Plant Cell Reports

Plant Cell Reports (1990) 9:233-236

9 Springer-Verlag1990

Transgenie Brassica napus plants obtained by coeultivation of protoplasts with Agrobacterium tumefaciens J.E. Thomzik and R. Hain Bayer AG, Agrochemicals Division, Development/Biotechnology, D-5090 Leverkusen-Bayerwerk, Federal Republic of Germany Received February 28, 1990/Revised version received May 14, 1990 Communicated by H. L6rz

ABSTRACT Hypocotyl oilseed

protoplasts

rape

double-low

of

(Brassica

quality

napus)

were

winter

lines

transformed

Agrobacterium

tumefaciens

3850:1103

(dimer)

neo

German

harbouring

containing

of

using pGV

chimaeric

tion

and

regeneration

cessfully

faciens

formed

protoplasts

hypocotyls

fertile

and

Transformation resistance,

was

blot

normal

confirmed

nopaline

phosphotransferase ern

regenerated

to

plants.

rape

several

using

cell

were

oilseed

with

kanamycin resistance reporter genes. Transphenotypically

used for Brassica

transgenic tained

systems

layer

cotyledonary

explants,

1987).

In

these

napus

L.

ssp.

progeny

from

obtumethin

segments, for

and

trans-

Fry et

et

et

Seed

A.

(Moloney et al. 1989;

Charest

hybridization.

of

as starting material

neomycinSouth-

Fertile

were

formation experiments

production,

and

suc-

petioles,

stem

kanamycin

activity,

napus.

plants

strains

by

II

being

al.

type B.

al.

1988;

al.

1987;

experiments oleifera

Pua

spring

cv.

Westar

was used and npt II as a selectable

marker

self-pollinated transformants expressed the

gene. Recently,

introduced kanamycin resistance as a Mende-

ported

lian trait.

tion of B. napus and B. oleracea cultivars.

ABBREVIATIONS

also

a

De Block

2.4D,

2,4-dichlorophenoxy

kanamycin; NAA,

MS, Murashige

s-naphthalene

Cf, ClaforanR;

acetic

acid;

and Skoog

acetic

(1962);

acid;

neomycinphosphotransferase;

npt

Km,

NPT

II,

II,

neo-

B.

been

nigra

report napus

by

A.

demonstrated (Sacristan

campestris 1988).

cv.

However, on

et

Emma

successful

has

vation

nopaline

fertile transgenic plants.

gene;

ocs,

gene;

IAA,

indole-3-acetic acid.

In ter

DNA

Agrobacterium mon way. generate

into

plant

tumefaciens

However,

transgenic

this

A.

plants

transfer

the

use

is the most

a prerequisite cells

or

of

to re-

tissues

to

There are some transforma-

Offprint requests to." J.E. Thomzik

and

B.

Eriksson

so

far

no

of

B.

paper

we

oilseed

regeneration

rape with of

describe

resulting the

protoplasts

(double-low

in

transof win-

quality)

A.

tumefaciens

and

these

protoplasts

to

by the fer-

tile transgenic plants.

com-

to obtain

is the capability

manipulated

fertile plants.

cells

to

been

tumefaciens

of hypocotyl

cocultivation

Among the different methods foreign

with

formation

INI[R~ODUCTION

and

transformation

synthase;

synthase

1989)

of

(winter type) protoplasts by coculti-

line

octopine

has

protoplasts

al.

II gene; NOS, nopasynthase

transforma-

(Ohlsson

there

re-

tumefaciens

for

mycinphosphotransferase nos,

(1989)

genotype-independent

Transformation BAP, 6-benzylaminopurine;

eta!.

