Plant regeneration from wheat leaf explants


Kopertekh, L.G.; Stribnaya, L.A.

Russian Journal of Plant Physiology 50(3): 365-368

2003


The factors affecting the callus formation and regeneration capacity of leaf explants of four genotypes of the genus Triticum, viz. T. aestivum cultivars Taezhnaya and Chinese Spring; T. durum cv. Kollektivnaya; and T. persicum [Aegilops triuncialis var. persica], were investigated. The process of callus formation did not depend on the explant genotype. Apical leaf segments were characterized by the lowest capacity of callus formation. In contrast, the rate of plant regeneration was correlated with the genotype and the explant developmental stage. The highest number of regenerants was obtained from a basal segment of three-day-old seedlings of T. aestivum cv. Taezhnaya. The yield of plants from one explant was doubled due to the use of maltose in the regeneration medium. The prospects of using leaf segments as the explants for the genetic transformation of wheat plants are discussed.

Russian
Journal
of
Plant
Physiology,
Vol.
50,
No.
3,
2003,
pp.
365-368.
Translated
from
Fiziologiya
Rastenii,
Vol.
50,
No.
3,
2003,
pp.
410-414.
Original
Russian
Text
Copyright
©
2003
by
Kopertekh,
Stribnaya.
Plant
Regeneration
from
Wheat
Leaf
Explants
L.
G.
Kopertekh
and
L.
A.
Stribnaya
Timiryazev
Institute
of
Plant
Physiology,
Russian
Academy
of
Sciences,
Botanicheskaya
ul.
35,
Moscow,
127276
Russia;
fax:
7
(095)
977-8018;
e-mail:
kopertekh@hotbox.ru
Received
June
28,
2001
Abstract—The
factors
affecting
the
callus
formation
and
regeneration
capacity
of
leaf
explants
of
four
geno-
types
of
the
genus
Triticum,
viz.
T
aestivum,
cvs.
Taezhnaya
and
Chinese
Spring;
T
durum,
cv.
Kollektivnaya;
and
T
persicum,
were
investigated.
The
process
of
callus
formation
did
not
depend
on
the
explant
genotype.
Apical
leaf
segments
were
characterized
by
the
lowest
capacity
of
callus
formation.
In
contrast,
the
rate
of
plant
regeneration
was
correlated
with
the
genotype
and
the
explant
developmental
stage.
The
highest
number
of
regenerants
was
obtained
from
a
basal
segment
of
three-day-old
seedlings
of
T
aestivum,
cv.
Taezhnaya.
The
yield
of
plants
from
one
explant
was
doubled
due
to
the
use
of
maltose
in
the
regeneration
medium.
The
pros-
pects
of
using
leaf
segments
as
the
explants
for
the
genetic
transformation
of
wheat
plants
are
discussed.
Key
words:
Triticum
-
leaf
-
callus
formation
-
plant
regeneration
INTRODUCTION
Modern
cereal
biotechnology
would
be
impossible
without
developing
a
cell
system
characterized
by
a
high
embryogenic
potential.
This
requirement
implies
the
choosing
of
an
explant,
which
would
have
a
good
ability
to
regenerate
and
to
maintain
the
embryogenic
potential
over
a
long
period
of
time.
In
the
case
of
cereal
plants,
the
immature
embryo
is
traditionally
used
as
an
explant.
Using
such
embryos,
the
wheat
[1,
2],
barley
[3],
rice
[4],
and
maize
[5]
cell
systems
having
a
high
regeneration
potential
were
developed.
An
embryo-derived
wheat
embryogenic
callus
was
suc-
cessfully
used
for
genetic
transformation
using
the
bal-
listic
transfection
[6]
or
Agrobacterium
tumefaciens
[7].
However,
growing
of
donor
plants
for
obtaining
immature
embryos
involves
the
expenditure
of
much
time
and
money.
The
leaf
explant
is
free
of
these
short-
comings,
and,
moreover,
makes
it
possible
to
rapidly
obtain
a
great
amount
of
uniform
material
regardless
of
season.
