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8/17/2019 Aloe vera transformation: The role of Amberlite XAD-4 resin and antioxidants during selection and regeneration
1/9
Aloe vera transformation: the role of Amberlite XAD-4 resin and antioxidants duringselection and regenerationAuthor(s): Margarita Velcheva, Zehava Faltin, Aliza Vardi, Uri Hanania, Yuval Eshdat, OdedDgani, Nachman Sahar and Avihai PerlSource: In Vitro Cellular & Developmental Biology. Plant, Vol. 46, No. 6 (November/December
2010), pp. 477-484Published by: Society for In Vitro BiologyStable URL: http://www.jstor.org/stable/40981336 .
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8/17/2019 Aloe vera transformation: The role of Amberlite XAD-4 resin and antioxidants during selection and regeneration
2/9
In
VitroCell.Dev.Biol.-
Plant
2010)
46:477^84
DOI
1
. 1007/s
1
27-01
-930
1-z
BIOTECHNOLOGY/GENETIC
RANSFORMATION/
UNCTIONAL
GENOMICS
Aloe vera ransformation:herole ofAmberlite AD-4resin
and antioxidants
uring
election nd
regeneration
Margarita
Velcheva Zehava
Faltin Aliza
Vardi
Uri
Hanania
•
Yuval Eshdat Oded
Dgani
NachmanSahar
•
Avihai
Perl
Received: 30 November
009
/Accepted:
7
July
010 /Published nline:
14
September
010 / Editor:T. J. Jones
©
The
Society
for
n Vitro
Biology
2010
Abstract
A
system
or
enetic
ransformationnd
ubsequent
plant egeneration
ia ndirect
rganogénesis
rom alluswas
developed
or loe vera.
Young eedlings
erved s
primary
expiants.
alluscultures ere stablishedn
Murashige
nd
Skoog 1962)
medium
upplemented
ith
mg
F1
benzy-
laminopurine
nd2
mg
F1
indole
cetic cid.A
protocol
as
developed
o switch rom he differentiated
tage, sing
n
vitro hoots r
young egeneratedlants,
ack
to
the
de-
differentiated
tage
f thecallus nd vice
versa.
Long-term
maintenance f this callus
paved
the
way
for
genetic
manipulation
fAloe vera.Calluseswerebombarded ith
plasmid ontaining
idA
and
hpt genes,
both
under he
control f the 35S promoter.ithiothreitolnd gibberellic
acid were found o
play
a
major
role
in
reducing
issue
necrosis
ollowing
ombardment.ransformedhootswere
regenerated
nder
stepwise
selection n
hygromycin-
containingiquid
medium
upplemented
ith
ifferentnti-
oxidants. mberliteAD-4
resinwas
embeddednto
lginate
beads and added to the
selectionmedium.Amberlite
as
best for
adsorbing
different
henolic compounds
nd
blocking xpiant
ecrosis. hoot
nitiationccurred
fter
transfer
f the
transformedells to
Murashige
nd
Skoog
medium
upplemented
ith 2.0
mg
F1
thidiazuron
nd
M. Velcheva Z. Faltin A. Vardi U. Hanania Y. Eshdat
O.
Dgani
N. Sahar
A. Perl
ÊE3)
Department
f Fruit
ree
Sciences,
Agricultural
esearch
Organization,
he Volcani
Center,
P.O. Box
6,
Bet-Dagan
50250,
Israel
e-mail:
PresentAddress:
M.
Velcheva
Alberta
Research
Council,
Hwy
16A & 75
Street,
.O.
Bag
4000,
Vegreville,
B
T9C
IT,
Canada
0.1
mg
1
l
indole
butyric
cid.
Murashige
nd
Skoog
medium
supplemented
ith 1
mg
F1
zeatin
riboside
promoted
hoot
longation.
ooting
nd
plant evelopment
were obtained n
Murashige
nd
Skoog
basal
medium
supplemented
ith15
mg
F1
hygromycin
acking rowth
regulators.
he
transgenic
ature f the
regeneratedlants
was verified
y
histochemical US
assay
and
Southern
blot
hybridization.
Keywords
Aloe Recurrent
allusogenesisRegeneration
Genetic ransformation
Introduction
The ast everal
ears
ave
witnessed boom
n
researchn
Aloe and
its
applications
for humans
and
mammals.
However,
mprovement
f
some valuable
traits uch
as
plant
morphology,
tress
esistance,
nd
modificationf
secondary
metabolitessed n
medicinend
cosmetics an
hardly
e achieved
y
conventional
reeding
ethods. his
is
mainly
ecause f
ytoplasmic
ale
terility,
ong egetative
generation,
ow
recombination
otential,
nd
embryo
bortion
(Velcheva
t al
2005).
