Plant Cell Reports

Plant Cell Reports (1989) 8:387-390

© Springer-Verlag 1989

Successful cocultivation of Brassica napus microspores and proembryos with Agrobacterium Paul M. Pechan *

University of Guelph, Guelph, Ontario, Canada N1G 2Wl Received July 14, 1989 Communicated by K. Hahlbrock

Abstract

transformation frequencies,

Brassica

napus

microspores

microspore-derived cocultivated with harbouring

a

and

proembryos Aqrobacterium

binary

were

tumefaciens

vector.

The

vector

c o n t a i n e d s e l e c t a b l e genes for k a n a m y c i n hygromycin

antibiotic

Microspores

and

antibiotic can

selection.

and

procedure

choosing

proper

the

allowing

of

the

d e p e n d e d l a r g e l y on

coculture

microspore

embryos

large

embryogenesis

inactivation

initiation,

obtained

Beversdorf

are

can

be pure

1985;

Pechan

-

studied,

and upon

be

1982; and

attempting

established

thus

homozygous

(Lichter

before

haploid

to

lines

Chuong

Keller to

culture s y s t e m for such studies, must

a

n u m b e r of embryos and p l a n t l e t s can be

However

conditions

culture

from m i c r o s p o r e s

culture

c h r o m o s o m e doubling, are

and

develop

w i t h i n 14 days after

genes

Success

cocultivation

studies:

transfer

plantlets

of

formation

a n u m b e r of c h a r a c t e r i s t i c s well gene

recessive

i n d i c a t e d up to 7.3%

activity.

has

subsequent

exhibited neomycin phosphotransferase

enzyme

system

suited for

obtained,

T h o u s a n d s of p l a n t l e t s

plant

i n a c t i v a t i o n studies (Schell 1987). The B r a s s i c a napus microspore

the

be r e g e n e r a t e d from a single experiment.

plants

while

survived

procedure

Biochemical analysis

II

resistance.

proembryos

cocultivation

and

and

a c o m b i n a t i o n d e s i r a b l e for gene

and

1988).

utilize this a

allow

procedure d e l i v e r y of

foreign genes into B r a s s i c a microspores. In this paper,

to for

proceed.

a

description

is

given

the s u c c e s s f u l c o c u l t i v a t i o n of B. napus

microspores

and p r o e m b r y o s with A q r o b a c t e r i u m

t u m e f a c i e n s c o n t a i n i n g a binary vector. Abbreviations:

Hm

=

hygromycin,

Km

= M a t e r i a l s and M e t h o d s

kanamycin.

Introduction

Plants of B. napus cv Topaz

A n u m b e r of gene

transfer

methods

can

m e d i a t e d e l i v e r y of foreign genes into plants (for

review

However,

see

none

of

Perani

et

al.

the gene t r a n s f e r m e t h o d s culture

system

at I0/5°C day/night temperature, -2 -1 s light intensity, 16 h light

m

highly

w i t h high

grown

300 pmol duration

and 50% r e l a t i v e humidity.

1986).

p r e s e n t l y used can c l a i m to combine a

were

Surface s t e r i l i z e d flower buds

(3-5

mm

in

length) were h o m o g e n i z e d w i t h a glass rod

in

a

B5

medium

(Gamborg

et

embryogenic

cell

* Present address.

Max-Planck-Institut f/ir Zfichtungsforschung, D-5000 K6ln 30, Federal Republic of Germany

supplemented

with

13%

al.

sucrose,

1968), and

the

388 released microspores additionally

washed

3x

kanamycin

(Km) 25 or 50 Bg/ml.

Embryos

were

w i t h B5 -13% sucrose m e d i u m by c e n t r i f u g a t i o n

subsequently

c u l t u r e d at 25°C and 16 h light

at

d u r a t i o n and

transferred

100

g

suspended

for

3

min.

in

a

modified

Microspores

medium

(Lichter 1982), w i t h o u t Difco p o t a t o and

g r o w t h regulators,

were

Lichter

extract

and p l a t e d at 1.25 ml

per 60 x 15 mm petri dish at

an

approximate

d e n s i t y of 2 x 104 m i c r o s p o r e s per ml et al. be

1988).

Two m i l l i o n

prepared

for

(Keller

microspores

culture

in

can

90

min.

after

3

weeks of culture.

chamber

6

weeks

selection. generated.

are

cocultivation

respectively,

with

A.

prior to

tumefaciens.

Microspores

c u l t u r e d for 8 h were d e f i n e d as

microspores;

those

cultured

d e f i n e d as proembryos. The A. tumefaciens GV3850

for

72 h

were

strain

used

was

and the v e c t o r was pCV730,

plasmid

originated

containing

genes.

from

Max-Planck-Institute,

K61n.

presented

of were

for tissue

a n a l y z e d for e x p r e s s i o n of the n e o m y c i n

essentially

II

(NPT

following

II)

the

gene

times,

treatment.

with

5

Controls

by

procedure

M c D o n n e l l et al. (1987). C o c u l t i v a t i o n experiments w e r e three

h y g r o m y c i n and k a n a m y c i n m a r k e r

plantlets

s e l e c t e d on 10 ~g/mi Hm and 50 Dg/ml Km only. Seventy-five randomly selected plants were

h,

medium

initiation

1000

Data

phosphotransferase

72

new

Rooted plantlets

after

Over

for

and

a

w e r e p o t t e d in soil and t r a n s f e r r e d to a mist

M i c r o s p o r e s were c u l t u r e d in d a r k n e s s at 32°C 8

to

of

repeated

replications

were

per

non-cultivated

m i c r o s p o r e s or proembryos.

