Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

ORIGINAL PAPER

Efficiency of direct and indirect shoot organogenesis, molecularprofiling, secondary metabolite production and antioxidantactivity of micropropagated Ceropegia santapaui

J. J. Chavan • N. B. Gaikwad • S. D. Umdale •

P. R. Kshirsagar • K. V. Bhat • S. R. Yadav

Received: 5 March 2013 / Accepted: 24 May 2013 / Published online: 31 May 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Ceropegias has acquired significant importance

due to their medicinal properties, edible tubers, and its

ornamental flowers. The aim of this study was to optimize

direct shoot organogenesis (DSO), indirect shoot organo-

genesis (ISO) and plant regeneration of threatened medicinal

plant Ceropegia santapaui, followed by analysis of genetic

status and biochemical characterization of micropropagated

plantlets. For optimization, cotyledonary nodes and cotyle-

dons were used as source of explants in DSO and

ISO respectively. The highest frequency of regeneration

(88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was

obtained from cotyledonary nodes cultured on Murashige

and Skoog’s (MS) medium containing 2.0 mg L-1 2iP. The

best response for callus induction and proliferation was

achieved with 1.5 mg L-1 PR (picloram) in which 97.5 % of

cultures produced an average of 913 ± 10.9 mg (fresh

weight) of callus. The highest frequency of shoot formation

(92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was

obtained when calli were transferred to MS medium sup-

plemented with 2.5 mg L-1 BAP and 0.4 mg L-1 IBA.

Regenerated shoots were best rooted in half-strength MS

medium with 2.0 mg L-1 NAA. Plantlets successfully

acclimatized were morphologically indistinguishable from

the source plant. Micropropagated plantlets subjected to

random amplified polymorphic DNA and inter simple

sequence repeats (ISSR) marker based profiling reveled

uniform banding pattern in DSO-derived plantlets which

was similar to mother plant. ISSR fingerprints of ISO-

derived plants showed low variation. Method of regenera-

tion, plant part and solvent system significantly affected the

levels of total phenolics, flavonoids and antioxidant capac-

ity. Assay of antioxidant activity of different tissues revealed

that significantly higher antioxidant activity was observed in

ISO-derived tissues than DSO-derived and mother tissues.

RP-HPLC analysis of micropropagated plantlets showed the

presence of three major phenolic compounds which were

similar to those detected in mother plant. Rapid multiplica-

tion rate, genetic stability and biochemical parameter

ensures the efficacy of the protocol developed for the prop-

agation of this threatened medicinal plant.

Keywords Antioxidant � Cerpegin � Ceropegia santapaui �Conservation � Flavonoids � ISSR � RP-HPLC � RAPD �Organogenesis � Phenolics � Threatened species

Abbreviations

BAP 6-Benzylaminopurine

Cerpegin 1,1-Dimethylfuro(3,4-C)pyridine-3,4

(1H, 5H)-dione

DPPH 2,2-Diphenyl-1-picrylhydrazyl

DSO Direct shoot organogenesis

FW Fresh weight

IAA Indole 3-acetic acid

IBA Indole 3-butyric acid

2iP 6-c,c-Dimethylallylaminopurine

ISO Indirect shoot organogenesis

ISSR Inter simple sequence repeats

MS Murashige and Skoog’s medium (1962)

J. J. Chavan � N. B. Gaikwad � S. D. Umdale �P. R. Kshirsagar � S. R. Yadav

Department of Botany, Shivaji University,

Kolhapur 416 004, Maharashtra, India

J. J. Chavan (&)

Department of Botany, Yashavantrao Chavan Institute

of Science, Satara 415 001, Maharashtra, India

e-mail: [email protected]

S. D. Umdale � K. V. Bhat

National Research Centre on DNA Fingerprinting, NBPGR,

New Delhi 110 012, India

123

Plant Growth Regul (2014) 72:1–15

DOI 10.1007/s10725-013-9830-7

NAA a-Naphthalene acetic acid

PGRs Plant growth regulators

PR Picloram (4-amino-3,5,6-trichloropicolinic acid)

RAPD Random amplified polymorphic DNA

RE Rutin equivalent

RP-HPLC Reverse phase-high performance liquid

chromatography

TAE Tannic acid equivalent

TPC Total phenolic content

TFC Total flavonoid content

Introduction

The genus Ceropegia L. (family–Asclepiadaceae) includes

herbs and climbers grows in tropical and sub tropical regions

of the World which comprises over 220 species (Bruyns

2003; Murthy et al. 2012). The maximum diversity of Ce-

ropegia occurs in South Africa followed by Kenya, Mada-

gascar, and India (Murthy et al. 2012). India alone harbors

over 50 species of which 35 species are endemic to country

(Murthy et al. 2012). Ceropegia santapaui Wadhwa and

Ansari is one of the twining herbs endemic to small pockets

in Western Ghats and considered as threatened because of

the small number of individuals (Yadav and Kamble 2008).

Ceropegias (including C. santapaui) are highly-prized for

their medicinal properties, edible tubers, and its ornamental

flowers. Owing to their ornamental potential, some species

are cultivated as horticultural crop (McNew 2002; Hodgkiss

2004; Reynolds 2006). The sweet–sour leaves are edible and

are considered to be tonic and digestive. The tubers of Ce-

ropegias are edible and contain starch, sugars, gum, alb-

uminoids, carbohydrates, fats, and crude fiber (Mabberly

1987; Jain and Defillips 1991). The ‘Cerpegin’ (1,1-dim-

ethylfuro[3,4-C]pyridine-3,4(1H,5H)-dione) present in the

root tubers (including C. santapaui) was isolated and iden-

tified as pyridine type alkaloid (Nadkarni 1976; Adibatti

et al. 1991; Phulwaria et al. 2013). The Cerpegin exhibited

promising hepatoprotective, antipyretic, anti-ulcer, analge-

sic, mast-cell stabilizing, tranquillsing and hypotensive

activities (Adibatti et al. 1991; Sukumar et al. 1995). The

tubers of many Ceropegia species are used by tribal women

to promote fertility and vitality, treatment of diarrhea and

dysentery, and treatment of urinary bladder stones (Khare

2007; Swarnkar and Katewa 2008).

The genus Ceropegia as a whole is under threat, owing

to either destructive collection or habitat degradation. Due

to pharmacological importance of ‘cerpegin’ C. santapaui

is over-exploited and current status is threatened species.

Since it is a seasonal plant, generally vegetative growth is

seen only after first rain by sprouting of dormant under-

ground tubers. Moreover, the species is difficult to propa-

gate by conventional methods of cuttings and seedlings

because of poor seed setting and limited seed viability

(Yadav and Kamble 2008). There is therefore an urgent

need for conservation action to ensure large scale propa-

gation. Plant tissue culture has remained valuable in terms

of their beneficial role in the production and conservation

of plant based resources (Vasil 2008; Amoo et al. 2012).

