Stevioside glycosides from in vitro cultures of Stevia rebaudianaand antimicrobial assay

Madhumita Kumari1 • Sheela Chandra1

Received: 30 January 2015 / Accepted: 6 July 2015 / Published online: 17 July 2015

� Botanical Society of Sao Paulo 2015

Abstract In this study, in vitro propagation protocols

have been developed through direct and indirect organo-

genesis from leaf and nodal explant for production of high

value of secondary metabolites in Stevia rebaudiana and

antimicrobial assay of in vivo and in vitro plant extract. A

combination of PGRs proves better than single for both

callusing and direct shoot multiplication from leaf

explants. Treatment of Kin ? IAA (1.5 mg L-1) each

showed best callusing response (85.5 ± 0.33 %). For shoot

proliferation, Kin (2.5 mg L-1) with IAA (1.5 mg L-1)

showed a maximum number of shoots (5.3 ± 0.3) prolif-

erating from callus with longest set of 9.03 ± 0.14 cm and

of direct organogenesis and Kin (1.5 mg L-1) with BAP

(2.5 mg L-1) gave maximum number (8.6 ± 0.33) of

shoots from leaf explant with longest shoot of

5 ± 0.11 cm. HPLC profiles showed that both the sec-

ondary metabolites (stevioside, 0.451 ± 0.001 mg g-1 and

Reb A, 0.131 ± 0.005 mg g-1) are higher in in vitro

shoots developed through organogenesis from callus. They

also proved better in antimicrobial activity with a maxi-

mum zone of inhibition against all tested bacteria including

B. subtilis (13.6 ± 0.3 mm), S. aureus (12.3 ± 0.33 mm),

P. fluorescence (8.6 ± 0.3 mm) and E. coli

(10.3 ± 0.1 mm).

Keywords Organogenesis � Rebaudioside A � Stevioside

Abbreviations

Ads Adenine sulphate

BAP 6-Benzyladenine

HPLC High performance liquid chromatography

IAA Indole-3-acetic acid

IBA Indole-3-butyric acid

Kin Kinetin

NAA Naphthalene acetic acid

PGRs Plant growth regulators

2,4-D 2,4-Dichlorophenoxyacetic acid

MS Murashige and Skoog

Reb A Rebaudioside A

ZI Zone of inhibition

Introduction

Stevia rebaudiana (Bertoni) is a small perennial herb,

which belongs to the Asteraceae family. It is native to

certain regions of Paraguay and Brazil in South America.

The plant is also known as a sweet herb of Paraguay, honey

leaf and candy leaf (Carakostas et al. 2008). S. rebaudiana

contains diterpene glycosides viz. stevioside and rebau-

diosides which are responsible for its sweet taste with zero

calories (Bridel and Lavielle 1931) and are estimated to be

250–300 times sweeter than sucrose. These glycosides

possess a number of therapeutics beyond their sweetness.

These regulates the blood glucose level by stimulating

insulin secretion so that it can be used as an alternative

sweetener by hyperglycemic patients (Kinghorn and Soe-

jarto 1985; Mizutani and Tanaka 2002). Steviol glycosides

can also be utilized as an antihypertensive (Ferri et al.

2006), anti-tumour (Yasukawa et al. 2002), vasodilator

& Sheela Chandra

schandra@bitmesra.ac.in

1 Department of Bio-Engineering, Birla Institute of

Technology, Mesra, Ranchi, Jharkhand 835215, India

123

Braz. J. Bot (2015) 38(4):761–770

DOI 10.1007/s40415-015-0193-3

(Bornia et al. 2008), antimicrobial (Jayaraman et al. 2008)

and neuroprotective drug (Xu et al. 2008). These are heat

and pH-stable with a good shelf life and can be added in

cooking, baking or in beverages. In a number of countries,

Stevia was approved as dietary supplements. It might be a

source of a number of pharmaceutical drugs. Since Stevia

is highly versatile as an additive, it has gained a great boost

in popularity in the past few years and is progressively

becoming the focal point of attention amongst food and

beverage producers. Thus, various therapeutics and

sweetening properties are the most important attributes of

Stevia, which makes it a commercially important plant.

