Note: Descriptions are shown in the official language in which they were submitted.
13 41524
MAX-PLANCK GESELLSCHAFT ZUR F(iRDERUNG DER WISSENSCHAFTEN e.V. Gdttingen
PROCESS FOR THE INTRODUCTION OF EXPRESSIBLE GENES INTO
PLANT CELL GENOMES AND AGROBACTERIUM STRAINS CARRYING
HYBRID Ti PLASMID VECTORS USEFUL FOR THIS PROCESS
Technical iield 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 sequen-
ces 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 sequen-
ces that code for products, e.g. amino acids or polypeptides, useful
for the growth of the p1_ant or for improving its quality as nutrient,
or for the production of valuable metabolites (e.g. alkaloids or steroid
precur5ors ).
In the description the following terms are used
bom site : reg:on of DNA where mob functions specifically interact and
initiate autonomous DNA transfer.
Border sequence : DNA sequence which contains the ends of the
T-DNA
Broad-host-rang? replicon : a DNA molecule capable of being trans-
ferred and maintained in many different host cells.
Callus tissue : a mass of unorganized and undifferentiated cells.
Cloning : the process of obtaining a population of organisms or DNA
sequences derived from one such organism or sequence by
_2- 13 4 1 5 2 4
asexual reproduction.
or better:
the process of isolating a particular organism or part there-
of, and the propagation of this subfraction as a homogenous
population.
Cloning 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 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 ampi-
cillin resistance. A cloning vehicle is often called vector.
Coding sequence : DNA sequence which determines the amino acid
sequence of a polypeptide.
Cointegrate : the structure resulting from a single cross-over event
bet,veen two circular DNA molecules.
Complementation in trans : process whereby a DNA molecule (replicon)
which is not physically linked to another replicon can
provide a diffusible substance which is missing and re-
quired by the other nonlinked replicon.
Conju ;~'lc~ : the process whereby DNA is transferred from bacteria
oT one type to another type during cell-to-cell contact.
Crossinc-over : the process of exchange of genetic material between
hor:zo;ogous DNA sequences.
Deletion su:cstitutio?: : removal of one DNA sequence and its replace-
ment by a di}ierent DNA sequence.
Differ ent:ation : the process whereby descendents of a cell achieve
and maintain specialization of structure and function.
DNA sec[uence or DNA segment : a linear array of nucleotides con-
nected osle to the other by phosphodiester bonds between
the 3' and 5' carbons of adjacent pentoses.
134152 4
- 3 -
Double cross-over : the process of resolution of a cointegrate struc-
ture 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 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 sequence : a sequence of nucleotides that controls
and regulates expression of structural genes when operative-
ly linked to those genes.
F-type plasmid : plasm-id 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
secruence for the gene product, and (2) the sequences in
the promoter region which control whether or not the gene
will be expressed.
Genomp : the entire DINA 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.
Genotype : 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 plasinid : a group of autotransferable plasmids of a different
incompatibility group than F.
4 13 41524
i
Incompatibility : Incapability of two DNA molecules of coexist-
ing in the same cell in the absence of selective pressure.
Insertion : addition of a DNA sequence within the DNA sequence of
another molecule.
Leader sequence : 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.
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 plasmid : a plasmid capable of providing diffusible
p~oducts which another plasmid lacks for transfer to an-
other host cell.
Nonconi,ugative 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 hetero-
cyclic base. The base is linked to the sugar moiety via a
glycosidic bond (1' carbon of the pentose) and that com-
bination 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").
13 415 2 4
Phenotype : the observable characteristics of an individual resulting
from the interaction between the genotype and the environ-
ment 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 plasmid is placed within a unicellular
organism, the characteristics of that organism are changed
or transformed as a result of the DNA of the plasmid. For
example, a plasmid carrying the gene for tetracycline resist-
ance (TcR) transforms a cell previously sensitive to tetra-
cycline into one which is resistant to it. A cell transformed
by a plasmid is called "transformant".
Polypentide : 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.
Promoter region : DNA sequences upstream to the start of. the coding
sequence which regulate transcription of the gene.
Promoter sequence : sequence at which RNA polymerase binds and
promotes the faithful transcription of downstream sequences.
Recombinant DNA molecule or hybrid DNA: a hybrid DNA sequence
cumprising at least two nucleotide sequences, the first
sequence not normally being found together in nature with
frie second.
Recor ~ir=~ ~n : the creation of a new association of DNA molecules or
parts of DNA. rr:olecules .
Region o= homolocT' : a region of DNA which shares the same DNA
seauence as that found in another region of. DNA.
Replicon : a self-replicating genetic element possessing a site for the
initiation of DNA replication and genes specifying the neces-
sary functions for controlling replication.
Restriction fragment : a DNA molecule resulting from double-stranded
cleavage by an enzyme which recognizes a specific target
DNA sequence.
RNA polymerase : enzyme which results in the transcription of DNA
into RNA.
6 13 4 1 5 2 4
- -
Selectable marker gene : a DNA sequence which, when expressed,
gives that cell a growth advantage over cells which do 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 sequen-
ces which are transferred to the plant cell genome.
Ti plasmid : large plasmids found in strains of Agrobacterium tume-
faciens containing the genetic information for tumor (crown
gall) induction on susceptible plants.
TL-DNA and TR-DN A: 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
coimmon with T-DNA of nopaline tumor cells whereas
TR-DNA does not.
tra (transfer functions) : both plasmid-encoded diffusible products
and sites of action utilized during DNA transfer between
ciiis, e. a. products required to make a bridge between two
cells and the site at which DNA transfer is initiated.
TranscL :-. tion : 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 comple-
mentary sequence of bases in an RNA chain.
Transfors,!ation : 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 - 13 4 1 5 24
during the synthesis of a polypeptide.
Undifferentiated phenotype : a homologous appearance of cells in a
tissue without any specialized parts.
Vector : a DNA molecule designed for transfer between different
host cells.
- s - 1~ 4 1 524
Background Art
The development of recombinant DNA techniques has made the genetic
engineering 'of microorganisms a challenging prospect: These tech-
niques 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 availabil-
ity 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 Agrobac-
terium tumefaciens. This organism has been shown to cause a neo-
plastic transformation, called "crown gall't, 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 cop_tain in their chromosomal DNA- a DNA segment called the
T-DNAwnich is homologous to DNA sequences in the Ti plasmid used
to indu,ce -Lb.e t~.~mor line. In all cases, this T-DNA corresponds to,
and is colMear with, a continuous stretch of Ti plasmid DNA which
is, therefore, called tile T-region.
Ti plasmids are classified according to the type of opine synthesized
in crown gall cells. Agrobacterium strains which induce the synthesis
of nopaline [N-u-(1,3-dicarboxypropyl)-L-arginine] in crown gall cells
are cailed nopaline strains, and strains which induce the synthesis of
octopine [N-cy-(iv'-i-carboxyethyl)-L-arginine] are called octopine
strains. These are the most commonly. used Agrobacterium strains.
9 13 41524
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 near the right border of the
T-DNA from the Ti plasmid of the strain Agrobacterium T37. Nopa-
line synthesis was eliminated in the tumors incited by agrobacteria
carrying this Ti plasmid: Furthermore, Southern blotting hybridiza-
tions 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 latter's
ability to be transferred to the plant cell genome.
