TRANSFORMATION OF ROSES WITH GENES FOR ANTIFUNGAL PROTEINS TO REDUCE THEIR SUSCEPTIBILITY TO FUNGAL DISEASES

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1 TRANSFORMATION OF ROSES WITH GENES FOR ANTIFUNGAL PROTEINS TO REDUCE THEIR SUSCEPTIBILITY TO FUNGAL DISEASES Andrea Dohm, Clarisse Ludwig, Dagmar Schilling and Thomas Debener Federal Centre for Breeding Research on Cultivated Plants Institute for Ornamental Plant Breeding Bornkampsweg Ahrensburg Germany Keywords: Agrobacterium mediated gene transfer, somatic embryogenesis, in planta transformation, ribosome inhibiting protein, chitinase, glucanase, T4- lysozyme, signal peptide Abstract In order to obtain partial resistance in roses to the major fungal diseases simultaneously, we followed the biotechnological approach of overexpressing genes for particular antifungal proteins. Via Agrobacterium mediated gene transfer of somatic embryos and subsequent adventitious shoot formation different combinations of antifungal defence genes were introduced into the garden rose cultivars Heckenzauber and Pariser Charme. The transformation frequency reached a maximum of 3 %. This number refers to the number of transgenic shoots obtained from transformed somatic embryos and was mainly determined by the regeneration capacity of the somatic embryos. The type of carbon source in the culture medium had a significant effect on adventitious shoot formation. Altogether, 80 true transgenic plants were analysed for expression of their transgenes and resistance to blackspot. Compared to non transgenic control plants, the susceptibility to blackspot did not decrease in the case of cytosolic expression of the antifungal proteins, although 80 % of these plants proved to express the transgenes by Northern analysis. The secretion of the ribosome inhibiting protein into the extracellular space, however, reduced the susceptibility against blackspot to 60 % on average. To avoid problems of standard in vitro transformation protocols, e.g. the use of antibiotics as selectable markers, we try to establish an in planta transformation system. 1. Introduction Due to their high economic value and their worldwide cultivation roses belong to the most important ornamental plants. The genus Rosa comprises numerous species and hybrids, which are cultivated out door as well as in glass houses for different purposes. Regardless of use or mode of cultivation rose growing is severily impaired by various fungal diseases (powdery mildew, downy mildew, blackspot, rust, botrytis blight). Therefore, breeding resistances against these diseases has become more and more important. During the last years it could be shown repeatedly, that induced overexpression of particular antifungal proteins may lead to enhanced pathogen resistance (Broglie et al., 1991; Logemann et al., 1992; Zhu et al., 1994; Jach et al., 1995). In roses Marchant et al. (1998a) introduced a rice chitinase gene under control of the constitutive CaMV 35S promotor. The resulting transgenic genotypes showed reduced susceptibility to blackspot. In contrast to the integration of resistance genes against particular pathogen species or even races into cultivars by conventional cross breeding, the biotechnological approach offers the opportunity to reduce the susceptibility to several pathogens, simultaneously. In Dohm et al. (2001a,b) we describe the production and analysis of transgenic Proc. XX EUCARPIA Symp. on New Ornamentals II Eds. J. Van Huylenbroeck et al. Acta Hort. 572, ISHS

2 roses with cdnas from barley, encoding a Class II chitinase, a Class II ß-1,3-glucanase and a Type I ribosome inhibiting protein (Jach et al., 1995) as well as with a lysozyme gene originating from the T4-phage (Düring, 1996). Transformation was performed by Agrobacterium mediated gene transfer of somatic embryos and subsequent regeneration to plants by adventitious shoot formation. Here, we present additional data on the regeneration and selection of transgenic shoots. Furthermore, we describe previous results on the establishment of an in planta transformation system for roses. 