For commercial cultivation, homogenous range of

improved plants is required and plants germinated from

seeds show variability. Also in field conditions, a wide

range of variation occurs due to external environmental

conditions such as plant pathogen, temperature, drought

and water logging, which leads to variance in composition,

and sweetening levels (Nakamura and Tamura 1985).

Seeds of Stevia have very poor germination potential

(Goettemoeller and Ching 1999; Macchia et al. 2007;

Abdullateef and Osman 2011). In today’s world production

of stress tolerant varities, enhanced plant biomass and

production of medicinally important secondary metabolites

are considerable issues. Earlier vegetative propagation is

generally used for cultivation of Stevia. Although this

technique is limited, numbers of explants were obtained

from a single plant and that may raise possibilities of

pathogen accumulation in tissues.

These in vitro tissue culture techniques might prove an

alternative tool to conventional methods for comparatively

rapid multiplication of elite medicinal platelets, which

gives disease-free resistant plant with high biomass and

secondary metabolites.

Antimicrobial resistance is a major problem expanding

worldwide. Studies revealed that one of the measures to

prevent the increasing rate of resistance on the long run is

to have a continuous investigation of new, safe and

effective antimicrobial compound (Hussein et al. 2010); so

plant secondary metabolites can be investigated as new

antimicrobial compounds. Significant correlation between

secondary metabolite content and antibacterial was recog-

nized (Li et al. 2006). Therefore, the main objective of the

present study is the high frequency regeneration of S.

rebaudiana through direct and indirect organogenesis for

production of healthy plants with high biomass and valu-

able secondary metabolites. In this study, quantitative

analysis of secondary metabolite was performed by high-

performance liquid chromatography, and then comparative

evaluation of antimicrobial activity of in vivo and in vitro

plant extract was done.

Materials and methods

Collection of plant material and surface sterilization

Stevia rebaudiana (Bertoni) plants were collected from the

Medicinal Plant Garden, Birla Institute of Technology

Mesra Ranchi, Jharkhand. Shoot tips and nodal segment

were separated from 3 to 4 months old in vivo grown

plants. Excised shoot tips were surface sterilized initially

by washing in running tap water to remove dirt, followed

by washing in Tween 20 (5 drops/100 mL) solution for

10 min to remove remaining dirt and spores. They were

then treated by 0.2 % bavistin (antifungal) solution for

8 min to remove fungal spores. Finally explants were

surface sterilized with 0.1 % mercuric chloride solution

inside laminar air flow chamber for 3 min, followed by

multiple rinsing with double distilled water to remove the

traces of mercuric chloride. Excess water was removed by

blot drying. For explant preparation, surface-sterilized

exposed ends were trimmed, nodal segments were cut into

0.5–0.8 cm in size and leaf explants were cut into small

disc.

Callus induction and multiplication

For callus induction and multiplication, selected explants

of leaf disc and nodal segments were inoculated in MS

medium (Murashige and Skoog 1962) supplemented with

3 % sucrose (w/v) in combination with different phyto-

hormones. In MS medium, kinetin (Kin) (0.5, 1, 1.5, 2, 2.5

and 3 mg L-1) was used alone and in a combination with

2, 4-dichlorophenoxyacetic acid (2, 4-D), Indole-3-acetic

acid (IAA) and Indole-3-butyric acid (IBA) in concentra-

tion of 0.5–3 mg L-1). The pH of the medium was

adjusted to 5.8 before autoclaving for 15 min at 121 �C.The cultures were maintained in a growth chamber with a

16/8 h light/dark photoperiod at 25 ± 2 �C with light

intensity of 25 lmol m-2 s-1 by cool-white fluorescent

lamps. After 21 days in culture, a number of explants

giving callus initiation (callus induction frequency) and

multiplication were recorded. The experiments were per-

formed at least three replicates, and the data of these

replicates were pooled for statistical analysis.

Shoot multiplication

Callus obtained from the induction medium was transferred

to MS media with plant growth regulators. Multiple com-

binations of Kin with BAP (6-bezyleadenine) and IAA

(0.5–3 mg L-1) were used for shoot induction and prolif-

eration. After 6 weeks, the mean number of shoots origi-

762 M. Kumari, S. Chandra

123

nated per callus and length of shoots were recorded. MS

medium without plant growth regulators serves as control.

