Agrobacterium

tumefaciens-mediated

transformation

of

poinsettia

Euphorbia

pulcherrima

with

virus-derived

hairpin

RNA

constructs

confers

resistance

to

Poinsettia

mosaic

virus

Agrobacterium-mediated

transformation

for

poinsettia

-LRB-

Euphorbia

pulcherrima

Willd

Ex

Klotzsch

-RRB-

is

reported

here

for

the

first

time

Internode

stem

explants

of

poinsettia

cv

Millenium

were

transformed

by

Agrobacterium

tumefaciens

strain

LBA

harbouring

virus-derived

hairpin

-LRB-

hp

-RRB-

RNA

gene

constructs

to

induce

RNA

silencing-mediated

resistance

to

Poinsettia

mosaic

virus

-LRB-

PnMV

-RRB-

Prior

to

transformation

an

efficient

somatic

embryogenesis

system

was

developed

for

poinsettia

cv

Millenium

in

which

about

of

the

explants

produced

somatic

embryos

In

experiments

utilizing

explants

independent

transgenic

lines

were

generated

An

average

transformation

frequency

of

-LRB-

range

-RRB-

was

revealed

Stable

integration

of

transgenes

into

the

poinsettia

nuclear

genome

was

confirmed

by

PCR

and

Southern

blot

analysis

Both

single

and

multiple-copy

transgene

integration

into

the

poinsettia

genome

were

found

among

transformants

Transgenic

poinsettia

plants

showing

resistance

to

mechanical

inoculation

of

PnMV

were

detected

by

double

antibody

sandwich

enzyme-linked

immunosorbent

assay

-LRB-

DAS-ELISA

-RRB-

Northern

blot

analysis

of

low

molecular

weight

RNA

revealed

that

transgene-derived

small

interfering

-LRB-

si

-RRB-

RNA

molecules

were

detected

among

the

poinsettia

transformants

prior

to

inoculation

The

Agrobacterium-mediated

transformation

methodology

developed

in

the

current

study

should

facilitate

improvement

of

this

ornamental

plant

with

enhanced

disease

resistance

quality

improvement

and

desirable

colour

alteration

Because

poinsettia

is

a

non-food

non-feed

plant

and

is

not

propagated

through

sexual

reproduction

this

is

likely

to

be

more

acceptable

even

in

areas

where

genetically

modified

crops

are

currently

not

cultivated

Introduction

Poinsettia

Euphorbia

pulcherrima

Willd

Ex

Klotzsch

is

a

contemporary

symbol

of

Christmas

in

most

parts

of

the

world

Since

it

was

introduced

to

the

United

States

in

from

Mexico

poinsettia

has

become

the

primary

potted

flower

produced

and

sold

in

North

America

Europe

Asia

and

Australia

-LRB-

Ecke

et

al.

Williams

-RRB-

Today

Europe

and

North

America

represent

the

largest

volume

of

production

and

sales

but

demand

is

growing

quickly

in

the

Australian

region

as

poinsettia

becomes

more

popular

each

year

-LRB-

Williams

-RRB-

Global

production

of

poinsettia

has

exceeded

hundreds

of

millions

and

is

still

expanding

indicating

its

economic

and

market

potential

for

the

floral

industry

Genetic

engineering

is

an

important

tool

for

breeding

ornamental

plants

with

addition

of

desirable

traits

such

as

novel

colour

better

quality

and

resistance

to

pathogens

and

insects

-LRB-

Mol

et

al.

Deroles

et

al.

Hammond

Hammond

et

al.

-RRB-

This

technology

has

been

successfully

utilized

in

the

production

of

a

number

of

important

ornamental

crops

e.g.

blue

roses

-LRB-

Yoshikazu

-RRB-

novel

carnations

-LRB-

-RRB-

transgenic

gladiolus

-LRB-

Kamo

et

al.

-RRB-

and

improvement

of

chrysanthemums

-LRB-

Teixeira

da

Silva

-RRB-

To

date

transgenic

ornamentals

of

over

genera

have

been

produced

by

different

transformation

approaches

-LRB-

Hammond

Hammond

et

al.

-RRB-

However

there

are

only

a

few

reports

describing

genetic

transformation

of

poinsettia

one

was

the

US

patent

-LRB-

Smith

et

al.

-RRB-

using

the

biolistic

transformation

approach

while

the

other

two

were

electrophoresis-based

transformation

attempts

-LRB-

Vik

et

al.

Clarke

et

al.

-RRB-

Biolistic

transformation

requires

the

use

of

a

gene

gun

device

-LRB-

Sanford

et

al.

-RRB-

and

tends

to

generate

transformants

with

a

high

transgene

copy

number

complex

transgene

loci

and

unpredictable

silencing

of

the

transgene

-LRB-

Herrera-Estrella

et

al.

-RRB-

Electrophoresis

of

DNA

into

meristems

on

a

living

plant

was

described

as

a

simple

method

to

generate

transformants

by

avoiding

tedious

tissue

culture

work

and

was

utilized

in

producing

transgenic

orchid

-LRB-

Griesbach

-RRB-

However

no

stable

transgenic

poinsettia

was

ever

produced

using

electrophoresis

regardless

of

the

strong

transient

expressions

that

were

detected

in

both

studies

-LRB-

Vik

et

al.

Clarke

et

al.

-RRB-

Thus

Agrobacterium-mediated

transformation

for

poinsettia

was

developed

in

the

present

study

Poinsettia

mosaic

virus

-LRB-

PnMV

-RRB-

is

a

single-stranded

positive-sense

RNA

virus

-LRB-

Bradel

et

al.

-RRB-

that

belongs

to

the

family

Tymoviridae

-LRB-

Dreher

et

al.

-RRB-

Infection

of

poinsettia

plants

with

PnMV

results

in

mosaic

symptoms

during

parts

of

the

growing

season

-LRB-

Fulton

and

Fulton

-RRB-

which

in

turn

decreases

the

commercial

value

of

this

ornamental

plant

Thus

growers

are

interested

in

the

potential

benefits

of

growing

PnMV-free

poinsettias

PnMV-free

poinsettia

plants

can

be

obtained

by

heat

treatment

or

in

vitro

culture

of

apical

meristems

which

are

time-consuming

and

cost-ineffective

methods

An

additional

problem

is

that

PnMV-free

poinsettia

tends

to

be

rapidly

reinfected

although

no

vector

is

known

-LRB-

Blystad

and

Fløistad

Siepen

et

al.

