WO2000001223A1

WO2000001223A1 - LECTINS TO CONFER RESISTANCE TO INSECTS IN $i(BRASSICA)

Info

Publication number: WO2000001223A1
Application number: PCT/SE1999/001209
Authority: WO
Inventors: Inger ÅHMAN; Margareta Melander; Kristofer Vamling
Original assignee: Plant Science Sverige Ab
Priority date: 1998-07-03
Filing date: 1999-07-02
Publication date: 2000-01-13
Also published as: SE512197C2; SE9802425D0; SE9802425L; AU5075399A

Abstract

The present invention relates to a transgenic Brassica plant resistant to Brassica-attacking insect pests, comprising in its genome at least one DNA sequence coding for a foreign lectin inhibitory to said insect pests together with a promoter active in the Brassica plant, as well as an expression cassette comprising a DNA clone which codes for said lectin (lectins), a method of imparting resistance to insects, and a new lectin; a method for protecting Brassica.

Description

ECTINS TO CONFER RESISTANCE TO INSECTS IN BRASSICA

TECHNICAL FIELD

The present invention relates to a transformed Brassica plant resistant to certain insect pests, an expression cassette containing a DNA, which codes sub- stantially for at least one lectin selected primarily from Concanavalin A (Con A) , modified Concanavalin A, and pea lectin; transgenic plant cells containing as foreign DNA at least one copy of the above-mentioned DNA; a new lectin derived from the jack bean lectin gene (Con A gene) ; a method of imparting resistance to insects selected from blossom beetles (synonymous with pollen beetles) of the genus Meligethes, flea beetles of the genus Phyllotreta , and root flies of the genus Delia of said genera; and a method for protecting a plant against infestation by insects selected from said genera. BACKGROUND OF THE INVENTION

Insect-resistant cultivars are essential in a sustainable agricultural system. In modern agriculture, however, such cultivars are rare, and insect control therefore mostly relies on the use of insecticides. For environmental reasons, a minimisation of the use of insecticides is desirable, and one way of reducing the use is to utilise plant breeding to impart insect resistance. During the last 10-15 years new tools have been de- veloped, which can be used to make breeding for resistance to insects more effective. These methods include isolation and cloning of single genes for transfer to the genome of crop plants. By such means, crops like potato, maize, rice and cotton have been transformed to include foreign genes conferring resistance to important insect pests. A great advantage of this technique is that the origin of the gene is not restricted to the species of the crop, not even to higher plants. The fact is that the most utilised genes for this application so far are genes from an insect-pathogenic bacterium, Bacillus thuringi ensis . However, also higher plant genes have been used. Genes coding for proteinase inhibitors, α-amylase inhibitors and lectins belong to this category. Plant lectins are defined as "all plant proteins that possess at least one noncatalytic domain that binds reversibly to a specific mono- or oligosaccharide" (Peumans & Van Damme 1995) . From the scientific literature (e.g. Peumans & Van Damme 1995) as well as from patent documents (e.g. EP-A2-0 , 351, 924 (Shell Internationale Research Maatschappij B.V.); EP-A2-0 , 427 , 529 (Pioneer Hi-Bred International, Inc.); O-A1-9202139 (Agricultural Genetics Company Limited) ; O-A1-9304177 (Agricultural Genetics Co Ltd); CA-A1-2 , 122 , 785 (DowElanco) ) it is known that lectins function as plant- defensive chemical compounds.

Thus, it is known from EP-A2-0 , 351 , 924 that lectins have pesticidal activity, especially insecticidal activity. A transgenic plant is described, which comprises a lectin gene to which a promoter regulating the gene has been attached. The lectin is foreign to the plant as found in nature. Preferably, the plant is a member of the family Solanaceae.

EP-A2-0, 427 , 529 discloses a method for protecting a plant against infestation by insects selected from

European corn borer, corn rootworm and cutworm, said method comprising inserting into the genome of the plant a sequence coding for at least one selected larvicidal plant lectin. However, Con A, pea, potato and peanut lectins were found not to have any significant activity. The plant is preferably a monocotyledonous species selected from corn, wheat, rice and sorghum. O-A1-9202139 discloses a transgenic plant containing and capable of expressing a gene coding for a lectin having specific mannose-binding ability, in particular a lectin from Amaryllidaceae or Alliaceae. The only example of a plant transformed with a lectin gene is a plant of Nicotiana species transformed with the lectin gene from Galanthus nivaliε . O-A1-9304177 relates to protection of plants by means of proteins having toxic effects on Homopteran insects only. Galanthus nivalis and wheat germ lectins are mentioned as examples of such proteins.

