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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
  • C12N15/8223—Vegetative tissue-specific promoters
  • C12N15/8227—Root-specific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

• the present invention relates to a plant promoter.
• Extensin is the best characterised structural protein in the walls of plant cells (Cassab and Varner, Ann. Rev. Plant Physiol. .39., 321-353, 1988). It is a member of a class of highly basic, hydroxyproline-rich glycoproteins present in a wide variety of plants. Extensin has been reported to be present at greatest abundance in sclerenchyma tissues and a variety of functional roles have been ascribed to the protein, including mechanical strengthening and organisation of the cell wall. Extensins vary in their primary structure in different plants, organs and tissues. In some cases, there are two different extensins within the same tissue or cell type, which may be function-related.
• mRNA species present in the roots of oilseed rape (Brassica napus L. ) were investigated, with the aim of isolating sequences abundant in root but not in other tissues.
• a family of cross- hybridising sequences present at highly enhanced levels in root has been isolated, characterised, and shown to encode proteins homologous to extensin. This has enabled us to identify a promoter capable of directing the expression of proteins in roots.
• the present invention provides a promoter which is capable of directing protein expression in roots of plants and which has:
• the invention also provides a DNA fragment comprising such a promoter operably linked to a gene, typically a heterologous gene encoding a protein.
• a vector which comprises a gene encoding a protein under the control of a promoter as above such that the gene is capable of being expressed in a plant cell transformed with the vector.
• a suitable vector is one in which the promoter is fused directly to the 5'-end of the gene.
• the vector may further contain a region which enables the gene and the promoter to be transferred to and stably integrated in a plant cell genome.
• the vector is generally a plasmid.
• Plant cells can be transformed with such a vector.
• the invention therefore further provides plant cells which harbour a promoter as above operably linked to a gene encoding a protein.
• Transgenic plants may be regenerated from such plant cells.
• a transgenic plant can be obtained which harbours in its cells a promoter as above operably linked to a gene encoding a protein. Seed may be obtained from the transgenic plants. Plants may in turn be grown from this seed.
• the invention additionally provides a method of producing a transgenic plant capable of producing a desired protein in the roots of the plant, which method comprises:
• Figure 1 shows the nucleotide sequence of gene extA, with the predicted amino acid sequence of the rape extensin polypeptide, and the sequence of the promoter of the invention from nucleotide -1616 to nucleotide -1.
• the complete sequence is the sequence of the 2.7kb HincII-HincII fragment shown in Figure 2.
• the predicted site of cleavage of the leader sequence is indicated by a colon (: ) .
• the transcription start point is indicated by a circumflex (T ⁇ S) , and a sequence similar to the consensus "TATA" control box is also indicated. Other sequence features are as indicated on the Figure.
• Figure 2 is a restriction map of the oilseed rape genomic clone lambdaB31 (12.7 kb) encoding gene extA.
• a restriction map of pRlambdaS4, a Hindlll-Aval fragment from lambdaB31 in pUCl ⁇ , and a sequencing map of the 2.7kb HincII-HincII fragment containing extA. ra and la, indicate right arm and left arm of the EMBL 3 lambda vector, respectively; indicates coding sequence.
• Sa/H2-Al/Sm/Xl fragment is a part of EMBL 3.
• Figure 3 shows a detailed restriction map of pRlambdaS4, a sub-clone from comprising a Hindlll-Aval fragment of lambdaB31 in pUCl ⁇ .M shows the location and direction of transcription of the extensin coding sequence. There are no sites in the insert for Pvul, Xhol, BscI, BamHI, Bglll and EcoRV.
• Figure 4 shows a simplified restriction map of the sub-clone pRlambdaS4;ESS1 indicates the position of the extensin coding sequence and V I indicates the region containing the promoter conferring root expression.
• Figure 5 is a map of the hybrid gene employed in Example 2; HHI indicates the nopaline synthase (NOS) terminator.
• NOS nopaline synthase
• Figure 6 is a map of. the hybrid gene from Figure 5 in the vector BIN 19.
• Figure 7 shows the rape extensin promoter on a Haelll fragment excised from lambdaB31, as described in Example 3.
• Figure 8 shows the fusion construct comprising the rape extensin promoter and the glucuronidase gene coding sequence, prepared in Example 3.
