Plant regeneration from leaf explants of Rhodiola fastigiata


Liu, H..J.; Xu, Y.; Liu, Y.J.; Liu, C.Z.

In Vitro Cellular and Developmental Biology - Plant 42(4): 345-347

2006


An efficient plant regeneration protocol for rapidly propagating Rhodiola fastigiata (Hk. f. et Thoms.) S.H.FU, a traditional Chinese medicinal plant, was developed. Shoot organogenesis occurred from the leaf explants inoculated on medium with appropriate supplements of plant growth regulators. Up to 5.3 shoots formed per leaf explant cultured on a medium containing 13.32 mu M 6-benzylaminopurine (BA) and 0.54 mu M 1-naphthaleneacetic acid (NAA). Regenerated shoots formed complete plantlets on a medium containing 1.48 mu M indole-3-butyric acid (IBA), and mature plants were established, acclimatized, and thrived in greenhouse conditions. The regeneration protocol developed in this study provides a basis for germplasm conservation and for further investigation of medicinally active constituents of the elite Chinese medicinal plant.

In
Vitro
Cell.
Dev.
Biol.—Plant
42:345-347,
July—August
2006
DOI:
10.1079/IVP2006773
©
2006
Society
for
In
Vitro
Biology
1054-5476/06
$18.00+0.00
PLANT
REGENERATION
FROM
LEAF
EXPLANTS
OF
RHODIOLA
FASTIGIATA
HAI-JUN
LIU
1
'
2
,
YAN
XU
3
,
YU-JUN
LIU
1
,
AND
CHUN-ZHAO
LIU
2
*
'College
of
Biological
Sciences
and
Biotechnology,
Beijing
Forestry
University,
Beijing
100083,
P.
R.
China
2
Sate
Key
Laboratory
of
Biochemical
Engineering,
Institute
of
Process
Engineering,
Chinese
Academy
of
Sciences,
Beijing
100080,
P.
R.
China
3
College
of
Landscape
Architecture,
Beijing
Forestry
University,
Beijing
100083,
P.
R.
China
(Received
21
June
2005;
accepted
17
March
2006;
editor
B.
M.
Reed)
SUMMARY
An
efficient
plant
regeneration
protocol
for
rapidly
propagating
Rhodiola
fastigiata
(Hk.
f.
et
Thorns.)
S.H.FU,
a
traditional
Chinese
medicinal
plant,
was
developed.
Shoot
organogenesis
occurred
from
the
leaf
explants
inoculated
on
medium
with
appropriate
supplements
of
plant
growth
regulators.
Up
to
5.3
shoots
formed
per
leaf
explant
cultured
on
a
medium
containing
13.32
pM
6-benzylaminopurine
(BA)
and
0.54
pilf
1-naphthaleneacetic
acid
(NAA).
Regenerated
shoots
formed
complete
plantlets
on
a
medium
containing
1.48
pilf
indole-3-butyric
acid
(IBA),
and
mature
plants
were
established,
acclimatized,
and
thrived
in
greenhouse
conditions.
The
regeneration
protocol
developed
in
this
study
provides
a
basis
for
germplasm
conservation
and
for
further
investigation
of
medicinally
active
constituents
of
the
elite
Chinese
medicinal
plant.
Key
words:
Chinese
traditional
medicinal
plant;
leaf
explant;
Rhodiola
fastigiata;
shoot
organogenesis.
INTRODUCTION
Rhodiola
fastigiata
is
a
perennial
herbaceous
plant
in
the
Crassulaceae
family
and
mostly
grows
in
Tibet.
It
has
been
used
in
Tibetan
traditional
medicine
for
promoting
blood
circulation
and
relieving
coughs.
Recently,
several
medicinally
active
constituents
of
R.
fastigiata
have
been
identified
including:
salidroside,
tyrosol,
and
fastigitin
A
(Peng
et
al.,
1996;
Yang
et
al.,
2002).
Studies
have
shown
that
salidroside
has
such
medicinal
properties
as
anti-anoxia,
anti-microwave
radiation,
anti-fatigue,
and
extending
human
life
(Kurkin,
1986).
A
growing
commercial
demand
for
R.
fastigiata
has
caused
a
serious
reduction
in
native
populations
and
as
a
consequence
of
over-
harvest
and
deforestation,
the
natural
resource
of
R.
fastigiata
is
on
the
edge
of
extinction.
In
vitro-propagation
methods
offer
powerful
tools
for
the
mass-multiplication
and
germplasm
conservation
of
many
threatened
plant
species
(Liu
et
al.,
2004;
Murch
et
al.,
2004).
Regeneration
in
