Note: Descriptions are shown in the official language in which they were submitted.
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PROCESS FOR THE INTRODUCTION OF EXPRESSIBLE GENES INTO PLANT
CELL GENOMES AND AGROBACTERIUM STRAINS CARRYING HYBRID TI
PLASMID VECTORS USEFUL FOR THIS PROCESS
Technical field of invention
This invention relates to recombinant molecules, processes for their
preparation, and
processes for their introduction into plant cells and the plant cells or
plants,
respectively, containing foreign DNA sequences in their genome. More
particularly,
the invention relates to DNA sequences to be expressed in appropriate host
plant
cells. The recombinant DNA molecules contemplated are characterized by
sequences that code for products, e.g. amino acids or polypeptides, useful for
the
growth of the plant or for improving its quality as nutrient, or for the
production of
valuable metabolites (e.g. alkaloids or steroid precursors).
In the description the following terms are used:
bom site : region of DNA where mob functions specifically interact and
initiate
autonomous DNA transfer.
Border seduence : DNA sequence which contains the ends of the T-DNA
Broad-host-range realicon : a DNA molecule capable of being transferred and
maintained in many different host cells.
Callus tissue: a mass of unorganized and undifferentiated cells.
Clonin : the process of obtaining a population of organisms or DNA
or better:
sequences derived from one such organism or sequence by asexual
reproduction.
the process of isolating a particular organism or part thereof, and the
propagation of this subfraction as a homogenous population.
Cloninct vehicle : a plasmid, phage DNA or other DNA sequences which are able
to
replicate in a host cell, characterized by one or a small number of
endonuclease recognition sites at which such DNA sequences may be
cut in a determinable fashion without attendant loss of an essential
X
__ 1 ~~+1 ~~
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biological function of the DNA, e.g. , replication, production of coat
proteins or loss of promoter or binding sites, and which contain a
marker suitable for use in the identification of transformed cells, e.g.,
tetracycline resistance, or ampicillin resistance. A cloning vehicle is
often called vector.
Coding seguence : DNA sequence which determines the amino acid sequence of
a polypeptide.
Cointegrate : the structure resulting from a single cross-over event between
two
circular DNA molecules.
Complementation ' tm tans : process whereby a DNA molecule (replicon) which is
not physically linked to another replicon can provide a diffusible
substance which is missing and required by the other nonlinked
replicon.
Coniuaation : the process whereby DNA is transferred from bacteria of one type
to another type during cell-to-cell contact.
Crossing-over : the process of exchange of genetic material between homologous
DNA sequences.
Deletion substitution : removal of one DNA sequence and its replacement by a
different DNA sequence.
Differentiation : the process whereby descendents of a cell achieve and
maintain
specialization of structure and function.
DNA seguence or DNA segment : a linear array of nucleotides connected one to
the other by phasphodiester bonds between the 3' and 5' carbons of
adjacent pentoses.
Double cross-over : the process of resolution of a cointegrate structure into
two
circular DNA molecules. This process is used to exchange genetic
information. One of the DNA circles has two regions of homology
with the target DNA through which recombination can occur; these
two regions bracket a nonhomologous DNA sequence which is to be
exchanged with the target DNA. If this second cross-over occurs in
the same region of DNA as the first, the original DNA circles will be
generated. If this second cross-over occurs in the second
1341419
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homologous region, genetic exchange will have occurred between
the two circles.
Expression : the process undergone by a structural gene to produce a
polypeptide. It is a combination of transcription and translation.
Expression control seguence : a sequence of nucleotides that controls and
regulates expression of structural genes when operatively linked to
those genes.
F-tvaeplasmid : plasmid carrying the F (fertility) factor which allows the
transfer of
a copy of the plasmid to a host not carrying the F-factor.
Gene : a DNA sequence composed of two parts, ( 1 ) the coding sequence
for the gene product, and (2) the sequences in the promoter region
which control whether or not the gene will be expressed.
Genome : the entire DNA of a cell or a virus. It includes inter alia the
structural
genes coding for the polypeptide(s), as well as operator, promoter
and ribosome binding and interaction sequences, including
sequences such as the Shine-Dalgarno sequences.
Genotvae: the sum total of the genetic information contained in an organism.
Homologous recombination : recombination between two regions of DNA which
contain homologous sequences.
I-type plasmid : a group of autotransferable plasmids of a different
incompatibility
group than F.
Incompatibility : Incapability of two DNA molecules of coexisting in the same
cell
in the absence of selective pressure.
Insertion : addition of a DNA sequence within the DNA sequence of another
molecule.
Leader seguence : the region of an mRNA molecule extending from the 5' end to
the beginning of the first structural gene; it also includes sites
important to initiate translation of the coding sequence of the
structural gene.
Meiosis : two successive divisions that reduce the starting number of 4 n
chromosomes to 1 n in each of four product cells. This process is
important in sexual reproduction.
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1 X41 41 9
-4-
mob (mobilization functions) : a set of products which promote DNA transfer
only
in combination with tra functions. Mob can promote transfer of
plasmids containing a bom site.
Mobilization : the process whereby one DNA molecule which is not able to
transfer
to another cell is helped to transfer by another DNA molecule.
Mobilization helper alasmid : a plasmid capable of providing diffusible
products
which another plasmid lacks for transfer to another host cell.
NoncoJuaative recombinant plasmid : a DNA molecule which is not capable of
being transferred by itself from its host cell to another host cell during
cell-to-cell contact. For transfer it will need further functions supplied
by other DNA, e.g. by (a) helper plasmid(s).
Nucleotide : a monomeric unit of DNA or RNA consisting of a sugar moiety
(pentose), a phosphate, and a nitrogenous heterocyclic base. The
base is linked to the sugar moiety via a glycosidic bond (1' carbon of
the pentose) and that combination of base and sugar is a nucleoside.
The base characterizes the nucleotide. The four DNA bases are
adenine ("A"), guanine ("G") , cytosine ("C") , and thymine ("T"). The
four RNA bases are A, G, C, and uracil ("U") .
Phenotyae : the observable characteristics of an individual resulting from the
interaction between the genotype and the environment in which
development occurs.
Plasmid : a nonchromosomal double-stranded DNA sequence comprising an
intact "replicon" such that the plasmid is replicated in a host cell. When
the piasmid is placed within a unicellular organism, the characteristics
of that arganism are changed or transformed as a result of the DNA of
the plasmid. For example, a plasmid carrying the gene for tetracycline
resistance (TcR) transforms a cell previously sensitive to tetracycline
into one which is resistant to it. A cell transformed by a plasmid is
called "transformant".
Polyaeptide : a linear series of amino acids connected one to the other by
peptide
bonds between the a-amino and carboxy groups of adjacent amino
acids.
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Promoter region : DNA sequences upstream to the start of the coding sequence
which regulate transcription of the gene.
Promoter seauence : sequence at which RNA polymerase binds and promotes the
faithful transcription of downstream sequences.
Recombinant DNA molecule or hybrid DNA: a hybrid DNA sequence comprising at
least two nucleotide sequences, the first sequence not normally being
found together in nature with the second.
Recombination: the creation of a new association of DNA molecules or parts of
DNA
molecules.
Region of homology : a region of DNA which shares the same DNA sequence as
that
found in another region of DNA.
Realicon : a self-replicating genetic element possessing a site for the
initiation of
DNA replication and genes specifying the necessary functions for
controlling replication.
Restriction fragiment : a DNA molecule resulting from double-stranded cleavage
by
an enzyme which recognizes a specific target DNA sequence.
RNA aolvmerase : enzyme which results in the transcription of DNA into RNA.
Selectable marker gene : a DNA sequence which, when expressed, gives that cell
a
growth advantage over cells which dg not contain that DNA sequence,
when all cells are in growth medium which can distinguish the two
types of cells. Commonly used selectable marker genes are those
which encode resistance to antibiotics.
Single cross-over : the process of recombining two circular DNA molecules to
form a
cointegrate larger circle.
Structural Gene : a gene which codes for a polypeptide.
T-DNA : portion of the Ti plasmid as it is found stably integrated into the
plant
cell genome.
T-region : portion of the Ti plasmid which contains the DNA sequences which
are
transferred to the plant cell genome.
Ti plasmid : large plasmids found in strains of Agrobacterium tumefaciens
containing the genetic information for tumor (crown gall) induction on
susceptible plants.
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TL-DNA and TR-DNA : octopine crown gall tumor cells can contain two T-DNA
sequences, a left T-DNA, i.e. TL-DNA, and a right TR-DNA. TL-DNA
contains sequences also found in common with T-DNA of nopaline
tumor cells whereas TR-DNA does not.
tr transfer functions, : both plasmid-encoded diffusible products and sites of
action
utilized during DNA transfer between cells, e.g. products required to
make a bridge between two cells and the site at which DNA transfer is
initiated.
Transcrietion : the process of producing mRNA from a structural gene
or : the process involving base paring whereby the genetic information
contained in DNA is used to order a complementary sequence of bases
in an RNA chain.
Transformation : genetic modification induced by the incorporation of
exogenous
DNA into the DNA complement of a cell.
Translation : the process of producing a polypeptide from mRNA
or : the process whereby the genetic information present in an mRNA
molecule directs the order of specific amino acids
-7- 1 341 41 9
during the synthesis of a polypeptide .
Undifferentiated pheno~e : a homologous appearance of cells in a
tissue without any specialized parts.
Vector : a DNA molecule designed for transfer between different
host cells .
~ 3t~1 41 9
Background Art
The development of recombinant DNA techniques has made the genetic engineering
of microorganisms a challenging prospect. These techniques might be extended
to
multicellular eukaryotes, if complete organisms could be regenerated from
single
somatic cells. The cells of some higher plants exhibit excellent regeneration
capacities and, therefore, are good materials for the genetic engineering of
higher
organisms.
A major problem for the genetic engineering of plants is the availability of a
system
for the introduction of exogenous (foreign) DNA into the plant genome. Such a
system is provided by the tumor-inducing (Ti) plasmids carried by the Gram-
negative
soil bacterium Agrobacterium tumefaciens. This organism has been shown to
cause
a neo-plastic transformation, called "crown gall", of wounded tissue of a very
wide
range of dicotyledonous plants. The proliferating neo-plasms synthesize novel,
Ti-
specified metabolites, called opines. The molecular basis of this
transformation is
the transfer and stable integration of a well-defined T-DNA (transferred DNA)
fragment of the Ti plasmid in the plant cell genome. In other words, crown
gall
tumors contain in their chromosomal DNA a DNA segment called the T-DNA which
is
homologous to DNA sequences in the Ti plasmid used to induce the tumor line.
In all
cases, this T-DNA corresponds to, and is colinear with, a continuous stretch
of Ti
plasmid DNA which is, therefore, called the T-region.
Ti plasmids are classified according to the type of opine synthesized in crown
gall
cells. Aarobacterium strains which induce the synthesis of nopaline [N-a-(1,3-
dicarboxypropyl)-L-arginine] in crown gall cells are called nopaline strains,
and
strains which induce the synthesis of octopine [N-a-(N-1-carboxyethyl)-L-
arginine]
are called octopine strains. These are the most commonly used Aarobacterium
strains.
The use of T-DNA as a vector for plant genetic engineering was demonstrated in
a
model experiment in which the 14 kb bacterial transposon Tn7 was inserted in
vivo
x
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9
near the right border of the T-DNA from the Ti plasmid of the strain
A4robacterium
T37. Nopaline synthesis was eliminated in the tumors incited by agrobacteria
carrying this Ti plasmid. Furthermore, Southern blotting hybridizations
revealed that
the entire Tn7 was present in the chromosomal DNA of these tumors as part of
an
otherwise normal T-DNA sequence (Hernalsteens et al., Nature 287 (1980), 654-
656; Holsters et al., Mol. Gen. Genet. 185 (1982), 283-289). Thus, the
introduction of
this 14 kb DNA fragment into the 23 kb T-DNA has not altered the tatter's
ability to
be transferred to the plant cell genome.
