Aloe vera transformation: The role of Amberlite XAD-4 resin and antioxidants during selection and...

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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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:

[email protected]

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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3/9

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




4/9

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




5/9

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




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




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




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

_{~|

-

j

g,

60

'

'

a

50-

- --

-

- -

-

i

S

40

-

.---3SJ .

3

30

.

-

_^._f

,

|

20-

i g j«

'"

n.7

^

r

| ;|

,

• £

$

/

/

f /

^

ï

Ï

12

3

4

5678 9 10 11 12

23.1

-

.

tü

t

mmmm

mm

9.4-

6.5-

mm

4.3-

2.3-




9/9

484

VELCHEVA ET

AL.

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