World Journal of Agricultural Sciences 5 (5): 651-661, 2009

ISSN 1817-3047
IDOSI Publications, 2009

Vegetative Propagation and Tissue Culture Regeneration of Hibiscus sabdariffa L. (Roselle)

J. Govinden-Soulange, N. Boodia, C. Dussooa, R. Gunowa, S. Deensah, S. Facknath and B. Rajkomar
Faculty of Agriculture, University of Mauritius, Rduit, Mauritius
Abstract: Hibiscus sabdariffa L. (Roselle) has gained popularity as an ornamental, medicinal, industrial and
food plant. These industries rely on the fast and guaranteed supply of clones to be cost-effective. Two vegetative
propagation methods for H. sabdariffa L. are proposed as a technique of ensuring maximum genetic stability.
Softwood and semi hardwood cuttings from two-month-old plants were rooted on a medium containing soil,
compost and rocksand after dipping in IBA (indole-3-yl-butyric acid) or NAA (a-naphthalene acetic acid) at (01.0g/l). Rooting was significantly (P< 0.05) affected by the type of cutting and the concentration of auxin used.
Softwood cuttings responded more positively to auxin treatment and lower levels (0.5g/l) of auxin stimulated leaf
and root formation. Rooting seemed to be more effective in cuttings treated with auxins than untreated cuttings.
Regeneration by tissue culture proved to be more successful by using nodal explants. Multiple shoots were
initiated on Murashige and Skoog 1962 (MS 1962) medium supplemented with various levels (0-2.0mg/l) of 6benzyl amino purine (BAP) and kinetin (KIN). Individual shoots with a minimum of two nodes were excised and
rooted on MS (1962) medium containing 1.5-2.5mg/l. Regenerants were acclimatized on a mixture (1:1) of
sterile peat and soil. They showed vigorous shoot growth (within 3 weeks) and after 5-6 months were suitable for
field planting.
Key words: Hibiscus sabdariffa L. roselle stem cutting tissue culture cytokinins auxins
include micropropagation,
micropropagation.
These
whereby new plants are
include direct methods such
INTRODUCTION
produced under aseptic
as axillary bud proliferation
and direct organogenesis
H. sabdariffa L. (Roselle), an annual shrub, is native ofconditions. Consequently,
and indirect techniques
Africa and is cultivated in many tropical and subtropical micropropagation by plant
culture
offers
involving an intermediate
regions of the world for stem fibers, paper pulp or edible tissue
possibilities.
callus
phase.
Direct
calyces, leaves and seeds. In some countries its flowers are promising
Moreover,
in
recent
years,
methods
of
plant
also used for decorative purposes [1]. The medicinal
the
application
of
plant
regeneration
usually
ensure
attributes of H. sabdariffa L. have also been reported [2-5].
culture
as
a
genetic stability whereas
In Mauritius, this plant is mainly recognized as being an tissue
micropropagation
technique
when plant tissues are
invasive weed and only a few people use the flower calyces
has
become
an
important
cultured via callus phase,
to make jams and pickles. With the drop in sugar prices in
biotechnological
tool
in
the
the
plants
that
are
Mauritius, Roselle is now regarded as a new crop with
multiplication
of
various
regenerated
may
exhibit
promising potential for intensive cropping systems owing to
variation [8]. Up to now,
its multifunctional attributes. Although most ornamentalplants that have a great
economic
importance.
research has focused mainly
Hibiscus species are vegetatively propagated, Roselle is
Likewise,
micropropagation
on the propagation of other
currently propagated by seeds [2]. Vegetative propagation
techniques
offer
additional
ornamental
Hibiscus
methods offer many benefits including ability to regenerate
advantages
such
as
the
species
such
as
H.
syriacus,
clones, convenience and ease of propagation, combination
[9-10] and H. rosa-sinensis
of genotypes and reduction of length of juvenile period [6]. rapid propagation rate, lack
of
seasonal
restrictions,
[11]. The only report on the
In addition, cuttings remain the most important means of
provision
of
disease
free
propagation
of
H.
propagating horticultural and ornamental crop species
plants,
maintenance
of
selfsabdariffa
L.
crop
relates
to
especially ornamental shrubs. If Roselle is to be proposed as
incompatible inbred lines
the effect of temperature on
an easy growing, alternative crop to farmers, it is crucial to
[7], international exchange
seed germination [12].
devise a method of rapidly supplying clean and genetically
of plant materials, culture
Tissue culture studies on H.
homogenous planting material to them. Traditional methods
systems
for
genetic
sabdariffa,
L.
have
of vegetative propagation also
transformation.
Different
involved
anthocyanin
pathways of regeneration
production in
can
be
adopted
in

Soulange, Faculty
Corresponding Author: Dr. J. Govinden- of Agriculture,

University of
Mauritius
Mauritius, Rduit,

651

World J. Agric. Sci., 5 (5): 651-661, 2009

propagaton
callus of Roselle
cultures namely
[13] and stem
genetic cuttings and
transfor micropropa
mation gation by
of
the direct and
indirect
crop
organogene
[14].
The use sis through
of shoot the use of
apices to nodal
micropro explants.
pagate
M
H.
A
sabdarif
T
fa
has
E
been
R
describe
I
d [15].
A
Convers
L
ely,
S
tissue
A
culture
N
studies
D
on other
Hibiscus
M
species
E
have
T
been
H
widely
O
reported
D
[16-22].
S
Incident
ally, all Stem
these
cuttings:
authors Cuttings
describe were taken
organog from twoenesis month-old
from the H.
sabdariffa
same
type of L. (Roselle)
explant plants cv.
namely Local
from
the
shoot
apices. farm of the
In this University
work we of
suggest Mauritius
early in the
two
methods morning.
Healthy
of
vegetativ branches
were
e
randomly

selected for
the excision
of cuttings
from
the
field.
Branches
were
separated
into
1722cm long/
4mm
diameter
soft wood
and
1820cm long /
6mm
diameter
semi-hard
wood
cuttings
with 45
slanting cut.
All
shoot
tips
were
removed
and
all
cuttings
were made
up of one
newly
formed leaf
with three
nodal
segments.
All cuttings
were
dipped in
0.05%w/v
Dithane