The borders of the T-DNA from the Ti plasmid of the nopaline strain
A4robacterium
T37 have been very precisely determined. It is only a portion, roughly 23 kb,
of the
entire nopaline Ti plasmid. Furthermore, the borders of the T-DNA are known;
the
nucleotide sequences which define the borders of the T-DNA have been
determined
and compared with the same region of the nopaline Ti plasmid (Zambryski et al.
,
Science 209 (1980), 1385-1391; Zambryski et al., J. Mot. Appl. Genet. 1
(1982), 361-
370). The borders of the T-region are most probably involved in the
integration of the
T-DNA into the plant cell genome.
Knowledge of the T-DNA sequences which define the borders of the transferred
DNA is a basic requirement for the use of the Ti plasmid as a vector for DNA
transfer
to plant cells. Thus, foreign DNA can be inserted within these borders to
ensure its
transfer to the plant cell genome. In addition, if one expects to utilize this
system it is
important that the transformed plant cells are normal rather than tumorous in
their
growth properties. To produce normal cells after T-DNA transfer requires
knowledge
of the functions encoded by the T-DNA itself. Thus, the T-region of the Ti
plasmid
has been subjected to intense genetic analysis to determine which regions are
responsible for the tumor phenotype.
The T-DNA encodes functions which are responsible for the crown gall
phenotype.
The genes have been localized to specific regions of the T-DNA (L-eemans et
al.,
EMBO J. 1 (1982), 147-152; Willmitzer et al., EMBO J_. 1 {1982), 139-146). In
general, there are at least 4 genes which control the undifferentiated
phenotype of
1341419
tumor callus tissue. Mutants in these genes can either lead to transformed
tissues
which appears shoot-like or root-like. The latter results are especially
important if
one hopes to transfer DNA to plants for expression in normal plant tissue
rather than
tumor tissue.
Recently, a mutant Ti plasmid was found to induce transformed shoots which
were
capable for regenerating into completely normal plants. These plants were
fertile
and even transmitted T-DNA specific sequences through meiosis, i.e. progeny
plants
still contained T-DNA specific sequences (Otten et al., Mol. Gen. Genet. 183
(1981 ),
209-213). However, the transformed plant tissue contained in its chromosomal
DNA
a T-DNA which was very much reduced in size due to the generation of a large
deletion which removed the region of the T-DNA controlling the tumor
phenotype. It
is not known whether the deletion occurred during the initial transformation
event or
as a subsequent event leading to shoot formation.
The Ti plasmids are large (200 kb) and many genes located at different sites
on the
Ti plasmid are involved in the transformation of plant cells. Therefore, it is
not
possible to construct a small Ti plasmid-derived cloning vector with unique
endonuclease recognition sites at appropriate locations within the T-region,
and
possessing all functions essential for T-DNA transfer and stable incorporation
into
the plant cell genome. One known way to introduce a chosen DNA fragment into
specific restriction enzyme cleavage sites of the T-region of a Ti plasmid has
been to
construct cloning plasmids which are able to replicate in Aprobacterium as
well as in
Escherichia coli, and which contain a chosen restriction fragment of the T-
DNA.
Such cloning plasmids were denoted "intermediate vectors". Such intermediate
vectors were used to analyze the functions encoded by the T-region (Leemans et
al.,
J. Mol. Aaal. Genet. 1 (1981 ), 149-164).
Disclosure of the invention
The present invention relates to a process for the introduction of expressible
genes
into plant cell genomes. It is one of the objects of the present invention to
provide
1341419
11
modified acceptor Ti plasmids which allow the introduction of any genes) of
interest
into these plasmids. The genes) of interest to be introduced is (are)
contained within
a novel intermediate cloning vector carrying a region of homology with a
corresponding region in the acceptor Ti plasmid. The intermediate cloning
vectors
are a further object of this invention.
The introduction of the genes) of interest into the acceptor Ti plasmids is
achieved
by a single cross-over event which occurs within the two homologous DNA
segments
of the acceptor Ti plasmid harbored in Agrobacterium and the intermediate
cloning
vector. The intermediate cloning vector is mobilized from Escherichia coli
where it is
propagated to Aarobacterium using helper plasmids. Such helper plasmids and
their
functions for mobilization are known (Finnegan et al., Mol. Gen. Genet. 185
(1982),
344-351).
The result of the single cross-over event in Aarobacterium is a hybrid Ti
plasmid
vector. Such hybrid Ti plasmid vectors are a further object of the invention
The resulting hybrid plasmid vectors harbored in Agrobacterium (hereinafter
called
vector composition) can be used directly to infect plant cells which are
subsequently
screened for the expression of the product{s) of the gene{s) of interest. This
process
for preparing transformed plant cells by infection of plant cells with the
vector
composition, and the transformed plant cells, as well as plants generated
therefrom
are further objects of this invention. This strategy is applicable to any of
the plant-
transferable plasmids of Aarobacterium.
Description of drawings
Fig. 1 illustrates one embodiment of an acceptor Ti plasmid of the invention
which is
the result of a deletion of the internal portion of the T-region, except for
the
border sequences (1 ) and (2). The border sequences are essential for the
integration of the T-region into a plant cell genome. The region (3) between
border sequences {1 ) and {2) is the DNA segment which will be transferred to
1 341 41 9
12
the plant. The acceptor Ti plasmid contains a DNA segment (3) with a DNA
sequence which is homologous with at least a part of a DNA sequence in an
intermediate cloning vector permitting integration of the intermediate cloning
vector via a single cross-over event. The Ti plasmid region (4) codes for
functions which are essential for the transfer by Agrobacterium of the T-
region into plant cell genomes. This region has been designated as vir-
region.
Fig. 2 illustrates an intermediate cloning vector of the invention to be
inserted by a
single cross-over event into the acceptor Ti plasmid of Fig. I. It contains a
cloning vehicle DNA segment (3') with a DNA sequence which is
homologous with a least part of the DNA segment (3) of the acceptor Ti
plasmid permitting the desired single cross-over event. Moreover, the
intermediate cloning vector contains a gene or group of genes (5) of
interest having its natural promoter sequence. In general plant genes can be
used in this construction as they are mare likely to be expressed. However, in
principle any natural gene of interest can be inserted. The
intermediate cloning vector may also contain a selectable marker gene
(6). This gene should contain a promoter sequence which permits
expression of the gene in plant cells. Plant cells containing this marker
gene should have a selective growth advantage over cells without this
,,
A. 4
-13- 1 341 41 9
gene; in this way plant cells which have been transformed
by DNA containing this marker gene can be distinguished
from untransformed plant cells .
Fig . 3 illustrates another embodiment of an intermediate cloning
vector of the invention which is similar to the intermediate
cloning vector of F'ig. 2, and is to be inserted by a single
cross-over event into the acceptor Ti plasmid of Fig. 1. It
contains the cloning vehicle DNA segment (3'), an exogen-
ous promoter sequence (8) allowing regulated expression of
the genes) of interest (7), and, if desired, a marker
gene (6). . .
Fig. 4 schematically illustrates the construction of hybrid Ti plas-
mid vectors of the invention from the acceptor Ti plasmid of
F ig _ 1, and the intermediate cloning vectors of Figs 2 and 3
by a single cross-over event.
Fig. 5 outlines the steps involved in the genetic transfer of an
in~errnedia~e cloning vector from E. coli to Aarobacterium
containing «r acceptor Ti plasmid. 'The first step is the
conjugation of the E. coll. strain (2) containing the inter-
me4iate cloning vector to another E. coli strain (2) which
cc:~tains ~wc helper plasmids far the later conjugation to
Agrobacter ium. One helper plasmid contains DNA sequences
irr_aortant for plasmid transfer (tra) and the other helper
pl«smid c3ntains sequences which are important for the
mobilization (mob). l~Vhen these helper plasmids are intro-
duced by conjugation into E. call strain (1), the inter-
mediate cloning vector contained therein will be capable of
being transferred to other b~icterial strains. The tra and
mob helper plasmids also contain antibiotic resistance mark-
ers Abr2 and AbrJ which are different from those :found in -
the intermediate cloning vector (Abr ). Thus, the pres-
ence of all the plasmids can be monitored on selective
1341419
14
media. A mobilizing strain (3) is obtained. This mobilizing strain (3) is
conjugated to an A. tumefaciens strain (4) which contains the acceptor Ti
plasmid of Fig. 1 followed by selection for antibiotic resistance markers) of
the intermediate cloning vector. As the intermediate cloning vector cannot
replicate in Agrobacterium, it can only be maintained if it has formed a
cointegrate with the recipient acceptor Ti plasmid; this cointegrate structure
in
Aarobacterium (5) is the final hybrid Ti plasmid used to transfer DNA into
plant cell genomes.
Fig. 6 illustrates the construction of a model acceptor Ti plasmid (A) which
is
analogous to that shown in Fig. 1. Here, a double cross-over event occurs
between a Ti plasmid and another plasmid containing a DNA sequence which
is to replace a portion of the original Ti plasmid. More specifically, the
smaller
plasmid contains the border sequences of the T-region (1; 2) in a cloning
vehicle (3). A double cross-over results in the deletion of the internal
portion
T of the T-region and its replacement by the cloning vehicle. The acceptor Ti
plasmid (A) obtained is capable of transferring DNA contained between the
border sequences (1; 2) into plant cell genomes. The resulting transformed
plant DNA will not produce tumoraus crown gall tissue as the genes
controlling neoplastic growth are deleted in the Ti plasmid (A). Ti plasmid
(A)
is a very generalized acceptor Ti plasmid for any intermediate cloning vector
with homology with cloning vehicle (3). The cloning vehicle (3) may be a
conventional plasmid, such as pBR322 (or its derivatives).
Fig. 7 illustrates schematically the steps leading to the construction of an
intermediate cloning vector in E. coli host cells. A gene of interest (5)
bracketed by restriction endonuclease site R1 and a selectable marker gene
(6) bracketed by .
-15- 1 341 41 9
restriction endonuclease site R2 are inserted into a cloning
vehicle (3') containing single restriction sites for the en-
zymes R1 and R2. All three molecules are digested with
restriction enzymes R:i and/or ft~ and ligated together using
DNA ligase to form the intermediate cloning vector. The
cloning vehicle 3' mu:~t also contain additional DNA sequen-
ces whi<:h code for antibiotic resistance (Abrl) to use as a
selectable marker for bacterial genetics . The gene of inter-
est (5) may be under the control of its natural promoter or
of an exogenous promoter as outlizned in Figs. 2 and 3.
Fig . $ illustrates the ~ construction of another embodiment of an
acceptor Ti plasmid (B) of the invention. Here, a double
cross-over event occurs between a 'l"i plasrnid and a cloning
- vehicle (3) which contains DNA sequences (9) and (10)
which are homologoLes to Ti sequences just outside the
border sequences (1) and (2) respectively. The double
cross-over went results in the deletion of the entire 'T-re-
gion T i.ncluying the border sequences (1) anal (2) and its
replaceme~ i with the cloning vehicle (3) . Ti plasmid (B ) is
an accep ~_or for intermediate cloning vectors which contain
ti~!e gene ef interest c:lor~ed- bet:weEen the border sequen-
ces (1) arid {2); (see Fig. 9).
