Plant Growth Regul

DOI 10.1007/s10725-014-9957-1

ORIGINAL PAPER

Plantlet regeneration of Paris polyphylla Sm. via thin cell layer

culture and enhancement of steroidal saponins in mini-rhizome
cultures using elicitors
Shiveirou Raomai Suman Kumaria
Mechuselie Kehie Pramod Tandon

Received: 23 April 2014 / Accepted: 24 June 2014

Springer Science+Business Media Dordrecht 2014

Abstract An efficient regeneration protocol for the

medicinal plant, Paris polyphylla Sm. was developed
through the formation of mini-rhizomes (MRs) using
transverse thin cell layer (tTCL) culture technique. MRs
were induced from tTCL explants derived from the basal
and middle stem portions while apical portion failed to
show any kind of response. Highest response percentage
(86.6 %) of MRs formation with a maximum fresh weight
(1.05 0.08 g) was achieved from basal sections cultured
on MS medium supplemented with 0.5 mg/l 6-benzylaminopurine (BAP). MRs transferred to plant growth regulator free medium gave rise to shoot buds that eventually
regenerated into plantlets and were successfully acclimatized with a survival percentage of more than 95 % under
greenhouse conditions. Quantification through reversephase HPLC showed 1.41-fold higher content of total steroidal saponins in MRs cultured on medium supplemented
with 0.5 mg/l BAP as compared to the field-grown rhizome. Elicitation of MRs liquid culture with chitosan,
salicyclic acid (SA) and yeast extract enhanced the production of steroidal saponins but resulted in reduced
growth rate. Highest total steroidal saponins content (87.66 1.66 mg/g DW) was achieved in cultures
treated with SA at 50 mg/l after 30 days of elicitation
which is 3.6 times higher than the in vivo rhizome. The
developed protocol would facilitate the conservation of this
valuable medicinal plant and could be used as a ready stock
to meet the demands of the pharmaceutical industry for
steroidal saponins productions.

S. Raomai
S. Kumaria (&)
M. Kehie
P. Tandon

Plant Biotechnology Laboratory, Department of Botany,
Centre for Advanced Studies, North-Eastern Hill University,
Shillong 793022, India
e-mail: sumankhatrikumaria@gmail.com

Keywords Elicitors
Medicinal plant
Mini-rhizome

Paris polyphylla
Steroidal saponins
Thin cell layer
Abbreviations
BAP
6-Benzylaminopurine
CHI
Chitosan
DW
Dry weight
FW
Fresh weight
HPLC High performance liquid chromatography
KIN
Kinetin
MR
Mini-rhizome
MS
Murashige and Skoog
SA
Salicyclic acid
TDZ
Thidiazuron
tTCL
Transverse thin cell layer
PGR
Plant growth regulator
YE
Yeast extract

Introduction
Paris polyphylla Sm. is a perennial herbaceous plant of the
family Trilliaceae which is distributed mainly in East Asia,
China and the Himalayas. In India, it is locally known as
Satwa and mainly used in Unani and ayurvedic medicine
preparations (Khare 2007). Its rhizomes are widely used in
Nepal as an antihelmintic, antispasmodic, digestive stomachic, expectorant and vermifuge (IUCN 2004, Bhattarai
and Ghimire 2006). In China, it is one of the famous
medicinal plants commonly known as Chonglou and
traditionally used not only as an anti-cancer, antibiotic and
anti-inflammatory drug, but also to treat snake bite, parotitis, mastitis, chronic bronchitis, injuries from fractures as

123

Plant Growth Regul

well as to stop bleeding (Zhou 1989) and for treating liver

cancer (Cheung et al. 2005). The rhizome of this plant has
been developed into patented Chinese medicines such as
Yunnan Bai Yao, Gong Xue Ning capsules and Jidesheng-she-yao-pian tablet which are used to treat dispersing blood stasis and hemostasis, activate blood circulation, alleviate pain, detoxification, reduce swelling,
inflammation and prevent bleeding (He et al. 2006).
Pharmacological and phytochemical investigations
revealed that the curative properties are associated with
steroidal saponins, present chiefly in the rhizome of the
plant (Zhang 2007). The steroidal saponins from P. polyphylla have been shown to have significant biological
activities that includes antitumor (Wu et al. 2004; Lee et al.
2005; Sun et al. 2007; Zhao et al. 2009; Man et al. 2013)
antifungal (Deng et al. 2008), antihelmintic (Devkota et al.
2007; Wang et al. 2010), antioxidant (Pan et al. 2004),
antimutagenic (Lee and Lin 1988), enhancement of
phagocytosis (Zhang et al. 2007), inhibition of gastric
lesion (Matsuda et al. 2003) and inhibitory activities
against abnormal uterine bleeding (Fu et al. 2008).
Due to its enormous medicinal use in China, there has
been an en masse trading of the dried rhizome from India to
China, through Indo-Myanmar border especially from
Manipur leading to the present endangered status of the
plant (Mao et al. 2009). At present, rhizomes collected
directly from the wild are the only source of raw material
for medicinal usage, with no cultivation measures reported
so far. In nature, the plant reproduces through seeds or
rhizome buds. However, its cultivation is difficult because
of long seed dormancy period (more than 18 months) and a
germination percentage of about 40 % (Li 1986). The other
limiting factor is slow growth, taking about 45 years from
seed to flowering and another 3 or 4 years to develop into
commercial size rhizome thus restricting the large scale
multiplication of this species for pharmaceutical purposes.
Therefore, there is an urgent need to develop an effective
technique for its rapid propagation and also efficient
strategies for metabolites production so as to conserve and
meet the ever increasing demand.
Plant tissue culture is a useful tool for the conservation
and rapid propagation of medicinally important and
endangered plants (Baskaran and Jayabalan 2008). It is the
only technology for the production of large quantities of
elite planting material so as to increase the biomass and
productivity (Kehie et al. 2013). The thin cell layer technique has been used for mass propagation of some
important medicinal plants species such as Panax ginseng
(Nhut et al. 2003) and Spilanthes acmella (Singh et al.
2009). The technique involves the use of small sized
explants excised from different plant organs either longitudinally (lTCL) or transversally (tTCL) (Silva 2003) and
was first described in Nicotiana tabacum (Van Tran Thanh