The borders of the T-DNA from the Ti plasmid of the nopaline strain
Agrobacterium T37 have been very precisely determined. It is only a
portion, roughly 23 kb, of the entire nopaline Ti plasmid. Further-
more, 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. Mol. Appl.
Genet. 1(1982) , 361-370). The borders of the T-region are most
probabiy i?wolved in the integration of the T-DNA,into the plant cell
genom-.
Knowle :ge oi 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 addi-tion, 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 it- elf . Thus, the T-region of the Ti plasmid has been sub-
jected to intense genetic analysis to determine which regions are
responsible for the tumor phenotype.
1341524
- 10 -
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 (Leemans 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 tumor callus
tissue. Mutants in these genes can either lead to transformed tissues
which appears shoot-like or root-like. The latter results are especial-
ly 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 sequen-
ces through meiosis, i.e. progeny plants still contained T-DNA-spe-
cific sequer_ces (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 gene-
ration of a large deletion which removed the region of the T-DNA
controilina the tumor phenotype.. It is not known, whether the dele-
tion occurred during the initial transformation event or as a subse-
quent event leading to shoot formation.
The Ti are large (200 kb) and many genes located at differ-
ent si'es or t-he Ti plasmid are involved in the transformation of plant
cells . T=? eMefore , it is not possible to construct a small Ti plasmid-
derived L=or~ing vector with unique endonuclease recognition sites at
appropriate locauop_s w-ithin 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 speciiic restr~ction enzyme cleavage sites of the T-region of a
Ti plasmid has been to construct cloning plasmids which are able to
replicate in Agrobacterium as well as in Escherichia co1i, and which
contain a chosen restriction fragnient of the T-DNA. Such cloning
plasmids were denoted "int-crmediate vectors". Such intermediate
vectors were used to analyze the functions encoded by the T-region
(Leemans et al., J. Mol. Appl. Genet. 1 (1981), 149-164).
13 41524
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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 modified acceptor Ti plasmids which
allow the introduction of any gene(s) of interest into these plasmids.
The gene(s) 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 gene(s) of interest into the acceptor Ti plas-
mids 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 propa-
gated to Agrobacterium using helper plasmids. Such helper plasmids
and their functions for mobilization are known (Finnegan et al., Mol.
Gen. Genet. 185 (10,82), 344-351).
The resu}.t of the single cross-over event in Agrobacterium is a
hybrid Ti plasmid vector. Such hybrid Ti plasmid vectors are a
further =h;e: t of the invention.
The resu3t;.ng hybrid plasmid vectors harbored in Agrobacterium
(hereinafter called vector composition) can be used directly to infect
plant ce.";s which are subsequently screened for the expression of the.
product(s) of the gene(s) of interest.. This process for preparing
trans?orr~ed plant cells by infection of plant cells with the vector
composition, and t.re transformed plant cells, as well as plants gener-
ated therefrom are further objects of this invention_ This strategy is
applicable to any of the plant-transferable plasmids of Agrobacterium.
_12_ 1341524
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 sequen-
ces (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 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 Agrobac-
Lerium 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. 1. It contains a cloning vehicle DNA
segment (3' ) with a DNA sequence which is homologous with
at least part oT the DNA segment (3) of the acceptor Ti
olas,d permittin,g the desired single cross-over event.
Moreover, the intermediate cloning vector contains a gene
or group of genes (5) of interest having its natural pro-
moter sequence. In general plant genes can be used in
this construction as they are more 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 contairi a
promoter sequence which permits expres.sion of the gene in
plant cells. Plant cells containing this marker gene should
have a selective growth advantage over cells without this
1341524
- 13 -
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 Fig . 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 gene(s) of interest (7), and, if desired, a marker
gene (6).
Fig. 4 schematically illustrates the construction of hybrid Ti plas-
mid vectors of the inverition from the acceptor Ti plasmid of
Fig . 1, and te intermediate cloning vectors of Figs 2 and 3
h
by a single cross-over event.
Fig. 5 outlines the steps involved in the genetic transfer of an
intermediate cloning vector from E. coli to Agrobacterium
containing an acceptor Ti plasmid. The first step is the
conjugation of the E. coli strain (1) containing the inter-
mediate cloniny vector to another E. coli strain (2) which
clntains tivc, helper plasmids for the later conjugation to
Agrobacterium. One helper plasmid contains DNA sequences
important for plasmid transfer (tra) and the other helper
piasmid contains sequences which are important for the
mobilization (mob). When these helper plasmids are intro-
duced by conjugation into E. coli strain (1), the inter-
mediate cloning vector contained therein will be capable of
being transferred to other bacterial strains. The tra and
mob helper plasmids also contain antibiotic =resistance mark-
ers Abr2 and Abr3 which are different from those found in
the intermediate clotiing vector (Abri ). Thus, the pres-
ence of all the plasmids can be monitored on selective
13 415~4
- 14 -
media. A mobilizing strain (3) is obtained. This mobiliz-
ing 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 marker(s) of the interme-
diate 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 Agrobacterium (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 specific-
ally, 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.
i i:z acceptor Ti plasmid (A) obtained is capable of trans-
fsrring DNA contained between the border sequences (1; 2)
?nto plant cell genomes. The resulting transformed plant
1~_,NA wil; not produce tumorous crown gall tissue as the
genes controlling neoplastic growth are deleted in the
Ti plasraid (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 illusLrates schematically the steps leading to the. construc=
tion of an intermediate cloning vector in E. coli host cells.
A gene of interest (5) bracketed by restriction endonucle-
ase site R1 and a selectable marker gene (6) bracketed by
13 4 1 5 24
- 15 -
restriction endonuclease site R2 are inserted into a cloning
vehicle (3') containing single restriction sites for the en-
zymes Rl and R2. - All three molecules are digested with
restriction enzymes R1 and/or R2 and ligated together using'
DNA ligase to form the intermediate cloning vector. The
cloning vehicle 3' must also contain additional DNA sequen-
ces which code for antibiotic resistance (Abri ) 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 outlined in Figs. 2 and 3.
Fig. 8 illustrates the construction of another embodiment of an
acceptor Ti plasmid (B) of the invention. Here, a double
cross-over event occurs between 'a Ti plasmid and a cloning
vehicle (3) whicll contains DNA sequences (9) and (10)
which are homologous to Ti sequences just outside the
border sequences (1) and (2) respectively. The double
cross-over event results in the deletion of the entire T-re-
gion T including the border sequences (1) and (2) and its
replacement with the cloning vehicle (3). Ti plasmid (B) is
an acceptor for intermediate cloning vectors which contain
the gene of interest cloned between the border sequen-
ces (1) and (2); (see Fig. 9).
Fig.. 9 i slustrates an intermediate cloning vector of- the invention to
I-)e inserted by a single cross-over event into the acceptor
Ti plasmid (B) of Fig. 8. It contains the border- sequen-
ces (1) and (2) which flank the gene(s) of interest (5). It
also contains a cloning vehicle sequence (3') which is at
least partially homologous to the cloning vehicle sequence
(3) in - tne -acceptor Ti plasmid (B) to allow homologous
recombina~ion between the two plasmids.