-RRB-

There

is

therefore

a

need

for

a

new

and

effective

alternative

approach

like

Agrobacterium-mediated

transformation

which

can

overcome

these

difficulties

RNA

silencing

is

a

mechanism

by

which

transcription

or

translation

of

a

gene

is

suppressed

It

is

known

to

occur

in

plants

fungi

and

animals

-LRB-

Fire

et

al.

Waterhouse

et

al.

Baulcombe

-RRB-

It

is

triggered

by

double

stranded

RNA

-LRB-

dsRNA

-RRB-

molecules

-LRB-

Meister

and

Tuschl

-RRB-

which

are

subsequently

recognized

and

cleaved

by

the

host-encoded

endoribonuclease

dicer

-LRB-

Bernstein

et

al.

-RRB-

into

small

interfering

RNA

-LRB-

siRNA

-RRB-

molecules

of

nucleotides

in

length

-LRB-

Hamilton

and

Baulcombe

-RRB-

These

siRNAs

in

conjunction

with

the

RNA-induced

silencing

complex

-LRB-

RISC

-RRB-

target

RNA

molecules

with

homologous

sequences

for

sequence-specific

degradation

-LRB-

Hammond

et

al.

Bernstein

et

al.

Tabara

et

al.

-RRB-

In

plants

RNA

silencing

can

be

achieved

by

genetic

transformation

with

gene

constructs

that

express

highly

transcribed

sense

anti-sense

or

self

complementary

hairpin

RNA

-LRB-

hpRNA

-RRB-

containing

sequences

homologous

to

the

target

gene

-LRB-

Smith

et

al.

Wesley

et

al.

Helliwell

and

Waterhouse

-RRB-

RNA

silencing

has

been

efficiently

used

to

generate

resistance

against

plant

viruses

in

many

plant

species

including

ornamentals

-LRB-

Metzlaff

et

al.

Tenllado

et

al.

Bucher

et

al.

Hammond

et

al.

-RRB-

In

this

study

we

report

the

development

of

an

A.

tumefaciens-mediated

transformation

method

for

poinsettia

which

has

never

previously

been

described

for

this

plant

species

This

method

could

facilitate

the

improvement

of

poinsettia

by

introducing

new

traits

into

existing

commercial

poinsettia

cultivars

in

order

to

meet

market

demands

Using

this

approach

in

combination

with

RNA

silencing

technology

transgenic

PnMV

resistant

poinsettia

plants

carrying

PnMV-derived

hpRNA

constructs

were

produced

for

the

first

time

Materials

and

methods

Plant

materials

Euphorbia

pulcherrima

poinsettia

cv

Millenium

plants

were

kindly

supplied

by

the

J.

Kristiansen

nursery

Grimstad

Norway

in

the

year

The

original

stock

plants

were

subjected

to

heat

therapy

to

eliminate

PnMV

-LRB-

Fløistad

and

Blystad

unpublished

-RRB-

PnMV-free

cv

Millenium

cuttings

were

grown

in

the

greenhouse

under

a

photoperiod

of

h

light

and

h

dark

with

a

temperature

of

°C

Internode

stem

explants

from

to

cv

Millenium

plants

were

used

for

somatic

embryogenesis

and

A.

tumefaciens-mediated

transformation

Somatic

embryogenesis

of

poinsettia

cv

Millenium

Internode

stem

explants

mm

long

from

cv

Millenium

plants

were

excised

and

used

in

the

establishment

of

somatic

embryogenesis

for

poinsettia

prior

to

transformation

In

Experiments

and

the

explants

were

surface

sterilized

for

min

in

NaOCl

and

then

rinsed

three

times

with

sterilized

deionized

and

autoclaved

for

and

min

according

to

Preil

-LRB-

-RRB-

Because

of

a

large

number

of

infections

encountered

in

Experiments

and

the

stem

explants

were

sterilized

in

Experiment

using

ethanol

for

min

followed

by

min

with

NaOCl

and

rinsings

with

sterile

deionized

and

autoclaved

water

for

and

min

a

modification

of

the

protocol

of

Preil

-LRB-

-RRB-

The

modified

protocol

was

repeated

twice

before

being

utilized

in

Experiment

and

also

in

subsequent

transformation

experiments

After

sterilization

stem

segments

of

ca.

mm

thickness

were

placed

on

callus

induction

-LRB-

CI

-RRB-

medium

-LSB-

MS

medium

-LRB-

Murashige

and

Skoog

-RRB-

supplemented

with

mg

l

BAP

and

mg

l

CPA

and

g

l

sucrose

Table

-RSB-

for

days

Embryogenic

calli

were

then

transferred

to

somatic

embryo

induction

-LRB-

SEI

-RRB-

medium

-LRB-

MS

medium

contains

mg

l

NAA

and

mg

l

and

g

l

sucrose

Table

-RRB-

for

somatic

embryogenesis

The

somatic

embryos

emerged

after

an

average

of

weeks

were

then

transferred

to

somatic

embryo

maturation

-LRB-

SEM

-RRB-

medium

-LRB-

MS

basal

medium

containing

mg

l

BAP

and

g

l

sucrose

Table

-RRB-

Shoots

and

plantlets

derived

from

somatic

embryos

were

subsequently

cultured

on

root

induction

-LRB-

RI

-RRB-

medium

consisting

of

strength

MS

mg

l

IAA

and

g

l

sucrose

or

on

hormone

free

strength

MS

-LRB-

HFMS

-RRB-

medium

supplemented

with

g

l

sucrose

for

root

induction

-LRB-

Table

-RRB-

Plants

with

well-developed

roots

were

transferred

to

soil

and

grown

in

the

greenhouse

at

°C

The

above

culture

conditions

and

media

compositions

for

somatic

embryogenesis

of

cv

Millenium

were

developed

according

to

Preil

-LRB-

-RRB-

with

a

number

of

modifications

described

above

Detailed

information

regarding

media

compositions

is

summarized

in

Table

Table

names

and

compositions

modified

according

to

Preil

-LRB-

-RRB-

MediumMSSucrose

-LRB-

-RRB-

CPA

-LRB-

mg

l

-RRB-

BAP

-LRB-

mg

l

-RRB-

NAA

-LRB-

mg

l

-RRB-

-LRB-

mg

l

-RRB-

IAA

-LRB-

mg

l

-RRB-

CIFull

SEIFull

SEMFull

CI

Callus

induction

medium

SEI

somatic

embryo

induction

medium

SEM

somatic

embryo

maturation

medium

RI

root

induction

medium

HFMS

hormone

free

MS

medium

for

root

induction

Light

conditions

were

μE

m

s

for

callus

and

somatic

embryos

and

μE

m

s

for

plantlets

in

RI

and

HFMS

media

with

a

h

photoperiod

Temperature

was

constant

at

°C

A

light

microscope

connected

to

a

digital

camera

was

used

to

follow

the

development

of

the

somatic

embryos

Agrobacterium

strain

and

hairpin

-LRB-

hp

-RRB-

RNA

constructs

The

disarmed

A.

tumefaciens

strain

-LRB-

Hoekema

et

al.