vitro
has
been
reported
for
a
growing
list
of
medicinal
and
aromatic
plants,
including
another
species
of
the
genus
Rhodiola
(Dimitrov
et
al.,
2003).
The
overall
objective
of
the
current
research
was
to
develop
an
efficient
protocol
for
in
vitro
multiplication
of
R.
fastigiata.
The
rapid
regeneration
system
established
here
provides
a
consistent
and
sterilized
tissue
resource
for
mass
micropropagation,
germplasm
conservation
as
well
as
further
evaluation
of
medicinally
active
constituents
of
this
important
Chinese
herb.
*Author
to
whom
correspondence
should
be
addressed:
Email
czliu@
home.ipe.ac.cn
MATERIALS
AND
METHODS
Wild
plants
of
R.
fastigiata
(Hk.
f.
et
Thoms.)
S.H.FU
were
harvested
from
Mountain
Segila
in
Linzhi
Prefecture
of
Tibet.
The
leaves
from
3-mo.-old
plants
were
surface
sterilized
by
dipping
in
70%
ethanol
for
30
s,
then
immersion
in
a
20%
aqueous
solution
of
5.4%
sodium
hypochlorite
in
water
for
20
min,
followed
by
three
rinses
with
sterile-
distilled
water.
The
aseptic
leaf
explants
(c.
0.5
X
0.5
cm
in
size)
were
cut
and
cultured
with
abaxial
sides
toward
the
MS
basal
medium
(Murashige
and
Skoog,
1962)
supplemented
with
BA
(4.44,
8.88,
13.32,
and
17.76
p,M)
alone
or
in
combination
with
NAA
(0.05,
0.54,
1.61,
and
2.69
pM).
After
1
mo.,
the
regenerated
shoots
were
transferred
onto
MS
medium
with
8.88
p,M
BA
and
0.54
p,M
NAA
for
shoot
elongation
and
proliferation.
Rooting
was
induced
on
half-strength
MS
solid
medium
supplemented
with
IBA
(0,
0.49,
1.48,
2.46,
or
4.92
pM).
Rooting
frequencies
were
recorded
after
1
mo.
of
rooting
culture.
After
2
mo.,
the
rooted
plantlets
were
removed
from
the
in
vitro
culture,
rinsed
in
water
to
remove
medium,
and
transferred
to
potting
soil
mixture
in
a
misting-bed
system
under
standard
greenhouse
conditions.
Survival
rates
of
transplanted
plantlets
were
investigated
after
1
mo.
All
cultures
were
incubated
in
a
controlled-environment
growth
room
with
a
16-h
photoperiod
under
cool
white
light
(40-60
p,mol
m
-2
s
-1
).
The
design
of
all
experiments
was
a
complete
randomized
block,
and
each
experiment
consisted
of
eight
explants
per
dish
and
10
replicate
culture
dishes
per
plant
growth
regulator
treatment.
All
the
experiments
were
repeated
twice,
and
the
data
were
analyzed
using
one-way
ANOVA
followed
by
Duncan's
multiple
range
test.
RESULTS
AND
DISCUSSIONS
Leaf
explants
of
wild
plants
of
R.
fastigiata
(Fig.
IA),
were
used
to
induce
shoot
regeneration
on
MS
medium
in
the
presence
and
absence
of
plant
growth
regulators.
Shoot
organogenesis
of
R.
fastigiata
was
not
induced
by
MS
medium
in
the
absence
of
plant
growth
regulator
or
with
BA
alone.
However,
application
of
BA
in
345
346
LIU
ET
AL.
.
I
"...Ps,
A
gr•
yes
*P
.
4,
%.•
:
r
i
4
4'
;""'.
'
41
,
k.
FIG.
1.
Plant
regeneration
from
leaf
explants
of
Rhodiolafastigiata.
A,
Wild
plant
of
R.
fastigiata
(bar
=
100
min).
B,
Callus
induction
from
leaf
explant
after
15
d
(bar
=
10
mm).
C,
Shoot
organogenesis
induced
by
the
combination
of
13.32
ILM
BA
and
0.54
ILM
NAA
from
leaf
explants
after
30
d
(bar
=
25
nun).
D,
Proliferation
and
elongation
of
regenerated
shoots
on
MS
medium
with
8.88
ILM
BA
and
0.54
ILM
NAA
(bar
=
15
nun).
E,
Rooting
of
regenerated
shoots
on
half-strength
MS
medium
supplemented
with
1.48
µM
IBA
(bar
=
25
nun).
F,
Mature
plant
grown
in
the
pot
after
1
mo.
of
transplantation
(scale
bar
=
25
nun).
combination
with
NAA
resulted
in
callusing
of
leaf
explants
on
day
15
(Fig.
1B),
and
subsequent
shoot
regeneration
after
30
d
of
culture
(Fig.
1C).
Significantly,
more
adventitious
shoots
were
observed
on
leaf
explants
exposed
to
13.32
pilf
BA
and
0.54
pilf
NAA
with
an
average
of