Fig. 10 schematically illustrates the construction of hybrid Ti plas-
mid vectors of the invention from the acceptor Ti plasmid
1~~+1524
- 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 inven-
tion.
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 plasrnid. This
clone pAcgB is used in the construction of acceptor plasmid
pGV3850, ari "A-like" acceptor plasmid as shown in Fig. 6.
It is ' obvious to those skilled in the art that a clone analo-
gous to clone pAcgB can be obtained by using the cloned
restrictiori fragments which contain the left and right bor-
der region of a wild-type Ti plasmid.
Fig. 12 1lluSt.ratas the T-region of nopaline Ti plasmid pGV3839.
The HindIII restriction endonuclease sites are indicated as
(I-.). Mutated HindliI fragment 19 is indicated (19'). The
acetylphosphotransferase gene providing kanamycin or
neo m.vcin resistance is indicated as apt 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 taiLen from example 1, and the two references cited.
Fig. 13 illus Lrates the construction of acceptor Ti plasmid pGV3850.
The piasm.id 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
la2 4
- 17 -
pBR322 by ApR. Part of the T-region of pGV3839 which is
shown in Fig. 12 is shown here; the HindIII 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 replicoii
containing the apt gene which is lost.
Fig. 14 illustrates schematically the construction of intermediate
cloning vector pGV700 which is given in detail in exam-
ple 2. The following abbreviations are used to indicate
restriction endonuclease sites : B, BamHI; Bg, Bc.lII; 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 on the bottom of the figure
which refer to TL-DNA indicate the RNA transcripts of this
region (4villrnitzer et al., EMBO J. 1 (1982), 139-146).
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 rela-
tive locations given in numbers as kilobase pairs (kb).
PstI sites are not indicated but there are 3 in the KmR/NmR
regior_, and one in the CbR gene. The right and left
borders are also indicated. The BqIII/BamHI and HpqI/Smal
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 resist-
-ance; CbR, carbenicillin -(analogous to ampicillin) resist-
ance, and KmR/NmR, kanamycin resistance/neomycin resist
ance.
= 134 1524
- 18 -
Fig. 16 illustrates the construction of the intermediate vector
pGV745 described in detail in example 3. pGV745 is used
in the construction of the acceptor plasmid pGV2260, a
"B-like" acceptor plasmid shown in Figure 8. Restriction
endonuclease sites are indicated as follows : B, BamHI; H,
HindIII; R, EcoRI. The ampicillin resistance gene is indic-
ated. 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 e- 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 (indic-
ated apt) wh?ch provides resistance to neomycin and kana-
rnycin. The intermediate vector pGV745 (Fig. 16) is depict-
ed in linearized form; it has been opened at the HindIII site
eT 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
ihe construczicn oI pGV2260 and the loss of the at gene.
Restriction endonuclease sites are indicated as follows : B,
B3SnHI; H, HindIIl; R, EcoRl.
Fig. 18 i~l=.:strates 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
reier 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.
1~4 1524
- 19 -
Fig. 19 illustrates the construction of the plasmid pAGV10 contain-
ing the complete octopine synthase (ocs) coding sequence
and its insertion in plasmid pLGV2381 (see also Fig. 18)
behind the nos promoter. The 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 nucleo-
tide 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 restric-
tion endonuclease sites are indicated, BamHI, HindIII, and
SacII. The 5' and 3' refer to the start and stop of tran-
scription. 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 ro Fig. 1, we have shown therein a simplified diagram of an
acceptor Ti p'asmid. This acceptor Ti plasmid contains the two
border seouences (1; 2) or regions of the wild-type tumor-inducing
(Ti) plGs,;d. The border sequences are essential for the integration
of the T-region ot 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 sequen-
ces.
The DNA sequence (3) of the acceptor Ti plasmid contains -a DNA
segment which is homologous with at least a part of a DNA sequen-
ce (3') oi an intermediate cloning vector illustrated in Figs. 2 and 3.
1341524
-20-
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 cointegra-
tes is determined essentially by the length of the region of homology.
In order to effect a homologous recombination event at a good fre-
quency, regions of 1 to 4 kb are normally used (Leemans et al.,
J. Mol. Appl. Genet. 1 (1981), 149-164).
The acceptor Ti plasmid furthermore contains sequences (4) which are
essential for the transfer by Agrobacterium 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 Gve have shown simplified diagrams of intermediate
cloning vectors for the cloning of any prokaryotic or eukaryotic
gene(s) of interest to be expressed, i.e. transcribed under the
control of a promoter and translated in plant cells. These interme-
diate clo~ing vectors contain a DNA segment (31) from a cloning
vehicle containing with a DNA sequence which is homologous with at
least a7~a-_t of the DwA segment (3) of the acceptor Ti plasmid thus
permi-::ng a single cross-over event. Moreover, the intermediate
cloning vectors contain at least one gene of interest (5; 7) which
contains a~zrher its natural or an exogenous promoter sequence. The
promoter. s? ;uence allows the expression of the inserted. gene sequen-
ce(s). The use of an exogenous promoter sequence (tailored promoter)
may be useful for directing the expression of the inserted gene(s) in
a regulated . fashion _
Examples of different types of regulation include the following :(i)
t}psue-sYecific 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.
93 4 1 5 21 24
- -
Examples of genes of interest for the intermediate cloning vectors
are : DNA fragments or sequences with the genetic information con-
trolling 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 resist-
ance 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.
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 conjuga-
tion steps involved in the isolation of Agrobacterium carrying the
hybrid i'i plasmid vectors. Since the intermediate cloning vector is
construr,ted in E. coli these steps are necessary to transfer the
intermediate cloning vector to the acceptor Ti plasmid in Agrobac-
teriutn.
The _',=~_-wn transfer process which was used to prepare modified
Ti plasm;-s i_n which a portion of the T-region was replaced by an
altered _Qe.y:zence involves many steps. Normally, most DNA. recom-
binant manipilations 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 Agrobacterium. 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 a!. , Gene 19 (1982), 361-364), capable of also replicating in
Agrobacterium. The manipulations are effected in E. coli. An
intermediate cloning vector is obtained.
13 41524
-22-
b) Conjugation of the E. coli strain carrying the intermediate clon-
ing vector containing the DNA of interest with another E. coli
strain carrying a helper plasmid which cannot replicate in Agro-
bacterium but which can mediate transfer of itself and other
DNAs to Agrobacterium.
c) Conjugation of E. coli obtained in step (b) -with Agrobacterium
containing a Ti plasmid. The helper plasmid is lost.
d) Since the intermediate cloning vector is capable of replicating
and existing in Agrobacterium as a separate replicon, the con-
jugants 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
cloning vector and the Ti plasmid where no cointegration has
occurred. In order to specifically isolate only the cointegrates,
a- second conjugation to another Agrobacterium strain without a
Ti plasmid must be performed. This transfer is mediated by
functions'- encoded by the Ti plasmid itself. Transfer of the
intermediate cloning vector into the second Agrobacterium strain
is ef;:ected 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., T. ':zol. Appl. Genet. .1 (1981), 149-164).
The orL,: other known method is essentially the same as outlined
above, ax-.,e~t that for step (d) another plasmid which is not com--
patible ~~_'~'i the intermediate cloning vector is introduced into A~c ro-
bacteriu.n; 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 et al., J. Mol.