Most
regeneration
ystems
orAloe
use
axillary
uds
(Keijzer
and
Cresti
1987;
Zhao
1990;
Corneanutal 1994;HirimburegamandGamage1995)or
inflorescences
Richwine
995;
Velcheva t
al
2005)
for n
vitro
mass
propagation.
ecent
tudies
avefocusedn the n
vitro
ropagation
f
Aloe
using
eitherhoot
multiplication
(Hashemabadi
nd Kaviani
008)
or somatic
mbryogenesis
(Garro-Monge
t al
2008).
For ransformation
ystems
ased
on the
development
fadventitious
hoots,
he
production
f
chimeric
lants
was described
everal
imes,
s
transgenic
plants
avenot
ecessarily
he ame
lonal
riginMatthews
t
al
1998;
Flachowsky
t
al.
2008).
This
means that he
Ô
Springer
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478 VELCHEVA
ET
AL.
development
f adventitioushoots oes not
necessarily
ead
to
plants
erived rom
single
ell
Schmulling
nd
Schell
1993). Very
ew
reportsRacchi 1988)
have described he
production
f rue allus rom loeor he
onditionsor
ong-
termmaintenancefthis allus.
Although
loe vera s
considered
prime
andidate or
genetic
ransformation,
ery
ew
tudies
ave
reportedene
transformationnd
subsequent egeneration
f
transgenic
plants
n
Aloe
(He
et al
2007).
Potential easons or his
include:
1)
browning
nd oxidative urst í Aloe
expiants,
mainly
n
liquid
ulture,
ften
eading
o
expiant
ell death
(Natali
t l
1990), 2)
cytokinins,
hich
re
usually
tilized
to
promote egeneration,
ave been
reported
o enhance
secretionf
phenols
n
Aloe
cells
Natali
t al
1990;
Meyer
and van Staden
1991;
Roy
and Sarkar
1991),
and
(3)
Agrobacterium
as been
found o be
an
additional actor
stimulatingxpiant
ecrosis
ollowing
o-cultivation.
oly-
phenol xcretion,hich eads to tissuenecrosis ndtarget-
explant
death,
has
been
previously eported
o
limit
transformation
apability
n
different
lants Lagrimini
1992;
Perl t al
1996;
Dan
2008).
It has been assumed hat
instead f
inducing
ell de-differentiationnd
competence,
wounded issues ccumulate
henols
nd
subsequently
ie
(Potrykus
991).
Pretreatment
ith
ntioxidantsas been
found o enhance
ell
viability
o minimize xidative urst
and to reduce ecrosis
Enriquez-Obregon
t al
1999;
Dan
2008).
Dithiothreitol
DTT)
is known
s a
reducing gent
(Apóstol
t al
1
89)
andhas been
xtensively
sed
n
plant
transformationnd
regeneration.
oreover,
TT has been
found o be more ffectivehan
many
ther
ntioxidants,
such as ascorbic cid,polyvinylpyrrolidonePVP), andL-
cysteine
Perl
t al
1996).
Amberlite
AD
adsorbent
esins
are
polymeric
dsorbents
ith
highly orous
tructure
n
which ts internal
urfaces an absorb
wide
variety
f
differentolecules
epending
n the
nvironment
n
which
they
reused nd he
ype
fresin sed.
Amberlite
AD-4 s
mainly
sed for he emoval f
aromatic
ydrocarbons
uch
as
phenols
nd
pesticides
rom
astes.
However,
hey
ave
alsobeenutilized
or oth xtractionf
specific
hytochem-
icals
produced
n vitro nd removal
f toxic
compounds
negativelyffectingxpiant
iability
n
plant
ell cultures
(Strobel
t al
1991;
Ui et al
1993;
Georgiev
t al
2006;
Mucciarellit
al
2009).
In thisstudy,we reporthedevelopmentf a highly
efficient
nd
reproducible
ystem
or
egeneration
n
Aloe
vera via callus
cultures
nd
procedures
or
the in vitro
long-term
maintenance
f such
morphogenetic
ells.
Successful
ransformationf
these
morphogenetic
alluses
by particle
ombardmentnd
subsequent
egeneration
f
transformed
lants
were
achieved.
Antioxidants
nd
Amberlite
esinwere
found o
play
a
major
role
in the
reduction
f necrosis
uring
he election
rocess
n
iquid
cultures.