CA-A1-2 , 122 , 785 discloses a transgenic maize plant resistant to attack by an insect selected from Western corn rootworm or Northern corn rootworm. This transgenic maize plant expresses an insect controlling amount of PNA lectin, amaranthin lectin or Con A.

However, the sensitivity to a particular lectin differs between insect species (see e.g. Czapla & Lang 1990; Rabhe et al 1995) and lectins belonging to the same class, based on sugar affinity, may differ widely in toxicity to a certain insect. Thus, with the present knowledge, it is not possible to predict which lectin will be adverse to a certain insect pest.

This is also elucidated by the test results in the different patent specifications mentioned above, and in particular the description of EP-A2-0 , 427 , 529 shows that the one skilled in the art cannot draw any conclusions whatsoever that an isolated lectin will be effective. Out of about 45 tested lectins, 11 had an anti-insect activity against neonate European cornborer and Southern corn rootworm larvae. One of two lectins from each of Griffonia simplici folia and Wistaria floribunda was insecticidal while one did not show any insecticial effect in the tests carried out. In sensitive insects, lectins impair growth and survival if applied in sufficiently high doses. There are various suggestions as to how lectins interfere with the performance of insects. Digestion may be impaired by lectins blocking the channels of the peritrophic membrane, lectins binding to glycoconjugates of the epithelial gut cells or lectins binding to digestive enzymes carrying sugar moieties (cf. e.g. Eisemann et al 1994; Harper et al 1995) .

Damage by insect pests is a limiting factor in Brassica oilseed production. The key pests differ with respect to geographical area of cultivation and whether the crop is a winter or spring type. In countries like Sweden and Denmark, blossom beetles of the genus Meligethes, especially the species Meligethes aeneus , are the major pests, and spring rape and spring turnip rape can hardly be grown without pesticide treatment (Nilsson 1987) . Even in cultivation of winter types, spraying with insecticide against blossom beetles is usually carried out .

The search for genotypes which may serve as sources for resistance genes in a breeding programme for resistance to M. aeneus has not been successful (Ahman 1993) . Furthermore, since there are high demands on seed quality and yield in oilseed rape, traditional breeding using resistance genes from agronomically primitive plants would require an extensive back-crossing programme.

The conditions are similar for resistance breeding to another important pest complex in Brassica oilseeds, viz. the flea beetles, Phyllotreta spp. These beetles cause severe problems in spring sown rape and turnip rape in northern Europe and also in Canada by feeding on seedlings, which may cause plant death. Thus, Brassica cultivation would profit from transgenic introduction of plant genes conferring resistance to these pests. Other important pests in Brassica cultivation are root flies of the genus Delia, such as Delia radicum and Delia floral is .

SUMMARY OF THE INVENTION

It has now been found possible to make Brassica plants resistant to insect pests by transforming them with DNA sequences coding for specific lectins which are inhibitory to said insect pests. Thus, in one of its aspects, the invention provides a transgenic Brassica plant resistant to Brassica- attacking insect pests, comprising in its genome at least one DNA sequence coding for a lectin inhibitory to said insect pests together with a suitable promoter active in the tissues of Brassica plants attacked by the pests.

In a further aspect of the invention, there is provided an expression cassette comprising DNA, which codes for one or more lectins operably linked to plant- regulating sequences, which results in expression of the DNA in the cells of Brassica species.

In another aspect, the invention relates to propagated plant material, derived in one or more generations, via cloning, selfing and/or hybridisation, of the transgenic Brassica plant mentioned above.

In another of its aspects, the invention provides a method of rendering a Brassica plant resistant to Brassica-attacking insect pests, which method comprises transforming the Brassica plant to express a lectin inhibitory to said insect pests. In an especially preferred embodiment, the invention relates to insect pests belonging to the Meligethes, Phyllotreta and Delia genera .

Lectins suitable for use in the transformation according to the invention are the following lectins, which represent three different saccharide-binding specificity groups: jack bean ( Canavalia ensiformis) lectin (Concanavalin A or Con A) , a new modified Con A lectin, obtained by site-specific mutation of the Con A gene, pea { Pisum sativum) lectin, all three belonging to the glucose/mannose-specific group, as well as wheat ( Tri ticum aestivu ) germ lectin belonging to the N- acetylglucosamine group, potato ( Solanum tuberosum) lectin which also belongs to the N-acetylglucosamine group and peanut {Arachis hypogaea) lectin belonging to the N-acetylgalactosamine/galactose group (Liener et al 1986) . None of the lectins mentioned is specifically mannose-binding (cf. WO-A-9202139 and the corresponding US-A-5,545,820.