Invitrogen

California

USA

-RRB-

was

utilized

throughout

the

study

Three

hpRNA

constructs

named

as

pCP

and

were

generated

Construct

pCP

targeted

the

viral

coat

protein

-LRB-

CP

-RRB-

whereas

constructs

and

targeted

two

distinct

regions

within

the

viral

RNA-dependant

RNA-polymerase

-LRB-

RdRp

-RRB-

-LRB-

Fig.

-RRB-

Briefly

constructs

pCP

and

were

generated

by

amplifying

the

corresponding

fragments

from

the

viral

genome

and

introducing

the

appropriate

restriction

sites

The

following

restriction

sites

were

introduced

XhoI

KpnI

-LRB-

for

sense

orientation

-RRB-

and

ClaI

XbaI

-LRB-

for

antisense

orientation

-RRB-

for

and

fragments

and

XhoI

EcoRI

-LRB-

sense

-RRB-

and

ClaI

XbaI

-LRB-

antisense

-RRB-

for

CP

fragment

The

primers

used

are

presented

in

Table

Each

amplified

fragment

was

bp

long

Subsequently

the

fragments

were

inserted

into

pHANNIBAL

vector

-LRB-

kindly

provided

by

CSIRO

Plant

Industry

Canberra

Australia

-RRB-

in

sense

and

antisense

orientations

interrupted

by

an

intron

pyruvate

orthophosphate

dikinase

-LRB-

pdk

-RRB-

gene

-LRB-

Fig.

-RRB-

as

described

by

Helliwell

and

Waterhouse

-LRB-

-RRB-

The

three

expression

cassettes

were

cloned

into

-LRB-

Gleave

kindly

provided

by

CSIRO

Plant

Industry

Canberra

Australia

-RRB-

a

binary

plasmid

vector

at

site

under

the

control

of

CaMV

promoter

and

with

ocs

terminator

-LRB-

Helliwell

and

Waterhouse

-RRB-

generating

hpRNA

constructs

pCP

and

-LRB-

Fig.

-RRB-

For

selection

the

neomycin

phosphotransferase

II

-LRB-

nptII

-RRB-

gene

conferring

kanamycin

resistance

was

used

under

the

control

of

nopaline

synthase

promoter

-LRB-

Nos-P

-RRB-

and

with

Nos

terminator

-LRB-

NosT

Fig.

-RRB-

in

the

binary

plasmid

expression

vector

Sequence

analysis

was

utilized

to

verify

the

junctions

and

orientations

in

the

expression

vectors

pCP

and

All

three

plasmid

vectors

were

introduced

into

A.

tumefaciens

strain

LBA

by

electroporation

according

to

the

manufacturer

's

instructions

-LRB-

Invitrogen

California

USA

-RRB-

Fig.

representation

of

PnMV

genome

-LRB-

a

-RRB-

and

hairpin

-LRB-

hp

-RRB-

RNA

constructs

used

in

the

current

study

-LRB-

b

-RRB-

The

locations

of

the

primers

used

to

generate

the

constructs

as

well

as

for

screening

purposes

are

presented

over

the

PnMV

genome

Sequences

of

the

primers

are

presented

in

Table

The

location

of

the

probe

used

to

detect

siRNA

is

presented

below

the

PnMV

genome

-LRB-

see

Materials

and

methods

-RRB-

CP

coat

protein

region

and

RNA-dependent

RNA-polymerase

-LRB-

RdRp

-RRB-

regions

of

the

PnMV

genomeTable

used

in

this

studyPrimer

nameRestriction

aPrimer

sequences

are

presented

in

the

orientation

Sequences

complementary

to

the

viral

genome

are

presented

in

uppercase

letters

whereas

non-complementary

restriction

sites

included

in

each

primer

are

presented

in

lowercase

letters

The

primers

used

for

screening

purposes

are

identical

to

the

ones

presented

but

do

not

include

the

non-complementary

restriction

sites

Transformation

of

poinsettia

stem

explants

A.