• the full length promoter of the invention is composed of the sequence upstream of the oilseed rape extA gene, from nucleotide -1616 to nucleotide -1 in Figure 1, base 1 being the adenine (A) base of the CAT transcriptional start codon for the extA gene.
• the promoter may be obtained from a clone, lambdaB31 by excision on a 4.5kb Hindlll-Aval fragment ( Figure 3) .
• Clone lambdaB31 was obtained from a genomic library prepared from oilseed rape (Brassica napus L) .
• Hindlll-Aval fragment was subcloned in p ⁇ C18 as pRlambdaS4 from which the promoter can be excised as a 0.96kb Haelll fragment.
• E. coli DH5 ⁇ harbouring pRlambdaS4 and bacteriophase lambdaB31 have been deposited at the National Collection of -Industrial and Marine Bacteria, Aberdeen, GB on 8 March 1990 under accession numbers NCIMB 40265 and NCIMB 40266.
• a part of the full length sequence from nucleotide -1616 to nucleotide -1 and modified forms of the full length or part sequence are alternative promoters according to the invention.
• the full length or part promoter sequence may be modified by one or more nucleotide substitutions, insertions and/or deletions and/or by an extension at either or both ends. Any part or modified promoter sequence must however still be capable of acting as a promoter in the roots of plants.
• the full length sequence or a part of the full length sequence i.e. an unmodified sequence
• is modified typically there is a degree of homology of at least 60% between a modified sequence and the unmodified natural sequence. The degree of homology may be at least 75%, at least 85% or at least 95%.
• a part of the full length promoter sequence may be obtained by use of restriction endonucleases and/or exonucleases.
• the promoter may be obtained as a 0.96 kb fragment by treating the sequence shown in Figure 1 with Haelll.
• a modified sequence may be obtained by introducing changes into an unmodified promoter sequence. This may be achieved by any appropriate technique, including restriction of the natural sequence with an endonuclease, insertion of oligonucleotide linker adapters, use of an exonuclease and/or a polymerase and site-directed mutagenesis.
• a part of the full length promoter sequence or a modified sequence is capable of acting as a promoter may be readily ascertained by experiment.
• the putative promoter sequence is fused to the glucuronidase coding sequence in the promoter-less binary vector pBHOl at the Smal site as described in Example 3. Following the procedure of Example 3, expression of glucuronidase is investigated in the transgenic hairy roots produced.
• the promoter may be operably linked to a gene, typically a heterologous gene, encoding a protein.
• the heterologous gene may encode any protein it is desired to express. "Heterologous" means that the gene is not naturally operably linked to the promoter, i.e. a heterologous gene is not the oilseed rape extA gene.
• the protein may additionally comprise a transit peptide sequence at its N-terminus, encoded within the "heterologous" gene sequence.
• the promoter is typically used to express proteins in the roots of plants.
• the protein whose expression is controlled by the promoter may be for example a protein conferring biological control of pests or pathogens, in particular pests or pathogens to which the roots of plants are susceptible.
• the promoter sequence may be fused directly to a gene or via a linker.
• the linker sequence may comprise an intron. Excluding the length of any intron sequence, the linker may be composed of up to 45 bases, for example up to 30 or up to 15 bases.
• DNA fragments and vectors can be prepared in which the promoter is operably linked to a gene, typically a heterologous gene.
• the fragments and vectors may be single or double stranded. Plant cells can be transformed by such fragment by direct DNA uptake, or by way of such a vector.
• the vector incorporates the gene under the control of the promoter.
• the vector contains regulatory elements capable of enabling the gene to be expressed in a plant cell transformed with the vector. Such regulatory elements include, besides the promoter, translational initiation and/or termination sequences.
• the vector typically also contains a region which enables the gene and associated regulatory control elements to be transferred to and be stably integrated in the plant cell genome.
• the vector is therefore typically provided with transcriptional regulatory sequences and/or, if not present at the 3'-end of the coding sequence of the gene, a stop codon.
• a DNA fragment may therefore also incorporate a terminator sequence and other sequences which are capable of enabling the gene to be expressed in plant cells.