Preferably the lectin is jack bean lectin (Concanavalin A) , modified Concanavalin A lectin and/or pea lectin.

For both Con A lectin and pea lectin there are closely related variants that can be isolated from related plant species. These variants are lectins that possess a biological activity that is substantially similar to the biological activity cf Con A or pea lectin. It is also possible to engineer variants of Con A, modified Con A, or pea lectin by different techniques used in molecular biology. For the purposes of the present invention, a variant is a protein, in which the active portions of the amino acid sequences are identical or equivalent to at least 80%, preferably at least 90%. Groups of equivalent amino acids are defined below, where an amino acid within a group can be substituted for another amino acid in that group: (1) glycine and alanine; (2) alanine, valine, leucine and isoleucine; (3) serine and threonine; (4) phenylalanine, tyrosine and tryptophan; (5) lysine, arginine and histidine; (6) aspartic acid and glutamic acid; and (7) asparagine and glutamine . The promoter is chosen such that the inserted DNA results in the production of the lectin in a suitable part or parts of the plant, plan:; parts that are subjected to damage by the specific insect to be controlled. Thus, to inhibit the growth and/or survival of Meligethes spp, the promoter should be active in stamens and/or other flower/bud parts. To inhibit the feeding of Phyllotreta spp, the promoter should be active in cotyledons, and to inhibit Delia spp, the promoter should be active in roots. The invention will now be described in more detail with reference to the following non-limiting examples. Provision of test chemicals

Con A, pea lectin, wheat germ lectin, potato lectin, and peanut lectin were used in feeding assays. The following lectins were purchased from Sigma Chemical Co.; jack bean C2010, wheat germ L9640, peanut L0881, and pea L5380.

Potato lectin was purified according to Matsumoto et al (1983) with some modifications. Approximately 0.5 kg washed potato tubers were homogenised in 0.5 1 of cold (4 °C) 3 M acetic acid, filtered through cheese-cloth and centrifuged at 9000 rpm for 30 min. at 4 °C in a Sorvall GSA rotor. To precipitate the proteins, ammonium sulphate was added to 60% saturation. This solution was kept at 4 °C overnight under constant stirring. The next day, after centrifugation as described above, the protein pellet was dissolved in 15 ml of water and dialysed against distilled water overnight at 4 °C. The protein solution was centrifuged in a Sorvall SA-600 rotor at 5000 rpm for 20 min. at 4 °C. The lectin was extracted from this protein mixture by affinity chromatography on a chitotriose-agarose column equilibrated with water and eluated by 0.2 M NH₃. Protein purity was secured by SDS- polyacrylamide gel electrophoresis, which showed a single band at 65 kD . Furthermore, protein-containing fractions from the column coincided with activity to agglutinate human erythrocytes . EXAMPLE 1 a Feeding assays with M. aeneus larvae

In larval feeding assays with M. aeneus, the above- mentioned five lectins were compared. The larvae were fed

Brassica napus oleifera (cv Katarina) stamens soaked in 1 or 10% (w/v) solutions of the respective lectins. The stamens were always taken from buds which had just started to burst. To further standardise the diet, groups of 9 stamens were soaked in 10 μl of the solutions. The exact concentrations of lectin in the pollen grains, the food of the larvae, have not been determined. The solvent was 0.1% Tween 20 in ultrafiltered, pyrogen-free water (Millipore ™) . This solvent was used as a control treatment .

The experiment started with larvae less than one day old. Eggs were collected from buds on plants in greenhouse cages described below. Under microscope, groups of up to 10 eggs were transferred to a damp cotton substratum in plastic-cap-sealed, ventilated wells of microtitre plates. Once a day the plates were searched for new lar- vae, which were transferred singly to other rearing plates, separate for each treatment. Every day each live larva was moved to an adjacent cotton-lined well provided with a fresh, impregnated stamen. The plates with eggs and larvae were kept in a Conviron E15 CMP 3244 growth chamber (temperature 15 °C, RH 80% and 18 h of light) . After 7 days, larval survival rate and live weight were recorded. Results

All the lectins significantly (as determined by chi²) decreased M. aeneus larval survival rate at the concentration of 10% lectin in the solutions used for treatment of stamens (Table 1, Figs 1, 2) . None of the larvae fed Con A or pea lectin-impregnated stamens survived day 4. At the 1% level of lectin in the solutions, larval survival rate was significantly lower when the lectin was wheat germ, Con A and pea lectin, but not potato. (Peanut lectin was not tested at this concentration) . As mentioned above, "10% Con A and pea lectin larvae" did not survive until the day for weighing. Larvae fed stamens impregnated with 1% pea lectin,