Recently,
a
great
interest
was
shown
in
using
the
leaf
explant
as
a
possible
alternative
to
the
immature
cereal
embryo.
In
this
regard,
promising
results
for
oat
[8,
9],
rye
[10],
and
sorghum
[11]
were
already
obtained.
Many
factors,
such
as
genotype,
a
develop-
mental
stage
of
the
donor
leaf,
and
culturing
conditions,
must
be
accounted
for
when
using
both
the
leaf
tissues
and
the
immature
embryos.
Only
few
studies
were
devoted
to
the
investigation
of
these
indices
in
several
species
and
varieties
of
the
Triticum
genus
[12-14].
The
objective
of
this
study
was
to
investigate
the
phys-
iological
and
genetic
factors
affecting
the
callus
initia-
tion
and
the
regeneration
of
plants
from
wheat
leaves.
Abbreviations:
BA—benzyladenine;
MS—Murashige
and
Skoog
nutrient
medium.
MATERIALS
AND
METHODS
Plant
material.
Four
genotypes
of
the
Triticum
genus,
such
as
T
aestivum,
cvs.
Taezhnaya
and
Chinese
Spring,
T
durum,
cv.
Kollektivnaya,
and
T
persicum,
were
used
in
this
work.
The
seeds
were
sterilized
with
2%
Na0C1
for
10
min
and
washed
three
times
with
sterile
distilled
water.
Subsequently,
the
seeds
were
ger-
minated
in
the
light
at
2510
for
two—six
days,
depend-
ing
on
the
experimental
conditions,
using
a
MS
hor-
mone-free
medium
[15].
The
leaf
basal
parts
of
young
shoots
were
cut
into
the
1-2-mm
segments,
which
were
placed
on
a
callus-formation
medium.
The
explants
were
cultured
at
2610
for
two
weeks
in
darkness.
Sub-
sequently,
the
explants
were
transferred
in
the
light
and
cultured
for
further
two—three
weeks.
For
morphogene-
sis
induction,
the
callus
was
placed
on
a
regeneration
medium,
and
the
plants
having
a
fully
developed
root
system
were
transferred
in
soil.
The
frequencies
of
cal-
lus
induction
and
plant
regeneration
were
determined
as
the
number
of
explants
that
formed
calli
or
regener-
ant
plants
as
the
percentage
of
total
number
of
explants.
Nutrient
media
and
culturing
conditions.
All
media
contained
2
mg/1
glycine,
1
mg/1
thiamine-HC1,
0.5
mg/1
pyridoxin,
0.5
mg/1
nicotinic
acid,
100
mg/1
meso-inos-
itol,
150
mg/1
L-asparagine,
20
g/1
sucrose,
and
8
g/1
agar,
as
well
as
MS
salts
and
micronutrients.
The
cal-
lus-forming
medium
contained
also
10
mg/1
AgNO
3
and
2
mg/12,4-D;
the
regeneration
medium,
1
mg/1BA,
0.5
mg/1
IAA,
and
10
mg/1
AgNO
3
;
and
the
rooting
medium,
1
mg/1
IAA.
Before
autoclaving,
the
pH
of
the
medium
was
5.6-5.8.
The
effects
of
maltose
on
the
callus
formation
and
the
regeneration
capacity
of
leaf
explants
were
investi-
gated
using
maltose
(40
g/1)
as
a
source
of
carbohy-
drates.
1021-4437/03/5003-0365$25.00
©
2003
MAIK
"Nauka
/Interperiodica"
366
KOPERTEKH,
STRIBNAYA
100
g
9
0
8
s
80
o
70
s)
g
V.
6
60
X
8
50
2
40
30
t:L
'
20
czt
10
0
Fig.
1.
Callus
formation
and
plant
regeneration
on
the
leaf
explants
of
various
ages
from
T
aestivum,
cv.
Taezhnaya.
(1)
Callus
formation;
(2)
plant
regeneration.
100
90
e
80
670
cd
E
60
X6
50
A
Ez
40
30
2
6.20
ett
c4
10
1
2
3
4
Leaf
segment
number
Fig.
