Note: Descriptions are shown in the official language in which they were submitted.
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Method of genetically modifying plants
The present invention relates to a novel method of inserting viral
DNA into plant material and to the products obtainable by the said
method.
It has already proved possible in many cases to insert selected DNA
fragments into viral DNA and then, together with the virus, to
introduce them into another organism. Although most plant viruses
are transmitted under natural conditions by insects that feed on
infected and uninfected plants, thereby causing fresh infection of
plants, this route is too inconvenient and difficult to control to
achieve a selective and systematic transmission of viruses. Thus,
for example, specially bred insect populations would be required for
such a method under contained conditions. In addition, it would be
very difficult to achieve a controlled virus infection, especially
of large amounts of plant material. The mechanical inoculation of
leaves with viruses, the method employed in genetic engineering, is
of only limited applicability, as cloned viral DNA will infect only
some plants but many others not yet. Although it is possible to
clone and study in bacteria a variety of types of viral genomea, for
example single strand DNA viruses which are obtained by cloning
double-stranded DNA forms (1~ Mullineaux, P.M. et al., 1984), many
viruses that are cloned in bacteria cannot be reintroduced into
plants or used for infecting plants. The use of methods such as in
vitro mutageneais and recombinant DNA technology are therefore ruled
out in basic studies as well as for exploiting such viruses as
carriers of selected foreign DNA. Such problems do not arise when
using the method of this invention as set forth hereinbelow.
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Within the scope of the present invention, the following definitions
apply:
transfer micro-organism: a micro-organism that is able to transfer a
part of its DNA into plant material
(e. g. Agrobacterium tumefaciens)
T-replicon: a replicon (2) Jacob, F. et al., 1963) that
can be transported in its entirety or in
part into plant cells by means of genes
that are localised on this replicon itself
or on another replicon present in the same
micro-organism (example: the Ti-plasmid of
Agrobacterium tumefaciens).
T-DNA border sequences: DNA sequences that, in one or more copies,
effect DNA transfer to plants by means of
microbial functions
cargo DNA: DNA inserted artificially into a DNA vector
plant cell cultures: cultures of plant units such as proto-
plasts, cell cultures, cells in plant
tissues, pollen, pollen tubes, egg-cells,
embryo-sacs, zygotes and embryos in
different stages of development.
completely trans-
formed plants: plants in which the genome of each cell is
transformed in the desired manner.
The present invention relates to a novel method of introducing viral
DNA into plant material, in particular into whole plants or plant
cell culture cells, which method is of general applicability. The method
comprises essentially:
a ~4a z9o
J
a) inserting viral. DNA, for example DNA of cauliflower
mosaic virus (CaMV), which viral DNA comprises more than one
viral genome or parts thereof which are still capable of
initiating a systemic infection in a host plant and which may
contain cargo DNA, into a T-replicon, for examp:Le a Ti-plasmid
or Ri-plasmid of an Agrobacterium, in the vicinity of one or
more T-DNA border sequences, the distance between said viral
DNA and the T-DNA sequence or sequences being chosen such that
viral DNA, including any cargo DNA present, is transferred to
plant material,
b) introducing the replir.on into a transfer micro-organism,
and
c) infecting plant material with the transfer micro-organism
that has been modified in accordance with b).
This method ensures that, after induction of the
microbial functions that promote the transfer of the plasmid
DNA to plants, the iiiserted DNA is also transferred, including
any cargo DNA that may be present. The transformed plant
material so obtained can be regenerated to completely
transformed plants.
Accordingly, the method of the present invention
essentially comprises the following partial steps:
a) isolating viral DNA or its equivalents (as described
hereinafter) of infected plants, for example of the Brassica
variety, and cloning said DNA in vectors of .a suitable
bacterium such as Escherichia coli;
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b) constructing a plasmid f=BaP) containing more than une
viral genome or parts thereof that are in the vicinity of one
or more T-DNA border sequences, the distanr_e between the viral
DNA and the T-DNA border sequence or sequences being chosen
such that said viral DNA, including any cargo DNA inserted
thereinto, is transferzved to plant material,
c} constructing a vector system by transferring the plasmid
BaP to a transfer micro-organism (for example Agrobacterium
turuefaciens or Agrubacterium rhizogenes}; and
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d) infecting plant material with the vector system constructed in
accordance with c).