123

Fig. 1 Plantlet regeneration via MRs formation from tTCL. a rhizome c

terminal bud, b exposed preformed shoot after removal of outer scales
from the terminal bud, c tTCL from apical part of the stem showing
phenolic accumulation, d initial response of tTCL from the middle
part of stem, e initial response of tTCL from the basal part of stem,
f well developed MRs from the basal section, g protuberances formed
on MR after transfer to PGR-free medium, h well developed shoot
buds with roots formed on MR, i histological section of in vivo
rhizome, j histological section of MR, k longitudinal section of
protuberances formed on MR, l complete plantlet developed from
shoot bud, m acclimatized plantlets under greenhouse conditions after
2 months. Scale bars: a, b, g, h, l = 1 cm; c, d, e, f, k = 1 mm; i,
j = 0.5 mm; m = 2.5 cm

1974). We have reported the micropropagation of P. polyphylla through somatic embryogenesis from immature
zygotic embryos (Raomai et al. 2014). In this report, we
describe the effect of cytokinins on MR formation from
tTCL followed by analysis of steroidal saponin production
in different concentrations of cytokinins. Also, the influence of chitosan (CHI), salicyclic acid (SA) and yeast
extract (YE) on growth and steroidal saponins production
in MR cultures is reported.

Materials and methods

Plant material
Fresh rhizomes of P. polyphylla were collected from their
natural habitat in Chazouba, Phek District of Nagaland,
India and maintained in the glass house of Plant Biotechnology Laboratory, Department of Botany, North-Eastern
Hill University, Shillong. Terminal buds (Fig. 1a) were
harvested during the month of MarchApril from 4 to
5 years old rhizomes growing in the glasshouse. They were
washed thoroughly under running tap water, treated with
1 % (w/v) bavistin for 15 min followed by several rinses
with sterile distilled water. The explants were then disinfected with 15 % sodium hypochlorite (4 % active chlorine
content) along with 23 drops of tween-20 for 15 min and
rinsed several times with sterile distilled water. The plant
material was further surface-sterilized by immersing in
0.2 % mercuric chloride (w/v) for 10 min followed by
several rinses with sterile distilled water. After removing
the outer scales, the preformed shoots (Fig. 1b) were
soaked in 5 % (v/v) plant preservative mixture (Plant Cell
Technology, USA) for 4 h. They were then dried with
sterile filter paper and inoculated on MS (Murashige and
Skoog, 1962) medium for 1 week to check contamination.
The contaminated shoots were re-sterilized with 0.1 %
mercuric chloride (w/v) for 10 min followed by several
rinses with sterile distilled water. The stem part of the
sterile preformed shoot was then sliced into three equal

Plant Growth Regul

123

Plant Growth Regul

portions viz., basal, middle and apical (Fig. 1b). These

portions were transversely sliced into pieces of about
0.5 mm in thickness and the slices were used as tTCL
explants for plant regeneration.
Media and culture conditions
Transverse thin cell layer explants were inoculated with
their original orientation on strength MS medium (halfstrength macro- and micro-elements) with 3 % (w/v)
sucrose and solidified with 0.8 % (w/v) agar. The culture
medium was fortified with various concentrations (0.25,
0.5 or 1.0 mg/l) of 6-benzylaminopurine (BAP), thiadiazuron (TDZ) and kinetin (KIN) individually for minirhizome (MR) induction. The pH of medium was adjusted
to 5.8 and autoclaved at 121 C, 1.05 kg/cm2 pressure for
15 min. All the cultures were incubated in the culture room
at temperature of 25 2 C in the dark. MR induction in
each treatment was evaluated 6 months after inoculation,
without any subculture. The percentage of explants forming MR, fresh weight (FW) of each MR in a treatment and
saponins content were simultaneously recorded after
6 months of culture. To induce shoot buds, MRs were
transferred to MS basal medium without plant growth
regulators (PGRs), with subcultures at three months intervals. The numbers of shoot buds formed per MR were
recorded after 5 months in PGR-free medium. Subsequently, individual shoot buds were detached from the
maternal MR tissue and either transferred to MS basal
medium for shoot sprouting or directly subjected to ex vitro
conditions in the greenhouse. The developed shoots were
kept under a 14/10h light photoperiod with a photosynthetic photon flux of 60 lmol/m2/s provided by cool-white
fluorescent lamps.
Acclimatization of regenerated plantlets
In vitro regenerated plantlets with well developed rhizome
and roots were washed thoroughly until traces of agar were
removed completely and then transferred to thermocol cups
containing potting mixtures of soil, compost and peat moss
in the ratio of 2:1:1 (w/w). The plantlets were maintained
under green house conditions with a temperature of
25 2 C with 12 h photoperiod and 60 5 % relative
humidity and were irrigated twice a week with MS
solution for the first 4 weeks.
Histological analysis
One year old rhizomes grown under greenhouse conditions,
6 months old in vitro MRs and MRs with protuberances
were gently washed with distilled water in order to remove