Appi. Genet. 1 (1981), 39-49).
We desc: i? e a novel and much simplified method for the introduction-
of intermediate cloning vectors into acceptor Ti plasmids of Agrobac-
terium. Briefly, this method is based on the finding that helper
plasmids of E. coli allow transfer of many of the commonly used
~3415 24
-23-
cloning plasmids (such as pBR322) directly to Agrobacterium. Since
none of these plasmids can replicate in Agrobacterium only those
which can cointegrate with the acceptor Ti plasmid will be retained.
In addition, we use this cointegrate in Agrobacterium as a vector
composition directly for infection of plant cells. In this manner we
have eliminated steps (d) and (e) described above which greatly in-
creases the possibilities for using the acceptor Ti plasmids as vectors
for DNA transfer to plant cell genomes by both decreasing the time
required for constructing modified hybrid Ti plasmids and increasing
the flexibility of the possible constructions.
Thus, as outlined in Figure 5, the introduction of the intermediate
cloning vector into the acceptor Ti plasmid is accomplished in two
steps. First, conjugation of E. coli strain (1) carrying the interme-
diate cloning vector to another E. coli strain (2) carrying two plas-
mids which will help to mobilize the intermediate cloning vector to
Agrobacterium. Typical and preferred examples of these helper
plasmids are R64drdll 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 func-
tions =ncUdnd by the other two plasmids to allow transfer to occur.
All F'a~-_ids should preferably contain antibiotic resistance markers to
detect _iõ presence. Secondly, the E. coli strain obtained, i.e. the
mobilizing strain (3) carrying all three plasmids is conjugated to
AgrobactLTium containing an acceptor Ti plasmid with a region of
homoloay 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 p1_asmid 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
13~1524
-24-
between a Ti plasmid and another plasmid carrying the border sequen-
ces (1) and (2) in a cloning vehicle (3). As illustrated in the fig-
ure, 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. How-
ever, 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 sequen-
ces (1) and (2). As also shown here in Figure 6, the double -cross-
over event also produces 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
cloni:?g vecTor of Figs 2 and 3. A gene of interest (5) and a select-
able na_ k~r gene 16), each bracketed by a restriction endonuclease
site Ri or R2, respectively, are inserted into a cloning vehicle se-
quence ;;3' j which contains unique restriction sites for enzymes R1
and R2 by digestion and ligation of all molecules. The recombinant
DNA mol eCule obtained is used to transform E. coli host cells and
transformants are selected for the antibiotic resistance marker (AbRi
)
of the cloning vehicle sequence (31).
Figure 8 schematically illustrates the DNA molecules used to construct
another e::ibodiment of the invention, i.e. acceptor Ti plasmid (B).
Here a double cross-over event occurs between a Ti plasmid and a
plasmid containing the cloning vehicle sequence (3) between the DNA
~341524
-25-
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 plas.mid (B) and another circular DNA molecule which is lost con-
taining the T-region from the original Ti plasmid plus the DNA se-
quences (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 sequen-
ces (1) and (2) which are contained in a cloning vehicle sequence
(3').
Figure 10 schematically illustrates how a single cross-over event
introduces the interrhediate clonirig vector of Fig. 9 iinto the acceptor
Ti plasmid (B). In this case,' selection for_ the antibiotic resistance
marker of the cloning vehicle sequence (3') of the intermediate clon-
ing vector ensures iha:. a hybrid Ti plasmid can be found which is
the result of a cointeg, ration between the two plasmids. A hybrid
Ti plasm-id ?s produced with the gene of interest contained within the
border 5--eUuences (1) and (2). The hybrid plasmid thus constructed
does r_o= '.cntain ir=. its T-region a sequence which is a direct repeat
(as for Lxar,-.ple tl-Le sequences. (3) and (3') in hybrid Ti plasmid of
Fig. 4, avoiding possible problems of instability of the hybrid vector
or of tble transformed DNA in the plant cell genome as a result of
intramoiecular recom~ination.
UnpubliS_.':ed result-s from our laboratory also indicate that for con-
struction of the intermediate vector in Fig. 9, it is not essential to
have boih border sequences 1 arid 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.
~341524
-26-
Knowledge of the restriction endonuclease maps of the Ti plasmids of
Agrobacterium, 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 abilitity to perform conventional recoin-
binant DNA techniques and basic bacterial genetic manipulations. The
present invention is unique in that it specifically proposes the de-
scribed 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 intro-
duce genes into the plant cell genome.
In order to further illustrate the disclosed acceptor Ti plasmids,
intermediate cloning 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 gene(s) integrated into the plant cell ge-
nome, the following examples are provided.
Example 1
Const_ruction of acceptor Ti plasmid pGV3850 (type A)
Starting strains and plasmids Aqrobacterium tumefaciens (rifampicin-resistant
strain C58C1, and
chlora:nphvnicol-eryt'i-romycin-resistant strain C58C1 derived from
wild-tvpe Agrobacterium)
T' plasmid = pGV3839 - -
Plasmid of Fig. 11 = pAcgB
1341524
-27-
The Ti plasmid pGV3839 is constructed from a nopaline plasmid
pTiC58trac (pGV3100; Holsters et al., Plasmid 3 (1980), 212-230). It
contains a deletion substitution mutation near the centre of the T-re-
gion; the SmaI 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 a~t (acetylphosphotransferase) 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 de-
fined 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 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 plas~;u, 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 descrined plasmid pAcgB carries a ColE1-specific bom site in the
pBR322 portion and can be mobilized from E. coli to Agrobacterium by
using the helper plasmids R64drdll and pGJ28. The plasmids R64drd11
and pGJ ?3 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), strepto-
piycin-resistant (frotr. R64drd11) and kanamycin-resistant (from pGJ28)
colonies.
_28- 1341524
The E. coli strain carrying all three plasmids is conjugated to Agro-
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 Agrobacterium 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
apt gene (kanamycin resistance) is replaced by the pBR322 sequences
of the clone pAcgB. Second recombinants are thus ampicillin-resist-
ant and kanamycin-sensitive. To increase the probability of isolating
a second recombinant, the rifampicin-resistant Agrobacterium carrying
the first recombinant (pAcgB :: pGV3839)- is conjugated with a second
chloramphznicol/erythromycin-resistant Agrobacterium strain without a
Ti plasmi'd. In this manner, a chloramphenicol/erythromycin-resistant
Agrobacterium pGV3850 can be obtained which is ampicillin-resistant
and kar.anmycin-sensititi e at a frequency of approximately one in 600
colonies.
Of coui se, there are other Ti plasmids which can be utilized to con-
struct p-GV3850-type acceptor Ti plasmids. Any Ti plasmid carrying a
selectah'_e marker gene near the centre of the T-region may be used
as a re~i~ient. Furthermore, a pAcgB-like plasmid may be construct-
ed by inserting the T-region border fragments into pBR322 in such a
way that- the pBR322 sequences lie in-between the left border frag-
ment an : the right border fragment in the orientation left border
fragment - pBR322 -right border fragment. For example, the left
and right border fragments of the nopaline Ti plasmid are HindIIl
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
~si+15 24
-29-
only requirement is that the DNA to be introduced contains an addi-
tional 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 marker 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 irsertion of dzfined intermediate cloning vectors containing a
pBP.39_2 se-auence by a single cross-over event into the = acceptor
Ti plas-T:u pGV3850; this acceptor Ti plasmid can also be used as a
recipien--- fcr= cloned :eanns of DNA in pBR322 or its derivatives in a
"shotgun''-typA experiment. The total population of hybrid plasmid
vectors in Agrobacterium can be used to infect plant cells and is
subsequently screenQd for expression of any selectable gene(s) of
interest. For example, one can easily select for genes encoding
amino acid syntl-tesis' by applying the total bank to plant cells which
are deficient in the chosen amino acid.
