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Industrial
Crops
and
Products
50 (2013) 517–
524
Contents
lists
available
at
ScienceDirect
Industrial
Crops
and
Products
journal
h
om
epage:
www.elsevier.com/locate/indcrop
A
reproducible
and
high
frequency
plant
regeneration
from
mature
axillary
node
explants
of
Gymnema
sylvestre
(Gurmur)—An
important
antidiabetic
endangered
medicinal
plant
M.
Thiyagarajan,
P.
Venkatachalam∗
Plant
Genetic
Engineering
and
Molecular
Biology
Lab,
Department
of
Biotechnology,
Periyar
University,
Periyar
Palkalai
Nagar,
Salem
636
011,
India
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
30
April
2013
Received
in
revised
form
19
July
2013
Accepted
6
August
2013
Keywords:
Anti
diabetic
Axillary
node
explants
Auxins
Cytokinins
Gymnema
sylvestre
Large
scale
propagation
MS
medium
a
b
s
t
r
a
c
t
Gymnema
sylvestre
is
an
important
medicinal
plant
used
in
different
systems
of
medicine
as
a
remedy
for
the
treatment
of
diabetes.
The
present
study
describes
an
efficient
and
rapid
protocol
for
large
scale
in
vitro
plant
regeneration
from
mature
axillary
node
explants
of
G.
sylvestre.
The
axillary
node
explants
were
cul-
tured
on
MS
medium
supplemented
with
different
concentrations
of
BAP
and
KIN
(0.5–3.0
mg/l)
for
shoot
bud
induction.
In
order
to
enhance
the
shoot
bud
multiplication,
regenerated
shoot
buds
were
further
subcultured
onto
MS
medium
fortified
with
different
concentrations
of
BAP
(0.5–3.0
mg/l)
in
combination
with
0.5
mg/l
of
NAA/IBA/IAA/KIN.
The
highest
frequency
(84.22%)
of
multiple
shoot
bud
regeneration
with
maximum
number
of
shoots
(14.20
shoots/explant)
was
noticed
on
MS
medium
supplemented
with
1.0
mg/l
BAP
and
0.5
mg/l
KIN
combination.
In
another
experiment,
in
vitro
derived
shoot
buds
were
cul-
tured
on
different
concentrations
of
GA3(0.5–2.0
mg/l)
and
various
concentrations
of
BAP
(0.5–3.0
mg/l)
in
combination
with
KIN
(0.5
mg/l)
and
GA3(1.0
mg/l)
for
shoot
bud
multiplications
as
well
as
elongation.
For
large
scale
plant
production,
in
vitro
derived
axillary
buds
were
cultured
on
MS
medium
fortified
with
BAP
(1.0
mg/l)
+
KIN
(0.5
mg/l)
+
GA3(1.0
mg/l)
combination,
in
which
about
418.72
shoots/explant
were
obtained
after
five
subcultures
on
the
same
media
composition.
Elongated
shoots
(>2
cm)
were
dissected
out
from
the
in
vitro
proliferated
shoot
clumps
and
were
cultured
on
half-strength
MS
medium
con-
taining
different
concentrations
of
various
auxins
(IAA,
IBA
and
NAA)
(0.5–2.0
mg/l)
for
root
induction.
Highest
frequency
of
rooting
(78%)
was
noticed
on
half-strength
MS
medium
augmented
with
2.0
mg/l
IBA.
The
rooted
plantlets
were
successfully
transferred
into
plastic
cups
containing
sand
and
soil
in
the
ratio
of
1:2
and
subsequently
they
were
established
in
the
greenhouse.
The
present
in
vitro
regeneration
protocol
would
facilitate
an
alternative
method
for
fast
and
large
scale
propagation
of
this
endangered
antidiabetic
medicinal
plant.
© 2013 Elsevier B.V. All rights reserved.
1.
Introduction
Diabetes
mellitus
(DM)
is
a
metabolic
disorder
characterized
by
hyperglycemia
resulting
from
defective
insulin
secretion,
resis-
tance
to
insulin
action
or
both
(Khan
et
al.,
2012).
Diabetes
is
a
deadly
disease
that
affected
an
estimated
285
million
people
world-
wide
in
2010
and
the
number
is
increasing
in
rural
and
poor
popu-
lations
throughout
the
world
and
is
projected
to
become
one
of
the
world’s
main
disablers
and
killers
within
the
next
25
years
(Shaw
et
al.,
2010).
Hyperglycemia
and
hyperlipidemia
are
two
impor-
tant
characters
of
diabetes
mellitus,
an
endocrine
based
disease.
Currently
available
therapy
for
diabetes
includes
insulin
and
var-
ious
oral
hypoglycemic
agents
such
as
sulfonylureas,
metformin,
∗Corresponding
author.
Tel.:
+91
9952609915;
fax:
+91
0427
2345124.
E-mail
addresses:
pvenkat67@yahoo.com,
pvenkatlab@yahoo.in
(P.
Venkatachalam).
glucosidase
inhibitors,
troglitazone,
etc.
(Kameswararao
et
al.,
2003).