This technique offers a way to multiply plants possessing a

special phenotypic character directly and rapidly, thereby

shortening the time needed, and providing an effective

technique for repopulation of the original locations of the

plants (Socorro et al. 1998). In response to degradation of

the habitat of these species, micropropagation systems have

been developed with several threatened species of Ce-

ropegia (Patil 1998; Beena et al. 2003; Nikam and Savant

2009; Chandore et al. 2010; Murthy et al. 2010; Chavan

et al. 2011a, b; Phulwaria et al. 2013).

The cryptic genetic defects arising via in vitro propaga-

tion in the regenerants seriously limits the utility of micro-

propagation system. However, it is important to assess the

genetic constitution and stability of in vitro micropropagated

plants before planning strategies for further propagation. The

progress made in DNA marker technology provided valuable

tools for the detection of somaclonal variation that was

reflected in the profiles of molecular genetic markers viz.

RFLPs, RAPDs and ISSRs (Devarumath et al. 2002; Bhatia

et al. 2011). However, none of the developed Ceropegia

micropropagation protocols has assessed the genetic fidelity

of micropropagated plants.

The present study was designed to optimize the direct

and indirect shoot organogenesis and plant regeneration in

C. santapaui. RAPD and ISSR markers were employed for

determining genetic stability of in vitro regenerated plants

and the reliability protocol for true to type cloning. Bio-

chemical parameters viz. total phenolic content, total fla-

vonoid content and antioxidant activity were investigated.

RP-HPLC analysis was also carried out for identification

and quantitation of phenolic compounds.

Materials and methods

In vitro seed germination and explant preparation

Mature plants and follicles of C. santapaui were collected

from the of Kumbharli locality of Northern Western Ghats,

India. Seeds separated from follicles, then washed and dried

for 3 days in the shade. Seeds were surface sterilized with

0.1 % HgCl2 for 5 min under aseptic conditions and washed

2–3 times with sterile distilled water. For germination, the

seeds were inoculated on MS medium without plant growth

regulators. Cultures were maintained in a growth room at

25 ± 1 �C in 16 h light and 8 h dark cycles with

50 lmol m-2 s-1 of light intensity provided by cool-white

2 Plant Growth Regul (2014) 72:1–15

123

fluorescent tubes. Cotyledonary nodes and cotyledons were

excised aseptically from 15 day old seedling were used as

explants for DSO and ISO respectively.

Optimization of direct shoot organogenesis (DSO)

Direct shoot organogenesis was optimized using cotyle-

donary nodes (about 1 cm) as source of explants with dif-

ferent plant growth regulators. The shoots were regenerated

on MS medium supplemented with 30 g L-1 sucrose,

0.1 g L-1 myo-inositol, different plant growth regulators

(BAP, KN, TDZ, 2iP, IBA) and solidified with 2 g L-1

ClariGel (Himedia, India). A control without any PGRs was

included. The pH of the medium was adjusted to 5.8 using

NaOH or HCl before autoclaving at 121 �C and 103 kPa for

20 min. All the cultures were maintained in a growth room at

25 ± 1 �C in a 16 h photoperiod and 8 h dark cycles with

50 lmol m-2 s-1 of light intensity. In order to assess the

influence of different PGRs on shoot proliferation from

cotyledonary nodes, following growth parameters were

measured: (1) the regeneration frequency (%), (2) average

number of shoots, and (3) average shoot length (cm).

Optimization of indirect shoot organogenesis (ISO)

Newly emerged cotyledons (more than 0.5 cm) were

aseptically excised from in vitro germinated seedlings were

cultured, abaxial side up, on MS media supplemented with

various concentrations of 2,4-D (0.5, 1.0, 1.5, 2.0,

2.5 mg L-1), PR (0.5, 1.0, 1.5, 2.0, 2.5 mg L-1), and NAA

(0.5, 1.0, 1.5, 2.0, 2.5 mg L-1). Frequency (%) of explants

that initiated callus was recorded after 4 weeks. Calli of

each treatment were sub-cultured in the same medium for

further proliferation every 4 weeks. Callus growth was

calculated by measuring its fresh weight of callus on each

auxin treatments. To optimize the effect of PGRs on shoot

regeneration, calli (approx. 200 mg) developed on opti-

mum callus induction medium were transferred to regen-

eration medium either BAP alone (0.5, 1.0, 1.5, 2.0, 2.5,

3.0 mg L-1) or in combination with IBA (0.2, 0.4, 0.6, 0.8,

1.0 mg L-1) or NAA (0.2, 0.4, 0.6, 0.8, 1.0 mg L-1). All

the cultures were incubated at 25 ± 1 �C under a 16 h light

and 8 h dark cycles with 50 lmol m-2 s-1 of light inten-

sity provided by cool fluorescent tubes (Philips, India).

Rooting and acclimatization

Regenerated shoots (both DSO and ISO-derived) from

optimal regeneration medium were transferred to half-

strength MS medium containing different auxins for in vitro

rooting. Auxins viz. IBA (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg L-1),

NAA (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg L-1), and IAA (0.5, 1.0,

1.5, 2.0, 2.5, 3.0 mg L-1) were tested either alone or their

combinations. To evaluate the influence of auxins on in vitro

rooting, following growth parameters were measured: (1) the

percentage of in vitro rooting (%), (2) average number of

roots, and (3) average root length (cm). After 4 weeks in

rooting medium, regenerated plants with well developed

roots were removed from culture tubes, washed free of

solidifying agent and transferred to plastic pots containing

sterile soil, sand and coco peat (1:2:1). Plants were accli-

matized for 2 weeks at 25 ± 1 �C in the growth room with

16-h photoperiod (50 lmol m-2 s-1 light intensity),

watered with half-strength MS liquid medium without

sucrose and then transferred to glasshouse conditions (temp:

30 �C, humidity: 70 %, 70 lmol m-2 s-1 light intensity).

Survival rate (%) was estimated after 60 days of transfer to

field conditions.

All the tissue culture experiments were set in a com-

pletely randomized block design and each experiment

repeated thrice with 20 tubes per treatment. Comparison

between the mean values of treatments with control were

made using Dunnett multiple comparison test (DMCT) at

ns, 0.05 and 0.01 level of significance.

RAPD and ISSR analysis

DNA was isolated from fresh young leaves of mother plant as

well as randomly selected in vitro raised plants (10 each from

DSO and ISO) were used for RAPD and ISSR analysis. The

genomic DNA was isolated following the protocol of Doyle

and Doyle (1990) with a little modification. Quality and

quantity of genomic DNA was assessed by 0.8 % agarose gel

electrophoresis with k uncut DNA. The final concentration

was made to 20 ng lL-1 and stored at -20 �C until further

use. A total of 45 random decamer primers (Genemed Syn-

thesis Inc, Texas, USA) were screened for RAPD analysis,

out of which 10 primers were selected on the basis of clarity

of banding patterns. The protocol for RAPD analysis was

adapted from that of Williams et al. (1990) with some

modification. DNA amplification was performed with reac-

tion volume of 25 lL containing 10 9 PCR buffer (2.5 lL),

2.5 mM MgCl2 (1.0 lL), 100 mM dNTPs (2.0 lL), primer

(2.0 lL), Taq polymerase (0.2 lL), 40 ng of template DNA

(2.0 lL), and 14. 3 lL sterile D.W. PCR was performed at

initial temperature of 94 �C (6 min, 1 cycle), followed by 40

cycles of 1 min at 94 �C, 1 min at 35 �C and 10 min at

72 �C, and a final cycle of 10 min at 72 �C.