The acceptor Ti plasmiw 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,
1341524-30-
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 plas-
mid 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 concen-
trations 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-t~ype" tumors are detected after two weeks).
This tissue does not grow on hormone-free media but can be
propagated further as sterile tissue culture on media containing
boLri auxin and cytokinin. This tissue is also shown to be
r:cctal ine-positive .
c) Fu= ~ ~r::ore, 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.
No_:-~a plants w?li be obtained by culturing the transformed cells
on conventional regeneration media (see also example 5).
To prove the useft;_ness of pGV3850 as an acceptor plasmid the follow-
ing experi.anent is performed. An intermediate cloning vector contain-
ing oncogenic functions of the octopine T-DNA in pBR325 is recom-
biped into Agrobacterium harboring pGV3850. The resulting hybrid
Ti plasmid in Agrobacterium obtained by a single cross-over event is
inoculated onto wounded.tobacco plants. Tumor .tissue develops after
13 41524
31 -
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 HindIIl site of
the broad-host range vector pGV1122 (Leemans et al., Gene 19 (1982),
361-364). The reco.mbinant plasmid pGV0201 contains the HindIII
fragment 1 inserted in the unique HindIII site of the multicopy vector
pBR322 (Bolivar et al., Gene 2 (1977), 95-113). pGV0201 and
pGV1122 DNA ?s prepared as described by Betlach et al., Fed. Proc.
35 (19706), 2037-2043. Two pg of pGV0201 DNA are totally digested
with 2 units of HindiIl (all restriction enzymes are purchased from
Boehrirg?r Mannheim) for 1 hour at 37 C in a final volume of 20 Nl.
The i":cw ;a~cn buf'er is described by O'Farrell et al., Mol. Gen.
Genet 179 (1980), 421-435. Two [ig of pGV1122 DNA are totally
digestcd wiz:ti HindII? under the same conditions.
One tentli-i pg of HindIII digested pGV0201 is ligated to 0.05 pg of
HindIII-digested pGV1122 with 0.02 units of T4 ligase (Boehringer
MannheiM) in a final volume of 20 pl. Incubation buffer and condi-
tions arF as recommended by the manufacturer (Brochure "T4 ligase",
Boehringer Mannhei~r~, August 1980, #10.M:880.486). Transformation of
the liga tior. mixture into competent E. coli K514 hsr hsm+ cells
(Colson e al., Genetics 52 (1965), 1043-1050) is carried out as de-
scribed by Dagert and Ehrlich, Gene 6 (1980), 23-28. Cells are
s3 41524
-32-
plated on LB medium (Miller, Experiments in Molecular Genetics (1972),
Cold Spring Harbor Laboratory, New York) supplemented with strep-
tomycin (20 pg/ml) and spectinomycin (50 pg/ml). Transformants
containing recombinant plasmids are screened for tetracycline sensi-
tivity (10 Ng/ml), due to the insertional inactivation of the gene
coding for tetracycline resistance (Leemans et al., Gene 19 (1982),
361-364). Streptomycin- and spectinomycin-resistant and tetracycline-
sensitive clones are physically characterized. Microscale DNA prepa-
rations are performed according to Klein et al.. (Plasmid 3 (1980),
88-91). The orientation of the HindIII fragment 1 in the HindlIi site
of pGV1122 is determined by SaII 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 electro-
phoresis. In the a-orientation there - are fragments of 0. 77 kb and
22.76 kb, whereas in the p-orientation there are fragments of
10.33 kb and 13 . 20 kb. A recombinant plasmid with the a-orientation
is used in subsequent cloning and called pGV1168.
A Bc lII-Sali fragment containing the left part of the TL-DNA (includ-
ing the lvit border sequence) is introduced in pGV1168, cut with
BgIII-Sa1I. This fragment is obtained from the recombinant _plasmid
pGV0153 (D e Vos et al., Plasrr:id. 6 (1981), 249-253), containing BamHI
fraginn-cnt E, from the T-region of pTiB6S3, inserted in the vector
pBR32_. n1 7V 01_53 and pGVllhB DNA is prepared according to Betlach
et al .(= ed. Proc. 35 (1976), 2037-2043). Ten pg of pGV0153 DNA are
completely digested with 10 units of Bc lII and 10 units of SalI for
1 hour at 37 C in final volume of 100 p1. The digestion mixture is
loaded on a preparative 0.8% agarose gel. The 2.14 kb B1gII-SalI
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 B9III 'and 2. units
SaII. One tenth pg of BgIII-Sa1I fragment DNA is ligated to 0.02 pg
of BÃ 1;II-Sall-digested pGV1168 with 0.02 units of T4 DNA ligase in a
final volume of 20 {al. The ligation mixture is transformed into compe-
tent E. coli K514 . hsr hsmt cells (Dagert and Erhlich, Gene 6(1980)
~31 41524
-33-
23-28) . Cells are plated on LB medium (Miller, Experiments in Molec-
ular Genetics (1972), Cold Spring Harbor Laboratory, New York),
supplemented with streptomycin (20 Ng/ml) and spectinomycin
(50 Ng/ml).
Microscale DNA preparations (Klein et al., Plasmid 3 (1980), 88-91)
are performed from streptomycin- and spectinomycin-resistant trans-
formants. Recombinant plasmids in which 2.14-kb-Bc lII-SaII fragment
is inserted in BgIII-Sall-digested pGV1168 are identified by Bc lII-Sall
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
pGV11711. 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.) (19178), 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 prepar-
ed according to Betlach et al. , Fed. Proc. 35 (1976), 2037-2043).
Two pg of each DNA are totally digested with 2 units of HindIII for
1 hour vt 37 C (incub tion buffer is described by O'Farrell et al. ,
lvIol..=-r . Genet. 179 (11980), 421-435). One tenth pg of HindIII-
digestv=_ -~~"ti'1~ 71 is ligaied to 0.05 pg of pBR325, linearized with
Hindlll , v+Ta.h 0.02 units T4 DNA ligase. Transformation of the ligation
mixture in competent E. coli K514 hsr hsm} is carried out as de-
scribed liby Dagert and Ehrlich, Gene 6 (1980), 23-28. Cells are plated
on LB r_<edium (':liller, Experiments in Molecular Genetics (1972), Cold
Spring Harbor Laboratory, New York), supplemented with carbenicillin
(100 pgi'ml ). Carbenicillin-resistant clones are screened for sensitiv-
ity to tetracycline (10 Ng/ml), due to insertional inactivation of the
gene coding for tetracycline resistance (Bolivar, Gene 4 (1978)
121-136). Carbeniciblin-resistant and tetracycline-sensitive clones are
physically characterized by restriction enzyme digestion of DNA pre-
pared from these clones by the microscale technique (Klein et al. ,
_34- 13 41524
Plasmid 3 (1980), 88-91). BamHI digestion gives 4 DNA fragments
in the -orientation fragments of 0.98 kb, 4.71 kb, 5.98 kb, and
7.02 kb are found whereas the p-orientation 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-Hpal
fragment coding for kanamycin resistance, for a 3.49-kb-Bc lII-Smal
fragment, encoding functions essential for oncogenicity, internal to
the TL-region inserted in pGV700. The BamHI-Hpal fragment encod-
ing kanamycin resistance is obtained from X: : Tn5 (Berg et aI., Proc.