2.
Effect
of
the
position
of
the
leaf
explant
on
(1)
callus
formation
and
(2)
plant
regeneration
of
wheat,
cv.
Taezh-
naya.
1
2
T
T
II
T
4
5
Explant
age,
days
0
100
90
o
e
80
6
c70
cd
E
60
1
T
2
14
112
71,
to
2
0
0)
°50
8
2
40
6
-
30
2
0
4
T
T
T
T
2
0
5
'
4
20
10
6
2
0
0
Callus
formation
Regeneration
2
3
4
5
Explant
age,
days
Fig.
3.
Effect
of
(1)
sucrose
and
(2)
maltose
on
callus
for-
mation
and
plant
regeneration
from
wheat
cv.
Taezhnaya
leaf
explants.
Callus
tissues
and
regenerated
plants
were
grown
in
the
light
using
luminescent
lamps
at
an
irradiance
of
100
W/m
2
,
a
photoperiod
of
16
h,
a
relative
humidity
of
70
±
5%,
and
a
temperature
of
25
±
11C.
The
data
presented
here
were
obtained
in
two
inde-
pendent
experiments
using
no
less than
4
petri
dishes
each
containing
20-25
explants.
In
Fig.
4,
no
less than
20
explants
were
used
for
each
experiment.
Means
and
their
standard
errors
are
presented.
RESULTS
AND
DISCUSSION
The
ability
of
cells
and
tissues
to
a
successful
in
vitro
culturing
is
determined
by
several
factors,
such
as
donor-plant
genotype,
epigenetic
characteristics
of
an
explant,
and
growing
conditions.
This
work
was
devoted
to
the
effects
of
all
these
factors
on
the
dedif-
ferentiation
and
differentiation
of
leaf
explant
cells.
Fig.
4.
Effect
of
(/)
sucrose
and
(2)
maltose
on
plant
regen-
eration
from
the
leaf
explants
of
wheat
cv.
Taezhnaya,
of
various
ages.
Genotype.
The
first
basal
leaf
segment
of
three-day-
old
seedlings
(situated
at
1
mm
from
the
seedling
stem)
of
all
four
wheat
genotypes
was
used
for
studying
the
effects
of
the
genotype
on
the
callus
formation
and
the
regeneration
of
plants
from
leaf
explants.
The
process
of
callus
formation
did
not
depend
on
the
genotype,
because,
in
all
varieties,
callus
was
formed
on
90-96%
of
explants
(table).
The
onset
of
callus
formation
was
always
observed
on
the
5th-7th
day
of
culturing.
The
callus
formed
was
transparent
and
light
in
color
and
had
an
amorphous
consistence.
The
transfer
of
callus
to
the
light
after
1.5-2
weeks
of
culturing
resulted
in
the
for-
mation
of
morphogenic
zones
in
some
explants.
Our
experiments
showed
that,
as
distinct
from
callus
forma-
tion,
plant
regeneration
on
leaf
explants
did
depend
on
the
genotype.
Regenerated
plants
were
obtained
in
all
four
genotypes;
the
highest
number
of
them
was
obtained
from
T
aestivum,
and
the
lowest
one,
from
T
persicum
(table).
Komatsuda
et
al.
[16]
used
a
dial-
RUSSIAN
JOURNAL
OF
PLANT
PHYSIOLOGY
Vol.
50
No.
3
2003
PLANT
REGENERATION
FROM
WHEAT
LEAF
EXPLANTS
367
lele
analysis
for
investigating
the
genetics
of
callus
pro-
liferation
and
the
subsequent
shoot
differentiation
in
the
tissue
culture
of
immature
barley
embryos.
Our
results
support
the
conclusions
of
these
authors
in
the
fact
that
these
indices
are
controlled
by
independent
genetic
mechanisms.
At
present,
a
search
for
molecular
and
biochemical
markers
of
embryogenesis
is
in
progress.