M45
fungicide
solution
prior
to
auxin
treatment.
The rooting
medium
consisted of
50%
soil
(Low
Humic
Latosols),
33%
compost
and
17%
rocksand in
cylindrical
black
polyethylen

e potting washed in
bags of running tap
40 x 27 water
containing
cm
dimensi one drop of
Tween 20
ons.
10
Cutt for
minutes and
ings
were rinsed
were
three times
dipped in
sterile
for five distilled
minutes water. After
in 0.5- an
1.0g/l overnight
soak
in
IBA,
sterile
(BHD
distilled
Limited water, seeds
Poole, were
England) disinfected
and 0.5- in a mixture
0.1%
1.0g/l of
NAA, Benomyl
(Sigma, and 0.07%
Aldrich) Dithane
and were M45 for
planted 10 minutes
to
a followed by
depth of rinsing
2cm in three
times
in
moist
sterile
rooting
medium. distilled
Controls water. The
seeds were
were
dipped then dipped
into
95%
in
distilled ethanol for
water. 10 seconds
and treated
The
rooting with
medium 1% sodium
was kept hypochlorit
e with 2
moist
of
througho drops
Tween 20
ut the
15
experime for
minutes
nt.
followed by
In vitro thorough
regener rinsing in
ation: sterile
Establish
distilled
ment of
for
aseptic water
seedling five times.
The seeds
s:
Mature coats were
Roselle aseptically
seeds
removed
were
(manually,

under
laminar
flow, using
sterilized
scalpels).
Seeds were
germinated
on
Murashige
and Skoog
(MS 1962)
[23]
medium
containing
3% sucrose
and
solidified
with 0.8%
agar. The
pH of the
medium
was
adjusted to
5.8 before
autoclaving
at
120C
and
0.138MPa
for
20
minutes.

Direct (1962)
organog medium
enesis: supplement
0-0.5cm ed with 1.5
mg/l-2.5
nodal
of
segment mg/l
s excised (Sigma,
from in Aldrich).
The
vitro
germinat percentage
(%) number
ed
shoots
seedling of
that
s were
developed
subcultured roots was
recorded.
on MS
(1962)
Indirect
media
organogene
containi
sis:
Calli
ng
were
various
induced
concentr
using
the
ations
leaves and
(0.1
stems from
mg/l-2.0
in
vitro
mg/l) of
germinated
6-Benzyl
seedlings as
Amino
explants.
Purine
Explants of
(BAP)
0.5 0.5
(Sigma,
cm
were
Aldrich)
inoculated
and
on
MS
Kinetin
(1962)
(KIN) medium
(Sigma, supplement
Aldrich) ed
with
.
The Thidiazuron
number (TDZ)
or
of leaves 2,4that
dicholrophe
develope noxy acetic
d from acid (2, 4the
D)
at
shoots in 0.1mg/l to
the
2.0
mg/l
different and
0.01
BAP and mg/l to 0.05
kinetin mg/l
cultures respectively
was
.
One
recorded explant was
every
cultured per
week. jar
and
Shoots callus
were
production
rooted was
on MS recorded

as
a
percentage
of explants
response.
The effect
of (4.0-5.0
mg/l),
6Benzyl
aminopurine
(BAP) (0.52.0
mg/l) and
TDZ (0.l1.5 mg/l)
was
compared
for shoot
regeneratio
n from the
calli.
Acclimatiza
tion
of
the
regenerate
d
plantlets:
Regenerate
d plantlets
were
acclimatize
d on a
mixture
(1:1)
of
sterile peat
and soil in
black
polythene
plastic
pots.
Culture
conditions
and
Media: All
in
vitro
work was
performed
on
MS
(1962)
medium
containing
3% sucrose
and
solidified

with
mental
0.8%
design and
agar.
measureme
The pH nts:
Ten
of the replicates
medium were used
was
for
each
adjusted treatment in
to 5.8
the cutting
before experiment.
autoclav Measureme
ing at
nts
were
120C recorded
for 20 over period
minutes.
of 4 weeks
The
and
each
cultures
treatment
were
incubate was
d in a replicated
culture five times
room at by
a
using
a
temperat completely
ure
of randomized
0
252 C design. At
and
a two weeks
photoper intervals, 3
iod of 16 out of the 5
hours. cuttings per
The light treatment
intensity were up
that was rooted
provided carefully, to
to
the record the
cultures fresh
was
weight,
2000
cutting
Lux and
diameter,
the
relative root
humidity number,
number of
was
23.5%. branches,
stem
and
dry
Experi root
652

weight.
Collected
data
was
treated by
ANOVA,
variations
and
interaction
effects were
compared
using
the
Dunnetts
test at the
5% level of
significance
using
MINITAB
13.1
and
Microsoft
Excel 2007
software.
For the
in
vitro
experiment,
all
treatments
were
replicated
five times
using
a
completely
randomized
design. The
number of
leaves that
initiated
from each
shoot was
noted. The
percentage
number of
shoots that

World J. Agric. Sci., 5 (5): 651-661, 2009

evaluated
the
develope using
d rootsMinitab
during 13.1
statistical
the
experime software at
nt wasP=0.05.
Least
also
recorded. significant
Collecte differences
d
data(LSD) were
computed at
were
analyzed the 5% level
by one-of
significance
way
ANOVA to compare
the
and
deviation treatment
s amongmeans.
means
were
20