Nati. Acad. Sci. USA 72 (1975), 3628-3632). Preparation of X: : Tn5 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 Bc lI and 2 units
SmaI. Two pg of A: : Tn5 DNA are totally digested with 2 units of
BamHi and 2 units Hnai. One pg of BamHI-HpaI digested X: : Tn5 is
ligated to 0.2 pg Bcll;-SmaI-digested pGV700 with 0.5 units of T4
DNA ligase in a final volume of 10 pl (conditions are as recommended
by the manufacterer). The ligation mixture is transformed in _ com-
pete~:~ ~. coli K514 lsr hsrrm+ cells (Dagert and Erhlich, Gene 6
(1980) 23=28) . Cells are plated on LB medium (Miller, Experiments
in Moipc::'=. _z_r Geneti cs (,1972 ), Cold Spring Harbor Laboratory, New
York), supplemented with carbenicillin (100 Ng/ml) and kanamycin
(25 pgim-'.,). CbR and KmR clones are physically characterized by
restricticrl enzyme analysis of DNA prepared according to the micro-
scale technique (Klein et al., Plasmid 3 (1980), 88-91). Bc lII/BamHI
double d-gests oi this DNA gives 3 fragments of 3.94 kb, 5.89 kb,
and 8.09 kb, whereas HindIll digests yields 3 fragments of 2.68 kb,
5.99 kb, and 9. 25 kb. A plasmid showing these digestion patterns is
ca~led pGV750 and is illustrated schematically in Figure 17.
13415 24
-35-
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; 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, fa,
6b) will cause shoot formation in transformed plants. pGV750 contains
even less of the T-region, i.e. only the octopine synthase gene. The
informati-on for- products 4, 6a, and. 6b has been substituted by the
antibiotic resistance marker gene encoding kanamycin (neomycin)
resistance.
pGV700 and pGV750 are examples of intermediate cloning vectors
which ca.n 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 Fia. 9 except that they do not contain a gene of
interes-L. A. gene of in tEerest can be easily inserted into these vectors
as thev contain single restriction endonuclease sites for cloning within
their mcuif?nd T-regions (see Figs. 14 and 15).
Example 3
~_:;nstruction oi acceptor Ti plasmid pGV2260 (type B)
Starting strains and plasmids :
Agrobacter?um tumeiaciens (r-ifampicin-resistant strain C58C1 and
erythrcr.-sycin-ch?oramph enicol-resistant strain C58C1, derived from
wild-type Agrobacter i 4im)
Ti plasmid = pGV2217
Intermediate vector (Fig. 16) = pGV745
-3s- 13 4 1 5 24
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. , Plasmid 6 (1981), 249-253) have been substituted by an EcoRl-
BamHI fragment of pKC7 (Rao & Rogers, Gene 7 (1979), 79-82) which
contains the apt (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
plasrnid pGV713 was derived from the * octopine Ti plasmid subclone
pGV0219 (De Vos et al., Plasmid 6 (1981), 249-253), containing
HindIII iragments 14, 18c, 22e and 38c in a-orientation. pGV0219 DNA
was digest-ed. to completion with BamHI and subsequently ligated under
conditions which favour self =ligation of the plasmid (final concentra-
tion of UNTh in ligation mixture < iF,g DNA/ml). Transformants were
selected for ampicillin resistance, and physically characterized by
restriction enzyme digestion. A clone, which no longer contains the
6.5 kb FsaaiHI fragment present in pGV0219, was isolated and called
pGV713. This clone pGV713 was used in subsequent cloning (see
below). The recombinant plasmid pGV738 'was derived from pGV0120
(De V::: al., P?asmuid 6(1981), 249-253), containing BamHI frag-
ment 2. puV0120 DNA was digested with EcoRI and self-ligated (as
above fc_- :~ie construction of pGV713). Transformants were selected
for amp_ci ~yn resi stan ce and analyzed by restriction enzyme digestion.
A clone :r~ which EcoRI fragments 20, 12, and a 2.95 kb EcoRI frag-
ment containing part of EcoR1 fragment 19a and part of pBR322 were
deleted, was used in further cloning and called pGV738. This plas-
mid stiil 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 HindIII and BamHI, and the
digest was applied onto a preparative agarose gel. After electro-
. 13 41524
- 3 7 -
phoresis the 2.30 kb HindIIl-BamHI fragment, contained within
pGV713, was purified by electroelution (as described by Allington et
al., Anal. Biochem. 85 (1975), 188-196). This fragment was ligated
to pGV738, digested to completion with HindIIl and BamHI. After
transformation, ampicillin-resistant clones were physically character-
ized by 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 Co1E1-specific bom site in the pBR322
portion and can be mobilized frorri E. coli to Agrobacterium by using
the helper plasmids R64drdll and pGJ28, as described in example 1
(construction of the acceptor Ti plasmid pGV3850).
pGV745 was mobilized to Agrobacterium strain C58C1 which is rifam-
picin-resistant and conta:ns the Ti plasmid pGV2217. The first cross-
over event was selected by using the ampicillin resistance of pBR322
in the same ,wav Gs described in example 1 (the construction of the
acceptor Ti plasm? d pG V3850) . By a second cross-over event the
deletion sub.stitution mutation present in pGV2217 is replaced by the
pBR32_2 sequences of the plasmid pGV745. Second recombinants were
picked u-~ 1-iv d3rectly screening the ampicillin-resistant transconju-
gants,~.1 hich resulted from cointegration of pGV745 with pGV2217, for
the loss of kanamycin resistance. In this way, a rifampicin Agrobac-
terium srr; an C58C1, containing pGV2260 (ampicillin-resistant, kana-
mycin-sensitivz), 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
~3 4~524
-38-
transfer of the intermediate cloning vector from E. coli to Agrobac-
terium as well as for its cointegration in the acceptor Ti plasmid
pGV2260.
For example, we have been able to show that Agrobacterium carrying
a cointegrate between pGV2260 and pGV700 is capable of transferring
the expected DNA 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 man-
ner we 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
Cor_struction of an intermediate cloning vector containing a
gene to be expressed in plants
Until the 1~resent invention, insertion of whole genes into more or less
random positions withir. 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 gene(s) of interestcan be linked to transcriptional
initiation and termination signals which are known to be functional in
the plani 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
39 - 1341524
-
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 20.