The use of vector systems as described in c), as well as novel
vector systems such as bacteria of the strain Agrobacterium tume-
faciens A136 (pTiBo542, pEAPI8::Ca305) and Agrobacterium tume-
faciens A136 (pTiBo542, pEAl) constitute further objects of the
present invention.
A particularly suitable T-replicon is a bacterial replicon such as a
replicon of Agrobacterium, preferably of a Ti-plasmid or Ri-plasmid
of an Agrobacterium.
Micro-organisms that contain a T-replicon will be understood as
meaning in particular bacteria, preferably soil bacteria and, of
these, first and foremost those of the genus Agrobacterium.
By viral DNA and its equivalents are preferably meant the following
DNA types:
- natural viral DNA (e. g. CaMV);
- double-stranded DNA forms of single strand DNA viruses
(e. g, gemini viruses);
- cDNA copies of viral RNA or viroid RNA (e. g. of tobacco mosaic
virus or cadang-cadang viroid);
- all lethal or viable mutants of viruses;
- cloned DNA under the influence of viral replication and/or
expression signals;
- parts of viral DNA;
- equivalents of the DNA types specified above in tandem farm; and
- equivalents of the DNA types specified above with inserted
cargo DNA.
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Suitable plant material comprises both whole plants as well as parts
of plants. Parts of plants are for example also protoplasts, cell
culture cells, cells in plant tissue, pollen, pollen tubes, egg-
cells, embryo-sacs or zygotes in different stages of development.
Plants and cell culture cells are preferred.
The method of this invention is suitable for infecting all plants
with virus. Of the systematic units Gymnospermae and Angiospermae
(including ornamentals), the latter are preferred.
Among the Angiospermae, plants of particular interest are, in
addition to deciduous trees and shrubs, plants of the following
families: Solanaceae, Cruciferae, Malvaceae, Compositae, Liliaceae,
Vitaceae, Chenopodiaceae, Rutaceae, Cucurbitaceae, Bromeliaceae,
Rubiaceae, Theaceae, Musaceae or Gramineae and of the order Legumi-
nosae, in particular of the family Papilianaceae. Preferred plants
are representatives of the Solanaceae, Cruciferae and Gramineae
families.
The high yield cultivated plants such as maize, rice, wheat, barley,
rye, oats or millet are to be singled out for special mention.
Target crops are for example those of plants of the genera Solanum,
Nicotiana, Gossypium (cotton), Brassica (rape), Beta, Pisum,
Phaseolus, Glycine, Helianthus, Allium, Triticum (wheat), Hordeum
(barley), Avena (oats), Setaria, Sorghum (millet), Oryza (rice), Zea
(maize), Cydonia, Pyrus, Malus, Rubus, Fragaria, Prunus, Arachis,
Secale, Panicum, Saccharum, Coffea, Camellia, Musa, Ananas, Yitis,
Citrus and Persea (avocado).
The method of infection preferably comprises combining the above
described transfer micro-organism with protoplasts or by wounding
whole plants or pieces of tissue and subsequently infecting them.
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The use of the method of this invention employing the above
described vector system affords numerous advantages compared with
the methods employed hitherto, viz.:
- inducing infectivity in viruses which it has so far not been
possible to make infectious by artificial means (for example
maize streak virus), by-passing natural vectors such as insects;
- the possibility of manipulating viral DNA in a bacterial system
such as E. cola;
- increasing the host range of viruses;
- simplifying inoculation by avoiding DNA purification and very
substantially reducing the amount of inoculum required for
inoculation;
- under control of bacterially coded functions, the T-DNA, including
the selected viral DNA, can become integrated into the host
genome. As regeneration of whole plants from single plant cells
after transformation with bacteria is possible, vfral DNA can be
introduced into the nuclear genome of every cell in a plant. Such
integrated virus genomes
- can then be transmitted sexually to offspring;
- can prevent infection by other viruses;
- can be a possible source of further copies of virus containing
selected cargo DNA and which escape from the integrated copy via
transcription, reverse transcription, homologous recombination
or other methods of modifying genetic material.
