Aloe vera transformation: the role of Amberlite XAD-4 resin and antioxidants during

selection and regeneration

Author(s): Margarita Velcheva, Zehava Faltin, Aliza Vardi, Uri Hanania, Yuval Eshdat, Oded
Dgani, Nachman Sahar and Avihai Perl
Source: In Vitro Cellular & Developmental Biology. Plant, Vol. 46, No. 6 (November/December
2010), pp. 477-484
Published by: Society for In Vitro Biology
Stable URL: http://www.jstor.org/stable/40981336 .
Accessed: 25/06/2014 01:57
Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at .
http://www.jstor.org/page/info/about/policies/terms.jsp

.
JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of
content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms
of scholarship. For more information about JSTOR, please contact support@jstor.org.

Society for In Vitro Biology is collaborating with JSTOR to digitize, preserve and extend access to In Vitro
Cellular &Developmental Biology. Plant.

http://www.jstor.org

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

In VitroCell.Dev.Biol.- Plant(2010) 46:477^84

DOI 10. 1007/s11627-010-9301-z

TRANSFORMATION/
BIOTECHNOLOGY/GENETIC
FUNCTIONALGENOMICS

theroleofAmberlite
XAD-4 resin
Aloe veratransformation:
and antioxidants
duringselectionand regeneration
MargaritaVelcheva Zehava Faltin Aliza Vardi
Uri Hanania Yuval Eshdat Oded Dgani
NachmanSahar AvihaiPerl

Received: 30 November2009 /Accepted:27 July2010 /Publishedonline: 14 September2010 / Editor:T. J. Jones

The SocietyforIn VitroBiology 2010

forgenetic
transformation
andsubsequent
AbstractA system
via
indirect
from
organognesis calluswas
plantregeneration
for
Aloe
vera.
developed
Youngseedlingsservedas primary
cultures
were
established
on Murashige
and
Callus
expiants.
with3 mg F1 benzySkoog(1962) mediumsupplemented
and2 mgF1 indoleaceticacid.A protocol
was
laminopurine
developedto switchfromthedifferentiated
stage,usingin
vitroshootsor youngregenerated
plants,back to the dedifferentiated
of
the
callus
and
vice versa.Long-term
stage
maintenanceof this callus paved the way for genetic
ofAloe vera.Calluseswerebombarded
witha
manipulation
uidA and hpt genes,both underthe
plasmidcontaining
controlof the 35S promoter.
Dithiothreitol
and gibberellic
acid were foundto play a majorrole in reducingtissue
necrosisfollowing
bombardment.
Transformed
shootswere
under
in
selection
regenerated
stepwise
hygromycinwithdifferent
anticontaining
liquidmediumsupplemented
oxidants.
Amberlite
XAD-4resinwas embedded
intoalginate
beads and added to the selectionmedium.Amberlite
was
best for adsorbingdifferent
phenolic compoundsand
occurredafter
blockingexpiantnecrosis.Shoot initiation
transfer
of thetransformed
cells to Murashigeand Skoog
mediumsupplemented
with 2.0 mg F1 thidiazuron
and
M. Velcheva Z. Faltin A. Vardi U. Hanania Y. Eshdat
O. Dgani N. Sahar A. Perl(E3)
of FruitTree Sciences,
Department
ResearchOrganization,The Volcani Center,
Agricultural
P.O. Box 6, Bet-Dagan 50250, Israel
e-mail:vhperl@agri.gov.il
PresentAddress:
M. Velcheva
AlbertaResearchCouncil,
Hwy 16A & 75 Street,P.O. Bag 4000, Vegreville,AB T9C IT,
Canada

0.1 mg 1 l indole butyricacid. Murashigeand Skoog

medium supplementedwith 1 mg F1 zeatin riboside
shootelongation.
promoted
Rootingandplantdevelopment
were obtainedon Murashigeand Skoog basal medium
with15 mg F1 hygromycin
supplemented
lackinggrowth
The transgenic
natureof theregenerated
regulators.
plants
was verifiedby histochemical
GUS assay and Southern
blothybridization.
Regeneration

KeywordsAloe Recurrent
callusogenesis
Genetictransformation
Introduction
The lastseveralyearshavewitnessed
a boominresearch
on
Aloe and its applicationsfor humans and mammals.
of some valuable traitssuch as
However,improvement
stressresistance,and modification
of
plantmorphology,
metabolites
used in medicineand cosmeticscan
secondary
methods.This
hardlybe achievedby conventional
breeding
is mainly
becauseofcytoplasmic
malesterility,
longvegetative
lowrecombination
andembryo
abortion
generation,
potential,
et
al
Most
(Velcheva
2005).
regeneration
systemsforAloe
use axillarybuds (Keijzer and Cresti 1987; Zhao 1990;
Corneanuet al 1994;Hirimburegama
and Gamage1995)or
inflorescences
(Richwine1995; Velchevaet al 2005) forin
vitromasspropagation.
Recentstudieshavefocusedon thein
vitropropagation
of Aloe usingeithershootmultiplication
and Kaviani2008) or somaticembryogenesis
(Hashemabadi
et al 2008). Fortransformation
based
(Garro-Monge
systems
on thedevelopment
of adventitious
the
of
shoots, production
chimericplantswas describedseveraltimes,as transgenic
thesameclonalorigin
et
plantshavenotnecessarily
(Matthews
al 1998; Flachowskyet al. 2008). This means thatthe
Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

