CA2089072A1

CA2089072A1 - Process for the gene manipulation of plant cells, recombinant plasmids, recombinant bacteria, plants

Info

Publication number: CA2089072A1
Application number: CA 2089072
Authority: CA
Inventors: Jean L. J. De Lafonteyne; Monika B. Sormann
Original assignee: Individual
Current assignee: Clovis Matton NV
Priority date: 1990-09-27
Filing date: 1991-09-26
Publication date: 1992-03-28
Also published as: NL9002116A; IE913359A1; EP0554273A1; JPH06501155A; WO1992006205A1

Abstract

A process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Agrobacterium tumefaciens with integration by T-DNA, which comprises introducing foreign DNA
into the plant cells by infecting the plant cells with one or more recombinant Agrobacterium tumefaciens strains which contain foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the plant cells, the foreign DNA located between the left- and right-hand ends of T-DNA comprising: (A) a transposase gene located in an expression cassette active in the plant cells but not beteween the left-and right- hand transposon ends necessary for integration in the DNA of the plant cells, and (B) a recombinant transposon comprising the left- and right- hand transposon ends necessary for integration in the DNA of the plant cells with an intermediate DNA
fragment to be integrated in the DNA of the plant cells, but containing no DNA fragment which can lead in the plant cells to expression of an active transposase.

Description

W092/0620~ PCI/EP91/018B3 ~ 2089072 Title: A process for the gene manipulation of plant cells, recombinant plasmids, recombinant bacteria, plants T~.is i~.ventio~. .elates to a process for the gene manipulatic of plant cells, which comprises introducing foreign DNA into plant cells by infecting the plant cells wlth one or more recombinant Aarobacter~um tum~aciens strains, which contain foreign DNA to be transferred to thé plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the plant cells.
The invention further relates to a recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA
insert, and to a recombinant Aarobact~rl~m tumefacie~s strain, which contains foreign DNA to be transferred to plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells.
Finally, the invention also relates to the plants deriving from the transformed cells and to the parts consisting of one or more cells or products thereof.
The Aarobacterium tumefaciens technology for the gene manipulation of plants, which technology has strongly developed and has been applied on a large scale since its first discovery, is in principle still limited, despite all progress made through the years, to the application in specific, substantially dicotyl plant species, such as tobacco, tomato, colza etc. Up to the present time a successful transformation of monocotyl plant species has essentially only been described for some Liliaceae, namely ~ Asparagus (Bytebier et al, Proc. Natl. Acad. Sci. USA 84, ; 5345-5349, 1987) and Narcissus ~Hooykaas et al, Nature ~11, ; -- 30 763-764, 1984), some Iridaceae (Graves et al, J. Bacteriol.
1~9, 1745-1746, 1987), some Dioscoreaceae (Schafer et al, ; Nature 3~7, 529-532, 1987), and some Gramineae, namely Zea mays (Graves and Goldman, Plant Mol. Biol. 7, ~3-50, 1986) and Oryza sativa (Raineri et al, Biotechnology 8, 33-38, 1990).
, ~, , ~' ,:

- ~ - - , ~ , ~ . .

, - . ' ' '. ,. : ' ' , ~ , , : , '. ' , . ' . , . ' ~ : .' W092/06205 PCT/EP9l/01883 ' ~ 2 ~ ~-Apart from these exceptions, one has not yet succeeded in realizing by means of the Aarobacterium ~umefaciens technology an efficient t-~nsform~ic.. of mv..ocotyl plant species, including the important cereals such as maize, wheat, barley, rye, oat, rice etc. and in producing stably transformed monocotyl plants. In a broader sense this applies to many species of plants, not only to the cereals belonging to the group of monocotyl plants but also to grasses such as ryegrass and fescue, leguminous plants such as leek, onion and garlic, ornamental plants such as tulipe, hyacinth, bromeliad, irls, orchid etc., and other plant species such as conifer etc.
There seem to be indications that the problem is not due to the transfer of the DNA. Thus it has been found possible to transmit viral genomes encoded in the form of DNA by means of Agrobacterium tumefaciens to maize (Grimsley et al, Biotechnology 6, 185-189, 1988) and wheat (Dale et al, Seventh International Wheat Genetics Symposium 1, 719-722, 1988).
Perhaps the problem is therefore due to the integration of the DNA in the genome: the integration system which Aarobacterium tumefaciens bacteria successfully use in dicotyl plants might not or not efficiently work in monocotyl plants. Generally, - however, it is assumed that a poor transfer of the T-DNA to the plant is responsible for the failure of the technology in monocotyl plants.
:
~i 25 In order yet to obtain the desired transformation in ; monocotyl plants, different alternative transformation techniques have been conceived and developed in which no use is made of Aarobacterium ~umefaciens bacteria for transmitting DNA to the plant cell. Examples thereof are microinjection with DNA, bombardment with DNA-carrying particles, electroporation of protoplasts with DNA etc.
These techniques have been successfully employed for the ~ introduction of DNA into plant cells, but generally only a - passing expression of the genes introduced could be observed.
As regards the use of integration systems, the selection made so far concerns either homologous genetic recombination, .~
'~:
., .
,, :-. .

W092/06~0~ PCT/EP91/01883 ,~ 2089~72 which is a hardly efficient system and remains limited inpractice to cells ln meiosis, or transposition, which is an efficiens s-ystem and is also active during the normal cell cycle but has the drawback that the genes introduced are not stable in the flrst generation and that it is necessary to wait one or more generations before the transposon and the genes introduced via transposition are separated by mendelizing out.
Transposons are, in fact, genetic elements which can - lO move within the genome of the host cells in which they are normally present. They have virtual ends, repeated border sequences which are in general invertedly repeated and may be rather different in length. At the location of their insertion into the genome a piece of host sequence is found back directly repeated to the left and the right of the transposon . sequence. There is generally no specific selection of the location of insertion. In case of excision either the original sequence is restored or a deletion occurs or part of the sequence remains at the relevant location (insertion). In its simplest form a transposon consists of a transposase gene located between terminal sequences which contain transposase binding sites and optional binding sites for other host enzymes and terminate with the repeated border sequence. These terminal sequences, together with the directly repeated sequences of the target DNA to the left- and right-hand of the border sequence, will also be referred to hereinbelow as left-and right-hand transposon ends.
The present invention provides a process which removes ., the main objections of the known methods and gives a very ` ' 30 efficient and stable transformation of cells of monocotyl .
, , plants, whlch has not been found possible so far. For this purpose use is made according to the invention of the ~ efficient transfer system of Aqrobacterium tumefaciens 5 ~ bacteria in combination with the efficient integration system ~ 35 of transposons.
~'' .
~, , .
t s, , .:: .

wo 9~/0620~ ~9 ~17~ PCT/EP91/01883 The invention relates in a first aspect to a process for the gene manipulation of cells of plants which, in essence, c~nnot be transformed by Aarobacterium tumeLaciens wi~h integration by T-DNA, with foreign DNA being introduced into the plant cells by infecting the plant cells with one or more recombinant Aarobacterium tumefaciens strains which contain foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the plant cells, the foreign DNA located between the left and right-hand ends of T-DNA comprising:
(A) a transposase gene located in an expression cassette active in the plant cells but not between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells, and (B) a recombinant transposon comprising the left- and right-- hand transposon ends necessary for integration in the DNA of the plant cells with an intermediate DNA fragment to be integrated in the DNA of the plant cells but containing no D~A
fragment which can lead in the plant cells to expression of an active transposase.
By the words "plants that, in essence, cannot be transformed by Aarobacterium tume~aciens with integration by T-DNA" are meant all those plant species in which ` 25 A~robacterium tumefaciens is not capable of effecting an efficient integration of the T-DNA in the plant genome, which ~ roughly means almost all the non-dicotyl plants. The invention is therefore eminently suited for the gene manipulatin of monocotyl plants and other non-dicotyl plants, preference being given according to the invention to gene manipulation of ~` plant cells of cereals such as maize, wheat, barley, rye, oat, rice etc., grasses such as ryegrass and fescue, leguminous j plants such as leek, onion and garlic, ornamental plants such '` ~ as tulipe, hyacinth etc. and other plant species such as ~ 35 conifer etc.

