduction of plant material into in vitro conditions has
been described earlier for several other plant species
(Luczkiewicz and Glod 2003, 2005; Lucchesini et al.
2009). The hormonal composition of the culture media is
known to be one of the main causes of morphological and
physiological modication in regenerated plantlets (Van
Staden et al. 2006), and in turn it could alter the plant
biomass capacity to produce secondary metabolites.
In summary, an efcient protocol for micropropagation of
an important medicinal plant, R. graveolens, was developed
in this study. An improved phytochemical constituent of
in vitro-raised plants was observed. The results will make the
enhanced production of furanocoumarin much easier.
Acknowledgments The authors are grateful to the Director, CSIR-
Institute of Minerals and Material Technology, Bhubaneswar for
providing facilities. One of the authors (S.B.) wishes to thank C.S.I.R.
for providing a fellowship (SRF).
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