Fig. ° illustrates an intermediate cloning vector of the invention
to
be inserted by a single cross-over event into the acceptor
Ti plasmid i8) of Fig. $. It contains the border sequen-
ces (1) and (2) which flank the genes) of interest (5). It
also contains a cloning vehicle sequence (3' ) which is at
least partially homologous to the cloning vehicle sequence
- - (3) in the acceptor Ti plasmid (B) to allow homologous
recombination between the two plasmids .
Fig. 10 schematically illustrat~a the construction of hybrid Ti plas-
mid vectors of the invention from the acceptor Ti plasmid
1 ~~1 41 9
16
(B) of Fig. 8 and the corresponding intermediate cloning vector of Fig.
9. A single cross-over event introduces the intermediate cloning vector
of Fig. 9 into the acceptor Ti plasmid (B) of Fig. 8.
The following figures 11 to 20 more specifically illustrate the invention.
Fig. 11 illustrates the insertion of the 5.2 kb HindIII fragment AcgB into
pBR322 (see also Zambryski et al., Science 209 (1980), 1385-1391 ).
This fragment AcgB contains the right and left border regions of the
nopaline Ti plasmid. This clone pAcgB is used in the construction of
acceptor plasmid pGV3850, an "A-like" acceptor plasmid as shown in
Fig: 6. it is obvious to those skilled in the art that a clone analogous to
clone pAcgB can be obtained by using the cloned restriction fragments
which contain the left and right border region of a wild-type Ti plasmid.
Fig. 12 illustrates the T-region of nopaline Ti plasmid pGV3839. The HindIII
restriction endonuclease sites are indicated as (H). Mutated HindIII
fragment 19 is indicated (19'). The acetylphosphotransferase gene
providing kanamycin or neomycin resistance is indicated as aft and is
located in the black box area. The borders of the T-region are
indicated by arrows. The nopaline synthase gene is indicated by nos.
The numbers refer to the sizes of the restriction fragments according
to Depicker et al., Plasmid 3 (1980), 193-211. The construction of the
Ti plasmid pGV3838 can be taken from example 1, and the two
references cited.
Fig. 13 illustrates the construction of acceptor Ti plasmid pGV3850. The
plasmid pBR322-pAcgB (Fig. 11 ) is depicted in a linearized form. The
pBR322 sequences are indicated by the cross-hatched area and the
ampicillin resistance gene of pBR322 by ApR. Part of the T-region of
pGV3839 which is shown in Fig. 12 is shown here; the HindIII
1341419
17
fragments (10) and (23) involved in homologous recombination with
pAcgB and the apt gene are indicated. Double cross-over events lead
to the construction of pGV3850 and another replicon containing the
aft gene which is lost.
Fig. 14 illustrates schematically the construction of intermediate cloning
vector
pGV700 which is given in detail in example 2. The following
abbreviations are used to indicate restriction endonuclease sites : B,
BamHI; Bg, B,~r III; E, EcoRI; H, HindIII; S, SaII; Sm , Smal. The
following abbreviations are used to indicate antibiotic resistance; Ap,
ampicillin; Cm, chloramphenicol; Sm, streptomycin; Tc, tetracycline.
The numbers an the bottom of the figure which refer to TL-DNA
indicate the RNA transcripts of this region (Willmitzer et al., EMBO J. 1
(1982), 139-14fi).
Fig. 15 illustrates the structure of the intermediate cloning vector pGV750.
Its
construction is described in example 2. The restriction endonuclease
sites are indicated with their relative locations given in numbers as
kilobase pairs (kb). Pstl sites are not indicated but there are 3 in the
KmR/NmR region, and one in the Cb~ gene. The right and left borders
are also indicated. The Bg-III/BamHI and HaaI/SmaI sites used in the
construction of pGV750 are indicated but are not present in pGV750.
The shaded area corresponds to TL-DNA, the dark area to the KmR/
NmR region, the white area to contiguous Ti plasmid sequences, and
the line to the cloning vehicle pBR325. Other abbreviations used are
Ocs, octopine synthase; CmR, chloramphenicol resistance; CbR,
carbenicillin (analogous to ampicillin) resistance, and KmR/NmR,
kanamycin resistancelneomycin resistance.
Fig. 16 illustrates the construction of the intermediate vector pGV745
described in detail in example 3. pGV745 is used in the construction of
V
4.
1341419
18
the acceptor plasmid pGV2260, a "B-like" acceptor plasmid shown in
Figure 8. Restriction endonuclease sites are indicated as follows : B,
BamHl; H, HindIII; R, EcoRI. The ampicillin resistance gene is
indicated by ApR. The cross-hatched region indicates DNA
homologous to the left side of the T-DNA region of the octopine Ti
plasmid and the white region indicates DNA homologous to the right
side of the T-DNA region of the octopine Ti plasmid; the physical
location and description of the starting plasmids pGV0219 and
pGV0120 can be found in De Vos et al. , Plasmid 6 (1981), 249-253 .
Fig. 17 illustrates the construction of the acceptor plasmid pGV2260. The
deletion substitution in pGV2217 is indicated as a black box containing
the acetyl phosphotransferase gene (indicated aft) which provides
resistance to neomycin and kanamycin. The intermediate vector
pGV745 (Fig. 16) is depicted in linearized form; it has been opened at
the Hindlll site of pGV745 shown in Fig. 16. The pBR322 sequence is
indicated as a cross-hatched section and the ampicillin resistance
gene by ApR. Double cross-over events lead to the construction of
pGV2260 and the loss of the aft gene. Restriction endonuclease sites
are indicated as follows : B, BamHL; H, HindIII; R, EcoRI.
Fig. 18 illustrates the construction of a plasmid pLGV2381 for the expression
of genes downstream of the promoter of the nopaline synthase gene
(nos). The 5' and 3' refer to the start and stop of transcription, and the
ATG and TAA refer to the codons used to start and stop translation.
The heavy black line indicates the nos promoter region, and the white
area indicates the nos coding region. ApR denotes ampicillin
resistance, and KmR kanamycin resistance.
Fig. 19 illustrates the construction of the plasmid pAGV10 containing the
complete octopine synthase (ocs) coding sequence and its insertion in
plasmid pLGV2381 (see also Fig. 18) behind the nos promoter. The
ix
1341419
19
heavy black lines refer to the promoter regions and the white area to
the ocs coding region . Other notations are as in Fig. 18.
Fig. 20 illustrates the nucleotide sequences around the promoter region of the
nopaline synthase (nos) gene and the nucleotide sequences around
the same region following fusion with the coding region of the octopine
synthase gene. The point of fusion is indicated by an asterisk (*).
Several restriction endonuclease sites are indicated, BamHI, HindIII,
and SacII. The 5' and 3' refer to the start and stop of transcription.
The ATG refers to the first codon used in translation and the TAA
refers to the stop codon used in translation. The large arrows indicate
the coding regions of the nopaline gene in white and the octopine
gene in stripes.
Detailed description of the invention
Referring to Fig. 1, we have shown therein a simplified diagram of an acceptor
Ti
plasmid. This acceptor Ti plasmid contains the two border sequences (1; 2) or
regions of the wild-type tumor-inducing (Ti) plasmid. The border sequences are
essential for the integration of the T-region of Ti plasmids into a plant cell
genome.
In other words, they are essential for the integration of any DNA sequence (3)
or T-
region into a plant cell genome located between these sequences.
The DNA sequence (3) of the acceptor Ti plasmid contains a DNA segment which
is
homologous with at least a part of a DNA sequence (3') of an intermediate
cloning
vector illustrated in Figs. 2 and 3. This homology is necessary for co-
integration by
a single cross-over event (homologous recombination) of the intermediate
cloning
vector with the acceptor Ti plasmid. The frequency of obtaining cointegrates
is
determined essentially by the length of the region of homology. In order to
effect a
homologous recombination event at a good frequency, regions of 1 to 4 kb are
normally used (Leemans et al., J. Mol. Appl. Genet. 1 (1981 ), 149-164).
C.:- , Al
'19S
1341419
Zo
The acceptor Ti plasmid furthermore contains sequences (4) which are essential
for
the transfer by Aarobacterium of the T-region of the Ti plasmids into plant
cell
genomes.
The construction of such acceptor Ti plasmids and their cointegration with
intermediate cloning vectors illustrated in Figs. 2 and 3 will be described
later in
connection with the discussion of Fig. 4.
In Figs. 2 and 3 we have shown simplified diagrams of intermediate cloning
vectors
for the cloning of any prokaryotic or eukaryotic genes) of interest to be
expressed,
i.e. transcribed under the control of a promoter and translated in plant
cells. These
intermediate cloning vectors contain a DNA segment (3') from a cloning vehicle
containing with a DNA sequence which is homologous with at least a part of the
DNA
segment (3) of the acceptor Ti plasmid thus permitting a single cross-over
event.
Moreover, the intermediate cloning vectors contain at least one gene of
interest (5;
7) which contains either its natural or an exogenous promoter sequence. The
promoter sequence allows the expression of the inserted gene sequence(s). The
use
of an exogenous promoter sequence (tailored promoter) may be useful for
directing
the expression of the inserted genes) in a regulated fashion.
Examples of different types of regulation include the following : (i) tissue-
specific
expression, i.e. leaves, roots, stem, flowers; (ii) level of expression, i.e.
high or low;
and (iii) inducible expression, i.e. by temperature, light, or added chemical
factors.
Examples of genes of interest for the intermediate cloning vectors are : DNA
fragments or sequences with the genetic information controlling the synthesis
of
products such as amino acids or sugars to modify the nutritive or growth
potential of
a plant; or products which provide protection against external pathogenic
agents
such as resistance to disease organisms or stressful environmental factors; or
products which provide information on basic plant processes which are to be
modified by genetic engineering techniques.
,:
1 341 41 9
21
Figures 2 and 3 each show intermediate cloning vectors which may contain
selectable marker genes (6). Examples of selectable marker genes are those
encoding resistance to antibiotics or toxic analogs (for example, amino acid
analogs), or genes which can supply a defect in the recipient host cell.
Figure 4 illustrates the structures involved in the construction of hybrid Ti
plasmid
vectors and Figure 5 describes the actual conjugation steps involved in the
isolation
of Aarobacterium carrying the hybrid Ti plasmid vectors. Since the
intermediate
cloning vector is constructed in E. coli these steps are necessary to transfer
the
intermediate cloning vector to the acceptor Ti plasmid in Agrobacterium.
The known transfer process which was used to prepare modified Ti plasmids in
which a portion of the T-region was replaced by an altered sequence involves
many
steps. Normally, most DNA recombinant manipulations are done in specially
designed cloning vehicles such as pBR322 (Bolivar, Gene 2 (1977), 75-93).
However, these cloning vehicles cannot transfer on their own to Aarobacterium.
In
the known process this problem is solved by
a) replacing the pBR cloning vehicle sequence by another wide-host-range
cloning vehicle, such as the mini-Sa plasmid (Leemans et al. , Gene 19
(1982), 361-364), capable of also replicating in Agrobacterium. The
manipulations are effected in E. coli. An intermediate cloning vector is
obtained.
b) Conjugation of the E. coli strain carrying the intermediate cloning vector
containing the DNA of interest with another E. coli strain carrying a helper
plasmid which cannot replicate in Aarobacterium but which can mediate
transfer of itself and other DNAs to Aarobacterium.
c) Conjugation of E. coli obtained in step (b) with Aarobacterium containing a
Ti
plasmid. The helper plasmid is lost.
d) Since the intermediate cloning vector is capable of replicating and
existing in
Aqrobacterium as a separate replicon, the conjugants obtained in step (c) are
a mixture of cells containing either the cointegrate between the intermediate
cloning vector and the Ti plasmid or other cells containing the intermediate
1341419
22
cloning vector and the Ti plasmid where no cointegration has occurred. In
order to specifically isolate only the cointegrates, a second conjugation to
another Aarobacterium strain without a Ti plasmid must be pertormed. This
transfer is mediated by functions encoded by the Ti plasmid itself. Transfer
of
the intermediate cloning vector into the second Aqrobacterium strain is
effected only in the form of a cointegrate with the Ti plasmid.
e) In order to obtain the final modified Ti plasmid with the desired
replacement a
second cross-over event is effected (Leemans et al., J. Mol. Aael. Genet. 1
(1981 ), 149-164).