478

VELCHEVA ET AL.

of adventitious
shootsdoes notnecessarily
lead
development
to plantsderivedfroma singlecell (Schmulling
and Schell
1993). Veryfewreports(Racchi 1988) have describedthe
oftruecallusfrom
Aloeortheconditions
forlongproduction
termmaintenance
ofthiscallus.
Aloe vera is considered
a primecandidatefor
Although
genetictransformation,
veryfewstudieshavereported
gene
transformation
and subsequentregeneration
of transgenic
plantsin Aloe (He et al 2007). Potentialreasonsforthis
include:(1) browning
and oxidativebursto Aloe expiants,
in
oftenleadingto expiantcell death
mainly liquidculture,
et
al
whichareusuallyutilized
(Natali
1990),(2) cytokinins,
to promoteregeneration,
have been reportedto enhance
secretion
ofphenolsinAloe cells(Nataliet al 1990;Meyer
and van Staden 1991; Roy and Sarkar 1991), and (3)
has been foundto be an additionalfactor
Agrobacterium
co-cultivation.
stimulating
expiantnecrosisfollowing
Polywhich
leads
to
tissue
necrosisand targetphenolexcretion,
explant death, has been previouslyreportedto limit
transformation
capabilityin different
plants (Lagrimini
1992;Perlet al 1996; Dan 2008). It has beenassumedthat
insteadof inducingcell de-differentiation
and competence,
die
woundedtissuesaccumulate
and
phenols
subsequently
with
Pretreatment
antioxidants
has
been
(Potrykus1991).
to minimizeoxidativeburst
foundto enhancecell viability
et al 1999; Dan
andto reducenecrosis(Enriquez-Obregon
2008). Dithiothreitol
(DTT) is knownas a reducingagent
used in plant
(Apstoletal 1989) andhas beenextensively
transformation
and regeneration.
Moreover,DTT has been
foundto be moreeffective
thanmanyotherantioxidants,
suchas ascorbicacid,polyvinylpyrrolidone
(PVP), and LXAD
resins
et
al
Amberlite
adsorbent
1996).
(Perl
cysteine
in
witha highlyporousstructure
adsorbents
are polymeric
whichits internalsurfacescan absorba wide varietyof
in which
on theenvironment
different
moleculesdepending
XAD-4
is
Amberlite
of
resin
used.
are
used
and
the
type
they
such
aromatic
used
for
the
removal
of
hydrocarbons
mainly
as phenolsand pesticidesfromwastes.However,theyhave
of specificphytochemalso beenutilizedforbothextraction
icals producedin vitroand removalof toxic compounds
affecting
expiantviabilityin plantcell cultures
negatively
(Strobelet al 1991; Ui et al 1993; Georgievet al 2006;
Mucciarelli
et al 2009).
In this study,we reportthe developmentof a highly
in Aloe
and reproducible
efficient
systemforregeneration
vera via callus culturesand proceduresforthe in vitro
long-termmaintenanceof such morphogeneticcells.
calluses
ofthesemorphogenetic
Successfultransformation
of
and subsequentregeneration
by particlebombardment
transformed
plants were achieved. Antioxidantsand
Amberliteresinwere foundto play a majorrole in the
ofnecrosisduringtheselectionprocessin liquid
reduction
cultures.

Materialsand Methods
ExpiantPreparationand CallusInduction.Aloe veraseeds
were sterilized(1% NaOCl, 15 min) and washed three
times with steriledistilledwater.Seeds (30 seeds per
Magenta box) were culturedon 50 ml Murashigeand
Skoog (MS; Murashigeand Skoog 1962) solid (0.25%
underlight(40 pinol
Gelrite)basalmediumforgermination
m2 s"1) at 25C. Two-to 3-wk-oldin vitroseedlingswere
used as primary
explants.Explantswerecut into0.7-1.0cm thicksectionsunderhalf-strength
MS liquid media,
with70 mgF1 DTT, washedthreetimes(3 x
supplemented
10 min) withthe same media,and transferred
to callus
induction
media(CIM). All mediawerebased on MS salts
and vitamins,
3% sucrose,70 mg F1 DTT, 0.25% Gelrite
and
different
(pH 5.2),
(mediadesignagrowthregulators
tion MSI to FRS3, Table 1). Calluses were subcultured
every4 wk on freshmediaandwerekeptin thedarkat 24
C beforetransferto shoot inductionmedia (SIM) for
regeneration.
Callus Re-initiation
and Long-term
Maintenanceof Callus
shoots
Cultures.In vzYro-initiated
shoots,in vzYro-elongated
or in vitroyoungplantsservedas expiantsforcallus reinitiation
studies.To re-initiate
callus fromtheseexpiants,
the proceduredescribedabove was applied,and expiants
withthe same
were culturedon MS media supplemented
callus was sub(Table 1). Re-initiated
growthregulators
cultured
everymo to freshmediumand callusfreshweight
To test
was monitored
duringthreesuccessivesubcultures.
calluses
after
of
the
re-initiated
themorphogenetic
potential
12 mo of successivesubcultures,
all callusesthatdeveloped
to
combinations
weretransferred
on all thegrowth
regulator
AV2 SIM (Table 1). The numberof regenerated
plantsper
calluswas monitored.
to SIM (desCallusesweretransferred
Plant Regeneration.
were
Table
SIM
to
AV0 AV3NAA,
1).
composedof
ignation
solidified(0.25% Gelrite)MS salts and vitamins,3%
70 mg F1 DTT (pH 5.8),
sucrose,0.5 g F1 myo-inositol,
acid (IBA),
and eitherzeatinriboside(ZR), indole-3
-butyric
combinations
acid (NAA) in different
or napthaleneacetic
Four wk aftershootinduction,
and concentrations.
regentoeither
eratedshootsweretransferred
0.5, 1,or2 mgF1 ZR
in MS mediaforshootelongation.
Elongatedplantswere
rootedon MS basal mediumlackinggrowthregulators.
in whitelightat 40 jimolm"2s"1
weremaintained
Cultures
to potscontaining
and 25C. Rootedplantsweretransferred
to the
and
for
mixture
standardsoil
hardening transferred
greenhouse.
were carriedout in five replicates.The
Experiments
werescoredevery4 wk.
numberof shootsand rgnrants
test.
difference
Data wereanalyzedusingtheleastsignificant

Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

ROLES OF AMBERLITE XAD-4 RESIN AND ANTIOXIDANTS ON ALOE VERATRANSFORMATION

Table 1. MS media supplementedwithdifferent
growth
regulatorsforcallus initiation
(CIM) or shootinduction(SIM)
of Aloe vera

Media designation
Callus Initiation
Media (CIM)

2,4-Da
MS I

IAA

l
Phytohormones
(mg 1 )

ZR

TDZ

BA

Kinetin
0.2

05

0.2

MS IV

NAA

MS n
MS III

Shoot Induction
Media (SIM)

IBA

479

FRSi

FRS2

FRS3

AV0

0.0

AVj

01

2
x

AV2

0.1

AV3

0.1

AVjNAA

0.1

AV2NAA

0.1

AV3NAA

0.1

BiolisticTransformation
ofAloe VeraCalluses.Forty-eight
h beforebombardment,
calluseswere transferred
to AV2
SIM withorwithout
70 mgF1 DTT and5 mgI"1GA3.The
thehptanduidAgenes,both
plasmidWAG1515containing
underthe controlof the 35 S cauliflowermosaic virus
promoter,was used for particle bombardment.Gold
particles(1.0 |nmin diameter)were coatedwithplasmid
DNA following
theprotocolofPerlet al (1992). Different
acceleration
pressures(450, 900 and 1,100psi) combined
withtwo targetdistances(8.5 and 11.0 cm) betweenthe
stoppingscreen and targetplate were tested.Optimal
bombardment
conditionswere evaluatedby scoringtransient-glucuronidas
(GUS) expression.

by autoclavingat 121C for20 min. AmberliteXAD-4

resins (Sigma) were sterilizedby immersingin 100%
methanolfor10 min,followedby a wash in 100% ethanol
(for 10 min),and finallythreewashes in steriledistilled
water.Amberlite
particleswere mixed(1:1 v/v)withthe
solution.
The alginate-Amberlite
beads were
Na-alginate
solidified
a
25-ml
by gentledropping
(using
pipette)of the
mixtureinto a sterile solution of
alginate-Amberlite
100 mM CaCl2. Following10 minincubation
in CaCl2 at
roomtemperature,
the solidifiedbeads werewashedwith
steriledistilledwater,and 5 g ofbeads was addedto 50 ml
liquid AV2 selectionmedium.Beads thatturnedbrown
werereplacedweeklywithfreshbeads upon subculture
to
freshAV2selectionmedium.Na-alginate
beads lackingany
SelectionProcess.Hygromycin
(0-15 mg F1) was utilized resinservedas controlsand were also replacedweekly
forselection
in solid(MagentaGA7 vessels)or liquidmedia duringthestudy.
Afterbombardment,
(25-mlliquidin a 250-mlErlenmeyer).
callusesweretransferred
to freshAV2mediumsupplemented Histochemical
GUS activity
was analyzedin
Investigation.
with3.75mgF1 hygromycin.
concentration
was
bombarded
and
Hygromycin
calluses,shoots, regenerated
plantsaccordelevatedin a stepwise
in
2-wk
from
3.75to
Jefferson
et
al
GUS
was
manner,
intervals,
ing
(1987).
activity visualized
7.5 mgF1, andfinally
15
F1.
Shoot
under
after
reaching mg
elongation
stereoscope
washingthe stainedexpiantsin a
was performed
on MS mediumsupplemented
with1 mg F1
mixtureof 70% ethanoland 30% glacial acetic acid,
ZR and 15 mg F1 hygromycin.
followedby a wash in 10% lacticacid.
Transgenic
plantsrootedon
hormone-free
MS mediumsupplemented
with 15 mg F1
In case anti-necroting
wereutilized, Plant DNA Analysis.From37 putativetransgenic
hygromycin.
compounds
plants,
the putativetransformed
calluseswere transferred
to AV2 12 werechosenrandomly
forDNA extraction
and Southern
with the following hybridization.
PCR was used to detectthepresenceof the
liquid or solid media supplemented
antioxidants:
PVP (0.5 and 1 g F1), L-cysteine
uidA
in
tissuesaccordingto Fultonet al.
(150 mg F1),
gene transgenic
acid
DTT
The
gallic
wereused:5'-CAGC(1.5 mg F1),
(70 mg F1), biopterin
(15 mg (1995).
following
oligonucleotides
F1), ascorbicacid (150 mgF1), andcitricacid (150 mgF1).
GAAGAGGCAGTCAACGGGGAA-3'
and 5'Calluseswerecultured
on a gyratory
shaker(110 rpm)under CATTGTTTGCCTCCCTGCTGCGGTT-3'
a 680-bp
yielding
cool whitefluorescent
and 5'-GGTTGGGCAGGCCAGCGTATC-3'
light(20 |nmolm~2s"1)at 25C.
and
fragment,
5'-TAACCTCTCTTTAGGCATTGG-3'
a
yielding 838-bp
Amber
lite Beads. Na-alginate fragment.
PCR productswere visualizedby running
Preparationof Alginateon a
(3%) was dissolvedin hot,distilledwaterand sterilized 1.5% agarosegel containing
ethidiumbromide.Southern
Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

VELCHEVA ET AL.

