Plant Cell Reports

Plant Cell Reports (1994) 13:587-593

9 Springer-Verlag1994

Agrobacterium-metfiated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting gene transfer efficiency during early transformation steps An De Bondt 1, Kristel Eggermont a, Philippe Druart 2, Maayke De Vii l, Inge Goderis 1, Jos Vanderleyden l, and Willem E Broekaert ~ 1 F. A. Janssenslaboratorium voor Genetica, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Heverlee, Belgium 2 Station des Cultures Fruiti6res et Maralch6res, Centre de Recherches Agronomiques de l'Etat, Chauss6e de Charleroi 234, B-5800 Gembloux, Belgium Received 29 September 1993/Revisedversion received 2 March 1994 - Communicated by M. R. Davey

ABSTRACT

INTRODUCTION

The factors influencing transfer of an intron - containing 13-glucuronidase gene to apple leaf explants were studied during early steps of an Agrobacterium tumefaciensmediated transformation procedure. The gene transfer process was evaluated by counting the number of 1~" glucuronidase expressing leaf zones immediately after cocultivation, as well as by counting the number of 13glucuronidase expressing calli developing on the explants after 6 weeks of postcultivation in the presence of 50 mg/1 kanamycin. Of three different tested disarmed A. tumefacJens strains, EHA101(pEHA101) was the most effective for apple transformation. Cocultivation of leaf explants with A. tumefaciens on a medium with a high cytokinin level was more conducive to gene transfer than cocultivation on media with high auxin concentrations. Precultivation of leaf explants, prior to cocultivation, slightly increased the number of ~-glucuronidase expressing zones measured immediately after cocultivation, but it drastically decreased the number of transformed calli appearing on the explants 6 weeks after infection. Other factors examined were: Agrobacterium cell density during infection, bacterial growth phase, nature of the carbon source, explant age, and explant genotype.

The production of transgenic plants by gene transfer techniques is expected to become an important tool for genetic improvement of agronomically important crop species. The transformation of plants depends on two essential requirements: the ability to stably introduce a desired gene into the plant genome and the ability to regenerate a fertile plant from the transformed cells. Transformation of apple by infecting wounded leaf explants with Agrobacterium tumefaciens has previously been reported by James et al. (1989), Maheswaran et aL (1992), and Norelli and Aldwinckle (1993). Agrobacterium rhizogenes has also been used successfully for genetic transformation of apple, but this procedure results in the cotransfer of genes of interest with bacterial genes that influence the hormonal balance of transformed plant tissues (Lambert and Tepfer 1992). The major problem with A. tumefaciens-mediated transformation remains the low efficiency of transformation (James and Dandekar 1991). As the efficiency of shoot regeneration from non-transformed leaves of many apple cultivars reaches close to 100%, with an average of over 10 shoots per leaf (Welander 1988, De Bondt et al. 1990, Dufour 1990, Theiler' He&rich and Theiler-Hedtrich 1990), further improvement of the transformation efficiency is more likely to be expected from the optimization of early gene transfer steps. To study the process of gene transfer, a binary transformation vector containing a chimeric gusA-intron gene has previously been constructed by Vancanneyt et al. (1990), The intron harbours three stop codons, one in each reading frame. In this way, the B-glucuronidase reporter gene can be expressed in plant cells but not in the bacterium (which cannot splice out introns) thus allowing to screen for gene transfer shortly after cocultivation of plant tissues with A. tumefaciens.

Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; CaMV35S, 35S RNA of cauliflower mosaic virus; EDTA, ethylenediaminetetraacetate, FeNaEDTA, ethylenediaminetetraacetate ferric-sodium salt; GusA, l~glucuronidase; gusA, l~-ghicuronidase gene of Escherichia coli; gusA-intron, B-glucuronidase gene containing an intron in the coding region; IBA, indole butyric acid; 2iP, N6-2-isopentenyl adenine; NAA, naphthaleneacetic acid; npt11, neomycinphosphotransferase II gene; X-Gluc, 5-bromo-4-chloro-3-indolyl B-D-glucuronide