MATERIALS AND METHODS Plant material Seeds of winter oilseed rape B. napus possessing double-low quality (Thomzik and Hain 1990) were kindly supplied by Kartoffelzucht Boehm KG, D 8301 Langquaid (line RII7) and Norddeutsche Pflanzen-

234 zucht Lembke KG, Holtsee (line R72 = cv. Ceres). Bacterial strain Protoplasts were inoculated with A. tumefaciens strain C58CI harbouring pGV3850::l103 neo dimer (Czernilofsky et el. 1986). This disarmed Tiplasmid contains the nos gene, two tandemly arranged chimaeric nptII genes flanked at the 5' end by the nos promotor, and at the 3' end, the 7 polyadenylation site of the ocs gene conferring resistance to Km in plants. Protoplast isolation and cocultivation Protoplasts of 2- to 3-day-old etiolated hypocotyls were isolated according to Thomzik and Hain (1988). Three days after protoplast isolation 5 ~i of an overnight culture of A. tumefaciens strain C58CI pGV3850::II03 neo (dimer) were added to 1 ml protoplast ~ulture medium (Glimelius 1984) containing 3 x 10- protoplasts (200-500 bacteria/ protoplast). After 48 - 72 hours of cocultivation at 18 ~ C in the dark or dim light free bacteria were removed by washing protoplasts twice in protoplast culture medium supplemented with 500 ~g/ml Cf. Protoplasts were embedded in 100 ~i droplets of culture medium with 500 ~g/ml Gf solidified with 0.6Z agarose and surrounded by liquid medium (Thomzik and Hain 1988) or cultured in liquid culture medium. Plant regeneration Protoplasts were regenerated to plants by the procedure of Thomzik and Hain (1988) with modifications for transformation experiments. Thirteen days after protoplast isolation, protoplast culture medium surrounding the agarose droplets was replaced by callus culture medium (GG medium) containing 500 ~g/ml Cf. The CC medium consists of MS medium supplemented with 0.i mg/l 2.4D, 0.I mg/l NAA, 0.5mg/l BAY and 20 g/l sucrose, pH 5.8. Km was added 2 to 7 days later to yield a final concentration of 25 ~g/ml. The ce medium was replaced by fresh CC meditun 18 days and 24 days after protoplast isolation and 35 ~g/ml Km was added to the medium. The cultures were kept in a 16 h photoperiod of 1,500 lux intensity. Microealli were separated and transferred onto 0,6Z agarose (Sigma, type I) solidified CC medium containing 50 ~g/ml Km. After 2 to 3 weeks on this medium calli of 1 to 3 mm diameter were finally transferred to a shoot regeneration medium (SR-medium; MS medium with 15 g/l sucrose, 0.I mg/l IAA, 1.0 mg/l BAY, 2.0 mg/l zeatin and 0.25 Z Gelrite, pH 5.8) containing 250 ~g/ml Cf. Km was omitted and the cultures illuminated with 3000 lux. The medium was replaced by fresh SR medium three times in 2 weeks intervals. Further growth and multiplication of shoots were obtained on SMI medium consisting of MS medium supplemented with 4 mg/l BAY, 0.2 mg/l NAA, 20 g/! sucrose, 250 ~g/ml Cf, and 50 ~g/ml Km, pH 5.8. Shoots were rooted on 1/2 MS medium containing 0.25 mg/l NAA in the presence of antibiotics as used before. Kanamycin resistance assay Leaf explants of individual regenerated plants were cultured on callus promoting MS medium supplemented with each 2 mg/l NAA and 2 mg/l Blip in the presence of 50 to 125 ~g/ml Km (MS/Km medium). The explants were kept on MS/Km medium in the light (3000 lux) at 26~ Nopalin assay Leaf tissue of transformants which produced a green callus on MS/Km medium was analyzed for nopalin according to Otten and Schilperoort (1978) and to Shaw et al. (1988). Nopalin was also detected in 5 ~i samples of tissue extracts separated by thin layer chromatography on silica gel 60 F25. (Merck, Germany) in acetone/ H90 (i:i) when staine~ with Sakaguchi's reagent (Saka~uchi 1950). Arginine (Rf=0.01) showing a crimson-pink colour is clearly