tumefaciens

strain

harbouring

the

plasmid

pCP

or

was

grown

overnight

in

ml

liquid

LB

medium

supplemented

with

mg

l

kanamycin

-LRB-

Sigma-Aldrich

St

Louis

USA

-RRB-

at

°C

with

shaking

at

rpm

until

an

of

was

reached

The

bacterium

suspension

was

collected

at

rpm

for

min

washed

twice

with

MS

basal

medium

supplemented

with

sucrose

-LRB-

-RRB-

and

resuspended

in

ml

The

internode

stem

explants

taken

from

to

poinsettia

plants

derived

from

cuttings

were

disinfected

as

described

above

excised

into

stem

segments

with

mm

thickness

and

inoculated

with

Agrobacterium

suspension

for

min

with

gentle

shaking

After

the

infection

stem

segments

were

blotted

briefly

with

sterile

filter

paper

and

placed

on

CI

medium

at

°C

in

the

dark

for

h

without

selection

After

co-cultivation

the

explants

were

blotted

gently

on

sterile

filter

paper

and

transferred

to

the

CI

medium

with

selection

and

light

conditions

as

described

above

Selection

and

regeneration

of

transgenic

plants

After

days

on

CI

medium

supplemented

with

mg

l

claforan

-LRB-

Aventis

Pharma

Ltd

Norway

-RRB-

and

mg

l

kanamycin

the

explants

were

transferred

to

SEI

medium

supplemented

with

mg

l

claforan

and

mg

l

kanamycin

for

somatic

embryogenesis

The

protocol

for

somatic

embryogenesis

is

detailed

above

The

somatic

embryos

obtained

after

about

weeks

were

then

transferred

to

the

SEM

medium

supplemented

with

mg

l

claforan

and

mg

l

kanamycin

Shoots

and

plantlets

derived

from

somatic

embryos

were

subsequently

cultured

on

RI

medium

or

on

hormone

free

strength

MS

-LRB-

HFMS

-RRB-

medium

-LRB-

Table

-RRB-

for

root

induction

and

were

subsequently

transferred

to

soil

and

grown

in

the

greenhouse

at

°C

Screening

of

transgenic

poinsettia

plants

Screening

of

transgenic

poinsettia

plants

was

carried

out

by

PCR

The

primer

pairs

used

to

amplify

the

CP

and

fragments

were

the

same

as

those

for

vector

constructions

but

did

not

include

any

additional

restriction

site

-LRB-

Table

-RRB-

HotStarTaq

PCR

kit

purchased

from

Qiagen

-LRB-

Valencia

California

-RRB-

was

used

in

PCR

screening

of

transformants

Twenty

micro

litres

of

reaction

mixture

containing

HotStarTaq

Mastermix

μM

of

each

primer

μg

template

DNA

and

were

subjected

to

PCR

amplification

under

the

following

conditions

min

at

°C

-LRB-

cycle

-RRB-

s

at

°C

s

at

°C

min

at

°C

-LRB-

cycles

-RRB-

and

a

final

extension

min

at

°C

-LRB-

cycle

-RRB-

according

to

the

manufacturer

's

instructions

All

the

PCR

amplification

was

performed

using

Applied

Biosystems

Thermal

Cycler

-LRB-

Applied

Biosystems

-RRB-

PCR

products

were

analysed

by

electrophoresis

on

-LRB-

W/V

-RRB-

agarose

gels

Southern

blot

analysis

To

confirm

the

stable

integration

of

transgenes

into

the

poinsettia

genome

and

to

evaluate

transgene

copy

number

Southern

blot

analysis

was

performed

basically

as

described

by

Sambrook

et

al.

-LRB-

-RRB-

Total

genomic

DNA

was

isolated

from

young

leaves

of

control

plant

and

the

putative

transformants

using

a

modified

CTAB

protocol

as

described

by

Rogers

and

Bendich

-LRB-

-RRB-

Ten

micrograms

of

genomic

DNA

was

digested

with

the

restriction

enzyme

HindIII

for

h

and

separated

on

a

-LRB-

W/V

-RRB-

TBE

agarose

gel

overnight

at

V

followed

by

transfer

onto

Gene

Screen

Transfer

membrane

-LRB-

NEN

Life

Science

Products

Inc.

Boston

MA

USA

-RRB-

The

HindIII

site

is

not

present

in

the

pCP

and

vectors

The

HindIII

site

from

the

pHANNIBAL

vector

was

removed

during

the

vector

construction

Membranes

were

hybridized

overnight

with

probe

of

kb

in

size

targeting

the

CP

and

regions

-LRB-

Fig.

-RRB-

The

probe

was

generated

by

amplifying

a

kb

fragment

that

included

the

and

CP

regions

used

to

generate

the

hairpin

RNA

constructs

and

pCP

This

allowed

us

to

use

one

probe

instead

of

three

probes

for

southern

blot

analyses

of

and

pCP

transformants

Northern

blot

hybridization

of

low

molecular

weight

RNA

Total

RNA

was

extracted

using

the

Trizol

reagent

-LRB-

Invitrogen

USA

-RRB-

as

recommended

by

the

manufacturer

Low

molecular

weight

-LRB-

LMW

-RRB-

RNA

was

separated

from

high

molecular

weight

RNA

by

precipitation

with

volume

of

M

at

°C

overnight

After

centrifugation

the

LMW

RNA-containing

supernatant

was

transferred

to

a

new

tube

and

precipitated

with

volume

of

isopropanol

LMW

RNA

concentrations

were

measured

using

a

GeneQuant

II

spectrophotometer

-LRB-

Amersham

Biosciences

UK

-RRB-

and

RNA

quality

was

verified

by

agarose

gel

electrophoresis

LMW

RNA

-LRB-

approximately

μg

-RRB-

was

mixed

with

volume

of

Tris-borate

EDTA

urea

sample

buffer

-LRB-

Biorad

USA

-RRB-

and

heated

at

°C

for

min

Subsequently

LMW

RNA

was

separated

on

a

polyacrylamid

tris-borate

EDTA

urea

gel

and

transferred

to

a

Hybond-N

membrane

-LRB-

Amersham

Biosciences

UK

-RRB-

overnight

The

membranes

were

hybridized

with

a

digoxigenin

-LRB-

DIG

-RRB-

labelled

-LRB-

Roche

Diagnostics

Germany

-RRB-

RNA

probe

-LRB-

Fig.

-RRB-

previously

cleaved

by

alkaline

hydrolysis

to

generated

fragments

of

approximately

bp

as

described

by

Hamilton

and

Baulcombe

-LRB-

-RRB-

After

hybridization

the

membranes

were

washed

at

room

temperature

min

in

SSC

SDS

and

min

SSC

SDS

and

then

exposed

to

films

Virus

inoculation

and

detection

Sap

from

poinsettia

and

N.