2. Materials and methods 2.1. Plant material The in vitro transformation experiments were performed on somatic embryos of the garden rose cultivars Heckenzauber and Pariser Charme. For transformation embryogenic callus was established by cultivation of in vitro leaf explants on MS basal medium (Murashige and Skoog, 1962) supplemented with 0.5 mg/l 2,4-D or 1 mg/l NAA for one culture period of four weeks followed by three further culture periods on MS medium with 4 mg/l zeatine. Regenerating embryogenic calli were separated from the original leaf explants and propagated on MS medium with 0.25 mg/l 1-NAA, 1.5 mg/l zeatine and 1.0 mg/l GA 3 according to Noriega and Söndahl (1991). Subcultures were carried out every 4 weeks. All cultures were kept at 25 C with a 16 h photoperiod. In planta infiltration experiments were carried out on in vitro shoots and greenhouse grown plants of the garden rose cultivars Heckenzauber and Pariser Charme, which were cultivated as described above Bacterial strains and plasmids For Agrobacterium mediated gene transfer of somatic embryos we used the disarmed strains EHA 105 and GV 2260, both containing the plasmid pbin 19 harbouring nptiii and nptii genes which provide resistance to the antibiotic kanamycine. The development of the transformation protocol was performed with the GUS(Int) gene (Vancanneyt et al., 1990) under control of the CaMV 35S promotor. Furthermore, combinations of cdnas encoding different antifungal proteins with a potato signal peptide controlling the transport of the antifungal proteins from the cytosol into the apoplast were introduced into pbin 19 (Table 1). All genes are driven by the CaMV 35S promotor. All bacterial strains were grown on LB agar plates (Sambrook and Russell, 2001) containing 50 mg/l kanamycine sulphate. For Agrobacterium infiltration experiments different wild strains of the Octopine or Nopaline type, which were isolated from infected rose plants, were kindly provided by C. Poncet, INRA, Antibes. These non disarmed strains were additionally transformed with pbin 19 containing GUS(Int) under control of the CaMV 35S promotor. Cultivation was carried out as described for the disarmed strains Agrobacterium mediated gene transfer into somatic embryos For transformation experiments a single bacterial colony was incubated in liquid YEP medium (Sambrook and Russell, 2001) supplemented with 50 mg/l kanamycine sulphate and cultivated at 29 C and 175 rpm for 24 h. The resulting bacterial suspension was diluted 1 : 20 in YEP medium or Minimal A medium according to Miller (1972) and incubated for 2 h at 29 C and 100 rpm. Fully developed somatic embryos were isolated from the callus at the end of a 4 week subculture. The selected embryos were immersed into the Agrobacterium suspension for 1 h. Subsequently, excess bacterial suspension was removed with sterile filter paper and the explants were incubated on MS basal medium supplemented with 0,01 mg/l IBA, 2 mg/l BAP and 0.1 mg/l GA 3 for shoot induction. Following a co- 106

3 cultivation period of 2 days for EHA 105 or of 6 days for GV 2260, the somatic embryos were transferred onto fresh medium containing 500 mg/l cefotaxime sodium and 50 mg/l carbenicilline to inhibit further bacterial growth. The cultures were grown at 25 C with a 16 h photoperiod. About 3 weeks after inoculation the immersed embryos were incubated on MS basal medium containing 0,01 mg/l IBA, 2 mg/l BAP and 0.1 mg/l GA 3 for shoot induction and supplemented with 150 mg/l TIMENTIN (Duchefa) to inhibit the bacterial growth as well as 60 mg/l kanamycine sulphate for selection. Regenerating shoots were separated from the original explants and transferred onto MS basal medium with mg/l 1-NAA, 1.0 mg/l BAP and 0.1 mg/l GA 3 containing 150 mg/l TIMENTIN and 60 mg/l kanamycine sulphate as well. Rooting of transgenic shoots was induced by subculture on half strength MS medium with 0.1 mg/l 1-NAA and 0.05 mg/l IBA. The regenerating somatic embryos as well as the shoots were incubated at 25 C with a 16 h photoperiod. For greenhouse adaptation the rooted plants were planted into a 1 : 1 mixture of standard pricking soil and perlite after removal of the agar based medium. Over the following 3 weeks air humidity was decreased stepwise and the plants were gradually transferred to 14 cm diameter containers and raised to flowering In planta transformation The bacterial suspensions were prepared as described for in vitro transformation. In vitro shoots were immersed into these suspensions, and the bacteria were forced into the shoots by short-term vacuum infiltration. Following this treatment the shoots were rooted, potted and adapted to greenhouse conditions as described above. Alternatively, greenhouse grown plants were infiltrated with Agrobacteria by injection of bacterial suspension into leaves with a blunt tipped syringe Analysis of transgenic plants Northern blot analysis Putative transgenic rose plants were screened with appropriate cdna probes by Northern blot analysis. Total RNA from young leaves was isolated using the Invitek RNA isolation kit (Invitek, Berlin). 