to discriminated from the fainter orange-pink coloured fast migrating nopalin (Rf=0.81). NPT II assay NPT II enzyme activity was determined in callus tissue and plant tissue according to the procedure described by Reiss et al. (1984). Southern blot and Northern blot hybridizations Plant DNA was isolated according to Taylor and Powell (1983) or Dellaporta et al. (1983). Ten ~g of total DNA were digested with EcoRI and HindIII in the appropriate restriction enzyme buffer and separated on IX agarose gels in TAE buffer (40mM Tris, 20mM sodium acetate, 18mM sodium chloride, and 2mM EDTA, pH 7.8). After electrophoresis DNA fragments separated were transferred to nitrocellulose filters. RNA was isolated and purified as previously described (Taylor and Powell 1983) and separated on a denaturing 1.5Z agarose/formaldehyde gel and transferred to nitrocellulose filters according to Maniatis et el. 1982. Hybridization probes (pLGVII03 neo or the EcoRI-SalI fragment containing the npt II coding r~gion) were synthesized by nick translation (> 10Vcpm/~g). Hybridization was for 24 h at 42~ and filters were washed at 50 ~ C as described by Hughes et al. (1978). Films were exposed at -70~ for 24 h using intensifying screens. Inheritance of Km resistance Seeds from self-pollinated transformants and non-transformed plants were surface-sterilized in 70 % v/v EtOH (I min) followed by continuous shaking in a 6 Z w/v solution of NaOCI for 20 min. The seeds were germinated on MS/Km medium or 1/2 MS medium without sucrose and growth regulators but containing 75~g/ml Km. Resistance or sensitivity to Km was scored 10 days after germination.

RESULTS

AND DISCUSSION

Proper

coculture

conditions

were

protoplasts calli

after

protoplast

30 % of

cells

rate

from

ture

was

stan

et

beginning

and

cessful

with

types

3 days

more

than

division

protoplast the

cul-

recovery

of

as d e s c r i b e d

by Sacri-

Cocultivation

directly

isolation

Eriksson

(1988)

both winter The

important

tion

optimal

was

when

to

(1989).

tested.

diminished

The

A high

of

for

napus

colonies

protoplast

Ohlson

plants.

isolation

B.

regenerate

cocultivation

essential

al.

after

to

had divided.

the

transformed

an

for

culture

to a l l o w

and

transgenic time

protoplast

necessary

to d i v i d e

and

starting

and

timing

factor.

the

dramatically.

Best

was

oilseed

to

less

suc-

rape

geno-

of K m s e l e c t i o n

Early

surviving

according

addition

protoplast results

of

is Km

populawere

ob-

tained

by starting

with

a low concentration

of

(25

when

microcolonies

Km

elongated tration Finally,

~g/ml) cells

was

were

then

putative

were

selected

dium

containing

on

formed.

The

increased

to

transgenic agarose

50 ~ g / m l

Km concen35

~g/ml.

microcalli

solidified Km.

with

CC m e -

Surviving

235 microcalli

grew

possessed

a

slowly

turned to g r e e n um.

The

on

this

brown-yellow after

presence

regeneration

transfer

of Km

medium

colour

to SR medi-

affected

negatively.

and

which

the

shoot

Therefore

no

Km

was u s e d in the SR medium. Three each

experiments

line.

erated RII7

in

all

three

(19 plants)

(2 plants).

200

calli

growing

plants

frequency

tumefaciens

line

of

cocultivation

shoots

after

was

80

to

containing

(2.5 x 105 p r o t o p l a s t s ) .

d e r i v e d callus

regen-

with

CC m e d i u m

50 ~ g / m l Km a p p e a r e d per

ation

were

Approximately

on

from

ex-

Regener-

protoplast

cocultivation with

lower

for

cv.

Ceres

A.

than

for line RII7 and s i g n i f i c a n t l y r e d u c e d for both

compared

to

experiments

when

no

co-

c u l t i v a t i o n was p e r f o r m e d

(Thomzik and H a i n

1988).

experiments

with

45 d i f f e r e n t

win-

Plant

regeneration

hypocotyl protoplasts

of

ter o i l s e e d rape g e n o t y p e s revealed

a

significant

capability

of

regenerate

shoots

ThUs,

Sometimes

a

appeared with violet concentration lin

with

and in one e x p e r i m e n t w i t h

Ceres

periment

performed

experiments

cv~

bleached.

was

yellow-brown

areas w h e n

callus

a lower Km

(50-75 ~g/ml) was used. Nopa-

detected

in

15

out

of

21

calli

tested.

were

Transformed

f o r m e d almost no callus and t u r n e d b r o w n or

II

enzyme

calli

MS/Km or

medium

no

NPT

leaves

of

Southern

activity

induced

from

(Fig. II

was leaf

2).

was

analysis

tissue

ments to RNA

of

the

(Fig.