ISSR analysis was performed following the procedure

described by Zietkiewicz et al. (1994). A factorial experiment

with varying concentrations of genomic DNA (30, 40 and

50 ng), MgCl2 (1.5, 2.0 and 2.5 mM) and Taq DNA poly-

merase (0.5, 1 and 1.5 U) was performed to optimize PCR

conditions. After preliminary screening with 32 ISSR primers

(UBC, Canada) for standardization of optimum annealing

temperature, 10 ISSR primers were selected on the basis of

Plant Growth Regul (2014) 72:1–15 3

123

sharpness and reproducibility. The amplification was per-

formed for 40 cycles, programmed as follows; initial dena-

turation at 94 �C for 6 min (one cycle), denaturating at 94 �C

for 1 min, annealing at 48, 50 and 52 �C (depending on primer

used) for 1 min and extension at 72 �C for 2 min (38 cycles)

and final extension at 72 �C for 12 min (one cycle). PCR

amplification was performed in Thermal Cycler (BIOER, XP

Cycler, China). Amplification products obtained from RAPD

and ISSR analysis were analyzed in 1.8 % agarose gel stained

with ethidium bromide (0.5 lg lL-1). The size of the

amplicons was estimated using a 100-bp ladder (GeneRular

100 bp plus ladder) and documented in the Gel documentation

system (BioRad, Hercules, CA).

Evaluation of secondary metabolites

Plant material and extract preparation

Fresh plant parts (tuber, stem, leaf) of mother as well as

in vitro raised plants (both DSO and ISO) were washed under

running tap water and blotted with tissue towel. one gram of

fresh tissue was homogenized in 25 mL of respective solvent

(methanol, ethanol, acetone and water) and extractions were

carried on orbital shaker (REMI, India) with constant stirring

at 150 rpm for 24 h. The mixtures were centrifuged at

10,000 rpm for 10 min and the supernatant was filtered

through Whatman filter paper (No. 1). Freshly prepared

extracts were used in all the experiments. Measurements of

biochemical parameters were taken on UV-190 double beam

spectrophotometer (Shimadzu, Japan).

Determination of total phenolic content (TPC)

The determination of total phenolic content was performed

by using Folin–Ciocalteu method (Singleton and Rossi

1965) with little modification. Briefly, 0.125 mL of extract

was mixed with 1.8 mL of Folin-Ciocalteu reagent (ten

fold diluted) and kept for 6 min at 25 �C. Then 1.2 mL of

15 % Na2CO3 was added to the reaction mixture and kept

for 90 min at room temperature. The absorbance of the

reaction was measured at 765 nm. The concentration of the

total phenolics was determined as milligrams of tannic acid

equivalents (TAE) per gram fresh weight by using an

equation obtained from tannic acid calibration curve.

Determination of total flavonoid content (TFC)

Total flavonoid content was determined by using the alu-

minium chloride colorimetric method (Chang et al. 2002).

Briefly, 0.5 mL of extracts, 1.5 mL of methanol, 0.1 mL of

aluminium chloride (10 %), 0.1 mL of potassium acetate

(1 M) and 2.8 mL of distilled water were mixed for 5 min

by vortexing. Reaction mixture was kept at room

temperature for 30 min and the absorbance was measured

at 415 nm. The calibration curve was prepared for rutin and

the results are expressed as milligrams of rutin equivalents

(RE) per gram fresh weight.

RP-HPLC analysis of phenolic compounds

Extract prepared for the HPLC analysis by using the proce-

dure described by Ghatge (2007) with slight modification.

Briefly, 1 g of fresh leaf tissue was crushed in 20 mL ethanol

and extract was centrifuged at 15,000 rpm at 4 �C for

10 min. The supernatant was filtered through a 0.22 lm filter

(Millipore, Westboro, MA, USA) before injection into

HPLC system. HPLC analyses were performed using a

Waters Chromatic system (Model no. HPLC-W6590)

equipped with a Waters 717 plus auto sampler and a Waters

2487 photodiode array detector. Separation was achieved on

a reverse phase C-18 column (Princeton SPHER, 5 lm,

3.9 mm 9 150 mm). The mobile phase was acetonitrile:

water (84:16, v/v) at a flow rate of 1.0 mL min-1, the

injection volume was set to 20 lL and the column was

retained at room temperature. Individual phenolic com-

pounds were identified at 280 nm by the retention time of

sample chromatographic peaks being compared with those

of authentic standards using the same operating conditions.

Antioxidant activity of micropropagated plants

DPPH free radical scavenging assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical

scavenging activity of the extracts was determined essen-

tially as described by Brand-Williams et al. (1995) and

modified by Jagtap et al. (2011). The stock reagent solution

was prepared by dissolving 24 mg of DPPH in 100 mL

methanol and stored at -20 �C until use. 100 lL of extract

was allowed to react with DPPH solution in the final

reaction volume of 3 mL. The mixture was shaken vigor-

ously and allowed to stand in the dark at room temperature.

The decrease in absorbance of the resulting solution was

then measured spectrophotometrically at 517 nm. The

control was prepared as above without any extract and

MeOH was used for the baseline correction. Radical

scavenging activity was expressed as the inhibition per-

centage and was calculated using the following formula,

% radical scavenging activity

¼ Control OD� sample OD=Control ODð Þ � 100:

FRAP (ferric reducing antioxidant power) assay

The FRAP assay was carried out according to procedure

described by Benzie and Strain (1996) and modified by


123

Chavan et al. (2012). Briefly the working FRAP reagent

prepared by combination of 0.3 M acetate buffer (pH 3.6),

10 mM 2, 4, 6-tripyridyl-s-triazine (TPTZ) in 40 mM HCL

and 20 mM FeCl3�6H2O in 10:1:1 ratio prior to use and

heated to 37 �C in water bath for 10 min. 100 lL of extract

was allowed to react with 2.7 mL of the FRAP reagent.

The final volume of the reaction mixture was made up to

3 mL with sterile distilled water. The reaction mixture was

kept in dark for 30 min. The absorbance of the colored

product (ferrous tripyridyltriazine complex) was then

recorded at 593 nm. A higher absorbance reading indicated

a higher reducing power.

