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: sumankhatrikumaria@gmail.com

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

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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

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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.

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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