Fig. 1: Number
0of leaves on
softwood
cuttings.
Vertical bars
indicatestanda
rd error (SE)
of means (n=3)

10
8

16

6
4

14
20

Stem
cuttings
Number of
leaves: The
number of
leaves
increased
significantly
with time in
both
softwood
and semihardwood
cuttings on
the different
treatments.
However, a

12

18

RESULTS

N
A
A
0.
5
g/
l

12

Week 2
10
18
8
16

N
A
A
1.
0
g/
Week
l 4

6
4

14

er
Fig
of
. 2:
lea
Nu
ve
mb
65

s hardwoo
on d
se cuttings.
mi Vertical
3

b
ar
s
in

dteSE
i of
cmeans
a(n=3)

World J. Agric. Sci., 5 (5): 651-661, 2009

6
3

5
2.5

4
2

3
1.5

2
1
1
0.5

Week 0

Week 0
Time
(Weeks)

Time
(Weeks)

CONTROL
NAA 0.5 g/l

Fig.

w
3:oo
Chan d
ge incu
lengt tti
h ofng
softw s.
ood Ini
and tia
semi- l
hard le
t
gre h
ater e
(P<
0.0 h
5) i
incrg
eas h
e ine
leaf s
nu t
mb
er n
wasu
not m
ed b
in e
the r
cas
e ofo
0.5 f
g/l
IB l
A e
for a
soft v
wo e
od s
cutt
ing a
s f
wh t
ere e
as r
the
con 4
trol
trea w
tme e
nt e
gav k
e s

IBA 0.5 g/l

NAA 1.0 g/l

ng mi-hardwood
th cuttings
were
of 19.572.02cm
so and
ft 19.421.96cm
w respectively.
oo LSD at 5% level
d of significance
an to compare any
d two treatment
se means = 3.02
g. 1 and 2). inc
l)
i
rea
of
nLength ofses
PG
cuttings: on
Rs
tData
0.5
wer
hanalysis g/l
e
eshowed
IB
use
that change
A
d.
in length of
c
(Fi
cuttings in
Di
a
g.
both
s
am
3).
softwood
e
ete
and semiFor
bot
r
hardwood
o
h
of
cuttings
f
typ
was
cut
significantl es
tin
sy (P<0.05)of
gs:
einfluenced cutt
All
m
by
theing
ilevel ands,
the
-type
tre
ofthe
hplant
ate
ove
agrowth
rall
d
rregulators cha
cut
d(PGRs)
nge
tin
w
used. Asin
gs
osuch,
len
sho
osoftwood
gth
dcuttings
we
wa
showed a
d a
s
cgeneral
sig
sm
uincrease in
nifi
alle
tlength for
st
can
tall
wh
t
itreatments,
en
(P
nwith
hig
<0.
gsignificantly
her
sin the case
05)
lev increase in
much greater
inc
els
(with
rea
Fsignificantl (1.
se
larger0g/
iy

IBA 1.0 g/l

in
dia
me
ter

w
i
t
h
t
i
m
e
i
r
r
e
s
p
e
c
t
i
v
e
o
f
t
h
e
t
r
e
a
t
m
e
n
t
s
u
s
e
d
(
F
i
g
.
4
)
.

C
<0.05)
con
haffected bycen
athe
trat
ntreatment ion
gemployed. of
eConversely aux
the
in
iincrease inuse
ndiameter d
was
(P<
dsignificantl 0.0
iy
much5) .
alower
inTh
m
the case ofe
ethe controlinte
ttreatments ract
efor
bothion
rsoftwood eff
and semiect
ohardwood bet
fcuttings. we
0.5g/l IBAen
cand/or
the
uNAA gavetyp
tsignificantl e of
ty (P<0.05)cutt
ihigher
ing
nincrease inand
gdiameter trea
sthan
thetme
higher
nt
w
levels ofon
aauxins
roo
s(1.0g/l), ast
depicted innu
aFig. 4.
mb
l
er
s
wa
oRoot
s
number: als
sBoth
o
isoftwood sig
gand seminifi
n-hardwood can
icuttings t
fproduced (P<
iroots in all0.0
ctreatments 5).
5aFor
a(Fig.
5b).soft
nand
tThe
wo
lnumber ofod
yroots wascutt
significantl ing,
(y affectedthe
thehig
Pby

hes
t
inc
rea
se
in
roo
t
nu
mb
er
wa
s
obt
ain
ed
wit
h
1.0
g/l
IB
A
and
the
low
er
con
cen
trat
ion
of
IB
A
(0.
5g/
l)
gav
e a
sup
eri
or
nu
mb
er
of
roo
ts
in
se
mi
har
dw
ood
cutt
ing
s.

Thee
hig r
hes
t o
nu f
mb
6

rat the endand

oof
the20
oexperiment for
twas 25 forse
ssoftwood micuttings har
54

dw
ood
cutt
ing
s.

World J. Agric. Sci., 5 (5): 651-661, 2009

3.5

2.5

2.5

1.5
1.5

1
1

0.5
0.5

0
Week 0

Week 2
Time (Weeks)

CONTROL

IBA 0.5g/l

NAA 0.5g/l

NAA 1.0g/l

Week 4

0
Week 0

Week 2
Time (Weeks)

CONTROL

IBA 0.5 g/l

NAA 0.5 g/l

NAA 1.0 g/l

Fig.