Metal chelating assay

The chelating effect of ferrous ions from different parts of

C. santapaui was estimated by the method of Dinis et al.

(1994). Briefly, 500 lL of plant extracts were added into

100 lL of 2 mM FeCl2. The reaction was initiated by

adding 40 lL of 5 mM ferrozine solution into the mixture,

which was then shaken vigorously and left standing at

temperature for 10 min. The absorbance of the reaction

mixture was measured at 562 nm. Three replicates were

made for each test sample. The ratio of inhibition of fer-

rozine–Fe2? complex formation was calculated as follows:

% inhibition ¼ Acontrol � Asample

� ��Acontrol � 100

where A = absorbance at 54 nm.

Results and discussion

Direct shoot organogenesis

Cotyledonary nodes were excised from in vitro grown

seedlings (Fig. 1a) were cultured on shoot multiplication

medium. Axillary buds started emerging after 7–10 days of

inoculation. MS medium lacking PGRs did not support the

induction of shoots. The number of shoots per cotyledonary

node explant was significantly affected by the type and

concentration of various PGRs (Table 1). Of the different

concentrations of KN, 1.0 mg L-1 elicited multiple shoots

in 82.5 % of cultures, with an average of 3.4 ± 0.5 shoots

per responsive explant and an average shoot length of

4.8 ± 0.6 cm (Table 1). TDZ induced a frequent formation

of short and fasciated shoots which were unsuitable for

in vitro rooting. BAP and TDZ were less effective in

inducing shoots when compared with KN and 2iP. None of

the concentrations of BAP and TDZ was effective in

enhancing shoot proliferation. The best frequencies of

shoot multiplication (88 %) and maximum numbers of

shoots (8.1 ± 0.6) were achieved on MS media containing

2.0 mg L-1 2iP (Table 1; Fig. 1b). Effectiveness of 2iP

has been previously reported in shoot multiplication of

Decalepis hamiltonii (Giridhar et al. 2005), and Caralluma

bhupenderiana (Ugraiah et al. 2011). In contrast, majority

of literature have reported BAP as the most active cyto-

kinin for shoot multiplication in various Asclepiadaceae

members (Patil 1998; Komalavalli and Rao 2000; Beena

et al. 2003; Amoo et al. 2009; Chavan et al. 2011a, b).

Topolins, naturally occurring aromatic cytokinins were

significantly altered the shoot multiplication in Huernia

hystrix, another member of Asclepiadaceae (Amoo and

Van Staden 2012) and Aloe arborescens (Amoo et al.

2012).

The combined treatments of cytokinins and auxins also

support shoot multiplication rates than the individual

treatments of the BAP, KN and TDZ. Addition of IBA

along with BAP or 2iP significantly boosted the multipli-

cation rate. MS medium supplemented with 3.0 mg L-1

BAP with 0.5 mg L-1 IBA produced an average of

4.0 ± 0.6 shoots per explant (Table 1). A combination of

2iP (2.0 mg L-1) and IBA (0.5 mg L-1) also produced an

average of 4.5 ± 0.6 shoots in 82 % cultures. The syner-

gistic effects of 2iP and auxins were reported in Gossypium

hirsutum cv. SVPR2, where a combination of these plant

Fig. 1 Regeneration in C. santapaui, a seed germination (MS basal medium), b direct organogenesis (MS ? 2iP 2.0 mg L-1), c formation of

embryogenic callus (MS ? PR 1.5 mg L-1) d indirect organogenesis (MS ? BAP 2.5 ? IBA 0.4 mg L-1)


123

growth regulators induced axillary bud sprouting at a

higher frequency (Ganesan and Jayabalan 2006). Baskaran

and Jayabalan (2005) also reported that when cotyledonary

nodes of Eclipta alba were cultured on MS medium con-

taining a combination of 2iP and auxins, the number of

shoots was increased per explant.

Indirect shoot organogenesis

Cotyledons from in vitro grown seedlings were cultured on

MS medium supplemented with 2,4-D, PR or NAA for

callus induction. Within 4 weeks of inoculation, callus was

proliferated from cut edges of the explants. Among the

auxins tested, PR was found more prominent than 2,4-D

and NAA for callus induction. About 82.5 % cultures

showed callusing at lower concentration of PR

(0.5 mg L-1). MS medium containing 1.5 mg L-1 PR was

best for callus induction (97.5 %) and growth (913

± 10.9 mg FW) of the callus (Fig. 2). Callus induced on

the same media composition was friable, granular and

comparatively fast growing than induced on other con-

centrations of PR, 2,4-D or NAA (Fig. 1c). Fitch and

Moore (1990) successfully used the picloram for callus

induction and long term maintenance of totipotent callus of

sugarcane.

The calli developed on PR (1.5 mg L-1) supplemented

medium were subcultured on MS medium supplemented

with various concentrations of BAP either alone or in

combination with IBA or NAA for shoot regeneration

(Table 2). After 2 weeks of culture, most of the calli

started to turn to light green, and they gradually became

dark green in the following week of culture. New white

calli were also developed from the callus mass. Similar

observation was also made in the Allium chinense (Yan

Table 1 Effect of different

plant growth regulators on shoot

regeneration from cotyledonary

nodes of C. santapaui

Values represents mean ± SE

of 20 replicates/treatment and

experiments were repeated

twice. Values are significantly

different at ns-non significant,

* P \ 0.05 and ** P \ 0.01

level according to Dunnett

multiple comparisons test

PGRs Concentration

(mg L-1)

Regeneration

frequency (%)

Number of shoots/explant

(mean ± SE)

Length of shoot (cm)

(mean ± SE)