In
particular,
several
proteins
were
identified
in
the
barley
cell
culture;
the
presence
and
absence
of
these
proteins
were
correlated,
respectively,
with
the
regeneration
capacity
(17.4
and
46
kD
proteins) and
with
its
loss
(85
kD
protein).
In
same
experiments,
two
molecular
markers
of
the
early
stages
of
embryogene-
sis,
B15C
and
pG22-69,
were
found
[17].
Several
experiments
demonstrated
the
effect
of
the
genotype
on
the
regeneration
capacity
of
cultured
oat
[18],
wheat
[1],
and
barley
[3]
cells.
At
the
same
time,
some
authors
reported
that
the
regeneration
of
plants
from
the
leaf
explants
of
oat
was
less
genotype-dependent
[8].
Our
experiments
showed
that
this
statement
did
not
apply
to
the
plant
regeneration
from
wheat
leaf
explants
(table).
Cv.
Tayezhnaya
was
characterized
by
the
highest
regeneration
capacity.
Therefore,
this
genotype
was
used
in
subsequent
experiments
for
investigating
the
effects
of
several
factors
on
the
dedifferentiation
and
differentiation
of
leaf
explant
cells.
Epigenetic
Characteristics
of
an
Explant
Developmental
stage
of
the
donor
material.
T
aesti-
vum,
cv.
Taezhnaya,
caryopses
were
germinated
on
the
nutrient
medium
for
two—six
days,
and
the
first
segment
of
the
basal
leaf
region
situated
at
1
mm
from
the
seed-
ling
stem
was
used
as
an
explant
(Fig.
1).
Up
to
88-
94%
of
leaf
segments
formed
callus
regardless
of
the
explant
age.
As
shown
in
Fig.
1,
basal
leaf
segments
from
three-day-old
seedlings
were
characterized
by
the
highest
regeneration
capacity
(36.3%),
and
those
from
the
six-day-old
seedlings,
by
the
lowest
one
(7.2%).
Explant
position
in
the
leaf
A
4-mm-long
leaf
of
the
three-day-old
seedling
of
T
aestivum,
cv.
Taezhnaya,
was
cut
into
four
segments,
1-mm-long
each,
and
the
first,
second,
third,
and
apical
segments
of
the
leaf
were
used
in
the
experiment.
The
apical
leaf
segment
was
characterized
by
the
lowest
shoot-forming
capacity,
and
no
plants
could
be
obtained
from
the
explants
of
this
type
(Fig.
2).
The
callus
obtained
from
such
explants
was
capable
only
to
rhizogenesis.
In
contrast,
in
the
case
of
a
basal
segment,
95%
of
explants
were
capable
of
callus
formation,
and
36%,
of
plant
regener-
ation.
Our
data
demonstrate
that
the
embryogenic
potential
of
the
wheat
leaf
tissue
is
determined
by
the
leaf
age
and
the
position
of
the
leaf
segment
(Fig.
2).
Such
gradient
of
the
embryogenic
capacity
was
demonstrated
for
the
leaf
explants
of
other
cereals,
such
as
rye
[10],
oat
[8],
barley
[19],
and
wheat
[14].
Some
authors
attempted
to
explain
the
existence
of
such
gra-
dient
by
changes
in
the
position
of
cells
in
the
cell
Callus
formation
and
plant
regeneration
on
the
leaf
explants
of
different
wheat
genotypes,
%
of
total
number
of
explants
Genotype
Frequency
of
callus
formation
Frequency
of
plant
regeneration
Triticum
aestivum
(Taezhnaya)
95
±
0.5
36
±
2
T.
aestivum
(Chinese
spring)
96
±
2
25
±
4
T.
durum
(Kollektivnaya)
92
±
4
14
±
0.5
T.
persicum
91
±
5
11
±
1
cycle,
from
the
leaf
base
to
its
apex
[9,
20].
Most
cells
in
the
basal
region
of
oat
seedlings
were
shown
to
be
at
the
G2
stage
of
the
cell
cycle
(4C).