• An enhancer or other element able to increase or decrease levels of expression obtained in particular parts of a plant or under certain conditions may be provided in the DNA fragment and/or vector.
• the vector is also typically provided with an antibiotic resistance gene which confers resistance on transformed plant cells, allowing transformed cells, tissues and plants to be selected by growth on appropriate media containing the antibiotic.
• Transformed cells are selected by growth in an appropriate medium.
• Plant tissue can therefore be obtained comprising a plant cell which harbours the gene under the control of the promoter , for example in the plant cell genome.
• the gene is therefore expressible in the plant cell.
• Plants can then be regenerated which include the gene and the promoter in their cells, for example integrated in the plant cell genome, such that the gene can be expressed.
• the regenerated plants can be reproduced and, for example, seed obtained. Root-specific expression of proteins can therefore be directed by the present promoter in plants.
• transformed roots may be grown in culture for the purposes of production of proteins, especially plant proteins.
• a preferred way of transforming a plant cell is to use A ⁇ robacterium tumefaciens containing a vector comprising the promoter operably linked to the gene encoding a protein it is wished to express.
• a hybrid plasmid vector may therefore be employed which comprises:
• the DNA to be integrated into the plant cell genome is delineated by the T-DNA border sequences of a Ti-plasmid. If only one border sequence is present, it is preferably the right border sequence.
• the DNA sequence which enables the DNA to be transferred to the plant cell genome is generally the virulence (vir) region of a Ti-plasmid.
• the gene and its transcriptional and translational control elements, including the promoter can therefore be provided between the T-DNA borders of a Ti-plasmid.
• the plasmid may be a disarmed Ti-plasmid from which the genes for tumorigenicity have been deleted.
• the gene and its transcriptional and control elements, including the promoter can, however, be provided between T-DNA borders in a binary vector in trans with a Ti-plasmid with a vir region.
• Such a binary vector therefore comprises:
• Acrrobacterium tumefaciens therefore, containing a hybrid plasmid vector or a binary vector .in trans with a Ti-plasmid possessing a vir region can be used to transform plant cells.
• Tissue explants such as stems or leaf discs may be inoculated with the bacterium.
• the bacterium may be co-cultured with regenerating plant protoplasts.
• Plant protoplasts may also be transformed by direct introduction of DNA fragments which encode the gene of interest and in which the promoter and appropriate other transcriptional and translational control elements are present or of a vector incorporating such a fragment. Direct introduction may be achieved using electroporation, polyethylene glycol, microinjection or particle bombardment.
• Plant cells from monocotyledonous or dicotyledonous plants can be transformed according to the present invention.
• Monocotyledonous species include barley, wheat, maize and rice.
• Dicotyledonous species include tobacco, tomato, sunflower, petunia, cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rapeseed) and poplars.
• Tissue cultures of transformed plant cells are propagated to regenerate differentiated transformed whole plants.
• the transformed plant cells may be cultured on a suitable medium, preferably a selectable growth medium. Plants may be regenerated from the resulting callus. Transgenic plants are thereby obtained whose cells harbour the promoter operably linked to the gene encoding the protein it is wished to express, for example integrated in their genome. The gene is consequently expressible in the cells. Seed from the regenerated plants can be collected for future use, and plants grown from this seed.
• a family of cross-hybridising cDNA clones was isolated from a cDNA library produced with poly(A)+RNA from the roots of oilseed rape (Brassica napus L. ) .
• the clones were selected as abundantly expressed in root by differential screening of the root cDNA library with cDNA probes prepared from root, green leaf, etiolated leaf and developing seed.
• mRNA species corresponding to the selected abundant clones were expressed in roots at levels of at least 400 x those in other organs, as shown by Northern blot analysis and RNase protection assays.
• One of the cDNA clones was designated pRR ⁇ 566.
• An extensin gene designated extA. was obtained from an oilseed rape (Brassica napus L.) genomic library screened with p Rt566 at high stringency.
• the gene is a member of a multigene family, consisting of about three members per haploid genome with strong homology to the probe, and a further twenty or so members of weaker homology.
• the isolated gene although not identical to the cDNA probe, was also found to be specifically expressed in roots, and was transcribed into a mRNA species of approximately 1300 nucleotides in size. A single transcription start was identified by SI mapping. A complete nucleotide sequence of the extA gene and its flanking regions was determined. This enabled the gene promoter to be identified.