Control 0.0 0.0 0.0 0.0

BAP 1.0 52.5 0.5 ± 0.1ns 5.0 ± 1.1**

2.0 75.0 1.3 ± 0.2* 6.4 ± 0.9**

3.0 77.5 2.1 ± 0.4** 7.0 ± 0.8**

4.0 67.5 1.1 ± 0.2** 6.3 ± 0.9**

5.0 62.5 1.0 ± 0.2** 6.1 ± 1.0**

KN 0.5 60.0 1.1 ± 0.2ns 4.3 ± 0.9**

1.0 82.5 3.4 ± 0.5** 4.8 ± 0.6**

1.5 72.5 1.9 ± 0.4** 5.4 ± 0.9**

2.0 70.0 1.7 ± 0.4** 4.3 ± 0.9**

2.5 62.5 1.4 ± 0.3* 4.5 ± 0.9**

TDZ 0.5 57.5 1.1 ± 0.3ns 3.4 ± 0.8**

1.0 72.5 1.8 ± 0.4** 4.1 ± 0.7**

1.5 82.5 2.6 ± 0.4** 3.5 ± 0.5**

2.0 75.0 2.1 ± 0.6** 3.2 ± 0.7**

2.5 57.5 1.3 ± 0.4ns 2.3 ± 0.5*

2iP 0.5 67.5 1.3 ± 0.2ns 6.2 ± 0.1**

1.0 75.0 2.4 ± 0.5** 6.3 ± 0.9**

1.5 85.0 3.6 ± 0.5** 6.1 ± 0.6**

2.0 88.0 8.1 ± 0.6** 6.5 ± 0.4**

2.5 72.5 2.9 ± 0.6** 6.2 ± 0.6**

BAP ? IBA 3.0 ? 0.1 65.0 2.1 ± 0.4** 6.2 ± 0.9**

3.0 ? 0.3 77.5 2.9 ± 0.5** 6.2 ± 0.8**

3.0 ? 0.5 85.0 4.0 ± 0.6** 6.5 ± 0.6**

3.0 ? 0.7 75.0 2.7 ± 0.6** 4.5 ± 0.6**

3.0 ? 1.0 62.5 2.3 ± 0.4** 4.2 ± 0.7**

2iP ? IBA 2.0 ? 0.1 75.0 1.9 ± 0.4** 6.1 ± 0.9**

2.0 ? 0.3 82.5 2.7 ± 0.4** 5.2 ± 0.7**

2.0 ? 0.5 82.0 4.5 ± 0.6** 5.3 ± 0.6**

2.0 ? 0.7 65.0 3.0 ± 0.5** 4.8 ± 0.7**

2.0 ? 1.0 60.0 1.4 ± 0.3ns 4.6 ± 0.9**


123

et al. 2009). In the present study, BAP alone could induce

shoot regeneration at the rate of 60 % when cultured on

medium with 2.5 mg L-1 BAP. These results agree with

the reports of Luciani et al. (2006), where BAP could

induce shoot regeneration from callus. The highest regen-

eration frequency (92.5 %) and maximum number of

shoots (19.7 ± 0.3) were achieved with combination of

2.5 mg L-1 BAP and 0.4 mg L-1 IBA (Table 2 and

Fig. 1d). The interactive effect of BAP and auxins on

multiple shoot differentiation has also been demonstrated

in Ceropegia spiralis (Murthy et al. 2010), and Ceropegia

bulbosa (Phulwaria et al. 2013).

Rooting and field establishment of plantlets

The success of in vitro regeneration relies on the rooting

percentage and survival of the plantlets in field conditions.

In vitro raised shoots (4–5 cm) with three to four nodes

were excised and implanted individually on half-strength

MS medium supplemented with different concentrations of

auxins (Table 3). The maximum frequency of root forma-

tion (90 %), number of roots (6.9 ± 0.8) and root length

(3.8 ± 0.4 cm) were achieved after 4 weeks when shoots

were cultured on half-strength MS medium supplemented

with 2.0 mg L-1 NAA. Similarly, NAA was the most

effective in promoting of rooting in Prunus insititia (Rossi

et al. 1993). In the present investigation, singular supple-

mentation of NAA resulted in good response towards root

induction, however beyond optimal level, slight callus

formation was observed at the basal cut ends. Similar

phenomenon was reported in medicinal plants viz. Rotula

aquatica (Martin 2003) and Cryptolepis buchanani (Prasad

et al. 2004). Rooted plantlets with 4–6 fully expanded

leaflets were successfully transferred into plastic cups

containing sterilized soil, sand and coco peat (1:2:1). The

acclimatized plantlets were established into earthen pots

containing garden soil and eventually established in the

field with 90 % survival. Micropropagated plants develop

normal inflorescence and appeared morphologically and

developmentally similar to the parent stock.

Molecular profiling of in vitro raised clones

Somaclonal variation has been frequently reported in

micropropagated plants. The occurrence of variations

during in vitro propagation depends upon the source of

explants and the pathway of regeneration (Goto et al.

1998). In the present study, plant regeneration involved

formation of adventitious shoots from direct (cotyledon-

ary nodes) and indirect shoot organogenesis (cotyledons).

The regenerated plantlets did not show any detectable

variation in morphology when compared with that of the

mother plant. However, sometimes some changes induced

by in vitro culture cannot be observed because the

structural difference in the gene product does not always

alter its biological activity to an extent for phenotypic

expression. Thus, validation of the similarity at genetic

level is essential by DNA analysis techniques. Therefore,

in this study two PCR-based marker systems (RAPD,

ISSR) were employed for genetic fidelity analysis of both

DSO and ISO-derived plantlets.

In this study, DNA of 20 randomly selected in vitro

regenerated plantlets (10 DSO-derived ? 10 ISO-

derived) as well as the mother plant was subjected to

RAPD and ISSR analysis to assess the genetic fidelity.

Out of the 45 RAPD decamer primers screened, only 10

resulted in clear and scorable bands in DSO as well as

ISO-derived plants. These 10 RAPD primers generated

48 and 52 amplicons ranging from 240 bp (primer 987)

to 2,100 bp (primer 985, 987) in DSO and ISO-derived

plants respectively. The number of bands in the selected

primers varied from 1 (primer 946) to 11 (primer 987),

with an average of 4.8 (DSO) and 5.2 (ISO) bands per

primer (Table 4). A total of 1,100 bands were generated

from the mother plant and 20 in vitro-raised clones (10

DSO ? 10 ISO). No polymorphism was detected during

Fig. 2 Effect of auxins on

callus induction and growth

from cotyledons of C. santapaui


123

the RAPD analysis of in vitro-raised clones (Fig. 3a, c).

The absence of genetic variation using RAPD has been

reported in micropropagated shoots of Pinus thunbergii

(Goto et al. 1998) and Gerbera jamesonii (Bhatia et al.

2011).

Ten ISSR primers generated 39 (DSO) and 44 (ISO)

amplicons ranging from 300 bp (UBC 809) to 2,300 bp

(UBC 813) in size. The number of bands for each primer

varied from 2 (UBC 814, UBC 823) to 7 (UBC 809), with

an average of 3.9 bands per primer (Table 5). A total of

924 distinct amplicons were generated from 10 ISSR

primers in mother plant and 20 in vitro-raised clones (10

DSO ? 10 ISO-derived plants). Out of 924 bands, 913

bands were monomorphic within in vitro raised clones and

the mother plant, whereas, 11 polymorphic bands were

detected with 3 ISSR primers (UBC 801, UBC 809 and

UBC 815) in ISO-derived clones. It might be due to the

application of PGRs during callus induction, shoot regen-

eration and prolonged culture passage. Similarly, ISSR

primers were capable to detect polymorphism in Camellia

chinensis (Devarumath et al. 2002) and Gerbera jamesonii

(Bhatia et al. 2009).

In this study, RAPD fails to detect polymorphic loci in

both DSO and ISO-derived plants, however polymorphism

was observed in the ISSR fingerprints of ISO-derived

plants. Devarumath et al. (2002) also revealed that ISSR

fingerprint is more capable to detect the polymorphic loci

compared to RAPD fingerprinting. The results of our study

also support the fact that axillary multiplication is the

safest mode of micropropagation to produce true to type

progeny. There are some reports in literature, which cite

similar results (Martins et al. 2004; Joshi and Dhawan

2007; Bhatia et al. 2011).

