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Plant Growth Regul DOI 10.1007/s10725-014-9957-1



Plantlet regeneration of Paris polyphylla Sm. via thin cell layer culture and enhancement of steroidal saponins in mini-rhizome cultures using elicitors

Shiveirou Raomai Suman Kumaria Mechuselie Kehie Pramod Tandon

Received: 23 April 2014 / Accepted: 24 June 2014 Springer Science+Business Media Dordrecht 2014

Abstract An efficient regeneration protocol for the medicinal plant, Paris polyphylla Sm. was developed through the formation of mini-rhizomes (MRs) using transverse thin cell layer (tTCL) culture technique. MRs were induced from tTCL explants derived from the basal and middle stem portions while apical portion failed to show any kind of response. Highest response percentage (86.6 %) of MRs formation with a maximum fresh weight (1.05 ± 0.08 g) was achieved from basal sections cultured on MS medium supplemented with 0.5 mg/l 6-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.

S. Raomai S. Kumaria (&) M. Kehie P. Tandon Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong 793022, India e-mail:


Elicitors Medicinal plant Mini-rhizome

Paris polyphylla Steroidal saponins Thin cell layer








Dry weight


Fresh weight


High performance liquid chromatography






Murashige and Skoog


Salicyclic acid




Transverse thin cell layer


Plant growth regulator


Yeast extract


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

Plant Growth Regul DOI 10.1007/s10725-014-9957-1 ORIGINAL PAPER Plantlet regeneration of Paris polyphylla Sm. via thin cell


Plant Growth Regul

well as to stop bleeding (Zhou 1989) and for treating liver cancer (Cheung et al. 2005). The rhizome of this plant has been developed into patented Chinese medicines such as ‘‘Yunnan Bai Yao’’, ‘‘Gong Xue Ning’’ capsules and ‘‘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 4–5 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

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

1974). We have reported the micropropagation of P. 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 March–April 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 2–3 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


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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/cm 2 pressure for

  • 15 min. All the cultures were incubated in the culture room

at temperature of 25 ± 2 C in the dark. MR induction in each treatment was evaluated 6 months after inoculation, without any subculture. The percentage of explants 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 growth regulators (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 basal medium for shoot sprouting or directly subjected to ex vitro conditions in the greenhouse. The developed shoots were kept under a 14/10–h light photoperiod with a photosyn- thetic photon flux of 60 lmol/m 2 /s provided by cool-white fluorescent lamps.

Acclimatization of regenerated plantlets

In vitro regenerated plantlets with well developed rhizome and roots were washed thoroughly until traces of agar were removed completely and then transferred to thermocol cups containing potting mixtures of soil, compost and peat moss in the ratio of 2:1:1 (w/w). The plantlets were maintained under green house conditions with a temperature of

  • 25 ± 2 C with 12 h photoperiod and 60 ± 5 % relative

humidity and were irrigated twice a week with MS

solution for the first 4 weeks.

Histological analysis

One year old rhizomes grown under greenhouse conditions, 6 months old in vitro MRs and MRs with protuberances were gently washed with distilled water in order to remove

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 (58–60 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 2.5–3 g FW were sus- pended in 50 ml MS medium containing 3 % sucrose supplemented with 0.5 mg/l BAP. Stock solution of SA and YE were prepared by dissolving in Milli-Q water. CHI was first dissolved in glacial acetic acid and then diluted with Milli-Q water. The pH of the elicitor stock solutions was adjusted to 5.8 and filter-sterilized before adding into the liquid medium. Elicitor solutions were added at the concentrations of 50, 100 and 200 mg/l immediately fol- lowing the inoculation of MRs in liquid medium. To the control variants, equal volumes of water were added. All flasks were shaken at 110–120 rpm on an orbital shaker at 25 ± 2 C in dark. The cultures were harvested after every 15 days for a period of 60 days for the analysis of MRs growth and steroidal saponins accumulation in order to identify the optimal exposure time and concentrations. At the end of each treatment period, the MR cultures were harvested, washed 2–3 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 = (W F - W 0 )7W 0 , where W 0 is the weight of inoculum at 0 day of inoculation and W F 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 the residue was re-dissolved in 5 ml methanol. Qualitative ste- roidal saponin profiling and quantification was carried out


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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 (0.1–1.0 mg/ml) in methanol and 20 ll each were injected to obtain standard curve plot of peak area with a run time of 12 min. The analyzed steroidal saponin content was expressed as mg steroidal saponin/g DW (dry weight). All the solvents (HPLC grade) were obtained from LOBA Chemie (Mumbai, India) and the standards were purchased from Shanghai Yaji Biological Technology Co., Ltd. (Shanghai, China).