benthamiana

plants

systemically

infected

with

PnMV

was

used

as

an

inoculum

Approximately

g

of

infected

leaf

tissue

was

ground

with

ml

of

inoculation

buffer

-LSB-

M

sodium

phosphate

buffer

-LRB-

pH

-RRB-

-RSB-

and

mechanically

inoculated

to

leaves

of

three

to

four-leaves

stage

plants

lightly

dusted

with

carborundum

Plants

were

grown

in

the

greenhouse

at

°C

Detection

of

PnMV

infection

by

double

antibody

sandwich

enzyme-linked

immunosorbent

assay

-LRB-

DAS-ELISA

-RRB-

was

carried

out

at

and

weeks

post-inoculation

basically

as

described

by

Clark

and

Adams

-LRB-

-RRB-

The

uppermost

fully

expanded

leaves

of

each

plant

were

collected

in

polyvinyl

bags

-LRB-

Bioreba

USA

-RRB-

Extraction

buffer

-LSB-

mM

phosphate

buffer

-LRB-

pH

-RRB-

mM

Tris

mM

NaCl

mM

KCl

PVP

kD

Tween

and

-RSB-

was

added

to

the

bags

-LRB-

w/v

-RRB-

and

the

samples

were

macerated

A

measure

of

μl

of

the

homogenates

were

transferred

to

wells

of

microtitre

plates

previously

coated

with

μl

of

PnMV

IgG

-LRB-

Agdia

USA

-RRB-

diluted

in

coating

buffer

-LSB-

mM

carbonate

bicarbonate

buffer

-LRB-

pH

-RRB-

and

-RSB-

followed

by

incubation

at

°C

overnight

Microtitre

plates

were

washed

with

washing

buffer

-LSB-

mM

phosphate

buffer

-LRB-

pH

-RRB-

mM

NaCl

mM

KCl

and

Tween

-RSB-

and

μl

of

alkaline

phosphatase

conjugated

PnMV

IgG

-LRB-

Agdia

USA

-RRB-

diluted

in

extraction

buffer

was

added

to

each

well

Microtitre

plates

were

incubated

at

°C

overnight

Subsequently

they

were

washed

with

washing

buffer

and

μl

of

substrate

buffer

-LSB-

M

diethanolamine

-LRB-

pH

-RRB-

and

-RSB-

containing

p-nitrophenyl

phosphate

-LRB-

mg

ml

-RRB-

was

added

Absorbance

values

were

recorded

at

nm

with

an

Expert

plus

microtitre

plate

reader

using

the

Kim

software

-LRB-

ASYS

HITECH

Austria

-RRB-

Values

were

considered

positive

when

the

value

was

twofold

higher

than

that

of

the

negative

control

Results

Establishment

of

somatic

embryogenesis

for

poinsettia

cv

Millenium

An

efficient

protocol

for

somatic

embryogenesis

of

poinsettia

cv

Millenium

was

developed

Stem

explants

of

cv

Millenium

responded

well

to

the

induction

media

and

a

large

number

of

somatic

embryos

were

effectively

induced

-LRB-

Table

Fig.

d

-RRB-

In

Experiments

and

somatic

embryos

could

only

be

induced

from

to

of

the

explants

due

to

infection

whereas

more

than

of

the

explants

produced

somatic

embryos

in

Experiment

after

the

modification

of

the

disinfection

protocol

The

average

numbers

of

somatic

embryos

on

each

embryogenic

callus

varied

from

and

for

Experiments

and

respectively

to

for

Experiment

-LRB-

Table

-RRB-

The

highest

induction

of

somatic

embryos

was

found

in

Experiment

with

somatic

embryos

on

a

single

callus

When

globular

stage

somatic

embryos

-LRB-

Fig.

arrowed

-RRB-

reached

the

mature

stage

-LRB-

Fig.

-RRB-

they

became

loosely

attached

to

the

calli

Shoots

derived

from

somatic

embryos

were

subsequently

cultured

on

RI

medium

or

HFMS

medium

-LRB-

Fig.

-RRB-

for

root

induction

and

were

transferred

into

the

greenhouse

when

the

roots

were

well

established

-LRB-

Fig.

-RRB-

The

three

experiments

conducted

and

the

efficiency

of

somatic

embryogenesis

are

summarized

in

Table

Table

and

efficiency

of

somatic

embryogenesisExperimentNo

of

explants

of

embryogenic

explantsRange

of

SE/embryogenic

explantMean

SE/embryogenic

explantaNo

of

regenerated

SE

somatic

embryosaValues

are

presented

with

their

corresponding

standard

deviationbRegenerated

plantlets

after

weeksFig

embryogenesis

in

poinsettia

cv

Millenium

a

embryogenic

structure

and

globular

stage

somatic

embryos

-LRB-

arrows

-RRB-

that

appeared

on

the

callus

-LRB-

bar

mm

-RRB-

b

cotyledonary

stage

of

somatic

embryos

-LRB-

bar

mm

-RRB-

c

plantlets

deriving

from

somatic

embryos

on

RIM

medium

and

d

regenerated

plants

established

in

the

greenhouse

The

RI

and

HFMS

media

used

for

root

induction

did

not

show

any

clear

differences

in

the

percentage

-LRB-

and

for

RI

and

HFMS

respectively

-RRB-

of

plants

that

developed

roots

weeks

after

being

transferred

to

the

rooting

media

Development

of

the

A.

tumefaciens-mediated

transformation

method

Prior

to

the

production

of

transgenic

poinsettia

plants

a

number

of

optimizations

were

carried

out

to

reveal

the

most

suitable

conditions

for

inoculation

and

co-cultivation

concentration

of

antibiotic

for

eliminating

A.