Surface-sterilized leaf explants were inoculated on MS in

combination of Kin (0.5–3 mg L-1) with BAP

(1–3 mg L-1) and Adenine sulphate (Ads) (5 and

10 mg L-1) for direct shoot multiplication.

Rooting and acclimatization of in vitro plantlets

After 8 weeks, when in vitro shoots were grown up to

2–8 cm, subcultured on MS medium supplemented with

various concentrations of IAA (0.5–3 mg L-1), IBA

(0.5–3 mg L-1) and Naphthalene acetic acid (NAA)

(0.5–3 mg L-1) for rooting. Numbers of roots developed

were recorded after 4 weeks of subculture into rooting

media. Rooted plantlets were removed from flask and

washed properly with distilled water and transferred to pots

containing soilrite:vermicompost (1:1 v/v). For 5 days,

plantlets were grown under a natural light environment at

24 ± 1 �C (day) and 18 ± 1 �C (night). Plants were then

transferred to greenhouse conditions (30–35 �C, relative

humidity 70 %) for acclimatization. After 2 weeks, estab-

lished plants were repotted in larger pots.

Extraction and estimation of secondary metabolites

In vivo and in vitro acclimatized plants were dried to con-

stant weight in room temperature ranging from 25 to 30 �Cfor 48 h. These sample materials are grounded to fine

powder. 1 g of each sample was taken in 250 mL flasks and

100 mL (1 % w/v) of methanol was added to it and left

overnight. In the next day, the sample mixture was filtered

using filter paper (Whatman No. 1). Filtrate was further

separated by using 250 mL separatory funnel with 25 mL of

diethyl ether to remove the green colour. Lower transparent

layer was collected and extracted again in separatory funnel

with Butanol (25 mL). Finally, the butanol extract (upper

layer) was collected and left overnight for drying. Dried

extracts of samples were stored for secondary metabolite

estimation. 1 mg of extract was dissolved in 1 mL water and

filtered through a 0.2-lm filter, and steviol glycosides were

separated using a Waters HPLC system (Waters Corpora-

tion, Milford, MA) in dC18 column (Waters Atlantis,

4.6 mm 9 150 mm; 4 lm particle size). 20 ll of extractswas injected in a HPLC system. The solvents optimized for

isocratic elution consisted of acetonitrile and Milli-Q with

0.1 % orthophosphoric acid in ratio of 20:30 with flow rate

of 0.5 mL min-1. The detector was set at 210 nm. The

compounds from samples were identified by comparing the

retention time with the corresponding retention time of

standards. Quantification of compounds was done using

standard curves. All experiments were repeated at least three

times. The results were presented as mg g-1 of extracts.

Antimicrobial assay

Four common food-borne bacterial strains (Bacillus subtilis

National Collection of Industrial Microorganism (NCIM)

2193, Staphylococcus aureus NCIM 2122, Pseudomonas

fluorescens NCIM 2100 and E. coli NCIM 2809) and two

fungal strains (Aspergillus niger NCIM 1056 and Asper-

gillus notatum NCIM 745) were selected for antimicrobial

assay. All cultures were kind gift from Microbiology lab-

oratory, dept. of Bio-Engineering, BIT Mesra, Ranchi.

Bacterial strains were grown in nutrient broth, and fungal

strains were grown in potato dextrose broth for 24 h at

37 �C and used for assay in logarithmic phase (cell count

approx. 1 9 106 CFU mL-1). Three different solvents

(methanol, petroleum ether and water) were used for

extraction of in vivo and in vitro S. rebaudiana.

100 lg mL-1 of extracts was used for comparative eval-

uation of antimicrobial potential along with standard

antibiotics by measuring diameter of zone of inhibition

(ZI). All concentrations of extract were subjected to

antimicrobial assay using well plate assay and incubated at

37 ± 2 �C. After incubation, ZI was determined in mil-

limeter. Experiments were carried out in triplicate and the

mean values were expressed as ± SEM.