123

soil debris and agar respectively. All samples were fixed in

2.5 % glutaraldehyde in 0.1 M phosphate buffer (pH 7.2)
and dehydrated through a graded ethanol series. They were
embedded in saturated paraffin wax (5860 C) and serially sectioned (10 lm thick) with a rotatory microtome
(Leica RM 2125RT). Sections were stained with 0.05 %
toluidine blue and mounted in DPX. They were observed
under a light microscope (Leica, Germany) and photographed using Sony digital camera (DSC-N1).
Liquid culture of MRs for elicitation
Six months old MRs maintained at 0.5 mg/l BAP were
used as liquid culture for elicitation treatment. Several
MRs collectively weighing about 2.53 g FW were suspended in 50 ml MS medium containing 3 % sucrose
supplemented with 0.5 mg/l BAP. Stock solution of SA
and YE were prepared by dissolving in Milli-Q water. CHI
was first dissolved in glacial acetic acid and then diluted
with Milli-Q water. The pH of the elicitor stock solutions
was adjusted to 5.8 and filter-sterilized before adding into
the liquid medium. Elicitor solutions were added at the
concentrations of 50, 100 and 200 mg/l immediately following the inoculation of MRs in liquid medium. To the
control variants, equal volumes of water were added. All
flasks were shaken at 110120 rpm on an orbital shaker at
25 2 C in dark. The cultures were harvested after every
15 days for a period of 60 days for the analysis of MRs
growth and steroidal saponins accumulation in order to
identify the optimal exposure time and concentrations. At
the end of each treatment period, the MR cultures were
harvested, washed 23 times with distilled water and dried
with Whatman filter paper to remove excess water. For
growth measurement, growth index was used in order to
minimize the differences in growth (FW increase) caused
by the variations in inoculum size, which was calculated
as; Growth Index = (WF - W0)7W0, where W0 is the
weight of inoculum at 0 day of inoculation and WF is the
weight of the MRs on the day of harvest.
Extraction and determination of steroidal saponins
Fresh MRs growing in medium containing different concentrations of cytokinins and elicitors were harvested,
washed with Milli-Q water and dried at 50 C until a constant weight was achieved. These were then ground into fine
powder using pestle and mortar. Sample powder (1 g) was
extracted from 80 ml of 90 % aqueous ethanol solution (v/v)
by refluxing for 3 h in Soxhlet apparatus. The volatile
component was evaporated to dryness at 50 C and the
residue was re-dissolved in 5 ml methanol. Qualitative steroidal saponin profiling and quantification was carried out

Plant Growth Regul

using high performance liquid chromatography (HPLC).

The liquid samples were centrifuged at 10,000 rpm for
10 min, filtered through a 0.22 lm microfiltration membrane (Rankem). The HPLC analysis was carried out by
using Waters 515 HPLC pump with Waters 2489 UV/visible
detector. The HPLC separation was performed on an analytical reverse phase column (Peco HCODS, C18,
150 9 4.6 mm, 5 lm) (Perkin Elmer) eluted with acetonitrile/water (47:53 v/v) at a flow rate of 1.0 ml/min and
detected at 203 nm. For calibration of standard curve, steroidal saponins including polyphyllin I, polyphyllin II,
polyphyllin VI and polyphyllin VII were prepared at various
concentrations (0.11.0 mg/ml) in methanol and 20 ll each
were injected to obtain standard curve plot of peak area with
a run time of 12 min. The analyzed steroidal saponin content
was expressed as mg steroidal saponin/g DW (dry weight).
All the solvents (HPLC grade) were obtained from LOBA
Chemie (Mumbai, India) and the standards were purchased
from Shanghai Yaji Biological Technology Co., Ltd.
(Shanghai, China).
Statistical analysis
All experiments were repeated thrice with three replicates
each and data were analyzed using one-way analysis of
variance (ANOVA) in JMPversion 7.0.1 (SASInstitute,
Cary, NC). The significant differences among the means
were assessed by Tukey HSD test at 5 % probability level.