~ .:

., .. ., . .. ... , ~ . . .

W092/0620~ PCT/EP91/01883 ~ 208~72 According to the invention there is used a transposase gene which is not located between the left- and right-hand transposon ends necessary fur ir,.egra~ior, in the DNA of the plant cells. According to the invention there is further used - 5 a recombinant transposon containing no DNA fragment which can lead in the plant cells to expression of an active transposase. This means that the ecombinant transposon is an inactive transposon. Consequently, a desirable high stability of the transformed plants can be obtained. The drawback of instability.normally connected with the use of transposons is, in fact, the result of the presence of a complete (or active) transposon, i.e. a transposon with a transposase gene located between the transposon ends. This gene is then co-incorporated with the transposon in the plant genome and may therefore give rise for several generations to the undesirable instability.
According to the present invention there is used an active transposase gene, namely in order to ensure efficient integration of the foreign DNA located in a transposon in the DNA of the plant cell (in the plant cell the transposase gene leads to the formation of transposase which, in cooperation with nuclear factors of the plant, is capable of effecting efficient integration of the transposon in the genome) but if this transposase gene is not within a transposon, in conformity with the preferred embodiment, it is not itself efficiently integrated in ~he genome in case of transformation of non-dicotylidons, such as in particular monocotylidons.
This means that the desired stability is obtained already in ~, the first generation of plants.
The transposase gene preferably consists of a genomic clone of the transposase gene, i.e. of genomic DNA which comprises not only the encoding exons but also the in~rons normally present. Although the use of a cDNA coding for ~he transposase is also possible, practice has shown that it gives a substantially less efficient integration of the recombinant transposon. The use of a genomic transposase gene does not mean that the natural promoter of the transposase gene should !

", ' "' ' ' :' ' , ' . '"' ' ' '' . ' ' '- :" ' ' ' ' " ' ' " ' '' '' ' ' :: ' ; ".' .' .,-, . , ' .: ' . '' ~ :. - ., '. ' ' .. ,, '' ,:' ' : ' ,""'-'~-," '' ' '. ., '. ,''" ' '' '.' ,' . , '' " ' ,'' " "'. ' ' '.............. :
.,' .- ' '' , . ', : ' ' ' . ' , ,,'', ' ' ' . ,' .: ' ' . " "' . ' :' . ' ' . . '- '' ' : . . .' ' . ' ;
' . ' . ,. . '' ,' , . ,. ' ' . ' .

W092/06205 PCT/EPg1/01883 be used too. Practice has shown that it is very advantageous to use, instead of the natural promoter, another promoter active in pla..ts, in particular a promoter that can errec~
efficient e~pression in cells from which germinative cells develop. A promoter suitable for this purpose is the known CaMV 35S promoter. The use of the pTRl' or pTR2' promoter is also posible and favorable if, as is also preferably done according to the invention, transfer occurs by microinjection with the apical meristem being mechanically damaged. As a result, the wound-inducible regulators of gene expression for these promoters are present in the meristematic cells. The use of such a construct of a genomic transposase gene under control of another promoter such as the CaMV 35S, the pTRl' or the pTR2' promoter, therefore constitutes a highly preferred embodiment of the invention.
According to the invention it is particularly preferred that between the left- and right-hand transposon ends the recombinant transposon comprises at least one foreign gene located in an expression cassette active in the plant cells.
By this is meant any gene that is to be incorporated in the genome of the plant for whatever reason. It may be a gene coding for a desirable property of the plant, such as resistance to herbicides, to pathogens, such as specific funai and insects, or to environmental factors, or properties, such . 25 as (in ornamental plant cultivation) a specific flower color, flower scent, flower size etc., or properties such as (in case of food crop) better nutritional properties, a higher yield - etc. It may also be a gene coding for a substance one wishes to produce on a large scale by cultivating a plant capable of ~ 30 forming the substance by means of gene manipulation. The gene i to be introduced into the plant may therefore be a gene within the scope of plant breedlng or a gene within the scope of biological production of specific products ~proteins and - enzymatic products, DNA, RNA etc.).
It is also possible that between the left- and right-, hand transposon ends the recombinant transposon contains a '`.
-i , . , , . :

.

720~9~7~ `
promoter active in plants, instead of a foreign gene in an expression cassette active in plants. Consequently, it becomes possible to alter the expression behavior of specifi~ genes and thus the phenotype.
S It is further preferred that also one or more selectable marker genes suitable for selection of transformed bacteria and located in an expresslon cassette active in the bacteria are located between the left- and right-hand ends of T-DNA.
This enables selection of Aarobacterium tumefacien,s bacteria with the desired DNA constructs. Marker genes suitable for this purpose are known to those skilled in the art. These are often genes the expression of which leads to a resistance tO
specific antibiotics, such as resistance to kanamycin, chloramphenicol, ampiclllln, tetracycline, spectinomycin etc.
It is particularly preferred here that one or more selectable marker genes suit~ble for selection of transformed bacteria and located in an expression cassette active in : bacteria form part of the recombinant transposon. This preferred embodiment has the advantage that the marker genes coupled to the foreign gene are co-incorporated in the genome of the plant so that in cases that as a result of the incorporation of the recombinant transposon in a gene of the ,~ host the transformed plant shows another phenotype (which is not due to the introduced genetic information as such) or that the foreign gene introduced proves to function differently in later generations than in the first generations (e.g. as a result of mutation) the recombinant transposon can be cloned for further examination from the plant genome into bacteria again by means of selection with the marker gene active in bacteria.
It is further preferred that also one or more selectable marker genes suitable for selection of transformed plant cells ` and located in an expression cassette active in the plant cells are located between the left- and right-hand ends of T-DNA. Also here it applies that many marker genes suitable for this purpose are known to those skilled in the art. Thus ,, .

,, :

~: .

9~ 8 ~' the resistance to, e.g., antibiotics or herbicides can be mentioned as a property which, as ls known to those skilled in the art, car. be used fo- a selec~ion cf ~uccessfully transformed plants.
It is particularly preferred here that one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active ln the plant cells form part of the recombinant transposon. This preferred embodiment has the advantage that the marker genes are co-inco~porated in the genome of the plant so that also later generations can be selected on the basis of the properties encoded by the marker genes.
A firs~ variant of the process according to the invention is characterized in that the plant cells are infected with a mixture of two different recombinant ~.
tume~faclens strains, one of which comprises between the left-and right-hand ends of T-DNA the transposase gene located in an expression cassette active in the plant cells and the other the recombinant transposon between the left- and right-hand , 20 ends of T-DNA. This variant has the advantage of having a wide ; range of applications, i.e. there can always be used the same '' Ag~,,ok~terlum strain supplying the transposase gene and only the second strain containing the recombinant transposon must be constructed for each new gene. In this second strain there is used a construct in which a multiple cloning site is located between the transposon ends so as to have a great freedom of choice with respect to the DNA fragments to be ' ~ incorporated in the transposon.
,:;!' In practice, in this first variant according to the ''' 30 invention both the first and the second A. tumefa~,i~n~ strain will comprise between the left- and right-hand ends of T-DNA
, at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells. It is preferred here that at least the marker genes for selection of transformed plant cells which carry the , two bacterial strains within the T-DNA ends are different from ,~ , .

s , ~ . ~., . - :

: ...... . . : , . , . . : -~, - '. - : ' . . ' ' ,: . : :' ;:. ~ ' , :

~ , 9 20~9~72 each other so as to recognize plant cells successfully transformed by the two strains.
.~n ltern~tivre embodlmcnt of the process according tG
the invention is characterized in that the plant cells are ` 5 infected with one recombinant ~. tumefaciens strain which comprises between the left- and right-hand ends of T-DNA both the transposase gene located in an e~pression cassette active in the plant cells and the recombinant transposon. This variant has the advantage of more chance of success of the transformation, i.e. the desired DNA transfer and integration will have taken place in a larger part of the treated plant cells.
In practice, in this second variant according to the invention the ~. tumefaciens strain will comprise between the lS left- and right-hand ends of T-DNA at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells.
, ~ The process according to the invention is of special ~ importance to the transformation of plant cells of a monocotyl * 20 plant species such as maize, wheat, barley, rye, oat, rice etc. An efficient transformation of such crops has not been found possible so far.
The invention can also be used, however, for the purpose j of altering phenotypic characteristics of plants resulting 25 from the presence of inactive transposons in one or more genes ~ of the plant by removing such a transposon from the gene in - which it is contained. For this purpose the invention provides a process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Aarobacterium tumefaciens 30 with integration by T-DNA, which comprises introducing foreign DN~ into the plant cells by infecting the plant cells with a ~ recombinant Aarobacterium tumefaciens strain which contains r foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a 35 transfer and is capable of transferring this foreign DNA to the plant cells, which process is characterized in that plant ., !