- In addition, superinfection of plants containing parts of viral
genomes integrated into the nuclear DNA may
a) permit the development of better viral vectors, as the
expression of viral genes from nuclear DNA could make it
possible to replace viral DNA with foreign DNA in the super-
infecting virus; and
b) contribute to a better understanding of host-parasite rela-
tionships and thus to substantially better protection of
plants.
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The method of this invention can also be utilised in
plant protection to "immunise" plants against virus attack by
means of a transfer micro-organism as described above by
transforming plants with a weakened non-pathogenic or only
slightly pathogenic virus.
By means of the method of this invention, cargo DNA
inserted into the virus genome can also be transported into plant
material in which it proliferates. The proliferation in plants of
the virus, and thus also of the foreign gene transported by it, is
especially advantageous whenever it is desired to propagate plants
asexually or to protect them direct and in the shortest possible
time against harmful influences (for example by inserting a gene
into the plants to impart resistance).
The method of this invention is in particular admirably
suitable for insinuating selected genes into plants, for example
adult plants, in which they then proliferate.
DNA transfer to plants can be effected by one of the
known systems, for example by the binary vector system described
by An, G. et al., 1985. This vector system can be improved by
inserting e.g. sequences for a homologous recombination of the DNA
to be transferred to the plant.
In Figure 1, which illustrates an embodiment of the
invention, is shown a map of a bacterial vectar pCa305 including
1.3 genomes of cauliflower mosaic virus (CaMV).
The following Example, in which CaMV is used as virus,
E. Coli as cloning bacterium and Agrobacterium tumefaciens as
vehicle bacterium, illustrates in more detail the construction and
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use of a suitable vector system. This Example can also be
performed in similar manner with Agrobacterium rhizogenes.
Preparation of the bacterial vector pCa305 containina 1.3-CaMV
aenomes (Fist. 1 y
a) The small area of the plasmid pHC79 (4)Hohn, B. et al., 1980)
between the EcoRI and the ClaI restriction sites is replaced with
the EcoRI-ClaI fragment originating from the plasmid pMON 30 (a
precursor of pMON 120; 5)Fraley, R.T. et al., 1983) and encoding
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spectinomycin/streptomycin resistance. The 2.5 kb fragment of the
resultant plasmid between the SalI and BstEII restriction sites is
replaced with the 3.3 kb fragment which has been excised from CaMV S
at the SaII to DetEII restriction sites (6)Hohn, B. et al., 1982), A
complete CaMV CM-41$4 genome is then inserted into the remaining
SalI restriction site of the plasmid pCa292 so obtained (~)Fiowarth,
A.J. et al., 1981) to give the plasmid pCa3U5 that contains
1.3 genomea of CaMV in tandem arrangement (P'ig. 1).
b) To establish that the transfer of the infectious cloned viral DNA
to the recipient plant is not caused by lyais of tine Agrobacterium
cells, the plasmid pGV1106 (8)Leemans, J. et al., 1982), which has a
broad host range, ie cut with the enzyme EcoRI and introduced into
the single SphI restriction site of pCa305. The resultant plaemid pEl~1 is
inserted into A. tumefaciens, where it replicates independently.
_Key to Fig. 1
R
Ap ~ ampicillin resistance
R R
Sp /Sm : epectinomcyin/etrepetomycin resistance
o n : origin of replication
bom : origin of mobilisation
BstEII, SphI, SalI: restriction sites
I-VII : open reading frame of cauliflower mosaic virus
kb : kilobases
Introduction of plaemida pCa305 and pLAl into Agrobacterium tume_
faciens
Plaemids pCa305 and pEAI are each transformed in bacteria of the
strain Eacherichia cola GJ23 (pGJ28; R64drd11) (9)van liaute,
E. et al., 1983). This E. coli strain permitB the conjugal transfer
of plasmida which have a bom restriction site to A, tumefaciena.
Recipients are two different strains of Agrobacterium, both of which
originate from A. tumefaciena A136 and contain the wild type
Ti-plasroid pTiDo542 (10)Hood, E.E. et al., 1984).
