A PROCESS FOR THE INCORPORATION OF FOREIGN DNA INTO THE GENOME OF MONOCOTYLEDONOUS PLANTS

Abstract: A process for the incorporation of foreign DNA into the genome of monocotyledonous plants with bacteria, which are provided with one or more tumour-inducing plasmides or derivatives thereof, initially originating from Agrobac­ terium, or of bacteria which have the T-DNA, originating from the above-meant plasmides, and/or the virulence genes, originating from the above-mentioned plasmides incorporated into the bacterial DNA elsewhere.

Description: A PROCESS FOR THE INCORPORATION OF FOREIGN DNA INTO THE GENOME OF MONOCOTYLEDONOUS PLANTS The invention relates to a process for the incorporation of foreign DNA into the genome of monotyledonous plants, by infecting the plants or incubating the plant protoplasts with bacteria suitable or made suitable for that purpose. A number of bacteria, such as Agrobacterium tumefaciens and Agrobacterium rhizogenes are by nature capable of manipulating plant cells genetically. The said bacteria adhere to the plant cell wall and subsequently introduce a fragment of DNA into the plant cell via a process still unexplained. This DNA fragment (the T-DNA) is as T-region in the bacteria a part of a large plasmid (120-150 Mdal.), which in case of A. tumefaciens is called Ti-plasmid and in case of A. rhizogenes Ri-plasmid. The T-DNA is then integrated into the nucleus genome of the plant cell (Thomashow, M.F., et al. (1980), Proc. Nat.Acad.Sci. USA 77: 6448-6452). Genes positioned in the T-DNA express therein and see to it that the plant cell will behave like a tumour cell (Ooms, C., et al (1981), Gene 14: 3-3-50). Meanwhile methods have been developed to couple no matter which DNA to the T-region in such a way that this can subsequently be introduced into the plant cell simultaneously with the T-region (Hille, J. et al (1983), J. Bacteriol. 154: 693-701 and Hoekema, A. et al (1983), Nature 303: 179-180). From the T-region the onc-genes can be removed (Hille J., et al (1983), Plant Mol. Biol. 2: 155-163 and Zambryski, P., et al (1983), EMBO J. 2: 2143-2150). This results in vectors which are indeed transmitted by the bacteria to the plant cell, but which are no longer oncogeneous (Hille, J., et al (1983), Plant Mol. Boil. 2: 155-163 and Zambryski, P., et al (1983), EMBO J. 2: 2143-2150). Plant cells transformed with these "tamedH vectors can regenerate to plants having new properties (encoded by the genes introduced into the T-region (Zambryski P., et al (1983), EMBO J. 2: 2143-2150). If Rhizobium bacteria are provided with plasmids such as meant above, or plasmids derived therefrom, they are also capable of the genetic manipulation of plant cells (Hooykaas et al 1977). Now, it has appeared from experiments with mutants of Agrobacterium and with strains of Rhizobium occurring in nature that in addition to the said plasmids also particular other sequences of the bacterial DNA are necessary, for instance the sequence responsible for the adherence of the bacteria to the cell wall. The above means that Agrobacterium is not the only organism capable of inserting of T-DNA into the plant genome. There are other bacteria which are capable hereof if they are provided with the plasmids or derivatives thereof ultimately originating from Agrobacterium. A conditio sine qua non is in this respect that they avail of the necessary attendant DNA-sequences such as described above, or functional analogues thereof and that no inherent impediments for the transfer of DNA are present. It also appeared in research work of our group performed earlier that it is not absolutely necessary that the plasmid genes for virulence and the T-DNA are actually positioned on plasmids. For instance the T-DNA may be situated on the bacterial chromosome, whilst the plasmid genes for cirulence are positioned on an independent plasmid. According to literature data the said systems can be used as a vector only for dicotyledonous plants, since only dicotyledonous plants respond on Agrobacteria with tumour formation (Lippincott, J.A. & Lippincott, B.B. (1975), Ann.Rev.Microbiol. 