0
4:
Ch
ang
e in
dia
met
er
of
soft
wo
od
and
se
mihar
dw
ood
cutt
ing
s. Time (Weeks)
Init
ial
dia
met
er
of
soft
wo
od
and
se
mihar
dw
ood
cutt
ing
s
wer
e
3.9
0
0.0
5
m
m
and
6.6
7
0.1

IBA 1.0 g/l

control

Fig. 5a: Effect of IBA and NAA on

means = 7.81
655

IBA 0.5 g/L

IBA 1.0 g/l

the root number of softwood

NAA 0.5 g/l

NAA 1.0 g/L

cuttings. LSD to compar

World J. Agric. Sci., 5 (5): 651-661, 2009

25

20

15

10

2 4 Time
(Weeks)

o and NAA on the root

f number of semi-hardwood
I cuttings. LSD to compare
Bany two treatment means =
A7.80

0.
25
C

0.
20

0.
15
0.
10
0.
05

0.
00

Week
2
Week 4

Time
(Weeks)

Week 2
Week 4
Time (Weeks)
CONTROL

IBA 0.5 g/l

NAA 0.5 g/l

NAA 1.0 g/l

s
Dry o
matt
er
accu
mula
tion:
Auxi
n
treat
ment
was
seen
to
have
a
signif
icant
effect
(P<0.
05)
on
dry
matte
r
accu
mulat
ion
depen
ding
on
the
type
of
cuttin
g
used.
As
such,
dry
weig
ht of
cuttin
g
incre
ased
signif
icantl
y in
both

1 illustrates
the number
of
elongated
shoots
(expressed
as
a
percentage)
that were
initiated
from
axillary
buds of in
vitro
germinated
seedlings of
H.
sabdariffa
on
MS
(1962)
medium
supplement
ed
with
various
levels
of
BAP. It can
be
noted
that 81% of
shoots were
produced in
the
hormone
free

IBA 1.0 g/l

656

Table

World J. Agric. Sci., 5 (5): 651-661, 2009

shoots
1.0
1: Shoot 1.5
(Plate 1b).
The
regenerat 2.0
ion from MeansS.E
axillary followed by
buds
H.

of the
letter

same
in

sabdariff column
a
on not

a
are

MS+BA significantly
P

different, as

Conc. of indicated by
BAP (mg/l)the LSD test
0
0.1
0.5
1.0

Table

(P=0.05)

medium
while only
33%
of
1.5
shoots
2.0
Means resulted in
the 2.0mg/l
S.E
of
BAP
followed
respectivel
by
the y.
same
The
letter in hormone
a column also had an
effect on
are not
the
significa
initiation of
ntly
elongated
different, (normal)
as
and stunted
indicated shoots.
by
the Interveinal
chlorosis,
LSD test
which
is
(P=0.05)
yellowing
of
the
2: Shoot leaves
regenerat between
ion from the veins,
axillary was
buds of observed in
cultures
H.
that
sabdariff
developed
a
on
normal
MS+KI
shoots
N
(Plate 1a)
Conc. of as well as
KIN (mg/l)in cultures
that
0
developed
0.1
stunted
0.5

developme
nt of the
normal
shoots
started
with
the
formation
of a small
mass
of
callus
at
the
cut
ends
but
after
elongation
no callus
was found
at the base.
Table
2 indicates
the% and
the number
of shoots
that were
initiated on
medium
with
Kinetin.
MS
medium
supplement
ed with 0.1
mg/l
of
kinetin and
MS (1962)
medium
without
any
hormone
gave
a
higher
response in
the shoot
initiation.
The
effect
of
KIN was
such that it
contributed
largely to
the
regeneratio
n of the
shoots
(normal

and
ng
stunted) concentrati
rather on
than to
the
formati
on
of
roots.
Moreov
er, the
roots
that
were
initiated
from
the
cultures
were
not
profusel
y
develop
ed
(Plate
1b).
Mi
croshoot
s
that
attained
a height
of 2-3
cm with
at least
one
node
were
individu
ally
excised
from the
shoot
cluster
and
selected
for
rooting.
Rooting
was
seen to
be
significa
ntly
more
efficient
with
increasi

Table 3: % Root initiation on MS+ IBA

1.5

28.10.6c

2.0

59.61.4b

2.5

86.20.0a

MeansS.E
followed
the
letter

by

same
in

column

a
are

not
significantly
different,

as

indicated by
the LSD test
(P=0.05)

Plate

1a:
Sh
oo
t
for
m
ati
on
fro
m
H.
sa
bd
ar
iff
a
ex
pl

ants on
MS+KI
N after 8
weeks

fa
ex
pl
an
ts
on
M
S+
KI
N
aft
er
ei
gh
t
w
ee
ks

of
IBA;
hence
the
Plate 1b: Shoot &
highest
Root
rooting
formatio
percentage
n from was
H.
obtained at
sabdarif 2.5mg/l IBA
(Table 3).
657

The
regenerated
plantlets
were
successfully
acclimatize
d in oven
-sterilized
soil under
100%
humidity
after
two
weeks
(Plate 2.).
Callus
initiation:
Callus
formation
was
observed on
both TDZ
and 2,4-D
on
leaf
explants of
H.
sabdariffa
(Plate 3).