6.0, probe.

isolated

revealed

4.3,

npt

callus tissue.

II

no

in callus expected

and

2.9

kbp

gene

in

diffe-

The

callus

weak

Nevertheless,

3).

Northern from

only

detected

revealed

rence of the t r a n s f e r r e d DNA leaf

in on

However,

activity

transformants.

blot

detected exp!ants

blot and

and

frag-

hybridized analysis leaf

expression

of

tissue only

in

T h e r e was no npt II m R N A

d e t e c t a b l e in leaves

(data not shown).

(incl. cv.Ceres)

difference

in

derived

calli

protoplast

NPT all

(Thomzik and H a i n

the to

1988).

o n l y w i n t e r o i l s e e d rape lines w i t h a

sufficient suitable

regeneration

for

capability will

transformation

of

be

protoplasts

w i t h A. tumefaciens. F r o m the first e x p e r i m e n t w i t h line RII7 12

shoots

These

were

shoots

regenerated

derived

calli g r o w i n g

on

from

on CC m e d i u m

SR

80

medium.

individual

in the p r e s e n c e

Figure i. Leaf segments of transformants (left) were scored for their ability to form callus on selective medium containing 125 ~g/ml kanamycin. Kanamycin sensitive controls formed no callus (right).

of 50 ~g/ml Km. Eight shoots r e m a i n e d g r e e n and

developed

plication

into

medium

plants

and

on

rooting

c o n t a i n i n g 50 ~g/ml

Km.

generants

reduced

showed

a

SMI

multi-

medium

Three

of

PT3

c

c

BnT

each

these re-

fertility.

Re-

cently, we have o b s e r v e d v a r i o u s degrees of female and m a l e s t e r i l i t y a m o n g r e g e n e r a n t s of

line

tion

RII7

derived

experiment

from

using

one

the

transforma-

same

strain

of

A.tumefaciens. All for

putative

expression

placing

leaf

Km-P transformants of

tissue

t a i n i n g 125 ~g/ml Km

the

npt

were II

tested

genes

onto M S / K m m e d i u m (Fig.

by con-

i). E x p l a n t s of

all r e g e n e r a n t s r e m a i n e d g r e e n and u n i f o r m ly

produced

a

dark

green

compact

callus.

T i s s u e from n o n - t r a n s f o r m e d c o n t r o l plants

Figure 2. Enzyme assay for NPT II activity in B. napus callus derived from leaves of regenerants (line Rl17). (Fr3) NPT II positive callus of Nicotiana tabacum served as positive control. (c) Nontransformed controls from leaf and leaf callus of line RII7. (BAT) Leaf callus from transformed regenerant (line RII7). (Km-P) Phosphorylated kanamycin.

236 REFKRKNCKS

FiKure 3. (I) Southern blot analysis of transformed Brassica napus (line RII7) callus (C), plant (P). (2) non-transformed controls.

Recently,

De

Block

that

transgenic

been

selected

N P T II

Km

organ

m o t o r has

been

co

(An et

was

highly

leaves This

and

in

older

weakly

expressed

t h a t the

seed

mants

144

sed Km leaves.

the

a n d 139

Our

express

our the

B. n a p u s this

rea-

medium.

with

Km

full

showed

transfor-

seedlings

posses-

green

progeny

Mendelian

were

resistance.

both

pirmary

plants

and bleached

formed

out.

Both

segregation

of

genes. show

transgenic

from protoplasts

faciens. ferent

leaves

results

regenerate

of

and developed

Non-transformed

the i n t r o d u c e d

in

For

of

be

explain

RII7)

inheritance

seedlings

primary

leaves.

self - p o l l i n a t i o n

seeds

transformants

type)

(line

lower

might

transgenic

medium.

germinated

resistance

violet

of

K m in o u r r e g e n e r a t i o n

following

tobac-

upper

can

pro-

promotor

regulated

of r e g e n e r a t i n g

for

200

nos

in

and

transformants

analyse From

nos

nos p r o m o t o r

Km containing

set

of the

The

in

develop-

tissues

specifically

had

negative and

transgenic

active

s o n we o m i t t e d Two

for

regenerants

difficulty on

expression

also

which

often

1988).

tissue

found

specific

al.

transgenic

plants

were

reported

indicates

also

(1989) plants

An

regulated

plants

al.

napus

on

assays.

mentally

et

B.