Data analysis

Data obtained from all experiments were presented as the

mean ± SE of three replications. Statistically significant

differences were determined by analysis of variance and

the Duncan multiple range test at a P\ 0.05 level of

significance.

Results and discussion

In the current investigation, complete regeneration was

successfully achieved from in vivo explants (internodes,

leaves) of S. rebaudiana through indirect (Figs. 1–9) and

direct organogenesis (Figs. 10–15). Surface-sterilized

in vivo explants induced callus on MS medium supple-

mented with Kin (0.5, 1.0, 1.5, 2.0 and 3.0 mg L-1) alone

and with combination of 2, 4-D (0.5, 1.0, 1.5, 2.0 and

3 mg L-1), IAA (0.5, 1.0, 1.5, 2.0, 3 mg - 1) and IBA

(0.5, 1.0, 1.5, 2.0 and 3 mg L-1) after 10 days.

Variation in plant growth regulators (PGRs), their con-

centration and type of explant influenced callus induction

per explant. Among different tested PGRs, Kin alone is

inadequate for callus induction and the increasing con-

centrations (3 mg L-1) have negative effects on callus

induction percentage. Only fewer explants showed callus-

ing with Kin supplement, whereas Kin in combination with

different auxins exhibits improved callus induction.

Stevioside glycosides from in vitro cultures of Stevia rebaudiana and antimicrobial assay 763

123

Combination of PGRs displays approx similar pattern of

callus induction with respect to their concentration, highest

concentration (3.0 mg L-1) was detrimental and interme-

diate concentration (1.5 mg L-1) was optimum in almost all

combinations (Fig. 16). Kin (1.5 mg L-1) in combination

with IAA (1.5 mg L-1) was found to be optimum for callus

induction and explant showed maximum (85 %) response

(Fig. 16) after 2 weeks of inoculation. Kin in combination

with 2, 4-D showed fastest response and callus induction

starts only after 5 days but its maximum response was 74 %

Figs. 1–9 1 Initial explant of intermodal segment inoculated on MS

media (Kin ? IAA, 1.5 mg L-1). 2 Callus induced on same media

after 2 weeks, subcultured for shooting. 3 Initiation of shoots after

2 weeks of subculture on MS supplemented with Kin 2.5 mg L-1 and

IAA 1.5 mg L-1, 4 and 5 on same media, shoots proliferated after

4 weeks, subcultured for rooting. 6 Roots after 4 weeks of subculture

on IBA 1 mg L-1. 7 Complete plant after 9 weeks. 8, 9 Acclimatized

plants in pot. Bar = 50 mm

764 M. Kumari, S. Chandra

123

(Fig. 16). Uddin et al. (2006) showed that internodal seg-

ment showed earlier response in 10 days for callus induction

and observed that highest concentration (5 mg L-1) of 2,

4-D produced poorest callus, whereas 3 mg L-1 2, 4-D pro-

duced best callus.

After 2 weeks of growth, the callus clumps were sub-

cultured to the regeneration medium. Kin (2 mg L-1) in

combination with BAP (2 mg L-1) was found to be best

(Fig. 4) for shoot multiplication with highest number of

shoots per callus (8.66 ± 0.3). In similar study on S.

rebaudiana, BAP (2 mg L-1) and IAA (1 mg L-1) were

optimum and 11 number of shoots regenerated from

internodal segment through direct regeneration (Sivaram

and Mukundan 2003). On many other plants, efficacy of

Kin and BAP for shoot regeneration has been demonstrated

earlier (Benne and Davies 1986; Rogers et al. 1998). Kin

alone (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mg L-1) and in combi-

