rao mai 2014
DESCRIPTION
regyrtggfTRANSCRIPT
-
ORIGINAL PAPER
Plantlet regeneration of Paris polyphylla Sm. via thin cell layerculture and enhancement of steroidal saponins in mini-rhizomecultures 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-benzyl-aminopurine (BAP). MRs transferred to plant growth reg-
ulator free medium gave rise to shoot buds that eventually
regenerated into plantlets and were successfully acclima-
tized with a survival percentage of more than 95 % under
greenhouse conditions. Quantification through reverse-
phase HPLC showed 1.41-fold higher content of total ste-
roidal saponins in MRs cultured on medium supplemented
with 0.5 mg/l BAP as compared to the field-grown rhi-
zome. Elicitation of MRs liquid culture with chitosan,
salicyclic acid (SA) and yeast extract enhanced the pro-
duction of steroidal saponins but resulted in reduced
growth rate. Highest total steroidal saponins con-
tent (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.
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 sto-
machic, 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, paro-
titis, mastitis, chronic bronchitis, injuries from fractures as
S. Raomai S. Kumaria (&) M. Kehie P. TandonPlant Biotechnology Laboratory, Department of Botany,
Centre for Advanced Studies, North-Eastern Hill University,
Shillong 793022, India
e-mail: [email protected]
123
Plant Growth Regul
DOI 10.1007/s10725-014-9957-1
-
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 Ji-
desheng-she-yao-pian tablet which are used to treat dis-
persing blood stasis and hemostasis, activate blood circu-
lation, 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. po-
lyphylla 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 tech-
nique 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 longi-
tudinally (lTCL) or transversally (tTCL) (Silva 2003) and
was first described in Nicotiana tabacum (Van Tran Thanh
1974). We have reported the micropropagation of P. po-
lyphylla 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 influ-
ence 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 Biotech-
nology 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 disin-
fected 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
cFig. 1 Plantlet regeneration via MRs formation from tTCL. a rhizometerminal bud, b exposed preformed shoot after removal of outer scalesfrom the terminal bud, c tTCL from apical part of the stem showingphenolic accumulation, d initial response of tTCL from the middlepart of stem, e initial response of tTCL from the basal part of stem,f well developed MRs from the basal section, g protuberances formedon MR after transfer to PGR-free medium, h well developed shootbuds with roots formed on MR, i histological section of in vivorhizome, j histological section of MR, k longitudinal section ofprotuberances formed on MR, l complete plantlet developed fromshoot bud, m acclimatized plantlets under greenhouse conditions after2 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
Plant Growth Regul
123
-
Plant Growth Regul
123
-
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 (half-strength 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), thiadi-
azuron (TDZ) and kinetin (KIN) individually for mini-
rhizome (MR) induction. The pH of medium was adjusted
to 5.8 and autoclaved at 121 C, 1.05 kg/cm2 pressure for15 min. All the cultures were incubated in the culture room
at temperature of 25 2 C in the dark. MR induction ineach treatment was evaluated 6 months after inoculation,
without any subculture. The percentage of explants form-
ing 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 growthregulators (PGRs), with subcultures at three months inter-
vals. The numbers of shoot buds formed per MR were
recorded after 5 months in PGR-free medium. Subse-
quently, individual shoot buds were detached from the
maternal MR tissue and either transferred to MS basalmedium 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 photosyn-
thetic photon flux of 60 lmol/m2/s provided by cool-whitefluorescent 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 % relativehumidity and were irrigated twice a week with MSsolution 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
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 seri-ally 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 photo-
graphed 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 sus-
pended in 50 ml MS medium containing 3 % sucrosesupplemented 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 fol-
lowing 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 every15 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 theweight 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 con-
centrations of cytokinins and elicitors were harvested,
washed with Milli-Q water and dried at 50 C until a con-stant 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 theresidue was re-dissolved in 5 ml methanol. Qualitative ste-
roidal saponin profiling and quantification was carried out
Plant Growth Regul
123
-
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 mem-brane (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 ana-
lytical reverse phase column (Peco HCODS, C18,
150 9 4.6 mm, 5 lm) (Perkin Elmer) eluted with acetoni-trile/water (47:53 v/v) at a flow rate of 1.0 ml/min and
detected at 203 nm. For calibration of standard curve, ste-
roidal 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 eachwere 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.