Further

it

B.

of

is

napus

possible

plants

transformed

experiments

agronomically genotypes

that

to

important winter

to

(winter

b y A. t u m e transfer

genes

oilseed

and

in d i f rape

are

in p r o g r e s s . ACKNOWLKDGEMENTS

The authors wish

to thank G.Werner,

B.Nelke,

C.Ebke

and R.Langebartels for excellent technical assistance.

An G, Costa MA, Mitra A, Ha SB, Marton L (1988) Organ-specific and developmental regulation of the nopaline synthase promotor in transgenic tobacco plants. Plant Physiol 88:547-552 Charest PJ, Holbrook LA, Gabard J, lyer VN, Miki BL (1988) A~robacterium-mediated transformation of thin cell layer explants from Brassica napus L. Theor Appl Genet 75:438-445 Czernilofsky AP, Hain R, Herrera-Estrella L, LSrz H, Goyvaerts E, Baker BJ, Schell J (1986) Fate of selectable marker DNA integrated into the genome of Nicotiana tabacum. DNA 5:101-113 De Block M, Brouwer DD, Tenning P (1989) Transformation of Brassica napus and Brassica oleracea using Agrobacterium tumefaciens and the expression of the bar and neo genes in transgenic plants. Plant Physiol 91:694-701 Dellaporta, SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Molecular Biology Reporter 1:19-21 Fry J, Barnason A, Horsch P~B (1987) Transformation of Brassica napus with Agrobacterium tumefaciens based vectors. Plant Cell Rep 6:321-325 Glimelius K (1984) High growth rate and regeneration capacity of hypocotyl protoplasts in some Brassicaceae. Physiol Plant 61:38-44 Hughes SH, Shank PR, Spector DH, Kung HJ, Bishop JM, Varmus HE, Vogt PH, Breitman ML (1978) Proviruses of avian sarcoma virus are terminally redundant, co-extensive with unintegrated linear DNA and integrated at many sites. Cell 15:13971410 Maniatis T, Frisch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold spring harbor laboratory press, N.Y. Moloney MM, Walker JM, Sharma KK (1989) High efficiency transformation of Brassica napus using Agrobacterium vectors. Plant Cell Rep 8:238-242 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473-497 Ohlson M, Eriksson T (1988) Transformation of Brassica campestris protoplasts with Agrobacterium tumefaciens. Hereditas 108:173-177 Otten LABM, Schilperoort RA (1978) Int Rev Cytol Suppl 16:169-189 Pua EC, Mehra-Palta A, Nagy F, Chua NH (1987) Transgenic plants of Brassica napus L. Bio/Technol 5:815-817 Reiss B, Sprengel R, Will H, Schaller H (1984) A new sensitive method for qualitative and quantitative analysis of neomycin phosphotransferase in crude cell extracts. Gene 30:217-223 Sacristan MD, Gerdemann-K-nSrck M, ~chieder 0 (1989) Inkorporation of hygromycin resistance in Brassica nigra and its transfer to B_~.napus through asymmetric protoplast fusion. Theor Appl Genet 78:194-200 Sakaguchi S "(1950) A new method for the colorimetric determination of arginine. J Biochem 37:231 Shaw ML, Conner AJ, Lancaster JE, Williams MK (1988) Quantitation of nopaline and octopine in plant tissue using Sakaguchi's reagent, Plant Mol Biol Rep 6:155-164 Thomzik JE, Hain R (1988) Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L. Theor Appl Genet 76:165-171 Thomzik JE, Hain R (1990) EinfOhrung einer Sencorresistenz in Deutschen Winterraps mit 00-Qualit~t. Pflanzenschutznachrichten 43, Bayer AG Taylor B, Powell A (1983) Isolation of plant DNA and RNA. BRL Focus 3, Gaithersburg, M_D, USA

Transgenic Brassica napus plants obtained by cocultivation of protoplasts with Agrobacterium tumefaciens.

Hypocotyl protoplasts of German winter oilseed, rape (Brassica napus) lines of double-low quality were transformed using Agrobacterium tumefaciens har...
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