tumefaciens

after

co-cultivation

and

kanamycin

selection

without

inhibiting

somatic

embryogenesis

and

regeneration

of

plants

Results

showed

that

the

optimal

inoculation

time

was

min

with

gentle

shaking

while

the

most

suitable

co-cultivation

time

was

h

-LRB-

Table

-RRB-

Using

this

combination

out

of

the

-LRB-

-RRB-

explants

tested

PCR

positive

after

inoculation

co-cultivation

and

weeks

on

claforan

-LRB-

Table

-RRB-

Those

explants

were

followed

and

showed

normal

embryogenesis

and

regeneration

as

compared

to

the

control

-LRB-

data

not

shown

-RRB-

Therefore

these

conditions

were

utilized

throughout

all

the

transformation

experiments

When

inoculation

time

exceeded

min

followed

by

a

co-cultivation

period

of

days

only

of

the

explants

survived

due

to

the

overgrowth

of

Agrobacterium

on

the

surfaces

of

the

explants

-LRB-

Table

-RRB-

Table

of

PCR

positive

explants

that

survived

weeks

after

Agrobacterium

inoculation

and

co-cultivation

treatments

A

total

of

explants

were

used

in

each

treatmentCo-cultivation

time

-LRB-

days

-RRB-

Inoculation

time

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

For

elimination

of

Agrobacterium

growth

after

co-cultivation

the

optimized

concentration

of

claforan

was

mg

l

for

the

CI

medium

whereas

mg

l

claforan

was

an

adequate

concentration

for

the

SEI

and

SEM

media

For

selection

of

transformed

cells

mg

l

kanamycin

was

found

optimal

for

the

SEI

medium

while

mg

l

kanamycin

was

optimal

for

the

CI

and

SEM

media

Production

of

transgenic

poinsettia

plants

molecular

analyses

and

resistance

assays

To

produce

transgenic

poinsettia

plants

transformation

experiments

including

stem

segment

explants

were

carried

out

using

the

established

inoculation

co-cultivation

and

selection

conditions

described

above

Of

those

explants

-LRB-

-RRB-

somatic

embryo-derived

plants

were

obtained

The

results

are

summarised

in

Table

Table

of

five

Agrobacterium-mediated

transformation

experiments

on

poinsettia

cv

Millenium

with

pCP

and

constructsExperimentaNo

of

explantsNo

of

regenerated

plantsNo

of

transformantsbTransformation

efficiency

-LRB-

-RRB-

-LRB-

CP

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

aCP

and

represent

transformation

experiments

with

constructs

pCP

and

respectivelybTransformants

verified

by

PCR

and

Southern

blot

analysiscNumber

of

transformants/total

number

of

explants

transformed

PCR

analysis

was

conducted

for

screening

the

putative

transformants

All

regenerated

plants

were

analysed

by

PCR

and

transformants

were

revealed

Of

these

three

possessed

pCP

constructs

while

six

and

nine

contained

and

constructs

respectively

-LRB-

Fig.

c

-RRB-

Transformation

frequency

varied

from

to

with

an

average

transformation

frequency

of

-LRB-

Table

-RRB-

Fig.

analysis

PCR

positive

transformants

detected

with

primer

pairs

for

-LRB-

a

-RRB-

CP

-LRB-

b

-RRB-

and

-LRB-

c

-RRB-

fragments

respectively

Lane

kb

marker

lane

non-transformed

plant

are

independent

CP-transgenic

lines

and

plasmid

control

Lane

kb

marker

lane

non-transformed

plant

lanes

are

lines

and

lane

plasmid

control

Lane

kb

marker

lane

non-transformed

plant

lanes

are

transgenic

lines

and

lane

plasmid

control

The

bp

band

of

the

bp

marker

is

depicted

with

an

arrow

Southern

blot

analysis

was

performed

on

selected

PCR

positive

plants

with

satisfactory

characteristics

for

studying

transgene

integration

and

estimating

the

transgene

copy

number

Results

of

Southern

blot

analysis

confirmed

the

stable

integration

of

transgene

and

both

single

and

multiple-copy

transgene

integration

into

the

poinsettia

genome

were

detected

among

the

transformants

-LRB-

Fig.

-RRB-

Of

the

eight

transformants

analysed

six

showed

single

copy

integration

of

transgene

-LRB-

lanes

and

in

Fig.

-RRB-

with

fragment

size

greater

than

the

length

of

intact

T-DNA

-LRB-

ca

kb

-RRB-

However

two

transformants

-LRB-

lanes

in

Fig.

-RRB-

showed

multiple-copy

transgene

integration

with

both

large

-LRB-

intact

T-DNA

-RRB-

and

small

fragments

-LRB-

partial

non

intact

T-DNA

-RRB-

Fig.

blot

analysis

of

selected

PCR

positive

transformants

carrying

pCP

and

constructs

The

HindIII-digested

total

genomic

DNA

was

probed

with

a

kb

probe

homologous

to

the

region

of

CP

and

fragments

-LRB-

Fig.

-RRB-

allowing

us

to

analyse

all

the

three

different

types

of

transformants

-LRB-

CP

and

transformants

-RRB-

at

the

same

time

plasmid

control

CP

transformants

and

transformants

lanes

transformants

negative

control

Furthermore

Northern

blot

analysis

of

low

molecular

weight

RNA

was

carried

out

on

the

transgenic

plants

harbouring

constructs

pCP

and

prior

to

inoculation

Transgene-derived

siRNA

molecules

of

nucleotides

length

were

detected

in

transgenic

lines

pCP

pCP

and

-LRB-

Fig.

-RRB-

This

data

indicated

that

the

transgene

had

been

transcribed

and

converted

into

siRNAs

-LRB-

Fig.

-RRB-

Due

to

limitations

in

the

amount

of

LMW

RNA

from

transgenic

lines

and

these

lines

were

not

included

in

the

experiment

Fig.

blot

analysis

of

low

molecular

weight

-LRB-

LMW

-RRB-

RNA

to

detect

small

interfering

RNAs

-LRB-

siRNA

-RRB-

in

non-inoculated

transgenic

plants

and

non-inoculated

control

aLane

control

lanes

CP

transgenic

lines

lanes

transgenic

lines

lane

transformant

Ribosomal

RNA

is

presented

in

-LRB-

b

-RRB-

to

indicate

presence

of

LMW

RNA

in

the

gel

Lane

control

lanes

and

PCP

transformed

plants

lanes

and

transformed

plants

lane

transformed

plant

Transgenic

plants

and

non-transformed

controls

were

mechanically

inoculated

with

PnMV

at

a

three

to

four

leaves

stage

The

upper

non-inoculated

leaves

were

tested

by

DAS-ELISA

at

and

weeks

post-inoculation

Results

showed

that

the

control

plants

were

systemically

infected

with

PnMV

whereas

the

tested

transgenic

lines

carrying

pCP

and

constructs

remained

virus

free

-LRB-

Table

-RRB-

However

of

the

five

transgenic

lines

studied

two

lines

remained

virus

free

while

the

remaining

three

lines

were

systemically

infected

-LRB-

Table

-RRB-

Table

detection

of

PnMV

on

non-transformed

and

transformed

poinsettia

plants

inoculated

with

PnMVConstructTransgenic

Control

Control

wpi

Weeks

post

inoculationaOne

plant

per

transgenic

line

was

challenged

in

this

assaybELISA

absorbance

values

-LRB-

nm

-RRB-

after

min

of

incubation

at

room

temperature

are

presented

Samples

with

twice

the

value

of

the

highest

negative

control

sample

were

deemed

positive

Discussion

Significance

of

genetic

engineering

in

control

of

virus

diseases

in

ornamentals

The

application

of

genetic

engineering

in

the

floriculture

industry

has

clearly

become

instrumental

and

rewarding

in

meeting

the

demand

for

novel

desirable

traits

A

number

of

genetic

engineering

approaches

have

been

developed

for

ornamental

plants

-LRB-

Griesbach

Kamo

et

al.