Results
Shoot regeneration via MRs derived from tTCL
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 per-
centage 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
Table 1 Effect of cytokinins onshoot regeneration via MR
formation from tTCL of
different stem portions in P.
polyphylla
* Data recorded after 6 months
** Data recorded after
5 months of transfer to PGR
free medium# Different letters within a
column indicate significant
differences at P B 0.05 by
Tukey HSD test
Portion of stem Cytokinins
(mg/l)
Response
(%)*#FW (g)/MR*# No. of shoot
buds/MR**#
0.0 0.0f 0.0i 0.0g
Basal BAP 0.25 83.7ab 0.94 0.05ab 5.1 0.5abc
0.5 86.6a 1.05 0.08a 5.6 0.4a
1.0 85.2b 0.95 0.05ab 5.2 0.6ab
KIN 0.25 65.2c 0.62 0.06cdef 3.3 0.3cdef
0.5 71.1c 0.64 0.06cdef 3.6 0.4bcdef
1.0 74.0bc 0.69 0.07bcde 3.9 0.3abcde
TDZ 0.25 83.7ab 0.93 0.05ab 5.0 0.4abc
0.5 85.9a 0.96 0.07ab 5.4 0.6a
1.0 82.9ab 0.90 0.05abc 4.6 0.4abcd
Middle BAP 0.25 36.3de 0.48 0.04efgh 2.7 0.4ef
0.5 44.4d 0.59 0.05def 3.1 0.3def
1.0 43.7d 0.55 0.04defg 2.9 0.3def
KIN 0.25 32.6e 0.20 0.01h 2.0 0.3f
0.5 36.3de 0.30 0.04gh 2.2 0.2ef
1.0 40.7de 0.38 0.05fgh 2.4 0.3ef
TDZ 0.25 38.5de 0.55 0.07defg 2.9 0.3def
0.5 43.7de 0.57 0.06defg 3.0 0.3def
1.0 37.8d 0.49 0.10efgh 2.8 0.3def
Plant Growth Regul
123
-
BAP (86.6 %) with maximum average FW
(1.05 0.08 g) followed by TDZ (Table 1). On subcul-
turing the MRs onto the same fresh medium with cytoki-
nins, 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 protuber-
ances 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 nat-
urally 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 poly-
phyllin I, polyphyllin II and polyphyllin VII but poly-
phyllin 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 dif-
ferent cytokinins concentrations. Table 2 shows the influ-
ence of different concentrations of cytokinins (BA, KIN
and TDZ) on steroidal saponin production in MRs har-
vested after 6 months of culture. Total steroidal saponins
(polyphyllin I ? polyphyllin II ? polyphyllin VII) accu-
mulation 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
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
Fig. 2 RP-HPLC chromatograms of steroidal saponin analysis in P.polyphylla. a HPLC profile of standard steroidal saponins, b HPLCprofile showing presence of polyphyllin I, polyphyllin II and
polyphyllin VII in field grown rhizome, c HPLC profile showingthe presence of polyphyllin I, polyphyllin II and polyphyllin VII in
MR cultures
Plant Growth Regul
123
-
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, poly-
phyllin 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 com-
pared 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
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 den-
sity 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. po-
lyphylla stem. BAP and TDZ were found to be more effec-
tive 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. Simi-
larly, 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
Table 2 Effect of cytokinins onin vitro production of steroidal
saponins in MR cultures derived
from tTCL
* Different letters within each
column represent significant
difference at P B 0.05 by
Tukey HSD test
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 0.75bcde 1.04 0.14cd 19.07 0.95bc 20.19 0.64cd
0.5 5.72 0.72ab 3.00 0.71b 25.12 0.85a 33.85 1.99a
1.0 4.39 0.45bc 2.21 0.40bc 22.24 1.24ab 28.84 1.90ab
KIN 0.25 0.35 0.04f 0.44 0.06d 13.70 0.74de 14.49 0.69d
0.5 1.20 0.36def 0.63 0.23cd 13.00 1.06de 19.66 0.97cd
1.0 0.95 0.05ef 0.82 0.17cd 17.89 1.07bcd 22.34 2.36bc
TDZ 0.25 1.20 0.79def 1.11 0.56cd 20.03 1.78abc 14.83 0.97d