1% and 10% wheat germ lectin and 10% peanut lectin weighed significantly less than the control larvae (as determined by ANOVA followed by Scheffe's test at the 5% significance level) . Table 1 M. aeneus larval survival and mean weight after 7 days of rearing on stamens impregnated with solutions of plant lectins as compared to control larvae fed stamens impregnated with solvent (0.1% Tween 20 in water) . ** = p<0.01, *** = p<0.001

Treatment ι e- 10^!

^a' ^b The control was the same for all lectins indicated. In the test where 10% pea lectin was found to be as efficient as Con A in killing the larvae (Fig. 2), there was also a treatment where larvae were given a choice between a Con A-treated and a solvent-treated stamen. Larval position and feeding preferences were recorded before new stamens were provided. There was no evidence for larvae being able to distinguish between the two treatments (Table 2), i.e. there is little risk for larvae damaging other plant parts if an anther-specific promoter controls the lectin gene.

Table 2 Preferences of Meligethes larvae (n = 41) given a choice between stamens impregnated with Con A or solvent only. No significant differences when tested by chi².

Assays with female M. aeneus

This test was carried out using potato lectin and Con A lectin to study whether larval reactions correspond to adult responses, potato lectin being a less potent larval inhibitor than Con A. Adults of M. aeneus were collected from daffodils and other spring flowers and were kept in net cages in a greenhouse for at least one week before the experiments started. The cages were provided with flowering oilseed rape in order to promote beetle sexual maturation and mating. Part of the collected beetles were stored in a refrigerator in ventilated 1.5 1 plastic jars partly filled with damp sand at a temperature of 4-8°C. These stored beetles were used later in the experiments and were then treated as described above. To sort out females from males, the beetles were caged singly with a raceme of spring rape.

The cages consisted of perforated 18 x 25 cm plastic bags (Cryovac) fixed to a plastic vial (diameter 1.5 cm; height 6 cm) by rubber bands. The racemes were inserted through a hole in the lid of the water-filled vial. After 2-3 days, the buds were searched for oviposition holes and eggs under microscope. Females which had oviposited were used in the experi- ments.

Females use predominantly 2-3 mm long buds for oviposition. The eggs are inserted into the bud through a hole made by the female with her mouthparts. The food consists of buds that are smaller or larger than these, and of pollen from stamens. Smaller buds are often devoured completely, leaving just the stalks. The effect of lectin on feeding as well as oviposition rate was assayed in the same type of plastic bag cages as the ones for sorting out females described above. To force the females to feed on treated plants only and to use as little lectin as possible, females were given two stamens to feed on for one day alternating with a raceme the next day and so on, during a period of 14 days. The stamens, which were placed on damp cotton on top of the lid, were treated in the same way as the stamens given to the larvae. The raceme was stripped of flowers and buds larger than 3 mm. The small buds (< 2 mm) at the tip of the raceme were painted with lectin or control solution. Buds for oviposition (2-3 mm) were not treated. The number of stamens and buds fed upon, the number of eggs laid and the number of buds oviposited on were recorded as were the weights of the females at the start and the end of the experiment. The final weights were recorded after the females had been killed in a freezer (-18°C) . Results

All the females survived the 14 -day period of testing. Female behaviour was quite variable. Some did not lay any eggs at all and data was analysed both with and without these females. In three comparisons, there were significant differences between control and Con A treatments; the number of buds eaten (all females), the number of eggs laid and the number of buds used for oviposition (ovipositing females) were significantly lower on Con A treated racemes (Table 3; as determined by t-test) . None of the variables were significantly different in the comparisons between control and potato-lectin-exposed females .