nation with IAA (1.0, 1.5, 2.0, 2.5, 3.0 mg L-1) also

showed good response of regeneration in higher concen-

tration and Kin (2.5 mg L-1) has 7.3 ± 0.3 numbers of

shoots proliferated. Optimized media for increasing length

of shoots were found to be Kin (2.5 mg L-1) with IAA

(2.5 mg L-1) and highest length of shoot was

9 ± 0.14 cm. Kin alone and in combination with IAA has

Figs. 10–15 10 In vivo leaf explant inoculated in MS media (Kin

1.5 mg L-1 ? BAP 2.5 mg L-1). 11, 12 Proliferated shoots from

leaf explant after 2 weeks and growing shoots after 3 weeks. 13

Multiple shoots after 5 weeks growing on Kin 2.5 mg L-1 ? BAP

1.5 mg L-1. 14 Root proliferation with IAA (1 mg L-1). 15 Accli-

matized plants in pots. Bar = 50 mm

Fig. 16 Effects of various

concentrations of plant growth

regulators in MS medium with

Kin alone and in combination

with 2,4-D, IAA and IBA on

callus induction of Stevia

rebaudiana. Values represent

mean ± SE of three replications

Stevioside glycosides from in vitro cultures of Stevia rebaudiana and antimicrobial assay 765

123

almost similar effect on shoot length (Fig. 17), whereas in

Kin with BAP, the number of shoots was higher but length

is lower (1.5 ± 0.28–3.6 ± 0.3 cm).

Direct organogenesis significantly reduces the total

number of stages in culture by omitting the callus and

embryoid stage and provides an efficient regeneration and

multiplication. Complete regeneration of S. rebaudiana

was obtained through direct organogenesis using leaf

explants. In vivo leaves multiply into number of shoots

when inoculated on MS medium supplemented with Kin

(0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mg L-1) alone and in

combination of BAP (1.0, 1.5, 2.0, 2.5, 3.0 mg L-1) and

Ads (5 and 10 mg L-1). Natural environment and con-

centration PGRs have impact on multiplication of shoots

from leaf explant. Among different concentrations of PGRs

tested, Kin (1.5 mg L-1) with BAP (2.5 mg L-1) were

found to be the most effective for shoot multiplication with

maximum 11 ± 0.57 numbers of shoots. Combined effects

of Kin with BAP were proved suitable for both shoot

multiplication and increase in length (Fig. 18). In similar

study (Sreedhar et al. 2008), combination of PGRs (BAP

2 mg L-1 with Kin 1 mg L-1) produced lesser number of

shoots (4.33 ± 0.45). Kin alone proved superior to Kin

with Ads in terms of shoot multiplication, but for shoot

length, both the combinations display similar pattern and

mean length not increases more than 3.5 cm (Maximum,

3.2 ± 0.14). Kin in combination with Ads showed minor

multiplication and shoots failed to elongate and further

leaves turn brownish after 2–3 weeks.

In vitro proliferated shoots and micro-cuttings ([3 cm)

were excised and cultured on MS medium containing dif-

ferent concentrations of IAA (0.5, 1.0, 1.5, 2.0,

2.5 mg L-1), IBA (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) for rooting. Among

different concentrations, 1 mg L-1 IBA was proved most

effective with maximum numbers (7.6 ± 0.3) of roots

(Fig. 19). 1 mg L-1 IAA was also effective with

6.3 ± 0.33 numbers of roots. IAA and IBA were estab-

lished PGRs for root induction and their effect was studied

in many plant species such as, Chrysanthemum morifolium

Ramat (Khan et al. 1994), Solanum trilobatum Linn

(Arockiasamy et al. 2002), Cajanus cajan Linn (Si-

vaprakash et al. 1994), Vitex negundo Linn (Thiruven-

gadam and Jayabalan 2000) and Psoralea corylifolia Linn

(Jeyakumar and Jayabalan, 2002). IBA, IAA and NAA

were also successfully established for rooting of S.

rebaudiana shoots (Sivaram and Mukundan 2003; Sreed-

har et al. 2008; Debnath 2008). Maximum root induction

(97.66 %) was observed in media supplemented with

0.5 mg L-1 IAA (Ahmed et al. 2007) and with the

increased concentration of auxin root induction decreased.

With 81 % rooting 0.5 mg L-1, NAA was found optimum

by Rafiq et al. (2007).

