(Triticum aestivum L.)

Diss. ETH No. 12411
Single-Stranded
DNA
as a
Tool
for Genetic Transformation in Wheat
(Triticum
aestivum
L.)
A dissertation submitted to the
SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH
for the
degree of
Doctor of Natural Sciences
presented by
Murielle UZE
DEA de
Biologie Moleculaire
Strasbourg,
born
May
et Cellulaire
France
27 1967
citizen of France
accepted
on
the recommendation of
Prof. Dr. I. Potrykus, examiner
Prof. Dr. B. Keller, co-examiner
PD. Dr. C.
Sautter, co-examiner
October 1997
II
SUMMARY
Wheat is
much effort has been put into
resistance
important cereals of
of the most
one
embryonic calli
bombardment to
can
work
frequency
develop
to
was
well
as
the
as
Agrobacterium would
method
a
embryos
from immature
efficiency
which
be the system of choice,
widely
is
used to
is still low. The aim of
transformation
the
increases
of gene transfer for
quality
disease
goal. Currently,
contribute to this
transform wheat. However, the transformation
my
(e.g. for
wheat
genetically improving
quality). Gene transfer
or
the world. Therefore,
single
copy insertions.
unfortunately,
its natural host
range is restricted to dicot's. However, different labs demonstrated efficient rice
by Agrobacterium, indicating
transformation
Agrobacterium tumefaciens
types have been screened
on
and A.
immature
that it
might
rhizogenes
work in wheat also.
strains of different
zygotic embryos
opine
from several wheat
varieties. A T-DNA transfer to wheat cells has been shown in transient with
pus-intron
regeneration
was
unsuccessful. In contrast, rice
before inoculation to
the
transfer,
play
an
cell free
a
Agrobacterium by using
{gus
and
bar)
important
of bacteria
case
tried
we
high efficiency
with
by Agrobacterium
In
were
cointroduced
single- (ss)
DNA
can
ssDNA.
or
double-stranded
integrate
be
ds
or ss
might
limiting,
be
we
a
compared:
It
was
efficiency
element
partially
in rice.
for
gene
the T-DNA of
DNA. Two marker genes
circular
(c)
or
in wheat
linear
(I)
as
discovered that linearized
high efficiency of
bar and
plant
plasmolysis
by microprojectile bombardment
were
but
have been transformed
limiting
single-stranded
form with
l-ssDNA showed
the
and mimicked
(ds) plasmid.
Although coexpression of
higher for dsDNA,
ssDNA
as
plants
role for gene transfer
approach
linearized
the gus gene
and I have shown callus
representing
cells. Four different DNA structures
a
expressed
Some sectors
marker gene.
a
up to 14% for I-
gus after cobombardment
was
higher cotransformation efficiency. Since
I-
good substrate for degradation, and its nuclear import might
suggest
agrobacterial proteins
to deliver l-ssDNA
similar to T-DNA.
fragments
with VirD2 and VirE2
Ill
RESUME
plus importantes
Le ble est une des
cereales
au
monde. Pour cela, de
nombreux efforts sont apportes dans
son
pour les resistances de maladie
qualite nutrionnelle).
contribuer
peut
a
zygotiques immatures
mon
objectifs.
ces
transformer le ble est
travail consistait
le
developper
a
efficacement
particules
son
Plusieurs souches
une
methode
il
ete
a
qui augmenterait
etre
d'opine
types
transitoirement
indiquant
d'Agrobacterium tumefaciens
ont
ete
criblees
en
sur
vers
des
et A.
frequence
systeme de
la classe
a
transforme
se
pourrait
la cellule
evidence par Putilisation du gene
ont ete obtenues et
j'ai
important
montre que la
plasmolyse
rhizogenes
embryons
aucune
a
ete mis
Des secteurs
plante
avec
de
zygotiques
vegetale
gftvs-intron.
regeneree. Au contraire, des plantes de riz transformers
role
la
methode
cette
que
bleus exprimant le gene gus ont ete obtenus mais
un
le
demontre que le riz
immatures de ble. Un transfert de T-DNA
joue
pour
embryons
des
sur
hote naturel de plantes est restreint
Agrobacterium,
par
utilisee
ble.
au
differents
technique actuellement
La
copie unique. Agrobacterium pourrait
de
Le transfert de gene
qualite de transfert de gene pour obtenir des
dicotyledones. Cependant,
s'etendre
genetique (par exemple
mais le rendement demeure encore faible. Le but de
transformation choisi mais
des
la
bombardement de
de transformation ainsi que la
insertions
ou
amelioration
n'a pu etre
Agrobacterium
des cals avant I'inoculation
pour I'efficacite de transfert de gene dans les cellules de
riz.
Dans le
gene,
cas ou
seconde approche
une
d'Agrobacterium
(gus
et
la bacterie serait le facteur limitant pour le transfert de
bar)
avec
de I'ADN
a
consiste
simple
a
imiter
partiellement
le
T-DNA
brin lineaire. Deux marqueurs de gene
ont ete cointroduits par cobombardement dans les cellules de ble.
Quatre differentes structures de I'ADN ont ete comparees: I'ADN circulaire (c),
ou
lineaire
(I)
en
tant que DB ou
tant que
simple (SB)
ou
double brin
(DB).
SB, semble integrer le genome bu ble
L'ADN lineaire,
avec
une
en
grande
IV
efficacite par rapport
des
coexpression
au
circulaire, jusqu'a 13.92% pour le SB. Bien que la
genes
bar
et
gus
cobombardement, plus elevee que le l-SB,
etait
ce
limitee,
degradation
nous
et que son
importation
suggerons de delivrer les
VirD2 et VirE2
d'Agrobacterium,
I'ADN
dernier montrait
efficacite de cotransformation. Comme I'ADN l-SB
pour la
pour
pourrait
vers
a
une
plus
apres
haute
etre un bon substrat
le noyau cellulaire serait
fragments d'ADN SB
similaire
l-DB,
avec
la structure du T-DNA.
les
proteines