Correspondence to: A. De Bondt

588

In this study, we have investigated the effect of different pre- and cocultivation conditions on the efficiency of Agrobacterium-mediated transfer of the gusA-intron marker gene to apple leaf cells and on the formation of transformed calli. MATERIALS AND

METHODS

Bacterial strains and vectors. Three Agrobacteriurn tumefaciens strains with different chromosomal background and different disarmed virulence plnsmids were used: LBA4404 (pAL4404) (Hoekema et al. 1983), C58C1 (pGV2260) (De Block et al. 1984) and EHA101 (pEHA101) (Hood et aL 1986), each of them provided with the binary expression vector pFAJ3000 (figure 1). This vector is a derivative of pGSC1702, which was kindly provided by Dr. J. Botterman (Plant Genetic Systems N.V., Gent, Belgium). pGSC1702 is a derivative of pGSC1700 (Cornelissen and Vandewiele 1989) containing an nptll-based expression cassette as selectable marker in plants and a gene coding for streptomycin/spectinomycin adenyl transferase as bacteria/ selection marker. A multiple cloning site was introduced in the unique BamHI site ofpGSC1702 situated between the right T-DNA border sequence and the plant selectable marker. AgusA-intron expression cassette was obtained as a 3kbp HindlII fragnent from the vector p35S GUS INT (Vancanneyt et al. 1990) and introduced into the unique HindlII site in the multiple cloning site to yield pFAJ3000. Introduction ofpFAJ3000 in the different A. tumefaciens strains was done by electroporation as described by Mattanovich et aL (1989). Plant tissue culture and transformation, Apple shoots were established by meristem-tip culture as previously described by Druart et al. (1982). Micropropagation of shoots was done by placing decapitated shoot explants horizontally on the surface of micropropagation medium 706 (Drnart 1988). Explants used for transformation were the youngest four fully expanded leaves of 5-week-old micropropagated shoots. The leaves were wounded by making three incisions perpendicular to the midrib taking care not to cut through the leaf edges (Drnart 1990). If a preeultivation was applied, the wounded leaves were cultured on apple shoot induction medium AS1 (solidified with 0.5% agar, unless stated otherwise) for 1 to 8 days in the dark at 23~ Medium AS1 contained the macro elements of Murashige and Skoog (1962), micro elements as reported by Druart (1980), 40 mg/l FeNaEDTA, 100 mg/l myo-inositol, 1 mg/1 thiamine-HCl, 0.3 mg/l indole-butyrie acid (IBA), 8 rag/1 N6-[2isopentenyl]adenine (2iP), 250 mg/l casein hydrolysate and 2% sucrose. To infect the leaves with A. tumefaciens, a bacterial suspension was made up as follows. A. tumefaciens was grown in YEB medium (10 g/l bacto peptone, 5 g/1 NaCI and 10 g/l yeast extract) supplemented with the appropriate antibiotics (300 mg/l streptomycin and 100 mg/1 spectinomycin) until an OD600 of 0.5 to 0.7 (midlog phase) was reached. The bacteria were spun down by centrifugation (4000g, 10 min) and resuspended in an equal volume of liquid AS 1 medium supplemented with 10 mM MgSO 4. The leaves were shaken gently in this suspension for about 1 minute and blotted on a sterile filter paper. They were then transferred to solid AS1 medium (unless stated otherwise), with their adaxial side in enntact with the culture medium and cocultivated during 4 days (unless stated otherwise) at 25~ in the dark. After eocultivation, the leaves were washed for 1 min in liquid ASI medium containing 500 mg/l cefotaxime, blotted on a sterile filter paper and transferred to solid AS 1 medium (unless stated otherwise) supplemented with 200 mg/l eefotaxime and 50 mg/1 kanamyein and solidified with 2.5 g/l gelrite. Gelrite was used in the postcultivation medium since agar inhibits the development of calli and shoots on selective medium (Uematsu et al. 1991; Maheswaran et aL 1992). Postcultivation of the leaves was done in the dark at 23~ GusA assay. Leaves were assayed for expression of the gusA-intron gene following the histochemicai staining procedure described by Jefferson (1987) with some modifications. The leaves were stained overnight at 37~ in a 50 mM sodium phosphate buffer (pH 7) containing 0.5 mM potassium ferricyanide, I0 mM Na2EDTA , 0.001% (v/v) Triton X-100 and 0.5 mg/ml 5-bromo-4-chloro-3-indolyl 13-D-ghicuronide (X-Glue). A_fferthe overnight staining, the leaves were cleared and fixed in FAA (5%