These
drugs
are
used
as
mono-therapy
or
in
combination
to
achieve
better
glycemic
control
but
they
have
their
limitations
and
are
known
to
produce
serious
side
effects;
therefore,
the
search
for
safer,
specific
and
effective
hypoglycemic
agents
has
continued
to
be
an
important
area
of
investigation
with
natural
extracts
from
readily
available
traditional
medicinal
plants
offering
potentials
for
discovery
of
new
antidiabetic
drugs
(Klein
et
al.,
2007).
Over
the
years,
various
medicinal
plants
and
their
extracts
have
been
reported
to
be
effective
in
the
treatment
of
diabetes.
Plants
are
rich
sources
of
antidiabetic,
antihyperlipedemic
and
antioxidant
agents
such
as
flavonoids,
gallotannins,
amino
acids
and
other
related
polyphenols
(Muruganandan
et
al.,
2005;
Miyake
et
al.,
2006).
The
major
concern
is
that
most
of
the
medicinal
plants
are
collected
from
the
wild
populations,
and
over
70%
of
the
plant
collection
involves
destructive
harvesting
mainly
because
of
the
use
of
plant
parts
like
leaf,
bark,
wood
and
whole
plants.
Medicinal
plants
are
of
great
concern
to
the
researches
in
the
field
of
plant
0926-6690/$
–
see
front
matter ©
2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.indcrop.2013.08.027
Author's personal copy
518 M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524
biotechnology
not
only
for
rapid
propagation
but
also
for
produc-
tion
of
valuable
secondary
metabolites.
Overexploitation
of
the
plant
for
its
various
potential
applications
in
the
indigenous
system
of
medicine
has
caused
a
serious
threat
to
the
existence
of
medic-
inal
plant
species.
The
harvest
of
medicinal
plants
on
a
mass
scale
from
the
natural
habitat
is
leading
to
a
depletion
of
plant
resources.
Gymnema
sylvestre
R.
Br.
(Asclepiadaceae),
a
vulnerable
species
is
a
slow
growing,
perennial
woody
climber
of
tropical
and
subtrop-
ical
regions
and
it
is
popularly
called
as
“Gur-mar”
for
its
distinctive
property
of
temporarily
destroying
the
taste
of
sweetness
and
is
used
in
the
treatment
of
diabetes.
The
leaves
of
the
plant
in
par-
ticular
are
used
as
antiviral,
diuretic,
antiallergic,
hypoglycemic,
hypolipidemic,
antibiotic,
in
stomach
pains
and
in
rheumatism.
In
addition,
it
possesses
antimicrobial,
anti-hypercholesterolemic,
hepatoprotective
and
sweet
suppressing
activities.
The
various
reports
on
its
multiple
uses
attracted
attention
for
utilization
of
the
plant
for
gymnemic
acid.
G.
sylvestre
species
is
threatened
with
extinction
due
to
its
indiscriminate
collection
for
commercial
pur-
poses
and
to
meet
the
requirements
of
the
pharmaceutical
industry.
Conventional
propagation
is
hampered
due
to
its
poor
seed
viabil-
ity,
low
rate
of
germination
and
poor
rooting
ability
of
vegetative
cuttings.
Inevitably,
therefore,
rapid
multiplication
of
this
impor-
tant
drug
yielding
genotype
is
imperative.
Alternatively,
in
vitro
micropropagation
would
be
beneficial
in
accelerating
large
scale
multiplication
and
conservation
of
this
important
plant
species.
In
vitro
culture
methods
through
multiple
shoot
induction
using
axillary
node
explants
have
proved
successful
for
rapid
propagation
of
a
number
of
medicinally
important
species
such
as
Santolina
canescens
(Casado
et
al.,
2002);
Bupleurum
fruticosum
(Fraternale
et
al.,
2002);
Rauvolfia
tetraphylla
(Faisal
et
al.,
2006).
In
vitro
culture
allows
maintaining
clonal
fidelity,
and
also
assures
the
consistent
production
of
true-to-type
plants
within
a
short
span
of
time.
There
are
few
reports
on
in
vitro
propagation
of
G.
sylvestre
using
axillary
node
explants.
In
vitro
plant
regeneration
from
nodal
explants
of
G.
sylvestre
was
reported
by
Komalavalli
and
Rao
(2000),
Sairam
Reddy
et
al.
(1998)
and
Subathra
Devi
and
Mohana
Srinivasan
(2008).
Though,
the
plant
regeneration
was
reported
in
G.
sylvestre
the
percent
of
shoot
bud
regeneration
was
found
to
be
low
and
may
not
be
useful
for
production
of
large
scale
plants
for
commercial
purpose/cultivation.
Therefore,
development
of
a
rapid
protocol
for
high
frequency
in
vitro
plant
regeneration
in
this
important
medicinal
herb
became
necessary
in
order
to
reduce
the
existing
pressure
on
natural
populations
and
continuous
supply
of
plant
materials
for
the
pharmaceutical
industry.
As
tissue
culture
tech-
nique
has
now
become
a
well
established
method
for
large
scale
plants
developed
for
commercial
utilization
of
several
endangered
medicinal
plants.
The
major
goal
of
the
present
investigation
was
to
standardize
the
best
media
composition,
growth
regulators
combi-
nations
for
high
frequency
plantlet
production
from
mature
axillary
node
explants
of
Gymenma
sylvestre
species.
In
order
to
enhance
the
shoot
bud
multiplication
rate,
various
growth
regulators
were
examined
to
identify
the
best
growth
hormone
combinations
for
maximum
number
of
shoot
buds
production.