Deroles

et

al.

Zuker

et

al.

Kamo

et

al.

Kishimoto

et

al.

Kim

et

al.

Teixeira

da

Silva

Hammond

and

the

current

study

-RRB-

to

facilitate

the

improvement

of

ornamental

crops

with

better

quality

and

enhanced

resistance

against

diseases

and

pests

Effective

resistance

against

viruses

conferred

by

transgenes

has

shown

significant

potential

because

no

chemical

can

be

applied

for

control

of

virus

diseases

Regardless

of

the

importance

only

a

few

reports

have

been

published

demonstrating

the

virus

resistance

obtained

in

ornamentals

through

genetic

engineering

-LRB-

Kamo

et

al.

review

by

Hammond

et

al.

-RRB-

Furthermore

many

of

the

ornamentals

were

transformed

only

with

marker

genes

like

gus

or

selectable

antibiotic

or

herbicide

resistant

markers

-LRB-

Deroles

et

al.

-RRB-

Using

the

Agrobacterium-mediated

transformation

protocol

developed

in

the

current

study

we

have

obtained

PnMV

resistant

transgenic

poinsettia

lines

expressing

viral

sequences

targeting

the

CP

and

RdRp

regions

of

the

PnMV

genome

To

our

knowledge

no

PnMV

resistance

has

previously

been

obtained

using

classical

or

molecular

breeding

approaches

demonstrating

the

potential

of

the

transgenic

lines

produced

in

the

present

study

for

the

poinsettia

industry

Somatic

embryogenesis

and

Agrobacterium-mediated

transformation

in

poinsettia

cv

Millenium

One

of

the

reasons

that

transformation

of

ornamental

plants

has

lagged

somewhat

behind

that

of

major

crops

is

the

lack

of

efficient

regeneration

systems

as

these

are

key

components

leading

to

the

successful

development

of

transformation

protocols

Moreover

both

transformation

and

regeneration

can

be

cultivar

or

even

genotype-dependent

Therefore

we

developed

an

efficient

somatic

embryogenesis

system

for

poinsettia

cv

Millenium

prior

to

transformation

although

a

number

of

somatic

embryogenesis

systems

were

reported

previously

for

old

cultivars

such

as

Angelika

Franzi

and

Diamond

-LRB-

Preil

and

Beck

Preil

Osternack

et

al.

-RRB-

A

recent

study

on

somatic

embryogenesis

of

cv

Freedom

Red

and

Freedom

White

demonstrated

the

influence

of

cultivar

and

even

plant

genotype

on

somatic

embryogenesis

in

poinsettia

-LRB-

Castellanos

et

al.

-RRB-

indicating

the

necessity

of

developing

an

effective

regeneration

system

for

the

individual

cultivar

prior

to

the

development

of

transformation

systems

Poinsettia

like

other

Euphorbia

species

produces

latex

a

milky

solution

exuded

from

cut

surfaces

When

explants

were

excised

from

greenhouse

grown

poinsettia

plants

the

latex

was

released

from

the

cutting

points

making

surface

sterilization

difficult

In

Experiments

and

a

very

low

percentage

of

explants

produced

somatic

embryos

due

to

a

severe

infection

found

in

the

callus

induction

culture

-LRB-

Table

-RRB-

This

is

likely

to

have

been

the

result

of

a

combination

of

latex

and

insufficient

sterilization

based

on

the

sterilization

procedure

described

for

cv

Angelika

Franzi

and

Diamond

-LRB-

Preil

-RRB-

which

might

not

be

optimal

for

cv

Millenium

-LRB-

Table

-RRB-

A

modified

protocol

was

thus

used

for

surface

sterilization

in

Experiment

as

well

as

in

the

five

transformation

experiments

and

the

efficiency

of

somatic

embryogenesis

was

greatly

improved

-LRB-

see

Table

-RRB-

demonstrating

the

significance

of

optimization

of

sterilization

and

regeneration

protocols

for

the

individual

cultivar

In

addition

to

the

availability

of

an

efficient

regeneration

system

successful

A.

tumefaciens-mediated

transformation

depends

on

several

factors

e.g.

inoculation

and

co-cultivation

times

with

A.

tumefaciens

antibiotic

selection

and

regeneration

The

optimized

infection

conditions

and

co-cultivation

times

in

our

study

-LRB-

Table

-RRB-

are

consistent

with

the

range

of

published

reports

on

ornamental

plants

where

min

infection

time

and

days

co-cultivation

have

been

reported

-LRB-

Aida

et

al.

Kishimoto

et

al.

Cui

and

Ezura

Kim

et

al.

Teixeira

da

Silva

-RRB-

However

in

our

study

days

co-cultivation

led

to

A.

tumefaciens

overgrowth

and

explant

death

thus

differing

from

the

results

for

Begonia

in

which

days

co-cultivation

is

required

-LRB-

Kishimoto

et

al.

-RRB-

These

data

indicate

that

co-cultivation

times

vary

among

different

plant

species

Antibiotic

selection

is

another

key

factor

influencing

the

establishment

of

a

transformation

protocol

To

eliminate

Agrobacterium

after

co-cultivation

several

antibiotics

such

as

cefotaxim

-LRB-

claforan

-RRB-

carbenecillin

and

timentin

have

been

used

in

previous

studies

The

influence

of

the

type

of

antibiotic

and/or

concentration

on

each

transformation

system

varies

greatly

-LRB-

Ishida

et

al.

Nauerbey

et

al.

Sunikumar

and

Rathore

-RRB-

In

the

present

study

optimised

concentrations

of

claforan

in

the

CI

SEI

and

SEM

media

effectively

eliminated

Agrobacterium

For

selecting

transformed

cells

the

nptII

gene

was

used

Although

selection

media

containing

and

mg

l

kanamycin

have

been

documented

in

the

production

of

transgenic

plants

-LRB-

Kishimoto

et

al.

Kim

et

al.

Wu

et

al.

-RRB-

those

concentrations

severely

inhibited

the

development

of

embryogenic

calli

and

somatic

embryos

in

poinsettia

In

our

study

the

optimal

concentrations

of

kanamycin

were

and

mg

l

In

the

case

of

Agrobacterium-mediated

transformation

of

Platanus

acerifolia

kanamycin

at

mg

l

was

found

effective

for

the

inhibition

of

regeneration

in

non-transformed

shoots

-LRB-

Li

et

al.