.,:, i-~ ~, ' '' ' .

W092/06205 ~ PCT/EP9l/01883 cells are infected which contaln one or more inactive transposons but no active transposase gene and that the foreisn DNA located between the lef~.- and right~ and end~ ~f T-DNA comprises a transposase gene located in an expression cassette active in the plant cells, which cassette is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.
The present invention is not subject to special limitations regarding the method of contacting the plant cells to be transformed with the Aarobacterium tumefaciens bacteria.
In order to be able to obtain several generations of transformed plants, it is particularly preferred, however, that cells of the apical meristem in germinating seeds are infected with the bacteria.
In connection with practical advantages, in particular a high production per time unit and a high precision, it is preferred that the infection is carried out by means of needleless high-pressure injection. By this is meant a bombardment directed to cells of the apical meristem under high pressure of germinating seeds of the plants to be transformed, with a liquid containing the Aarobacte~i_m ~umefaciens bacteria. Instruments suitable for such a bombardment, such as a high-pressure '`pistol", are known per :
se.
The invention, however, is not limited to such a method and comprises the possibility of carrying out the infection by means of microinjection. Moreover, also other meristematic or embryogenic tissues or cells can be used for introducing a foreign gene.
In another aspect the invention relates to a recombinant ~`~ plasmid consisting of a bacterial vector plasmid and at least one DNA insert, which recombinant plasmid is characterized in that at least one DNA insert comprises a transposase gene located in an expression cassette active in plant cells, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-, . .. ~, - . : . , : . :- ., . , ., :
.
, . . . . .
:

W092/06205 PCT/EP9~/01883 ~ 2~89072 ;''''`'' ;' hand transposon ends necessary for integration in the DNA of plant cells.
The invention further l-elatcs .o c recombinant piasmid consisting of a bacterlal vector plasmid and at least one DNA
S insert, which recombinant plasmid is characterized in that at least one DNA insert comprises a recombinant transposon which contains the left- and right~hand transposon ends necessary for integration in the DNA of plant cells but no DNA fragment which can lead in plant cells to expression of an active transposase.
A preferred embodiment of such a recombinant plasmid is characterized in that between the left- and right-hand transposon ends the recombinant transposon comprises one or more restriction endonuclease recognition sequences suitable as cloning site. Such a plasmid has a wide range of applications as a carrier for genes that are to be introduced into the genome of plants.
A special preferred embodiment of such plasmids is characterized in that the recombinant transposon comprises ~; 20 between the left- and right-hand transposon ends at least one foreign gene located in an expression cassette active in plant cells.
In such recombinant plasmids it is further preferred that the recombinant transposon comprises between the lefl-2S and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed plant cells and , located in an expression cassette active in plant cells, and that the recombinant transposon comprises between the left-and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria.
In yet another aspect the invention provides a , recombinant A~robacterium tumefaciens strain which contains foreign DNA to be transferred to plant cells between the left-and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant , .
, ' ;' 7'~ `~
~: , !. . ~ ,,' '' : : . ' ' ' .
:', ' ~ , ~ , ' :

W092/06205 ~ PCT/EP91/01883 ~ 12 cells, which strain is characterized in that a transposase gene located in an expression cassette active in plant cells is l^cafed between. the left- and right-hand ends of T-DN.~, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.
As regards this aspect, the invention also provides a recombinant Aarobacterium tumefaciens strain which contains foreign DNA to be transferred to plant cells between the left-and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, which strain is characterized in that between the left-and right-hand ends of T-DNA a recombinant transposon is located, which comprises the left- and right-hand transposon - ends necessary for integration in the DNA of plant cells but no DNA fragment which can lead in plant cells to expression of an active transposase.
For such a recombinant Aarobacterium tumefaciens strain it applies again that the recombinant transposon may comprise between the left- and right-hand transposon ends at least one foreign gene located in an expxession cassette active in plant cells and/or one or more seiectable marker genes suitable for selection of transformed plant cells and located in an ; 2S expresslon cassette active in plant cells, and/or one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in ; bacteria.
A special embodiment of such a recombinant Aarobacteri~m tumefacien$ strain is characterized in that also a transposase , gene located in an expression cassette active in plant cells is located between the left- and right-hand ends of T-DNA, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the ; DNA of plant cells.

,, ~ :, -.-.. ~ -: . . .

W092/0620~ PCT/EP91/01883 13 2 ~8 ~ 0 72 The inventlon is finally also contained in plants and parts consisting of one or more cells or products thereof, which pl~nts derive from plant cells ~ransformed by ~he process according to the invention. By parts and products of plants is meant, e.g., the bulb, the tuber or the root, the flowers, the seed and the meal made therefrom etc.
The invention will be further explained with reference to the accompanying drawings, which diagrammetically show in Fig. 1 a T-DNA construct comprising both a foreign gene - 10 X included in an inactive transposon and a transposase gene not located in an active transposon, Fig. 2a a T-DNA construct comprising a foreign gene X
included in an inactive transposon, Fig. 2b a T-DNA construct which can be used in combination with that of Fig. 2a and comprises a transposase gene not located in an active transposon, Fig. 3a a T-DNA construct comprising a foreign gene X
included in an inactive transposon which comprises a marker gene interrupted by a second inactive transposon for selecting transformed plant cells, Fig. 3b a T-DNA construct which can be used in ` combination with that of Fig. 3a and comprises a transposase gene not located in an active transposon, Fig. 4a a T-DNA construct comprising a foreign gene X
included in an inactive transposon, which foreign gene X is ;~
interrupted by a second inactive transposon, Fig. 4b a T-DNA construct which can be used in combination with that of Fig. 4a and comprises a transposase gene not located in an active transposon, Fig. 5a the structure of the recombinant plasmid pMH10, pMH10-LTS and pMH10-DsB, respectively a plasmid having a complete transposon, a transposase gene with only the left transposon end and a transposon without transposase gene, Fig. 5b an example of a T-DNA construct with plasmid pMH10-LTS according to the invention, ~ ' "'' .: -., .
. , .

., : . :

W092/0620~ PCT/EP91/01883 i 14 Fig. 6a the structure of the recombinant plasmid pMHlODsl5GUS, ';
Fis. 6b an e~ample of a .-DNA corstruct -with this plasmid according to the invention, Fig. 7 the structure of the recombinant plasmid pMHlODs34CAT, Fig. 8 the structure of the recombinant plasmid pMHlODsl2IAA, Fig. 9 the structure of the recombinant plasmid pMXllDs4IPTkan, Fig. lO the structure of the recombinant plasmid pUCSSAc9Dsl9, suitable for cloning foreign genes between the transposon ends of Ac9, Fig. lla the structure of the recombinant plasmid pUCSS19tpnAc9P35S, Fig. llb the structure of the recombinant plasmid pUCSS19tpnAc9PTR1, Fig. llc a T-DNA construct with the recombinant plasmid pUCSS19tpnAc9P35S according to the invention.
Fig. 12a the structure of the recombinant plasmid ~ pUCSSDsl9BTKA, - Fig. 12b a T-DNA construct with this plasmid according to the invention, ~ Fig. 13a a cointegrate plasmid with the constructs -` 25 pMHllDs4IPTkan and pUCtpnAc9P35S, and Fig. 13b an example of a T-DNA construct with the cointegrate plasmid pMHllDs4IPTkan/pUCtpnAc9P35S.
In the drawings the symbols have the following meanings:
X represents a foreign gene located in an expression ` - 30 cassette, not separately shown, active in plant cells; if the `~ gene is interrupted by an insert, the component parts of the gene are indicated by X' and X", respectively;
~ S1 represents a marker gene which can be used for the ,~ selection of transformed plant cells; here, too, the component parts are indicated by S1' and S1", respectively, when the gene is interrupted by an insert;

., .