World J. Agric. Sci., 5 (5): 651-661, 2009

Table 4: %
Callus
formation
on leaf
explants of
H.
sabdariffa
using

Plate

3:
Ca
llu
TDZ and 2,4-D
s
Conc.of
ini
TDZ mg/l
tia
0.1
tio
0.5
n
1.0
1.5
fro
2.0
m
lea
f
ex
pla
nts
of
H.
sa
bd
ar
iff
a
cul
tur
ed
on
T
D
2:
H.
Z
sabdarif
fa
plantlet
on
a
mixture
(1:1)
peat and
soil

Plate

However,
1.5 mg/l
TDZ
showed a
higher
response in
callus
formation
(80%)
(Table 4);
nevertheles
s the lowest
callus
formation
was
observed
with
0.1
mg/l
of

TDZ. On
the other
hand, all
levels
of
2,4-D
(0.010.05mg/l)
produced
equally
high
percentage
of callus
(80%).
Conversely
, these calli
could not
be
regenerate
d further as

only
d after four
it
profuse weeks,
seems
that
chloroph
softwood
yll
formatio cuttings
are
more
n
responsive
(organog
to
auxin
enic
treatment
calli)
than semi
was
hardwood
observed cuttings of
after two H.
weeks sabdariffa.
culture Several
factors can
on
the
different affect
levels of number of
leaves
KIN
produced
(0.15.0mg/l) including
the type of
.
cuttings
used;
the
DISCUSSI
plant
growth
ON
regulators
utilised,
Stem
cuttings temperature,
:
This dry matter
content of
study
demonst the cuttings
rates that before
sticking in
H.
sabdarif the medium
the
fa can be and
propagat health status
of the plant
ed
It
through [24].
the use seems
of both however
softwoo that
d
and compared to
other
semi
hardwoo hibiscus
species, H.
d
cuttings sabdariffa
especiall L. produced
y
by new leaves
faster (than
using
some other
auxin
treatmen Hibiscus
species such
t.
However as H. rosa
, based sinensis (27
on the days) [25].
our
number In
of leaves experiment,
14
produce after

days,
Roselle
already had
3
fully
expanded
leaves
in
semihardwood
cuttings and
4 in soft
wood
cuttings.
The
leaves
produced in
the
experiment
varied with
the
treatment
given and
the type of
the cuttings
used. Fig 1
and Fig 2
also
indicate that
the amount
of
dry
matter
originally
present in
the
semi
hardwood
cuttings had
a
more
significant
effect than
that of the
auxin
treatment as
the
semi
hardwood
cuttings had
an
initial
weight of
1.5120.36
6g and the
softwood
cuttings
were lighter
(0.6770.13
3g) . The
number of
leaves
produced is
also

determin d only 14
ed
by leaves. For
the
the case of
initial semi
amount hardwood
of dry cuttings, the
matter in highest
number of
the
cuttings leaves was
[11, 26]. produced in
the control.
The
number Softwood
of leaves cuttings
in
the seemed to
cuttings respond
is very more
importan positively to
t
for auxin
successf treatment
ul post than semi
propagat hardwood
ion. As with respect
to
the
such,
number
of
softwoo
leaves.
d
Similar
cuttings
both
produce ly,
d a total softwood
of
16 and semileaves hardwood
cumulati cuttings
vely for witnessed
the
4 the highest
weeks increase in
period length and
diameter
with
0.5g/l with lower
levels
of
IBA
whereas auxin
(0.5g/l IBA
the
&
0.5g/l
semiNAA).
The
hardwoo
increase
in
d
length
of
cuttings
produce cuttings and
658

increase in
diameter
seemed to
be due to
the use dry
matter for
the
shoot
growth. The
smallest
increase in
diameter
was
observed
with 1.0g/l
NAA
in
both types
of cuttings
probably
due to the
diversion of
the cuttings
food
reserves
towards root
formation
[27]. Auxin
treatment
had
a
significantly
positive
effect
on
root number
of
H.
sabdariffa
cuttings as
the
untreated
cuttings
produced a
lower
number of
roots in both
species after
four weeks.

World J. Agric. Sci., 5 (5): 651-661, 2009

reported [6,
28-29] and
The
treatmen also seem to
t giving be species
dependent
the
As
highest [28].
such,
dry
matter although
same
accumul the
ation in auxin (IBA)
the roots produced
was IBA similar
1.0g/l number of
roots
in
for
both
types
softwoo
d
and of cuttings,
it seemed
semi
hardwoo that,
softwood
d
cuttings. cuttings
Roo required a
higher
t
producti concentratio
on in H. n of IBA for
sabdarif maximum
fa was root
production
faster
.
(within (1.0g/l)
This could
two
weeks) be due to
than in dry matter
H. rosa- content
sinensis which was
higher
in
L.
semi
cultivar
Paramar hardwood
cuttings
ibo
which batch
took 21 initially and
days to at the end
the
produce of
the first experiment.
visible The effect
roots
of
initial
[11]. The carbohydrat
effect of e
content
various (dry matter)
coon rooting
factors
ability has
includin
previously
g
endogen been
described
ous
auxins [30-31].
on
vitro
rooting In
regeneratio
has been
formerly n: In vitro

regeneratio
n from H.
sabdariffa
seemed to
be
more
successful
by axillary
bud
regeneratio
n than by
indirect
organogene
sis. As such
more
efficient
shoot
regeneration
from
axillary
buds of H.
sabdariffa
was
obtained on
MS media
with
low
levels of
cytokinins
than
on
media
supplement
ed
with
higher
concentratio
ns of BAP
or KIN. The
optimum
concentratio
n
of
cytokinin
for
successful
shoot
regeneratio
n
was
between 00.1mg/l for
both growth
regulators.
These
findings are
in
accordance
with those
of Samanthi
et al. [20],
on
tissue
culture of
H.

