Genetic Engineering of Poinsettia with the Aim of Enhancing its Resistance to Poinsettia Mosaic Virus

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1 Genetic Engineering of Poinsettia with the Aim of Enhancing its Resistance to Poinsettia Mosaic Virus J.L. Clarke ¹, S.S. Klemsdal ¹, E. Fløistad ¹, A.K. Hvoslef-Eide 2, S. Haugslien ¹, R. Moe ², and D.R. Blystad ¹ 1 Plant Protection Center, The Norwegian Crop Research Institute, Høgskoleveien 7, N-1432 Ås, Norway 2 Department of Plant and Environmental Sciences, The Agricultural University of Norway Abstract Poinsettia (Euphorbia pulcherrima) is a major ornamental pot plant in many countries. Two viruses, poinsettia mosaic virus (PnMV) and poinsettia cryptic virus, often infect modern poinsettia cultivars. To produce virus-resistant poinsettia plants, genetic engineering has been considered. Since there is no established method available for the transformation of poinsettia, we have chosen an electrophoresisbased transformation method to generate poinsettia transformants. The main advantage of this method is that we can avoid the time-consuming tissue culture and regeneration procedure. To develop a reliable transformation method, the green fluorescent protein (GFP) gene has been used as a reporter gene. Here we report our preliminary results. INTRODUCTION Poinsettia, Euphorbia pulcherrima Willd & Klotsch, is a contemporary symbol of Christmas in many parts of the world, and the most important pot plant in production in Norway (Bævre et al., 1994). In the commercial cultivars of poinsettia, two viruses, poinsettia mosaic virus (PnMV) and poinsettia cryptic virus (PnCV), can cause diseases and worldwide losses (Preil, 1994; Lee et al., 1997). PnMV gives visible symptoms in poinsettia during parts of the growing season, and is often associated with PnCV and a phytoplasma (Bradel et al., 2000). Growers show great interest in the potential benefits of growing PnMV-free poinsettias. Our previous results indicate that PnMV-free poinsettias have better commercial quality than PnMV-infected plants. Traditionally, PnMV-free poinsettia plants were obtained by in vitro culture of apical meristems. However, this is a time-consuming method and the regenerated new PnMV-free poinsettia plants have lost the branching characteristic that is important for poinsettia (Lee et al., 1997). With the help of genetic transformation, we could overcome the difficulties encountered using the traditional approach. However, there is little information describing transformation of poinsettia using electrophoresis (Vik et al., 2001) and the information regarding in vitro culture and regeneration of poinsettia is also very limited, although this has been described by Preil (1994). Thus, we have chosen the electrophoresis-based approach to transform poinsettia. The advantage of this method is that it allows us to transform intact plants and no regeneration is needed. The electrical field can draw a negatively charged DNA into cells (Ahokas, 1989; Griesbach, 1993, 1994). In the current study, poinsettia cv. Millenium has been transformed using electrophoresis. Here we report the results obtained so far and the ongoing southern analysis. MATERIALS AND METHODS Plant Materials, Gene Constructs and Electrophoresis Poinsettia cv. Millenium was used in electrophoresis. The apical meristems of sixweek-old poinsettia plants were subjected to the electrophoresis treatment (Fig. 1 and 2). Plasmid ppn93 containing the GFP gene controlled by a 35S promoter (Fig. 3) was constructed by Dr. Simon Deroles s lab (Food and Crop Research, New Zealand) and used in the current study. The conditions for electrophoresis are listed in Table. 1. Proc. XI th IS on Virus Diseases in Ornamentals Ed. C.A. Chang Acta Hort. 722, ISHS