Materials nd Methods
Expiant reparation
nd
Callus nduction. loe vera
eeds
were sterilized
1%
NaOCl,
15
min)
and washed three
times withsteriledistilledwater.Seeds (30 seeds per
Magenta
box)
were cultured n 50 ml
Murashige
nd
Skoog (MS; Murashige
nd
Skoog
1962)
solid
(0.25%
Gelrite)
asal medium
or
ermination
nder
ight
40 pinol
m2
s"1)
at25°C. Two- o 3-wk-oldn vitro
eedlings
ere
used as
primaryxplants.
xplants
were
cut nto0.7-1.0-
cm thick
ectionsunder
half-strength
S
liquid
media,
supplemented
ith 0
mg
F1
DTT,
washed hree imes
3
x
10
min)
with
he same
media,
and
transferredo callus
induction
edia
CIM).
All
mediawerebased on
MS salts
and
vitamins,
%
sucrose,
0
mg
F1
DTT,
0.25%
Gelrite
(pH
5.2),
and different
rowth egulatorsmediadesigna-
tion MSI to
FRS3,
Table
1).
Calluses were subcultured
every wkon freshmedia nd werekeptnthedark t24°
C before ransfero shoot inductionmedia
(SIM)
for
regeneration.
Callus Re-initiationnd
Long-term
aintenance
f
Callus
Cultures.
n vzYro-initiated
hoots,
n
vzYro-elongated
hoots
or in vitro
oungplants
erved
s
expiants
or allus re-
initiationtudies. o re-initiateallus
from hese
xpiants,
the
procedure
escribed bove was
applied,
nd
expiants
were cultured n MS media
supplemented
ith he same
growth egulators
Table 1).
Re-initiated
allus was sub-
cultured
very
mo to freshmedium
nd callusfresh
eight
was monitored
uring
hree uccessive
ubcultures.
o test
themorphogeneticotentialfthe e-initiatedalluses fter
12 mo of
successive
ubcultures,
ll calluses
hat
eveloped
on
all
the
growth
egulator
ombinationsere
ransferred
o
AV2
SIM
(Table
1).
The numberf
regenerated
lants
er
calluswas monitored.
Plant
Regeneration.
alluseswere
ransferred
o
SIM
(des-
ignation
V0
o
AV3NAA,
able
1).
SIM
were
omposed
f
solidified
0.25%
Gelrite)
MS salts and
vitamins,
%
sucrose,
.5
g
F1
myo-inositol,
0
mg
F1
DTT
(pH
5.8),
and either
eatin iboside
ZR),
indole-3
butyric
cid
IBA),
or
napthaleneacetic
cid
(NAA)
in different
ombinations
and concentrations.
our wk
after hoot
nduction,
egen-
erated hootswere ransferredo either.5, 1,or2 mgF1 ZR
inMS mediafor hoot
longation.
longated lants
were
rooted n MS basal
medium
ackinggrowth
egulators.
Cultures
eremaintained
n white
ight
t
40
jimol
m"2 "1
and 25°C.
Rooted
lants
were ransferred
o
pots
ontaining
standardoil
mixtureor
hardening
nd
transferred
o
the
greenhouse.
Experiments
ere
carried
ut in five
replicates.
he
number f shoots
nd
régénérants
ere
cored
very
wk.
Data were
nalyzed
sing
he
east
ignificant
ifferenceest.
â
Springer
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ROLES OF
AMBERLITE
XAD-4
RESIN
AND
ANTIOXIDANTS ON ALOE
VERA
TRANSFORMATION 479
Table 1. MS media
supple-
mented
withdifferent
rowth
regulators
or
allus initiation
(CIM)
or shoot nduction
SIM)
of Aloe
vera
a
Phytohormones
mg
1
l
)
Media
designation
2,4-Da
IAA IBA NAA ZR
TDZ BA
Kinetin
Callus
Initiation MS I 1
0.2
Media
(CIM)
MS n 05 2
MS III 0.2
1
MS
IV 1
FRSi
2 2
FRS2
2
3
FRS3
3
2
Shoot nduction
AV0
0.0 2
Media
(SIM)
AVj
01
x
AV2
0.1 2
AV3
0.1 3
AVjNAA
0.1 1
AV2NAA
0.1 2
AV3NAA
0.1 3
Biolistic
ransformationf
Aloe VeraCalluses.
Forty-eight
h
before
ombardment,
alluses
were transferredo
AV2
SIMwith rwithout0
mg
F1 DTT
and
5
mg
"1
GA3.
The
plasmid
WAG1515
ontaining
he
hpt
nduidA
genes,
oth
under he control f the 35
S cauliflowermosaic virus
promoter,
as used for
particle
bombardment. old
particles1.0
|nm
n
diameter)
ere coatedwith
plasmid
DNA
following
he
protocol
f Perl t
al
(1992).
Different
acceleration
ressures450,
900 and
1,100
psi)
combined
with
wo
target
istances
8.5
and 11.0
cm)
between
he
stopping
creen and
target late
were
tested.