The

C. Koncz, Bacteria

were

Results and D i s c u s s i o n

made c o m p e t e n t for t r a n s f o r m a t i o n by a d d i t i o n of a c e t o s y r i n g o n e LB m e d i u m

(Engstr~m et al.

(200 pg/ml)

this m e d i u m o v e r n i g h t at 28°C. subsequently

resuspended

in twenty

culture

petri

to

to

B a c t e r i a were

L i c h t e r m e d i u m (NLN) and added

1987)

and g r o w i n g b a c t e r i a in

each

a

modified

pl

of

dish

two m i c r o s p o r e s or proembryos.

an

every

Cocultivation

and

proembryos

and plants e x h i b i t i n g NPTII

can

enzyme

r e g e n e r a t e d at high frequencies

the

at

a p p r o x i m a t e ratio of one b a c t e r i u m for

Microspores

activity

(Table I).

The success of the p r o c e d u r e is dependent

on

the

ability

to

cocultivate

namely,

to

control

the

growth

in

the

tumefaciens

dark

It

was

u n d e r g o embryo formation. M i c r o s p o r e s should be suspended

25°C

terminated

for

when

microspores

or

about

approximately proembryos

Microspores/proembryos washed min., ~g

2x

by

24 h.

25%

had

aggregated.

were

subsequently

c e n t r i f u g a t i o n at 200 g for 3

r e s u s p e n d e d in fresh NLN c o n t a i n i n g i00

per

ml

Canada), petri

of

Claforan

(Roussel, Montreal,

p l a t e d at 1.25 ml per

dish

60

x

at

32°C

initiation. transferred

15

mm

at an a p p r o x i m a t e d e n s i t y of 2 x

104 m i c r o s p o r e s per ml and incubated dark

of

up

to

day

Petri to

a

the

14 after culture

dishes 25°C

under c o n s t a n t light

in

were

incubator

then and kept

(50 Dmol m -2 s -1) for

2

Sixteen days embryos

were

after

culture

transferred

w i t h o u t g r o w t h regulators, with

while

liquid medium, ease

of

1% agar,

2% sucrose,

A__~.

once

was

5

or

a

microspore

globular

for

corresponds

to

10

Bg/ml,

for

is

culture

are

not

medium

formed:

for

incubated

at

This incubation

period

the m i n i m u m time r e q u i r e d to

acceptable

levels Only

of

small

microspore amounts

of

c o n c e n t r a t e d A__~.t u m e f a c i e n s w e r e added to the of m e d i u m c o m p o s i t i o n and

(Hm)

NLN

8 h prior to c o c u l t i v a t i o n w i t h A__~.

but

hygromycin

the

embryos

t u m e f a c i e n s at 250C.

induce

to

culture

It

NLN m e d i u m can be utilized. M i c r o s p o r e cultures were 32°C

in

A__~.

B5 liquid m e d i u m or one-half d i l u t e d

microspore

supplemented

microspores

tumefaciens.

n e c e s s a r y to use NLN as

example,

of

an important c o n s i d e r a t i o n for

onto a B5 medium, 500 ~g/ml C l a f o r a n

and s e l e c t a b l e antibiotics: concentration

initiation

allowing

washing

containing

embryogenesis.

days.

largely

m i c r o s p o r e s or p r o e m b r y o s with A__u.tumefaciens

was c a r r i e d out on a shaker (50 rpm) at

be

s u c c e s s f u l l y c o c u l t i v a t e d with A__u.tumefaciens

culture m e d i u m to m i n i m i z e change

microspore

interference

embryogenesis.

with

Microspore

cultures w e r e w a s h e d 24 h after c o c u l t i v a t i o n by

repeated

centrifugation

at

200 g

for

389 3 min.

A higher g

allow

embryogenic

Unattached

force

bacteria

supernatant. bacteria

If

used

recommended,

was

necessary

microspores

to

remained the

is

to

pellet. in

the

concentration

higher

additional

than

washing

of that

B5

medium containing

did so f o l l o w i n g 50 (Fig.

i,

embryos

developed

8 h

containing

microspores/proembryos

into

terminated began

to

when

aggregate.

originating

Table i).