2 DNA Extraction and PCR Analysis Genomic DNA from putative transgenic poinsettia was extracted from leaves using DNeasy Plant Mini and Maxi Kits (Qiagen Inc., Valencia, CA, USA) according to the manufacturer s instructions. To verify the presence of the transgene, the GFP gene was amplified by PCR using the primer pairs PosF1/PosR1 (sequences can be obtained on request). The expected fragment size is 500 bp. PCR amplifications were performed with denaturation at 95 C for 2 min; 30 cycles of 95 C 30 sec, 60 C 1 min and 72 C 1min followed by a final extension step at 72 C for 7 min. All the PCR reactions were carried out in a 25 µl reaction volume using GeneAmp PCR System 9700 Thermal Cycler (Applied Biosystems, Drive Foster City, CA, USA). Southern Blot Analysis of Transgenic Plants Ten micrograms genomic DNA of each PCR-positive plant was digested with EcoRI and subsequently separated on 1% agarose gels and transferred onto Gene Screen Transfer membranes (NEN TM Life Science Products, Inc. Boston, MA, USA). A PCR fragment of GFP gene (500 bp) amplified with PosF1/PosR1 primer pair was used as hybridization probe and labeled with (α- 32 P) dctp using a rediprimer TM II random prime labeling system (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, England). RESULTS AND DISCUSSION In total, 78 plants, including 8 controls, were transformed with the GFP construct driven by the 35S promoter under varying conditions. The results are summarized in Table 1. GFP fluorescent screening was performed three weeks after the transformation using microscopy. Fig. 4 shows some of the putative transformants with green fluorescence. However, auto-fluorescent immersion was observed among controls. This was due to the lack of an effective filter that could discriminate GFP from autofluorescence. The PCR analysis conducted revealed that 22 of the 78 plants were positive, showing the expected fragments (Fig. 5a and b). To verify our PCR results, southern analysis has been performed. Our preliminary result revealed that 4 of the 22 (18%) PCR positive plants have shown the expected 1009 bp EcoRI fragment indicating the stable integration of transgene into the poinsettia genome (data not shown). Due to the weak southern hybridization, further verification is being carried out. The result is in progress. To compare the results from PCR and southern analyses, 82% of the PCR positive plants did not show any hybridization to the probe indicating the losses of transgene in those plants. Such phenomenon has been reported in many other plant species (Finnegan and McElroy, 1994). The explanations could be several. a) The transformation method used has led to the loss of the GFP transgene. b) The conditions used in electrophoresis were not optimal and c) the transformed cells were out- competed by non-transformed cells. FUTURE WORK 1) Complete ongoing southern analysis on the PCR-positive plants. 2) Transform Poinsettia with a new plasmid containing the hairpin RNA targeting the PnMV replicases to obtain durable and effective protection of poinsettia. 3) To carry out Agrobacterium-mediated transformation in poinsettia in the very near future. Literature Cited Ahokas, H Transfection of germinating barley seed electrophoretically with exogenous DNA. Theor. Appl. Genet. 77: Bævre, OA, Fjeld, T. and Moe, R Poinsettia production in the North. In Strømme E (ed) The scientific basis of poinsettia production. Agricultural University of Norway, ISBN: , pp Bradel, G.B., Preil, W. and Jeske, H Sequence analysis and genome organization of Poinsettia mosaic virus (PnMV) reveal closer relationship to marafiviruses than to 322

3 tymoviruses. Virology 271: Finnegan, J. and McElroy, D Transgene inactivation: plants fight back! Bio Technol. 12: Griesbach, R.J Genetic transformation of ornamentals through in vivo electrophoresis. Proceed. XVIIth Eucarpia Symposium Creating Genetic Variation in Ornamentals. Sanremo, Italy, pp Griesbach, R.J An improved method for transforming plants through electrophoresis. Plant Sci. 102: Lee I.-M., Klopmeyer M., Bartoszyk I.M., Gundersen-Rindal D.E., Chou T.-S., Thomson K.L. and Eisenreich R Phytoplasma induced free-branching in commercial poinsettia cultivars. Nature Biotechnology 15: Preil, W The study of chimerism, elimination of virus, and the induction of mutagenesis in poinsettia. In Strømme E (ed) The scientific basis of poinsettia production. Agricultural University of Norway, ISBN: Vik, N.I., Gjerde, H., Bakke, K. and Hvoslef-Eide, A.K Stable transformation of Poinsettia via DNA-electrophoresis. Acta Hort. 560: Tables Table 1. Summary of experiments conducted. 323

4 Figures Fig. 1. Fig. 2. Figs. 1 and 2. Electrophoresis-based transformation: 1) overview and 2) an apical meristem subjected to electrophoresis. 35S promoter GFP Nos terminator Fig. 3. Schematic presentation of gene construct. Fig. 4a-d. Green fluorescent immersion: a) untransformed control and b-d) putative transformants plant. 6, 7, and

5 Fig. 5. PCR screening of transformants. P: plasmid control; C: negative control; Numbers: PCR-positive plants. 325