The only other known method is essentially the same as outlined above, except
that
for step (d) another plasmid which is not compatible with the intermediate
cloning
vector is introduced into Aarobacterium: in this case, the cointegration
(single cross-
over event) can be selected for since all the intermediate cloning vectors
which
remain as independent replicons will be lost (Matzke ~t al., J. Mol. Appl.
Genet. 1
( 1981 ), 39-49).
We describe a novel and much simplified method for the introduction of
intermediate
cloning vectors into acceptor Ti plasmids of Agrobacterium. Briefly, this
method is
based on the finding that helper plasmids of E_. coli allow transfer of many
of the
commonly used cloning plasmids (such as pBR322) dir_ ectly to A~qrobacterium.
Since
none of these plasmids can replicate in Aarobacterium only those which can
cointegrate with the acceptor Ti plasmid will be retained. In addition, we use
this
cointegrate in Aprobacterium as a vector composition directly for infection of
plant
cells. In this manner we have eliminated steps (d) and (e) described above
which
greatly increases the possibilities for using the acceptor Ti plasmids as
vectors for
DNA transfer to plant cell genomes by bath decreasing the time required for
constructing modified hybrid Ti plasmids and increasing the flexibility of the
possible
constructions.
.,
23 1341419
Thus, as outlined in Figure 5, the introduction ofi the intermediate cloning
vector into
the acceptor Ti plasmid is accomplished in two steps. First, conjugation of E.
coli
strain (1 ) carrying the intermediate cloning vector to another E. coli strain
(2)
carrying two plasmids which will help to mobilize the intermediate cloning
vector to
Agrobacterium. Typical and preferred examples of these helper plasmids are
R64drd11 containing mob functions and pGJ28 containing tra functions (Finnegan
et
al., Mol. Gen. Genet. 185 (1982), 344-351 ). A bom site (Warren et al., Nature
274
(1978), 259-261 ) on the cloning vehicle of the intermediate cloning vector is
recognized by the functions encoded by the other two plasmids to allow
transfer to
occur. All plasmids should preferably contain antibiotic resistance markers to
detect
their presence. Secondly, the E. coli strain obtained, i.e. the mobilizing
strain (3)
carrying all three plasmids is conjugated to A4robacterium containing an
acceptor Ti
plasmid with a region of homology with the intermediate cloning vector. A
single
cross-over event between the intermediate cloning vector and the acceptor Ti
plasmid is detectable by selection for the antibiotic resistance marker of the
intermediate cloning vector.
Figure 6 schematically illustrates the DNA molecules used to construct the
acceptor
Ti plasmid shown in Figure 1. We call this acceptor Ti plasmid (A) to
distinguish it
from the other acceptor Ti plasmid (B) (Fig. 8). The construction requires a
double
cross-over event between a Ti plasmid and another plasmid carrying the border
sequences (1 ) and (2) in a cloning vehicle (3). As illustrated in the figure,
the cloning
vehicle sequence (3) lies between border sequence (1 ) on the left and (2) on
the
right; in order to show the correct polarity of the DNA strands, this can be
drawn on a
circle. However, to show the regions of homology used in the double cross-over
event, this circle has been broken as indicated. This is an important subtlety
to
realize and it is also used in the construction of acceptor Ti plasmid (B) in
Figure 8,
i.e., if border sequences (1 ) and (2) were simply inserted within cloning
vehicle
sequence (3), a double cross-over event would produce a Ti plasmid with a
deleted
T-region but without the cloning vehicle sequence between the border sequences
(1 )
-. and (2). As also shown here in Figure 6, the double cross-over event also
produces
1 341 41 9
2~
a circular DNA molecule which contains the original T-region between border
sequences (1 ) and (2); as this is not a replicon, it will be lost. Genetic
selection for
these events can be achieved by, for example, selecting for the loss of an
antibiotic
resistance marker contained within the T-region of the Ti plasmid and
selecting for
an antibiotic resistance marker contained within the cloning vehicle sequence
(3).
Figure 7 schematically illustrates the construction of an intermediate cloning
vector
of Figs. 2 and 3. A gene of interest (5) and a selectable marker gene (5),
each
bracketed by a restriction endonuclease site R1 or R2, respectively, are
inserted into
a cloning vehicle sequence (3') which contains unique restriction sites for
enzymes
R1 and R2 by digestion and ligation of all molecules. The recombinant DNA
molecule obtained is used to transform E. coli host cells and transformants
are
selected for the antibiotic resistance marker (AbR' ) of the cloning vehicle
sequence
(3').
Figure 8 schematically illustrates the DNA molecules used to construct another
embodiment of the invention, i.e. acceptor'Ti piasmid (B). Here a double cross-
over
event occurs between a Ti plasmid and a plasmid containing the cloning vehicle
sequence (3) between the DNA sequences (9) and (10), which are located just
outside the border sequences (1 ) and (2); in order to illustrate the
homologous
regions used in the cross-over event the small plasmid has been broken (as in
Fig.
6). The product of the double cross-over event is acceptor Ti plasmid (B) and
another circular DNA molecule which is lost containing the T-region from the
original
Ti plasmid plus the DNA sequences (2), (10), (9), and (1 ). Genetic selection
can be
achieved as described for Figure 6.
Figure 9 schematically illustrates an intermediate cloning vector to be used
in
combination with the acceptor Ti plasmid B of Figure 8. Here, the gene of
interest (5)
is inserted between the border sequences (1 ) and (2) which are contained in a
cloning vehicle sequence (3').
Figure 10 schematically illustrates how a single cross-over event introduces
the
:~
134419
intermediate cloning vector of Fig. 9 into the acceptor Ti plasmid (B). In
this case,
selection for the antibiotic resistance marker of the cloning vehicle sequence
(3') of
the intermediate cloning vector ensures that a hybrid Ti plasmid can be found
which
is the result of a cointegration between the two plasmids. A hybrid Ti plasmid
is
produced with the gene of interest contained within the border sequences (1 )
and
(2). The hybrid plasmid thus constructed does not contain in its T-region a
sequence
which is a direct repeat (as for example the sequences (3) and (3') in hybrid
Ti
plasmid of Fig. 4, avoiding possible problems of instability of the hybrid
vector or of
the transformed DNA in the plant cell genome as a result of intramolecular
recombination.
Unpublished results from our laboratory also indicate that for construction of
the
intermediate vector in Fig. 9, it is not essential to have both border
sequences 1 and
2; it is sufficient but essential to have at least the right border sequence
(2) (see Fig.
1 and Fig. 9), in order to obtain integration of the desired DNA sequence into
the
plant genome.
Knowledge of the restriction endonuclease maps of the Ti plasmids of
Ag~robacterium, e.g. nopaline or octopine Ti plasmids (Depicker et al.,
Plasmid 3
(1980), 193-211; De Vos et al., Plasmid 6 (1981 ), 249-253), plus the
knowledge of
the restriction fragments which contain the T-DNA border regions (Zambryski et
al.,
J. Mol. Appl. Genet. 1 (1982), 361-370; De Beuckeleer et al., Mol. Gen. Genet.
183
(1981 ), 283-288) enables now any investigator to construct acceptor Ti
plasmids
according to the process of this invention. The only additional skill required
is the
ability to perform conventional recombinant DNA techniques and basic bacterial
genetic manipulations. The present invention is unique in that it specifically
proposes the described acceptor Ti plasmids which have been shown to be
effective
for the construction of hybrid Ti plasmid vectors; furthermore these acceptor
Ti
plasmids are designed to form part of a process to introduce genes into the
plant cell
genome.
In order to further illustrate the disclosed acceptor Ti plasmids,
intermediate cloning
K
g
.. . . ,
1 X41 41 9
2s
vectors, hybrid Ti plasmid vectors and vector compositions, and to demonstrate
the
effectiveness of the vector compositions in providing transformed plant cells
and
plants showing expression of the foreign genes) integrated into the plant cell
genome, the following examples are provided.
Example 1
Construction of acceptor Ti plasmid pGV3850 (type A)
Starting strains and plasmids
Agrobacterium tumefaciens (rifampicin-resistant strain C58C1, and
chloramphenicol-
erythromycin-resistant strain C58C1 derived from wild-type Aarobacterium)
Ti plasmid = pGV3839
Plasmid of Fig. 11 = pAcgB
The Ti plasmid pGV3839 is constructed from a nopaline plasmid
pTiC58tra°
(pGV3100; Holsters et al., Plasmid 3 (1980), 212-230). It contains a deletion
substitution mutation near the centre of the T-region; the Smal fragment 24
internal
to HindIII fragment 19 (Depicker et al., Plasmid 3 (1980), 193-211 ) has been
substituted by a HindII fragment of pKC7 (Rao et al., Gene 7 (1979, 79-82)
which
contains the aft, (acetylghosphotransferase) gene of Tn5. This gene codes for
resistance to the aminoglycosides neomycin and kanamycin. Figure 12 gives a
restriction map of the T-region of pGV3839.
The plasmid pAcgB is an insert of AcgB in pBR322 which contains only the
borders
of the T-DNA (see Fig. 11 ). The borders are defined as the ends of the T-DNA
and
these regions play a role in the stable integration of the T-DNA into the
plant cell
genome. The origin and analysis of this clone has been described in detail
(Zambryski et al. , Science 209 (1980), 1385-1391 ). This clone was obtained
by re-
isolating portions of the T-DNA from transformed tobacco DNA. pAcgB contains
the
junction of two T-DNA copies which are arranged in tandem so that it contains
the
:>~
Cx
1
1 ~~1 41 9
27
left and right borders of the T-DNA. In addition, pAcgB contains the nopaline
synthase gene since this genetic information maps very close to the right T-
DNA
border. The plasmid pAcgB is used for the construction of an "A-like" acceptor
Ti
plasmid, pGV3850. Figure 6 outlines the structures involved and Figure 13
gives
more precisely the regions of DNA involved in the double cross-over events
leading
to pGV3850.
The described plasmid pAcgB carries a ColEl-specific bom site in the pBR322
portion and can be mobilized from ~. coli to Aarabacterium by using the helper
plasmids R64drdll and pGJ28. The plasmids R64drdll and pGJ28 contained in E.
coli
are introduced into the E. coli strain carrying pAcgB by conjugation.
Transconjugants
are selected as ampicillin-resistant (from the pBR322 sequences of pAcgB),
streptomycin-resistant (from R64drdll) and kanamycin-resistant (from pGJ28)
colonies.
The E. coli strain carrying all three plasmids is conjugated to A roq
bacterium stain
C58C1 which is rifampicin-resistant and which contains the Ti plasmid pGV3839.
The ampicillin-resistance of pBR322 is used to select for the first single
cross-over
event with the nopaline Ti plasmid. The only way that the ampicillin
resistance can
be stabilized in Aprobacterium is a cross-over event upon homologous
recombination with pGV3839 through one of the homology regions near the T-
region
borders. By a second cross-over event through the other homology region, the
central portion of the T-region of pGV3839 including the aft gene (kanamycin
resistance) is replaced by the pBR322 sequences of the clone pAcgB. Second
recombinants are thus ampicillin-resistant and kanamycin-sensitive . To
increase the
probability of isolating a second recombinant, the rifampicin-
resistantAgrobacterium
carrying the first recombinant (pAcgB::pGV3839) is conjugated with a second
chloramphenicol/erythromycin-resistant Aarobacterium strain without a Ti
plasmid. In
this manner, a chloramphenicollerythromycin-resistant Agirobacterium pGV3850
can
be obtained which is ampicillin-resistant and kanamycin-sensitive at a
frequency of
approximately one in 600 colonies.