480

was performed
to Perlet al (1996)
according
hybridization
withsomemodifications.
Aloe DNA was extracted
Briefly,
from3 g ofin vitrogrownplantsas described
(Hananiaetal
DNA
About
10
of
was
2004).
digestedwith Not!,
|LLg
in
to
0.8%
electrophoresed
agarose gel and transferred
N+
Gemembrane
NJ).
(Amersham,
Piscataway,
Hybond
witha 0.8-kbSstllfragment
of
nomicDNA was hybridized
DNA containing
partof the3' regionof the35S cauliflower
as wellas partofthe5' regionofthe
mosaicviruspromoter,
hygromycin
hptgene.

the typeof callus and its regenerative

potentialdiffered
to thegrowth
used.Rapid
significantly
according
regulators
in FRSi
of
callus
was
observed
on
cultured
growth
expiants
medium.However,thesecallusesfailedto regenerate
on all
SIM combinations
tested.Similarobservations
weremade
forcallusesdevelopedon FRS3,MS I, MS III, and MS IV
media.Callusesoriginated
on thesephytohormone
combinationsformedonlyrootson all SIM combinations
tested,
whileno shootregeneration
was observed.MS II medium,
withNAA andZR, inducedcallusformation,
supplemented
on the same medium,
but aftersubsequentsubcultures
necrosiswas visible,leadingto callusdeath.The onlycallus
thatwas foundto have a morphogenetic
potentialwas
Resultsand Discussion
a soft,
initiated
on FRS2 medium.Undertheseconditions,
were
pale greencallusdeveloped(Fig. la). Shootprimordia
ProtocolUsingAloe Vera visible4 wk aftertransfer
Establishment
ofthiscallusfromFRS2 to AV0ofa Regeneration
within2-3 wk withno
withN-phenyl-N'-l
Calluses. Seedlingsgerminating
2,3-thiadiazolAV3mediasupplemented
IBA
werecut into0.7-1.0-cmthickstem 5-yiurea[thidiazuron]
and
visiblecontamination
(TDZ)
(Fig. 'b). TDZ alone
Four
wk
cultured
on
different
CIM
and
to
was
unable
(Table 1).
(AV0 medium)
promoteshootregeneration.
expiants
all expiantsexhibitedcallus proliferation Seventy
afterinitiation,
of
the
calluses
(48 outof61) gaveriseto an
percent
to the originalpositionof the section averageof 14.6 shootsper callus on AV2 medium.Lower
withoutcorrelation
in
as inAVi mediumwerelesseffective
used as the explanton the germinated
seedling.Calluses TDZ concentrations
CIM
for12 mo on thedifferent
TDZ concentrations
shootregeneration.
weresubcultured
higher
monthly
promoting
SIM combinations. than2.0 mgF1, as intheAV3medium,
to different
reducedsignificantly
combinations
beforetransfer
of abnormal
all
CIM
the
on
initiation
and
induced
were
obtained
calluses
shoot
combinations,
regeneration
Although

callus
Figure 1. Morphogenetic
obtainedon FRS2 MS medium
of Aloe
(a). Shoot regeneration
to
vera calli followingtransfer
AV2 MS mediumsupplemented
with2 mg F1 TDZ and 0.1 mg
F1 IBA (b). Matureelongated
shooton MS mediumsupplementedwith 1 mg F1 ZR (c).
RootedAloe vera planton MS
medium(d).
hormone-free

Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

ROLES OF AMBERLITE XAD-4 RESIN AND ANTIOXID ANTS ON ALOE VERATRANSFORMATION

481

and differentiation
shootshavingsomemorphological
disorders
suchas fused Table 2. De-differentiation
efficiencyof calluses
fromdifferent
originated
expiants
thickenedleaf primordiasurrounded
by granulateddeep
callus.
These
shoot
failed
to developany Primaryin vitro
green
primordia
Callus freshweightincrease(g)
Average
"normal"
aloe
shoots.
Shoot
number
was
expiantsused for
morphological
regeneration
callus re-initiation Subculture
of plants
reducedwhen indoleacetic acid (IAA) was
dramatically
regenerated
with
NAA
replaced
(AV1NAA-AV3NAA
media).Optimal
I
II
III
percallusa
shootelongation
(calculatedas meannumberof elongated
shoots per expiant) was observedusing MS medium Germinated
14.10.9 16.671.9 18.220.9 13.41.3
with1 mgf1 ZR. On thismedium,initiated seedlings
supplemented
17.151.1 18.051.6 17.671.5 14.32,7
shootsbeganto elongateandthefirst
leaves Initiatedshoots
well-developed
shoots
14.561.6 19.670.8 18.920.5 11.61.4
were visible(Fig. 'c). Some tendencytowardsecondary Elongated
fromsingleshootswas visiblewhenhigher Regeneratedplants 16.012.1 19.231.7 19.041.3 12.82.2
organognesis
concentrations
of ZR wereused. An average78.7% of the a
Regenerated
plantswerescoredfollowing12 mo of successivemonthly
initiated
shoots(877 outof 1,113)elongatedand developed subcultures
in FRS2 CIM. At theend of thesesubcultures,
calluseswere
to AV2SIM
intovigorousplantsthatrootedsuccessfully
on MS basal transferred
mediumlackinggrowth
regulators
(Fig. Id).
In numerous
monocotspecies,embryo-derived
expiants
havebeenfoundtobe optimal
forin-vitro
regeneration
(Wuet
al 1990).Severaltypesofcallusesweredevelopeddepending ofswitching
froma differentiated
stageto a de-differentiated
on thecombination
ofgrowth
in
used.Groenewald
et stage Aloe vera and vice versa withoutany apparent
regulators
al. (1976), Racchi (1988), and Roy and Sarkar(1991)
in themorphogenetic
reduction
of thecallusline.
potential
observedthat2,4-D can promoteboth callus and shoot The regeneration
herewas foundto be
protocolreported
inductionin different
Aloe species. In our study,2,4-D highlyefficient,
and has been successfully
synchronous,
cell division,
butregeneration
was neverobserved, maintained
forthelast4 yr.
promoted
as was also reported
by Nataliet al. (1990) and Meyerand
van Staden (1991), suggestingthat2,4-D can probably Aloe Bombardment
and Selectionof TransgenicPlants.
of
some
Aloe
Calluses
wereused as a targetforparticlebombardment.
promoteregeneration only
In
species. The
combination
of IAA and BA was foundto promotecallus preliminary
oftransient
GUS
studies,thehighestefficiency
formation
fromall expiantsutilizedin this study.Similar expressionwas observedwhen the accelerationpressure
results
havebeenreported
forother
to was set to 1,100 psi and the distanceof the targetwas
plantspeciesbelonging
thefamilyLilaceae(Wanget al 1998). Shootregeneration 8.5 cm (data not shown).Undertheseconditions,
41% of
fromcallusesinAloeveraappearedto dependmainlyon the thecallusestransiently
rise
to
more
expressedGUS, giving
concentrations
of TDZ and IAA. WhenTDZ was applied than200 GUS-positivespotsperPetriplate(Fig. Id). Three
alonewithout
occurred.
bombardedcallusesturned
anyauxin,no regeneration
Onlythe to 4 d following
bombardment,
combination
of TDZ andan auxinsourcepromoted
and necrosiswas visible.It shouldbe notedthat
regener- brownish,
forAloe regeneration necrosiswas visiblealso in callusesbombardedwithonly
ation,as has beenpreviously
reported
thatnecrosismightbe correlated
(Nataliet al. 1990; Meyerand van Staden1991). ZR was
gold particles,indicating
foundto promoteshoot elongation.Shoot degeneration withthewoundingeffectof theprocedure.
We studiedthe
occurred
iftheinitiated
shootsweretransferred
to MS basal effectsof pretreatment
of thecalluseswithDTT and GA3
mediumlackinggrowth
on callus survivaland transient
GUS expression(Table 3).
regulators
(Richwine1995).
The additionof 70 mg I"1 DTT significantly
improved
Re-callusingofAloe InVitroExpiants.Once a regerenative expiantviability,as well as transientGUS expression
calluswas initiated,
FRS2 mediumwas foundto be optimal followingbombardment.
GA3 had onlya moderateeffect
to supportthe long-term
maintenance
of thiscallus while on improvingtransientGUS expression.However,the
its morphogenetic
retaining
potential.FRS2 mediumbest combinationof DTT and GA3 had an additiveeffect,
callusre-initiation
bothparameters
promoted
usingin vitroelongatedshoots improving
Chemotaxonomic
significantly.
or young plantletsas expiants.The efficiencyof de- studieshave indicatedthatphenols(mostlyflavonoids)are
as wellas subsequent
from majorcompoundsin speciesbelongingto thegenusAloe
differentiation,
plantregeneration
thesecalluses,was similarwithall expiantsused (Table2).
(Viljoen et al 1998). GA3 has been reportedto inhibit
of
the
initial
Sixty-three
one of thekeyenzymesin anthocyanin
percent
expiantsproducedregener- chalconesynthase,
able callus,enablingtheregeneration
ofup to 14 shootsper biosynthesis
(Ilan and Dougall 1994). In thisreport,
GA3
of calluscultureis
explant.As themorphogenetic
enhancedtransformation
potential
and reducedexpiant
efficiency
thisstudydemonstrated
thepossibility necrosis.We cannotrule out the possibilitythatother
usuallytime-limited,

Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

VELCHEVA ET AL.