Fully grown in vitro plantlets (6–8 cm) with roots were

carefully detached from culture medium and then trans-

ferred to soilrite:vermicompost (1:1 v/v) and after 5 days

transferred to greenhouse conditions (30–35 �C, relative

humidity 70 %) for acclimatization. Plantlets were suc-

cessfully acclimatized to ex vitro conditions. Micropropa-

gation allows rapid production of high quality, disease-free

and uniform plants. However, a major limitation in large-

scale application of this technology is high mortality

experienced by micropropagated plants during or following

laboratory to land transfer. Plantlets should be slowly

Fig. 17 Effects of various

concentrations of PGRs in MS

medium with Kin alone and in

combination with IAA and BAP

on mean number of shoots per

explants of Stevia rebaudiana

callus. Values represent

mean ± SE of three replications

766 M. Kumari, S. Chandra

123

acclimatized to ex vitro conditions with high light intensity

and low humidity conditions to overcome the problem.

Carbohydrate concentration plays role in acclimatization as

plantlets are transferred from heterotrophic condition to

autotrophic conditions. (Chandra et al. 2010).

For qualitative and quantitative analysis of plant sec-

ondary metabolites, two standard compounds viz. ste-

vioside and rebaudioside A (Reb A) purchased from

Sigma-aldrich were used for study Fig. 20. Previously,

in vitro culture of S. rebaudiana was established in number

of studies using different explants (Uddin et al. 2006;

Ahmed et al. 2007; Satpathy and Das 2010; Das et al.

2011). Quantitative and qualitative analysis of secondary

metabolites from in vitro plantlets through HPLC was not

much explored. Sivaram and Mukundan (2003) established

in vitro culture and done TLC and HPTLC for identifica-

tion of sweetening metabolites and collectively called those

as ‘Active principles’ but not specified as stevioside or

rebaudiosides.

In this study, Fig. 21 shows quantity of stevioside and Reb

A from plantlets grown in three different conditions, in vivo

plantlets, in vitro plantlets grown fromcallus regeneration and

in vitro plantlets directly grown from leaf explants. Highest

amount of stevioside (0.451 ± 0.001 mg g-1) and Reb A

0.131 ± 0.0057 mg g-1) found in plantlets regenerated

indirectly from callus. Both the secondary metabolites are

significantly higher in in vitro plantlets than in vivo plantlets,

in which stevioside is 0.274 ± 0.002 mg g-1 and Reb A is

0.01 ± 0.002 mg g-1.

Comparative analysis of steviol glycoside content in the

Stevia plant grown from seeds, cuttings and stem tip culture

was studied (Tamura et al. 1984) and no significant differ-

ence was observed for growth and chemical composition of

plants grown from seeds and stem tip culture. As for the

contents of sweet glycosides, clonal plants showed smaller

variation than sexually propagated plants, and they were

almost homogenous. Through these results, they concluded

that clonal propagation of stem tip culture is an effective

method of obtaining uniform plants for production of sweet

diterpene glycosides. Reis et al. (2011) established adven-

titious root culture of S. rebaudiana Bertoni in a roller bottle

system and screened the root extract for the presence of

steviol glycosides through HPLC. They showed similar

results with Yamazaki and Flores (1991) and concluded that

the methanolic extracts of wild plant roots and established

root culture of both extracts presented very similar profiles

and both showed absence of known steviol glycosides;

Fig. 19 Effects of various concentrations of auxins in MS medium

with IAA, IBA and NAA on mean number of roots per explants of

Stevia rebaudiana shoots. Values represent mean ± SE of three

replications

Fig. 18 Effects of various

concentrations of PGRs in MS

medium with Kin alone and in

combination with IAA and BAP

on mean number of shoots per

leaf explants of Stevia

rebaudiana. Values represent

mean ± SE of three replications

Stevioside glycosides from in vitro cultures of Stevia rebaudiana and antimicrobial assay 767

123

however, mass spectroscopic analysis showed the presence

of some unknown mass which further needs complete

identification of unknown compounds. In contrast, Khalil

et al. (2014) showed that roots from in vivo grown plants

were producers of steviol glycosides even higher than seeds,

stem and floral parts; however, significantly higher amount

of steviol glycoside content was observed in in vitro shoots

and in vivo leaves when compared with seeds floral parts

and stem parts. They studied the effect of different propa-

gation techniques (in vitro and in vivo) and gamma irradi-

ation on steviosides and Reb A content in S.rebaudiana and

concluded that leaves possess higher steviol glycosides than

other parts of plants. Similar results were shown in other

studies. (Bondarev et al. 2003; Ladygin et al. 2008)

Secondary metabolites play an important role in thera-

peutic potential of a plant. Stevia rebaudiana plant extract

possesses antimicrobial activity against plethora of

microorganisms (Table 1).