• the insert of pRR- ⁇ 566 hybridised to a large number of genomic fragments in all restriction digests, but the strength of hybridisation to all fragments was not equal; 1-2 fragments in all digests hybridised at an intensity at least five times that of the other bands.
• the weakly hybridising fragments were present at less than one estimated copy per haploid genome, and must therefore represent genes that are only partly homologous to the extensin probe.
• the total sequences detected by this probe make up a large multigene family, of which three genes, determined to be closely homologous to pRR-t-566, form a sub-family.
• hybridisation of this probe to the strongly hybridising bands was identical to that using the whole cDNA insert as a probe, except hybridisation to the pRRt566 weakly hybridising bands was largely eliminated.
• An oilseed rape genomic library constructed by inserting random Sau 3A fragments of genomic DNA, into lambda EMBL3, was screened at low stringency with a probe prepared from the insert of This cDNA clone was isolated from a library prepared from rape root mRNA, and encoded part of an extensin polypeptide. Forty positive phage plaques were identified, and were extensively plaque- purified. The isolated clones were then re-screened under high stringency conditions (0.1 x SSC, 0.1% SDS at 65°C) , when only one phage clone gave continued strong hybridisation to the cDNA probe. This clone, lambdaB31, was selected for further study.
• DNA from the selected phage was isolated and characterised by restriction and Southern blotting, using pR -t566 as a probe.
• the restriction map of lambdaB31 obtained is shown in Fig. 2, and shows that the clone contains an insert of 12.7 kb.
• the region of DNA hybridising to the cDNA probe was localised to a fragment of 1.0 kb (Nsi I-Pst I) adjacent to the left arm of the vector.
• restriction with Dde I and Rsa I was carried out, followed by Southern blotting and probing with the cDNA as before.
• the first 23 residues of the sequence encode a leader sequence, when compared with other extensin sequences and as defined by the rules of von Heijne (1965, J. Mol. Biol. 164. / 99-105).
• the remainder of the coding sequence encodes a highly proline-rich polypeptide of 276 amino acids.
• the promoter of the invention is the 5' flanking sequence of the extA gene as shown in Figure 1.
• This sequence contains a sequence (CTATATAAA) closely homologous to the consensus "TATA" box for plants seventy-four bases 5' to the initiation codon. Apart from this no significant features can be recognised.
• Comparison of this 5 1 flanking sequence with that of the carrot extensin gene pDC5Al (Chen and Varner, EMBO J. 4., 2145-2151, 1985) reveals no strongly conserved sequence regions, apart from the "TATA" box.
• extA represents an expressed gene, and to determine its transcription start, a series of SI mapping experiments were carried out. Fragments of extA 5 1 end-labelled at a Dde I site (base 71) and extending in a 5' direction to the Nsi I site (base -74) or the Dde I site (base -237) were isolated and hybridised to poly(A)+RNA from rape roots. After treatment with SI nuclease, the protected fragments were sized by polyacrylamide gel electrophoresis. In both cases, protected fragments of 66-75 bases were obtained with the strongest band corresponding to the underlined base in the sequence TAAGAGCATCAAAC, which was designated base +1 (indiated in Figure 1 by T ⁇ S) .
• fragments of the gene were used as probes on Northern blots of RNA from different rape organs.
• a probe consisting of the complete coding sequence of the gene, and extending to base -74, was hybridised to RNA from four rape organs: root, green leaf, etiolated leaf and developing seed. This probe hybridised to two mRNA species in root, of approx. 1300 and 1480 bases, and hybridised only very weakly to a mRNA species in developing seeds, of approx. 1600 bases.
• pNOP-NEO is a clone containing - the NOS promoter linked to the Neomycin Phosphotransferase (NPT) gene which encodes kanamycin resistance, linked to the NOS terminator as published by Bevan, Nucleic Acids Research 12, 8711-6721 (19 ⁇ 4) .
• NPT Neomycin Phosphotransferase
• the extA gene excised from pRlambdaS4 on a 4.75kb Hindlll-Sall fragment, was then ligated into the NOS terminator pUCl ⁇ clone, also restricted with Hindlll-Sall, and cloned in E. coli DH5 ' ⁇ .