In
addition,
in
vitro
culture
passages
were
also
standardized
to
maximize
the
number
of
shoot
buds
without
losing
its
vigour
and
viability
for
commercial
scale
application/utilization.
2.
Materials
and
methods
2.1.
Preparation
of
explants
G.
sylvestre
plants
were
collected
from
ABS
medicinal
garden,
Salem,
Tamil
Nadu
and
maintained
in
the
Greenhouse,
Department
of
Biotechnology,
Periyar
University,
Salem
11.
For
shoot
bud
induc-
tion,
mature
axillary
node
explants
were
collected
from
3
months
old
plants
and
were
washed
in
running
tap
water.
Explants
were
washed
with
few
drops
of
10%
(v/v)
Tween-20
to
remove
the
super-
ficial
dust
particles
including
microbes.
Then,
they
were
surface
sterilized
with
0.1%
(w/v)
mercuric
chloride
solution
for
10
min
fol-
lowed
by
rinsing
them
for
five
times
with
sterile
distilled
water.
Sterilized
nodal
explants
were
used
for
in
vitro
culture
studies
as
described
below.
2.2.
Culture
media
and
growth
conditions
The
culture
medium
consisted
of
MS
(Murashige
and
Skoog’s,
1962)
salts,
vitamins,
3%
(w/v)
sucrose
and
the
pH
of
the
media
was
adjusted
to
5.6
with
0.1
N
NaOH
or
HCl
before
adding
0.7%
(w/v)
agar.
Media
(15
ml)
were
poured
into
25
mm
×
150
mm
cul-
ture
tubes
(Borosil,
Mumbai)
and
autoclaved
at
121 ◦C
for
15
min.
The
cultures
were
maintained
at
24
±
2◦C
under
16/8
h
(light/dark
cycle)
photoperiod
(60
E
m−2s−1)
and
irradiance
provided
by
cool-white
fluorescent
tubes
(Philips,
India).
2.3.
Shoot
bud
initiation
Surface
sterilized
mature
axillary
node
explants
were
cultured
on
MS
medium
supplemented
with
different
concentrations
of
BAP
and
KIN
(0.5–3.0
mg/l)
for
shoot
bud
induction.
After
two
weeks
of
culture,
direct
shoot
bud
initiation
from
the
axillary
node
explants
was
noticed.
2.4.
Induction
of
multiple
shoots
In
this
experiment,
axillary
node
explants
derived
in
vitro
regen-
erated
shoot
buds
as
explants
source
were
cultured
on
MS
medium
fortified
with
different
concentrations
of
BAP
(0.5,
1.0,
1.5,
2.0
and
3.0
mg/l)
in
combination
with
0.5
mg/l
IAA/IBA/NAA/KIN
individu-
ally
for
multiple
shoot
bud
development.
2.5.
Shoot
bud
elongation
and
multiplication
To
achieve
shoot
bud
multiplication
and
elongation,
two
exper-
iments
were
adopted.
In
the
first
experiment,
the
in
vitro
derived
shoot
buds
were
cultured
on
MS
medium
supplemented
with
dif-
ferent
concentrations
of
GA3(0.5–2.0
mg/l)
alone
for
shoot
bud
elongation.
In
another
experiment
the
shoot
buds
were
cultured
on
different
concentrations
of
BAP
(0.5–3.0
mg/l)
in
combination
with
0.5
mg/l
KIN
and
1.0
mg/l
GA3for
shoot
bud
multiplication
and
elongation.
2.6.
Large
scale
production
of
shoot
buds
For
large
scale
plant
production,
in
vitro
regenerated
shoot
buds
were
subcultured
onto
MS
medium
supplemented
with
1.0
mg/l
BAP
+
0.5
mg/l
KIN
+
1.0
mg/l
GA3combination.
The
cultures
were
subcultured
onto
the
fresh
same
media
composition
once
in
2
weeks
interval.
This
process
was
repeated
for
another
five
sub-
cultures
(each
15
days)
to
examine
the
effect
of
subculture
on
production
of
large
scale
shoot
buds.
After
75
days
of
culture
multi-
ple
shoots
were
counted
for
analysis
of
total
number
of
regenerated
shoot
buds.
2.7.
Rooting
of
elongated
shoots
and
acclimatization
The
elongated
shoots
(>2.0
cm
height)
were
transferred
to
half-
strength
MS
medium
fortified
with
different
concentrations
of
IAA
or
IBA
or
NAA
(0.5–2.0
mg/l)
for
root
induction.
Plantlets
with
well-
developed
roots
were
removed
from
the
culture
tubes
and
gently
washed
under
running
tap
water
to
remove
adhering
medium.
Sub-
sequently,
they
were
transferred
to
plastic
cups
containing
sterile
Author's personal copy
M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524 519
Table
1
Effect
of
different
concentrations
of
two
cytokinins
on
initiation
of
shoot
buds
from
mature
axillary
node
explants
of
Gymnema
sylvestre.