Table 1 Effect of cytokinins on

shoot regeneration via MR
formation from tTCL of
different stem portions in P.
polyphylla

Portion of stem

Cytokinins
(mg/l)
0.0

Basal

BAP 0.25

83.7ab

65.2

71.1

1.0

74.0

bc

TDZ 0.25

83.7ab

0.5
1.0

Different letters within a

column indicate significant
differences at P B 0.05 by
Tukey HSD test

0.0f

85.2

0.5

Response
(%)*#

KIN 0.25

** Data recorded after

5 months of transfer to PGR
free medium

Transverse thin cell layer explants cultured in the absence

of cytokinins did not show any kind of response but
gradually turned brown and died subsequently. However,
in the presence of all the three types of cytokinins used, the
explants become swollen and enlarged. Response percentage was also significantly influenced by the parts of the
stem from which the explant was derived (Table 1).
Explants from the basal section showed higher percentage
of response than the middle parts while no response was
observed in apical part. Though slight response was
observed initially in the explant derived from the apical
part, it died as a consequence of oxidative browning
(Fig. 1c). Slight accumulation of phenolic compounds was
also observed in the middle part as well (Fig. 1d) resulting
in lower response percentage as well as FW compared to
basal part (Table 1). Explants from the basal part of the
stem grew in size without any sign of oxidation and
eventually form white or cream coloured nodular, smooth
surfaced growth which was designated as MR (Fig. 1e, f).
The response of the tTCL explants of P. polyphylla to
various concentrations of cytokinins is shown in Table 1.
Of the three types of cytokinins tested, BAP and TDZ were
found to be more effective than KIN. Frequency of MR
formation was highest on medium containing 0.5 mg/l

86.6

1.0

* Data recorded after 6 months

Shoot regeneration via MRs derived from tTCL

0.5

Middle

Results

85.9

82.9

ab
de

FW (g)/MR*#

No. of shoot
buds/MR**#

0.0i

0.0g

0.94 0.05ab

5.1 0.5abc

5.6 0.4a

ab

5.2 0.6ab

cdef

3.3 0.3cdef

cdef

3.6 0.4bcdef

bcde

0.69 0.07

3.9 0.3abcde

0.93 0.05ab

5.0 0.4abc

1.05 0.08
0.95 0.05
0.62 0.06
0.64 0.06

ab

5.4 0.6a

abc

4.6 0.4abcd

efgh

0.96 0.07
0.90 0.05

BAP 0.25
0.5

36.3
44.4d

0.48 0.04
0.59 0.05def

2.7 0.4ef
3.1 0.3def

1.0

43.7d

0.55 0.04defg

2.9 0.3def

32.6

36.3

de

40.7

de

TDZ 0.25

38.5

de

0.5

43.7de

KIN 0.25
0.5
1.0

1.0

37.8

2.0 0.3f

gh

2.2 0.2ef

fgh

2.4 0.3ef

defg

0.55 0.07

2.9 0.3def

0.57 0.06defg

3.0 0.3def

efgh

2.8 0.3def

0.20 0.01
0.30 0.04
0.38 0.05

0.49 0.10

123

Plant Growth Regul

BAP
(86.6 %)
with
maximum
average
FW
(1.05 0.08 g) followed by TDZ (Table 1). On subculturing the MRs onto the same fresh medium with cytokinins, it increased in size whereas transferring them onto
PGR-free medium resulted in the appearance of small
protuberances after about 3 months on its surface (Fig. 1g).
These structures developed into mature shoot buds with
roots being simultaneously formed while still attached to
the maternal MRs (Fig. 1h). The highest numbers of shoot
buds were obtained on MRs previously induced at 0.5 mg/l
of BAP (5.6 0.4) followed by 0.5 mg/l of TDZ
(5.4 0.6). Larger the MRs size, more were the number of
shoot buds induced per MR (Table 1). Comparison of
histological section between the MRs and in vivo rhizome
showed close resemblance in their anatomical details
(Fig. 1i, j). Further, longitudinal section of the protuberances revealed shoot primordia with vascular strand and
root apical meristem (Fig. 1k). The shoot buds on isolating
and subculturing to PGR-free medium eventually sprouted
into a complete plantlet (Fig. 1l). Both isolated shoot buds
and sprouted plantlets when transferred to the soil, under
green house conditions showed more than 95 % survival
with morphological characters comparable to that of naturally propagated plants (Fig. 1m). Plants or shoot buds
transferred to soil in the previous year sprouted again the
next year after over-wintering.
Effect of cytokinins on steroidal saponin production
The presence and accumulation of steroidal saponins were
analyzed using HPLC in relation to the concentrations of
different cytokinins. The HPLC profiles of both MRs and
in vivo rhizome extracts showed the presence of polyphyllin I, polyphyllin II and polyphyllin VII but polyphyllin VI was found to be absent when compared to the
standard HPLC profile (Fig. 2). Synthesis of steroidal
saponins in MRs was observed in all the treatments. The
content of each steroidal saponins differed between different cytokinins concentrations. Table 2 shows the influence of different concentrations of cytokinins (BA, KIN
and TDZ) on steroidal saponin production in MRs harvested after 6 months of culture. Total steroidal saponins
(polyphyllin I ? polyphyllin II ? polyphyllin VII) accumulation was recorded highest in 0.5 mg/l BAP
(33.85 1.99 mg/g DW) which was 1.41-fold higher than
the in vivo rhizome (Table 2).
Effect of elicitors on biomass accumulation
and steroidal saponin production in MRs
In CHI treated MRs cultures, maximum accumulation of
polyphyllin I (14.65 0.55 mg/g DW) and polyphyllin II
(11.40 0.52 mg/g DW) were recorded in MRs treated

