Agrobacterium tumefaciens mediated transformation of Cyclamen persicum Mill.

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1 Plant Science 148 (1999) Agrobacterium tumefaciens mediated transformation of Cyclamen persicum Mill. Ryutaro Aida *, Yukio Hirose 1, Sanae Kishimoto, Michio Shibata National Research Institute of Vegetables, Ornamental Plants and Tea, Ano, Mie , Japan Received 27 October 1998; received in revised form 15 April 1999; accepted 23 April 1999 Abstract A method for Agrobacterium-mediated transformation of Cyclamen persicum Mill. is reported. Etiolated petiole segments were infected with Agrobacterium tumefaciens strain AGL0 or LBA4404. These strains have a binary vector plasmid, pig121hm, that includes the -glucuronidase (GUS) gene with an intron as reporter gene, and the neomycin phosphotransferase II gene and the hygromycin phosphotransferase gene as selection markers. Explants were cultured on Murashige and Skoog medium supplemented with 1.0 mg/l thidiazuron, 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 300 mg/l ticarcillin, and 5 mg/l hygromycin or 100 mg/l kanamycin (selection medium) for regeneration. Transformation was confirmed by histochemical assays of GUS activity in plant tissues, and by Southern blot analysis of the GUS gene. Through five experiments, 103 independent GUS-positive plants were obtained from 920 explants Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cyclamen persicum Mill.; Transformation; Ornamental plants; Agrobacterium tumefaciens; Regeneration 1. Introduction Genetic transformation can create novel cultivars. Several attempts have been made to improve ornamental plants by genetic engineering [1 3]. Transformation of ornamental plants is important, especially for the modification of ornamental characteristics such as flower color, shape and longevity. The genus Cyclamen, the family Primulaceae, contains about 19 species, most of which occur in the Mediterranean region [4]. C. persicum Mill., commonly known as cyclamen, is the only commercially important species in the genus and is one of the most important ornamental pot plants in the world. Although tissue culture of cyclamen has * Corresponding author. Fax: address: ryu@nivot.affrc.go.jp (R. Aida) 1 Present address: Ehime Agricultural Experiment Station, Hojo, Ehime , Japan been well reported [5], and transgenic cyclamen plants have been obtained by Agrobacterium rhizogenes-mediated transformation (Dr Masahiro Mii, Chiba University, personal communication), transformation of cyclamen has not been documented until now. A transformation system for cyclamen would be useful for breeding; for example, to modify flower color or to improve disease resistance. Flower colors of cyclamen range from white to scarlet, salmon, and pale pink [4]. A new color, pale yellow, has been reported recently [6], but blue cyclamens do not exist. Genetic transformation would be a powerful tool for producing deep yellow- or blue-flowered cyclamens. Resistance to diseases could also be introduced by genetic transformation. In this paper, we describe an Agrobacterium tumefaciens-mediated transformation system for cyclamen, the first formal report of the transformation of cyclamen /99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S (99)