Secondary metabolite production

Total phenolics and flavonoids

The chemical composition of different parts of C. santa-

paui is essential in order to establish a potential relation-

ship and understanding its role with different beneficial

biological activities enhancing human health benefits. The

phenol and flavonoid compounds present in plants are

known for their positive effects on inflammation (Xanth-

opoulou et al. 2010), cardiovascular diseases (Alothman

et al. 2009), besides having antioxidant activities (Jagtap

et al. 2011). Table 6 shows the total phenolic and flavonoid

contents of different parts viz. tuber, stem and leaf of

mother plant, DSO-derived plant and ISO-derived plants.

The results indicated that donor mother plant and DSO-

derived plants have lower levels of phenols and flavonoids

compared to ISO-derived plants. Methanolic ISO-derived

leaf extract exhibited the highest phenolic content

(6.8 ± 1.1 mg TAE/g FW) whereas, aqueous tuber extract

of donor plant showed lowest phenolic content

(1.1 ± 0.4 mg TAE/g FW). Similarly, the methanol had its

effect on the extraction of TPC from fruit pulp of Salacia

chinensis (Chavan et al. 2012).

The higher extractability of flavonoid (3.1 ± 0.4 mg

RE/g FW) was recorded in the methanolic leaf extract

of ISO-derived plants while, lowest flavonoid contents

were recorded in the aqueous tuber extract of donor and

Table 2 Effect of BAP either

alone or in combination with

auxins on shoot regeneration

from cotyledon-derived callus

of C. santapaui



experiments were repeated

twice. Values are significantly

different at ns- non significant,

* P \ 0.05 and ** P \ 0.01

level according to Dunnett

multiple comparisons test

PGRs Concentration

(mg L-1)

Regeneration

frequency (%)

Number of shoots

(mean ± SE)

Length of shoot

(cm) (mean ± SE)

Control 0.0 0.0 0.0 0.0

BAP 0.5 22.5 0.7 ± 0.2ns 2.1 ± 1.1**

1.0 30.0 2.0 ± 0.5* 2.8 ± 0.8**

1.5 45.0 2.7 ± 0.3** 3.1 ± 1.0**

2.0 52.5 4.0 ± 1.0** 3.8 ± 1.4**

2.5 60.0 7.8 ± 0.5** 4.0 ± 1.0**

3.0 47.5 5.1 ± 0.9** 3.5 ± 0.6**

BAP ? IBA 2.5 ? 0.2 60.0 10.3 ± 1.1** 3.7 ± 0.6**

2.5 ? 0.4 92.5 19.7 ± 0.3** 4.1 ± 1.0**

2.5 ? 0.6 77.5 15.5 ± 0.2** 3.0 ± 0.3**

2.5 ? 0.8 57.5 9.3 ± 0.6** 2.8 ± 0.9**

2.5 ? 1.0 52.5 5.8 ± 0.4** 2.0 ± 0.9**

BAP ? NAA 2.5 ? 0.2 57.5 6.3 ± 0.8** 1.1 ± 0.8*

2.5 ? 0.4 50.5 5.6 ± 1.2** 1.3 ± 1.0**

2.5 ? 0.6 45.0 4.4 ± 0.6** 1.5 ± 1.1**

2.5 ? 0.8 37.5 3.1 ± 0.3** 1.0 ± 0.5*

2.5 ? 1.0 30.0 2.1 ± 0.3* 1.0 ± 0.5*


123

DSO-derived plants (Table 6). The solubility of flavonoids

was significantly affected by the solvent used for extraction

and these findings are in accordance with the results

obtained for Asimina tribloba (Harris and Brannan 2009)

and Salacia chinensis (Chavan et al. 2012). The fluctuation

in the phenolic and flavonoid contents in the different parts

might be due to the hormonal content, specific metabolic as

well as endogenous physiological changes taking place in

the plants. Similarly, the variation of phenol and flavonoid

contents within the plant parts was reported in 12 medicinal

plants of family Asclepiadaceae and Periplocaceae (Sur-

veswaran et al. 2010). Present findings indicated that,

exogenous supply of different PGRs during in vitro prop-

agation, pathway of regeneration was markedly influenced

the in vitro production of phenolics and flavonoids. Simi-

larly, influence of PGRs on in vitro production of bioactive

secondary metabolites is reported in Aloe arborescens

(Amoo et al. 2012). The present study has shown the total

phenolic and flavonoid contents in C. santapaui in

decreasing order as: ISO-derived plant [DSO-derived

plants [Donor mother plant.

Identification of phenolic compounds

Leaves obtained from donor and micropropagated (both

DSO and ISO) C. santapaui was subjected to RP-HPLC

analysis to evaluate its phenolic composition (Fig. 4a–d).

Total phenolics and flavonoids content was recorded

highest in the leaves, hence considered for further analysis.

The major peaks identified by comparison with authentic

standards (Fig. 4a). The analysis of leaves showed the

presence of 3 major phenolic compounds viz. gallic acid,

cathechol and vanillin. The compounds identified in donor,

DSO-derived and ISO-derived leaves were quite similar to

each other, however the amount was significantly affected

by the method of regeneration. Gallic acid comprised the

maximum content followed by cathechol and vanillin in all

three sources (Fig. 4b–d). The highest amount of gallic

Table 3 Effect of different

auxins on in vitro rooting of

regenerated shoots of

C. santapaui on half-strength

MS medium



all the experiments were

repeated twice. Values are

significantly different at ns-non

significant, * P \ 0.05 and

** P \ 0.01 level according to

Dunnett multiple comparisons

test

Auxin Concentration

(mg L-1)

Rooting

frequency (%)

Number of roots/shoot

(mean ± SE)

Root length (cm)

(mean ± SE)