123

Fig. 2 RP-HPLC chromatograms of steroidal saponin analysis in P.

polyphylla. a HPLC profile of standard steroidal saponins, b HPLC
profile showing presence of polyphyllin I, polyphyllin II and
polyphyllin VII in field grown rhizome, c HPLC profile showing
the presence of polyphyllin I, polyphyllin II and polyphyllin VII in
MR cultures

with 100 mg/l CHI for 60 days while polyphyllin VII was
highest at 100 mg/l CHI treated for 45 days
(46.74 0.83 mg/g DW). Total steroidal saponin content

Plant Growth Regul

Table 2 Effect of cytokinins on
in vitro production of steroidal
saponins in MR cultures derived
from tTCL

PGRs (mg/l)

Polyphyllin I
(mg/g DW)*

Polyphyllin II
(mg/g DW)*

Polyphyllin VII
(mg/g DW)*

Total saponin
(mg/g DW)*

Rhizome

6.94 0.27a

5.49 0.21a

11.47 0.69e

23.89 1.04bc

BAP 0.25

3.44 0.75

1.04 0.14

19.07 0.95

20.19 0.64cd

0.5

5.72 0.72ab

3.00 0.71b

25.12 0.85a

33.85 1.99a

1.0

bc

KIN 0.25
0.5
1.0
* Different letters within each
column represent significant
difference at P B 0.05 by
Tukey HSD test

bcde

4.39 0.45

0.35 0.04

def

1.20 0.36

ef

0.95 0.05

bc

2.21 0.40

0.44 0.06

cd

0.63 0.23

cd

0.82 0.17

ab

28.84 1.90ab

de

14.49 0.69d

de

19.66 0.97cd

bcd

22.34 2.36bc

abc

22.24 1.24
13.70 0.74
13.00 1.06
17.89 1.07

1.20 0.79

1.11 0.56

20.03 1.78

14.83 0.97d

0.5

2.28 0.54cdef

1.61 0.19bcd

21.64 0.84ab

25.53 1.45bc

bcd

3.73 0.40

was maximum (69.73 1.06 mg/g DW) in MRs treated for

45 days at 100 mg/l CHI which was 2.05-folds higher
compared to the control (Table 3). Treatment with CHI at all
concentrations resulted in the decreased growth of MRs
compared to the control (Fig. 3).
In case of SA treatment, 30 days of elicitation with
50 mg/l SA resulted in the highest content of polyphyllin I
(16.01 0.99 mg/g DW) and polyphyllin VII (65.14
1.65 mg/g DW) whereas polyphyllin II accumulation
was highest at 100 mg/l SA for 45 days (11.24
0.62 mg/g DW). Total steroidal saponin content was highest
in cultures treated with 50 mg/l SA for 30 days (87.66
1.66 mg/g DW). This content is 2.58-fold higher than the
untreated cultures (Table 4). SA also affected MRs growth
as indicated by the decrease in growth index (Fig. 3).
In YE treated cultures, treatment with 100 mg/l YE for
30 days elicited the highest production of all the steroidal
saponins (polyphyllin I = 14.33 0.37 mg/g DW, polyphyllin II = 9.38 0.57 mg/g DW and polyphyllin
VII = 47.75 3.11 mg/g DW) (Table 5). Thus the total
steroidal saponin content (71.46 4.09 mg/g DW) was
2.1-fold higher than the control (Table 5). YE also affected
the MRs growth in a concentration and treatment period
dependant manner resulting in lower growth index compared to the control (Fig. 3).
Overall, highest content of total steroidal saponins was
achieved in cultures treated with SA at 50 mg/l for 30 days
(87.66 1.66 mg/g DW) which is 3.6 times higher than
the in vivo rhizome.

Discussion
The present study revealed that MRs formation from tTCL
was significantly influenced by different portions of the
stem. From the results, it can be suggested that higher levels
of phenolic compounds in the explant led to the loss of its

cd

bc

TDZ 0.25
1.0

def

cd

cd

0.95 0.18

cde

15.50 0.90

23.55 0.80bc

regenerative ability. Therefore, differential accumulation of

phenolic compounds in the different parts of the stem might
be the reason for its variation in the explant response.
Another possible reason could be due to the increased density of vascular tissue in the basal portion. Pence and Soukup
(1993) described MRs in Trillium grandiflorum and T.
erectum from stem and leaf sections and discussed that
differences in the response percentage between different
explants could be due to their developmental stage. Higher
regenerative potential of basal sections have also been
observed in other plant species (Mata-Rosas et al. 2010;
Scherwinski-Pereira et al. 2010).
Further, types and concentrations of cytokinins also had a
profound influence on MR induction from tTCL of P. polyphylla stem. BAP and TDZ were found to be more effective than KIN. Stronger physiological effects of BAP and
TDZ on organ formation have also been reported in other
studies (Takayama and Misawa 1982; Nhut et al. 2001).
However, FW of MRs was found to be higher in medium
supplemented with 0.5 mg/l BAP compared to TDZ. Similarly, Han et al. (2005) also observed that the FW of bulblets
formed per bulb scale was larger on medium with BAP than
TDZ. BAP has been one of the most successfully used
cytokinins for in vitro tuberization in several other species
(Piao et al. 2003; Omokolo et al. 2003; Poornima and
Ravishankar 2007; Cousins and Adelberg 2008). It has been
reported that BAP can be metabolized more easily than other
synthetic growth regulators by plant tissues and has the
ability to induce production of natural hormones such as
zeatin within the tissue (Zaerr and Mapes 1982). Contrary to
our studies, BAP has been reported to have an inhibitory
effect on in vitro microrhizome production in turmeric
(Shirgurkar et al. 2001). Cytokinins have been considered to
be involved in the development of the storage organ by
promoting cell division in the growing tuber (Fernie and
Willmitzer 2001). Sarkar et al. (2006) found that potato
tubers grown in the presence of cytokinins increased starch