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a) The strain A. tumefaciens A136 (pTiBo542, pEAPl$) is used as
recipient for the plasmid pCa305. pEAPl8 is a binary vector which is
constructed by replacing the 6.7 kb EcoRI-BamHI fragment of the
plasmid pGA472 (3)An, G. et al., 1985) with the 2.7 kb EcoRI-BglII
fragment of the plasmid pHC79 (4)Hohn, B. et al., 1980), which
contains between T-DNA border sequences a region for homologous
recombination with the plasmid pCa305. As plasmid pCa305 is unable
to replicate in A. tumefaciens, selection of the exconjuganta on
rifampicin, spectinomycin and streptomycin affords the new strain
A. tumefaciena A136 (pTiBo542, pEAPlB::pCa305) in which the plas-
mid pCa305 has been integrated into the binary vector pEAPI8 by
homologous recombination (11)Leemans, J. et al., 1981).
b) The strain A. tumefaciens A136 (pTiBo542) is used as recipient
for the plasmid pEAl. The introduction of the plasmid pEAl, which
replicates independently in A. tumefaciens, results in the formation
of the new strain A. tumefaciens A136 (pTiBo542, pEAl). Selection of
the exconjugants is made on rifampicin (selective for A. tumefac-
iens), kanamycin, spectinomycin and streptomycin.
The plasmida of the strains obtained as described in a) and b) above
are tested by DNA isolation and restriction mapping.
Transfer of the Agrobacterium strains containing the plasmida pCa305
or pEAl to plants of the species Brassica raga
Agrobacteria of the strains A. tumefaciens Al3b (pTiBo542,
pEAPl8::pCa305) or A. tumefaciens A136 (pTiBo542, pEAl) are grown
for 40 hours in a YEB medium (1 litre of water containing 5 g each
of bacto-beef extract, peptone and sucrose, 1 g of bacto-yeast
extract and 2mM of MgS04) which contains the antibiotics spectino-
mycin and/or streptomycin suitable for selection of the plasmids.
Subsequent enrichment of the bacteria is effected by centrifuging
and resuspending the culture in 1/100 volumes of YEB (based on the
amount of YEB previously employed). The leaves and petioles of
3-week-old Brassica raga c.v. Just Right plants are inoculated with
p~, and 20 p.,~ respectively of this suspension. Inoculation is made
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either by wounding two leaves of each plant with celite or by boring
a hole with a sterilised toothpick through a cluster of petioles
close to the surface of the soil. The above indicated amount of
Agrobacterium suspension is then inoculated into the injury site.
Results
Whereas inoculation with the control strain of A. tumefaciens A136
(pTiBo542, pEAl) does not produce viral infection, systemic infect-
ion with CaMV using the test strain A. tumefaciens A136 (pTiBo542,
pEAPl8::pCa305) is achieved, usually after a period of about 3 weeks.
As both strains contain 1.3 CaMV genomes in the same arrangement, it
may be deduced from the differing infectivity that the infection of
the plants with the test strain is a consequence of the virus
situated between the T-DNA border sequences escaping from the
plasmid, with the transfer of the virus to the plants being promoted
by bacterially coded functions, and that the infection of the plants
cannot be ascribed to lysis of the Agrobacterium cells, accompanied
by generation of DNA.
Literature
1. Mullinesux, P.M., EMBO J. 3, No. 13, 3063-3068, 1984
2. Jacob, F, et al., Cold Spring Harbor Symp. 28, 329, 1963
3. An, G. et al., EMBO J. 4, No. 2, 277-284, 1985
4. Hohn, B. et al., Gene 11, 291-298, 1980
5. Fraley, R.T. et al., Proc.Natl.Acad.Sci. USA 80. 4803-4807, 1983
6. Hohn, T. et al., Current Topics of Microbiology 96, (ed. Henle
et al.), 193-236, Springer-Verlag Berlin, 1982
7. Howarth, A.J. et al., Virology 112, 678-685, 1981
8. Leemans, J. et al., Gene 19, 361-364, 1982
9. Van Haute, E, et al., EMBO J. 2, No. 3, 411-417, 1983
10. Hood, E.E. et al., Bio/Technology, August 1984
11. Leemans, J. et al., Genet. 1, 149-164, 1981