Optimal
bombardment
onditions
ere
evaluated
y
scoring
ran-
sient -glucuronidasGUS) expression.
Selection rocess.
Hygromycin0-15
mg F1)
was
utilized
for
election
n
solid
Magenta
A7
vessels)
r
iquid
media
(25-ml
iquid
n
a
250-ml
rlenmeyer).
fter
ombardment,
calluseswere
ransferredofresh
V2
medium
upplemented
with
.75
mg
F1
hygromycin.ygromycin
oncentration
as
elevatedn a
stepwise
anner,
n
2-wk
ntervals,
rom
.75-
7.5
mg
F1,
and
finally
eaching
5
mg
F1. Shoot
longation
was
performed
n
MS medium
upplemented
ith
mg
F1
ZR
and 15
mg
F1
hygromycin.
ransgeniclants
ooted n
hormone-freeS
medium
upplemented
ith
15
mg
F1
hygromycin.
n
case
anti-necroting
ompounds
ere
tilized,
theputativeransformedallusesweretransferredo AV2
liquid
or
solid
media
supplemented
ith
the
following
antioxidants:VP
(0.5
and
1
g F1),
L-cysteine150
mg F1),
gallic
cid
1.5
mg F1),
DTT
(70
mg F1),
biopterin15
mg
F1),
ascorbic
cid
150
mg F1),
and
citric
cid
150
mg F1).
Calluses
were
ultured
n a
gyratory
haker
110
rpm)
nder
cool
white
luorescent
ight20 |nmol
m~2
"1)
at
25°C.
Preparation
of
Alginate-
mberite
Beads.
Na-alginate
(3%)
was
dissolved
n
hot,
distilled
water
nd
sterilized
by
autoclaving
t 121°C for 20 min.
Amberlite AD-4
resins
Sigma)
were
sterilized
y
immersing
n
100%
methanol or 0
min,
ollowed
y
a
wash
n
100%
ethanol
(for
10
min),
nd
finally
hree
washes
n
sterile
istilled
water.
Amberlite
articles
were
mixed
1:1
v/v)
with
he
Na-alginate
olution. he
alginate-Amberlite
eads
were
solidified
y
gentle ropping
using
25-ml
ipette)
f the
alginate-Amberlite
ixture
nto
a
sterile
solution
of
100 mM
CaCl2.
Following
0
min
ncubationn
CaCl2
at
room
emperature,
he
solidified eads were
washedwith
sterile
istilled
ater,
nd 5
g
of
beads was
added o
50
ml
liquid
AV2
selection
medium.
Beads that
turned
rown
werereplacedweeklywith resh eadsuponsubcultureo
fresh
V2
electionmedium.
a-alginate
eads
acking
ny
resin
served s
controls nd
were
also
replaced
weekly
during
he
tudy.
Histochemical
nvestigation.
US
activity
as
analyzed
n
bombarded
alluses,
hoots,
nd
regenerated
lants
ccord-
ing
to
Jeffersont
al
(1987).
GUS
activity
as
visualized
under
tereoscope
fter
washing
he
stained
xpiants
n
a
mixture f
70% ethanol
and
30%
glacial
acetic
acid,
followed
y
a
wash
n
10% lactic cid.
Plant
DNA
Analysis.
rom37
putative
ransgenic
lants,
12were hosen andomlyor NA extractionndSouthern
hybridization.
CR
was used
to
detect he
presence
fthe
uidA
gene
n
transgenic
issues
ccording
oFulton t al.
(1995).
The
followingligonucleotides
ere sed:
5'-CAGC-
GAAGAGGCAGTCAACGGGGAA-3'
and 5'-
CATTGTTTGCCTCCCTGCTGCGGTT-3'
ielding
680-bp
fragment,
nd
5'-GGTTGGGCAGGCCAGCGTATC-3'nd
5'-TAACCTCTCTTTAGGCATTGG-3'
ielding
838-bp
fragment.
CR
products
ere
visualized
y
running
n a
1.5%
agarose
gel
containing
thidium
romide.
outhern
Ô
Springer
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480 VELCHEVA
ET
AL.
hybridization
as
performed
ccording
o Perl t al
(1996)
with omemodifications.
riefly,
loe
DNA was extracted
from
g
of n vitro
rown lants
s described
Hanania
t l
2004).
About 10
|LLg
f
DNA
was
digested
with
Not ,
electrophoresedn 0.8% agarose gel and transferredo
Hybond
N+
membrane
Amersham,iscataway,
J).
Ge-
nomic
NA was
hybridized
ith 0.8-kb stll
fragment
f
DNA
containing
art
f
the
'
region
f the35S cauliflower
mosaic
irus
romoter,
s well s
part
fthe '
region
f the
hygromycinpt
ene.