29: 377-407). The fact that the cell wall of monocotyledonous plant differs from that of dicotyledonous plants would be the cause (Lippincott, J.A. & Lippincott, B.B. (1980). In "Bacterial Adherence in Receptors and Redognition" (E.H. Beachley, ed.), pages 377-396 and Rao, S.S. et al (1982), Physiol. Plant. 56: 374-380). So, the prevailing opinion is that the said plasmids are not suitable as a vector for the genetic manipulation of monocotyledonous plants (Flavell, R. & Mathias, R. (1984), Nature 307: 108-109). But among the monocotyledonous plants there are highly important agricultural and horticultural crops, such as the grains and bulbous plants like tulip, daffodil and lily. The research performed described hereinunder was carried out in order to see whether the prevailing opinion about the absence of possibilities to use the Ti-plasmid as a vector for monocotyledonous plants was correct indeed. In this research various species of monocotyledonous plants were infected with A. tumefaciens strans, which either possessed various types of Ti- or Ri-plasmids separately or accomodated a combination of such plasmids. Surprisingly, it appeared from the experiments that cells of monocotyledonous plants were positively transformed by A. tumefaciens. This showed for the first time that the Ti-plasmid (or plasmids derived therefrom) is suitable for the genetic manipulation of monocotyledonous plants. Experiment: In the T-DNA in addition to onc-genes, which see to phytohormone synthesis coombs G., et al (1981), Gene 14: 33-50 and Garfinkel D.J., et al (1981), Cell 27: 143-153, there are also genes which encode for enzymes which see to the biosynthesis of "opines". These are tumour specific amino acidcerivatives, such as octopine and nopaline (tempt, J. & Goldman, A. (1982) in: "Molecular Biology of Plant Tumours" (G. Kahl & J. Schell, eds.), pages 427-449. Opines do not occur in normal plant cells and so can be considered as specific indicators for incorporation and expression of T-DNA in the plant cell, even if there would not be question of transformation leading to formation of tumour cells. There are various types of T-DNA which carry genes for enzymes which see to the biosynthesis of different opines. The most well-known are the octopine and resp. nopaline T-DNA's. A great number of tests was now carried out in which monocotyledonous plants from the family of Liliaceae (Chlorophytum capense) and the family of Amaryllidaceae (Narcissus cv. Paperwhite) were wounded and subsequently infected with various A. tumefaciens strains. The strains used were: LBA288 (avirulent; no Ti-plasmid), LBA1010 (contains an octopine Ti-plasmid), LBA2318 (contains a nopaline Ti-plasmid), LBA1020 (contains a Ri-plasmid), LBA2347 (contains a nopaline Ti-plasmid + Ri-plasmid), LBA1516 (contains an octopine Ti-plasmid with a Vir-mutation; avirulent). After a few weeks plant material was removed from the infection sites and analysed for opines (Otten, L.A.B.M. & Schilperoort, R.A. (1978) Biochim. Biophys. Acta 527: 497-500). It is to be remarked that after infection with virulent strains often a slight thickening around the wound was to be observed. Thick tumours were not formed. In the case of infection with avirulent strains no thickening was observed at all. Plant material isolated from non-wounded or wounded, but noninfected monocotyledonous plants appeared to contain neither octopine nor nopaline. Opines were not discovered either in plant material isolated from plants infected with the avirulent strains of LBA288 or LBA1516. However, plant cells obtained from wound sites infected with virulent A. tumefaciens strains appeared to contain nearly always opines. After infection with a strain with an octopine Ti-plasmid (LBA1010) octopine appeared to be present, whilst after infection with strains with a nopaline Ti-plasmid (LBA2318, LBA2347) nopaline appeared to be present. The results obtained with strain LBA2347 gave the most reproducible positive results. All these data together tender evidence that A. tumefaciens is capable of transmitting T-DNA to monocotyledonous plant cells, and furthermore that T-DNA genes (at any rate those involved for the enzymes in che biosynthesis of opines) express in monocotyledonous plant cells Consequently, it can be