Table 3

Oviposition and feeding rates of females exposed to treated feeding buds and stamens. Mean sums per female over 14 days. Control treatment = solvent (0.1% Tween 20 in water) ; potato lectin, Con A = 10% lectin in solvent

* = p<0.05

EXAMPLE lb

Feeding assays with Phγllotreta spp. adults

Phyllotreta spp. adults were attracted to cotyledons of rape (cv. Katarina) growing in trays placed outdoors on the ground. From these, beetles were collected with an exhaustor. The live beetles were sorted according to species and randomly assigned to one of the two treatments, i.e. control or Con A. In the experiment, the adults were kept singly in 9-cm-diameter plastic Petri dishes on moist filter paper in a climate chamber under the same conditions as M. aeneus . The Petri dishes with Phyllotreta were placed on damp sand in plastic trays (41 x 62 x 11 cm) , with the two treatments randomly placed. Every third day the beetles were provided with one fresh cotyledon of spring rape (cv. Katarina) painted on both sides with either 10% Con A or the control solution of water with 0.1% Tween 20 as a detergent. The feeding damage to the four cotyledons exposed to each beetle over the period of 12 days was recorded, as well as their final weight and survival rate. Results

In all the three Phyllotreta spp., Con A treatment resulted in significantly less damage to the leaves (Table 4: t-test). In P. undulata, the dominating

Phyllotreta species in Swedish spring rape, also the mean weight of Con-A-fed beetles was significantly lower than that of the controls. Survival rates did not differ between the two treatments. Table 4

Feeding rate and body weight of Phyllotreta spp. fed cotyledons treated with Con A (10%) or control solution

(0.1% Tween in water) . Mean sums of leaf area consumed per beetle and beetle weight after 12 days of treatment.

** = p<0.01, *** = p<0.001

CONCLUSIONS The conclusions from these larval and adult tests with M. aeneus, P. undulata, P . nigripes och P . vi tata are that primarily Con A and pea lectin are lectins of potential use as resistance factors in transgenic Brassica . Accordingly, the corresponding genes were isolated from Canavalia ensiformis and Pisum sativum . Example 2a

Gene isolation, mutation, cloning and transformation with Con A gene Gene isolation Canavalia ensiformis plants were grown from seed

(Sigma Chemical Co.), and from developing seeds (200 mg) mRNA was purified using a QuickPrep micro mRNA Purification Kit (Pharmacia Biotech, Uppsala, Sweden) . First strand cDNA was prepared from this mRNA with reverse transcription using oligo dT and random hexamers .

This was further amplified by PCR with the following primers which were designed according to a sequence published by Carrington et al . 1985, with the inclusion of an EcoRI and a Pstl site, respectively, 5'-TCA GAA TTC

GTA GCA AGC AGC-3' and 5' -GAC CTG CAG TGG ATT ACA CAG C -

3'. A DNA fragment corresponding to nucleotides 1-986 of the published sequence was obtained by PCR using these primers. The PCR reaction was carried out with Pwo DNA polymerase (Boehringer Mannheim GmbH, Germany) . The amplified fragment was inserted into EcoRI and Pstl of pBluescript SK- (Stratagene, La Jolla, CA, USA). The fragment was subcloned and sequenced (Fig. 3) . The isolated sequence differed from the published one in 7 positions. The plasmid with the Con A gene in pBluescript is named pConA. The amino acid sequence of the Con A is shown in Fig. 4. Mutation The Con A protein is posttranslationally processed in a very unique way. Glycosylation occurs at amino acid 152 (asparagine) but for the lectin to be active, this glycosylation has to be removed again. As it is uncertain if the enzymes responsible for this deglycosylation are present in rapeseed, a mutated version of the Con A gene was produced. The mutation was introduced by PCR of the complete pCon A using Tth DNA polymerase (Clonetech Laboratories, Inc., Palo Alto, CA, USA) for the PCR reaction. A PCR primer pair was used where the backward primer replaced a triplet AAT with TCT at the same time creating a Bglll site: 5'- TAG TGG TTC CAC TGG AAG G-3' and 5'- GGA AGA TCT GAT AAC ATT TGC -3' . This will give a protein in which asparagine 152 is replaced with a serine thus disrupting the glycosylation signal. The Bglll site was used for cutting and religating the mutated pConA which was then named pConAmut . The mutated Con A gene was sequenced and the introduced mutation confirmed (Fig. 5) . In addition to the expected mutation, one additional base change was introduced by the PCR reaction but this will not alter the amino acid sequence (Fig. 6) .