20 µg RNA each was separated elektrophoretically on a denaturing agarose gel, transferred to a Hybond-N membrane (Amersham) and hybridized to the radioactively labelled probes according to standard protocols (Sambrook and Russell, 2001) Blackspot bioassay Rose plants were inoculated with a single spore isolate of Diplocarpon rosae. Conidia suspension was sprayed onto the leaves at a density in the range of conidia/ml. With the appearance of first symptoms due to blackspot infection about ten days after inoculation the further development of infection was assessed every four days. Each transgenic genotype was tested in three independent trials with at least ten plants. From these data disease indices were determined for the transgenic genotypes and the control plants GUS-Assay of Agrobacterium infiltrated plants All plants were raised till flowering and various vegetative as well as floral parts were analysed for GUS-expression in a histochemical assay according to Jefferson et al. (1987). 107

4 3. Results 3.1. Agrobacterium mediated gene transfer and regeneration of transgenic plants Somatic embryos of the garden rose cultivars Heckenzauber and Pariser Charme were transformed via Agrobacterium mediated gene transfer with different combinations of genes encoding antifungal proteins. Table 1 summarises the current stock of independent transgenic plants. With a maximum transformation frequency of 3 % referred to the incubated somatic embryos forming transformed shoots, flowering transgenic plants can be produced routinely within nine months. The regeneration system combines somatic embryogenesis and adventitious shoot formation: First somatic embryogenesis is induced on leaf explants. The resulting embryogenic callus serves as a source for somatic embryos as a target for transformation. As the germination of somatic embryos occured with a low frequency, transgenic shoots had to be regenerated by adventitious shoot formation of the somatic embryos. The regeneration capacity is strongly effected by the sugar composition of the culture medium (Table 2). Adventitious shoot formation of somatic embryos of the cultivar Pariser Charme, for example, is enhanced by xylose compared to sucrose, which is routinely used in rose tissue culture. Somatic embryos originating from different callus lines, however, respond differently to the tested carbon sources, although preculture conditions were identical and all of them belong to the same rose genotype (Table 2) Analysis of transgenic plants All putative transgenic plants were analysed by Northern blot hybridisation to prove the expression of the introduced transgenes. Altogether, 80 % of the tested genotypes showed expression of the transgenes. The susceptibility to blackspot could not be reduced by cytosolic expression of the antifungal proteins obtained with the constructs pgj40, pgj42 and T4-LYS (Table 1). Those plants carrying the antibacterial T4-lysozyme gene performed even worse than the control plants. However, the secretion of the ribosome inhibiting protein into the apoplast resulted in a significant reduction of the disease index down to 60 % compared to control plants (Dohm et al., 2001a) Development of an in planta transformation system Up to now six infiltration experiments were performed. Regardless of the infiltration method all of them resulted in plants which reproducibly display transformation events in anthers or pistils. On average about 50 % of the analysed flowers showed GUS-expression of pollen or pistils, although the frequencies varied between independent plants and infiltration experiments. GUS-expression of vegetative parts was only occasionally detected. Furthermore, abnormalities of various morphological characters due to Agrobacterium infection were observed, for example tumor formation, decreased plant growth, abnormal branching types as well as deviating leaf and flower morphology. 4. Discussion Here, we present an efficient protocol for Agrobacterium mediated gene transfer of roses. In contrast to previous reports on rose transformation (Firoozabady et al., 1994, Marchant et al., 1998b, van der Salm et al., 1997) somatic embryos are used as target for transformation. Furthermore, transgenic plants are regenerated via adventitious shoot formation instead of germination of somatic embryos. Transformation efficiency is significantly influenced by the regeneration capacity of the transformed somatic embryos. Studies on the effectiveness of various carbon sources on adventitious shoot formation of 108