2 2 R. Aida et al. / Plant Science 148 (1999) Materials and methods 2.1. Plant materials for transformation Cyclamen cv. Anneke was used as plant material. This cultivar is known to have high embryogenic potential [7]. Seeds were soaked in 70% ethyl alcohol for 15 sec, surface-sterilized with 1% sodium hypochlorite for 30 min, and then rinsed twice with sterilized distilled water for 10 min each time. They were germinated on Murashige Skoog medium with half-strength minerals (1/2 MS) [8], solidified with 0.2% (w/v) gellan gum. Cultures were maintained in the dark at 20 C to obtain etiolated petioles, which have high regenerative potential [9]. Two to three months after germination without any subculture, etiolated petioles were cut into 8-mm segments and used as explants for transformation experiments Bacterial strain and ector plasmids A. tumefaciens strains AGL0 [10] and LBA4404 (Clontech, Palo Alto, CA, USA), both of which harbor the binary vector plasmid pig121hm (Fig. 1) [11], were used for experiments. pig121hm contains the neomycin phosphotransferase II (NP- TII) gene (nos promoter), the -glucuronidase (GUS) gene with a modified intron from the castor bean catalase gene [12] (35S promoter), and the hygromycin phosphotransferase (HPT) gene (35S promoter). Fig. 1. Structure of the binary vector pig121hm [11]. The chimeric genes were inserted between the right and left border sequences of T-DNA. The length in the figure does not correspond to the actual length. The GUS probe was used for Southern blot analysis. BR and BL=right and left border sequences of T-DNA; Pnos and Tnos=promoter and terminator of nopaline synthase gene; 35S=promoter of CaMV 35S RNA gene; NPTII=coding region of neomycin phosphotransferase II gene; Intron-GUS = coding region of -glucuronidase gene with an intron; HPT=coding region of hygromycin phosphotransferase gene; H, X, B, S, and Sc= restriction sites of HindIII, XbaI, BamHI, SalI, and SacI, respectively. Plasmid-bearing Agrobacterium cells were inoculated into liquid YEB medium (sucrose 5 g/l, beef extract 1 g/l, yeast extract 1 g/l, peptone 1 g/l) containing 50 mg/l kanamycin and shaken for 48 h at 28 C. The cells were pelleted by centrifugation and resuspended in 10 mm magnesium sulfate solution to a density of cells/ml for plant infection Preculture and coculture Precultures and cocultures of the Cyclamen explants were maintained in the dark at 20 C. Segments of etiolated petioles were precultured on MS medium solidified with 0.2% (w/v) gellan gum and containing 1.0 mg/l thidiazuron (TDZ), 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), and 100 M acetosyringon for 6 days. Acetosyringon activates the virulence genes of Agrobacterium and enhances the transfer of foreign genes into a plant genome [13]. After preculture, the explants were incubated in the Agrobacterium suspension for 5 min, then blotted dry on sterilized filter paper. Explants were placed on another sterilized filter paper on the same medium for 6 days Selection and growing culture After cocultivation, the explants were transferred to solid MS medium (0.2% gellan gum) containing 1.0 mg/l TDZ, 1.0 mg/l 2,4-D, 300 mg/l ticarcillin, and 5 mg/l hygromycin or kanamycin (selection medium) for regeneration. The selection medium was changed every 2 weeks. Ando and Murasaki [9] reported the stimulative effects of darkness during culture of etiolated petioles on shoot regeneration, so we maintained cultures in the dark at 20 C. About 3 months after infection, explants that formed shoots were transferred to solid 1/2 MS medium (0.2% gellan gum) containing 300 mg/l ticarcillin (growing medium) and cultured under a 16-h photoperiod regime under fluorescent light (70 mol/m 2 /s) at 20 C for regenerated plants to grow. After 2 to 3 months culture on the growth medium, a single regenerated plant was excised from each explant and cultured on sterilized Metro-Mix 350 (bark ash product; Scotts-Sierra Horticultural Products Company, Marysville, OH, USA) containing 1/2 MS solution with 300 mg/l carbenicillin (Metro-Mix medium) for rooting and further growth (16 h light, 20 C).