(v/v) formaldehyde, 5% (v/v) acetic acid, ethanol), first with 20% (v/v) ethanol then with 50% (v/v) ethanol for at least 20 rain each. The leaves are preserved in FAA containing 80% (v/v) ethanol. The number ofgusA expressing units was determined by use of a binocular. Transient expression was measured immediately after cocultivation of the leaf explants (at least 10 explants per tested parameter) by counting the number of gusA expressing leaf zones (either individual cells or cell clusters) appearing as blue spots on a white background after the staining procedure. The number of transformed calli developed on selective medium, 6 weeks after infection, was determined by counting blue calli after X-Glue staining of the leaf explants.

RESULTS AND DISCUSSION

Vector construction. GusA-intron was chosen as a marker gene to score DNA transfer events, since it allows rapid histochemical detection in intact plant tissues and shows no background expression in Agrobacterium tumefaciens (Vancanneyt et al. 1990). p35S GUS INT, the gusA-intron containing binary vector originally constructed by Vancanneyt et al. (1990), carries an npt11based bacterial selection marker. The presence of this marker complicates the introduction of the vector into the disarmed A. tumefaciens strain EHA101 (pEHA101) which has a kanamycin resistant genotype (Hood et al. 1986). Therefore, the gusA-intron expression cassette was excised as a HindlII fragment from p35S GUS INT and inserted in the binary vector pGSC1702 harbouring a streptomycin/spectinomycin adenosyl transferase gene as bacterial selection marker, yielding the plasmid pFAJ3000 (Figure 1).

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Figure 1: Schematic representation of the binary vector pFAJ3000. The region external to the ToDNA border sequence is identical to that of pGSC1700 (Cornelissen and Vandewiele 1989). Abbreviations: Ampr, ampicillin resistance gene; GUS int, coding region of the B-glueuronidase gene provided with an intron; LB, left border of T-DNA; nptIl, coding region of the neomycinephosphotransferase gene; OriColE1, origin of replication of Escherichia cell plasmid ColE1; OriPVS1, origin of replieation of the Pseudomonas aeruginosa plasmid PVS1; p35S, promoter of CaMV35S; pNos, promoter of the nopaline synthase gene; RB, right border of T-DNA; Sm/Sp r, streptomycin/ spectinomycin resistance gene; t35S, termination signal of CaMV35S; tOes, termination signal of the octopine synthase gene

589 Influence of A. tumefaciens strain, cell density, growth phase and.period of COCultivation. The evaluation of the transformation efficiency was primarily based on histochemicai analysis of the number of gusA expressing zones immediately after the cocultivation period. Figure 2A shows a typical pattern of gusA expression 4 days after infection of apple (cv. Jonagold) leaves with A. tumefaciens strain EHA101 (pEHA101/pFAJ3000). Expression of gusA was found to be scattered along the wounded sites. In some cases, half of the explants was further postcultivated on selective medium during 6 weeks after infection, whereafter the number of transformed calli was determined using the histochemical GusA assay. Figure 2B shows a leaf explant stained for detection of GusA activity (6 weeks after infection), showing both transformed (blue) and untransformed (white) calli,

In a first comparative experiment, wounded leaves were infected with each of three different A. tumefaciens strains and cocultivated for 2, 3, 4 or 5 days on the apple shoot induction medium AS 1. As can be seen in figure 3A, strain EHA101 (pEHA101/pFAJ3000) yields at least twice as much gusA expressing leaf zones as strain LBA4404 (pAL4404/pFAJ3000), whereas with strain C58C1 (pGV2260/pFAJ3000) virtually no gusA expressing leaf zones were observed. The number of gusA expressing leaf zones raised with increasing cocultivation time and reached a maximum of approximately 12 zones per leaf explant after 4 days (strain EHA101). In practice, however, the length of cocultivation is limited to a maximum of 4 days since longer cocultivation times result in abundant proliferation of A. tumefaciens EHA101 (pEHA101/pFAJ3000) on apple leaf explants and subsequent losses during postcultivation due to infection by A. tumefaciens.