• the final extA - NOS terminator construct is shown in Figure 5.
• the whole extA - NOS terminator construct was then excised on an intact 5.01kb Hindlll-EcoRI fragment and ligated into the binary vector pBIN19 (Bevan, 1984) restricted with Hindlll-EcoRI, and cloned into E. coli MC1022.
• the final hybrid gene construct designated pBIN19:extA is shown in Figure 6 and contains additionally the endogenous kanamycin resistance gene (NPT) located between the T-DNA borders.
• NPT endogenous kanamycin resistance gene
• pBIN19:extA was triparentally mated into A. tumefaciens LBA4404 and used to transform leaf discs of Nicotiana tabacum (SRI) .
• SRI Nicotiana tabacum
• the vector plasmid pBIN19 in Escherichia coli strain MC1022 (Bevan, Nucl. Acids Res. 12., 8711-8721, 1984) , the mobilising E. coli strain HB101/pRK2013 (Ditta et al, PNAS USA 72, 7347-7351, 1980) and the host Agrobacterium tumefaciens strain LBA4404/pAL4404 (Hoekema et al, Nature 303. 179-180, 1983) were used.
• kanamycin acid sulphate 50 ⁇ g.ml ⁇ (kan) ; rifampicin, lOO ⁇ g.ml “ (rif) ; streptomycin sulphate, 500 ⁇ g.ml " (strep).
• the recombinant vector BIN19:ext A was mobilized in a triparental mating by mixing 200 ⁇ l each of overnight cultures of E. coli MC1022/BIN19 (kan R , strep s , rif s ) , E. coli HB101/pRK2013 (kan R , ⁇ trep s , rif s ) and A.
• tumefaciens LBA4404 (kan s , strep R , rif R ) , and incubating at 27°C on a LB plate without antibiotic selection for 15h.
• the cell mixture was then diluted with 10 mM MgS0 4 ; plated out on minimal medium containing kanamycin and incubated at 27°C for 4 days.
• the A. tumefaciens LBA4404 colonies were checked for the correct antibiotics resistance markers (kan , strep R , rif R ) , and further analysed by colony hybridisation and Southern analysis to confirm that the npt and ext A genes were present in an intact, unrearranged form in the BIN19:ext A plasmid.
• Root-induction medium comprising half-strength MS salts, lOO ⁇ g.ml " carbenicillin, lOO ⁇ g.ml -1 kanamycin and 8 mg.ml-1 agar.
• Tissues from the transgenic plants were harvested into liquid air for extraction of DNA, for genomic analysis to assess the integrity of the transferred gene, and RNA, for analyses of extensin gene expression.
• the potted plants flowered after about five weeks, and following self-pollination, the seeds from each plant were collected at dehiscence. Extraction of DNA and Southern Analysis
• the DNA from N. tabacum leaves was extracted and purified.
• the DNA probes for hybridization were:
• RNAs from tissues of the transgenic N. tabacum plants were isolated by the procedure of Logemann et al. (Anal. Biochem. 163. 16-20, 1987). To estimate the size of RNAs specifically hybridizing to the rape extensin probe, lO ⁇ g of each sample RNA was glyoxalated, run on agarose gels, and transferred to nitrocellulose. Northern blots were washed to high stringency (15 mM NaCl, 1.5 mM Na 3 citrate, 0.1% sodium dodecyl sulphate (SDS) , 25 min, 50°C) , and the filters autoradiographed for 2 weeks as described above.
• SDS sodium dodecyl sulphate
• transgenic tobacco plants were regenerated and grown from the initial transformation experiment. Samples of leaf and root were collected for analyses by Southern and Northern blotting. The majority of the transgenic plants regenerated contained intact, unrearranged copies of the introduced hybrid extensin gene. Estimates of the copy number of the introduced gene in the various independent transformants varied from one copy to five copies. Only transgenic tobacco plants containing intact, unrearranged copies of the introduced gene were used for further analysis of extensin expression.
• Seeds were collected from the initial batch of transformed tobacco, and were sown in compost. Plants were grown from this seed.