cannabi regulators
nus
were
whereby observed. In
the
the present
optimum study,
we
concentr also found
ation of that kinetin
BAP
was more
was
responsive
1.98mg/l than BAP in
for shoot inducing
regenera shoot
tion
formation in
from
contrast to
nodal
the findings
explants of
with
Christensen
negative et al.
results [33] who
on using obtained
higher efficient
cytokini shoot
n
regeneratio
concentr n from
ations. nodal
Similar cuttings of
type of H. rosa
response sinensis L.
was
on 0.5mg/l
publishe BAP. The
d
by incidence of
Jorge et abnormal
al. [32] shoots and
who also interveinal
reported chlorosis
that
during
cytokini tissue
n
is culture of
directly hibiscus
responsi species has
ble for formerly
reprogra been
mming reported by
apical Samanthi et
meriste al. [20] on
m axes H.
of cotton cannabinus
towards and on
the
H.
rosa
multiplic sinensis by
ation of Christensen
buds and et al. [33].
also
Our study
fewer
shows that
shoots single node
with
explants
higher can also be
dose ofused for the
growth in
vitro

regeneratio
n of Roselle
even with
very
low
levels
or
even in the
absence of
cytokinins.
Moreover,
our results
differ from
the report of
GomezLeyva et al.
[15], who
used shoot
apices with
cytokinins
to
induce
sprouting in
that
we
have made
use
of
different
explants.
We
infer
that single
nodes
probably do
not suffer
from
the
apical
dominance
effect
exerted by
the apical
meristem
and

hence observed
also
on
were
able to shoots
sprout cultured in
without both shoot
or with initiation
very low and shoot
levels of growth
growth media
regulator (MS+Cytok
inin). The
s.
Likewise positive
effect
of
,
on
Gomez- IBA
Leyva et rooting of
vitro
al. [15] in
regenerated
highlight
ed
the shoots has
significa been widely
nt role of reported
BAP and [34-36].
Indirec
mt
topolin
organogene
in
from
reprogra sis
leaf
mming
the cells explants of
in shoot H.
apices of sabdariffa
Roselle proved to
more
towards be
sproutin intricate in
that
g.
In although
callus
vitro
rooting formation
was
of
initiated observed on
shoots both TDZ
and 2,4-D
was
efficientl supplement
ed medium,
y
obtained these calli
after a could not
period of be
8 weeks regenerated.
either on Our results
confirm the
MS
medium significant
of
containi effect
genotype
in
ng
regeneratio
2.5mg/l
n by tissue
IBA.
However culture
methods
,
simultan [37-40].
Similarly
eous
rooting other
Hibiscus
was

species
namely H.
syriacus
could
be
efficiently
regenerated
from nodal
explant on
MS media
supplement
ed
with
TDZ [41].
Likewise,
Jenderek et
al.
[19]
have
succeeded
in
regenerating
in
vitro
derived
callus from
H. syriacus
seedling
fragments
on
MS
medium
supplement
ed
with
BAP
or
KIN.
CONC
LUSIO
NS
This
study
has
shown
for the
first
time
that H.
sabdariffa
can
be
propagated
by
vegetative
methods
both in vitro
and ex vitro.
Although
auxins were
not a sine
qua
non
condition
for effective

rooting efficient
of
method for
softwoo in
vitro
d
or regeneratio
semi
n of H.
hardwoo sabdariffa.
d
In addition,
cuttings, by making
the use use
of
of
single node
exogeno explants,
us
the number
auxins of
accelerat micropropa
ed and gules used
ensured in
one
uniformi tissue
ty of the culture
rooting multiplicati
process. on
cycle
The
offer
the
present advantage
findings of
being
indicate more
also the abundant
possibili than
ty
of through the
using
use of shoot
micropro apices. The
pagation fact that no
methods or
low
for the levels
of
in vitro cytokinins
regenera induced
tion of higher
H.
sprouting
sabdarif efficiency
fa. The also reduces
use
of the risks of
direct
somaclonal
organog variation as
enesis as a result of
suggeste the use of
d in this
plant
work
growth
proved
to be a regulators
in
tissue
quick
culture.
and
659

REFER
ENCES

1.

Sanche
zMendo
za, J.,
A.
Domin
guezLopez,
S.
Navarr
oGalind
o and
J.A.
LopezSandov
al,
2007.
Some
physica
l
propert
ies of
Roselle
(Hibisc
us
sabdar
iffa L.)
seeds
as
a
functio
n
of
moistur
e
content
.
Journal
of
Food
Engine
ering,
87:
391397.

World J. Agric. Sci., 5 (5): 651-661, 2009

2.

3.

Mor
ton,
J.,
198
7.
Ros
elle,
Hibi
scus
sab
dari
ffa
L.
In:
Frui
ts of
War
m
Cli
mat
es, 4.
Ed.,
Mor
ton,
J.F.
Crea
tive
Res
ourc
e
Syst
ems,
Inc.,
pp:
281286.
Aki
nda
huns
i,
A.A
.
and
M.T
.
Olal
eye,
200
3.
Toxi
colo
gica
l
inve
stiga

tion of
aqueou
s
methan
olic
extract
of
calyces
of
Hibisc
us
sabdar
iffa L.
Journal
of
Ethnop
harmac
ology,
89:
161164.
Herrera
Arellan
o, A.,
S.
FloresRomer
o,
M.A.
Chavez
-Soto,
J.
Tortori
ello,
2004.
Effecti
veness
and
tolerabi
lity of
standar
dized
extract
from
Hibisc
us
sabdar
iffa in
patient
s with
mild to
modera
te
hyperte
nsion:
a

5.

control
led and
random
ized
clinical
trial.
Phyto
medici
ne, 11:
375382.
Hirunp
anich,
V., A.
Utaipat
, N.P.
Morale
s, N.
Bunya
praphat
sara, H.
Sato,
A.
Heruns
ale and
C.H.
Suthisi
sang,
2006.
Hypoc
holeste
romic
and
antioxi
dant
effects
of
aqueou
s
extracts
from
the
dried
calyx
of
Hibisc
us
sabdar
iffa L.
in
hyperc
holeste
romic
rats. J.
of
Ethnop

har
mac
olog
y,

103:

ntific
Eds.,
Guilfor
d.