Briefly, the restriction fragment HindIII-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 (JL'ig. 18). Ten pg of pGV0422 (a pBR322 derivative carrying
the HinuIII-23 fragment which contains the complete nos gene;
Depicker et al., Plasmid (1980), 193-211) are digested with Sau3A and
the 350 bp fragraent carrying the nos promoter is isolated from a
preparative 5% pclvacr ylamide gel. The promoter fragment is ligated
to Bgl?I-cut pKC7 (Rao et al., Gene 7 (1979), 79-82) previously
treated with bacterial alkaline phosphatase (BAP) to remove 5'-termi-
nal phosp Inate groups. Twenty pg of the resulting plasmid (pLGV13)
are di ge: :e d wit:.~ BglII an d treated with 7 units of the Ba131 exonu-
clease (3-:ciabs, New England) for 4 - 10 minutes in 400 pl of 12 mNI
MgCl21 12 CaCi21 0.6 M NaCl1 1 mM EDTA, and 20 mM Tris-HCI,
pH 8.0, at 30 C. During this time approximately 20 - 50 bp of DNA
are remfl-vid. Tne 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. P lasmids 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-Sacll fragment of several candi-
dates ar e estimated in a 6% urea-polyacrylamide gel, and the nucleo-
tide sequences of the candidates with sizes ranging between 200 - 280
- 4 0 - 113 4 1 5 24
nucleotides are determined. The clone pLGV81 containing the SacII-
BamHI fragment of 203 bp carrying the promoter is used to substitute
the Sacll-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 ocs which is contained on a
BamH1 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 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 pg of the resulting
plasmid, pAGV828, is digested with BamHI, treated with the exonu-
clease Bal31 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 nucleo-
tide sequences of several candidates are determined, and a candidate
haviny only 7 bp rem--ining of the 5'-untranslated leader sequence is
chosen ~or further work (pOCS ) . In order to bracket the ocs
sequenca with Bam?-iI sites, the ClaI-Rsal fragment is filled-in and
subclo~; zl :s_to tl?e Bai? site of pLC236 (Remaut et al., Gene 15 (1981),
81-93; . _'_e resulting piasmid pAGV40 is digested with BamHI, the
fragment -_arrying the ocs sequence isolated by electroelution from a
preparative 1% agarose gel, and ligated to pLGV2381 previously cut
with Bam and treated with BAP (bacterial alkaline phosphatase).
The insertion of the ocs sequence in pLGV2381 is obtained in both
orientaticns (pi\;O-i and pNO-2).
The nuc:eotide sequences showing the exact junction point in the
nos : ocs fusion are shown in Fig. 20.
1341~4
- 41 2
-
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 di-
hydrofolate reductase gene is contained on a BamHI fragment as de-
scribed (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 describ-
ed, 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 Agrobacterium 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 tvinfect plants. The 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 pro-
moter described above, and their transfer and expression in trans-
far:ned plant cells following cointegration with the Ti plasmid of Agro-
bacterium provides evidence that foreign genes can be transferred
and expressed in plant cells according to the processes of this inven-
tion.
Example 5
2sc?ation of plant cells and plants containing the desired
gene(s) inserted in their chromosomes
We have ebtained plant cells and whole plants transformed with non-
oncogeni acceptor Ti plasmid derivatives (e.g. pGV3850) using any
of the following three methods
1~41524
-42-
(1) inoculation in vivo of whole plants followed by subsequent cul-
ture 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 Agrobacterium
strains which results in the production of crown gall tissues. Since
pGV3850 is a non tutnor=producing (nononcogenic) Agrobacterium
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 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 oi nopaline.
We have cbtained pGV3$50-transformed calli and shoots derived from
decapi=: -iEed tobacco plantilets of Nicotiana tabacum Wisconsin 38 using -
the follc..irig protocol (all manipulations are done under sterile condi-
tions in a las~m-nar 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, Physiol. Plant. -15 (1962) 473-497)
conLai.ning 0.8% agar.
(2) Remove youngest top leaves with a scalpel and discard.
(3) Inoculate wound surface with a spatula or toothpick containing
Agrobacterium derived from a fresh plate culture grown under
selective conditions (e.g. for the rifampicin-resistant, ampicillin
43 41~24
= resistant Agrobacterium strain containing Ti plasmid pGV3850,
YEB medium containing 100 pg/ml rifampicin and 100 Ng/ml car-
benicillin are used; YEB medium : 5 g/l Bacto beef extract,
1 g/l Bacto yeast extract, 5 g/l peptone, 5 g/l sucrose, 2 x
3 M MgSO4, pH 7.2, and 15 g/l 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, Physiol. Plant. 18
(1965) 100-127) with auxins and cytokinins (1 mg/l NAA,
0.2 mg/i 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 one in four
plants produce a nopaline-positive wound callus.
(7) Tr-ansfer nopaline-positive calli to agar plates containing regenera-
tion medium : LS medium as above + 1% sucrose and 1 mg/1 BAP
cy tokinin .
(8) Good-sized shoots (1 cm high) appear after about 4-6 weeks.
Tra7i-fer the shoots to fresh agar plates containing LS medium
+14s'ucrose x,ritlhout hormones to allow further growth and root
fcrTa;.ion.
(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 nopa-
line.
(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 for infected tissues contain 500 Ng/ml of
the antibiotic cefotaxime (Claforan , Hoechst) as a selection against
Agrobac terium containing pGV3850. This drug works well to prevent
growth of all Agrobacteria (including those which are carbenicillin-
resistant).
13 41524
-44-
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 Agrobacterium
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 32 (1983) 1057-1067).
Often the induced shoots are composed of completely normal untrans-
formed cells. Thus, we tried to inoculate plants with a mixture of
two different Agrobacteria, 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 wh?ch have been transformed with pGV3850. We have inoculat-
ed 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 nopa-
line-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 b:. transferred to soil for propagation. This coinfection pro-
ceaure snculd be pa. ticularly useful for transforming plant species
which ar:_:~ not readil-y amenable to tissue culture. Thus, a whole
range ci agrcnomically and economically important plants, such as
legumes, meciici r.al plants, and ornamentals will be able to be engi-
neered b1 Agrohacterium.
The third procedure allows the isolation of Nicotiana tabacum proto-
plasts ar_d the sc.ection of hormone-independent T-DNA-transformed
cell clones after co-cultivation of the protoplast-derived cells with
oncogenic Agrobacterium strains. An analogous technique can be
used for the selection of transformed cells when other dominant select-
ive markers are used, such as antibiotic resistance genes constructed
in such a way as to be expressed in higher plant cells (see example 3).
1341524
- 4 5 -
In this case, however, the conditions for selection have to be optimized
in each case (concentration of the selective agent, time between
transformation 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, Physiol. 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 Leemans et al., J. Mol.
Appl. Genet. 1 (1981), 149-164) about 660 colonies can be found,
obtained after infection with pGV2217, a Nicotiana tabacum SRl cell
line which synthesizes the TR-encoded opine mannopine (N2-(1-man-
nityl)-glutan, ine). lNunerous shoots are formed after incubation of
callus pieLes of this ce?1 line on regeneration medium (Murashige and
Skoog mcuium with BA P(6-benzylaminopurine) (1 mg/litre) as the
sole plant errowth regulator). All 20 shoots analyzed are still able to
synthesizG mannopine. After transfer onto hormone-free Murashige
and SkUog medium, the shoots grow as morphologically normal tobacco
plants S7_-i~l containirg marrapine.