3 Table 1 Transformation efficiency in Cyclamen with binary vector pig121hm R. Aida et al. / Plant Science 148 (1999) Experiment Agrobac- Antibiotics for selec- No. of No. of No. of GUSregenerants Efficiency (transforterium strain tion (mg/l) explants 1 2 positive mants/explants) plants 3 1 AGL0 Hygromycin % 2 AGL0 Hygromycin % 3 AGL0 Kanamycin % 4 LBA4404 Hygromycin % 5 LBA4404 Kanamycin % 1 Segments of etiolated petiole were used as explants. 2 Only a single regenerant was collected from each explant to obtain independent transformants. 3 Transformation was confirmed by histochemical GUS assay GUS assay and Southern blot analysis Histochemical GUS activity was examined by the procedure reported by Jefferson et al. [14] using 5-bromo-4-chloro-3-indolyl- -D-glucuronic acid (X-GLUC) as a substrate. The GUS assay buffer used in this experiment contained 20% methyl alcohol to eliminate endogenous GUS activity, as reported by Kosugi et al. [15]. The samples were incubated at 37 for 16 h. Total DNA was extracted from leaf tissue with a Phytopure plant DNA extraction kit (Amersham, Little Chalfont, England) according to the manufacturer s instructions. About 20 g ofdna digested with HindIII was electrophoresed in a 0.6% agarose gel and transferred to a nylon membrane. HindIII cuts the plasmid at a single site outside the coding region of the GUS gene. The coding region of the GUS gene was used as a probe. Southern hybridization was carried out with a DIG-High Prime and DIG Luminescent Detection Kit for nucleic acids (Boehringer- Mannheim, Germany). Blots were finally washed with 0.2 SSC, 0.1% SDS, at 68 C. 3. Results 3.1. Transient GUS assay after Agrobacterium infection In a preliminary examination, we compared the ability of A. tumefaciens strains AGL0 and LBA4404 to transfer genes by looking for the transient GUS activity that represents early infection by Agrobacterium. Five explants were assayed for each strain. All the examined explants, regardless of strain, showed at least one blue precipitation at the cut surface, which corresponds to GUS activity (Fig. 2). GUS activity of explants inoculated with AGL0 seemed to be stronger than that of plants inoculated with LBA4404. Vector pig121hm contains a modified GUS gene [12] that can be expressed only in plant cells. This result shows that the GUS gene was transferred to the cyclamen cell and was successfully expressed Regeneration One to two months after Agrobacterium infection, adventitious buds appeared at the cut surface of explants (Fig. 3A). After transfer to the growing medium, the buds became green and grew gradually (Fig. 3B). By 5 months after infection, we obtained 161 independent plants from 920 explants through five experiments (Table 1). Regenerated plants grew normally (Fig. 3C) after being transplanted to the Metro-Mix medium GUS assay We examined 161 regenerated plants for GUS activity. The assay showed that 103 plants were GUS-positive (Fig. 4) and confirmed that nontransformed control plants never showed GUS activity, suggesting that the GUS-positive plants were transgenic. Transformation efficiency (transformants/explants) was higher with AGL0 (9.0% 19.0%) than with LBA4404 (0% 2.5%) (Table 1). AGL0 might have a higher infection ability than LBA4404, as shown by the transient GUS assay after Agrobacterium infection (Fig. 2).

4 4 R. Aida et al. / Plant Science 148 (1999) 1 7 Fig. 2. Transient GUS assay after Agrobacterium infection. After coculture with Agrobacterium strain AGL0 (lower) or LBA4404 (upper) harboring a binary vector plasmid, pig121hm, that contains the GUS gene with an intron, all explants showed at least one blue precipitation, representing GUS activity, at the cut surface. Fig. 3. Regeneration of putative transgenic cyclamen. (A) Regenerated buds from explants on solid MS medium (0.2% gellan gum) containing 1.0 mg/l TDZ, 1.0 mg/l 2,4-D, 300 mg/l ticarcillin, and 5 mg/l hygromycin (selection medium). (B) Growing plants on solid 1/2 MS medium (0.2% gellan gum) containing 300 mg/l ticarcillin (growing medium). (C) Normal cyclamen plants growing on the Metro-Mix medium (8 months after Agrobacterium infection). Fig. 4. GUS assay of the regenerated plants. Non-transformed control plants never showed blue precipitation (top row). Some regenerated plants showed blue precipitation, which represents GUS activity (lower rows).