0.5 2.28 0.54cdef 1.61 0.19bcd 21.64 0.84ab 25.53 1.45bc
1.0 3.73 0.40bcd 0.95 0.18cd 15.50 0.90cde 23.55 0.80bc
Plant Growth Regul
123
-
accumulation. Besides, exogenously applied cytokinins
have been reported to effectively promote tuberization and
yield of underground storage organs in many other mono-
cotyledonous 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 regen-
erated 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 hard-
ened. 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 char-
acteristic of MRs could contribute to cost-effective storage
as the cultures could be stored at 25 2 C in the form ofrhizomes for extended period without involving compli-
cated techniques as in other storage methods where
chemical or physical methods were applied. In vitro rhi-
zome 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
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 contin-
ued 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
Table 3 Effect of CHI andduration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL
* Different letters within each
column represent significant
difference at P B 0.05 by Tukey
HSD test
Days CHI
(mg/l)
Polyphyllin I
(mg/g DW)*
Polyphyllin II
(mg/g DW)*
Polyphyllin VII
(mg/g DW)*
Total saponin
(mg/g DW)*
0 0 5.72 0.72f 3.02 0.53d 25.14 0.85k 33.88 1.99h
15 0 5.75 0.37f 3.04 0.05d 26.11 1.72jk 34.91 1.37h
50 6.31 0.39ef 4.44 0.32cd 28.85 0.07ghijk 39.60 0.60fgh
100 8.73 0.49cdef 7.24 0.53abcd 34.39 0.71bcdef 50.36 1.65cde
200 7.44 0.87def 6.52 0.40bcd 29.53 0.41fghijk 43.48 0.64efg
30 0 5.79 0.43f 3.06 0.16d 26.84 0.33ijk 35.68 0.91h
50 7.92 0.35cdef 6.20 0.56bcd 31.79 1.74efghi 45.91 2.58def
100 9.81 0.44bcd 8.15 0.59abc 38.52 1.30b 56.48 0.66bc
200 8.43 0.33cdef 7.89 0.31abc 32.89 1.07cdefg 49.21 1.06cde
45 0 5.81 0.32f 3.08 0.07d 27.07 0.58ijk 35.96 0.44h
50 8.97 1.49cdef 8.51 0.81abc 37.84 1.05bc 55.33 1.17bc
100 12.72 0.23ab 10.27 0.67ab 46.74 0.83a 69.73 1.06a
200 10.17 0.42bcd 9.59 0.49ab 36.82 0.48bcd 56.58 0.71bc
60 0 5.84 0.27f 3.09 0.04d 27.71 1.17hijk 36.64 1.35h
50 9.67 1.14bcde 9.24 3.02ab 32.32 0.54defgh 51.23 3.36cd
100 14.65 0.55a 11.40 0.52a 36.33 0.58bcde 62.38 1.08ab
200 11.08 0.81bc 9.37 1.00ab 31.03 0.85fghij 51.48 0.57cd
Plant Growth Regul
123
-
accumulation in MR cultures is significantly affected by
different concentrations of cytokinins, thus establishing the
fact that there exists a strong relationship between cytoki-
nins 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 sec-
ondary 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 (Vinter-
halter 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 sec-
ondary 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 metab-
olites (Zhao et al. 2005). The accumulation of polyphyllins
is also significantly affected by YE and CHI. The elicita-
tion effect of biotic elicitors is most likely due to the oli-
gosaccharides present in them which have been reported as
potent signalling molecules that regulate growth, devel-
opment 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; Vee-
rashree et al. 2012). CHI has also been reported to act as an
elicitor for the improved production of secondary metab-
olites 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 metabo-
lism 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
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
Plant Growth Regul
123
-
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
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 metab-
olites compared to both cell suspension and biomass pro-
duction 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.