~.
;~ ~a ,
134~~119
28
Of course, there are other Ti plasmids which can be utilized to construct
pGV3850-
type acceptor Ti plasmids. Any Ti plasmid carrying a selectable marker gene
near
the centre of the T-region may be used as a recipient. Furthermore, a pAcgB-
like
plasmid may be constructed by inserting the T-region border fragments into
pBR322
in such a way that the pBR322 sequences lie in-between the left border
fragment
and the right border fragment in the arientation left border fragment - pBR322
-right
border fragment. For example, the left and right border fragments of the
nopaline Ti
plasmid are HindIII fragment 10 and 23, respectively (Depicker et al., Plasmid
3
(1980), 193-211 ).
A single cross-over event will introduce an intermediate cloning vector
containing
any gene of interest which is inserted in pBR322 (or its derivatives) into the
modified
T-DNA region of pGV3850. The only requirement is that the DNA to be introduced
contains an additional resistance marker gene to those already found in pBR322
to
use as a means to select for the transfer of the intermediate cloning vector
from E.
coli to Agrobacterium. This resistance market can be contained either within
the pBR
sequences (such as CmR for pBR325 or KmR for pKC7) or within the DNA which is
to
be tested in the plant cell. In addition, in the acceptor Ti plasmid pGV3850
the ApR
gene pBR322 can be replaced by another resistance marker gene such as KmR; in
this way even pBR322-containing intermediate cloning vectors which are ApR can
be
directly mobilized to this pGV3850-like acceptor Ti plasmid.
An added advantage of the pGV3850-type acceptor Ti plasmid is that it does not
produce tumors in transformed plant cells. Cells which have been "transformed"
by
pGV3850 can easily be screened from untransformed cells by assaying for the
presence of nopaline as the shortened T-DNA region of pGV3850 still contains
the
gene encoding nopaline synthase. Of course, if the intermediate cloning vector
which is cointegrated into the acceptor Ti plasmid pGV3850 contains a marker
gene
it can also be directly screened or selected for.
Besides insertion of defined intermediate cloning vectors containing a pBR322
sequence by a single cross-over event into the acceptor Ti plasmid pGV3850,
this
~X
1 X41 41 9
29
acceptor Ti plasmid can also be used as a recipient for cloned banks of DNA in
pBR322 or its derivatives in a "shotgun"-type experiment. The total population
of
hybrid plasmid vectors in Aqrobacterium can be used to infect plant cells and
is
subsequently screened for expression of any selectable genes) of interest. For
example, one can easily select for genes encoding amino acid synthesis by
applying
the total bank to plant cells which are deficient in the chosen amino acid.
The acceptor Ti plasmid pGV3850 has two phenotypic characteristics: (i) the
inability to produce tumors, and (ii) the ability to synthesize nopaline if T-
DNA
transfer occurs into the plant cell genome. Thus, several experiments are
performed
to check for these characteristics in various plant tissues infected with
Agrobacterium containing pGV3850.
a) Tests with potato and carrot discs
Inoculation of potato and carrot slices with the acceptor Ti plasmid pGV3850
results in the production of a small amount of callous tissue. This tissue is
tested for the presence of nopaline and is found to be positive. It is
interesting
that this mutant is able to produce small callous tissue; however, it can only
be obtained if the discs are grown in media containing low concentrations of
both auxins and cytokinins.
b) Inoculation of whole plants with acceptor Ti plasmid pGV3850
Tobacco and petunia plantlets growing on sterile agar media (without
hormones) are inoculated with pGV3850. A small amount of tissue growth can
only be observed after several months (normally "wild-type" tumors are
detected after two weeks.) This tissue doss not grow on hormone-free media
but can be propagated further as sterile tissue culture on media containing
both auxin and cytokinin. This tissue is also shown to be nopaline-positive.
c) Furthermore, since pGV3850 "transformed" cells are not tumor-like, these
cells are capable of regenerating into normal plants which still contain in
their
genome the transferred DNA segment. Normal plants will be obtained by
:~~.. ,~
1341419
culturing the transformed cells on conventional regeneration media (see also
example 5).
To prove the usefulness of pGV3850 as an acceptor plasmid the following
experiment is performed. An intermediate cloning vector containing oncogenic
functions of the octopine T-DNA in pBR325 is recombined into A~,robacterium
harboring pGV3850. The resulting hybrid Ti plasmid in Aarobacterium obtained
by a
single cross-over event is inoculated onto wounded tobacco plants. Tumor
tissue
develops after about two weeks. This is evidence that the tumor-inducing DNA
is
reintroduced into pGV3850 and is expressed properly in transformed plant
cells.
Example 2
Construction of intermediate cloning vectors
pGV700 and pGV750
An overview of the constructions is represented schematically in figure 14.
HindIII
fragment 1, representing the right part of TL-DNA of the octopine Ti plasmid
B6S3,
and present in pGV0201 (De Vos et al. , Plasmid 6 (1981 ), 249-253), is
inserted first
in the HindIII site of the broad-host range vector pGV1122 (Leemans et al.,
Gene 19
(1982), 361-364). The recombinant plasmid pGV0201 contains the HindllIfragment
1 inserted in the unique HindIII site of the multicopy vector pBR322 (Bolivar
et al.,
Gene 2 (1977), 95-113). pGV0201 and pGV1122 DNA is prepared as described by
Betlach et al., Fed. Proc. 35 (1976), 2037-2043. Two pg of pGV0201 DNA are
totally
digested with 2 units of HindIII (all restriction enzymes are purchased from
Boehringer Mannheim) for 1 hour at 37°C in a final volume of 20 N1. The
incubation
buffer is described by O'Farrell et al., Mol. Gen. Genet 179 (1980), 421-435.
Two Ng
of pGV1122 DNA are totally digested with HindIII under the same conditions.
One tenth Ng of HindII1 digested pGV0201 is ligated to 0.05 Ng of HindIII-
digested
pGV1122 with 0.02 units of T4 ligase (Boehringer Mannheim) in a final volume
of 20
øX
1341419
31
NI. Incubation buffer and conditions are as recommended by the manufacturer
(Brochure "T4 ligase", Boehringer Mannheim, August 1980, #10.M.880.486).
Transformation of the ligation mixture into competent E. coli K514 hsr~ hsm'
cells
(Colson et al., Genetics 52 (1965), 1043-1050) is carried out as described by
Dagert
and Ehrlich, Gene 6 (1980), 23-28. Cells are plated on LB medium (Miller,
Exaeriments in Molecular Genetics {1972), Cold Spring Harbor Laboratory, New
York) supplemented with streptomycin (20 Ng/ml) and spectinomycin (50 pg/ml).
Transformants containing recombinant plasmids are screened for tetracycline
sensitivity (10 Ng/ml), due to the insertionai inactivation of the gene coding
for
tetracycline resistance (Leemans et ~I. , Gene 19 (1982), 361-364).
Streptomycin-
and spectinomycin-resistant and tetracycline-sensitive clones are physically
characterized. Microscale DNA preparations are performed according to Klein et
al.
(Plasmid 3 (1980), 88-91 ). The orientation of the HindIII fragment 1 in the
HindIII
site of pGV1122 is determined by Sall digestion. Digestion (conditions
according to
O'Farrell et al., Mol. Gen. Genet. 179 (1980), 421-435) of recombinant
plasmids
gives 2 fragments after agarose gel electrophoresis. In the a-orientation
there are
fragments of 0.77 kb and 22.76 kb, whereas in the ~i-orientation there are
fragments
of 10.33 kb and 13.20 kb. A recombinant piasmid with the a-orientation is used
in
subsequent cloning and called pGV1168.
A B~c III-SaII fragment containing the left part of the TL-DNA (including the
left border
sequence) is introduced in pGV1168, cut with BgIII-SaII . This fragment is
obtained
from the recombinant plasmid pGV0153 (De Vos et al . , Plasmid 6 (1981 ), 249-
253),
containing BamHi fragment 8, from the T-region of pTiB6S3, inserted in the
vector
pBR322. pGV0153 and pGV1168 DNA is prepared according to Betlach et al. (Fed.
Proc. 35 (1976), 2037-2043). Ten Ng of pGV0153 DNA are completely digested
with
units of BgIII and 10 units of SaII for 1 hour at 37°C ire final volume
of 100 NI. The
digestion mixture is loaded on a preparative 0.8°~ agarose gel. The
2.14 kb BIgII-
SaII fragment is recovered from this gel by electroelution as described by
Allington
et al., Anal. Biochim, 85 (1978), 188-196. Two pg of pGV1168 DNA are totally
digested by 2 units of BgIII and 2 units Sali. One tenth pg of BgLII-SaII
fragment
,~.
1 341 ~+ 1 9
32
DNA is ligated to 0.02 pg of BgIIII-SaII-digested pGV1168 with 0.02 units of
T4 DNA
ligase in a final volume of 20 NI. The ligation mixture is transformed into
competent
E. coli K514 hsr hsm+ cells (Dagert and Erhlich, Gene 6 (1980) 23-28). Cells
are
plated on LB medium (Miller, Experiments in Molecular Genetics (1972), Cold
Spring
Harbor Laboratory, New York), supplemented with streptomycin (20 pg/ml) and
spectinomycin (50 Ng/ml).
Microscale DNA preparations (Klein et al., Plasmid 3 (1980), 88-91 ) are
performed
from streptomycin- and spectinomycin-resistant transformants. Recombinant
plasmids in which 2.14-kb-BgIII-SaII fragment is inserted in Bc~III-SaII-
digested
pGV1168 are identified by B~c III-SaII digestion, yielding 2 fragments of 2.14
kb and
21.82 kb. A plasmid with a digest pattern corresponding to these molecular
weights
(2.14 kb and 21.82 kb) is used for further cloning and called pGV1171. A 12.65-
kb-
fragment from pGV1171 contains the right and left TL-DNA border sequences (De
Beuckeleer et al., in Proceedings IVth International Conference on Plant
Pathogenic
Bacteria, M. Ride (ed.) (1978), I.N.R.A., Angers, 115-126), as well as genes
which
permit oncogenic proliferation (Leemans et aL,EMBO J. (1982), 147-152). This
HindIII fragment is inserted into the plasmid pBR325 (Bolivar, Gene 4 (1978),
121-
136). pGV1171 and pBR325 are prepared according to Betlach et al., Fed. Proc.
35
(1976), 2037-2043). Two Ng of each DNA are totally digested with 2 units of
HindIII
for 1 hour a 37°C (incubation buffer is described by O'Farrell et al.,
Mol. Gen. Genet.
179 (1980), 421-435). One tenth Ng of HindIII-digested pGV1171 is ligated to
0.05
Ng of pBR325, linearized with HindIII, with 0.02 units T4 DNA ligase.
Transformation
of the ligation mixture in competent E. coli K514 hsr~ hsm+ is carried out as
described
by Dagert and Ehrlich, Gene 6 (1980), 23-28. Cells are plated on LB medium
(Miller,
Experiments in Molecular Genetics (1972), Cald Spring Harbor Laboratory, New
York), supplemented with carbenicillin (100 Ng/ml). Carbenicillin-resistant
clones are
screened for sensitivity to tetracycline (10 Ng/ml), due to insertional
inactivation of
the gene coding for tetracycline resistance (Bolivar, Gene 4 (1978) 121-136).