concluded that the Ti-plasmid is suitable as a vector for monocotyledonous plant cells. Furthermore, the conclusion can be drawn that the regulator zones which see to the expression of the opine synthesis are function regions and so suitable as buildingstones for making chimaeric genes which should express in monocotyledonous plant-cells. Consequently, the invention relates to a process for incorporating foreign DNA into monocotyledonous plants by infecting the plants or incubating plant protoplasts with bacteria suitable or made suitable for that purpose which avail of one or more tumourinducing plasmids, for instance Ti- or Ri-plasmids, or derivatives thereof, or parts thereof, originally originating from Agrobacterium bacteria. Preferably, use is made of the Agrobacterium bacteria, which has at least one plasmid which has the Vir-region of a Ti (tumour inducing) plasmid but no T-region and at least one other plasmid which has a Ti-region with built in it foreign DNA but no Virregion. For the Agrobacterium bacteria, which are suitable for the process according to the invention as well as for the way in which these bacteria can be obtained rights were already claimed in the non-prepublished European patent application 84200239.6. For expressing foreign genes in monocotyledonous plant cells use can be made of the regulator regions of T-DNA-genes, in particular the genes for octopine synthesis and nopaline synthesis.

Claims: CLAIMS 1. A process for the incorporation of foreign DNA into the genome of plants by infecting these plants or incubating the plant protoplasts with bacteria suitable or made suitable for that purpose, characterized in that monocotyledonous plants are infected or monocotyledonous plant protoplasts are incubated with bacteria suitable or made suitable for that purpose, which are provided with one or more tumour-inducing plasmids or derivatives thereof, originally originating from Agrobacterium, or from bacteria, which have the T-DNA, originating from the above-meant plasmids and/or the virulence genes originating from the above mentioned plasmids incorporated elsewhere in the bacterial DNA. 2. A process according to claim 1, characterized in that for the infection or incubation use is made of Agrobacterium or Rhizobium bacteria. 3. A process according to claim 1 or 2, characterized in that bacteria are used which are provided with one or more Ti- or Riplasmids or derivatives thereof. 4. A process according to claim 3, characterized in that the bacteria used are provided with a stable cointegrate plasmid constructed from a plasmid R772 and a plasmid pTiB6 with foreign DNA incorporated into the T-region of the latter. 5. A process according to any of the preceding claims, characterized in that bacteria are used, which contain at least one plasmid, which has the Vir-region of a Ti (tumour inducing) plasmid but no T-region, and at least one other plasmid, which has a T-region with built in therein foreign DNA but no Vir-region. 6. Monocotyledonous plant and plant cells obtained after that by applying the process according to any of the preceding claims the genetic properties of the original plants c.q. plant cells have been modified. 7. A process for the preparation of chemical and/or pharmaceutical products, characterized in that cells obtained by application of the process according to any of the claims 1-5 are cultivated and the desirable substance is isolated. 8. A process according to claim 7, characterized in that the cultivating is carried out by means of fermentation and optionally subsequent immobilisation. 9. A process according to any of the claims 1-5 incl., 7 or 8, characterized in that the regulator regions positioned before and after the protein encoding regions of T-DNA-genes in particular the genes for octopine synthesis and nopaline synthesis are used for expressing foreign genes in monocotyledonous plant cells.

Inventors: HOOYKAAS, PAUL JAN JACOB, DR., SCHILPEROORT, ROBBERT ADRIAAN, PROF. DR., VAN SLOGTEREN, GEERTRUI MARTHA SIBRIG, DR.
Applicants: RIJKSUNIVERSITEIT LEIDEN, SCHILPEROORT, ROBBERT ADRIAAN, PROF. DR.
Application year: 1984
Main classification: C12N15
IPC codes: C12N15, C12P1, A01H1, C12R1, C12N5