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In all explants examined, more than 50% of developing calli expressed gusA. Leaves cocultivated with A. tumefaciens strain EHA101 (pEHA101) which did not contain plasmid pFAJ3000 did not exhibit Gus A activity in the histochemical assay (results not shown).

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cell density (xl09cells/ml) Figure 3: Transient gus,4 expression assayed immediately following eoenltivation of wounded leaves with A. tumefaciens: influence of the bacterial strain and the period of cocultivation (A) or the bacterial cell density (B). The leaves were eocultivated with three different strains: EHA101 (pEHA101/pFAJ3000) (abbreviated as E3000), LBA4404 (pAL4404/pFAJ3000) (abbreviated as L3000) and C58C1 (pGV2260/pFAJ3000) (abbreviated as C3000) for periods varying from 2 to 5 days with initial cell density of0.5xl09 eells/ml (A) or for 4 days with initial cell densities of 0.1 to 2.5x109 cells/ml (B). The expression is represented as the average number of gus,4 expressing zones per leaf explant. Vertical bars denote standard error (n=20),

In the second experiment, comparing three inoculum densities (0.1, 0.5 and 2.5x109 cells/ml), EHA101 (pEHA101/pFAJ3000) clearly outperformed LBA4404

590 (pAL4404/pFAJ3000) and C58C1 (pGV2260/ pFAJ3000) (Figure 3B). Highest numbers of gusA expressing leaf zones were obtained upon inoculation of the leaves with a suspension containing 0.5 or 2.5x109 cells/ml of EHAI01 (pEHA101/pFAJ3000). Tests performed with all three strains collected from either lag phase, midlog phase or stationary phase cultures confirmed the superiority of EHA 101 (pEHA101/pFAJ3000) and, additionally, showed no significant effect of the bacterial growth phase on the efficiency of DNA transfer (results not shown). Finally, the efficiency of the three tested A. tumefaciens strains was also assessed by comparing the average number ofgusA expressing calli formed after 6 weeks of explant postcultivation (Figure 4).

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Influence of explant precultivation. As shown above, longer cocultivation periods significantly increase the efficiency of transformation. We investigated whether further increase in transformation efficiency could be gained from the introduction of a precultivation step preceding cocultivation. The precultivation period was varied from 0 to 8 days, followed by a 3 days cocultivation period (both on AS I medium) and a variable PoStcultivation period (8 to 0 days on AS1 medium supplemented with 200mg/l cefotaxime). The postcultivation step was introduced so that all explants had received an equal period of treatment (pre-, co- and postcultivation adding up to 11 days) and had a comparable physiological status at the end of the experiment. After the l 1-days treatments, leaves were analysed histochemically to determine the number of gusA expressing zones. Precultivation clearly enhanced the number of gene transfer events with a maximum reached at 6 days (Figure 5A). After 6 days of precultivation, the number ofgusA expressing zones was more than two-fold higher compared with the nonprecultivated leaves.

A E3000 L3000 C3000 bacterial strain Figure 4: Stable gusA expression assayed following kanamycin selection (50mg/I during 6 weeks) aider cocultivation of wounded leaves with A. tumefaciens: influence of the bacterial strain. The leaves were cocultivated with three different strains (cf. Figure 3). The expression is represented as the average number of gusA expressing calli per leaf explant. Vertical bars denote standard error (n=50).

Using EHA101 (pEHA101/pFAJ3000) for infections, the number of transformed calli was more than 5 fold higher than those obtained with strains LBA4404 (pAL4404/pFAJ3000) or C58C1 (pGV2260/pFAJ3000), confirming previous observations. Therefore, A. tumefaciens strain EHA101 (pEHA101/pFAJ3000) was used in all subsequent experiments. Strain EHA101 (pEHA101) has previously been shown to be most effective, relative to other A. tumefaciens strains, for transformation of alfalfa (Chabaud et al. 1988) and pea (Lulsdorf et aL 1991); In addition, Martin et al. (1990) and Maheswaran et al. (1991) have shown that the wild type strain A281, from which EHA101 (pEHA101) was derived, is more virulent on apple stems than other wild type strains such as C58, Ach5 or A348. The hypervirulence of strain A281 is believed to result from a high level of virG expression (Jin et al. 1987), whose gene product is necessary for the activation of inducible virulence genes (Hooykaas and Schilperoort 1992).