~: -. . . ,.. , : - . .

W092/0620~ PCT/EP91/01883 (` " ` ~ ` , ~" 15 2 0 g g 7 ~
S2 and S3 also represent marker genes suitable for the selection of successfully transformed plant cells, further -efer-cd lo as plan. aelcc~lon genes;
sl and s2 represent marker genes suitable for the selection of transformed bacteria, further referreà to as bacteria selection genes;
tpn represents a genomic clone of a transposase gene located in an expression cassette, not separately shown, which can function in plant cells;
LTA and RTA represent respectively the left end of T-DNA
and the right end of T-DNA, which ends are necessary for transferring the T-DNA by the Aarobacte~ium L~meL~s~D~
bacteria tO the plant cell;
LTS and RTS represent respectively the left end of a transposon and the right end of an active or inactive transposon,;
Wx represents the sequence of the waxv gene of maize from which Ac9 was cloned;
Fig. 1 shows a T-DNA construc~ according to the invention, one plasmid containing between the T-DNA ends LTA
and RTA both an inactive transposon having a foreign gene X
~ and a plant selection gene Sl contained therein and a ; transposase gene tpn not located on an active transposon. The selection genes shown in Fig. 1 are facultative, for that matter, in particular one of the bacteria selection genes and '~ J one of the plant selection genes being dispensable, if required. As regards the transposase gene, it is important that it is not located within the transposon ends LTS and RTS.
This does not mean that the transposon ends must be completely absent: often removal of (a significant portion of) one of the transposon ends will be sufficient to prevent efficient ; incorporation of the transposase gene in the genome of the transformed plant cells. Thus, for instance, one transposon end could be retained in Fig. 1 beside the transposase gene (see Fig. 5a).

. ~, `: :
, . ~, .

:. ~
~'"'' .
~ . . . : .
, , ~ - . . . ,, :

.- - . : . . ..
., . , - .. .. . :
: ~ . . ': '' , ' .

W092/06205 PCT/EP9l/01883 ~S~ 16 Figs. 2a and 2b show a preferred embodiment consisting of an analogous construct, the elements being distributed over .wo dl~ferent plasmids. The inactive transposon having a foreign gene X and a plant selection gene Sl contained therein is located on a first plasmid the T-DNA of which i5 shown in Fig. 2a. Fig. 2b shows the T-DNA of a second plasmid which contains a transposase gene and a plant selection gene S2. As shown in Figs. 2a and 2b, each of the two T-DNA constructs also contains a bacteria selection gene ~sl and s2, respectively).
For clarity's sake, reference is made to the fact that the present invention is in no way limited to the constructs concretely shown. Thus only one bacteria selection gene will easily do in the construct of Fig. 1. Also the location of the different elements can be varied, e.g. in Fig. 1 a bacteria - selection gene can be placed in the transposon or the location of the plant selection genes can be altered with respect to the other elements. Also T-DNA constructs according to the invention may contain additional or other elements than are shown in the drawings, e.g. one or more additional foreign genes, one or more additional bacteria or plant selection genes etc.
Figs. ~a ana 3b show a preferred embodiment resembling those of Figs. 2a and 2b. An inactive transposon having a foreign gene X and a plant selection gene S1 contained therein is located on a first plasmid, the T-DNA of which is shown in Fig. 3a. In this case, however, the plant selection gene S1 is interrupted by a second inactive transposon which, if ` required, contains its own plant selection gene S3 as shown in Fig. 3a. Fig. 3b shows the T-DNA of a second plasmid ` containing a transposase gene and a plant selection gene S2.
;- This plasmid is identical with the plasmid shown in Fig. 2b.
Figs. 9a and 9b show a preferred embodiment corresponding to those of Figs. 3a and 3b, with the ` 35 understanding that not plant selection gene Sl but foreign gene X is interrupted by the second inactive transposon.
~ .

,, E; '~ `

: - - ~ .. . . . .
-, - , : ~ ..

:. ~ . . . : : .
. .. . . .

. 20~9072 el-Figs. 5 through 13 show DNA constructs which will be further illustrated by the Examples.

EXAMPLES
Example 1 Elimination of insertion mutations of sequences (Ds) derived from transposon Ac (Activator) in maize.

10 By introducing an active transposon Ac into the genome of maize by means of crossbreeding, the activity of this transposon can be observed in the next generations because of the development of mutations (insertion into a gene) or through the elimination of a mutation owing to ~c insertion lS (excision from a gene). In the example described here the AC
transposase gene is used for eliminatlon of insertion mutations (in this case Ds) from genes of maize. In conformity with the above described technology Aarobacterium is used herein to introduce the transposase gene into cells of the apical meristem of maize.
The plasmid pMH10 (Fig. Sa) contains the PstI clone of ~transposon Ac9 in the waxy gene of maize (Pohlman et al, 1984, ; !,Muller-Neumann et al, 1984), a ~LI-BamHI fragment or plasmid pBR322 (Bolivar et al, 1977) with a functional QLL sequence ~. 25 for replication in gram-negative bacteria, but a non-.,functional ~m~ and tet resistance gene and moreover a BamHI-.PstI fragment of plasmid pR702 (Hedges and Jacobs, 1974, and Leemans et al, 1981) containing the sequence of an ~/spc resistance gene. The plasmid was obtained from Laboratorium voor 5enetika RUG, Ledeganckstraat 35, B9000 Ghent, Belgium.
In this plasmid the BamHI fragment containing the pBR32 sequence of the right Ac9(wx) transposon end was replaced by a ~ mHI fragment containing the complete pUC8 plasmid (Vieira ; and Messing, 1982). The resulting plasmid is called pMHlOlLTS
(Fig. Sa). The pMHlOlLTS plasmid is transmitted by conjugation to a E~ resistance Aarobacterium tumefaciens (A. ~m-) C58 , . .

.
,.~ ~,j .
, . . . . . . . .
, ,- , ", .. ... .
.: , .- . : . ' .. ,', ~ - . ;
¢', ~'', ' '. . , '', ' ,'. . ' '., ': ' . :

W092/06205 ~ ~ PCT/EP91/01883 v strain containing the Ti plasmid pGV3850 (Zambryski et al, 1983), in which p3R322 is cloned as a HindIII fragment into the T-~MA ^f pTi C58 frcm which, furthermore, .~ll HindIII
fragments were cut, except the Left ~order (LB) fragment S (~indIII-10) and the Right Border (RB) fragment (~indIII-23) containing the nopaline synthase gene (nos). The plasmid pGV3850 was obtained from Laboratorium voor Genetika RUG, Ledeganckstraat 35, B9000 Ghent, Belgium.
The method of conjugation was described by Van Haute et al (1983). By selection of transconjugants for rif, ~/spc and amp resistance a cointegrate was selected, pMHlOlLTS being integrated between the borders of the T-DNA.
Maize lines obtained by inbreeding for at least ten years were made available, in the form of seed, by the firm of Clovis Matton, Avelgem-Kerkhove, Belgium. These lines contain no active transposon Ac, which, inter alia, could be proved by breaking down the DNA of young leaves with PvuII and hybridizing it with the internal ~indIII fragment of Ac (Ac9(wx): bp: 1270-2877) : no hybridizing band was observed corresponding to the 2626 bp internal PvuII fragment of Ac (Ac9(wx): bp: 719-3345); different bands were found indeed but the smallest band e~ceeded 4 kb. Crossings with test lines for Ac activity have never sho~n transposition phenomena in the lines used. The lines are used for the production of commercial hybrids.
Seeds of ten maize lines were disinfected with NaClO 2%
and then laid on sterile wet sand to germinate on sterile sand at 25C in the dark. Three days after germination 50 nl ~~ induced A. ~m. culture were integrated with the construction pMH10 LTS by genetic recombinantion between the borders of the ` T-DNA (Fig. 5b), injected into the apical meristem by '~ microinjection.
'' r~ The induction medium has been described by Vernade et al (1988). After one week at 20C the young plants were cultured together with untreated plants in large pots in an air-,~ conditioned greenhouse in a mixture of compost and sandy clay 1.