5 R. Aida et al. / Plant Science 148 (1999) Southern blot analysis We selected two GUS-positive and three GUSnegative plants from experiment one (Table 1) for Southern blot analysis. Analysis showed that the GUS-positive plants had the GUS gene in their genome but that the GUS-negative plants did not (Fig. 5). Digestion of pig121hm DNA with HindIII cuts the plasmid at a single site outside the coding region of the GUS gene (Fig. 1). The presence of contaminating plasmid in the tissues should be detected by the presence of a single 15.6-kb band. The GUS-positive plants showed one or more bands of differing sizes, indicating single- or multiple-copy integration of the GUS gene into the genome. We considered the GUSpositive plants to be transformants. 4. Discussion There have been many reports of tissue culture of cyclamen. Most early studies used tuber tissue as explant material, but contamination caused by parasitic microorganisms living in the tuber was a serious problem [5]. The use of tissue of seedlings grown from surface-sterilized seeds can overcome Fig. 5. Southern blot analysis of 2 GUS-positive (GUS+) and 3 GUS-negative (GUS ) plants. The coding region of the GUS gene was used as a probe. The GUS-positive plants showed one or more bands of differing sizes, indicating singleor multiple-copy integration of the GUS gene into the genome. The GUS-negative plants showed none. this problem. Ando and Murasaki [9] reported that etiolated petioles of cyclamen could be regenerated, but that normal (not etiolated) petioles could not. This method also avoids damage to stock plants. We therefore, decided to use etiolated petioles of aseptic seedlings as explants for the transformation experiments. We based the characterization of gene transfer on GUS gene expression. We used vector pig121hm [11], which contains GUS with a modified intron from the castor bean catalase gene in the coding region [12]. Prokaryotes cannot express the intron GUS combination, so the GUS expression in the explants and regenerated plants was a result of transgene expression in the plant cells. There was no detectable endogenous GUS activity in the cyclamen tissues (Fig. 4, top row). The experiment used selection medium containing TDZ and 2,4-D as plant growth regulators for bud regeneration. TDZ is highly efficient at shoot regeneration in a wide variety of plants [16], and 2,4-D is known to stimulate adventitious organogenesis from cyclamen tissue [17]. Adventitious buds appeared at the cut surface of explants 1 to 2 months after Agrobacterium infection (Fig. 3A), and regenerated plants grew normally (Fig. 3C). The combination of 1.0 mg/l TDZ and 1.0 mg/l 2,4-D used in this study seemed to be effective for organogenesis of adventitious buds from etiolated petioles of cyclamen. Embryogenesis was never observed under this culture condition. In other experiments, we tried to obtain cyclamen transformants through embryogenic calluses under culture conditions required for embryogenesis, but failed to obtain any embryos on selection medium with antibiotics (data not shown). We considered that kanamycin and hygromycin would have negative effects on cyclamen embryogenesis. We examined 161 independent regenerated plants for GUS activity; 103 showed GUS activity (Fig. 4), thus showing the existence of the GUS transgene. Two putative transformants and 3 putative non-transformants were subsequently analyzed by Southern blot analysis using the GUS coding region as a probe. The analysis confirmed the integration of the GUS gene into the GUSpositive plants only. However, the GUS-negative plants might be non-transformants there is a possibility that they have only the HPT (and NP- TII) gene and lack the GUS gene. Further investigation will serve to clarify the nature of the