Table 4 Effect of SA andduration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL
* Different letters within each
column represent significant
difference at P B 0.05 by
Tukey HSD test
Days SA (mg/l) Polyphyllin I
(mg/g DW)*
Polyphyllin II
(mg/g DW)*
Polyphyllin VII
(mg/g DW)*
Total saponin
(mg/g DW)*
0 0 5.72 0.72g 3.02 0.53ef 25.14 0.85j 33.88 1.99i
15 0 5.75 0.37g 3.04 0.05ef 26.11 1.72j 34.91 1.37i
50 12.70 1.79abc 4.36 0.52ef 58.37 1.15ab 75.43 2.51bc
100 8.78 0.30defg 7.45 0.60cdef 48.70 1.01cde 64.93 1.49de
200 6.55 0.30fg 4.00 0.21ef 37.71 1.70fg 48.27 1.97gh
30 0 5.79 0.43g 3.06 0.16ef 26.84 0.33ij 35.68 0.91i
50 16.01 0.99a 6.51 0.83def 65.14 1.65a 87.66 1.66a
100 11.50 0.58bcd 11.24 0.62bc 53.22 1.69bcd 75.95 1.00bc
200 9.40 0.22cdef 6.14 0.29def 44.67 1.78ef 60.20 1.41def
45 0 5.81 0.32g 3.08 0.07ef 27.07 0.58hij 35.96 0.44i
50 13.99 0.56ab 10.11 0.79bcd 55.22 0.82bc 79.32 1.00ab
100 9.42 0.31cdef 16.85 2.22a 42.12 2.05ef 68.39 4.29cd
200 7.51 0.26efg 12.37 1.14ab 33.82 1.05ghi 53.70 1.70fg
60 0 5.84 0.27g 3.09 0.04ef 27.71 1.17hij 36.64 1.35i
50 10.98 0.78bcde 6.80 0.41cdef 46.66 2.36de 64.44 2.12de
100 8.62 0.30defg 13.53 1.65ab 34.50 1.33gh 56.65 0.33efg
200 6.69 0.72 fg 7.55 0.72cde 28.63 0.46hij 42.86 0.55hi
Table 5 Effect of YE andduration of elicitation on in vitro
production of steroidal saponins
in MRs liquid cultures derived
from tTCL
* Different letters within each
column represent significant
difference at P B 0.05 by
Tukey HSD test
Days YE (mg/l) Polyphyllin I
(mg/g DW)
Polyphyllin II
(mg/g DW)
Polyphyllin VII
(mg/g DW)
Total saponin
(mg/g DW)
0 0 5.72 0.72f 3.02 0.53f 25.14 0.85g 33.88 1.99f
15 0 5.75 0.37f 3.04 0.05f 26.11 1.72fg 34.91 1.37ef
50 7.24 1.03ef 4.13 0.58cdef 29.03 1.58efg 40.40 1.22def
100 11.35 0.49abc 5.23 0.43cdef 36.10 1.96bcdef 52.68 1.93bc
200 8.16 0.31cdef 4.65 0.24cdef 30.05 1.14defg 42.86 1.14cdef
30 0 5.79 0.43f 3.06 0.16f 26.84 0.33fg 35.68 0.91ef
50 10.78 0.78bcd 5.29 1.14cdef 34.48 1.15cdefg 50.56 2.14bcd
100 14.33 0.37a 9.38 0.57a 47.75 3.11a 71.46 4.09a
200 11.88 0.80ab 6.48 0.28bc 35.98 0.55bcdef 54.34 1.12b
45 0 5.81 0.32f 3.08 0.07f 27.07 0.58fg 35.96 0.44ef
50 7.92 0.58def 6.12 0.51bcd 38.18 3.99abcde 52.22 4.32bc
100 10.65 0.66bcd 7.83 0.41ab 44.79 2.82ab 63.27 2.94ab
200 9.32 0.59bcde 4.96 0.45cdef 39.84 2.42abcd 54.13 2.67b
60 0 5.84 0.27f 3.09 0.04f 27.71 1.17fg 36.64 1.35ef
50 8.19 1.25cdef 4.03 0.42def 32.69 2.35cdefg 44.90 2.81bcde
100 8.65 0.29bcdef 5.62 0.30bcde 40.46 1.36abc 54.73 0.87b
200 7.21 0.24ef 3.66 0.24ef 34.21 0.99cdefg 45.08 0.85bcde
Plant Growth Regul
123
-
Conclusion
The protocol described here for the medicinally important
and endangered plant, P. polyphylla provides a novel sys-
tem for mass propagation, storage and production of sec-
ondary 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 interna-
tional 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. Bhattacharjeeand Ms. B. J. Mylliemngap, Department of Biotechnology and Bio-
informatics, 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.