Cytokinin
concentration
(mg/l)
Percent
of
shoot
bud
initiation
(Mean
±
SE)
Number
of
shoots/explants
(Mean
±
SE)
Shoot
length
(cm)
(Mean
±
SE)
BAP
KIN
0.5
-
44.12
±
3.2g*4.50
±
0.65c*1.48
±
0.54e*
1.0 –
65.56
±
2.5a7.30
±
0.79a2.21
±
0.28a
1.5
–
56.66
±
1.8c6.80
±
0.68a2.11
±
0.11b
2.0
–
54.20
±
3.3d5.40
±
0.91b2.09
±
0.38a
3.0
–
39.76
±
2.7h4.20
±
0.87c1.11
±
0.11g
–
0.5
62.83
±
1.6b3.71
±
0.91d2.01
±
0.27c
–
1.0
51.55
±
1.1e3.42
±
1.02d1.65
±
0.28d
–
1.5 48.28
±
1.7f3.14
±
0.98d1.11
±
0.93g
–
2.0 45.56
±
1.7g2.66
±
0.95e1.31
±
0.44f
–
3.0
36.23
±
2.1i1.57
±
0.61f1.13
±
0.55g
*Mean
values
within
the
column
followed
by
the
same
letter
in
superscript
are
not
significantly
different
at
P
<
0.05
level.
sand
and
soil
mixture
in
1:2
ratio.
The
potted
plantlets
were
initially
maintained
in
the
controlled
environment
for
two
weeks
and
sub-
sequently
they
were
shifted
to
the
greenhouse.
After
three
weeks,
the
plantlets
were
successfully
established
in
the
field.
2.8.
Statistical
analysis
Experiments
were
set
up
in
a
completely
randomized
block
(CRB)
design
and
each
experiment
had
three
replicates.
The
cul-
tures
were
observed
periodically
and
percent
of
response
for
shoot
bud
regeneration,
multiple
shoots
development
and
rooting
were
recorded.
The
frequency
of
shoot
induction,
mean
number
of
shoots/explant
and
mean
shoot
length
(cm)
were
recorded
after
four
weeks
of
culture.
A
total
number
of
shoots
as
well
as
roots
were
also
noticed
by
visual
observations.
The
analysis
of
variance
(ANOVA)
was
performed
using
SAS
programme.
The
differences
among
mean
values
were
determined
by
Student–Newman–Keuls
Test
at
5%
significance
level.
3.
Results
and
discussion
3.1.
Shoot
bud
induction
Mature
axillary
node
explants
from
G.
sylvestre
plants
were
cultured
on
MS
medium
supplemented
with
different
concen-
trations
of
BAP
and
KIN
(0.5,
1.0,
1.5,
2.0
and
3.0
mg/l)
alone
for
shoot
bud
induction.
After
7
days,
the
shoot
buds
emerged
directly
from
the
axillary
node
explants.
The
regenerated
shoot
buds
were
subcultured
onto
fresh
medium
containing
the
same
concentrations
of
growth
regulators
for
shoot
bud
multiplica-
tion.
Among
the
different
concentrations
of
cytokinin
(BAP/KIN)
tested,
highest
percent
(65.56%)
of
shoot
bud
regeneration
was
noticed
on
MS
medium
containing
1.0
mg/l
BAP
while
62.83%
shoot
bud
regeneration
was
observed
on
MS
medium
fortified
with
0.5
mg/l
KIN.
The
morphogenetic
responses
of
axillary
node
explants
to
different
concentrations
of
cytokinins
are
summarized
in
Table
1.
Though,
shoot
bud
regeneration
was
noticed
in
all
the
concentrations
of
BAP
and
KIN
tested,
maximum
percent
of
shoot
regeneration
was
observed
on
medium
containing
BAP
compared
to
KIN.
The
number
of
axillary
shoots/explant
was
significantly
higher
on
BAP
(7.30
±
0.79
shoots/explant)
containing
media
than
KIN
(3.71
±
0.91
shoots/explant).
In
general
by
increasing
concen-
trations
of
cytokinins,
the
frequency
of
shoot
bud
induction
as
well
as
the
number
of
multiple
shoot
bud
was
increased.
Increasing
the
concentration
of
either
BAP
(above
1.0
mg/l)
or
KIN
(above
0.5
mg/l)
in
the
culture
media
had
resulted
in
reduction
of
shoot
bud
regen-
eration.
Shoot
bud
regeneration
was
gradually
decreased
when
the
cytokinin
concentration
was
increased
beyond
optimum
level.
Analysis
of
variance
revealed
that
mean
shoot
number
as
well
as
shoot
length
was
significantly
affected
by
the
concentrations
and
types
of
cytokinin
tested
(Table
1).
Maximum
number
of
shoots
(7.30
±
0.79
shoots/explant)
(Fig.
1A)
and
shoot
length
(2.21
cm)
were
recorded
on
MS
medium
supplemented
with
1.0
mg/l
BAP.
The
superiority
of
BAP
over
KIN
in
shoot
bud
regeneration
has
been
well
documented
in
Pterocarpus
marsupium
(Chand
and
Singh,
2004).
Similarly
the
influence
of
BAP
on
induction
of
multiple
shoot
buds
from
nodal
explants
has
been
reported
in
various
plant
species,
including
Eclipta
alba
(Dhaka
and
Kothari,
2005),
Quercus
euboica
(Kartsonas
and
Papafotiou,
2007),
Ulmus
parvifo-
lia
(Thakur
and
Karnosky,
2007),
Sarcostemma
brevistigma
(Thomas
and
Shankar,
2009),
Clitoria
ternatea
(Singh
and
Tiwari,
2010)
and
Stevia
rebaudiana
(Thiyagarajan
and
Venkatachalam,
2012).
