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
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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Extract (crude) Type of shoots Test organisms (zone of inhibition in mm)
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
Stevioside glycosides from in vitro cultures of Stevia rebaudiana and antimicrobial assay 769
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