References
Aitsu Y, Yokoyama M, Yanagi M (1992) Production of ginsenosides
by culture of transformed ginseng. Jpn Kokai Tokkyo Koho
JPO4341194
Ali MB, Yu KW, Hahn EJ, Paek KY (2006) Methyl jasmonate and
salicylic acid elicitation induces ginsenosides accumulation,
enzymatic and non-enzymatic antioxidant in suspension culture
Panax ginseng roots in bioreactors. Plant Cell Rep 25:613620
Baskaran P, Jayabalan N (2008) Effect of growth regulators on rapid
micropropagation and psoralen production in Psoralea corylifo-
lia L. Acta Physiol Plant 30:345351
Bhattarai KR, Ghimire MD (2006) Cultivation and sustainable
harvesting of commercially important medicinal and aromatic
plants of Nepal. Heritage Research and Development Forum,
Nepal, pp 369372
Cheng XY, Zhou HY, Cui X, Ni W, Liu CZ (2006) Improvement of
phenylethanoid glycosides biosynthesis in Cistanche deserticola
cell suspension cultures by chitosan elicitor. J Biotechnol
121:253260
Cheung JYN, Ong RCY, Suen YK, Ooi V, Wong HNC et al (2005)
Polyphyllin D is a potent apoptosis inducer in drug-resistant
HepG2 cells. Cancer Lett 217:203211
Cho JS, Kim JY, Kim IH, Kim DI (2003) Effect of polysaccharide
elicitors on the production of decursinol angelate in Angelica
gigas Nakai root cultures. Biotechnol Bioproc Eng 8:158161
Costa F, Yendo ACA, Fleck JD, Gosmann G, Fett-Neto AG (2013)
Accumulation of a bioactive triterpene saponin fraction of
Quillaja brasiliensis leaves is associated with abiotic and biotic
stresses. Plant Physiol Biochem 66:5662
Cousins MM, Adelberg JW (2008) Short-term and long-term time
course studies of turmeric (Curcuma longa L.) microrhizome
development in vitro. Plant Cell Tissue Organ Cult 93:283293
Deng DW, Lauren DR, Cooney JM, Jensen DJ, Wurms KV, Upritchard
JE, Cannon RD, Wang MZ, Li MZ (2008) Antifungal saponins
from Paris polyphylla Smith. Planta Med 74:13971402
Devkota KP, Tareq HKM, Ranjit R, Lannang AM, Samreen,
Choudhary MI (2007) Tyrosinase inhibitory and antileishmanial
constituents from the rhizomes of Paris polyphylla. Nat Prod Res
21:321327
Fernie AR, Willmitzer L (2001) Update on tuber formation,
dormancy and sprouting: molecular and biochemical triggers
of potato tuber development. Plant Physiol 127:14591465
Fu YL, Yu ZY, Tang XM, Zhao Y, Yuan XL, Wang S, Ma BP, Cong
YW (2008) Pennogenin glycosides with a spirostanol structure
are strong platelet agonists: structural requirement for activity
and mode of platelet agonist synergism. J Thromb Haemost
6:524533
Ghosh S, Ghosh B, Jha S (2007) In vitro tuberisation of Gloriosa
superba L. on basal medium. Sci Hortic 114:220223
Gopal J, Minocha JL, Dhaliwal HS (1998) Microtuberization in
potato (Solanum tuberosum L.). Plant Cell Rep 17:794798
Hahn MG (1996) Microbial elicitors and their receptors in plants.