Results nd Discussion
Establishment
f Regeneration
rotocol
Using
Aloe Vera
Calluses.
Seedlingsgerminating
ithin
-3 wk
with no
visible ontaminationerecut nto0.7-1. -cm hick tem
expiants
nd
culturedn differentIM
(Table
1).
Four wk
after
nitiation,
ll
expiants
xhibited
allus
proliferation
without
orrelation
o the
original osition
f the section
used as
the
explant
n the
germinated
eedling.
alluses
were ubcultured
onthly
or12 mo on the
differentIM
combinationseforeransfer
o different
IM combinations.
Although
alluses
were btained
n all CIM
combinations,
the
type
of callus
and its
regenerativeotential
iffered
significantlyccording
o the
growthegulators
sed.
Rapid
growth
f calluswas observed n
expiants
ultured
n
FRSi
medium.
owever,
hese alluses ailed o
regenerate
n all
SIM combinationsested. imilar bservations eremade
for
alluses
eveloped
n
FRS3,
MS
I,
MS
III,
and MS
IV
media.Calluses
originated
n these
phytohormone
ombi-
nations ormed
nly
roots n all SIM combinations
ested,
whileno shoot
egeneration
as observed.MS
II
medium,
supplemented
ithNAA and
ZR,
induced allus
formation,
but after
ubsequent
ubcultures
n the same
medium,
necrosis as
visible,
eading
o callus
death. he
only
allus
that
was found o
have a
morphogenetic
otential
was
initiatedn
FRS2
medium.
nder hese
onditions,
soft,
pale
green
allus
developedFig.
la).
Shoot
rimordia
ere
visible wk afterransferf
this allusfrom
RS2
to
AV0
AV3
media
upplemented
ith
N-phenyl-N'-l
,3-thiadiazol-
5-yiureathidiazuron]TDZ) and BA (Fig. b). TDZ alone
(AV0 medium)
was unable
o
promote
hoot
egeneration.
Seventy ercent
f he alluses
48
out f
61)
gave
rise o an
average
f
14.6 shoots
er
callus
on
AV2
medium. ower
TDZ
concentrations
s
in
AVi
medium ere
ess effective
n
promoting
hoot
regeneration.
DZ concentrations
igher
than .0
mg
F1,
as
in
the
AV3
medium,
educed
ignificantly
shoot
nitiationnd induced he
regeneration
f abnormal
Figure
1.
Morphogenetic
allus
obtained
n
FRS2
MS medium
(a).
Shoot
regeneration
f Aloe
vera calli
following
ransfero
AV2
MS medium
upplemented
with
mg
F1 TDZ and
0.1
mg
F1 IBA
(b).
Mature
longated
shoot
on MS medium
upple-
mented
with
1
mg
F1 ZR
(c).
Rooted
Aloe
vera
plant
n MS
hormone-free
edium
d).
â
Springer
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8/17/2019 Aloe vera transformation: The role of Amberlite XAD-4 resin and antioxidants during selection and regeneration
6/9
ROLES
OF AMBERLITE
XAD-4
RESIN AND
ANTIOXID
ANTS ON
ALOE VERA
TRANSFORMATION
481
shoots
aving
ome
morphological
isorders
uch as
fused
thickenedeaf
primordia
urrounded
y granulated
eep
green
allus.
These shoot
primordia
ailed o
develop ny
morphological
normal"loe shoots.
hoot
egeneration
as
dramaticallyeducedwhen ndoleaceticacid (IAA) was
replaced
withNAA
(AV1NAA-AV3NAA
edia).
Optimal
shoot
longation
calculated
s mean
number f
elongated
shoots
per
expiant)
was
observed
using
MS medium
supplemented
ith
mgf1
ZR. On this
medium,
nitiated
shoots
egan
o
elongate
nd the
irst
ell-developed
eaves
were
visible
Fig.
'c).
Some
tendency
oward
econdary
organogénesis
rom
ingle
hootswas
visiblewhen
higher
concentrationsf ZR
wereused. An
average
8.7%
of
the
initiated
hoots
877
out
of
1,113)
longated
nd
developed
into
vigorous
lants
hat
ooted
uccessfully
n
MS basal
medium
acking rowth
egulators
Fig. Id).
In
numerous onocot
pecies,
mbryo-derived
xpiants
have een oundobeoptimalorn-vitroegenerationWuet
al
1990).
everal
ypes
f
alluseswere
eveloped
epending
on
the
ombination
f
growth
egulators
sed.
Groenewald
t
al.
(1976),
Racchi
(1988),
and
Roy
and
Sarkar
1991)
observed
hat
2,4-D
can
promote
othcallus
and
shoot
induction
n
different
loe
species.
n
our
study,
,4-D
promoted
ell
division,
ut
egeneration
as
never
bserved,
as was
also
reported
y
Natali t al.