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0.5~.___ 0 2 4 6 8 precultivation period (days) Figure 5: Influence of preeultivation of explants on gusA expression assayed either immediately after eocultivation of wounded leaves with A. tumefaciensstrain EHA101 (pEHA101/pFAJ3000)(A) or after 6 weeks of kanamycin selection (50 mg/I) after cocultivation. The expression is represented as the average number ofgus.4 expressing zones (A) and ealli (B) per leaf explant. Vertical bars denote standard error (n=15 and n=60 in A and B respectively).

591 A s e c o n d set o f l e a v e s w a s t r e a t e d e q u a l l y as d e s c r i b e d

above except that the postcultivation period was extended to 6 weeks (on ASI supplemented with 50mg/l kanamycin and 200rag/1 cefotaxime) after which the number of transformed caUi was counted (Figure 5B). Surprisingly, precultivation of the leaf explants, even for a short 2-days period, appeared to drastically decrease the number of transformed calli, with a further decrease down to zero after 6 days or more of precultivation. It should be noted that the blue spots assessed histochemically after cocultivation represent zones of transient expression (mainly from non-integrated DNA copies), whereas the gusA expressing calli most probably derive from cells where DNA integration has occurred (stable expression). The discrepancy between the results obtained by measurement of transient expression and stable expression in figure 5 may indicate that the physiological status of a tissue optimal for DNA uptake (transient expression) is not necessarily optimal for integration of foreign DNA in the host genome (stable expression). DNA uptake has often been suggested to be cell cycle dependent, whereas DNA integration may primarily rely on the availability of DNA repair enzymes. Influence of carbon-source. Sucrose, glucose, fructose, galactose and sorbitol were compared for their influence on the efficiency of gene transfer and regeneration of gusA expressing calli. Except for their main carbon source, all media had the same composition (see materials and methods). The reference medium contained 2% sucrose, the other media contained either 2% glucose, 2% fructose, 2% galactose or 2% sorbitol. The number ofgusA expressing zones per leaf, measured immediately after cocultivation, is depicted in figure 6A. In figure 6B, the number of gusA expressing calli obtained after 6 weeks on selective medium is represented. The carbon source appears to influence both gene transfer and regeneration of transformed tissue. Fructose and galactose are clearly inhibitory for both processes. In addition, galactose caused necrosis of the explants after 6 weeks of postcultivation, an effect that was not observed with the other carbon sources. Highest frequencies of transformation were obtained with sucrose and glucose and, to a somewhat lesser extent, with sorbitol. Influence of hormonal compositionl To test whether plant hormones affect the ability of apple leaf ceils to accept foreign genes from A. tumefaciens, gene transfer efficiencies were compared after cocultivation on three different media, varying in hormonal composition. The first medium, AS1, contains a high concentration of cytokinin (8 mg/1 2iP) and a lower concentration of auxin (0.3 mg/l IBA). The second medium, ASll4, has 5 mg/1 2,4-1) and no cytokinin and the third medium, ASll5, contains 5 mg/1 NAA and no cytokinin. As shown in

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gusA expression assayed either immediately after cocultivation of wounded leaves with A. turnefaciens strain EHA101 (pEHA101/pFAJ3000) (A) or ai~er 6 weeks of kanarnycin selection (50 rag/l) after cocultivation (B). The expression is represented as the average number of gusA expressing zones (A) and calli (B) per leaf explant. Vertical bars denote standard error (n=10 and n=50 in A and B respectively). Abbreviations: sue, sucrose; glu, glucose; fru, fructose; gal, galactose; sor, sorbitol

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AS1 ASl14 ASl15 cocultivation medium Figure 7: Transient gusA expression assayed immediately aRcr cocultivation of wounded leaves with A. tumefaciens strain EHAI01 (pEHA101/pFAJ3000): influence of the hormonal composition of the cocultivation medium. The expression is represented as the average number of gusA expressing zones per leaf explant. Vertical bars denote standard error (n=20).