5 somatic embryos revealed the highest regeneration frequency for xylose. The original intention of these experiments was to find alternatives to selectable markers based on antibiotics. For example Haldrup et al. (1998) successfully used the enzyme xylose isomerase for selection of transgenic potato shoots. In maize phosphomannose-isomerase was used as selection marker (Negrotto et al., 2000). As one prerequisite for this strategy plants must be unable to metabolise a particular sugar as carbon source. Our studies, however, demonstrate, that roses can use D-xylose as a sole carbon source, it even enhances the regeneration capacity of somatic rose embryos compared to sucrose. The complete substitution of sucrose by mannose in fact inhibits adventitious shoot formation of somatic rose embryos. On the other hand a partial substitution achieves an overall higher regeneration frequency. Furthermore, the varying response of somatic embryos originating from different calli complicates the determination of an appropriate media formulation for selection. As a consequence, the use of enzymes for sugar metabolism as an alternative selection strategy in roses seems to be difficult. The susceptibility of the different transgenic genotypes to blackspot was analysed with a quantitative bioassay, because only partial resistance could be expected (Jach et al., 1995). Reaction of the transgenic genotypes to blackspot infection varied due to the integrated transgene. A significant reduction of susceptibility down to 60 % compared to control plants could only be achieved by secretion of the barley ribosome inhibiting protein into the apoplast. In contrast to the findings of Marchant et al. (1998a) the cytosolic expression of the antifungal proteins did not result in decreasing susceptibility. Although Stahl et al. (1998) observed enhanced fungal resistance in T4-lysozyme transgenic tomato and oilseed rape, in the current study expression of the T4-lysozyme gene even increased susceptibility to blackspot. An explanation for this observations is not available so far. The method of in planta transformation was first described for Arabidopsis thaliana by Feldman and Marks (1987). In the meantime various protocols have been developed, which enable the production of thousands of independent transgenic lines within several months (reviewed by Bent, 2000). Although numerous efforts have been made to adapt the in planta transformation method to plant species other than Arabidopsis, successful protocols have only been published for a handful of different species. Detailed studies on the mechanism of in planta transformation of Arabidopsis with GUS(Int) as target gene revealed, that ovules are the primary target of transformation (Desfeux et al., 2000, Ye et al., 1999). Although reproducible GUS-expression of pollen cells was detected, the transgene was only transmitted in crosses using infiltrated plants as the pollen recipient, but not as the pollen donor. Therefore, the expression of a transgene in germ cells does not guarantee its stable transmission in crosses. In running experiments the infiltrated plants are used in crossing experiments. As a result of reduced male and female fertility only 70 % of the pollinated flowers formed hips. At present, the hips are grown to maturity. The resulting seeds will be harvested, sawn and analysed for GUS-expression. Acknowledgements We want to thank Dr. G. Jach (MPI, Cologne) for kindly providing the pbin 19 constructs containing the cdnas from barley, and we are grateful to Dr. K. Düring (MPB Cologne), who put the T4-lysozyme gene to our disposal. References Bent A.F., Arabidopsis in planta transformation. Uses, mechanism, and prospects for transformation of other species. Plant Physiology 124: Broglie K., Chet I., Holliday M., Cressman R., Biddle P., Knowlton S., Mauvais C.J., and Broglie R., Transgenic plants with enhanced resistance to the fungal pathogen Rhizoctonia solani. Science 254: Desfeux C., Clough S.J., and Bent A.F., Female reproductive tissues are the 109