~. .
~'' - , , ; : ~ :, , ,,, " . . . ~

,: ' ' ' ' :' W092/06205 PCT/EPg1/01883 20~9072 in their normal growing season (May to October). Each plant was fertilized manually, both the plume and the ear being kept ln small bags. Each plant was individ~aally ~.atered in ~he F^'-The ears were harvested in September and October and after drying the seed was stored in paper bags during winter at 6C.
The next year the plants were sown on ear lines in the soil in the greenhouse and again fertilized manually and evaluated by corn growers for their characteristics both during growth and in surmaturity.
The fe,llowing improvements over the original lines were observed in some ear lines:
- plume more branched - 30 - 50 cm higher plants - stronger stem - significant increase in the number of grain rows per ear - remains green for a longer time - absence of fusariosis - better anchoring by secondary roots ~ Seeds of plants having deviating features as well as of : 20 control plants were germinated in the laboratory under sterile conditions and DNA was prepared from the young leaves, after ~- which the plants were planted in the greenhouse in large pots, The DNA of each plant was then broken down such as for ` `1 RFLP mapping with different restriction enzymes and hybridized with the internal HindIII fragment of Ac9. The control plants `~ showed the same band pattern, as regards the prominent bands, as the original line. In plants with deviating features either disappearance of one or more fragments hybridizing with the DNA probe or the disappearance of a fragment and the appearance of a new fragment with a larger molecular weight . was observed.
;
Example 2 (comparative e~ample) Insertion of a foreign gene into the genome of maize by using ` 35 Aarobacterium tumefaciens as transfer system and transposon Ac ~: as integration system.
!, 1 , . . ~' ~' ' .

G
. '''' '' : ' ' ', ~ ' , . . . .

,, : '., ' , , :

W092/0620~ ~ PCT/EP91/01883 For the purpose of~comparison an active transposon Ac9(wx) is used here. By using the integration properties of the transposon Ac and the transfer properties of A. ~m. a S foreign gene located between the recogni~ion sequences of the transposon can be incorporated in the genome of maize by means of transposase of the same transposon.
As described, two A. tumefaciens strains are used by way of example. The first contains the ~-glucuronidase gene of Escheric~ coli located between the left- and right-hand transposon ends of Ac9(wx), which, in turn, is located between the Left and Right Border ~LB and RB) of the T-DNA on a non-oncogenic Ti plasmid. The second contains a complete transposon Ac9(wx) located between LB and RB of the T-DNA.
The ~. ~m. strain containing the transposon Ac9 in the T-DNA is shown in Fig. 5a. The other A. ~m. strain was obtained in the following manner:
Plasmid pBI 221 contains the ~-glucuronidase gene of ~. coli under the control of the CaMV 35S promoter (Cauliflower Mosaic Virus 35S RNA promoter) and the mRNA distal end of the nopaline synthase gene of the Ti plasmid of ~. tum. T37. This plasmid was available from Dr R. Jefferson, c/o AFRC
Institute, Colney Lane, Norwich NR4 7UA, U.K.
The plasmid pMH10 shown in Fig. Sa was cut with EcoRI
~; 25 and partially with ~indIII and the 10.1 ~b fragment was ~ ligated with the 3 kb ~SQRI-~1ndIII fragment of pBI 221 containing the sequence of the chimeric ~1~ gene of this last plasmid. The resulting plasmid pMHlODsl5GUS contains the chimeric ~i~ gene in a transcription orientation contrary to that of the (partially) deleted transposase gene.
~m- bacteria with this plasmid, introduced in the ` manner described in Example 1, show ~-glucuronidase activity.
; The construction is shown in Figs. 6a and 6b.
i The same maize lines described in Example 1 were used for the transformation. In three-day-old germs the apical meristem was infected by microinjection with 50 nl of a ~, ~;

j . .

, , : . , ', `::
... . . . .. . .
. : , : . ' i ' : '' :--'~

W092/0620~ PCT/EP91/01883 20~9~72 mixture of the two above-described ~. tum. strains induced for transfer. In a first phase some hundreds of germs were tested afte- l o weeks ~or ~-glucuronidase activity by incubating a piece of tissue containing the apical meristem with X-gluc (5-bromo-4-chloro-3-indolyl-D-glucuronide). In about 7.5% of the tissues blue staining occurred. With a dark field microscope it was clearly visible that the cells in the tissue cuts and not the remaining bacteria were the origin of the purple color. In particular small cells around the vascular ; 10 bundles were.stained. With an electron microscope the precipitate of dimeric bromo-chloro indole was found ln cells which were removed from the place of injection up to ten cell layers. Sporadically, cut bacteria were visible which were '. only present in the extracellular spaces but showed no precipitate.
Other treated germs were grown in the greenhouse in a normal growing season, manually fertilized and the seed was ` ~ harvested as described in Example 1.
During the winter months these seeds were germinated in !,; 20 sterile conditions and the proteins were isolated from the . young leaves and subjected to polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditlons. Beta-glucuronidase activity was detected by covering the gel with a thin agarose gel containing the substrate methyl-umbelliferyl-D-glucuronide which fluoresces in UV light if ~he ~-glucuronidase enzyme causes release of methyl umbelliferone.
A fluorescent band was found in about 1~ of the tested plants. DNA was prepared from leaves and pieces of roots of these plants and, as in RFLP mapping, hybridized with a 2.9 kb ~1ndIII-EcoRI fragment containing the chimeric ~1~ gene. In , ~ the lane of the agarose gel containing maize DNA broken down with ~ndIII and EcoRI hybridization with the probe was found ~ on a level with the place where about 3 kb fragmen~s migrate.
-~ Of young F1 plants pieces of leaves and roots were incubated in X-gluc as described by Jefferson (GUS gene fusion ; system user's manual, 1987). Blue stains, -ones and sectors `~, . .
, .
~-` , , ' . ' ' . ' '`

. ' ` ~ ~ ,.",."~.,."~
~'~` ` ~ ` ' ' ` " ` '' ` .

W092/06205 ~ PCT/EPgl/01B83 were found in the pieces of tissues of PAGE-~-glucuronidase positive plants: around the vascular bundles, around the s~omata, V-shaped segments of the roots, etc.
Light microscopic and electron microscopic analyses confirmed the expression of the ~-glucuronidase enzyme in the plant cells.
This example clearly shows that by using an active - transposon in combination with a chimeric transposon containing a foreign gene but no transposase gene between the transposon ends expression is only obtained in specific cells still containing the chimeric transposon, while most of the plant cells show no expression. The same phenomenon can be observed after transformation of plants such as tobacco which are efficiently transformed by Aarobacterium but in this case instability occurs after transformation with a chimeric transposon, both in the presence of an active transposon and in the presence of a transposase gene. This shows the ; difference between plants in which Aarobacterium can efficiently integrate foreign DNA in the plant genome and plants in which ~arobacterium cannot.
. . .
. Example 3 Generation or mutations in barley by using a transposase gene ; and a chimeric transposon.
, .
.i In this example the mutagenic properties of a transposon - are used by integrating a chimeric transposon containing a plant selection gene but no transposase gene in the genome of barley by means of a transposase gene that is not located between transposon ends.
Use is made of two Aarobacterium strains for the . transfer: the first strain contains the chimeric transposon ~ i between the T-DNA ends and the second strain contains the c ' transposase gene between the T-DNA ends. This example ~`~ 35 corresponds with Figs. 3a and 3b.