Control 0.0 0.0 0.0 0.0

IBA 0.5 57.5 0.7 ± 0.1ns 2.0 ± 0.4**

1.0 67.5 2.4 ± 0.6** 2.4 ± 0.6**

1.5 82.5 3.2 ± 0.4** 2.4 ± 0.3**

2.0 70.0 2.2 ± 0.4** 2.2 ± 0.4**

2.5 62.5 1.9 ± 0.3** 2.0 ± 0.3**

3.0 50.0 1.6 ± 0.3** 1.9 ± 0.3**

NAA 0.5 72.5 1.8 ± 0.3* 2.8 ± 0.4**

1.0 80.0 2.3 ± 0.4** 3.1 ± 0.4**

1.5 82.5 3.4 ± 0.5** 3.4 ± 0.5**

2.0 90.0 6.9 ± 0.8** 3.8 ± 0.4**

2.5 62.5 2.6 ± 0.6** 2.6 ± 0.4**

3.0 62.5 1.8 ± 0.3* 2.7 ± 0.4**

IAA 0.5 40.0 0.5 ± 0.1ns 1.2 ± 0.4**

1.0 57.5 1.1 ± 0.2* 1.1 ± 0.3**

1.5 72.5 1.7 ± 0.3** 1.5 ± 0.3**

2.0 65.0 2.1 ± 0.5** 1.3 ± 0.3**

2.5 52.5 1.5 ± 0.3** 1.1 ± 0.3*

3.0 47.5 1.3 ± 0.8* 1.1 ± 0.4*

NAA ? IBA 2.0 ? 0.2 75.0 1.9 ± 0.4** 2.9 ± 0.4**

2.0 ? 0.4 82.5 5.1 ± 0.7** 4.0 ± 0.5**

2.0 ? 0.6 75.0 3.0 ± 0.7** 3.0 ± 0.4**

2.0 ? 0.8 72.5 2.4 ± 0.5** 2.7 ± 0.5**

2.0 ? 1.0 60.0 2.0 ± 0.5* 2.4 ± 0.5**

NAA ? IAA 2.0 ? 0.2 57.5 1.3 ± 0.4* 1.3 ± 0.4**

2.0 ? 0.4 75.0 2.6 ± 0.5** 2.0 ± 0.3**

2.0 ? 0.6 82.5 3.3 ± 0.5** 2.0 ± 0.3**

2.0 ? 0.8 85.0 4.1 ± 0.5** 1.8 ± 0.3**

2.0 ? 1.0 85.0 4.5 ± 0.6** 1.7 ± 0.2**


123

acid (16.6 ppm) present in the ISO-derived leaves, fol-

lowed by of DSO-derived (14.8 ppm) and donor plant

(14.6 ppm) respectively. The gallic acid is a naturally

abundant plant phenolic compound, constituent of poly-

phenols which are known to be the principle compounds

related to the antioxidant and free radical scavenging

properties (Paixao et al. 2008). Likewise, levels of cathe-

chol and vanillin were recorded highest in leaves of ISO-

derived plants.

Antioxidant activity

Scavenging activity towards DPPH free-radical

The free radical scavenging activity of the different parts

was tested through DPPH method and the results are pre-

sented in the (Fig. 5a). The essence of DPPH method is

that the antioxidants react with the stable free radical i.e.,

2,2-diphenyl-b- picrylhydrazyl (deep violet colour) and

Fig. 3 DNA fingerprinting patterns generated by RAPD and ISSR

analysis in DSO raised plants (a RAPD primer 946, b ISSR primer

UBC 813) and ISO raised plants (c RAPD primer 961, d ISSR primer

UBC 809). Lane L: 100 bp ladder, Lane M: Mother plant, Lane 1–10:

Micropropagated plants, ;: Missing band

Table 4 List of primers, their sequences, number and size of the amplified fragments generated by RAPD primers in C. santapaui

Sr.

no.

Primer

code

Primer

sequence

(50–30)

Number of bands/primer Total number of bands amplified

DSO ISO

DSO ISO Mono-

morphic

Poly-

morphic

Size

range (bp)

Mono-

morphic

Poly-

morphic

Size

range (bp)

1 946 CTCAGGGCAT 1 1 11 – 1,700 11 – 1,700

2 961 ACTACGCGAT 5 5 55 – 300–1,700 55 – 550–1,700

3 967 GTGTGGTGAT 6 6 66 – 380–1,050 66 – 360–1,000

4 971 AGGCTGACTT 7 9 77 – 330–1,900 99 – 260–1,900

5 985 GCT CGTCGTT 4 3 44 – 650–2,100 33 – 840–1,700

6 987 TGTCGAGGTT 10 11 110 – 240–2,000 121 – 240–2,100

7 988 GAATGGGGTT 3 3 33 – 600–960 33 – 600–960

8 994 CGCACTCAAA 3 3 33 – 600–1000 33 – 600–1000

9 995 GTGTCG GAAA 5 6 55 – 360–1,500 66 – 280–1,500

10 OPC–11 AAAGCTGCGG 4 5 44 – 450–1,200 55 – 300–1,250

Total 48 52 528 – 572 –

DSO direct shoot organogenesis derived plants, ISO indirect shoot organogenesis derived plants


123

convert it to 2,2-diphenyl-b- picrylhydrazine with discol-

ouration. The degree of discolouration indicates the scav-

enging potentials of the sample antioxidant. In the present

study the extracts of different parts were able to decolou-

rise DPPH. Among various solvents and plant parts tested,

methanolic leaf extract of ISO-derived plant showed the

highest DPPH activity (67.0 ± 4.9 %), while aqueous stem

extract of donor plant exhibited lowest radical scavenging

activity (10.0 ± 3.1 %). It has been reported that the

highest antioxidant activity in methanol extract from the

peel of pineapple (Hossain and Rahman 2011) and fruit

pulp of Salacia chinensis (Chavan et al. 2012). The ISO-

derived tissues were found to have a significantly higher

antioxidant potential than the DSO-derived and donor tis-

sues. Rising radical scavenging activity was also observed

in the regenerated shoots of Piper nigrum (Ahmad et al.

2010) and Aloe arborescens (Amoo and Van Staden 2012).

It appears that the extracts from the different plant parts of

C. santapaui possess hydrogen donating capabilities to act

as antioxidant.

Table 5 List of primers, their sequences motifs, nature, annealing temperatures, number and size of the amplified fragments generated by ISSR

primers in C. santapaui

Sr.

no.

Primer

code

50–30

motif

Annealing

temp. (�C)

Number of bands/primer Total number of bands amplified

DSO ISO

DSO ISO Mono-

morphic

Poly-

morphic

Size

range (bp)

Mono-

morphic

Poly-

morphic

Size

range (bp)

1 UBC-801 (AT)8T 49 5 5 55 – 550–2,100 55 3 550–2,100

2 UBC-808 (AG)8C 52 3 4 33 – 340–950 44 – 320–1,000

3 UBC-809 (AG)8G 50 7 7 77 – 300–1,200 77 4 300–1,300

4 UBC-811 (GA)8C 48 4 5 44 – 400–1,050 55 – 400–1,050

5 UBC-813 (CT)8T 51 4 4 44 – 1,100–2,300 44 – 1,000–2,100

6 UBC-814 (CT)8A 48 2 3 22 – 580–700 33 – 600–850

7 UBC-815 (CT)8G 50 5 5 55 – 600–1,600 55 4 450–1,800

8 UBC-818 (CA)8G 52 3 4 33 – 500–1,500 44 – 550–1,500

9 UBC-822 (TC)8A 52 4 4 44 – 600–1,500 44 – 600–1,500

10 UBC-823 (TC)8C 52 2 3 22 – 400–980 33 – 340–1,100

Total 39 44 429 – 484 11

DSO Direct shoot organogenesis derived plants; ISO Indirect shoot organogenesis derived plants

Table 6 Total phenolics and flavonoids content in different parts of C. santapaui with respect to different solvent systems (phenolics: mg TAE/g

FW; flavonoids: mg RE/g FW)