Antimicrobial activity of in vivo grown Stevia extract

was available on few studies (Tadhani and Subash 2006;

Ghosh et al. 2008) but only one study available in com-

parison with in vitro extract. Debnath (2008) screened

in vivo and in vitro explants for antimicrobial activity and

found that both have similar activity in methanol and

chloroform extract. In our study, in vitro methanol extract

was found most effective against all bacteria including B.

subtilis, S. aureus, P. fluorescence and E. coli with zone of

inhibition 13.6 ± 0.3, 12.3 ± 0.33, 8.6 ± 0.3 and

10.3 ± 0.1 mm, respectively. Methanol and petroleum

ether extract showed activity against all tested microor-

ganisms except A. notatum. Aqueous extract showed

activity against B. subtilis (7 ± 0.28 mm), S. aureus

(5.6 ± 0.33 mm) and P. fluorescence (6.3 ± 0.3 mm)

only. Our study revealed that in vitro extract has almost

similar inhibitory activity against bacteria in comparison to

standard antibiotic (ciprofloxacin) and their antifungal

Fig. 20 HPLC profiles of a Standards, Reb A (4.5 min) and Stevioside (4.7 min Reb A 4.4), b methanol extract of in vivo shoots, c in vitro

shoots grown from callus regeneration, d in vitro shoots directly grown from leaf explants

Fig. 21 Quantity of stevioside and Reb A in, methanol extract of

in vivo shoots, in vitro shoots grown from callus regeneration and

in vitro shoots directly grown from leaf explants

768 M. Kumari, S. Chandra

123

activity is lower than standard antifungal drug (Griseoful-

vin). The solvents used for extract preparation showed no

activity.

Conclusion

Findings from the current study highlighted the direct and

indirect organogenesis protocol via leaf and internodal

explants of S. rebaudiana. In addition to this, comparative

estimation of secondary metabolites in in vitro developed

plantlets and their antimicrobial assay was performed in

relation with secondary metabolite content. In vitro plant-

lets regenerated from callus were proved to be efficient in

terms of secondary metabolite content (variety and con-

centration) and their antimicrobial potential. The protocol

developed could be successfully employed for large-scale

multiplication, conservation of germplasm and isolation of

valuable secondary metabolites from S. rebaudiana.

Acknowledgments Mrs. Madhumita Kumari gratefully acknowl-

edges the Centre of Excellence (TEQIP II), Department of Bio-

Engineering, Birla Institute of Technology, Mesra, Ranchi, for pro-

viding the fellowship and infrastructure facilities.

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B. subtilis S. aureus P. fluorescence E.coli A. notatum A. niger

Methanol In vivo 13 ± 0.33 11.1 ± 0.16 8.3 ± 0.33 9.6 ± 0.57 – 7 ± 0

In vitro 13.6 ± 0.1 12.3 ± 0.33 8.6 ± 0.33 10.3 ± 0.1 – 6.6 ± 0.33

Petroleum ether In vivo 7.1 ± 0.16 6.6 ± 0.33 7.6 ± 0.3 7.3 ± 0.3 – 5.1 ± 0.57

In vitro 8.6 ± 0.33 7 ± 0.28 7 ± 0.57 6.6 ± 0.5 – 5.6 ± 0.33

Aqueous In vivo 7 ± 0 6.3 ± 0.33 6.6 ± 0.3 – – –

In vitro 7 ± 0.28 5.6 ± 0.33 6.3 ± 0.3 – – –

Ciprofloxacin 14 ± 0.00 14 ± 0.33 12 ± 0.00 12 ± 0.00

Graciofulvin 10 ± 0.00 12 ± 0.00

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