(1990)
and
Meyer
nd
van
Staden
1991),
suggesting
hat
2,4-D
can
probably
promote
egeneration
f
only
some
Aloe
species.
The
combinationf
IAA
and BA
was found o
promote
allus
formationrom
ll
expiants
tilized n
this
study.
imilar
results
avebeen
eported
or
ther
lant
pecies
elonging
o
thefamilyilaceae Wang t al 1998).Shootregeneration
from
alluses n
Aloe
vera
ppeared
o
depend
mainly
n
the
concentrations
f TDZ
and IAA.
When
TDZ
was
applied
alone
without
ny
uxin,
o
regeneration
ccurred.
nly
he
combinationf
TDZ and an
auxin
ource
romoted
egener-
ation,
s has been
previouslyeported
or
Aloe
regeneration
(Natali
t al.
1990;
Meyer
nd
van
Staden
1991).
ZR
was
found o
promote
hoot
elongation.
hoot
degeneration
occurredf
the
nitiated
hoots
were
ransferred
oMS
basal
medium
acking
rowth
egulatorsRichwine
995).
Re-callusing
f
Aloe
nVitro
xpiants.
nce
a
regerenative
callus
was
nitiated,
RS2
medium
as
found
o be
optimal
to supporthe ong-termaintenancefthis alluswhile
retaining
ts
morphogenetic
otential. RS2
medium
est
promoted
allus
re-initiation
sing
n
vitro
longated
hoots
or
young plantlets
s
expiants.
The
efficiency
f
de-
differentiation,
swell
s
subsequent
lant
egeneration
rom
these
alluses,
was
similar
ith
ll
expiants
sed
Table
2).
Sixty-three
ercent
f
the
nitial
xpiants
roduced
egener-
able
callus,
nabling
he
egeneration
f
up
to 14
shoots
er
explant.
s the
morphogenetic
otential
f
callus
culture
s
usually
ime-limited,
his
tudy
emonstrated
he
possibility
Table 2.
De-differentiation
nd
differentiation
fficiency
f calluses
originated
rom ifferent
xpiants
Primary
n vitro
Callus fresh
weight
ncrease
g)
Average
expiants
sed for
number
callus
re-initiation
Subculture of
plants
regenerated
I
II
III
percallusa
Germinated
14.1±0.9
16.67±1.9
18.22±0.9
13.4±1.3
seedlings
Initiated hoots
17.15±1.1
18.05±1.6
17.67±1.5
14.3±2,7
Elongated
hoots 14.56±1.6
19.67±0.8
18.92±0.5
11.6±1.4
Regenerated
lants
16.01±2.1
19.23±1.7
19.04±1.3
12.8±2.2
a
Regenerated
lants
were cored
ollowing
2
mo of
successive
monthly
subculturesn
FRS2
CIM. At
the nd of
these
ubcultures,
alluseswere
transferred
o
AV2
SIM
of
witching
rom
differentiated
tage
o a
de-differentiated
stage
n
Aloe
vera
and
vice versa
without
ny
apparent
reduction
n
the
morphogeneticotential
f
the
callus ine.
The
regeneration
rotocol
eported
erewas
found
o be
highly
fficient,
ynchronous,
nd
has
been
successfully
maintainedor
he ast4
yr.
Aloe
Bombardment
nd
Selection
f
Transgenic
lants.
Calluses
were
used
as a
target
or
article
ombardment.n
preliminary
tudies,
he
highest
fficiency
f
transient
US
expression
was
observed
when
the
acceleration
ressure
was set
to
1,100
psi
and
the
distance f the
target
was
8.5 cm datanotshown).Under hese onditions,1% of
the
alluses
ransiently
xpressed
US,
giving
ise omore
than
00
GUS-positive
pots
er
Petri
late
Fig. d).
Three
to
4
d
following
ombardment,
ombarded
alluses
urned
brownish,
nd
necrosis
was
visible. t
should e
noted hat
necrosis
was
visible
lso in
calluses
bombarded
ith
nly
gold
particles,
ndicating
hat
ecrosis
might
e
correlated
with
he
wounding
ffect f
the
procedure.
We
studied he
effects f
pretreatment
f
the
calluseswith
DTT
and
GA3
on
callus
survival
nd
transient
US
expression
Table
3).
The
addition f 70
mg
I"1
DTT
significantlymproved
expiant
viability,
s
well
as
transient
US
expression
following
ombardment.