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Plant Growth Regul

Table 3 Effect of CHI and
duration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL

Days
0
15

CHI
(mg/l)

* Different letters within each

column represent significant
difference at P B 0.05 by Tukey
HSD test

25.14 0.85k

33.88 1.99h

3.02 0.53d

5.75 0.37

3.04 0.05

26.11 1.72

50

6.31 0.39ef

4.44 0.32cd

28.85 0.07ghijk

39.60 0.60fgh

34.39 0.71

bcdef

50.36 1.65cde

29.53 0.41

fghijk

43.48 0.64efg

26.84 0.33

ijk

35.68 0.91h

31.79 1.74

efghi

45.91 2.58def

56.48 0.66bc

cdef

8.73 0.49

def

7.44 0.87

abcd

7.24 0.53

bcd

6.52 0.40

5.79 0.43

3.06 0.16

cdef

7.92 0.35

bcd

bcd

6.20 0.56

abc

jk

100

9.81 0.44

8.15 0.59

38.52 1.30

200

8.43 0.33cdef

7.89 0.31abc

32.89 1.07cdefg

5.81 0.32

3.08 0.07

cdef

abc

34.91 1.37h

49.21 1.06cde

ijk

35.96 0.44h

bc

37.84 1.05
46.74 0.83a

55.33 1.17bc
69.73 1.06a

27.07 0.58

50
100

8.97 1.49
12.72 0.23ab

8.51 0.81
10.27 0.67ab

200

10.17 0.42bcd

9.59 0.49ab

36.82 0.48bcd

56.58 0.71bc

5.84 0.27f

3.09 0.04d

27.71 1.17hijk

36.64 1.35h

0
50
100
200

bcde

9.67 1.14

9.24 3.02
11.40 0.52

bc

11.08 0.81

ab
a

14.65 0.55

accumulation. Besides, exogenously applied cytokinins

have been reported to effectively promote tuberization and
yield of underground storage organs in many other monocotyledonous plants (Suri et al. 1999; Sharma and Singh
1995; Ghosh et al. 2007).
The advantage of the present method over caulogenesis
or callogenesis is the direct formation of the desired organ
i.e. the MRs which could develop shoot buds that regenerated into a complete plantlet with shoot, rhizome and
roots. In the present study, a rhizome induction stage is
found to be more important than rooting stage. Once the
rhizomes are established, cultures could be easily hardened. The MRs could be maintained for more than
6 months on the same medium without subculture and with
periodical subculture of 60 days, it can be maintained for
more than 3 years or longer. On repeated subculturing of
MRs on medium with 0.5 mg/l BAP, MRs grew in size and
attained a FW of approximately 35 g after about
18 months without producing shoot buds (data not shown).
Thus, from a single preformed shoot about 1015 MRs,
each weighing about 45 g could be obtained within
2 years which is not possible in vivo. This growth characteristic of MRs could contribute to cost-effective storage
as the cultures could be stored at 25 2 C in the form of
rhizomes for extended period without involving complicated techniques as in other storage methods where
chemical or physical methods were applied. In vitro rhizome production has been successfully used for storage
purpose under normal temperature in other tuberous plants
such as Zingiber officinale (Tyagi et al. 2006). Induction of

123

Total saponin
(mg/g DW)*

50

60

Polyphyllin VII
(mg/g DW)*

5.72 0.72f

200

45

Polyphyllin II
(mg/g DW)*

100
30

Polyphyllin I
(mg/g DW)*

ab

9.37 1.00

32.32 0.54

defgh

51.23 3.36cd

36.33 0.58

bcde

62.38 1.08ab

31.03 0.85

fghij

51.48 0.57cd

in vitro storage organs have been proven as a potent

method for conservation of ginger (Sharma and Singh
1995), potato (Gopal et al. 1998) and yams (Jean and
Cappadocia 1991). Natural rhizomes under storage are
known to be infected with many pathogens. Therefore,
MRs could be a good source of disease-free material for
planting in the field. Storage and transport of MRs will also
be easier, facilitating germplasm exchange across national
borders.
Generally, shoot induction precedes rhizome formation
which requires several steps before the final product could
be obtained. Our protocol however has much more
advantage in that rhizome was first induced directly in the
presence of cytokinins from a comparatively small explant
which can be made to grow further on the same medium.
Moreover, shoot buds production can be induced by
transferring the MRs to PGR-free medium as and when
required. Further, when shoot buds at their initial stage
were maintained in cytokinins containing medium, they
developed into MRs producing shoot buds. Also, the stems
of in vitro preformed shoots when used as tTCL explants
readily formed MRs and hence the cycle could be continued repeatedly for MRs production using this technique
(data not shown).
The histological details and morphology of MRs were
found to be similar to those of field-grown rhizomes.
Therefore, we hypothesized that MRs might offer potential
value for secondary metabolite production, and hence MRs
were used to study the biosynthesis of steroidal saponin.
The present study showed that steroidal saponin