Carbenicillin-resistant and tetracycline-sensitive clones are physically
characterized
by restriction enzyme digestion of DNA prepared from these clones by the
~- t
o . i
~!Mv
1 341 4 1 9
33
microscale technique (Klein et al., Plasmid 3 (1980), 88-91 ). BamHI digestion
gives
4 DNA fragments : in the a-orientation fragments of 0.98 kb, 4.71 kb, 5.98 kb,
and
7.02 kb are found whereas the (3-orientatian gives fragments of 0.98 kb, 4.71
kb,
1.71 kb, and 11.20 kb. A recombinant plasmid corresponding to the a-
orientation is
obtained, called pGV700, and used for further experiments.
pGV750 is derived from pGV700 by substituting a 2.81 kb BamHI-H~aI fragment
coding for kanamycin resistance, for a 3.49-kb-BgIII-Smal fragment, encoding
functions essential for oncogenicity, internal to the TI_,-region inserted in
pGV700.
The BamHI-H~aI fragment encoding kanamycin resistance is obtained from ~: :TnS
(Berg et al., Proc. Natl. Acad. Sci. USA 72 (1975), 3628-3632). Preparation of
a::TnS
is as described (Miller, Experiments in Molecular Genetics (1972), Cold Spring
Harbor Laboratory, New York). pGV700 DNA is prepared according to Betlach et
al.
(Fed. Proc. 35 (1976), 2037-2043). Two pg of pGV700 DNA are totally digested
with
2 units of B~c II and 2 units Smal. Two Ngof A: :TnS DNA are totally digested
with 2
units of BamHI and 2 units H .,~LaI. One pg of BamH1-Haa1 digested A: :TnS is
ligated
to 0.2 Ng BgIII-SmaI-digested pGV700 with 0.5 units of T4 DNA ligase in a
final
volume of 10 p1 (conditions are as recommended by the manufacturer). The
ligation
mixture is transformed in competent E. c~oli K514 hsr hsm+ cells (Dagert and
Erhlich,
Gene 6 (1980) 23-28). Cells are plated on I_B medium (Miller, Experiments in
Molecular Genetics (1972), Cold Spring Harbor Laboratory, New York),
supplemented with carbenicillin (100 Nglml) and kanamycin (25 Ng/ml) . CbRand
KmR clones are physically characterized by restriction enzyme analysis of DNA
prepared according to the micro-scale technique (Klein et ai., Plasmid 3
(1980), 88-
91 ). BgIII/BamHI double digests of this DNA gives 3 fragments of 3.94 kb,
5.89 kb,
and 8.09 kb, whereas HindII1 digests yields 3 fragments of 2.68 kb, 5.99 kb,
and
9.25 kb. A plasmid showing these digestion patterns is called pGV750 and is
illustrated schematically in Figure 17
pGV700 and pGV750 are two different intermediate cloning vectors which contain
the left and right border sequences of TL-DNA of the octopine Ti plasmid
pTiB6S3;
~ 3~1 41 9
34
in addition, the internal T-region is deleted to different extents in each of
the two
plasmids. pGV700 contains a shortened T-region with genetic information for
octopine synthase (transcript 3), and 3 other products, 4, 6a, and 6b (see
Willmitzer
et al., EMBO J. 1 (1982), 139-146, for a description of T-region products).
The
combination of these three products (4, 6a, 6b) will cause shoot formation in
transformed plants. pGV750 contains even less of the T-region, i.e. only the
octopine synthase gene. The information for products 4, 6a, and 6b has been
substituted by the antibiotic resistance marker gene encoding kanamycin
(neomycin)
resistance.
pGV700 arid pGV750 are examples of intermediate cloning vectors which can be
used with acceptor Ti plasmids of the B-type (see Fig. 8 and example 3 below);
these vectors are partially analogous to the one shown in Fig. 9 except that
they do
not contain a gene of interest. A gene of interest can be easily inserted into
these
vectors as they contain single restriction endonuclease sites for cloning
within their
modified T-regions (see Figs. 14 and 15).
Example 3
Construction of acceptor Ti plasmid pGV2260 (type B)
Starting strains and plasmids
Aarobacterium tumefaciens (rifampicin-resistant strain C58C1 and erythromycin-
chloramphenicol-resistant strain C58C1, derived from wild-type Agrobacterium)
Ti plasmid = pGV2217
Intermediate vector (Fig. 16) = pGV745
The construction of the Ti plasmid pGV2217 has been described in detail
(Leemans
et al. , EMBO J. 1 {1982), 147-152). It contains a deletion substitution
mutation of the
entire TL-region of the octopine Ti plasmid : the BamHI fragments 8, 30b, 28,
17a
and the left 3.76 kb BamHI-EcoRI fragment of the BamHI fragment 2 (De Vos et
al. ,
'~ r
r -'~,.
~~141 9
Piasmid 6 (1981 ), 249-253) have been substituted by an EcoRI-BamHI fragment
of
pKC7 (Rao & Rogers, Gene 7 (1979), 79-82) which contains the a~,t. (acetyl
phosphotransferase) gene of Tn5. This gene codes for resistance to the
aminoglycosides, neomycin and kanamycin.
The construction of the intermediate vector pGV745 is represented
schematically in
Fig. 16 and is described as follows. The recombinant plasmid pGV713 was
derived
from the octopine Ti plasmid subclone pGV0219 (De Vos et al., Plasmid 6 (1981
),
249-253), containing HindIII fragments 14, 18c, 22e and 38c in a-orientation.
pGV0219 DNA was digested to completion with BamHI and subsequently ligated
under conditions which favour self ligation of the plasmid (final
concentration of DNA
in ligation mixture < INg DNAImI). Transformants were selected for ampicillin
resistance, and physically characterized by restriction enzyme digestion. A
clone,
which no longer contains the 6.5 kb BamHI fragment present in pGV0219, was
isolated and called pGV713. This clone pGV713 was r~sed in subsequent cloning
(see below). The recombinant plasmid pGV738 was derived from pGV0120 (De Vos
et al., Plasmid 6 (1981 ), 249-253), containing BamHI fragment 2. pGV0120 DNA
was digested with EcoRI and self-ligated (as above for the construction of
pGV713).
Transformants were selected for ampicillin resistance and analyzed by
restriction
enzyme digestion. A clone in which EcoRI fragments 20, 12, and a 2.95 kb EcoRI
fragment containing part of EcoRI fragment 19a and part of pBR322 were
deleted,
was used in further cloning and called pGV738. This plasmid still contains a
5.65 kb
EcoRI-BamHI fragment from the right part of BamHI fragment 2 (De Vos et al.,
Plasmid 6 (1981 ), 249-253).
Next, pGV713 DNA was digested with HindITI and BamHI, and the digest was
applied onto a preparative agarose gei. After electrophoresis the 2.30 kb
HindIII-
BamHI fragment, contained within pGV713, was purified by electroelution (as
described by Allington et al., Anal. Biochem. 85 (1975), 188-196). This
fragment was
figated to pGV738, digested to completion with HindIII and BamHI. After
transformation, ampicillin-resistant clones were physically characterized by
1 ~~+1 41 9
36
restriction enzyme digestion. For example, EcoRl-BamHI digestion should give 2
fragments of respectively 3.98 kb (= vector part) and 7.95 kb (= insert part).
A
recombinant plasmid with these characteristics was called pGV745 and used as
intermediate vector for the construction of the acceptor Ti plasmid pGV2260.
The plasmid pGV745 contains a ColEl-specific bom site in the pBR322 portion
and
can be mobilized from E. coli to Ac~robacterium by using the helper plasmids
R64drd11 and pGJ28, as described in example 1 (construction of the acceptor Ti
plasmid pGV3850).
pGV745 was mobilized to Agrobacterium strain C58C1 which is rifampicin-
resistant
and contains the Ti plasmid pGV2217. The first cross-over event was selected
by
using the ampicillin resistance of pBR322 in the same way as described in
example
1 (the construction of the acceptor Ti plasmid pGV3850) . By a second cross-
over
event the deletion substitution mutation present in pGV2217 is replaced by the
pBR322 sequences of the plasmid pGV745. Second recombinants were picked-up
by directly screening the ampicillin-resistant transconjugants, which resulted
from
cointegration of pGV745 with pGV2217, for the loss of kanamycin resistance. In
this
way, a rifampicin Aarobacterium strain C58C1, containing pGV2260 (ampicillin-
resistant, kanamycin-sensitive), was obtained.
This Ti plasmid pGV2260 will be used as an acceptor plasmid (type B) for
intermediate cloning vectors of the pGV700- or pGV750-type. These are composed
of (i) a DNA fragment carrying the ampicillin resistance gene, the origin of
replication
and the bom site of pBR322 ; (ii) a DNA fragment containing the left and right
border
sequences of the TL-DNA, and an additional resistance marker to those already
present on pBR322, which will enable the genetic selection for the transfer of
the
intermediate cloning vector from ~. coli to Ag~robacterium as well as for its
cointegration in the acceptor Ti plasmid pGV2260.
For example, we have been able to show that Aprobacterium carrying a
cointegrate
between pGV2260 and pGV700 is capable of transferring the expected DNA
y~: ..'~""'
1 X41 41 9
37
sequences (those contained between the T-DNA borders) to plant cell genome.
The
transformed plant cells exhibit the expected phenotype i.e. tumors which
produce
shoots, given that pGV700 contains the genetic information for three products
(4, 6a,
6b; see Willmitzer et al., EMBO J. 1 (1982), 139-146). In this mannerwe have
shown
that an acceptor Ti plasmid of the B-type is capable of transferring DNA to
plant cells
when used as a cointegrate with an intermediate cloning vector of the type
illustrated
in Fig. 9 and given in example 2.
Example 4
Construction of an intermediate cloning vector containing a gene to be
expressed
in plants
Until the present invention, insertion of whole genes into more or less random
positions within the T-region of Ti plasmids has not resulted in expression of
the
foreign sequence following transfer to the plant genome. According to the
process of
this invention, the coding region of (any) foreign genes) of interest can be
linked to
transcriptional initiation and termination signals which are known to be
functional in
the plant cell. The usefulness of this approach is demonstrated according to
the
present invention by experiments involving the DNA sequences encoding the
nopaline synthase gene. The entire sequence of this gene and the exact start
and
stop of transcription are known (Depicker et al., J. Mol. Appl. Genet. 1
(1982), 561-
574). According to the present invention, the protein-coding region of any
foreign
gene can be inserted adjacent to the nos promoter. As an example of a foreign
gene
sequence, the coding region of the octopine synthase gene (De Greve et al., J.
Mol.
Appl. Genet. 1 (982), 499-512) is inserted adjacent to the nos promoter. This
construction is mobilized into an acceptor Ti plasmid and used to infect
plants. The
resulting tumor tissue is assayed for the presence of octopine, and is found
to be
positive.
The construction of the intermediate cloning vector containing the chimeric
nopaline
promoter : octopine synthase structural gene is shown and described in figures
18 to
i
_ w
38 ~ 3~+~ 41 9
20.