1%

preferable

the

proembryos

survived

the

(Table i).

to

plantlets.

forming

non-cocultivated 2.5%

of

embryos

the when

procedure

Table

embryos

controls.

microspores

a

B5

medium

and 50 Dg/ml of Km of

the

embryos

selection

selection

clarity of

(Fig. i,

of

potentially

was visual

transformed

in

However, developed

only into

s u b j e c t e d to the c o c u l t i v a t i o n

without

tumefaciens

7.5% of the

which

A. tumefaciens

on

Hm

Hygromycin for

identification

microspores

plantlets zg/ml

antibiotic

A__~. t u m e f a c i e n s

contrasts

selection and 15% of

proembryos

with

None

c o c u l t i v a t i o n p r o c e d u r e with This

Km

from n o n - c o c u l t i v a t e d m i c r o s p o r e s

s u r v i v e d the

One and a half percent of the m i c r o s p o r e s and of

i0

respectively.

was

from

cocultivation

m i c r o s p o r e culture should be done w i t h i n

of

Twenty-nine

originating

survived

the

Cocultivation

zg/ml

Table I).

of

of i n i t i a t i o n of coculture.

i0 zg/ml of Hm and 1.2%

the

addition

of

A__~.

.

I

Cocultivation of Brassica napus microspores and proembryos with Agrobacterium tumefaciens.

Dmbryo formation a

Resistant embryosb Hm

Km

Gene . c expres slon (NPT II) Figure

1

Microspores

I. 5

6

I. 2

40

S e l e c t i o n of embryos on agar with

Proembryos

I. 0

29

14.7

25

or kanamycin.

Control A d

2.3

0

0

0

Upper row:

0

n o n - c o c u l t i v a t e d embryos.

Control B e

7.5

0

0

antibiotics,

..................................................

a

c o c u l t i v a t e d embryos, A =

lower row:

agar

without

B = agar w i t h 5 Dg/ml of Hm, C =

agar w i t h 10 Dg/ml of Hm, D = Percentage of microspores and proembryos developing

hygromycin

agar

with

25

~g/ml of Km, E = agar w i t h 50 Dg/ml of Km.

into embryos. b

Percentage of ~mbryos resistant to 10 Dg/ml Hm and 50 ~g/ml Km.

C

Percentage of plants derived from Hm and Km Of the

resistant embryos exhibiting NPT II activity. d

plants tested,

Microspores were subjected to the cocultivation

from

to culture.

proembryos

activity Microspores were not subjected to cocultivation

(Fig.

of all embryos procedure,

Six p e r c e n t of embryos o r i g i n a t i n g

from

microspores which survived cocultivation with A__~.t u m e f a c i e n s d e v e l o p e d into p l a n t l e t s on

a

of

the

selected

plants

exhibited

2, Table i).

of the plantlets,

procedure.

randomly

40% of the plants d e r i v e d from

m i c r o s p o r e s and 25%

procedure, except that no A. tumefaciens was added

e

seventy-five

NPT Thus

II upto

It

7.3%

surviving

the

cocultivation

e x h i b i t e d NPT II activity.

delivered appears

enzyme

e x p r e s s e d as the p e r c e n t a g e

It is not u n d e r s t o o d how and w h e n was

derived

into

that

the

T-DNA

microspores/proembryos. time

of

embryogenic

390 References

•A

0A Chuong PV, Beversdorf a

Figure

b

leaf

extracts

napus plants embryos

II dot blot assay

of i0 randomly selected B__~.

originating

which

from

(1985)

Plant

Sci.

on i0 Dg/ml of Hm or 50 ~g/ml control;

of

control;

selection ~m.

b

=

a

swelling

- thus

stretching

out the exine and intine and further exposing apertures)

formation,

establishment well

and

initiated

of

active

Moreover,

microspores division

undergo about

initiation

8-48 h

(Pechan

cocultivating

after

when microspores

or

of

M

phase

positively

cell

contributed

into the cells

is

nuclear culture

Keller

microspores

tumefaciens,

Van

Mol.

Montagu Biol.

M, 197:

635-645

Gamborg OL, Miller RA, Ojima 50:

K

(1968)

Exp.

151-158

Keller WA, Fan F, Pechan PM, Long N, Grainger J (1988) Poland,

7th International Vol i, pp.

Rapeseed Congress,

152-157

Pflanzenphysiol.

Lichter R (1982) Z.

105:

427-434

embryogenic

first

and

P,

J.

cocultivation

since the

Zambryski (1987)

wall

observation),

suited for A__u.tumefaciens

experiments.

cell

within 48 h of culture

(unpublished

S

Cell Res.

(doubling in size within

72 h of culture initiation

P,

Stachel

=

positive

A plants with NPT II activity.

microspore

Engstr6m

cocultivated

survived antibiotic

non-cocultivated

pore

WD

219-226

2

Neomycin phosphotransferase in

39:

with might

RE, Clark RD, Smith WA, Hinchee

(1987) Plant Mol.

Rep.

5:

MA

380-386

1988), A__~.

were at the G2

cycle,

McDonnell

Okada K, Takeba I, Nagata T (1986) Mol. Genet.

205:

Gen.

398-403

have

the delivery of T-DNA

(Okada et al.

1986).

Pechan PM, Keller WA (1988) Physiol. 74:

Plant.

377-384

Acknowledgement Perani K, Radke S, Wilke-Douglas (1986) Physiol. This without

project the

Biochemistry

would

generosity Dept., Univ.

not of

have S.

Plant.

68:

M, Bossert M

566-570

started

Fabijanski,

of Ottawa,

Canada.

Schell J (1987) Science 237:

1176-1183