A3
had
only
a
moderate
ffect
on improvingransient US expression.However, he
combination
f DTT
and
GA3
had an
additive
ffect,
improving
oth
parameters
ignificantly.
hemotaxonomic
studies
ave
ndicated hat
henols
mostly
lavonoids)
re
major
ompounds
n
species
belonging
o the
genus
Aloe
(Viljoen
et
al
1998).
GA3
has
been
reported
o
inhibit
chalcone
ynthase,
ne
of
the
key
enzymes
n
anthocyanin
biosynthesis
Ilan
and
Dougall
1994).
In
this
report,
A3
enhanced
ransformation
fficiency
nd
reduced
xpiant
necrosis.
We
cannot
rule out
the
possibility
hat
other
Ô
Springer
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8/17/2019 Aloe vera transformation: The role of Amberlite XAD-4 resin and antioxidants during selection and regeneration
7/9
482
VELCHEVA
ET AL.
Figure
2.
TransientGUS
ex-
pression
n Aloe vera
calli 48
h
after ombardment
a).
Stable
GUS
expression
f
Aloe vera
calluses
following
ulture
n
50
ml
liquid
FRS2
selection
medium upplemented ith5 g
alginate
eads
containing
Amberlite
AD4
resin,
15
mg
F1
hygromycin,
mg
F1 BA
and
2
mg
F1 IAA
(b).
GUS
expression
n callus
and
regen-
eration
hoot
primordia
SP)
on
MS
medium
upplemented
ith
15
mg
F1
hygromycin,
mg
F1
TDZ and0.1
mg
I"1
IBA
(c).
Stably
ransformed
loe
vera
plant
n
MS
elongation
medium
supplemented
ith
1
mg
F1
ZR
and 15
mg
F1
hygromycin
d).
mechanisms
re
responsible
or he
eneficial
ffect
f
GA3
on
Aloe
vera
transformation.
In
preliminary
tudies,
election
n
solid
AV2
SIM
supplemented
ith
15
mg
F1
hygromycin
esulted
n a
significant
umber
f
escapees
data
not
shown).
Thus,
bombardedallusesweretransferredor election oAV2
liquid
SIM
supplemented
ith
3.75
mg
F1
hygromycin.
Hygromycin
oncentrations
ere
ncreased
tepwise
ver
2-wkintervals
o
7.5
mg
F1,
and
finally
o 5
mg
F1.
Proliferation
f
putatively
ransformed
alluses
was
ob-
served
within
wk
following
ombardment.
alluses
were
positively
tained
orGUS
(Fig.
2b).
Unfortunately,
evere
necrosis
asobserved
t this
tage
fthe
election
rocess
in
liquid
hampering
he
regeneration
f
transgenic
hoots.
Within
-5
d,
the
iquid
AV2
medium
urned
ark
brown
indicating
he
presence
f
phenolic
ompounds
ecreted
from
the
putative
transgenic
alluses.
Control
non-
bombarded
alluses
didnot xhibit
ny
necrotic
ymptoms
when
ultured
n
AV2
iquid
medium.
We
studied
he
ffect
ofreducingecrosis fputativelyransformedloecalli
n
liquid
ultures
uring
election
nd
regeneration.
oderate
callus
survival
was
observed
when
AV2
medium
was
supplemented
itha combination
f
70
mg
F1
DTT,
1.5
mg
F1
gallic
acid,
and
150
mg
F1 citric
cid.
AV2
medium
upplemented
ith
ither
VP or
biopterin
as
unable
to
inhibit
rowning
data
not
shown).
Amberlite
beads,
containing
he
XAD-4
resin,
were
found
o
best
promote
allus
viability
nd
survival
uring
election
n
Table 3.
Effects
f
DTT
and
GA3
as
pretreatments
n transient
US
expression
nd
expiant
iability
AV2
medium
upplement
ith
24-h
treatment)
No
supplement
DTT
(70mg
I"1)
GA3
(5mg
F1)
DTT
(70mg
F1)
+
GA3
(5mg
F1)
Mean
number
f
transientUS
events3
27±4ac
148±6b
39±5c
204±9d
Expiant
viability%)b
3.1iQ.7e
50.7±6.4f
11.4*3.2
g
87.6±9.2
h
a
GUS-positive
vents
were cored
er
bombarded
late
b
Expiants
were
maintained
ollowing
ombardment
n
solid
AV2
medium
without
ny
ntibiotics.
iability
green
ealthy
xpiant
s. brown
ecroting
ones)
was
scored
wk after
ombardment
cEach
experiment
as
repeated
or
hree
imes.
aluesfollowed
y
the ame
etter
re
not
ignificantly
ifferent
t/?
0.05,
(Turkey,
953)
â
Springer
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8/9
ROLES OF
AMBERLITE XAD-4
RESIN AND ANTIOXIDANTS ON ALOE VERA
TRANSFORMATION
483
AV2
medium
Fig.