3.2.
Shoot
bud
multiplication
In
vitro
developed
shoot
buds
from
axillary
node
explants
were
placed
on
MS
medium
supplemented
with
different
con-
centrations
of
BAP
(0.5–3.0
mg/l)
in
combination
with
0.5
mg/l
IAA/IBA/NAA/KIN
for
shoot
bud
multiplication.
Among
the
various
combinations
tested,
BAP
(1.0
mg/l)
+
KIN
(0.5
mg/l)
combina-
tion
was
found
to
be
best
for
multiple
shoot
bud
induction
(14.20
shoots/explants).
The
highest
percent
(84.22%)
of
multiple
shoot
bud
formation
was
noticed
on
MS
medium
fortified
with
1.0
mg/l
BAP
and
0.5
mg/l
KIN
combination
which
was
statistically
significant
at
5%
level
(Table
2).
Among
the
combinations
tested,
BAP
+
KIN
combination
was
found
to
be
superior
for
induction
of
highest
percent
of
multiple
shoot
bud
development
(84.22%),
followed
by
1.0
mg/l
BAP
+
0.5
mg/l
IAA
(80.82%)
and
1.0
mg/l
BAP
+
0.5
mg/l
IBA
(78.57%)
combinations.
The
BAP
and
KIN
com-
bination
was
found
to
be
superior
for
shoot
bud
multiplications
than
BAP
and
IAA/IBA/NAA
combination.
Similar
results
were
also
reported
earlier
in
Pogostemon
heyneanus
(Hembrom
et
al.,
2006);
Alpinia
calcarata
(Asha
et
al.,
2012);
Calamus
ravancoricus
(Krishna
Kumar
et
al.,
2012).
The
reduction
of
shoot
buds
on
medium
con-
taining
BAP
and
NAA
may
be
due
to
the
appearance
of
profuse
callusing
at
the
basal
end
of
the
proliferated
shoots.
3.3.
Multiple
shoot
bud
induction
and
elongation
In
vitro
regenerated
shoot
buds
were
cultured
on
MS
medium
containing
different
concentrations
of
gibbrelic
acid
(GA3)
(0.5–2.0
mg/l)
for
shoot
bud
elongation.
After
seven
days
of
cul-
ture,
the
shoot
bud
elongation
was
observed.
The
maximum
length
(5.25
cm)
of
shoot
bud
elongation
was
noticed
on
1.0
mg/l
GA3.
When
GA3concentration
was
increased
above
(1.0
mg/l)
the
length
of
the
shoot
bud
elongation
was
decreased
inversely
(Table
3).
If
the
GA3was
not
added
in
the
medium,
the
shoot
growth
was
reduced,
and
this
result
suggested
that,
GA3had
vital
role
Author's personal copy
520 M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524
Fig.
1.
Large
scale
in
vitro
propagation
of
Gynema
sylvestre
using
mature
axillary
node
explants.
(A)
Shoot
bud
initiation.
(B)
Multiple
shoot
bud
induction.
(C)
Multiple
shoot
bud
regeneration
with
elongation.
(D)
Root
initiation
from
the
elongated
shoot.
(E)
Regenerated
plantlets
with
well
developed
roots.
(F)
In
vitro
derived
plantlets
growing
in
plastic
cups.
(G)
Acclimatized
tissue
culture
plant
growing
in
the
field.
of
in
shoot
bud
elongation
as
reported
earlier
for
other
plants
such
as
E.
alba
(Dhaka
and
Kothari,
2005)
and
Andrographis
pan-
iculata
(Purkayastha
et
al.,
2008).
In
addition,
shoot
buds
were
cultured
on
MS
medium
fortified
with
different
concentrations
of
BAP
(0.5–3.0
mg/l)
in
combination
with
KIN
(0.5
mg/l)
and
GA3
(1.0
mg/l)
for
multiple
shoot
bud
induction
as
well
as
elongation.
After
two
weeks
of
culture,
the
maximum
number
of
multiple
shoot
buds
was
noticed
(16.80
shoots/explants)
on
1.0
mg/l
BAP
+
0.5
mg/l
KIN
+
1.0
mg/l
GA3combination
(Table
3)
(Fig.
1C).
The
results
strongly
suggest
that
cytokinin
was
essential
for
shoot
bud
ini-
tiation
from
axillary
node
explants
and
GA3became
necessary
for
large
scale
shoot
bud
regeneration
as
well
as
elongation.
The
combination
of
GA3and
cytokinins
not
only
promoted
shoot
bud
elongation
but
also
enhanced
multiple
shoot
bud
proliferation
rate.
Similarly
Thirugnanasampandan
et
al.
(2010)
described
that
GA3in
combination
with
cytokinin
promotes
shoot
bud
elongation
as
well
as
multiple
shoot
bud
development
in
Isodon
wightii.
3.4.
Large
scale
production
of
shoots
In
order
to
produce
large
scale
plants,
the
optimum
concen-
trations
of
growth
hormones
combination
was
selected
based
on
above
results
and
the
subculture
was
repeated
as
passage
for
five
times
as
described
in
Fig.
2.
Shoot
bud
multiplication
was
generally
achieved
by
splitting
of
the
shoots
into
2–3
small
clumps
(4–5
shoots/clump).