Statistical analysis

All experiments were repeated thrice with three replicates each and data were analyzed using one-way analysis of variance (ANOVA) in JMP version 7.0.1 (SASInstitute, Cary, NC). The significant differences among the means were assessed by Tukey HSD test at 5 % probability level.


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 on shoot 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




(%)* #

FW (g)/MR* #

No. of shoot buds/MR** #




BAP 0.25



KIN 0.25



TDZ 0.25



BAP 0.25



KIN 0.25



TDZ 0.25









































0.0 i 0.94 ± 0.05 ab

1.05 ± 0.08 a

0.95 ± 0.05 ab 0.62 ± 0.06 cdef

0.64 ± 0.06 cdef

0.69 ± 0.07 bcde 0.93 ± 0.05 ab

0.96 ± 0.07 ab

0.90 ± 0.05 abc 0.48 ± 0.04 efgh

0.59 ± 0.05 def

0.55 ± 0.04 defg 0.20 ± 0.01 h

0.30 ± 0.04 gh

0.38 ± 0.05 fgh 0.55 ± 0.07 defg

0.57 ± 0.06 defg

0.49 ± 0.10 efgh

0.0 g 5.1 ± 0.5 abc

5.6 ± 0.4 a

5.2 ± 0.6 ab 3.3 ± 0.3 cdef

3.6 ± 0.4 bcdef

3.9 ± 0.3 abcde 5.0 ± 0.4 abc

5.4 ± 0.6 a

4.6 ± 0.4 abcd 2.7 ± 0.4 ef

3.1 ± 0.3 def

2.9 ± 0.3 def 2.0 ± 0.3 f

2.2 ± 0.2 ef

2.4 ± 0.3 ef 2.9 ± 0.3 def

3.0 ± 0.3 def

2.8 ± 0.3 def


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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

Plant Growth Regul BAP (86.6 %) with maximum average FW (1.05 ± 0.08 g) followed by

Fig. 2 RP-HPLC chromatograms of steroidal saponin analysis in P. polyphylla. a HPLC profile of standard steroidal saponins, b HPLC profile showing presence of polyphyllin I, polyphyllin II and polyphyllin VII in field grown rhizome, c HPLC profile showing the presence of polyphyllin I, polyphyllin II and polyphyllin VII in MR cultures

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



100 mg/l




45 days

(46.74 ± 0.83 mg/g DW). Total steroidal saponin content


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Table 2 Effect of cytokinins on in 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

Polyphyllin II

Polyphyllin VII

Total saponin

(mg/g DW)*

(mg/g DW)*

(mg/g DW)*

(mg/g DW)*


6.94 ± 0.27 a

5.49 ± 0.21 a

11.47 ± 0.69 e

23.89 ± 1.04 bc

BAP 0.25

3.44 ± 0.75 bcde

1.04 ± 0.14 cd

19.07 ± 0.95 bc

20.19 ± 0.64 cd


5.72 ± 0.72 ab

3.00 ± 0.71 b

25.12 ± 0.85 a

33.85 ± 1.99 a


4.39 ± 0.45 bc

2.21 ± 0.40 bc

22.24 ± 1.24 ab

28.84 ± 1.90 ab

KIN 0.25

0.35 ± 0.04 f

0.44 ± 0.06 d

13.70 ± 0.74 de

14.49 ± 0.69 d


1.20 ± 0.36 def

0.63 ± 0.23 cd

13.00 ± 1.06 de

19.66 ± 0.97 cd


0.95 ± 0.05 ef

0.82 ± 0.17 cd

17.89 ± 1.07 bcd

22.34 ± 2.36 bc

TDZ 0.25

1.20 ± 0.79 def

1.11 ± 0.56 cd

20.03 ± 1.78 abc

14.83 ± 0.97 d


2.28 ± 0.54 cdef

1.61 ± 0.19 bcd

21.64 ± 0.84 ab

25.53 ± 1.45 bc


3.73 ± 0.40 bcd

0.95 ± 0.18 cd

15.50 ± 0.90 cde

23.55 ± 0.80 bc

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.


