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.
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