Briefly, the restriction fragment HindlII-23 containing the nos gene is
engineered in
vitro to remove most of the nos coding sequence, and retain the nos promoter
adjacent to the restriction endonuclease site BamHI (Fig. 18). Ten Ng of
pGV0422 (a
pBR322 derivative carrying the HindlIl-23 fragment which contains the complete
nos
gene; Depicker et al. , Plasmid (1980), 193-211 ) are digested with Sau3A and
the
350 by fragment carrying the nos promoter is isolated from a preparative 5%
polyacrylamide gel. The promoter fragment is ligated to Bg~III -cut pKC7 (Rao
et al.,
Gene 7 (1979), 79-82) previously treated with bacterial alkaline phosphatase
(BAP)
to remove 5'-terminal phosphate groups. Twenty pg of the resulting plasmid
(pLGVl3) are digested with B~c III and treated with 7 units of the Ba131
exonuclease
(Biolabs, New England) for 4 -10 minutes in 400 NI of 12 mM MgC1 Z, 12 mM
CaC12,
0.6 M NaCI, I mM EDTA, and 20 mM Tris-HCI, pH 8.0, at 30°C. During this
time
approximately 20 - 50 by of DNA are removed. The Ba131-treated molecules are
digested with BamHI, and incubated with the Klenow fragment of DNA polymerase
and all four deoxynucleoside triphosphates {at 10 mM each) to fill in the
ends.
Plasmids are screened for a regenerated BamHI site derived from the ligation
of a
filled-in BamHI end and the end of the Ba131 deletion. The sizes of the BamHI-
SacII
fragment of several candidates are estimated in a 6°~ urea-
polyacrylamide gel, and
the nucleotide sequences of the candidates with sizes ranging between 200 -
280
nucleotides are determined. The clone pLGV81 containing the SaCII-BamHI
fragment of 203 by carrying the promoter is used to substitute the SacII-BamHI
fragment in the nos gene in pGV0422; the final promoter vector is called
pLGV2381.
All the recombinant plasmids are selected by transformation of the E_. coli
strain
HB101.
The plasmid vector containing the engineered nos promoter is digested with
BamHI
and the coding sequence for oc which is contained on a BamHI fragment is
inserted
into this site. The ocs coding sequence is also engineered in vitro to be
bracketed by
the BamHI restriction endonuclease site as described in Fig. 19. Ten pg of
BamHI
1341419
39
fragment 17a of the octopine Ti plasmid B6S3 (De Vos et al., Plasmid 6 (1981
), 249-
253), are digested with BamHI and Smal, the fragment containing the ocs-coding
sequence isolated from a 1 % agarose gel, and ligated to the large BamHI-PvuII
fragment of pBR322; 20 Ng of the resulting plasmid, pAGV828, is digested with
BamHI, treated with the exonuclease Ba131 as described in Figure 18,
subsequently
digested with HindIII, and the ends are filled-in and self-ligated. The sizes
of the
Ba131 deletions are estimated in a 6% polyacrylamide gel. The nucleotide
sequences of several candidates are determined, and a candidate having only 7
by
remaining of the 5'-untranslated leader sequence is chosen for further work
(pOCS~). In order to bracket the ocs sequence with BamHI sites, the CIaI-RsaI
fragment is filled-in and subcloned into the BaII site of pLC236 (Remaut et
al. , Gene
15 (1981 ), 81-93). The resulting plasmid pAGV40 is digested with BamHI, the
fragment carrying the ocs sequence isolated by electraelution from a
preparative 1
agarose gel, and ligated to pLGV2381 previously cut with BamHI, and treated
with
BAP (bacterial alkaline phosphatase). The insertion of the ocs sequence in
pLGV2381 is obtained in both orientations (pN0-1 and pN0-2).
The nucleotide sequences showing the exact junction point in the nos:ocs
fusion are
shown in Fig. 20.
Further, the plasmid vector containing the engineered nos promoter is used to
insert
DNA from the plasmid R67 which encodes the enzyme dihydrofolate reductase. The
coding sequence containing the dihydrofolate reductase gene is contained on a
BamHI fragment as described (O'Hare et al. , Proc. Natl. Acad. Sci. USA 78 (
1981 ),
1527-1531 ), and thus is easily inserted into the nos promoter vector
containing the
BamHI site adjacent to the promoter region as described above. This gene is an
example of a selectable marker gene (see for example Figs 2, 3, 4, 5, and 7)
since
when expressed it provides resistance to the antibiotic methotrexate. When
this
intermediate cloning vector is mobilized into an Aprobacterium containing a
wild-type
nopaline acceptor Ti plasmid, a single cross-over event occurs and a hybrid Ti-
plasmid vector is obtained. The vector composition is used to infect plants.
The
W
i
1~414i9
a0
resulting tumor tissue is found to be capable of sustained growth in the
presence of
0.5 Ng/ml methotrexate.
The construction of the intermediate cloning vectors containing the ocs and
dihydrofolate reductase coding regions behind the nos promoter described
above,
and their transfer and expression in transformed plant cells following
cointegration
with the Ti plasmid of Agrobacterium provides evidence that foreign genes can
be
transferred and expressed in plant cells according to the processes of this
invention.
example 5
Isolation of plant cells and plants containing the desired
genes) inserted in their chromosomes
We have obtained plant cells and whole plants transformed with non-oncogenic
acceptor Ti plasmid derivatives (e.g. pGV3850) using any of the following
three
methods:
(1 ) inoculation in vivo of whole plants followed by subsequent culture in
vitro on
media which allow the regeneration of shoots;
(2) coinfection in vivo of whole plants in the presence of other Agrobacteria
strains which directly induce shoots at the wound site;
(3) cocultivation in vitro of single plant cell protoplasts.
We will describe each of these methods below.
The first method is based on a modification of methods normally used to obtain
infection of whole plant tissues with wild-type Aprobacterium strains which
results in
the production of crown gall tissues. Since pGV385(7 is a non tumor-producing
(nononcogenic) A~4robacterium derivative, no tumorous growth is observed at
the
infected site. However, if the infected tissue is removed and propagated in
tissue
culture, transformed tissues can easily be obtained. After an initial culture
period
(simply to increase the mass of the tissue) the wound site tissue is grown
under
..
1 341 4 1 9
41
conditions which allow shoots to form. Both untransformed and pGV3850-
transformed cells will produce shoots. The transformed shoots can easily be
distinguished by a simple assay for the presence of nopaline.
We have obtained pGV3850- transformed calli and shoots derived from
decapitated
tobacco plantlets of Nicotiana tabacum Wisconsin 38 using the following
protocol (all
manipulations are done under sterile conditions in a laminar flow hood) .
(1 ) Use 6-week old tobacco seedlings grown in small jars (10 cm diameter x 10
cm height) on solid Murashige & Skoog (MS) medium (Murashige and Skoog,
Phvsiol. Plant. 15 (1962) 473-497) containing 0.8% agar.
(2) Remove youngest top leaves with a scalpel and discard.
(3) Inoculate wound surface with a spatula or toothpick containing
Aprobacterium
derived from a fresh plate culture grown under selective conditions (e.g. for
the rifampicin-resistant, ampicillin resistant Aqrobacterium strain containing
Ti
plasmid pGV3850, YEB medium containing 100 pg/ml rifampicin and 100
Ng/ml carbenicillin are used; YEB medium : 5 g/1 Bacto beef extract, 1 g/1
Bacto yeast extract, 5 g/1 peptone, 5 g/1 sucrose, 2 x 10-3 M MgS04, pH 7.2,
and 15 g/1 agar). Inoculate at least 8 plantlets for each pGV3850
construction.
(4) Incubate 2 weeks; there should be little or no response at the site of
inoculation; sometimes very tiny calli appear.
(5) Remove a thin section less than 1 mm thick from the wound surface.
Incubate
this section of the wounded surface on a plate containing Linsmaier and
Skoog (LS) agar medium (Linsmaier and Skoog, P_hvsiol. Plant. 18 (1965)
100-127) with auxins and cytokinins (1 mgl1 NAA, 0.2 mg/l BAP) and 1
sucrose.
(6) After about 6 weeks callus should be large enough, about 5 mm diameter at
least, to test a portion for the presence of nopaline. Not all wound calli
produce nopaline; approximately ane in four plants produce a nopaline-
positive wound callus.
(7) Transfer nopaline-positive calli to agar plates containing regeneration
" ~ medium : LS medium as above + 1 % sucrose and 1 mg/1 BAP cytokinin.
A
-,
~ 3~~ 4~ 9
42
(8) Good-sized shoots (1 cm high) appear after about 4-6 weeks. Transfer the
shoots to fresh agar plates containing LS medium + 1 °!o sucrose
without
hormones to allow further growth and root formation.
(9) Let shoots grow 1 or 2 weeks such that a portion (one or two small leaves)
can be removed to test for the presence of nopaline.
(10) Transfer nopaline-positive shoots to larger vessels (10 cm jars as above
containing MS medium as in (1 ) to grow further.
N.B. All plant culture media far infected tissues contain 500 Ng/ml of the
antibiotic
cefotaxime (Claforan~, Hoechst) as a selection against A~arobacterium
containing
pGV3850. This drug works well to prevent growth of all A-.ac irobacteria
(including
those which are carbenicillin-resistant).
Another method to obtain transformed and shooting tissues has been recently
developed in our laboratory. This method is based on the observation that
certain
mutant Ti plasmid strains of Aqrobacterium induce crown gall tumors which
produce
shoots. Such shoot-inducing (shi) mutants map to a particular region of the T-
DNA
(transferred DNA segment) of the Ti plasmids of A. tumefaciens (Leemans et
al.,
EMBO J. 1 (1982) 147-152; Joos et al., Cell ~2 (1983) 1057-10fi7). Often the
induced shoots are composed of completely normal untransformed cells. Thus, we
tried to inoculate plants with a mixture of two different Aorobacteria, one
carrying an
octopine Ti plasmid shooter mutant and the other carrying pGV3850. In this
manner,
there is a good chance that the octopine shooter mutation can induce shoots
which
have been transformed with pGV3850. We have inoculated plants with
Agrobacterium containing Ti plasmid pGV3850 and an octopine shoot inducing Ti
plasmid in a 5:1 ratio. In this way we have obtained pGV3850-transformed
shoots;
these shoots are easily screened by assaying for the presence of nopaline.
This
method avoids the need for any elaborate tissue culture methods. The nopaline-
positive shoots are transferred to media containing simple salts and sugar
with any
growth-regulating hormones to allow further growth. After the shoots have
reached a
sufficient size they can easily be transferred to soil for propagation. This
coinfection
procedure should be particularly useful for transforming plant species which
are not
~;
. ~."r 1
1 341 41 9
43
readily amenable to tissue culture. Thus, a whole range of agronomically and
economically important plants, such as legumes, medicinal plants, and
ornamentals
will be able to be engineered by Aarobacterium.
The third procedure allows the isolation of Nicotiana tabacum protoplasts and
the
selection of hormone-independent T-DNA-transformed cell clones after co-
cultivation
of the protoplast-derived cells with oncogenic Ac~obacterium strains. An
analogous
technique can be used for the selection of transformed cells when other
dominant
selective markers are used, such as antibiotic resistance genes constructed in
such
a way as to be expressed in higher plant cells (see example 3).
In this case, however, the conditions for selection have to be optimized in
each case
(concentration of the selective agent, time between transformatian and
selection,
concentration of the protoplast-derived cells or cell colonies in the
selective
medium). If no selection for the transformed cells is possible, e.g. because
avirulent
T-DNA mutants are used, such as e.g. pGV3850 or pGV2217 (Leemans et al.,
EMBO J. 1 (1982), 147-152), it is possible to cultivate the cells after
genetic
transformation on auxin- and cytokinin-containing medium (e.g. Murashige and
Skoog medium (Murashige and Skoog, P_ hysiol. Plant 15 (1962), 473-497) with 2
mg/litre NAA (a-naphthalene acetic acid) and 0.3 mg/litre kinetin), and to
identify the
transformed colonies by their opine content. In this way, after
electrophoretic
analysis for agropine and mannopine synthesis (method see t_eemans et al., J.
Mol.