The nucleotide sequence of the thus mutated Con A is shown in SEQ ID No. 2. This codes for the amino acid se- quence as shown in SEQ ID No . 5, where the mutation of the nucleotide sequence has been indicated by underlining, and the mutation of the corresponding amino acid sequence has been indicated with an underlined Ser (serine) . The invention also comprises insecticidal vari- ants and homologues of said amino acid sequence as well as of said nucleotide sequence. By variants and homologues are here meant sequences which deviate from the indicated sequences in one or more positions but are functionally equivalent to the indicated sequences. Construction of transformation vectors

(cf. Fig. 7) pConA and pConAmut were cleaved with Kpnl and Ncol . The BnSD promoter of Brassica napus was excised from pBSBnSD2.1-GUS (kindly provided by Dr D J Murphy, John Innes Centre, Norwich, UK) with the same enzymes and ligated in front of the Con A/ConAmut gene. The combined BnSD promoter - Con A/ConAmut gene was excised with Xbal and ligated into the Xbal site of pPTV-pA. pPTV-pA is a derivative of the binary vector pGPTV-kan (Becker et al . 1992) in which the GUS gene has been replaced with the polylinker of pUC19. The plasmids with correct gene orientation resulting from these ligations were named pins: 3 containing the Con A gene and pins: 4 containing the ConAmut gene . Transformation of Acτrojbacteriu.m

Agrobacterium tumefaciens, strain LBA4404, was transformed with pins: 3 and pins: 4 by direct transformation of competent cells. The presence of the binary vector was verified by plasmid DNA preparation and its restriction enzyme digestion pattern after agarose gel electrophoresis . Transformation of plant material

4 days old seedlings of spring rape, cv Westar, were used as a source for transformation explants. In the transformation method used (Moloney 1989, with slight modifications) , the petioles of the cotyledons were dipped into a solution with Agrobacterium tumefaciens and thereafter transferred to a co-cultivation dish. After 2- 3 days of co-cultivation, the bacteria were killed by transferring the explants to a culture substrate with 500 mg/1 carbencillin and 25 mg/1 kanamycin for selection of transformed cells. After 4 to 10 weeks, green shoots emerged from the cut ends of the cotyledons. The green shoots were transferred to a shoot culture substrate and tested for successful transformation by a PCR-based method. PCR test

Total DNA was prepared from 5 mm0 leaf disks. A PCR test was performed using the primers 5 ' -CAGACAATCGGCTGCTCTGATG-3 ' and 5' -AGCAAGGTGAGATGACAGGAAGATC-3' , amplifying a fragment of about 300 bases in the NPTII gene of pPTV-pA. The presence of the fragment was analysed by agarose gel electrophoresis . EXAMPLE 2b

Gene isolation, cloning and transformation with pea lectin gene

Gene isolation Total DNA from leaves of pea, cv Lincoln, was extracted with DNeasy Plant Mini kit (QIAGEN GmbH,

Hilden, Germany) . From this DNA, the pea lectin gene was isolated by PCR using the primers 5'- CAT GAA TTC AAC CGA

ACA ACC TCG AAG-3' and 5' -GGG TCG ACA ACA AGG TGT CTC TGC CC-3'. The primers were selected according to a published sequence (Hoedemaeker et al . 1994) with an EcoRI and a Sail site incorporated, respectively. Pwo DNA polymerase was used for the PCR reaction. The isolated PCR fragment was ligated into EcoRI and Sail of pBluescript SK- . The fragment was subcloned and sequenced (Fig. 8) . The sequence of the isolated gene was identical with the published one. The lectin is identified by the amino acid sequence shown in Fig. 9.

Construction of transformation vector (cf. Fig. 10) pGEM4Z/Sta44-4 (kindly provided by Dr L.S. Robert,

Agriculture and Agri-Food Canada, Ottawa, Canada) containing the promoter Sta44-4 of Brassica napus (Robert et al . 1997) was linearised with Smal. The pea lectin gene was cut from pBart with Sphl and Ndel, was made blunt -end and ligated into the Smal site of pGEM4Z/Sta44-4. The combined Sta44-4 promoter/pea lectin gene was excised with HindiII and Kpnl and ligated into the same sites of pPTV-pA. pPTV-pA is a derivative of the binary vector pGTPV-Kan (Becker et al . 1992) in which the GUS gene has been replaced with the polylinker of pUC19. The plasmid resulting from these ligations was named pins : 5.

Transformation of Agrobacterium, transformation of plant material and PCR test of this material were carried out in the same way as described for the Con A gene in

Example 2a.

EXAMPLE 3

M. aeneus larvae fed genetically modified oilseed rape The following tests using transformed plants were identical to the tests with larvae fed stamens soaked in protein solutions except that no treatment of the stamens was necessary in this case. The larval weights on several of the transformants, transformed with the Con A- , ConAmut- or the pea lectin gene, were significantly lower than on the control, as determined by ANOVA followed by Scheffe's test (Table 5) .

Table 5 Mean weights of pollen beetle larvae after 7 days of feeding on stamens of the rapeseed cultivar Westar compared to after feeding on Westar transformed with the Con A (R9) , ConAmut (R10) or pea lectin (Rll) gene. Means followed by the same letter are not significantly different in Scheffe's test, n = initial number of larvae per treatment.