8.

252-260.

6.

7.

Hart
man
,
B.H.
,
D.E.
Kest
er
and
F.T.
Dav
ies,
199
4.
Plan
t
Prop
agat
ion: 9.
Prin
cipl
es
and
Tec
hniq
ues.
Pren
tice
Hall
,
New
Jers
ey.
Coll
in,
H.A 10.
.
and
S.
Edw
ards
,
199
8.
Plan
t
Cell
Cult
ure.
Bios
Scie

Lindse
y, K.
and
M.G.K.
Jones,
1989.
Plant
Biotec
hnolog
y
in
Agricul
ture.
Open
Univer
sity
Press
Ed.
Milton
Keynes
, UK.
Grange
, R.I.
and K.
Loach,
1985.
The
effect
of light
on
rooting
of leafy
cutting
s. Sci.
Hort.,
27:
105111.
Jendere
k,
M.M.
and
A.J.
Olney,
1999.
High
regener
ation
ability
of
benzyl
adenin
e and
napthal

ene
acetic
acid
induce
d callus
of
eastern
Hibisc
us.
Hortsci
ence,
34 (3):
458.

11. Bertra
m Lise,
1991.
Vegetat
ive
propag
ation of
Hibisc
us
rosaSinensi
s L. in
relation
to
nutrien
t
concent
ration
of
propag
ation
mediu
m. Sci.
Hort.,

48: 13
1139
.

12. Manso
ur,
B.M.M
., 1974.
Effect
of
temper
ature
and
day
length
on
growth
and
floweri
ng of

rose
lle
Hibi
scus
sab
dari
ffa
L.
Sci.
Hort
., 3:
129135.

13. Baja

14.

j,
Y.S.
,
199
9.
Biot 15.
echn
olog
y in
Agri
cult
ure
and
Fore
stry
24,
Med
icin
al
and
Aro
mati
c
Plan
ts.
V.
Spri
nger
Verl
ag,
Berl
in.
Gas
sam
aDia,
Y.K.
, D.
San
,
M.
Ndo

ye,
2004.
Direct
genetic
transfo
rmatio
n
of
Hibisc
us
sabdar
iffa L.
African
Journal
of
Biotec
hnolog
y,
3:
226228.
Gomez
-Leyva,
J.F.,
L.A.
Martin
ezAcosta,
I.G.
LopezMurair
a, H.
SilosEspino,
F.
Ramire
zCervan
tes and
I.
Andrad
eGonzal
ez,
2008.
Multipl
e shoot
regener
ation of
Roselle
(Hibisc
us
sabdar
iffa L.)
from a
shoot
apex
culture

system.
Internat
ional
Journal
of
Botany,
4: 326330.

16. Zap

ata, 17.
C.,
M.
M.
Sriv
atan
akul
,
S.H.
Park
,
B.M
.
Lee,
M.G
.
Sala
s
and
R.H.
Smit
h,
199
9.
Imp
rove
men
ts in
shoo
t
18.
apex
rege
nera
tion
of
two
fiber
crop
s:
Cott
on
and
Ken
af.
Plan
t
Cell
Tiss
ue
Org
an
Cult
ure,

56:
1851
9

1.
McLea
n, K.S.,
G.W.
Lawren
ce and
N.A.
Reiche
rt,
1992.
Callus
inducti
on and
adventi
tious
organo
genesis
of
kenaf
(Hibisc
us
cannab
inus
L.).
Plant
Cell
Report
s, 11:
532534.
Srivata
nakul,
M.S.,
S.
Park, J.
Sander
s, M.
Salas
and R.
Smith,
2000.
Multipl
e bud
regener
ation of
kenaf.
(Hibisc
us
cannab
inus
L.)
from
bud
apex
culture
system.
Plant
Cell.

19.

20.

Rep.,
19:
11651170.
Jendere
k, M.,
R.
Olney
and
C.A.
Fresno,
2001.
Hibisc
us
syriacu
s plant
regener
ation
from
callus.
Internat
ional
Pl.
Propag
ators
Proc.,
50:
565568.
Samant
hi,
P.H., S.
Takayu
ki and
K.
Hattori,
2004.
Multipl
e shoot
regener
ation
from
young
shoots
of
Kenaf
(Hibisc
us
cannab
inus),
Plant
Cell,
Tissue
and
Organ
Culture
,
77:
49-53.

21. Ada

22.

mso
n,
W.C
.
and
J.E.
OB
ryan
,
198
1.
Plan
t
diffe
renti
atio
n of
Hibi
scus
hier
anu
s
fro
m 23.
call
us
gro
wn
fro
m
emb
ryo
expl
ants.
Envi
ron
men
tal
and
Exp
erim
enta
l
Bota
ny,
21:
434435. 24.
Sak
han
okh
o,
H.
and
C.
Pou
nder

s,
2006.
Evaluat
ion of
growth
regulat
ors on
in vitro
Hibisc
us
shoots
regener
ation.
Southe
rn
Nurser
y
Associ
ation
Procee
dings,
51:
346349.
Murash
inge, T.
and F.
Skoog,
1962.
A
revised
mediu
m for
rapid
growth
and
bioassa
ys with
tobacc
o tissue
culture
s.
Physiol
. Plant.,
15:
473497.
Richar
ds, M.
(1985).
Prepar
ation
of
mother
plants
for
both
cutting

25.

26.

and
graftin
g.
Acta.
Hort.,
166:
41-44.
Bertra
m Lise,
1992.
The
relation
betwee
n
adventi
tious
root
formati
on and
the
post
propag
ation
growth
of
Hibisc
us rosa
-sinens
is
L.
Acta
Hort.,
314:
105111.
Sarang
a,
J.
and R.
Camer
on,
2006.
Advent
itious
root
formati
on in
Anacar
dium
occide
ntale
L.
in
respons
e
to
phytoh
ormone
s and
remova
l
of
roots.