3).
Within ̂ d of
culture,
he
lginate
beads
containing
he resin urned ark
brown,
ndicating
that
they
were able
to absorb some of the
phenolic
compounds
ecretednto he medium. eads
lacking ny
resin ervings control id not hangencolor nd did not
reduce allus necrosis.
egenerating
alluseswere tained
blue
during
he election
rocess,
nd heGUS
staining
as
also visible t the
differentiating
hoot
primordia
n the
calli
Fig.
2c).
Approximately
.7%
GUS-positive
egener-
ating lants
were bserved fterransfero shoot
longation
medium
upplemented
ith1
mg
F1 ZR
and 15
mg
F1
hygromycin.
US
staining
n
mature
hootswas located
mainly
n
the tems nd
n
the eafveins
Fig. 2d).
Thirty-
seven
putative-transformedlants riginated
rom ifferent
calluses ooted n
MS
hormone-free
edium
upplemented
with 5
mg
F1
hygromycinFig.
Id).
PCR
amplification
f
genomic
NA
was
performed
or
2
putative
ygromycin-
resistantlants. heDNA of all of theseplants ontained
the
expected
ands of 680
bp
and
838
bp.
These bands
were not
present
n
the
genomic
DNA
fromnon-
transformedontrol
lants.
CR
analysis
learly
onfirmed
that Aloe
vera
plants regenerated
nder
hygromycin
selection
were
positive
for the
inserted
ene (data
not
shown).
outhern lot
hybridization
as
performed
sing
these
12
plants
Fig.
4).
Analysis
f transformed
lants
confirmedtable
ntegration
f the
hptgene
nto he
plant
genome.Transgenic lants
containedfrom
1-3
bands
hybridizing
ith
hpt,
whereas
non-transgeniclants
did
not
yield ny
bands
not
hown).
Although
he
plants
were
selected
randomly,
t seems that
multiple
ransformants
(namely lantdesignated -4, 5-6, 8-9, and 10-11)may
have
originated
rom
ingle
ransformation
vents,
espec-
tively. lthough ene
transfer
y particle
ombardments
widely
sed n
monocots,
everal
actors
ere ound
o
play
a
major
ole
pecifically
n
Aloe vera
ransformation:
1)
the
appropriatexpiants
eing
competent
or
transformation
Figure
3.
The
effect f
different
ntioxidants
nd
Amberlite AD-4
resin n
the
viability
f
Aloe
vera calli
cultured n
AV2
MS
medium
supplemented
ith
mg
F1
TDZ and
0.1
mg
F1
IBA.
Figure
4. Southern
ybridization
f Noti
digested
NA isolated rom
transgenic
ygromycin-resistant
ature loe
plants.
Genomic DNA
was
hybridized
o a 0.8 kb
Sstll
fragment
f
DNA
containing art
f
the 35S
cauliflowermosaic
virus
promoter
nd
the
hygromycin pt
gene.
and
regeneration,
2)
pretreatment
f the
expiants
sing
DTT
and
GA3
before
ombardment,
3)
establishmentf n
appropriate
tepwise
ncreased
iquid
election
ystem,
nd
(4)
post-bombardment
ecoveryrocedure
tilizing
edium
supplemented
ith
amberliteXAD
resin
entrapped
n
alginate
eads.
Although
mberlite
s
mainly
sed to
trap
and
extract
ifferent
econdary
metabolite
roduce
n
vitro
(Falket al. 1990),wecannot ule ut he ossibilityhathe
beneficialffect
f
Amberlitean
be
attributedlso to
the
possibility
that
medium-organic
ompounds
are
also
trapped
y
the
mberlite
esin
Mucciarelli
t al.
2009).
Conclusion
Here
we
report,
or he first
ime,
he
successful
iolistic
transformation
fAloe
vera
nd the
production
f
transgenic
Aloevera
plants.
n
v/iro-germinated
eedlings
erved
s the
initial
xpiants
n
this
study.
The
regeneration
rotocol
reportederewas foundo be highlyfficientndsynchro-
nous,
nd
enabled
witching
rom
differentiated
tage
o a
non-differentiated
tage
nd
vice
versa.
The
morphogenetic
potential
four
callus
cultures
as
been
successfully
ain-
tained or
he ast
yr.
To the
best of
our
knowledge,
his s the
first
eport
suggesting
mberliteAD
resin s a tool
o
mprove
xpiant
viability
nd
reduce
necrosis
during
ntibiotic
election
following
ransformation.
Ö
Springer
80 n
™£
70
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g,
60
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484
VELCHEVA ET
AL.
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Rapid
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uring
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Dan Y.
Biological
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2008.
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Prieto-Samsonov.
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