A
decrease
in
the
proliferative
activity
of
cells
in
the
apical
region
of
a
seedling
was
accompanied
by
a
decrease
in
the
number
of
4C
nuclei
in
these
cells
[9].
In
contrast,
no
differ-
ences
in
the
content
of
nuclear
DNA
were
found
in
basal
and
apical
leaf
segments
of
maize
[20].
The
authors
of
these
works
suggest
that,
in
the
leaf
cells
of
oat
and
maize,
the
cell
cycle
stage
and
the
mitotic
activ-
ity
can
determine
the
proliferative
capacity
of
cells
rather
than
their
embryogenic
capacity.
Use
of
Maltose
as
a
Source
of
Carbohydrate
Nutrition
Culturing
conditions,
such
as
a
source
of
carbohy-
drate
nutrition,
are
one
of
the
factors
affecting
the
regenerative
capacity
of
the
in
vitro
cultures.
Two
series
of
experiments
were
performed.
In
the
first
one,
mal-
tose
was
used
as
a
source
of
carbohydrates
in
both
the
callus
formation
medium
and
in
the
regeneration
medium
(Fig.
3).
This
resulted
in
a
decrease
in
the
pro-
liferative
activity
of
cells,
the
callus
size,
and
the
regen-
erative
capacity
from
36
to
9%.
In
the
second
series
of
experiments,
when
calli
were
induced
on
a
sucrose
medium,
and
then
the
callus
tissue
was
transferred
onto
a
regeneration
medium
containing
maltose
(Fig.
4),
a
greater
number
of
regenerants
per
one
explant
was
obtained.
This
trend
was
retained
in
the
experiments
on
the
segments
of
different
age.
A
possi-
bility
of
obtaining
a
greater
number
of
regenerants
from
one
explant
is
particularly
important
for
performing
gene-engineering
investigations
using
cereals,
because,
for
these
species,
there
is
no
technique
of
clonal
micro-
propagation.
The
use
of
maltose
in
the
medium
affected
induction,
yield,
and
maturation
of
embryoides
in
Med-
icago
sativa
[21],
Malus
domestica
[22],
and
Abies
alba
[23].
Some
investigators
suggest
that
maltose
is
here
not
only
a
source
of
carbohydrates,
but
also
exerts
an
osmotic
effect
[21].
Norgaard
[24]
suggests
that
maltose
and
sucrose
differ
in
the
rate
of
metabolization,
and
maltose
is
metabolized
much
slower
than
sucrose.
A
rapid
metabolization
of
sucrose
seems
to
result
in
a
certain
hypoxia
and
an
accumulation
of
ethanol
in
cells,
RUSSIAN
JOURNAL
OF
PLANT
PHYSIOLOGY
Vol.
50
No.
3
2003
368
KOPERTEKH,
STRIBNAYA
whereas
a
slow
metabolization
of
maltose
does
not
lead
to
such
consequences.
This
can
positively
affect
cell
viability
and
subsequent
development
of
somatic
embryoids.
Thus,
genotype
and
epigenetic
characteristics
of
an
explant
must
be
taken
into
account
for
a
successful
ini-
tiation
of
the
callus
tissue
and
the
regenerant
plants
from
the
wheat
leaf
tissues.
Our
experiments
showed
that
the
basal
leaf
segment
of
three-day-old
seedlings
is
most
appropriate
to
attain
these
ends.
Among
the
geno-
types
studied
here,
the
best
result
was
obtained
for
the
T
aestivum,
cv.
Taezhnaya.
Using
maltose
in
the
regen-
eration
medium
can
increase
the
yield
of
plants
from
one
explant.
ACKNOWLEDGMENTS
The
authors
are
grateful
to
Dr.
S.E.
Zorinyants,
Institute
of
Plant
Physiology,
RAS,
Moscow,
for
the
helpful
advises
in
the
course
of
the
editorial
preparation
of
this
manuscript
and
to
Dr.
A.A.
Solov'
ev,
Timiryazev
Agricultural
Academy,
Moscow,
for
kindly
placing
at
our
disposal
wheat
seeds
used
in
the
experiments.
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