6 primary target of Agrobacterium-mediated transformation by floral-dip method. Plant Physiology 123: Dohm A., Ludwig C., Schilling D., and Debener T., 2001a. Transformation of roses with genes for antifungal proteins. Acta Horticulturae 547:27-34 Dohm A., Ludwig C., Nehring K., and Debener T., 2001b. Somatic embryogenesis in roses. Acta Horticulturae 547: Düring K., Genetic engineering for resistance to bacteria in transgenic plants by introduction of foreign genes. Molecular Breeding 2: Feldman K.A., and Marks M.D., Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol.Gen.Genet. 208:1-9 Firoozabady E., Moy Y., Courtney-Gutterson N., and Robinson K., Regeneration of transgenic rose (Rosa hybrida) plants from embryogenic tissue. Bio/Technology 12: Haldrup A., Petersen S.G., and Okkels F.T., Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry. Plant Cell Reports 18:76-81 Jach G., Goernhardt B., Schell J., Pinsdorf E., Mundy J., Logemann J., and Maas C., Synergistically enhanced resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. The Plant Journal 8: Jefferson R.A., Kavanagh T.A., and Bevan M.W., GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal 6: Logemann J., Jach G., Tommerup H., Mundy J., and Schell J., Expression of a barley ribosome-inactivating protein leads to increased fungal protection in transgenic tobacco plants. Bio/Technology 10: Marchant R., Davey M.R., Lucas J.A., Lamb C.J., Dixon R.A., and Power J.B., 1998a. Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf). Molecular Breeding 4: Marchant R., Power J.B., Lucas J.A., and Davey M.R., 1998b. Biolistic transformation of rose (Rosa hybrida L.). Annals of Botany 81: Miller J.H., Experiments in Molecular Genetics. Cold Spring Harbor Press Murashige T., and Skoog F., A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: Negrotto D., Jolley M., Beer S., Wenck A.R., and Hansen G., The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Reports 19: Noriega C., and Söndahl M.R., Somatic embryogenesis in hybrid tea roses. Bio/Technology 9: Salm T.P.M. van der, van der Toorn C.J.G., Bouwer R., Hänisch ten Cate C.H., and Dons H.J.M., Production of ROL gene transformed plants of Rosa hybrida L. and characterisation of their rooting ability. Mol. Breed. 3:39-47 Sambrook J., and Russell D.W., Molecular Cloning A Laboratory Manual, 3 rd edition. Cold Spring Harbor Laboratory Press Stahl D.J., Maser A., Dettendorfer J., Holtschulte B., Thomzik J.E., Hain R., and Nehls R., Increased fungal resistance of transgenic plants by heterologous expression of bacteriophage T4 lysozyme gene. Abstracts, 7 th International Congress of Plants Pathology, 9 th 16 th August 1998, Edinburgh Vancanneyt G., Schmidt R., O Connor-Sanchez A., Willmitzer L., and Rocha-Sosa M., Construction of an intron-containing marker gene: Splicing of the intron in transgenic plnts and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol.Gen.Genet. 220: Ye G.-N., Stone D., Pang S.-Z., Creely W., Gonzales K., and Hinchee M., Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration 110

7 transformation. The Plant Journal 19: Zhu Q., Maher E.A., Masoud S., Dixon R.A., and Lamb C.J., Enhanced protection against fungal attack by constitutive co-expression of chitinase and glucanase genes in transgenic tobacco. Bio/Technology 12: Table 1: Number of transgenic rose genotypes harbouring genes for antifungal proteins construct gene origin number of independent transgenic plants pgj 42 Type I RIP + Class II chitinase barley 36 pgj 40 Class II ß-1,3-Glucanase barley 12 + Class II chitinase pgj 28 Type I RIP + signalpeptide barley 27 pgj 36 Class II chitinase + signalpeptide barley 13 T4-LYS Lysozyme T4 phage 5 Table 2: Regeneration of somatic embryos (%) of the rose Pariser Charme in relation to the sugar composition of the culture medium Sugar composition Somatic embryos originating from callus (%) Mean 3 % sucrose % sucrose % xylose % sucrose % xylose % mannose % mannose % sucrose % glucose % glucose % sucrose % mannitol % mannitol % sucrose % fructose % fructose sucrose % sucrose + 3 % PEG Numbers printed in bold represent the highest regeneration frequency of the particular callus lines. 111