482
Figure 2. TransientGUS expressionin Aloe vera calli 48 h
afterbombardment
(a). Stable
GUS expressionof Aloe vera
calluses followingculturein
50 ml liquid FRS2 selection
with5 g
mediumsupplemented
alginatebeads containing
AmberliteXAD4 resin,15 mg
3 mg F1 BA
F1 hygromycin,
and 2 mg F1 IAA (b). GUS
expressionin callus and regenerationshootprimordia(SP) on
with
MS mediumsupplemented
2 mg F1
15 mg F1 hygromycin,
TDZ and 0.1 mg I"1 IBA (c).
Aloe vera
Stablytransformed
planton MS elongationmedium
with 1 mg F1 ZR
supplemented
and 15 mg F1 hygromycin
(d).

ofGA3
effect
forthebeneficial
areresponsible
mechanisms
on Aloe veratransformation.
In preliminary
studies,selectionon solid AV2 SIM
resultedin a
with 15 mg F1 hygromycin
supplemented
numberof escapees (data not shown). Thus,
significant
forselectionto AV2
bombardedcallusesweretransferred
3.75
with
SIM
mg F1 hygromycin.
supplemented
liquid
increased
were
concentrations
stepwiseover
Hygromycin
2-wk intervalsto 7.5 mg F1, and finallyto15 mg F1.
calluses was obof putativelytransformed
Proliferation
Calluseswere
bombardment.
servedwithin3 wk following
severe
stainedforGUS (Fig. 2b). Unfortunately,
positively
necrosiswas observedat thisstageof theselectionprocess
shoots.
of transgenic
theregeneration
in liquidhampering

Within4-5 d, theliquidAV2 mediumturneddarkbrown

indicatingthe presenceof phenoliccompoundssecreted
from the putative transgeniccalluses. Control noncallusesdid notexhibitanynecroticsymptoms
bombarded
in AV2liquidmedium.We studiedtheeffect
whencultured
Aloe calli in
transformed
of putatively
necrosis
of reducing
Moderate
and
selection
regeneration.
liquidculturesduring
was
medium
callus survivalwas observedwhen AV2
with a combinationof 70 mg F1 DTT,
supplemented
1.5 mg F1 gallic acid, and 150 mg F1 citricacid. AV2
witheitherPVP or biopterinwas
mediumsupplemented
unable to inhibitbrowning(data not shown).Amberlite
beads, containingthe XAD-4 resin,were foundto best
promotecallus viabilityand survivalduringselectionin

GUS expressionand expiantviability

on transient
Table 3. Effectsof DTT and GA3 as pretreatments
AV2 mediumsupplementwith(24-h treatment)
No supplement
GUS events3
Mean numberof transient
Expiantviability(%)b

DTT (70mg I"1)

274ac

1486b

3.1iQ.7e

50.76.4f

GA3 (5mg F1)

DTT (70mg F1) + GA3 (5mg F1)

395c
11.4*3.2 g

2049d
87.69.2 h

a
eventswerescoredperbombarded
plate
GUS-positive
b
on solidAV2mediumwithoutanyantibiotics.
Viability(greenhealthyexpiantvs. brownnecroting
bombardment
maintained
were
following
Expiants
ones)was scored2 wk afterbombardment
different
1953)
at/?<0.05, (Turkey,
was repeatedforthreetimes.Valuesfollowedby thesame letterarenotsignificantly
cEach experiment

Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

ROLES OF AMBERLITE XAD-4 RESIN AND ANTIOXIDANTS ON ALOE VERATRANSFORMATION

12

5678

483

9 10 11 12

thealginate
AV2medium(Fig. 3). Within3^ d of culture,
the resinturneddarkbrown,indicating
beads containing
23.1 .
that they were able to absorb some of the phenolic
compoundssecretedintothemedium.Beads lackingany
resinservingas controldidnotchangein colorand didnot
reducecallusnecrosis.Regenerating
calluseswerestained
9.4theselectionprocess,andtheGUS staining
blueduring
was
also visibleat the differentiating
shootprimordiaon the
mm
6.5calli (Fig. 2c). Approximately
8.7% GUS-positiveregenerto shootelongation
atingplantswereobservedaftertransfer
mediumsupplemented
with 1 mg F1 ZR and 15 mg F1
4.3GUS stainingin matureshootswas located
hygromycin.
mainlyin thestemsand in theleafveins(Fig. 2d). Thirtysevenputative-transformed
fromdifferent
plantsoriginated
callusesrootedon MS hormone-free
mediumsupplemented
with15 mgF1 hygromycin
of
(Fig. Id). PCR amplification
2.3DNA
12
was
for
genomic
performed
putativehygromycinresistant
plants.The DNA of all of theseplantscontained
4. Southernhybridization
ofNoti digestedDNA isolatedfrom
the expectedbands of 680 bp and 838 bp. These bands Figure
maturealoe plants. Genomic DNA
transgenichygromycin-resistant
were not present in the genomic DNA from non- was hybridizedto a 0.8 kb Sstll fragmentof DNA containingpartof
transformed
controlplants.PCR analysisclearlyconfirmed the 35S cauliflowermosaic viruspromoterand the hygromycinhpt
that Aloe vera plants regeneratedunder hygromycin gene.
selectionwere positivefor the insertedgene (data not
blothybridization
was performed
of the expiantsusing
shown).Southern
(2) pretreatment
using and regeneration,
these 12 plants(Fig. 4). Analysisof transformed
DTT
and
before
ofan
GA3
bombardment,
plants
(3) establishment
confirmed
stableintegration
of thehptgene intotheplant appropriate
and
stepwiseincreasedliquidselectionsystem,
medium
genome. Transgenicplants containedfrom 1-3 bands (4) post-bombardment
recovery
procedure
utilizing
withhpt,whereasnon-transgenic
hybridizing
plantsdid supplementedwith amberliteXAD resin entrappedin
notyieldanybands(notshown).Although
theplantswere alginatebeads. Althoughamberlite
is mainlyused to trap
selectedrandomly,
it seems thatmultipletransformantsand extractdifferent
metabolite
secondary
producein vitro
and
et
al.
we
cannot
rule
out
the
thatthe
1-4,
5-6,
8-9,
(namelyplantdesignated
10-11) may (Falk
1990),
possibility
have originated
fromsingletransformation
beneficial
effect
of
Amberlite
can
be
attributed
also
to the
events,respectransfer
bombardment
is
that
tively.Althoughgene
by particle
possibility
medium-organiccompounds are also
severalfactors
werefoundtoplay trappedby theamberlite
resin(Mucciarelliet al. 2009).
widelyusedinmonocots,
a majorrolespecifically
inAloe veratransformation:
(1) the
appropriate
expiantsbeing competentfor transformation