~' , , ~
,, . . ....... , , ~ , . ~ , , . , .:

F - ;~

W092/0620~ PCT/EP91/01883 ~ r~ 2 1~ 8 ~ 0 7 2 The employed plasmids are pMHlODsl5GUS and pMHlOLTS. As described before, they are conjugated from Escherichi~ CQli to Aqrobac~rium and integ-ation 1,. ~r'~,T35~ scle_'cd by also adding spectinomycin to the medium.
Seeds of two- and six-rowed winter barley ear lines ~F7-F8) were supplied by the plant breeding station of the firm of Clovis Matton, Avelgem-Kerkhove, Belgium.
Seeds of 12 ear lines were disinfected with 6~ NaClO and the seeds laid on moist sand to germinate at 4C. Germinating seeds were further incubated at room temperature under sterile conditions.
In 3-5-day-old seedlings the apical meristem was infected by microinjection with Agr3b~cterium strains containing pMHlO~slSGUS or pMHlOLTS. Transfer was induced as described bv Vernade e~ al (1988). Infections were conducted both with the strain containing pMG15 (control) and with the strain containing pMHlOLTS (control) or with both of them (experiment). About 300 seedlings were treated per ear line: a hundred per experiment. Further control experiments were conducted on tobacco, for rapid tests of expression.
After 1 week at 22C the young plants were cultured further in a greenhouse and the seed was harvested after self-pollination.
In the next generation the plants were sown again in a greenhouse and selected by barley growers for deviating features.
The following variants were traced:
- difference in height of the plant during growth - difference in time of growth - mutations from erectant to nutant or conversely - difference in number of grains per ear - difference in flattening . ,. ,-, .
. - better or poorer phytosanitary condition.
Of 37 ear lines with clear morphological or - 35 phytosanltary differences and 110 other ear lines with less . --.

, :.
,: -, .
, ~ ' : : ` ' ..
f ` :, ~ ,,` .
`: . -:

', :,. ,, ` . i W092/06205 ~ ~ PCT/EP91/01883 ~Q,oG9 ~ ~

clear deviations five ripe ears were further examined for expression of the uidA gene in the chimexic transposon.
~ !e-r'~ hc '? plants with clear differences over the original ear line express the ~1~ gene to a greater or lesser degree. No ~-glucuronidase activity could be demonstrated in the controls, except in the case of bacterial infection. Only 9 plants with less clear deviations show ~i~ expression.
In tobacco ~-glucuronidase activity was found when the ; gene was present ln the infection, with or without additional infection with the transposase gene.

Example 4 Construction of recombinant plasmids with plant selection ~` genes or genes with industrial applications between left- and right-hand transposon ends in the plasmid pMH10.
i, .
In this example the construction of some plant selection genes was shown by using unique restriction sites in the sequence of Ac9 in the plasmid pMH10.
The chloramphenicol acetyl transferase gene in an . . expression cassette consisting of the 35S promoter of CaMV and the nos-terminator of the T-DNA of Aarobacterium tumefaciens is available as plasmid pCAMVCN from Pharmacia-LKB, Bromma, Sweden. The complete chimeric gene was cut as Xbal fragment.
,:~`' 25 The plasmid pMHl0 was partially cut with ~S11 (position 1530 in Ac9Wx) and BamHl (position 4498 in Ac9Wx) and the 8.1 kb fragment was ligated back. This plasmid, called pMHlODsB, contains unique restriction sites in the remaining Ac9 ~: sequence: ~il (five sites at position 571, 972, 1003, 1183, 30 and 1251 of AC9WX), ~hQl (at position 1125 of Ac9Wx) and Xbal ~^ (at position 1257 of AC9WX) ~Fig. 5a). The ~at expression cassette was cloned into the unique XbaI site of Ac9. The resulting plasmid was called piMHlODs34CAT (Fig. 7), conjugated ` to Aarobacte_ium and integrated in the plasmid pGV3850. By -~ 35 transformatio~ of tobacco the expression of the cat gene was :~ proved.

.~
.
:, ''' ; . ~ ,., , . , . .. - . .. .

The genes iaaM and iaaH from the T-DNA of Aarobacterium tumefaciens Ach5 (Gielen et al, 1984) were cloned from the plasmlds pGV819 (~Q~ l clone of ~ uda_ et 31, 1986) and pGV829 (HindIII clone of ia~H) (Budar et al, 1986). The S complete construct with the genes iaaM and iaaH was first cloned into pBR322 (Bolivar et al, 1977) by a threefold ligation of the 3.6 kb pBR322 ~ I fragment, the 3.7 kb EcoR1-ClaI fragment of pGV814 and the 2.7 kb partial PstI-EcoR1 fragment of pGV824. From this was cloned the 6.4 kb ~I fragment in the ~iI sites (position 571 and 2881 of Ac9Wx) of pMH10. The resulting plasmid pMHlODs12IAA (Fig. 8) was conjugated to Aarobacterium and integrated in the plasmid pGV3850 by additional selection with spectinomycin. The usability of the construction was tested by transformation of tobacco and expression of the L~ genes.
The gene ie~ from the T-DNA of Aqrobacterium tumefaciens CS8 together wi~h the bacterial n~tII gene of transposon Tn5 of plasmid pG4L ~Inzé et al, 1989) was cloned as a BamHI
fragment in pUC19 and isolated therefrom as a partial ~hl-EcoR1 fragment of 3.7 kb and cloned into pMH11 cut with EcoR1 and partially with SDhl. pMH11 is a derivative of pMH10 obtained by cutting in the vector plasmid of pMH10 the 0.8 kb ~hl fragment located between the str/s~c resistance gene and the (partially deleted) ~ resistance gene, blunt ending the ends with S1 nuclease and ligating back the vector. The PstI
clone of Ac9Wx was again incorporated in the resulting vector in the unique ~stI site. The orientation, however, is different from that in pMH10.
The resulting plasmid pMHllDs4IPTkan (Fig. 9) was 30 conjugated to Aarobacterium and integrated in pGV3850 by additional selection with spectinomycin and then with kanamycin. The usability of the construction was proved by transformation of tobacco and expression of the gene 12~-,~
Example 5 ~'~.
~-. ~
, .

.

W092/0620S ~ ~ PCT/EP9l/0l883 Construction of a recombinant transposon with a multiple cloning site between the transposon ends.

The ~hI~ I fragment from mini-Sa, a derivative of pR702 ~Leemans et al, 1983) containing the sm-sp adenosyl transferase gene encoding resistance to streptomycin and spectinomycin was blunt ended with T4 DNA polymerase and cloned into the plasmid pUC19 (Yanisch-Perron et al, 1985), blunt end cut with ~I. This plasmid was 4330bp and will be referred to hereinbelow as pUCSS19.
Both orientations of the sm/sp resistance gene are suitable. However, the work was further done with the plasmid in which the amp resistance gene and the ~m/~ resistance gene have the same transcription orientation. pUCSSl9 was blunt end cut with PvuII and both the 4030bp vector fragment and the 300bp ~CS fragment were separated and stored.
. The PstI clone of Wx-Ac9 was cut from pMH10 (Fig. 5a) ; and blunt ended with T4 DNA polymerase. This fragment was , cloned into the 4030bp ~II fragment of pUSS19. This plasmid ,;
was 8840bp and will be referred to hereinbelow as pUCSSAc9.
pUCSSAc9 was cut with NsiI and ~I and the NsiI end was blunt ended with T4 DNA polymerase. This fragment was 4980bp `~ and contained the ends of transposon Ac9Wx. Into this was ' cloned the 300bp ~II fragment of pUC19. One of the orientations is shown in Fig. 10. The plasmid was 5280 bp and will be referred to as pUCSSAcDsl9. It can be used for cloning numerous genes with their expression cassette. It can be '`~ conjugated to Aarobacterium with plasmid pGV3850 as described in the first example by additional selection with spectinomycin.