Annu Rev Phytopathol 34:387412
Han BH, Yae BW, Yu HJ, Peak KY (2005) Improvement of in vitro
micropropagation of Lilium oriental hybrid Casablanca by the
formation of shoots with abnormally swollen basal plates. Sci
Hortic 103:351359
Hanafy MS, Abou-Setta LM (2007) Saponins production in shoot and
callus cultures of Gypsophila paniculata. J Appl Sci Res
3:10451049
He J, Zhang S, Wang H, Chen CX, Chen SF (2006) Advances in
studies on and uses of Paris polyphylla var. yunnanensis. Acta
Botanica Yunnanica 28:271276
Jean M, Cappadocia M (1991) In vitro tuberization in Dioscorea alata
L. Brazo fuerte and Florida and D. abyssinica Hoch. Plant Cell
Tissue Organ Cult 26:147152
Jeong GT, Park DH (2005) Enhancement of growth and secondary
metabolite biosynthesis: effect of elicitors derived from plants
and insects. Biotechnol Bioprocess Eng 10:7377
Kang SM, Jung HY, Kang YM, Yun DJ, Bahk JD, Yang JK, Choi MS
(2004) Effects of methyl jasmonate and salicylic acid on the
production of tropane alkaloids and the expression of PMT and
H6H in adventitious root cultures of Scopolia parviflora. Plant
Sci 166:745751
Karwasara VS, Jain R, Tomar P, Dixit VK (2010) Elicitation as yield
enhancement strategy for glycyrrhizin production by cell
cultures of Abrus precatorius Linn. In vitro Cell Dev Biol Plant
46:354462
Kehie M, Kumaria S, Tandon P (2013) In vitro plantlet regeneration
from cotyledon segments of Capsicum chinense Jacq. cv. Naga
King Chili, and determination of capsaicin content by high
performance liquid chromatography. Sci Hortic 164:18
Khare CP (2007) Indian medicinal plantsan illustrated dictionary.
Springer, New Delhi, pp 16, 463464
Korsangruang S, Soonthornchareonnon N, Chintapakorn Y, Saralamp
P, Prathanturarug S (2010) Effects of abiotic and biotic elicitors
on growth and isoflavonoid accumulation in Pueraria. candollei
var. candollei and P. candollei var. mirifica cell suspension
cultures. Plant Cell Tissue Organ Cult 103:333342
Lee H, Lin JY (1988) Antimutagenic activity of extracts from
anticancer drugs in Chinese medicine. Mutat Res 204:229234
Lee MS, Yuet-Wa JC, Kong SK, Yu B, Eng-Choon VO, Nai-Ching
HW, Chung-Wai TM, Fung KP (2005) Effects of polyphyllin D,
a steroidal saponin in Paris polyphylla, in growth inhibition of
Plant Growth Regul
123
-
human breast cancer cells and in xenograft. Cancer Biol Ther
4:12481254
Li YC (1986) Vegetative propagation of Paris polyphylla var.
yunnanensis. Acta Bot Yunnan 8:429435 (in Chinese)
Lipsky AK (1992) Problems of optimization of plant cell culture
processes. J Biotechnol 26:8397
Ludwig-Muller J, Georgiev M, Bley T (2008) Metabolite and
hormonal status of hairy root cultures of Devils claw (Harpag-
ophytum procumbens) in flasks and in a bubble column
bioreactor. Process Biochem 43:1523
Man SL, Wang YL, Li YY, Gao WY, Huang XX, Ma CY (2013)
Phytochemistry, pharmacology, toxicology and structure-cyto-
toxicity relationship of Paridis Rhizome saponin. Chin Herbal
Med 5:3346
Mao AA, Hynniewta TM, Sanjappa M (2009) Plant wealth of
northeast India with reference to ethnobotany. Indian J Tradit
Knowl 8:96103
Mata-Rosas M, Baltazar-Garca RJ, Moon P, Hietz P, Luna-Monter-rojo VE (2010) In vitro regeneration of Lycaste aromatica
(Graham ex Hook) Lindl. (Orchidaceae) from pseudobulb
sections. Plant Biotechnol Rep 4:157163
Matsuda H, Pongpiriyadacha Y, Morikawa T, Kishi A, Kataoka S,
Yoshikawa M (2003) Protective effects of steroid saponins from
Paris polyphylla var. yunnanensis on ethanol- or indomethacin-
induced gastric mucosal lesions in rats: structural requirement
for activity and mode of action. Bioorg Med Chem Lett
13:11011106
Merkli A, Christen P, Kapetanidis I (1997) Production of diosgenin
by hairy root cultures of Trigonella foenum-graecum L. Plant
Cell Rep 16:632636
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473497
Nhut DT, Le BV, Fukai S, Tanaka M, Van TT (2001) Effects of
activated charcoal, explant size, explant position and sucrose
concentration on plant and shoot regeneration of Lilium longi-
florum via young stem. Plant Growth Regul 33:5965
Nhut DT, Teixeira da Silva JA, Le BV, Van Tran Thanh K (2003)