mmmm

mm

Conclusion

80 n

70
g, 60

- --

50-

- -

_{~| '

40

| 20-7i g j '" n.7 ^

.---3SJ

. - _^._f!

3 30

j
'

r
/

;| ,

antioxidantsand AmberliteXAD-4
Figure 3. The effectof different
resinon the viabilityof Aloe vera calli culturedin AV2 MS medium
with2 mg F1 TDZ and 0.1 mg F1 IBA.
supplemented

Here we report,forthe firsttime,the successfulbiolistic

transformation
ofAloe veraand theproduction
of transgenic
Aloe veraplants.In v/iro-germinated
seedlingsservedas the
initialexpiantsin this study.The regeneration
protocol
herewas foundto be highlyefficient
and synchroreported
froma differentiated
nous,and enabledswitching
stageto a
non-differentiated
and
vice
versa.
The
stage
morphogenetic
of our calluscultures
has been successfully
mainpotential
tainedforthelast4 yr.
To the best of our knowledge,this is the firstreport
Amberlite
XAD resinas a toolto improve
suggesting
expiant
viabilityand reduce necrosisduringantibioticselection
transformation.
following

Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions

484

VELCHEVA ET AL.

References

Meyer H. J.; van Staden J. Rapid in vitro propagationof Aloe

barbadensis Mill. Plant Cell Tiss Organ Cult 26: 167171Mucciarelli M, Bertea CM, Camusso W (2009) Sugar
of an oxidative
ApstolL; HeinsteinP. F.; Low P. S. Rapid stimulation
metabolismof Vetiveria
zizanioidescells culturedwithAmberlite
burstduringelicitationof culturedplantcells. Plant Physiol90:
XAD-4. Uptakeof radiolabelledsugars,cell wall invertaseactivity
109-116; 1989.
and gene expression.J Plant Interactions
4: 23-31; 1991.
CorneanuM; CorneanuG.; Badica C; Minea R.; Bica D.; Vekas L.
Mucciarelli M.; Bereta CM.; Camusso W. Sugar metabolismof
In vitroorganognesisat Aloe arborescens (Liliaceae). Revue
Vetiveriazizaniodes cells cultured with Amberlite XAD-4.
Roumainede Biologie 39: 45-52: 1994.
of radiolabelledsugars,cell wall invertaseactivityand
Uptake
Dan Y. Biological functions
of antioxidants
in planttransformation.
In
J Plant Interactions4: 23-31; 2009.
gene
expression.
VitroCell Dev Biol Plant 44: 149-161; 2008.
F. A revised medium for rapid growthand
T.;
Murashige
Skoog
G. A.; Prieto-Samsonov
D. L.; de la Riva G. A.; Perez
Enriquez-Obregon
withtobacco tissueculture.PhysiolPlant 15: 473^97;
bioassay
M.; Selman-HouseinG.; Vasquez-PadronR. I. Agrobacterium1962.
mediatedJapnicarice transformation:
a procedureassistedby an
Natali L.; Sanchez I. C; Cavallini A. In vitro culture of Aloe
antinecrotic
treatment.
Plant Cell Tiss Organ Cult 59: 159-168;
barbadensisMill: micropropagation
fromvegetativemeristems.
1999.
Plant Cell Tiss Organ Cult 20: 71-74; 1990.
Falk K. L.; GershenzonL.; CroteauR. Metabolismof monoterpenes
PerlA.; Kless H.; BlumenthalA.; Galili G.; Galun E. Improvement
of
in cell cultureof commonsage (Salvia offcinallis).
Plant Physiol
and GUS expressionin scutellarwheatcalli by
plant
regeneration
93: 1559-1567; 1990.
Mol Gen Genet235: 279-284; 1992.
optimization.
FlachowskyH.; Riedel M.; Reim S.; Hanke M. V. Evaluationof the
Perl A.; Lotan O.; Abu-Abied M.; Holland D. Establishment
of an
and stabilityof T-DNA integration
and gene expresuniformity
mediatedtransformation
Agrobacteriumsystemforgrape (Vitis
sion in transgenicapple plants.ElectronicJournalof BiotechviniferaL.): the role of antioxidantsduring the grape-^4gronology[online]. 15 January2008, vol. 11, no. 1 Available from
bacteriuminteractions.
Nat Biotechnol14: 624-628; 1996.
Internet:httpV/www.ejbiotechnology.info/content/voll
1/issuel/
I. Gene transferto plants: assessment of published
Potrykus
full/1
0/index.html.
ISSN 0717-3458; 2008.
approachesand results.Ann Rev Plant Physiol Plant Mol Biol
FultonT. M.; ChunwongseJ.; TanksleyS. D. Microprepprotocolfor
42: 205-225; 1991.
extractionof DNA fromtomatoand otherherbaceous plants.
Racchi M. L. Using in vitrocultureto studythe biosynthesisof