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


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Table 3 Effect of CHI and duration 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



Polyphyllin I

Polyphyllin II

Polyphyllin VII

Total saponin


(mg/g DW)*

(mg/g DW)*

(mg/g DW)*

(mg/g DW)*



5.72 ± 0.72 f

3.02 ± 0.53 d

25.14 ± 0.85 k

33.88 ± 1.99 h

  • 15 3.04 ± 0.05 d


5.75 ± 0.37 f

26.11 ± 1.72 jk

34.91 ± 1.37 h


6.31 ± 0.39 ef

4.44 ± 0.32 cd

28.85 ± 0.07 ghijk

39.60 ± 0.60 fgh


8.73 ± 0.49 cdef

7.24 ± 0.53 abcd

34.39 ± 0.71 bcdef

50.36 ± 1.65 cde


7.44 ± 0.87 def

6.52 ± 0.40 bcd

29.53 ± 0.41 fghijk

43.48 ± 0.64 efg

  • 30 3.06 ± 0.16 d


5.79 ± 0.43 f

26.84 ± 0.33 ijk

35.68 ± 0.91 h


7.92 ± 0.35 cdef

6.20 ± 0.56 bcd

31.79 ± 1.74 efghi

45.91 ± 2.58 def


9.81 ± 0.44 bcd

8.15 ± 0.59 abc

38.52 ± 1.30 b

56.48 ± 0.66 bc


8.43 ± 0.33 cdef

7.89 ± 0.31 abc

32.89 ± 1.07 cdefg

49.21 ± 1.06 cde

  • 45 3.08 ± 0.07 d


5.81 ± 0.32 f

27.07 ± 0.58 ijk

35.96 ± 0.44 h


8.97 ± 1.49 cdef

8.51 ± 0.81 abc

37.84 ± 1.05 bc

55.33 ± 1.17 bc


12.72 ± 0.23 ab

10.27 ± 0.67 ab

46.74 ± 0.83 a

69.73 ± 1.06 a


10.17 ± 0.42 bcd

9.59 ± 0.49 ab

36.82 ± 0.48 bcd

56.58 ± 0.71 bc

  • 60 3.09 ± 0.04 d


5.84 ± 0.27 f

27.71 ± 1.17 hijk

36.64 ± 1.35 h


9.67 ± 1.14 bcde

9.24 ± 3.02 ab

32.32 ± 0.54 defgh

51.23 ± 3.36 cd

  • 100 11.40 ± 0.52 a

14.65 ± 0.55 a

36.33 ± 0.58 bcde

62.38 ± 1.08 ab

  • 200 9.37 ± 1.00 ab

11.08 ± 0.81 bc

31.03 ± 0.85 fghij

51.48 ± 0.57 cd

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 3–5 g after about 18 months without producing shoot buds (data not shown). Thus, from a single preformed shoot about 10–15 MRs, each weighing about 4–5 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 of rhizomes 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

Plant Growth Regul

Plant Growth Regul Fig. 3 Effect of elicitors on the growth of MRs. a effect of

Fig. 3 Effect of elicitors on the growth of MRs. a effect of CHI, b effect of SA, c effect of YE. Growth Index = (W F - W 0 )7W 0 , where W 0 is the weight of inoculum at 0 day of inoculation and W F is the weight of the MRs on the day of harvest

accumulation in MR cultures is significantly affected by different concentrations of cytokinins, thus establishing the fact that there exists a strong relationship between 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


Plant Growth Regul

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

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

Table 5 Effect of YE and duration 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


SA (mg/l)