5.3
shoots
per
leaf
explant
and
86.1%
frequency
of
shoot
regeneration,
compared
to
those
exposed
to
other
treatments
(Table
1).
In
general,
the
stimulation
of
tissue
growth
to
form
adventitious
roots
and
shoots
depends
on
the
relative
ratios
of
auxin
to
cytokinin
in
the
culture
medium
(Skoog
and
Miller,
1957).
The
synergistic
effect
of
NAA
in
combination
with
BA
on
promotion
of
shoot
multiplication
was
also
observed
in
shoot
regeneration
of
another
Rhodiola
species
and
other
plant
species
(Kantia
et
al.,
2002;
Dimitrov
et
al.,
2003;
Prakash
et
al.,
2004).
A
combination
of
10-15
pM
6-BA
and
1-1.5
pM
NAA
was
also
proved
suitable
for
shoot
regeneration
from
immature
leaf
explants
of
Rhodiola
rosea
(Yin
et
al.,
2004).
Calluses
with
regenerated
shoots
were
subcultured
on
MS
medium
with
8.88
pM
BA
and
0.54
pM
NAA
for
shoot
proliferation
and
elongation
which
occurred
after
18
d,
as
shown
in
Fig.
1D.
The
individual
regenerated
shoots
(>4
cm
in
height)
were
excised
from
the
subcultured
callus
and
inoculated
into
rooting
media.
Rooting
occurred
on
half-strength
MS
solid
medium
in
the
absence
of
plant
growth
regulators,
however,
the
roots
were
relatively
slim
and
fragile.
In
contrast,
healthy
and
strong
roots
were
observed
on
A
A
Rooting
Survival
B
BC
C
B
A
B
D
100
80
co
co
'p
c
60
rn
0
0
0
a)
20
0
O
40
DEVELOPMENTAL
BIOLOGY/MORPHOGENESIS
347
TABLE
1
EFFECTS
OF
DIFFERENT
LEVELS
OF
COMBINATIONS
OF
NAA
AND
BA
ON
SHOOT
REGENERATION
FROM
LEAF
EXPLANTS
OF
R.
FASTIGIATA
AFTER
1
MO.
OF
CULTURE
NAA
(µM)
BA
(IA
Percentage
explants
producing
shoots
Number
of
shoots
per
explant
0 0
4.44
8.88
13.32
17.76
0
0
0
0
0
0
0
0
0
0
0.05
4.44
40.8
±
13.5
E
1.6
±
0.6
C
8.88
43.4
±
10.4
E
2.5
±
1.2
C
13.32
66.7
±
0.0
B
3.5
±
1.4
BC
17.76
52.6
±
19.4
C
3.2
±
0.4
C
0.54
4.44
27.1
±
7.2
EF
1.7
±
0.5
C
8.88
33.3
±
16.7
EF
3.0
±
1.4
C
13.32
86.1
±
13.9
A
5.3
±
5.5A
17.76
38.9
±
14.7
EF
4.2
±
2.8
B
1.61
4.44
30.6
±
19.5
EF
1.4
±
0.6
D
8.88
29.5
±
6.6
EF
1.6
±
0.3
CD
13.32
50.0
±
9.6
D
2.2
±
0.6
C
17.76
30.6
±
2.8
EF
1.6
±
0.4
C
Values
followed
by
the
same
letter
within
columns
are
not
significantly
different
at
P
<
0.05.
half-strength
MS
solid
medium
containing
IBA
(Fig.
1E).
As
shown
in
Fig.
2,
the
best
rooting
frequencies
of
82-83.3%
were
achieved
using
medium
with
0.49-1.48
µM
IBA.
Complete
plants
were
obtained
after
2
mo.,
and
66.6%
of
the
rooted
plantlets
survived
after
transplantation.
The
acclimatized
plantlets
exhibited
morpho-
logically
normal
development
(Fig.
1F).
It
must
be
emphasized
that
in
vitro
regenerated
plantlets
of
R.
fastigiata
need
to
be
acclimatized
carefully
due
to
the
large
number
of
adventitious
roots
which
were
easily
injured
during
transplantation.
The
results
of
these
studies
have
provided
an
efficient
regeneration
system
for
mass-propagation
of
R.
fastigiata
plantlets
for
different
purposes.
Plantlets
derived
from
leaf
explants
can
provide
masses
of
tissue
for
the
biochemical
characterization
of
medicinally
active
constituents,
and
selection
and
cloning
of
superior
individual
genotypes.
Mass-production
of
R.
fastigiata
in
controlled-environment
bioreactors
is
currently
underway.
This
in
vitro
regeneration
system
may
also
be
useful
for
crop
improvement
through
genetic
engineering
and
cell
culture
techniques.
Together
these
approaches
may
result
in
novel
uses
for
this
valuable
species.
0.49
1.48
2.46
4.92
IBA
concentration
(ccM)
FIG.
2.
Effect
of
IBA
concentration
on
the
rooting
and
survival
of
regenerants
of
R.
fastigiata.
Values
within
categories
(rooting
and
survival)
with
different
letters
were
significantly
different
at
P
<
0.05.
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