Plant Mol Biol Rep 13: 207-209; 1995.
secondary products in Aloe Ferox. Rivista di Agrocultura
Garro-MongeG.; Gatica-Arias A. M.; Valdez-Melara M. Somatic
e Tropicale82: 707-714; 1988.
Subtropicale
in
and acemannandetection
embryogenesis,
plant regeneration
RichwineA. Establishment
of Aloe, Gasteriaand Haworthiashoot
aloe {Aloe barbadensisMill). AgronomiaConstarricense32: 41culturesfrominflorescenceexplants.Hort Sci 30: 1443-1444;
52; 2008.
1995.
of
GeorgievM.; Kovacheva E.; MarchevaN.; Llieva M. Purification
and micropropagation
of
Roy S. C; SarkarA. In vitroregeneration
rosmarinicacid extractsfromLavandula vera MM cell biomass.
Aloe vera L. Sci Hort 47: 107-113; 1991.
Food Chem 94: 111-114; 2006.
SchmullingT.; Schell J. Transgenictobacco plantsregeneratedfrom
GroenewaldE. G.; Koeleman A.; Wessels D. C. J. Callus formation
leaf disks can be periclinalchimeras.Plant Mol Biol 21: 705and plantregeneration
fromseed tissueof Aloe pretoriensisPole
708; 1993.
75: 270-272; 1976.
Evans. Z Pflanzenphysiol
StrobelJ.;Hieke M.; GrogerD. Increasedanthraquinone
productionin
Hanania U.; VelchevaM.; SaharN.; Perl A. An improvedmethodfor
Galium vernumcell culturesinducedby polymericadsorbents.
the isolationof highqualityDNA fromVitisvinifera.Plant Mol
Plant Cell Tiss Organ Cult 24: 207-210; 1991.
Biol Rep 22: 173-177; 2004.
TurkeyJ. W. The problem of multiplecomparisons.PhD thesis,
ofAloe vera L.
HashemabadiD.; Kaviani B. Rapid micro-propagation
PrincetonUniversity.,
Princeton;1953.
via shootmultiplication.
AfricanJ Biotech7: 1899-1902; 2008.
Ui S.; Yamada K.; Hyuga S.; Seki H. Effectsof additionwithvarious
He C; Zhang J.; Chen J.; Ye X.; Du L.; Dong Y; Zhao H. Genetic
adsorbentson protoplastcultureof grapes.Plant Tiss Cult Lett
of Aloe barbadensis Miller by Agrobacterium
transformation
10: 172-175; 1993.
J GenetGenomics34: 1053-1060; 2007.
tumefaciens.
Velcheva M.; FaltinZ.; VardiA.; Eshdat Y; Perl A. Regenerationof
of Aloe vera
K.; Gamage N. In vitromultiplication
Hirimburegama
Aloe arborescens via somaticorganognesisfromyoung inflormeristemtips formass propagation.Hort Sci 27: 15-18; 1995.
escences. Plant Cell Tiss Organ Cult 83: 293-304; 2005.
lian A.; Dougall D. K. Effectsof gibberellicacid and uniconazoleon
and
Viljoen A. M.; van Wyk B. E.; van HeerdenF. R. Distribution
the activitiesof some enzymesof anthocyaninbiosynthesisin
mAloe (Asphodelaceae).
offlavanoids
chemotaxonomic
significance
carrotcell cultures.J Plant GrowthReg 13: 213-219; 1994.
PlantSystemEvol 211: 31-12; 1998.
R. A.; Kavanagh T. A.; Bevan M. W. GUS fusions: Jefferson
Wang L.; Ming B. X.; Huang B. Q.; Shui H.; Wang L.; Bao X. M.;
glucuronidaseas a sensitiveand versatilegene fusionmarkerin
Huang B. Q.; Hao S. Somatic embryogenesispotentialdetermined by the morphologicalpolarityof the expiant in tissue
higherplants.EMBO J 6: 3901-3907; 1987.
culturesof Freesia refracta.Acta Bot Sinica 40: 138-143; 1998.
Keijzer C. J.; Cresti M. A compositionof anthertissue of Aloe
of
and regeneration
Wu R.; KemmerE.; McElroy D. Transformation
pretoriensisPole Evans. Z Pflazenphysiol75: 270-272; 1987.
L. M. Wound-induced
depositionofpolyphenolsintransgenic
cropplants:rice as themodel systemformonocots.In:
important
Lagrimini
II.
GustafsonJ. P. (ed) Gene manipulationin plantimprovement
peroxidase.Plant Physiol96: 26-31; 1992.
plantsoverexpressing
9th StadlerGeneticsSymposium.Missouri,Columbia,pp 251MatthewsH.; Dewey V.; WagonerW.; BestwickR. K. Molecularand
263: 1990.
cellularevidenceof chimaerictissuesin primarytransgenicsand
J Shenyang
ofAloe varieties
Zhao M. Rapidpropagation
eliminationof chimaerismthroughimprovedselectionprotocols.
bytissueculture.
AgrUniv 21: 339-340; 1990.
TransgenicRes 7: 123-129; 1998.

4y Springer

This content downloaded from 91.229.229.13 on Wed, 25 Jun 2014 01:57:38 AM

All use subject to JSTOR Terms and Conditions