Polyphyllin I

Polyphyllin II

Polyphyllin VII

Total saponin

(mg/g DW)*

(mg/g DW)*

(mg/g DW)*

(mg/g DW)*



5.72 ± 0.72 g

3.02 ± 0.53 ef

25.14 ± 0.85 j

33.88 ± 1.99 i



5.75 ± 0.37 g

3.04 ± 0.05 ef

26.11 ± 1.72 j

34.91 ± 1.37 i


12.70 ± 1.79 abc

4.36 ± 0.52 ef

58.37 ± 1.15 ab

75.43 ± 2.51 bc


8.78 ± 0.30 defg

7.45 ± 0.60 cdef

48.70 ± 1.01 cde

64.93 ± 1.49 de


6.55 ± 0.30 fg

4.00 ± 0.21 ef

37.71 ± 1.70 fg

48.27 ± 1.97 gh



5.79 ± 0.43 g

3.06 ± 0.16 ef

26.84 ± 0.33 ij

35.68 ± 0.91 i


16.01 ± 0.99 a

6.51 ± 0.83 def

65.14 ± 1.65 a

87.66 ± 1.66 a


11.50 ± 0.58 bcd

11.24 ± 0.62 bc

53.22 ± 1.69 bcd

75.95 ± 1.00 bc


9.40 ± 0.22 cdef

6.14 ± 0.29 def

44.67 ± 1.78 ef

60.20 ± 1.41 def



5.81 ± 0.32 g

3.08 ± 0.07 ef

27.07 ± 0.58 hij

35.96 ± 0.44 i


13.99 ± 0.56 ab

10.11 ± 0.79 bcd

55.22 ± 0.82 bc

79.32 ± 1.00 ab


9.42 ± 0.31 cdef

16.85 ± 2.22 a

42.12 ± 2.05 ef

68.39 ± 4.29 cd


7.51 ± 0.26 efg

12.37 ± 1.14 ab

33.82 ± 1.05 ghi

53.70 ± 1.70 fg



5.84 ± 0.27 g

3.09 ± 0.04 ef

27.71 ± 1.17 hij

36.64 ± 1.35 i


10.98 ± 0.78 bcde

6.80 ± 0.41 cdef

46.66 ± 2.36 de

64.44 ± 2.12 de


8.62 ± 0.30 defg

13.53 ± 1.65 ab

34.50 ± 1.33 gh

56.65 ± 0.33 efg


6.69 ± 0.72 f g

7.55 ± 0.72 cde

28.63 ± 0.46 hij

42.86 ± 0.55 hi



YE (mg/l)

Polyphyllin I

Polyphyllin II

Polyphyllin VII

Total saponin

(mg/g DW)

(mg/g DW)

(mg/g DW)

(mg/g DW)



5.72 ± 0.72 f

3.02 ± 0.53 f

25.14 ± 0.85 g

33.88 ± 1.99 f

  • 15 3.04 ± 0.05 f


5.75 ± 0.37 f

26.11 ± 1.72 fg

34.91 ± 1.37 ef


7.24 ± 1.03 ef

4.13 ± 0.58 cdef

29.03 ± 1.58 efg

40.40 ± 1.22 def


11.35 ± 0.49 abc

5.23 ± 0.43 cdef

36.10 ± 1.96 bcdef

52.68 ± 1.93 bc


8.16 ± 0.31 cdef

4.65 ± 0.24 cdef

30.05 ± 1.14 defg

42.86 ± 1.14 cdef

  • 30 3.06 ± 0.16 f


5.79 ± 0.43 f

26.84 ± 0.33 fg

35.68 ± 0.91 ef


10.78 ± 0.78 bcd

5.29 ± 1.14 cdef

34.48 ± 1.15 cdefg

50.56 ± 2.14 bcd


14.33 ± 0.37 a

9.38 ± 0.57 a

47.75 ± 3.11 a

71.46 ± 4.09 a


11.88 ± 0.80 ab

6.48 ± 0.28 bc

35.98 ± 0.55 bcdef

54.34 ± 1.12 b

  • 45 3.08 ± 0.07 f


5.81 ± 0.32 f

27.07 ± 0.58 fg

35.96 ± 0.44 ef


7.92 ± 0.58 def

6.12 ± 0.51 bcd

38.18 ± 3.99 abcde

52.22 ± 4.32 bc


10.65 ± 0.66 bcd

7.83 ± 0.41 ab

44.79 ± 2.82 ab

63.27 ± 2.94 ab


9.32 ± 0.59 bcde

4.96 ± 0.45 cdef

39.84 ± 2.42 abcd

54.13 ± 2.67 b

  • 60 3.09 ± 0.04 f


5.84 ± 0.27 f

27.71 ± 1.17 fg

36.64 ± 1.35 ef


8.19 ± 1.25 cdef

4.03 ± 0.42 def

32.69 ± 2.35 cdefg

44.90 ± 2.81 bcde

  • 100 5.62 ± 0.30 bcde

8.65 ± 0.29 bcdef

40.46 ± 1.36 abc

54.73 ± 0.87 b

  • 200 3.66 ± 0.24 ef

7.21 ± 0.24 ef

34.21 ± 0.99 cdefg

45.08 ± 0.85 bcde

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-Mu¨ ller 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.

Plant Growth Regul


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. Bhattacharjee and 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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