Thin cell layer studies of vegetable, leguminous and medicinal
plants. In: Nhut DT, Van Tran Thanh K, Le BV, Thorpe T (eds)
Thin cell layer culture system: regeneration and transformation
applications. Kluwer, Dordrecht, pp 387426
Omokolo ND, Boudjeko T, Tsafack Takadong JJ (2003) In vitro
tuberization of Xanthosoma sagittifolium L. Schott: effects of
phytohormones, sucrose, nitrogen and photoperiod. Sci Hortic
(Amsterdam) 98:337345
Pan Y, Liang Y, Wang H, Liang M (2004) Antioxidant activities of
several Chinese medicine herbs. Food Chem 88:347350
Pence VC, Soukup VG (1993) Factors affecting the initiation of mini-
rhizomes from Trillium erectum and T. grandiflorum tissues
in vitro. Plant Cell Tissue Organ Cult 35:229235
Piao XC, Chakrabarty D, Hahn EJ, Paek KY (2003) A simple method
for mass production of potato microtubers using a bioreactor
system. Curr Sci 84:11291132
Poornima GN, Ravishankar RV (2007) In vitro propagation of wild
yams, Dioscorea oppositifolia (Linn) and Dioscorea pentaphylla
(Linn). Afr J Biotechnol 6:23482352
Qian ZG, Zhao ZJ, Xu Y, Qian X, Zhong JJ (2006) Novel chemically
synthesized salicylate derivative as an effective elicitor for
inducing the biosynthesis of plant secondary metabolites.
Biotechnol Prog 22:331333
Radman R, Saez T, Bucke C, Kesahvarz T (2003) Elicitation in plants
and microbial cell systems. Biotechnol Appl Biochem 37:91102
Raomai S, Kumaria S, Tandon P (2014) Plant regeneration through
direct somatic embryogenesis from immature zygotic embryos of
the medicinal plant, Paris polyphylla Sm. Plant Cell Tissue
Organ Cult. doi:10.1007/s11240-014-0496-2
Roat C, Ramawat KG (2009) Elicitor-induced accumulation of
stilbenes in cell suspension cultures of Cayratia trifolia (L.)
Domin. Plant Biotechnol Rep 3:135138
Sarkar D, Pandey SK, Sharma S (2006) Cytokinins antagonize the
jasmonates action on the regulation of potato (Solanum tubero-
sum) tuber formation in vitro. Plant Cell Tissue Organ Cult
87:285295
Scherwinski-Pereira JE, da Guedes RS, Fermino PCP Jr, Silva TL,
Costa FHS (2010) Somatic embryogenesis and plant regenera-
tion in oil palm using the thin cell layer technique. In Vitro Cell
Dev Biol-Plant 46:378385
Sharma TR, Singh BM (1995) In vitro microrhizome production in
Zingiber officinale Rosc. Plant Cell Rep 15:274277
Shirgurkar MV, John CK, Nadgauda RS (2001) Factors affecting
in vitro microrhizome production in turmeric. Plant Cell Tissue
Organ Cult 64:511
Silva JAT (2003) Thin cell layer technology in ornamental plant
micropropagation and biotechnology. Afr J Biotech 2:683691
Singh SK, Rai MK, Asthana P, Sahoo L (2009) An improved
micropropagation of Spilanthes acmella L. through transverse
thin cell layer culture. Acta Physiol Plant 31:693698
Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil
Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012)
Optimization of elicitation conditions with methyl jasmonate and
salicylic acid to improve the productivity of withanolides in the
adventitious root culture of Withania somnifera (L.) Dunal. Appl
Biochem Biotechnol 168:681696
Sudha G, Ravishankar GA (2002) Involvement and interaction of
various signalling compounds on the plant metabolic events
during defense response, resistance to stress factors, formation of
secondary metabolites and their molecular aspects. Plant Cell
Tissue Organ Cult 71:181212
Sun J, Liu BR, Hu WJ, Yu LX, Qian XP (2007) In vitro anticancer
activity of aqueous extracts and ethanol extracts of fifteen
traditional Chinese medicines on human digestive tumor cell
lines. Phytother Res 21:11021104
Suri SS, Jain S, Ramawat KG (1999) Plantlet regeneration and bulbil
formation in vitro from leaf and stem explants of Curculigo
orchioides, an endangered medicinal plant. Sci Hortic
79:127134
Takayama S, Misawa M (1982) Regulation of organ formation by
cytokinin and auxin in Lilium bulb scales grown in vitro. Plant
Cell Physiol 22:461467
Tyagi RK, Agrawal A, Yusuf A (2006) Conservation of Zingiber
germplasm through in vitro rhizome formation. Sci Hortic
108:210219
Van Tran Thanh K (1974) Direct flower neoformation from
superficial tissue of small explants of Nicotiana tabacum L.