Appl. Genet. 1 (1981 ), 149-164) about 660 colonies can be found, obtained
after
infection with pGV2217, a Nicotiana tabacum SRI cell line which synthesizes
the TR-
encoded opine mannopine (N2-(1-mannityl)-glutamine). Numerous shoots are
formed
after incubation of callus pieces of this cell line on regeneration medium
(Murashige
and Skoog medium with BAP (6-benzylaminopurine) (1 mg/litre) as the sole plant
growth regulator). All 20 shoots analyzed are still able to synthesize
mannopine.
After transfer onto hormone-free Murashige and Skoog medium, the shoots grow
as
morphologically normal tobacco plants still containing mannopine.
The protoplast isolation and transformation described here for N. tabacum can
also
1 3~1 41 9
44
be used for N. plumbaginifolia.
2. Experimental procedures
2.1. Shoot culture conditions
Nicotiana tabacum shoot cultures are maintained in 250 ml glass jars on
hormone-free Murashige and Skoog medium (Murashige and Skoog, Physiol.
Plant 15 (1962), 473-497) under sterile conditions in a culture room (16 hour
day, 1500 lux white fluorescent light ("ACEC LF 58 W/2 4300°K
Economy"),
24°C, 70% relative humidity). Five week old shoot cultures are used for
protoplast isolation.
2.2. Protoplast isolation
Aseptic techniques are used far all steps in protoplast isolation and culture.
The protoplasts are isolated by a mixed enzyme procedure. All leaves, except
for very young leaves smaller than 2 cm, can be used for protoplast isolation.
The leaves are cut in strips, about 2 - 3 mm wide, with a sharp scalpel knife.
Two to three grams of leaf material is incubated 18 hours at 24°C in
50 ml
enzyme mixture in the dark without agitation. The enzyme mixture consists of
0.5% cellulose Onozuka* R-10 and 0.2% macerozyme Onozuka R-10 in
hormone-free K3 medium (Nagy and Malign, Z. Pflanzenphvsiol. 78 (1976),
453-455). The mixture is filter-sterilized through a 0.22 Nm pore membrane,
and can be stored for at least 6 months at -20°C without notable loss
of
activity.
2.3. Protoplast culture
After 18 hours incubation the mixture is agitated gently in order to release
the
protoplasts. The mixture is subsequently filtered through a 50 Nm sieve, and
the filtrate is transferred to 10 ml centrifuge tubes. After centrifugation
for 6
minutes at 60 - 80 g in a swinging bucket rotor the protoplasts form a dark
;,
t
a,y
/rp.
*trade mark
45 ~ 3 4 ~ 4 ~ 9
green floating band. The liquid underlying the protoplasts, and the debris
which forms the pellet, are removed using a capillary tube connected to a
peristaltic pump. The protoplasts are pooled in one tube and washed 2 times
with culture medium. The culture medium is the K3 medium (Nagy and
Maliga, Z. Pflanzenahysial. 78 (1976), 453-455) with NAA (0.1 mg/litre) and
kinetin (0.2 mg/litre) as growth regulators. The medium is adjusted to pH 5.6
and sterilized through 0.22 Nm filter membrane. After the second wash, the
protoplasts are counted using a Thoma hemacytometer (obtained from
"Assistant", F.R.G.) , and resuspended in culture medium at a final density of
105 protoplasts/ml. The protoplasts are cultured in a volume of 10 ml per 9 cm
diameter tissue culture quality petri dish. The dishes are sealed with
Parafilm~ and incubated for 24 hours in the dark, and thereafter in dim light
(500 - 1000 lux) at 24°C.
2.4. Transformation by co-cultivation
The protoplast cultures are infected 5 days after isolation. Agrobacterium
cultures are grown for 18 hours in liquid LB medium (Miller, Experiments in
Molecular Genetics (1972). Cold Spring Harbor Laboratory, New York), and
resuspended in K3 culture medium at a density of 2 x 109 cells/ml. Fifty NI of
this suspension is added to the plant protoplast cultures and after sealing
with
Parafilm~, the cultures are incubated under the same conditions as described
under 2.3. After 48 hours the cultures are transferred to 10 ml centrifuge
tubes and centrifuged in a swinging bucket rotor at 60 - 80 g for 6 minutes.
The floating band and pellet are pooled and resuspended in 10 ml of K3
medium (Nagy and Maliga, Z. Pflanzen~~h~~ol. 78 (1976), 453-455)
supplemented with an antibiotic (carbenicillin 1000 Ng/ml or cefotaxime 500
N9/ml).
After two weeks of incubation, the protoplast-derived micro-calli are
centrifuged and resuspended in K3 medium (Nagy and Maliga, Z.
. Pflanzenahysiol. 78 (1976), 453-455) with the same growth regulator and
antibiotic concentrations as before, but 0.3 M sucrose instead of 0.4 M. The
1 X41 41 9
46
cell density in this medium is adjusted to about 2 5 x 103 micro-calli per ml.
After two more weeks of incubation under the same conditions the calli are
transferred to K3 medium with the same antibiotic concentrations as before,
but with reduced sucrose (0.2 M) and growth regulators (NAA 0.01 mg/litre
and kinetin 0.02 mg/litre). After two or three more weeks of incubation, the
putative transformants can be recognized by their light green and compact
aspect, and better growth. These colonies are then transferred to hormone-
free Linsmaier and Skoog medium (Linsmaier and Skoog, P, hysiol. Plant. 18
(1965), 100-127) solified with 0.6% agar and containing reduced antibiotic
concentrations (carbenicillin 500 pg/ml or cefotaxime 250 Ng/ml).
Opine tests can be done on the putative transformants which grow on this
hormone-free medium when they reach 3 - 4 mm in diameter. Half of each
colony is used for the detection of octopine and nopaline (Aerts et al., Plant
Sci. Lett. 17 (1979), 43-50) or agropine and mannopine (Leemans et al., J.
Mol. Appl. Genet. 1 (1981 ), 149-164). This test allows to confirm the
transformed nature of the colonies selected on hormone-free medium.
Afterwards, the selected colonies can be cultured on antibiotic-free medium.
2.5. Co-cultivation without selection on hormone-free media
When selection for transformed cells is not possible (e.g. because avirulent
T-DNA mutants are used) or is not required because a dominant selectable
marker such as an antibiotics resistance gene is present in the T-DNA, the
treatment of the protoplast-derived cells can be simplified (the hormone
reduction steps are no longer necessary). The protoplasts are treated as
described previously until the infection step. Forty-eight hours after
addition
of the bacteria the protoplast-derived cells are centrifuged (6 minutes, 60-80
g), and resuspended in medium AG (Caboche, Planta 149 (1980), 7-18)
which is able to support cell growth at very low density. The cells are
counted
using a Fuchs-Rosenthal counting chamber (obtained from "Assistant", F. R .
G.), and resuspended at the density required for subsequent work. If the
colonies must be manipulated individually for opine tests, plating at low cell
density (100 protoplast-derived cells and cell colonies per ml) gives large
cell
X41 41 9
47
colonies after one month of incubation. If drug selection for the transformed
cells is possible, the cells are incubated at a higher density (103 -104/ml),
and
the selective agent used is added to the medium in a concentration and at a
time which has to be optimized for each type of selection.
2.6. Regeneration of whole plant from callous tissue
Normal plants are easily obtained from callous tissue (for example either
derived from protoplast transformation or from whole plant inoculation (see
2.7). The callous tissue is grown an Murashige and Skoog medium containing
1 mg/ml BAP; this medium induces shoot formation after 1 - 2 months. These
shoots can be transferred to medium without hormones so that roots form and
a complete plant is produced.
2.7. Tumor induction on tobacco seedlings
Tobacco seeds (e.g. cultivar Wisconsin 38) are surface sterilized by
treatment with 70% denaturated ethanoI/Ha0 for 2 minutes; followed by 10%
commercial bleach and 0.1 % sodium docecyl sulfate (SDS); further rinsed 5
time with sterile H20.
The sterile seeds are sown in large (25 mm wide) test tubes containing the
salts of Murashige and Skoog medium solidified with 0.7% agar and covered
with polycarbonate tops. Then the tubes are incubated in culture room
(12,000 lux, 16 hours light/8 hours dark; 70% relative humidity; 24°C).
After4
- 6 weeks the plants are ready to use. They remain optimal for at least
another month.
Plantlets should be at least 3 cm high and have four or more leaves . The
plants are then decapitated transversally through the youngest internode with
a new sterile scalpel blade; the upper part of the plant removed from the
tube,
and bacteria from an agar plate culture applied on the wound surtace with a
flamed microspatula.
Tumors appear after 2 weeks for the wild-type and after a longer time for
some of the attenuated mutant strains. This method is used inoculate tobacco
(Nicotiana tabacum), Nicotiana plumbaainifolia and Petunia h_ybrida.
4~ 1 ~~1 41 9
Concluding remarks
The present invention offers for the first time the possibility to transform
plants with
Aprobacterium harboring a hybrid Ti plasmid vector which lacks the oncogenic
functions of the T-region of wild-type Ti plasmids. Since the influence of the
oncogenic functions of the T-region on the transfer of DNA from the Ti plasmid
to
plant cells was not known, it is surprising that nevertheless transfer of the
modified
T-region containing (a) genes) of interest to plant cells occurs. There is
cointegration and stable maintenance of this transferred DNA in the plant cell
genome. Furthermore, expression of chosen genes) of interest can be achieved
provided the genes) either contain - or are constructed to contain - suitable
promoter sequences. The concept of effecting a single cross-over event between
an
intermediate cloning vector containing the chosen genes) of interest with an
especially designed acceptor Ti plasmid greatly simplifies the construction of
any
hybrid Ti plasmid vector useful for the transformation of plant cells. The
especially
designed acceptor Ti plasmids contain the DNA segment of a conventional
cloning
vehicle such that any genes) of interest (which has been inserted into the
same or a
related cloning vehicle as part of an intermediate cloning vector) can form a
cointegrate by a single cross-over event. The two segments of the cloning
vehicles)
provide the necessary regions of homology for recombination.
Microorganisms and intermediate cloning vectors, acceptor Ti plasmids and
hybrid
plasmid vectors prepared by the processes of this invention are exemplified by
cultures deposited in the German Collection of Microorganisms (DSM),
Gottingen,
on December 21 st, 1983, and identified there as
(1) intermediate vector plasmid pAcgB in Escherichia coli K12 HB101;
(2) Aprobacterium tumefaciens C58C1 rifampicin-resistant strain carrying
carbenicillin-resistant acceptor Ti plasmid pGV3850;
(3) intermediate vector plasmid pGV700 in Escherichia coli K12 strain K514
(thr
leu thi lac hsdR);
(4) intermediate vector plasmid pGV750 in Escherichia coli K12 strain K514 (as
~- ,
3
1 X41 41 9
49
above in (3));
(5) Aprobacterium tumefaciens C58C1 rifampicin-resistant strain carrying
carbenicillin-resistant acceptor Ti plasmid pGV2260;
(6) intermediate vector plasmid pN0-1 carrying the octopine synthase coding
region under the control of the nopaline promoter, in Escherichia coli K12
HB101;
(7) strain used in mobilization of intermediate vectors to A rg obacterium =
GJ23
carrying mobilizing plasmids pGJ28 and R64drdll (Van Haute et al., EMBO J.
2 (1983), 411-418); GJ23 is Escherichia coli K12, JC2926, a recA derivative
of AB1157 (Howard-Flanders et al. , Genetics 49 (1964), 237-246).
These cultures were assigned accession numbers 2792. (1 ), 2798 (2), 2796 (3),
2797. (4), 2799 (5), 2833 (6), and .2793. (7), respectively.
While we have hereinbefore presented a number of embodiments of this
invention,
it is apparent that our basic constructions can be altered to provide other
embodiments which utilize the processes and compositions of this invention.
.~ ~,~