Plant Growth Regul

Fig. 3 Effect of elicitors on the growth of MRs. a effect of CHI,

b effect of SA, c effect of YE. Growth Index = (WF - W0)7W0,
where W0 is the weight of inoculum at 0 day of inoculation and WF is
the weight of the MRs on the day of harvest

accumulation in MR cultures is significantly affected by

different concentrations of cytokinins, thus establishing the
fact that there exists a strong relationship between cytokinins and steroidal saponins biosynthesis in MRs which is
consistent with the observation in Gypsophila Paniculata
(Hanafy and Abou-Setta 2007). Maximum amount of total
steroidal saponins observed on medium containing 0.5 mg/l
BAP could be due to its strong effect on growth and
differentiation resulting in the higher production of secondary metabolites. For instance, enhancement of saponin
production by the addition of BAP has been observed in
transformed root of Panax ginseng (Aitsu et al. 1992) and
xanthones in Gentianella austrica shoot cultures (Vinterhalter et al. 2008).

Results on experiments with the influence of elicitors on

steroidal saponin production showed significant increase in
the accumulation of steroidal saponin in MR cultures
treated with CHI, SA and YE at optimum concentrations
when compared to the control. Exogenous addition of
biotic or abiotic elicitors in culture was considered to be
one of the most promising strategies for the increased
production of secondary metabolites (Radman et al. 2003).
Elicitors are generally defined as molecules that stimulate
any defense response of plants, including the formation of
phytoalexins (Hahn 1996). The induction mechanism of
elicitor is generally regarded as inducing the expression of
defense-related genes and activating defense-related secondary metabolic pathways (Qian et al. 2006). In the
present study, the response to elicitation is dependent on
the type and concentration of elicitors as well as the
duration of treatment. Abiotic elicitor, SA was found to be
a more effective elicitor than the biotic elicitors, CHI and
YE. SA has been shown to elicit higher accumulation of
secondary metabolites in plant cell/organ cultures of many
plant species (Ali et al. 2006: Roat and Ramawat, 2009;
Sivanandhan et al. 2012; Costa et al. 2013). Positive
response of cultures to SA elicitation is possibly associated
with the fact that SA accumulates locally at the site of
infection and then it spreads to other parts of the plant,
mostly as methyl salicylate inducing a range of defense
responses, including the biosynthesis of secondary metabolites (Zhao et al. 2005). The accumulation of polyphyllins
is also significantly affected by YE and CHI. The elicitation effect of biotic elicitors is most likely due to the oligosaccharides present in them which have been reported as
potent signalling molecules that regulate growth, development and defense mechanisms in plants (Sudha and
Ravishankar 2002). In contrast to our study, YE was found
to be more effective than SA in some previously reported
studies (Karwasara et al., 2010; Zhao et al. 2010; Veerashree et al. 2012). CHI has also been reported to act as an
elicitor for the improved production of secondary metabolites in many other medicinal plants such as Trigonella
foenum-graecum (Merkli et al. 1997), Panax ginseng
(Jeong and Park 2005), Cistanche deserticola (Cheng
et al. 2006) and Salvia miltiorrhiza (Zhao et al. 2010).
Hence, it can be inferred that the effects of various elicitors
on secondary metabolite production in plant tissue culture
are dependent on specific secondary metabolites. Though
steroidal saponin accumulation was enhanced by elicitor
treatment, reduced growth of MRs was observed. Zhang
et al. (2002) suggested that this phenomenon might be due
to switching of primary metabolism to secondary metabolism in the cells. The present result is in agreement with
other previously reported studies (Cho et al. 2003; Kang
et al. 2004; Zhao et al. 2010; Korsangruang et al. 2010).
One of the major problems in the adoption of plant cell

123

Plant Growth Regul

Table 4 Effect of SA and
duration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL

Days
0
15

SA (mg/l)

3.02 0.53ef

25.14 0.85j

33.88 1.99i

5.75 0.37

3.04 0.05

ef

26.11 1.72

34.91 1.37i

50

12.70 1.79abc

4.36 0.52ef

58.37 1.15ab

75.43 2.51bc

0
100
200

Table 5 Effect of YE and

duration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL

fg

6.55 0.30

5.79 0.43

16.01 0.99

bcd

11.50 0.58

9.40 0.22cdef
g

5.81 0.32

4.00 0.21

ef

3.06 0.16

ef

6.51 0.83

def

11.24 0.62

6.14 0.29def
3.08 0.07

ef

35.68 0.91i

87.66 1.66a

bcd

53.22 1.69

75.95 1.00bc

44.67 1.78ef

60.20 1.41def

26.84 0.33
65.14 1.65

hij

35.96 0.44i

bc

27.07 0.58

200

7.51 0.26efg

12.37 1.14ab

33.82 1.05ghi

53.70 1.70fg

5.84 0.27g

3.09 0.04ef

27.71 1.17hij

36.64 1.35i

200

YE (mg/l)