Planta 115:8790
Veerashree V, Anuradha CM, Kumar V (2012) Elicitor-enhanced
production of gymnemic acid in cell suspension cultures of
Gymnema sylvestre R. Br. Plant Cell Tissue Organ Cult 108:2735
Vinterhalter B, Jankovic T, Sabikin K, Nicolic R, Vinterhalter D
(2008) Propagation and xanthone content of Gentianella austri-
aca shoot cultures. Plant Cell Tissue Organ Cult 94:329335
Wang GX, Han J, Zhao LW, Jiang DX, Liu YT, Liu XL (2010)
Anthelmintic activity of steroidal saponins from Paris polyphy-
lla. Phytomedicine 17:11021105
Wink M (1989) Genes of secondary metabolism: differential expres-
sion in plants and in vitro cultures and functional expression in
genetically transformed microorganisms. In: Kurz WGW (ed)
Primary and secondary metabolism of plant cell cultures I.
Springer, Berlin, pp 239251
Wu SS, Gao WY, Duan HQ, Jia W (2004) Advances in studies on
chemical constituents and pharmacological activities of Rhizoma
paridis. Chin Tradit Herbal Drugs 35:344347
Plant Growth Regul
123
-
Zaerr JB, Mapes MO (1982) Action of growth regulators. In: Bonga
JM, Durzan DJ (eds) Tissue culture in forestry. Martinus Nijhoff/
Dr. W. Junk Publishers, The Hague/Boston/London, pp 231255
Zhang CH, Wu JY, He GY (2002) Effects of inoculum size and age
on biomass growth and paclitaxel production of elicitor-treated
Taxus yunnanensis cell cultures. Appl Microbiol Biotechnol
60:396402
Zhang X, Cui Y, Huang J, Zhang Y, Nie Z et al (2007) Immuno-
stimulating properties of diosgenyl saponins isolated from Paris
polyphylla. Bioorg Med Chem Lett 17:24082413
Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction
leading to production of plant secondary metabolites. Biotechnol
Adv 23:283333
Zhao Y, Kang LP, Liu YX, Liang YG, Tan DW, Yu ZY (2009)
Steroidal saponins from the rhizome of Paris polyphylla and
their cytotoxic activities. Planta Med 75:356363
Zhao JL, Zhou LG, Wu JY (2010) Effects of biotic and abiotic elicitors
on cell growth and tanshinone accumulation in Salvia miltiorrhiza
cell cultures. Appl Microbiol Biotechnol 87:137144
Zhou J (1989) Some bioactive substances from plants of West China.
Pure Appl Chem 61:457460
Plant Growth Regul
123
Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitorsAbstractIntroductionMaterials and methodsPlant materialMedia and culture conditionsAcclimatization of regenerated plantletsHistological analysisLiquid culture of MRs for elicitationExtraction and determination of steroidal saponinsStatistical analysis
ResultsShoot regeneration via MRs derived from tTCLEffect of cytokinins on steroidal saponin productionEffect of elicitors on biomass accumulation and steroidal saponin production in MRs
DiscussionConclusionAcknowledgmentsReferences