, Example 6.
Construction of recombinant plasmids containing plant selection genes or genes with industrial applicaticns and located between the transposon ends of pUCSSAcDsl9.
,;
::, , ..
., .
~, ,,, ,,, -- - ,. . :' ' , . ~ - ': -, ' 1~ ',: . _ - ' ,, , ' ' ' ,, ' . , ., . ' , ' . ', . .
; : :. . .. . :: . . . .. . .. . .
~. . . .. . ... .. .. ... . ... .
;.................... . . . . . .
: . , ~ . , , . . ,, :

27 2 ~ 8 9 ~ 72 The delta endotoxin gene of the 42 MDa plasmid of Bacillus thurin~iensis var. Berliner 1715 (Klier et al, 1982), 'he se~uence of which was published by Hofte et al, 1986, was obtained from S.A. Solvay, Brussels, Belgium, as plasmid pBT424. The strain is available from the Institut Pasteur in Paris, France.
From pBT924 the BamHl~ I fragment containing the start codon of the delta endotoxin gene and the first 1939 bp, necessary for expression of the toxin, was cut and cloned downstream of the pTRl' promoter of plasmid pPCV520 (Koncz and Schell, 1986), cut with BamHl and ~glII. The correct orientation was verified with BamHl and PstI (0.7 kb in the correct orientation). The complete double expression cassette pTRl'-BT~Q~ terminator gene 4/pTR2'-nptII terminator ocs was cut again with KonI and partially with PstI as a 5.7 kb fragment and cloned into the corresponding restriction sites of pUCSSAcDsl9. The terminator of gene 4 contains different stop codons in the three reading frames upstream of the ~ polyadenylation signal.
- ~ 20 The resulting plasmid (pUCSSDsl9BTKA) (~ig. 12a) was conjugated to Aaroba~terium and integrated in pGV3850 by - additional selection with spectinomycin as described berore (Fig. 12b).
. Resistance to kanamycin could be shown after transformation to tobacco.
The construct was used for making maize lines resistant to Pyralidae, in the manner as described in Comparative :~ Example 2, with the difference that use was made of a second A~robacterium strain containing a transposase gene without ~ 30 transposon ends (pMHlOLTS).

,i Example 7.
- Construction of a recombinant plasmid containing the transposase gene of Ac under control of the CaMV 35S promoter or of the pTRl' promoter of ~grobact~ium Ach5 T-DNA.

~, -, , ,, , W092/~6205 ~ ~ PCT/EP91/01883 ~ 28 The plasmid pMH10 (Fig. 5a) was cut with PstI and the 4810bp Ac9 wx fragment, c~oned into pUC19, cut wit ~I. This plasmiu was 7500bp and will be referred ~o hereinbel~w d~
pUC19Ac9wx. The work was further done with the orientation 1, namely the one in which the BamHI sites are remotest from each other.
The Ac9 wx fragment was cut again, as ~mHI fragment.
This fragment was partially cut with Cfr1QI and two fragments were stored: the 3.4 kb CfrlOI fragment and the 4.2 kb BamHI-. 10 CfrlOI fragment. The first contained a transposase genewithout a promoter and the second a transposase gene with a promoter. The 4.2 kb fragment was cloned into pUCSS19, cut with XmaI and BamHI. This plasmid (8.5kb) contained the normal transposase gene of Ac but no transposon ends (pUCSS19tpnAc9PAc9) and could be transmitted by conjugation to Aarobacterium and integrated in the T-DNA of pGV3850 by - additional selection of spectinomycin resistance. The 3.4 kb fragment was cloned into pUC19, cut with ~m~I, and the orientation was determined using the ~L~I site in the MCS and in the transposase gene (position 1260 of Ac9Wx). This plasmid (7.7 kb) is called pUCSS19tpnAc9cds) and may serve for cloning new promoters upstream of the transposase gene.
;
For further use pUCSS19tpnAc9cds was taken in orientation 1, the ~k~I fragment from this plasmid being 2.5 kb. This plasmid was cut with PstI and BamHI and the CaMV 35S
promoter fragment from pBI221, cut with Ps I and BamHI, was cloned into it.
The resulting plasmid is called pUCSS19tpnAc9P35S
(Fig. lla). The pTRl'2' double promoter of the T-DNA of Aarobacterium Ach5 (Velten et ~1, 1984) was obtained as a ........................................................................... .
~ BamH1-Ps~I fragment from the plasmid pPCV520 (Koncz and ; ~ Schell, 1986) and was cloned into the BamHl and PstI sites of :. .
pUCSS19tpnAc9cds. The resulting plasmid (pUCSS19tpnAc9PTRl') -contains the transposase gene under controi of the pTRl' promoter (Fig. llb).
Both plasmids can be conjugated, as described before, to '~ ~'', . .

,' '`' '' ~ .,' .'', ~' ,' ''.''' '. ' ~.', :,'' :
,, ; . , .. . ;. : , . , ~

W092/0620~ PCT/EP9l/01883 29 2 ~ 89 0~
Aarobacterium and integrated in pGV3850 by additional selection for spectinomycin (Fig. llc). The use of the pTR1' promoter has the advantage tha. in CâSe OL mic.oinject Gr; or bombardment of Aarobacte_ium under high pressure this promoter strongly expresses in the wounded tissue.

Example 8.
Construction of recombinant plasmids containing a chimeric transposon and a transposase gene without transposon ends.
, Recombinants between â pMH10 derivative with a chimeric transposon and pUC19 derivatives with a chimeric transposase gene were selected in vivo in MC1051 bacteria after transformation with both plasmids and selection for ampicillin and spectinomycin, followed by alternating growth cycles with : selection for ampicillin and then spectinomycin. This results in cultures in which recombinant cointegrates of pMH10 and pUC
~: derivatives occur in 50% of the bacteria.
` The constructs tpnP35S and tpnPTRl' were cloned as KpnI-~0 ~I from their respective pUCSS plasmids into pUCl9 containing the ampicillin resistance gene. The employed pMH10 derivatives were: pMHlODslSGUS, pMHlODs3qCAT, pMHlODsl2IAA and pMHllDs4IPTkan and are all carriers of a spectinomycin resistance gene. The cointegrate of the recombinant plasmids pMHllDs4IPTkan and pUCSS19Ac9P35S is shown in Fig. 13 by way . of example. These cointegrate plasmids were transmitted by - transformation to the strain HB101 and after selection for ampicillin and spectinomycin they were characterized by plasmid preparation and restriction enzyme analysis. Then ~he plasmids were transmitted by conjugation to Aarobacterium, as ~` described before.
The resulting Aqrobacterium strains are an example of the use shown in Fig. 1.
~: The resulting recombinant AarobactQrlum strains were tested on tobacco ror expression of respectively nopaline ' synthase (pGV3850 marker), uidA, cat, -oot ~ormation and shoot ~' ';
.~

, ; . ': : ' . ': .: .

~,~ . : . . : - . .
' ' ` ' .. : , ' :

W092/0620S ~ PCT/EP91/01883 ~ 30 formation and finally for transposition, before they were used on cereals.

Literature Bolivar, F. et al., Gene 2: 95-113 (1977) Budar, F. et al., Plant Science 46: 195-206 (1986) Gielen, J. et al. EMBO J. ~: 835-846 (1984) -Hedges, R.W. and Jacob, A., Mol. Gen. Genet. 132: 31-40 (1974) Hofte, H. et al., Eur. J. Biochem. 161, 273-280 (1986) Inzé, D. et al., Mol. Gen. Genet. 194: 265-274 (1984) Klier, A. et al., EMBO J. 1, 791-799 (1982~
Konc~, C. and Schell, J., Molec. Gen. Genet. 2Q~:3&3-396 ('986) Leemans, J. et al., J. Mol. Appl. Genet. 1:149-164 (1981) - 15 Muller-~eumann, M., Yoder, I. and Starlinger, P., Mol. Gen.
Genet. 198: 19-24 (1984) Pohlman, R.F. et al., Cell 37: 635-643 ~1984) Van Haute, E. et al., EMBO J. ~: 411-418 (1983) Velten, J. et al., EMBO J. 3: 2723-2730 (1984) Vernade, D. et al., J. Bacteriol. 170: 5822-5829 (1988) Vieira, J. and Messing, J., Gene 1~: 259-268 (1982) Yanisch-Perron, C., Vieira, J. and Messing, J., Gene 33: 103-^ 119 (1985) Zambryski, P. et al., EMBO J. 2: 2143-2150 (1983) . ;:
... .