592 figure 7, the number of gusA expressing zones per leaf from medium AS1 is about three times higher than for the media with high auxin content, AS114 and AS115. These results are in line with the fact that cytokinins stimulate cellular division and with the assumption that mitotic ceils are more suseptible to Agrobacterium. In contrast, however, Lu et al. (1991) have found that for carnation, high auxin levels in the cocultivation medium resulted in high transformation efficiencies compared with media with high cytokinin contents. Influence of explant age. Another factor that may influence transformation efficiency is the age of the micropropagated shoots used as source of leaf explants.

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numbers of gusA expressing leaf zones were obtained using explants derived from 25-days old shoots, but did not decrease significantly until the shoots were older than 40 days. We can conclude that for maximal transformation and regeneration efficiency of apple leaf explants, the shoot culture must be between 20 and 40 days in age. Influence of explant genotype. The influence of the genotype on the efficiency of transformation, was assessed by comparing six different apple cultivars: Jonagold, Elstar, Gala, Braeburn, Merlijn and Fuji. Each cultivar was tested on 2 different media: AS1 and AS37.

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Figure 8: Influence of explant age on shoot regeneration efficiency from wounded leaves (without precedent transformation) after 6 weeks cultivation on ASI (A) or on transient gusA expression immediately after cocultivation of wounded leaves with A. tumefaciens strain EHA101 (pEHA101/pFAJ3000)(B). The expression is represented as the average number of gusA expressing zones per leaf explant. Vertical bars denote standard error (n=10 and n=50 in A and B respectively);

We have tested leaves from shoots of 21 to 45 days after micropropagation for their shoot regeneration efficiency (without precedent transformation) (figure 8A) and for their ability to accept DNA from A. tumefaciens (figure 8B). The total number of regenerated buds per leaf explant varied with its physiological age: it raised from about 10 for 21 days old shoots to a value between 20 and 30 for 25 to 40 days old shoots, and dropped drastically down to 1 for 45 days-old shoots. Similarly, highest

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Figure 9: Influence of genotype on gusA expression assayed either immediately after cocultivation of wounded leaves with A. tumefaciens strain EHA101 (pEHA101/pFAJ3000) (A) or after 6 weeks ofkanamyein selection (50 rag/l) after cocultivation (]3). The growth media AS1 and AS37 have the same basal content (see materials and methods) supplemented with plant growth regulators: 8 mg/l 2iP and 0.3 mg/l IBA for AS1 and 8 mg/l 2iP and 1.5 rag/1 IBA for AS37. The expression is represented as the average number ofgusA expressing zones (A) and calli (B) per leafexplant. Vertical bars denote standard error (n=10 and n=50 in A and B respectively). Abbreviations: Jo, Jonagold; El, Elstar; Ga, Gala; Br, Braeburn; Me, Merlijn; Fu, Fuji

Both media have the same composition (see materials and methods), except for different amounts of plant growth regulators: 8 mg/l 2iP and 0.3 mg/1 IBA for AS1 and 8 mg/l 2iP and 1.5 mg/1 IBA for AS37. As shown in figure 9A, gusA is expressed in all cultivars, but the number of gusA expressing zones differed significantly. Highest transient expression was observed for the cultivars Braeburn, Jonagold and Gala. The genotype

593 effect was also determined after a postcultivation of 6 weeks on selective medium. In this case, the number of gusA expressing calli was highest for the cultivars Merlijn, Jonagold and Elstar, whereas cultivars Gala, Braeburn and Fuji yielded 5 to 10-fold less transformed calli (Figure 9B). Again, there was no good correlation between transient and stable expression for the different cultivars. This suggests that genotype dependent factors may affect DNA uptake and DNA integration differentially. These results also demonstrate that care must be taken in interpreting data based solely on the analysis of early expression of a marker gene, as is now frequently done in protocol optimisation for Agrobacterium-mediated gene transformation (Janssen and Gardner 1989; Higgins 1992; Van Wordragen et aL 1992). For all cultivars tested, the number of GusA expressing calli obtained on medium AS1 was at least 10-fold higher than on medium AS37 confirming the negative effect of high auxin levels on the formation of transformed callus.