• the rape promoter from lambdaB31 was fused in a translational fusion to the coding sequence of the glucuronidase (GUS) gene in a binary vector variant of BIN19.
• the clone containing the construct was mated with an Agrobacterium strain (LBA 9402) containing an oncogenic Ri plasmid, pRi 1885 (Constantino et al, Plasmid 5_, 170-182, 1981) . Inoculation of this strain into rape seedlings produces transgenic hairy roots, which are then easily assayable for GUS activity. Whole plants can be regenerated from excised transgenic hairy roots.
• the rape extensin promoter was excised from pRlambdaS4 as a l.Okb Haelll-Haelll fragment. This fragment was blunt-end ligated into a modified pBIN19 binary vector, pBIlOl containing a promoterless glucuronidase (GUS) gene, restricted with Smal at the start of the GUS coding sequence (see Jefferson, Plant Molecular Biology Reporter 5_, 387-405, 1987) ( Figure 8) .
• GUS promoterless glucuronidase
• the rape promoter expression from this translational fusion will give a glucuronidase enzyme with either 7 or 11 extra amino acids at the N- terminus, depending on which rape ATG is used as the translation initiation codon.
• extA-GUS hybrid gene in pBIlOl was cloned in E. coli MC1022 and then transferred into Agrobacterium tumefaciens or Agrobacterium rhizogenes by tri-parental atings with the appropriate Agrobacterium strains. Preliminary results to test for expression from this construct, using the.
• X-Gluc (5-bromo-4-chloro-3- indolyl-3-D-glucuronide) histochemical assay of Jefferson (Plant Molecular Biology Reporter 5, 387-405, 1987) in rape •hairy roots' transformed with Agrobacterium rhizogenes containing pBIlOl:extA-GUS, have clearly shown that the promoter is active in rape root tissue.
• Rape Tissue culture and transformation protocols Transformation experiments using A. rhizogenes
• Hairy roots arising from the site of inoculation can be excised and grown in a culture medium (1 x MS salts and vitamins, 4% sucrose, 2 g/1 CaCl 2 .H 2 0, 0.1 mg/1 NAA, 2.5 mg/1 BAP) 0.1 mg/1 thiamine, 200 mg/1 cephotaxime, 50 mg/1 kanamycin, 8 g/1 Bacto Difco agar, pH 5.8, to proliferate the hairy roots.
• a culture medium (1 x MS salts and vitamins, 4% sucrose, 2 g/1 CaCl 2 .H 2 0, 0.1 mg/1 NAA, 2.5 mg/1 BAP) 0.1 mg/1 thiamine, 200 mg/1 cephotaxime, 50 mg/1 kanamycin, 8 g/1 Bacto Difco agar, pH 5.8, to proliferate the hairy roots.
• Regeneration can be achieved as follows: 0.5 cm sections of the terminal 2 - 3 cm of hairy roots are excised, treated with 2,4 - D at a concentration of 3 mg/1, and plated on shooting medium containing kanamycin at 50 mg/1 to select for tissues which contained the T-DNA kanamycin gene of the fusion construct of Figure 8.
• Shoots arising from these hairy roots are excised and grown on medium F of Pelletier et al (Mol. Gen. Genet. 191, 244-250 (1983)) for 2-3 weeks. They are then transferred to rooting medium G, and when a sufficient root system has developed they are transferred to soil and grown to maturity in a containment growth room.
• Transgenic winter rape plants are vernalised to induce flowering by growing them at 4°C with a 8h daylight period. Flowers are emasculated before anthesis and outcrossed with wild type rape pollen. Seeds obtained after pod set are collected, sown in soil and the progeny plants grown to maturity. The progeny plants are initially scored visually with regard to the degree of the hairy root syndrome being exhibited. Kanamycin resistance in the leaves of the progeny is assayed by transferring leaf discs to callus inducing media (see below) containing kanamycin at either 50 or 150 mg/1. Both the Ri and the kanamycin resistance traits can be further confirmed by Southern hybridisation analysis.
• the media used in the Examples are: Media
• D solution, RCC medium, F and G media are as described by Pelletier et al. (1983) and Guerche et al. (Mol. Gen. Genet. 206, 382-386, 1987) .
• a level of Cephotaxime of 200 mg/1 is maintained in all these media at all times.

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