Plant part Solvent Donor DSO ISO

Phenolics Flavonoids Phenolics Flavonoids Phenolics Flavonoids

Tuber Methanol 3.6 ± 0.3 1.0 ± 0.1 3.6 ± 0.2 0.9 ± 0.3 4.1 ± 0.1 1.8 ± 0.6

Ethanol 2.3 ± 0.2 0.7 ± 0.1 2.5 ± 0.4 0.9 ± 0.6 3.0 ± 0.6 1.2 ± 0.4

Acetone 1.9 ± 0.3 0.7 ± 0.1 2.0 ± 0.3 0.6 ± 0.2 2.5 ± 0.2 1.2 ± 0.3

Water 1.1 ± 0.4 0.3 ± 0.2 1.3 ± 0.4 0.3 ± 0.3 1.8 ± 0.2 0.7 ± 0.6

Stem Methanol 4.0 ± 0.3 2.4 ± 1.3 4.1 ± 0.2 2.5 ± 1.0 4.7 ± 0.1 3.0 ± 0.8

Ethanol 4.3 ± 1.4 2.2 ± 0.5 4.5 ± 0.7 2.3 ± 0.8 4.9 ± 0.6 3.1 ± 1.1

Acetone 2.9 ± 1.0 1.1 ± 0.5 3.1 ± 0.8 1.4 ± 0.3 3.7 ± 0.9 2.0 ± 0.8

Water 1.7 ± 0.9 0.7 ± 0.6 2.1 ± 0.6 0.7 ± 0.8 2.5 ± 1.0 1.0 ± 0.6

Leaf Methanol 6.3 ± 1.0 2.5 ± 0.2 6.4 ± 1.1 2.7 ± 0.4 6.8 ± 1.1 3.1 ± 0.4

Ethanol 4.0 ± 0.4 2.0 ± 0.3 3.9 ± 0.3 2.3 ± 0.6 5.1 ± 0.5 2.9 ± 0.6

Acetone 2.8 ± 0.3 1.8 ± 0.9 2.9 ± 0.3 1.9 ± 0.4 3.4 ± 0.5 2.4 ± 0.7

Water 2.1 ± 0.3 1.3 ± 1.1 2.0 ± 0.2 1.3 ± 0.4 2.8 ± 0.1 1.5 ± 0.2

DSO Plants generated through direct shoot organogenesis, ISO Plants generated through indirect shoot organogenesis, TAE Tannic acid

equivalent, RE Rutin equivalent, FW Fresh weight; Values represents mean ± SE of three replicates


123

Ferric reducing antioxidant power (FRAP)

The FRAP assay is commonly used in routine analysis for

evaluation of antioxidant capacity, since it is simple, rapid,

sensitive and inexpensive. The reducing capacity of a

compound might serve as a significant indicator of its

potential antioxidant capacity. The antioxidant capacity of

different parts using FRAP assay, is shown in Fig. 5b. The

extent of reduction in terms of absorbance values was

observed at 593 nm. The highest ferric reducing capacity

was found in methanolic leaf extract of ISO-derived plant

(0.550 ± 0.02 O.D.) followed by methanolic leaf extract of

DSO-derived plants (0.538 ± 0.01 O.D.) and methanolic

leaf extract of donor plant (0.513 ± 0.02 O.D.), while the

remaining extracts reduced a smaller amount Fe3? to

Fe2?. There was no any significant difference observed in

reducing capacity of different parts of donor plant and

DSO-derived plants, however, ISO-derived plants showed

rising radical scavenging activity compared to donor and

DSO-derived plants. These results indicated that tuber and

leaf parts of ISO-derived plants have a good reducing

potentiality.

Metal chelating activity

Iron is essential for life because it is required for oxygen

transport, respiration and activity of many enzymes.

However, iron is an extremely reactive metal and will

catalyze oxidative changes in lipid, protein and other cel-

lular components. The study of the chelating effects on the

ferrous ions is beneficial since ferrous ions are the most

active pro-oxidants in the food system (Yamaguchi et al.

1988). In the presence of samples possessing chelating

activity, the formation of Fe2? complexes is decreased.

Fig. 4 RP-HPLC chromatograms of phenolic compounds in leaves of

C. santapaui, a Standards (conc. 100 ppm), b leaves of mother plant,

c leaves of DSO raised plant, d leaves of ISO raised plant (DSO

Direct shoot organogenesis raised plant; ISO Indirect shoot organo-

genesis raised plant). GA Gallic acid, Cat Cathechol, Van Vanillin


123

Therefore, measurement of the rate of color reduction helps

to estimate the chelating activity of the samples. As shown

in Fig. 5c, the methanolic tuber extract of ISO-derived

plant showed a better inhibitory effect with 65 ± 1.8 % of

inhibition, followed by methanolic tuber extract of DSO-

derived plant (61 ± 2.5 %) and methanolic tuber extract of

donor plant (59 ± 7.7 %). In the current study, metal

chelating activity significantly affected by the solvent

system employed. Similar observations were made in the

Helicteres isora and Ceiba pentandra (Loganayaki et al.

2011). These results indicate that C. santapaui tuber

extracts showed good metal chelating activity.

The increasing quantity of total phenolics, flavonoids,

antioxidant activity and phenolic compounds in DSO and

ISO-derived plants was might be due to the exogenous

supply of the plant growth regulators during in vitro mul-

tiplication. Similarly, exogenous supply of the PGRs sig-

nificantly alters the quantity of secondary metabolites in

the micropropagated Aloe arborescens (Amoo et al. 2012).

Conclusion

An efficient micropropagation protocol via direct shoot

organogenesis (DSO) and indirect shoot organogenesis

(ISO) has been developed for C. santapaui, a threatened

medicinal plant of Western Ghats of India. This protocol

imparts a successful and rapid technique that can be uti-

lized for the commercial propagation and ex situ conser-

vation of this plant. The frequency of regeneration was

Fig. 5 Antioxidant activity of

C. santapaui, a DPPH activity,

b FRAP activity, c Metal

chelating activity (DSO direct

shoot organogenesis raised

plant, ISO indirect shoot

organogenesis raised plant)


123

significantly higher in ISO compared to DSO. Considering

the importance of genetic stability in germplasm conser-

vation programme, our protocol appears to be highly

effective and to the best of our knowledge it is the first

report on genetic fidelity testing in micropropagated plants

of genus Ceropegia. Protocol described in this study may

be used for an efficient genetic transformation of this

valuable medicinal asclepiad for quality improvement. The

pathway of regeneration, plant part, solvents and PGRs

greatly influenced the in vitro secondary metabolites and

antioxidant capacity of C. santapaui.

Acknowledgments Junior research fellowship (JRF) to JJC and

financial support for this work from Department of Biotechnology

(DBT), Govertment of India, New Delhi is gratefully acknowledged.

Authors are also thankful to Dr. V. A. Bapat, Emeritus Scientist from

Plant cell culture technology section, Nuclear agriculture and bio-

technology division, Bhabha Atomic Research Centre (BARC)

Mumbai, India for his constructive comments on the manuscript.

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