Shoot
clusters
were
subcultured
on
the
same
hormonal
combination
for
shoot
bud
multiplication
at
15
days
interval.
At
the
end
of
the
first
subculture,
the
maximum
number
of
shoots
(29.4
shoots/clump)
was
obtained.
The
number
of
shoots
in
a
cluster
was
increased
with
subsequent
passage
of
Author's personal copy
M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524 521
Table
2
Effect
of
different
concentrations
and
combinations
of
auxins
and
cytokinins
on
multiple
shoot
bud
induction
from
mature
axillary
node
explants
of
Gymneme
sylvestre.
Hormone
concentration
(mg/l)
Percent
of
multiple
shoot
bud
developments
(Mean
±
SE)
Number
of
shoots/explants
(Mean
±
SE)
Shoot
length
(cm)
(Mean
±
SE)
BAP
KIN
NAA
IAA
IBA
0.5
–
0.5
–
–
63.65
±
1.52g*4.42
±
0.94g*1.11
±
0.55f*
1.0 –
0.5 –
–
72.13
±
1.18d6.12
±
1.41e1.31
±
0.64e
1.5
–
0.5
–
–
68.84
±
1.82f5.92
±
1.25f1.27
±
0.95e
2.0
–
0.5
–
–
65.76
±
1.59g4.78
±
0.64g1.18
±
0.49f
3.0
–
0.5
–
–
59.44
±
1.39i3.69
±
1.26h1.10
±
0.65f
0.5
–
–
0.5
–
77.19
±
1.70c7.33
±
1.14d1.77
±
0.45c
1.0
–
–
0.5
–
80.82
±
1.32b8.53
±
0.86c1.81
±
0.74b
1.5 –
–
0.5 –
75.55
±
1.49c6.13
±
1.59d1.65
±
0.89c
2.0 –
–
0.5 –
71.78
±
1.95e5.85
±
1.40f1.56
±
0.65d
3.0
–
–
0.5
–
62.29
±
1.79h4.16
±
0.91g1.50
±
0.75d
0.5
–
–
–
0.5
59.05
±
1.92i4.09
±
1.38g1.96
±
0.35b
1.0
–
–
–
0.5
78.57
±
1.27c6.90
±
1.51e2.18
±
0.34a
1.5
–
–
–
0.5
74.79
±
1.59d5.88
±
1.01f2.07
±
0.95a
2.0
–
–
–
0.5
70.86
±
1.31e5.09
±
0.94f1.65
±
0.29d
3.0
–
–
–
0.5
70.25
±
1.25e4.50
±
1.43g1.40
±
0.85d
0.5
0.5
–
–
–
71.23
±
2.20e9.80
±
1.23d2.11
±
0.45a
1.0
0.5
–
–
–
84.22
±
2.60a14.20
±
0.56a2.31
±
0.94a
1.5 0.5 –
–
–
82.60
±
1.80a10.23
±
1.25b1.50
±
0.65d
2.0
0.5
–
–
–
78.23
±
1.50c8.50
±
0.91c1.27
±
0.85e
3.0
0.5
–
–
–
69.66
±
1.70f6.20
±
0.54d1.15
±
0.39f
*Mean
values
within
the
column
followed
by
the
same
letter
in
superscript
are
not
significantly
different
at
P
<
0.05
level.
Table
3
Effect
of
different
concentrations
of
GA3and
various
concentration
of
BAP
(0.5–3.0
mg/l)
in
combination
with
0.5
mg/l
KIN
and
1.0
mg/l
GA3for
and
multiple
shoot
bud
induction
as
well
as
shoot
bud
elongation
of
Gymenma
sylvestre.
Hormone
concentration
(mg/l) Percent
of
response
(Mean
±
SE) No.
of
shoots/culture
(Mean
±
SE)
Shoot
length
(cm)
(Mean
±
SE)
BAP
KIN
GA3
–
–
0.5 77.5
±
1.75d*3.42
±
2.1g*4.95
±
0.61b*
–
–
1.0
82.5
±
2.56c4.62
±
1.7f5.25
±
0.50a
–
–
1.5
75.0
±
3.20d2.66
±
2.5h4.25
±
0.46b
–
–
2.0
71.0
±
2.00e2.12
±
1.7h3.54
±
0.54c
0.5
0.5
1.0
78.4
±
1.11c11.30
±
1.5d3.70
±
1.21c
1.0
0.5
1.0
86.7
±
2.34a16.80
±
1.6a5.82
±
1.88a
1.5
0.5
1.0
80.3
±
1.98b13.20
±
1.2b5.20
±
1.22a
2.0 0.5 1.0 76.0
±
1.93d10.40
±
1.1c4.80
±
0.23b
3.0
0.5
1.0
68.4
±
1.67f7.70
±
1.8e3.60
±
0.78c
*Mean
values
within
the
column
followed
by
the
same
letter
in
superscript
are
not
significantly
different
at
P
<
0.05
level.
subcultures.
The
shoot
bud
multiplication
was
performed
upto
5
passages
and
the
shoot
buds
were
also
elongated
on
the
same
media
composition.
The
elongated
shoots
were
dissected
out
individually
and
used
for
rooting.