The -oretonlast iso>ation and transformation described here for N.
tabacuc: can also 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 fluor-
escent light ("ACEC LF 58 W/2 4300 K Economy"), 24 C, 70%
13 4 1 5 2 4
-46-
relative humidity). Five week old shoot cultures are used for
protoplast isolation.
2.2. Protoplast isolation
Aseptic techniques are used for 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 o cellulase Onozuka R-10 and
0.2% macerozyme Onozuka R-10 in hormone-free K3 medium (Nagy
and 114aliga, Z. Pflanzenphysiol. 78 (1976), 453-455). The mix-
ture is filter-sterilized through a 0.22 pm pore membrane, and
can be stored for at least 6 months at -20 C without notable loss
of activity.
2.3. Protopiast culture
After 18 hours incubation the mixture is agitated gently in order
to reiease the protoplasts. The mixture is subsequently filtered
through a 50 um sieve, and the filtrate is transferred to 10 ml
cen:rifuge tubes. After centrifugation for 6 minutes at 60 - 80 g
in a swinging bucket rotor the protoplasts form a dark green
fioa tiny band. The liquid underlying the protoplasts, and the
debris which forms the pellet, are removed using a capillary
tuLe connected to a-peristaltic pump. The protoplasts are
pooted in one tube and washed 2 times with culture medium.
The culture medium is the K3 medium (Nagy and Maliga, Z.
Pflanzenphysiol. 78 (1976), 453-455) with NAA (0.1 mg/litre) and
ki ietin (0.2 mg/litre) as growth regulators. The medium is
adjusted to pH 5.6 and sterilized through 0.22 pm filter mem-
brane. 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
~"r r' GuE ~ rY~a r l'L
1341524
-47-
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. N1 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 centri-
fuged
in a swinging bucket rotor at 60 - 80 g for 6 minutes.
The floating band and pellet are pooled and resuspended in
ml of K3 medium (Nagy and Maliga, Z. Pflanzenphysiol. 78
(1976), 453-455) supplemented with an antibiotic' (carbenicillin
1000 pg/ml or cefotaxirrie 500 Ng/ml).
After tivo weeks of incubation, the protoplast-derived micro-calli
are centrifuged and resuspended in K3 medium (Nagy and Maliga,
Z. P_1_anzenphysiol. 78 (1976), 453-455) with the same growth
reguiator and antibiotic concentrations as before, but 0.3 M
sucrose instead of 0.4 M. The 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 concentra-
tions as before, but with reduced sucrose (0.2 M) and growth
regulators (~ AA 0.01 ma/litre and kinetin 0.02 mg/litre). After
two or three more weeksof incubation, the putative transfor-
mant's can be recognized by their light green and compact as-
pect, and better growth. These colonies are then transferred to
hormone-free Linsmaier and Skoog medium (Linsmaier and Skoog,
48 _ 1341-524
Physiol. Plant. 18 (1965), 100-127) solified with 0.61-o agar and
containing reduced antibiotic concentrations (carbenicillin
500 Ng/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 thedetection of octo-
pine and nopaline (Aerts et al., Plant Sci. Lett. . 17 (1979),
43-50) or agropine and mannopine (Leemans et al., T. 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 w-ithout selection on hormone-free media
When selection for transformed cells is not possible (e.g. be-
cause 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
centr;=uged (6 minutes, 60 - 80 g), and resuspended in medium
AG (Caboche, Planta 149 (1980), 7-18) which is able to support
cell g:rowth at very low density. The cells are counted using a
Fuc :s-Rosenthai counting chamber (obtained from "Assistant",
F. R. G_ ), and resuspended at the density required for subse-
quent 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 colonies after one
month of incubction. 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 opti-
mized for each type of selection.
= _ 13 4 1 5 24
-49-
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 on Murashige
and Skoog medium containing 1 mg/ml BAP; this medium induces
shoot formation after 1 - 2 months. These shoots can be trans-
ferred 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 ethanol/H20 for 2 minutes ;
followed by 10% cemmercial bieach and 0.1% sodium docecyl sul-
fate (SDS); further rinsed 5 time with sterile H20.
The sterile seeds are sown in large (25 mm wide) test tubes
conta?ning 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:-1ours dark; 70% relative humidity; 24 C). After 4 - 6 weeks
the plants are ready to use. They remain optimal for at least
ancth; r month.
PlanLiets should be at least 3 cm high and have four or more
?na L es . The plants are then decapitated transversally through
the LL-cungest iniernode with a new sterile scalpel blade; the
up-per part of the plant removed from the tube, and bacteria
frcm an agar plate culture applied on the wound surface with a
flamed _microspatul_a.
Tumol-s 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
plumbaginifolia and Petunia hybrida.
-50- 13 4 1 ~ 24
Concluding remarks
The present invention offers for the first time the possibility to
transform plants with Agrobacterium 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) gene(s) of interest to plant cells
occurs. There is cointegration and stable maintenance of this trans-
ferred DNA in the plant cell genome. Furthermore, expression of
chosen gene(s) of interest can be achieved provided the gene(s)
either contain - or are constructed to contain - suitable promoter
sequences. The concept of effecting a single cross-over event be-
tween an intermediate cloning vector containing the chosen gene(s) of
interest with an especially designed acceptor Ti plasmid greatly sim-
plifies the construction of any hybrid Ti plasmid vector useful for the
transformatton of plant cells. The especially designed acceptor
Ti plas, ia,s contain the DNA segment of a conventional cloning vehicle
such that any gene(s) of interest (which has been inserted into the
same or ca 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 vehicle(s) prrovide the necessary regions
of homc?cc-, for recorr:.0i,nation.
Microorcan?sms anu intermediate cloning vectors, acceptor Ti plasmids
and hybrid piasmaid vectors prepared by the processes of this inven-
tion are exernplified by cultures deposited in the German Collection of
Microorganisms (DSM), Gottingen, on December 21st, 1983, and
identified there as :
(1) intermediate vector plasmid pAcgB in Escherichia coli K12 HB101;
(2) Agrobacterium tumefaciens C58C1 rifampicin-resistant strain
carrying carbenicil_lin-resistant acceptor Ti plasmid pGV3850;
(3) intermediate vector plasmid pGV700 in Escherichia coli K12 strain
K514 (thr leu thi lac hsdR);
~ 1341524
- 51 -
(4) intermediate vector plasmid pGV750 in Escherichia coli K12 strain
K514 (as above in (3));
(5) Agrobacterium tumefaciens C58C1 rifampicin-resistant strain
carrying carbenicillin-resistant acceptor Ti plasmid pGV2260;
(6) intermediate vector plasmid pNO-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 Agrobac-
terium = GJ23 carrying mobilizing plasmids pGJ28 and R64drdll
(Van Haute et al., EMBO J. 2 (1983), 411-418); GJ23 is Esche-
richia coli K12, JC2926, a recA derivative of AB1157 (Howard-
Flanders et al., Genetics 49 (1964), 237-246).
These cu? tlures were assigned accession numbers
..279.8.. (2), . . 2.7.9G . . (3), .2797... (4), (5),
2$33... (6), and ..2193... (7), respectively.
While we have hereinbLfore presented a number of embodiments of this
invention, it is apparent that our basic constructions can be altered
to prcvAUe other embo::?ments which utilize the processes and composi-
tions of th ls invention.