References

- Ahman, I. 1993. A search for resistance to insects in spring oilseed rape. OPBC/WPRS Bulletin 16(5):36-46.

- Becker, D., Kemper, E., Schell, J. & Masterson, R. 1992. New plant binary vectors with selectable markers located proximal to the left T_DNA border. Plant Mol . Biol. 20:1195-1197.

- Carrington, D.M., Auffret, A. & Hanke, D.E. i985. Polypeptide ligation occurs during post-translational modification of Concanavalin A. Nature 313:64-67.

- Czapla, T.H. & Lang, B.A. 1990. Effect of plant lectins on the larval development of European corn borer

(Lepidoptera: Pyralidae) and Southern corn rootworm (Coleoptera: Chrysomelidae) . J. Econ. Entomol . 83: 2480- 2485.

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- Liener, I.E., Sharon, N. & Goldstein, I.J. 1986. The lectins: properties, functions and applications in biology and medicine. Academic Press, Orlando, Florida, 600 pp. - Matsumoto, I., Jimbo, A., Mizuno, Y. & Seno, N. 1983.

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- Moloney, M.M., Walker, J.M. & Sharma, K.K. 1989. High efficiency transformation of Brassica napus using

Agrobacterium vectors. Plant Cell Rep. 8: 238-242.

- Nilsson, C. 1987. Yield losses in summer rape caused by pollen beetles {Meligethes spp.) Swedish J. Agric. Res. 17: 105-111. - Peumans, W.J. & Van Damme, E.J.M. 1995. Lectins as defence proteins. Plant Physiol . 109:347-352.

- Rabhe, Y., Sauvion, N. , Febvay, G. , Peumans, W.J. & Gatehouse, A.M.R. 1995. Toxicity of lectins and processing of ingested proteins in the pea aphid Acyrthosiphon pisum . Entomol. Exp. appl. 76:143-155.

- Robert L.S., Gerster J.L., Hong H.P. 1997. BRASSICA SP. POLYGALACTURONASE GENE PROMOTER U.S Patent No. 5,689,053.

Figure leαends

Fig. 1. Survival rates of Meligethes larvae fed stamens soaked in 10% solutions of plant lectins as compared to the solvent . Fig. 2a. Survival rates of Meligethes larvae fed stamens soaked in 10% solution of pea lectin as compared to Con A- and solvent-treated stamens. In addition, there was a treatment where larvae had a choice between a Con A-treated and a solvent -treated stamen. Fig. 2b. Survival rates of Meligethes larvae fed stamens soaked in 1% solution of pea lectin as compared to Con A- and solvent-treated stamens.

Fig. 3. Sequence of isolated Con A gene. Start and stop codons are underlined. Fig. 4. Amino acid sequence of Con A lectin.

Fig. 5. Sequence of the mutated Con A gene. Start and stop codons are underlined and the introduced mutation is in bold type.

Fig. 6. Amino acid sequence of modified Con A lectin.

Fig. 7. Construction of pins : 3 and plns:4. Con A = Con A gene, ConAmut = ConAmut gene, BnSD-P = BnSD promoter, nos-P = nopaline synthase promoter, NPT II = neomycin phosphotransferase II for kanamycin resistance, pA = polyA sequence, RB = right border, LB = left border, and Kan = bacterial kanamycin resistance.

Fig. 8. Sequence of the isolated pea lectin gene. Start and stop codons are underlined.

Fig. 9. Amino acid sequence of pea lectin. Fig. 10. Construction of plns:5. Pea lectin = pea lectin gene, Sta44-4-P = S a44-4 promoter, nos-P = nopaline synthase promoter, NTPII = neomycin phosphotransferase II for kanamycin resistance, pA = polyadenylation sequence, RB = right border, LB = left border, Kan = bacterial kanamycin resistance.

Claims

1. A transgenic Brassica plant resistant to Brassica- attacking insect pests, selected from the group consisting of the genera Meligethes, Phyllotreta and Delia, said transgenic Brassica plant comprising in its genome at least one DNA sequence coding for a foreign lectin inhibitory to said insect pests, said foreign lectin being se- lected from the group consisting of jack bean lectin (Concanavalin A) , modified jack bean lectin (modified Concanavalin A) , pea lectin, wheat germ lectin, potato lectin and peanut lectin, or insecticidal variants or homologues or combinations thereof, together with a pro- moter active in the Brassica plant.