In
this
study,
it
is
quite
possible
to
regenerate
multiple
shoots
continuously
without
showing
any
decrease/reduction
and
a
total
number
of
shoots
obtained
was
418.7
shoots/explant
at
the
end
of
the
5th
subculture
(105
days)
(Table
4).
On
contray,
Sairam
Reddy
et
al.
(1998)
and
Subathra
Devi
and
Mohana
Srinivasan
(2008)
reported
that
in
vitro
regenerated
multiple
shoots
of
Gymnema
did
not
survive
even
after
subcultur-
ing
on
the
same
media
combinations
whereas,
Komalavalli
and
Rao
(2000)
described
that
it
was
possible
to
regenerate
multiple
shoots
2–3
subcultures
(3
months)
and
reduced
thereafter.
Also
Sairam
Reddy
et
al.
(1998)
reported
that
GA3and
KIN
did
not
improve
the
number
of
multiple
shoot
bud
proliferation
in
Gymnema.
The
present
results
clearly
suggested
that
the
development
of
multiple
shoots
could
be
successful
without
showing
any
decline
in
number
of
shoots
up
to
5
subcultures
or
even
beyond
(data
not
shown).
In
addition,
it
is
interesting
to
note
that
the
number
of
multiple
shoots
obtained
was
418.7
from
single
axillary
node
explant
after
5
subcultures.
Subathra
Devi
and
Mohana
Srinivasan
(2008)
reported
that
maximum
number
of
shoots
regenerated
was
45
shoots/explant
on
MS
medium
fortified
with
BA
(1.0
mg/l)
+
IAA
(0.5
mg/l)
+
100
mg/l
B2vitamin
combinations
after
30
days
of
culture.
Sairam
Reddy
et
al.
(1998)
demonstrated
that
induction
of
multiple
shoots
obtained
was
7.4
shoots/explant
on
MS
medium
Table
4
Large
scale
plant
production
in
Gymneme
sylvestre
using
in
vitro
regenerated
shoot
buds
as
explant
source.
Hormone
concentration
BAP
(1.0
mg/l)
+
KIN
(0.5
mg/l)
+
GA3(1.0
mg/l)
No.
of
shoot
clumps
cultured
for
multiplication
Total
number
of
shoots
Clump
1
Clump
2
Clump
3
1st
Subculture
(15
days
interval)
27.70
±
2.02*24.50
±
1.20*29.4
±
1.85*81.60
2nd
Subculture
(15
days
interval)
45.20
±
1.20
41.75
±
2.33
48.25
±
2.41
135.20
3rd
Subculture
(15
days
interval)
78.23
±
3.93
70.20
±
1.66
82.56
±
2.40
230.99
4th
Subculture
(15
days
interval)
94.66
±
1.20
92.50
±
2.18
98.32
±
3.45
285.48
5th
Subculture
(15
days
interval)
138.12
±
2.40
135.3
±
2.21
145.3
±
4.26
418.72
Total
No.
of
days
at
5th
passage
of
culture
–105
days
*Number
of
shoots/clump
represents
mean
±
SE
in
three
independent
experiments.
Author's personal copy
522 M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524
Fig.
2.
Schematic
representation
of
large
scale
in
vitro
plantlet
production
from
mature
axillary
node
explants
of
Gymnema
sylvestre.
containing
BAP
(5.0
mg/l)
and
NAA
(0.2
mg/l)
combination
after
5–6
weeks
of
culture.
The
maximum
number
of
multiple
shoots
noticed
was
Gymnema
57.2
shoots/explants
in
Gymnema
by
Komalavalli
and
Rao
(2000).
The
present
results
on
induction
of
multiple
shoots
from
were
found
to
be
superior
over
earlier
reports,
because
the
number
of
multiple
shoots
obtained
was
7
times
higher.
As
a
results
of
the
present
study,
it
indicated
that
enhancement
of
multiple
shoot
bud
induction
is
feasible
using
defined
media
with
specific
growth
regulator
combinations.
In
this
study
enhanced
shoot
multiplication
rate
was
achieved
on
MS
medium
fortified
with
BAP
(1.0
mg/l)
+
KIN
(0.5
mg/l)
+
GA3(1.0
mg/l).
3.5.
Rooting
In
vitro
regenerated
shoots
were
excised
individually
and
trans-
ferred
to
half-strength
MS
medium
supplemented
with
various
concentrations
of
IAA
or
IBA
or
NAA
(0.0,
0.5,
1.0,
1.5
and
2.0
mg/l)
for
root
induction.
Shoots
produced
roots
after
two
weeks
of
culture
Author's personal copy
M.
Thiyagarajan,
P.
Venkatachalam
/
Industrial
Crops
and
Products
50 (2013) 517–
524 523
Table
5
Effect
of
different
concentrations
of
three
auxins
on
root
induction
from
elongated
shoots
of
Gymnema
sylvestre.