James DJ, Dandekar AM (1991) In: Lindsey K. (ed) Plant Tissue Culture Manual, vol B8. Kluwer Academic Publishers, pp 1-18 Janssen B-J, Gardner RC (1989) Plant Mol Biol 14:61-72 Jefferson, RA (1987) Plant Mol Biol Rep 5:387-405 Jin S, Komari T, Gordon MP, Nester EW (1987) J Bacteriol 169: 44174425 Lambert C, Tepfer D (1992) Theor Appl Genet 85:105-109 Lu C-Y, Nugent G, Wardley-Richardson T, Chandler SF, Young R, Dalling MJ (1991) Bio/Technol 9:864-868 Lulsdorf M. M., Rempel H., Jackson J. A., Baliski D. S., Hobbs S. L. A. (1991) Plant Cell Reports 9:479-483 Maheswaran G., Welander M., Hutchinson J. F., Graham M. W., Richards D. (1992) J Plant Physiol 139:560-568 Martin G. C., Miller A. N., Castle L. A., Morris J. W., Morris R. O., Dandekar A. M. (1990) J Am Soc Hort Sci 115:686-691 Mattanovich D., Rfiker F., da Camara Machado A., Laimer M., Regner F., Steinkellner H., Himmler G., Katinger H. (1989) Nucleic Acids Res 17: (16) Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Norelli J, Aldwinckle H. (1993) J Amer Soc Hort Sci 118 (in press)

ACKNOWLEDGEMENTS This work was supported in part by the 'Vlaams Actieprogramma Biotechnologie' of the Flemish Government (Project VLAB 014) and by Jo Nicolai N.V. W.F. Broekaert is Research Associate of the Belgian q',/ationaal Fonds voor Wetenschappelijk Onderzoek'. The authors wish to thank Dr. J. Botterman (Plant Genetic Systems N.V., Gent, Belgium) for providing the plant transformation vector pGSC1702, Dr. J. Keulemans and J. Nicolai for stimulating support and Dr. B. Cammue and Dr. W. Broothaerts for critically reading the manuscript. REFERENCES Chaband M, Passiatore JE, Cannon F, Buchanon-Wollaston V (1988) Plant Cell Reports 7, 512-516 Cornelissen M, Vandewiele M (1989) Nucleic Acids Res 17:19-29 De Block Mo Herrera-Estrella L, Van Montagu M, Schell J, Zambryski P (1984) EMBO J 3:1681-1689 De Bondt A, Druart P, Vanderleyden J, Cammue BPA, Broekaert WF (1990) Arch Int Physiol Biochem 98:PP32 Druart P (1980) Scientia Hortic 12:339-342 Druart P (1988) Acta Hort 227:369-380 Druart P. (1990) Acta Hort 280:117-124 Druart P, Kevers C, Boxus P, Gaspar T (1982) Z Ptlanzenphysiol Bd 108S: 429-436 Dufour M (1990) Acta Hort 280:51-58 Higgins ES (1992) AgBiotech News and Information 4:341-346 Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) Nature 303:179-180 Hood EE, Helmer GL, Frayley RT, Chilton M-D (1986) J Bacteriol 168:1297-1301 Hooykaas PJJ and Schilperoort RA (1992) Plant Mol Biol 19:15-38 James DJ, Passey AJ, Barbara DJ, Bevan MW (1989) Plant Cell Reports 7:658-661

Theiler-Hedtrich C, Theiler-Hedtrich R (1990) Acta Holt 280:195-199 Uematsu C, Murase M, Ichikawa K, Imamura J (1991) Plant Cell Reports 10:286-290 Vancanneyt G, Schmidt R, O'Connor-Sanchez A, Willmitzer L, RochaSosa M (1990) Mol Gen Genet 220:245-250 Van Wordragen MF, De Jong MJ, Schornagel MJ, Dons HJM (1992) Plant Sci 81:207-214 Welander M (1988) J Plant Physiol 132:738-744