:' :

...
.! ' ':^. '~
. ., .

, ~

, : : : ' . `- : : - : ` ' '`~! ` .

Claims

1. A process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Agrobacterium tumefaciens with integration by T-DNA, which comprises introducing foreign DNA into the plant cells by infecting the plant cells with one or more recombinant Agrobacterium tumefaciens strains which contain foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and are capable of transferring this foreign DNA to the plant cells, the foreign DNA located between the left- and right-hand ends of T-DNA comprising:
(A) a transposase gene located in an expression cassette active in the plant cells but not between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells, and (B) a recombinant transposon comprising the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells with an intermediate DNA fragment to be integrated in the DNA of the plant cells, but not comprising a DNA fragment which can lead in the plant cells to expression of an active transposase.

2. A process according to claim 1, which comprises the gene manipulation of plant cells of cereals such as maize, wheat, barley, rye, oat, rice etc., grasses such as ryegrass and fescue, leguminous plants such as leek, onion and garlic, ornamental plants such as tulipe, hyacinth etc. and other plant species such as conifer etc.

3. A process according to claim 1 or 2, which comprises using a genomic transposase gene.

4. A process according to claim 3, which comprises replacing the natural promoter of the genomic transposase gene by another promoter which can give expression in plant cells from which germinative cells develop.

5. A process according to any of the preceding claims, characterized in that between the left- and right-hand transposon ends the recombinant transposon comprises at least one foreign gene located in an expression cassette active in the plant cells.

6. A process according to any of the preceding claims, characterized in that also one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria are located between the left- and right-hand ends of T-DNA.

7. A process according to claim 6, characterized in that one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria form part of the recombinant transposon.

8. A process according to any of the preceding claims, characterized in that also one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in the plant cells are located between the left- and right-hand ends of T-DNA.

9. A process according to claim 8, characterized in that one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in the plant cells form part of the recombinant transposon.

10. A process according to any of the preceding claims, characterized in that the plant cells are infected with a mixture of two different recombinant A. tumefaciens strains, one of which comprises between the left- and right-hand ends of T-DNA the transposase gene located in an expression cassette active in the plant cells and the other comprises the recombinant transposon between the left- and right-hand ends of T-DNA.

11. A process according to claim 10, characterized in that between the T-DNA ends each of both strains contains at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells.

12. A process according to claim 11, characterized in that between the T-DNA ends both strains each contain at least one different marker gene for selection of transformed plant cells.

13. A process according to any of claims 1-9, characterized in that the plant cells are infected with one recombinant A. tumefaciens strain which comprises between the left- and right-hand ends of T-DNA both the transposase gene located in an expression cassette active in the plant cells and the recombinant transposon.

14. A process according to claim 18, characterized in that the strain between the T-DNA ends contains at least one marker gene for selection of transformed bacteria and at least one marker gene for selection of transformed plant cells.

15. A process according to any of the preceding claims, characterized in that meristematic or embryogenic cells or tissues are infected with the bacteria.

16. A process according to claim 15, characterized in that cells of the apical meristem in germinating seeds are infected with the bacteria.

17. A process according to any of claims 15-16, characterized in that the infection is carried out by means of needleless high-pressure injection.

18. A process according to any of claims 15-16, characterized in that the infection is carried out by means of microinjection.

19. A recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA insert comprising a transposase gene located in an expression cassette active in plant cells, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.

20. A recombinant plasmid according to claim 19, characterized in that it contains a genomic transposase gene.

21. A recombinant plasmid according to claim 20, characterized in that the natural promoter of the genomic transposase gene is replaced by another promoter which can give expression in plant cells from which germinative cells develop.

22. A recombinant plasmid consisting of a bacterial vector plasmid and at least one DNA insert comprising a recombinant transposon which contains the left- and right-hand transposon ends necessary for integration in the DNA of plant cells but no DNA fragment which can lead in plant cells to expression of an active transposase.

23. A recombinant plasmid according to claim 22, characterized in that between the left- and right-hand transposon ends the recombinant transposon comprises one or more restriction endonuclease recognition sequences suitable as cloning site.

24. A recombinant plasmid according to claim 22 or 23, characterized in that between the left- and right-hand transposon ends the recombinant transposon comprises at least one foreign gene located in an expression cassette active in plant cells.

25. A recombinant plasmid according any of claims 22-24, characterized in that the recombinant transposon comprises between the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in plant cells.

26. A recombinant plasmid according any of claims 22-25, characterized in that that the recombinant transposon comprises between the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria.

27. A recombinant Agrobacterium tumefaciens strain, which contains foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, characterized in that between the left- and right-hand ends of T-DNA a transposase gene is located in an expression cassette active in plant cells, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.

28. A recombinant Agrobacterium tumefaciens strain according to claim 27, characterized in that the transposase gene is a genomic transposase gene.

29. A recombinant Agrobacterium tumefaciens strain according to claim 28, characterized in that the natural promoter of the genomic transposase gene is replaced by another promoter which can give expression in plant cells from which germinative cells develop.

30. A recombinant Agrobacterium tumefaciens strain, which contains foreign DNA to be transferred to plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer to the plant cells and is capable of transferring this foreign DNA to the plant cells, characterized in that between the left- and right-hand ends of T-DNA a recombinant transposon is located which comprises the left- and right-hand transposon ends necessary for integration in the DNA of plant cells but contains no DNA fragment which can lead in plant cells to expression of an active transposase.

31. A recombinant Agrobacterium tumefaciens strain according to claim 30, characterized in that between the left-and right-hand transposon ends the recombinant transposon comprises at least one foreign gene located in an expression cassette active in plant cells.

32. A recombinant Agrobacterium tumefaciens strain according to claim 30 or 31, characterized in that the recombinant transposon comprises between the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed plant cells and located in an expression cassette active in plant cells.

33. A recombinant Agrobacterium tumefaciens strain according any of claims 30-32, characterized in that the recombinant transposon comprises between the left- and right-hand transposon ends one or more selectable marker genes suitable for selection of transformed bacteria and located in an expression cassette active in bacteria.

34. A recombinant Agrobacterium tumefaciens strain according any of claims 30-33, characterized in that between the left- and right-hand ends of T-DNA there is also present a transposase gene located in an expression cassette active in plant cells, which expression cassette active in plant cells and filled with a transposase gene is not located between the left- and right-hand transposon ends necessary for integration in the DNA of plant cells.

35. Plants and parts consisting of one or more cells or products thereof, which plants derive from plant cells transformed by the process according to any of claims 1-18.

36. A process for the gene manipulation of cells of plants which, in essence, cannot be transformed by Agrobacterium tumefaciens with integration by T-DNA, which comprises introducing foreign DNA into the plant cells by infecting the plant cells with a recombinant Agrobacterium tumefaciens strain which contains foreign DNA to be transferred to the plant cells between the left- and right-hand ends of T-DNA necessary for such a transfer and is capable of transferring this foreign DNA to the plant cells, characterized in that plant cells are infected which contain one or more inactive transposons but no active transposase gene and that the foreign DNA located between the left- and right-hand ends of T-DNA comprises a transposase gene located in an expression cassette active in the plant cells, which expression cassette is not located between the left- and right-hand transposon ends necessary for integration in the DNA of the plant cells.

37. A process according to claim 36, which comprises the gene manipulation of plant cells of cereals such as maize, wheat, barley, rye, oat, rice etc., grasses such as ryegrass and fescue, leguminous plants such as leek, onion and garlic, ornamental plants such as tulipe, hyacinth etc. and other plant species such as conifer etc.

38. A process according to claim 36 or 37, which comprises using a genomic transposase gene.

39. A process according to claim 38, which comprises replacing the natural promoter of the genomic transposase gene by another promoter which can give expression in plant cells from which germinative cells develop.