Hormone
concentration
(mg/l)
Percent
of
rooting
(%)
Number
of
roots
per
shoot
(Mean
±
SE)
IAA
IBA
NAA
0.0
0.0
0.0
–
–
0.5
–
–
36
±
0.78g*3.89
±
2.12g*
1.0
–
–
57
±
1.32e5.25
±
1.77f
1.5 –
–
61
±
1.56d5.58
±
1.97f
2.0
–
–
68
±
0.89b7.50
±
1.34d
–
0.5
–
43
±
2.33f6.21
±
1.87e
–
1.0
–
54
±
1.56c7.30
±
1.08c
–
1.5
–
61
±
1.22d9.58
±
2.10b
–
2.0
–
78
±
1.72a12.8
±
2.30a
–
–
0.5 31
±
8.56h3.89
±
1.12g
–
–
1.0 27
±
0.82g2.85
±
1.77h
–
–
1.5
18
±
1.38h2.58
±
1.97h
–
–
2.0
0.00
(callusing)
0.00
(callusing)
*Mean
values
within
the
column
followed
by
the
same
letter
in
superscript
are
not
significantly
different
at
P
<
0.05
level.
and
the
data
were
recorded.
The
first
roots
emerged
directly
from
the
basal
part
of
the
shoots
after
21
days
of
culture
with
no
interven-
ing
callus.
The
highest
percent
of
root
induction
(78%)
was
observed
on
half-strength
MS
medium
fortified
with
2.0
mg/l
IBA
followed
by
IAA
and
maximum
number
of
roots
obtained
was
12.8
roots/shoot
(Fig.
1D
and
E)
(Table
5).
The
rooting
percent
was
significantly
increased
by
the
increasing
concentrations
of
IBA
and
IAA.
Similar
observation
was
reported
by
Thiruvengadam
and
Jayabalan
(2000),
Arockiasamy
et
al.
(2002),
Raman
and
Jaiwal
(2000)
and
Jeyakumar
and
Jayabalan
(2002).
However,
the
rooting
response
was
nega-
tively
correlated
with
NAA
concentrations
and
no
root
initiation
was
noticed
at
higher
concentration
2.0
mg/l
NAA
(Table
5).
Results
of
this
study
were
corroborated
by
the
findings
of
previous
reports
(Anitha
and
Pullaiah,
2002)
and
(Thiyagarajan
and
Venkatachalam,
2012).
Among
the
three
auxins
used,
maximum
percent
of
rooting
was
obtained
on
half-strength
MS
medium
fortified
with
2.0
mg/l
IBA
which
was
found
to
be
superior
auxin
for
rooting
over
other
two
auxins
(IAA
and
NAA).
In
general,
IBA
was
showed
a
strong
root-
ing
response;
in
a
wide
range
of
plant
species.
Similar
observation
has
been
reported
in
Ceropegia
candelabrum
(Beena
et
al.,
2003),
Mucuna
pruriens
(Faisal
et
al.,
2006)
and
Jatropho
curcas
(Kumar
et
al.,
2008).
3.6.
Acclimatization
The
rooted
plantlets
with
expanded
leaves
were
successfully
transferred
into
plastic
cups
containing
sand
and
soil
in
the
ratio
of
1:2
and
covered
with
polythene
bags
to
ensure
high
humidity.
Ini-
tially
the
plantlets
were
kept
in
the
controlled
environment
for
two
weeks
and
the
polybags
were
gradually
removed
in
order
to
accli-
matize
the
plantlets
under
greenhouse
conditions.
Subsequently
they
were
transferred
to
the
field
conditions
and
the
survival
rate
noticed
was
85%
(Fig.
1F
and
G).
Regenerated
plants
grew
well
and
phenotypically
similar
to
the
parental
stock.
4.
Conclusion
In
conclusion,
this
report
describes
a
two
step
culture
protocol
for
large
scale
multiple
shoot
bud
regeneration
from
axillary
nodal
explants
of
G.
sylvestre.
The
maximum
shoot
bud
induction
(84.2%)
as
well
as
shoot
number
(14.2)
without
callus
formation
occurred
on
MS
medium
augmented
with
BAP
and
KIN
in
the
first
step,
whereas,
the
combination
of
BAP
(1.0
mg/l)
+
KIN
(0.5
mg/l)
+
GA3
(1.0
mg/l)
produced
418.7
shoots/explant
after
5th
subculture
in
the
second
step.
The
in
vitro
regeneration
protocol
described
here
is
different
from
that
of
G.
sylvestre
reported
earlier.
Thus
the
cul-
ture
of
shoot
clumps
in
the
presence
of
GA3containing
medium
increased
the
number
of
multiple
shoots
in
Gymnema.
The
present
study
strongly
suggested
that
the
superiority
of
BAP
and
KIN
in
combination
with
GA3was
found
to
be
necessary
for
rapid
multiple
shoot
bud
induction
as
well
as
enhanced
rate
of
shoot
prolifera-
tion
in
Gymnema.
Half-strength
MS
medium
supplemented
with
2.0
mg/l
IBA
was
found
to
be
best
for
maximum
rooting
response
and
regenerated
plantlets
were
successfully
established
in
the
field
condition
with
85%
survival
rate.
To
the
best
of
our
knowledge,
this
in
the
first
report
on
production
of
large
scale
in
vitro
plantlets
from
this
important
medicinal
plant.
The
protocol
described
herein
can
be
applied
for
commercial
scale
plant
production
as
well
as
conservation
of
this
endangered
medicinal
plant
from
the
possible
extinction
in
the
near
future.
Acknowledgement
Dr.
P.
Venkatachalam
gratefully
acknowledges
University
Grants
Commission
(UGC),
New
Delhi
for
financial
assistance
pro-
vided
under
UGC
Major
Project
No.
37-297/2009
(SR).
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