-RRB-

suggesting

the

variability

of

antibiotic

selection

in

each

transformation

system

and

the

necessity

of

optimization

of

antibiotic

selection

for

each

plant

species

Although

transgenic

PnMV

resistant

poinsettias

were

produced

using

the

Agrobacterium-mediated

transformation

protocol

developed

in

the

current

study

the

transformation

frequency

was

fairly

low

-LRB-

Table

-RRB-

suggesting

that

further

improvement

is

required

This

will

be

carried

out

in

the

near

future

Southern

blot

analysis

confirmed

the

integration

of

transgenes

into

the

poinsettia

genome

and

detected

both

single

and

multiple-copy

integration

of

the

transgenes

among

the

transformants

-LRB-

Fig.

-RRB-

The

majority

-LRB-

six

out

of

eight

-RRB-

of

the

transformants

showed

single

copy

transgene

integration

with

fragment

sizes

greater

than

that

of

the

intact

T-DNA

-LRB-

ca

kb

-RRB-

indicating

that

the

intact

T-DNA

was

integrated

into

the

poinsettia

genome

However

the

bands

with

sizes

around

kb

smaller

than

that

of

intact

T-DNA

were

also

found

in

the

transgenic

lines

with

multiple

insertions

-LRB-

lanes

and

in

Fig.

-RRB-

According

to

Afolabi

et

al.

-LRB-

-RRB-

and

Zhu

et

al.

-LRB-

-RRB-

insertion

of

non-intact

T-DNAs

were

found

in

more

than

of

the

transgenic

rice

lines

causing

of

the

loci

to

contain

only

part

of

the

T-DNA

due

to

T-DNA

truncation

Moreover

T-DNA

rearrangement

is

well

documented

in

many

plant

species

including

both

dicots

and

monocots

-LRB-

Deroles

and

Gardner

Puchta

et

al.

Azhakanandam

et

al.

Yin

and

Wang

Rai

et

al.

-RRB-

Almost

of

the

transgenic

rice

lines

studied

showed

rearrangement

of

T-DNA

inserts

according

to

Rai

et

al.

-LRB-

-RRB-

Consequently

such

T-DNA

truncations

and/or

rearrangements

could

also

possibly

occur

in

our

study

causing

partial

T-DNA

insertion

Since

Agrobacetrium-mediated

transformation

of

poinsettia

has

never

been

described

before

there

is

no

available

information

regarding

T-DNA

transfer

and

integration

into

the

poinsettia

genome

The

hp

RNA

constructs

and

PnMV

resistance

in

transgenic

poinsettia

cv

Millenium

A

number

of

effective

approaches

have

been

utilized

to

control

viral

diseases

-LRB-

Goldbach

et

al.

-RRB-

Among

these

coat

protein-mediated

and

RNA

silencing-based

resistance

have

been

shown

to

be

efficient

transgenic

approaches

to

engineer

virus

resistance

in

plants

-LRB-

Powell-Abel

et

al.

Barker

et

al.

Waterhouse

et

al.

Smith

et

al.

Domínguez

et

al.

Lu

et

al.

-RRB-

Furthermore

according

to

previous

studies

hpRNA-induced

RNA

silencing

is

more

efficient

than

sense

or

antisense-mediated

silencing

-LRB-

Chuang

and

Meyerowitz

-RRB-

In

the

current

study

the

establishment

of

Agrobacterium-mediated

transformation

enabled

us

to

transform

poinsettia

plants

with

intron-containing

hpRNA

constructs

homologous

to

the

PnMV

genome

PnMV

resistant

transgenic

poinsettia

lines

showing

no

systemic

infection

were

identified

after

mechanical

inoculation

as

determined

using

the

DAS-ELISA

assay

-LRB-

Table

-RRB-

The

presence

of

transgene-derived

siRNA

molecules

in

non-inoculated

transgenic

poinsettia

plants

carrying

pCP

and

constructs

and

the

absence

of

these

in

the

non-transformed

control

plants

indicates

that

the

transgenes

were

expressed

and

subsequently

cleaved

This

data

indicates

that

RNA

silencing

underlies

the

PnMV

resistance

observed

in

our

study

-LRB-

Fig.

-RRB-

As

mentioned

previously

LMW

Northern

analysis

included

only

one

line

-LRB-

-RRB-

due

to

the

limitations

in

the

amount

of

RNA

available

for

the

remaining

lines

Therefore

the

underlying

reason

for

lack

of

resistance

in

some

of

the

transformants

-LRB-

Table

-RRB-

needs

to

be

clarified

by

further

studies

Poinsettia

is

a

non-food

non-feed

and

vegetatively

propagated

ornamental

plant

Pollen

mediated

transgene

outflow

is

restricted

a

significant

advantage

over

sexually

propagated

plants

The

combination

of

genetic

engineering

and

RNA

silencing

technologies

for

the

generation

of

virus-resistant

transgenic

ornamentals

is

therefore

appealing

especially

in

an

industry

in

which

quality

is

so

important

Transgenic

vegetatively

propagated

ornamentals

like

PnMV

resistant

transgenic

poinsettia

are

likely

to

be

more

acceptable

even

in

areas

where

genetically

modified

crops

are

currently

not

cultivated

In

summary

we

have

developed

an

A.

tumefaciens-mediated

transformation

method

for

poinsettia

Using

this

approach

transgenic

PnMV

resistant

transgenic

lines

expressing

PnMV-derived

hpRNA

constructs

were

produced

Phytoplasma

the

poinsettia

branch-inducing

factor

for

good

branching

-LRB-

free-branching

-RRB-

and

for

a

compact

growth

habit

-LRB-

Lee

et

al.

-RRB-

is

currently

being

reintroduced

into

the

transgenic

poinsettias

Candidate

transgenic

lines

with

PnMV

resistance

and

desired

ornamental

values

will

be

selected

for

further

study

with

the

future

goal

of

possible

commercialization

The

methodology

developed

here

could

facilitate

the

future

engineering

of

poinsettia

to

meet

customers

expectations

for

new

colours

and

other

desirable

traits

O O B O O O B I O O O O O O O O O B I I O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I I O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O B O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O B I O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

euphorbia pulcherrima
somatic embryogenesis
transformation
agrobacterium tumefaciens
poinsettia mosaic virus