bcde

10.98 0.78

defg

8.62 0.30

6.69 0.72 f

Polyphyllin I
(mg/g DW)

bcd

48.27 1.97gh

ij

37.71 1.70

79.32 1.00ab
68.39 4.29cd

ab

bc

64.93 1.49de

fg

48.70 1.01

55.22 0.82
42.12 2.05ef

6.80 0.41
13.53 1.65
7.55 0.72

cdef
ab
cde

de

64.44 2.12de

gh

56.65 0.33efg

hij

42.86 0.55hi

46.66 2.36
34.50 1.33
28.63 0.46

Polyphyllin II
(mg/g DW)

Polyphyllin VII
(mg/g DW)

Total saponin
(mg/g DW)

5.72 0.72f

3.02 0.53f

25.14 0.85g

33.88 1.99f

15

5.75 0.37f

3.04 0.05f

26.11 1.72fg

34.91 1.37ef

100
200
30

11.35 0.49
8.16 0.31cdef

50

10.78 0.78bcd
14.33 0.37

11.88 0.80

ab

5.81 0.32

50

7.92 0.58

def

100

10.65 0.66bcd

200
60

abc

5.79 0.43f

200
45

7.24 1.03

ef

0
100

0
50
100
200

9.32 0.59

bcde

5.84 0.27

8.19 1.25

cdef

8.65 0.29

bcdef

7.21 0.24

ef

cultures as an industrial process is that of process cost and

hence, productivity (Lipsky 1992). Therefore, it can be
suggested that in vitro production of MRs which is an
organ culture could be an ideal approach for secondary
metabolite production. Moreover, there have been reports
of the failure of callus to produce secondary metabolite
since callus cultures consist of undifferentiated tissues, in
which gene expression pattern markedly differ from those

123

7.45 0.60

cde

10.11 0.79
16.85 2.22a

50

* Different letters within each

column represent significant
difference at P B 0.05 by
Tukey HSD test

8.78 0.30

cdef

13.99 0.56
9.42 0.31cdef

100

Days

defg

50
100

50

* Different letters within each

column represent significant
difference at P B 0.05 by
Tukey HSD test

Total saponin
(mg/g DW)*

50

60

Polyphyllin VII
(mg/g DW)*

5.72 0.72g

200

45

Polyphyllin II
(mg/g DW)*

100
30

Polyphyllin I
(mg/g DW)*

4.13 0.58

cdef
cdef

efg

40.40 1.22def

bcdef

29.03 1.58

5.23 0.43
4.65 0.24cdef

36.10 1.96
30.05 1.14defg

52.68 1.93bc
42.86 1.14cdef

3.06 0.16f

26.84 0.33fg

35.68 0.91ef

5.29 1.14cdef

34.48 1.15cdefg

50.56 2.14bcd

9.38 0.57

6.48 0.28

bc

3.08 0.07

6.12 0.51

bcd

7.83 0.41ab
4.96 0.45

cdef

3.09 0.04

4.03 0.42

def

5.62 0.30

bcde

3.66 0.24

ef

71.46 4.09a

bcdef

54.34 1.12b

fg

35.96 0.44ef

abcde

38.18 3.99

52.22 4.32bc

44.79 2.82ab

63.27 2.94ab

47.75 3.11
35.98 0.55
27.07 0.58

abcd

54.13 2.67b

fg

36.64 1.35ef

cdefg

44.90 2.81bcde

abc

54.73 0.87b

cdefg

45.08 0.85bcde

39.84 2.42
27.71 1.17
32.69 2.35
40.46 1.36
34.21 0.99

in whole plant, so genes involved in the production of

desirable secondary metabolites may be even repressed
(Wink 1989). Ludwig-Muller et al. (2008) showed that
organ culture can be a major source of secondary metabolites compared to both cell suspension and biomass production in the field. Therefore, this experiment identifies
the merit of MRs as a constant source of medicinally
important compounds, in high amounts, all the year round.

Plant Growth Regul

Conclusion
The protocol described here for the medicinally important
and endangered plant, P. polyphylla provides a novel system for mass propagation, storage and production of secondary metabolites. The procedure is simple and practical
that can be efficiently used for year-round production of
MRs independent of the growing season and for international germplasm distribution or exchange. These results
further showed that high levels of steroidal saponins can be
achieved in a reduced period of time by using elicitors.
Moreover, bioreactor technique can be applied for large
scale production of steroidal saponins. Therefore, this
research represents a direct contribution to the germplasm
conservation which will greatly reduce pressures on wild
populations of this valuable natural resource. Apart from
these, thin cell layer method can be efficiently applied for
genetic transformation of P. polyphylla.
Acknowledgments The authors acknowledge Dr. A. Bhattacharjee
and Ms. B. J. Mylliemngap, Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong for providing the
HPLC facilities and valuable help. The authors would also like to
thank Prof. N. Venugopal, Department of Botany, North-Eastern Hill
University, Shillong for permission to use microtome. SR is thankful
to University Grant Commission, India for awarding her Rajiv Gandhi
National Fellowship for SC/ST.

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