Sci
Hort
.,

111:

28.

164-172.
27. Oko
ro,
O.O
.
and
J.
Gra
ce,
197
5.
The
phy
siol
ogy
of
rooti
ng
Pop
ulus
cutti
ngs:
Car
boh
ydra
tes
and
phot
osy 29.
nthe
sis.
Phy
siol.
Plan
660

t., 36:
133138.
Carpen
ter,
W.J.
and
J.A.
Cornell
, 1992.
Auxin
applica
tion
duratio
n and
concen
tration
govern
rooting
of
hibiscu
s stem
cutting
s.
J.
Americ
an
Society
for
Hort.
Sc.,
117:
68-74.
Day,
J.S.
and
B.R.
Loveys

, 1998.
Propag
ation
from
cutting
s
of
two
woody
orname
ntal
Austral
ian
shrubs,
Boroni
a
megast
igma,
Nees
(brown
boronia
) and
Hypoc
alymm
a
augusti
folium,
Endl.
(white
myrtle)
.
Austral
ian J.
Exper.
Agric.,
38:
201206.

World J. Agric. Sci., 5 (5): 651-661, 2009

30. Bul
daw
a, F.
and
K.
Ki
Sun,
199
4.
Effe
cts
of
irrig
atio
n
freq
uen
cy
on
root
for
mati
on
and
sho
ot
gro
wth
of
spra
y
gro
wth
chry
sant
hem
um
cutti
ngs
in
32.
sma
ll
jute
plug
s.
Sci.
Hort
.,
60:
125138.

31. Yon
g
Kw
eon,

Y. and
K. Ki
Sun,
1996.
Season
al
variatio
n
in
rooting
ability,
plant
hormo
nes,
carboh
ydrate,
nitroge
n,
starch
and
soluble
sugar
content
s
in
cutting
s
of
white
forsyth
ia
(Abelio
phyllu
m
distich
um,
Nakai).
J. Kor.
Soc.
Hort.
Sci.,
37:
554560.
Jorge,
L.M.,
H.R.
Permin
geat,
M.V.C.
Romag
noli,
M.
Heister
borg
and
H.V.
Ruben,
1998.

Multipl
e shoot
inducti
on and
plant
regener
ation
from
embryo
nic
axes of
cotton.
Plant
Cell
Tiss.
Org.
Cult.,
54:
131136.

33. Christe
nsen,
B., S.
Sriskan
darajah
,
M.
Serek
and R.
Muller,
2008.
In vitro
culture
of
Hibisc
us rosa
sinensi
s
L.:
Influen
ce of
iron,
calciu
m and
BAP
on
establis
hment
and
multipl
ication.
Plant
Cell,
Tissue
and
Organ
Culture

, 93:
151161.
34.
Aktar,
S.,
K.M.N.
Asiruddi
n and
H.H. Uq,
200
7.
In
vitr
o
root
for
mati
on
in
Den
dro
biu
m
orch
id
plan
tlets
with
IBA
. J.
Agri
c.
Rur
al
Dev.
,
5:
4851.

35. Hat
zila
zaro
u,
S.,
H.
Gra
mm
atik
os,
A.S.
Eco
nom
ou,
N.
Rifa
ki

and F.
Ralli,
2003.
Rootin
g
in
vitro
and
acclim
atizatio
n
of
Myrtus
commu
nis
microc
uttings.
Acta
Hot.,
616:
259264.

36. Rout,
G.R.,
S.
Samant
aray
and P.
Das,
1999.
Root
inducti
on in
micros
hoots
of
Simaro
uba
glauca
L. in
vitro:
Peroxi
dase as
a
marker
for
rooting
. Silvae
genetic
a., 48:
14-17.

37. Mathia
s, R.J.
and
E.S.
Simpso
n,
1986.
The
interact
ion of
genoty
pe and
culture
mediu
m on
the
tissue
culture
respons
es of
wheat
(Triticu
m
aestivu
m
L.
em.
Thell.)

call
us.
Plan
t
Cell
,
Tiss
ue
and
Org
an
Cult
ure,
7:
3137.

38. Wol
ford
,
D.S.
and
K.H
.
Que
senb
erry,
199
2.
Tiss
ue
cult
ure
rege
nera
tion
of 40.
Des
mod
ium.
Cro
p
Sci.,

32:
2662
6
8
.

39. Nie
der
wie
ser,
J.G.
and
J.
Van

Staden,
1990.
The
relatio
nship
betwee
n
genoty
pe,
tissue
age
and
endoge
nous
cytoki
nin
levels
on
adventi
tious
bud
formati
on on
leaves
of
Lache
nalia.
Plant
Cell,
Tissue
and
Organ
Cultur
e, 22:
223228.
Colom
ba,
E.L.,
K.
Grunb
erg, S.
Griffa,
L.
Ribitta,
L.
Mrogi
nski
and E.
Biderb
ost,
2006.
The
effect
of
genoty
pe and

culture
mediu
m on
somati
c
embry
ogenes
is and
plant
regener
ation
from
mature
embry
os of
fourtee
n
apomic
tic
cultiva
rs of
buffel
grass
(Cench
rus
ciliaris
L.).
Grass
and
Forage
Sci.,

61: 28.

41. West,
T. and
J.E.
Preece,
2004.
Effects
of
thidiaz
uron
and
nutrien
t salt
formul
ations
on
microp
ropagat
ion of
hardy
hibiscu
s
(Hibis
cus

mos
che
utos

L.).
Acta
Hort.,

630:
293297.

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