2. A transgenic Brassica plant according to claim 1, wherein the lectin is a jack bean lectin (Concanavalin A) or an insecticidal variant or homologue thereof.

3. A transgenic Brassica plant according to claim 1, wherein the lectin is a modified jack bean lectin or an insecticidal variant or homologue thereof.

4. A transgenic Brassica plant according to claim 1, wherein the lectin is pea lectin or an insecticidal variant or homologue thereof.

5. A transgenic Brassica plant according to one or more of claims 1-4, wherein the jack bean lectin, the modified jack bean lectin and/or the pea lectin are present in combination with one or more of wheat germ lectin, potato lectin and peanut lectin, or an insecti- cidal variant or homologue of any one thereof.

6. A propagated plant material from the transgenic Brassica plant as defined in any one of claims 1-5, derived by cloning, selfing and/or hybridisation in one or more generations.

7. An expression cassette comprising a DNA clone, which codes for at least one lectin inhibitory to Brassica-attacking insect pests, selected from the group consisting of the genera Meligethes, Phyllotreta and Delia, said lectin being selected from the group consisting of jack bean lectin, modified jack bean lectin, pea lectin, wheat germ lectin, potato lectin and peanut lectin, or insecticidal variants, homologues or combinations thereof, operably linked to regulatory sequences in plants expressing at least one of said lectins in buds, pollen, cotyledons, and/or roots of Brassica, which cause the expression of the DNA clone in cells of Brassica species.

8. An expression cassette according to claim 7, wherein the lectin is Concanavalin A or an insecticidal variant or homologue thereof .

9. An expression cassette according to claim 7, wherein the lectin is modified Concanavalin A or an insecticidal variant or homologue thereof.

10. An expression cassette according to claim 7, wherein the lectin is a pea { Pisum sativum) lectin or an insecticidal variant or homologue thereof.

11. An expression cassette according to claim 7, wherein the jack bean lectin, the modified jack bean lectin and/or pea lectin or an insecticidal variant or homologue thereof is combined with one or more of wheat germ lectin, potato lectin and peanut lectin or an in- secticidal variant or homologue of any one thereof.

12. Transgenic plant cells containing as foreign DNA at least one copy of the DNA sequence of an expression cassette according to one or more of claims 7-11.

13. A lectin having insecticidal effect against insect pests, selected from the group consisting of the genera Meligethes, Phyllotreta and Delia , in plants of Brassica species and comprising the amino acid sequence as shown in SEQ ID No. 5 or an insectidal variant or homologue thereof .

14. A method for protecting a Brassica plant against infestation by insect pests, selected from the group consisting of the genera Meligethes, Phyllotreta and Delia, comprising inserting into the genome of the Brassica plant at least one DNA sequence coding for at least one foreign lectin inhibitory to said insect pests, said lectin being selected from the group consisting of jack bean lectin (Concanavalin A), modified jack bean lectin (modi- fied Concanavalin A) , pea lectin, wheat germ lectin, potato lectin and peanut lectin or insecticidal variants, homologues or combinations thereof, together with a promoter active in the Brassica plant.

15. A method according to claim 14, wherein the lec- tin is Concanavalin A or an insecticidal variant or homologue thereof .

16. A method according to claim 14, wherein the lectin is modified Concanavalin A or an insecticidal variant or homologue thereof .

17. A method according to claim 14, wherein the lectin is pea { Pisum sativum) lectin or an insecticidal variant or homologue thereof.

18. A method according to one or more of claims 14- 17, wherein at least one more DNA sequence is introduced, which codes for one or more lectins selected from the group consisting of wheat germ lectin, potato lectin and peanut lectin or insecticidal variants or homologues of any one thereof .

19. A method according to one or more of claims 14-

18, comprising the steps of a) selecting a Brassica species, b) culturing cells or tissues from said plant species, c) introducing into the cells of the plant cell or tissue culture at least one copy of the expression cassette defined in any one of claims 7-11, comprising a sequence coding for the lectin, variant, homologue or combination of lectins, and d) regenerating resistant whole plants from the cell or tissue culture.

20. A method according to any one of claims 14, 16, 18 and 19, wherein the DNA sequence comprises the nucleotide sequence which is shown in SEQ ID No. 2 and which codes for modified jack bean lectin (modified

Concanavalin A) , or a functionally equivalent variant or homologue thereof.

21. A method according to one or more of claims 14- 20, comprising the further step of propagating the plant material by cloning, selfing and/or hybridisation in one or more generations in such a manner that at least one copy of the sequence given in the expression cassette is present in the cells of the offspring.