6 6 R. Aida et al. / Plant Science 148 (1999) 1 7 GUS-negative plants. Following the results of the preliminary experiment, we used 5 mg/l hygromycin or 100 mg/l kanamycin for selection of transformants, both of which suppressed organogenesis from non-infected segments of etiolated petioles (data not shown). We obtained transformants with both as selective agents, regeneration of non-transformed escapees could be reduced with more appropriate selective pressure. Recently, there have been many reports of modification of flower color by genetic transformation; for example, in petunia [18 26], Arabidopsis [27], gerbera [28], lisianthus [29,30], chrysanthemum [31], and rose [32]. Our transformation system could be useful for creating cyclamens with new colors, such as deep yellow or blue. Other characteristics, such as resistance to disease, could be improved by this system. Tabei et al. [33] reported that transgenic cucumber plants expressing a rice chitinase gene showed increased resistance to gray mold (Botrytis cinerea). This disease is one of the most serious for cyclamen. Introduction of a chitinase gene into cyclamen might enhance its resistance to B. cinerea. Other disease resistance genes could improve cyclamen in the future. Acknowledgements We thank Dr Kenzo Nakamura, Nagoya University, for providing the plasmid pig121hm, and Dr Robert A. Ludwig, University of California, Santa Cruz, for providing the Agrobacterium strain AGL0. This work was supported by a grant from the Ministry of Agriculture, Forestry and Fisheries, Japan. References [1] J.F. Hutchinson, V. Kaul, G. Maheswaran, J.R. Moran, M.W. Graham, D. Richards, Genetic improvement of floricultural crops using biotechnology, Aust. J. Bot. 40 (1992) [2] K.E.P. Robinson, E. Firoozabady, Transformation of floriculture crops, Scientia Horticulturae 55 (1993) [3] A. Zuker, T. Tzfira, A. Vainstein, Genetic engineering for cut-flower improvement, Biotechnol. Adv. 16 (1998) [4] A.J. Huxley, M. Griffiths, M. Levy, The New Royal Horticultural Society Dictionary of Gardening, Macmillan Press Limited, London and Basingstoke, 1992, pp [5] T. Geier, H.W. Kohlenbach, G. Reuther, Cyclamen. Chapter 15, in: P.V. Ammirato, D.A. Evans, W.R. Sharp, Y.P.S. Bajaj (Eds.), Handbook of Plant Cell Culture, Volume 5, Ornamental Species, McGraw-Hill, New York, 1990, pp [6] I. Miyajima, T. Maehara, T. Kage, K. Fujieda, Identification of the main agent causing yellow color of yellowflowered cyclamen mutant, J. Japan. Soc. Hort. Sci. 60 (1991) [7] T. Takamura, I. Miyajima, E. Matsuo, Somatic embryogenesis of Cyclamen persicum Mill. Anneke from aseptic seedlings, Plant Cell Reports 15 (1995) [8] T. Murashige, F. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plant. 15 (1962) [9] T. Ando, K. Murasaki, In vitro propagation of cyclamen by the use of etiolated petioles. Technical Bulletin of the Faculty of Horticulture, Chiba University, 32 (1983) 1 5. [10] G.R. Lazo, P.A. Stein, R.A. Ludwig, A DNA transformation-competent Arabidopsis genomic library in Agrobacterium, Bio/Technology 9 (1991) [11] Y. Hiei, S. Ohta, T. Komari, T. Kumashiro, Efficient transformation of rice (Oryza sati a L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J. 6 (1994) [12] S. Ohta, S. Mita, T. Hattori, K. Nakamura, Construction and expression in tobacco of a -glucuronidase (GUS) reporter gene containing an intron within the coding sequence, Plant Cell Physiol. 31 (1990) [13] S.E. Stachel, E. Messens, M.V. Montagu, P. Zambryski, Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens, Nature 318 (1985) [14] R.A. Jefferson, T.A. Kavanagh, M.W. Bevan, GUS fusions: -glucuronidase as a sensitive and versatile gene fusion marker in higher plants, EMBO J. 6 (1987) [15] S. Kosugi, Y. Ohashi, K. Nakajima, Y. Arai, An improved assay for -glucuronidase in transformed cells: methanol almost completely suppresses a putative endogenous -glucuronidase activity, Plant Sci. 70 (1990) [16] C.A. Huetteman, J.E. Preece, Thidiazuron: a potent cytokinin for woody plant tissue culture, Plant Cell. Tissue and Organ Culture 33 (1993) [17] T. Takamura, M. Tsutsui, M. Tanaka, Micropropagation of yellow-flowered Cyclamen through adventitious organogenesis in medium containing 2,4-dichlorophenoxyacetic acid. Technical Bulletin of the Faculty of Agriculture, Kagawa University, 48 (1996) [18] P. Meyer, I. Heidmann, G. Forkmann, H. Saedler, A new petunia flower colour generated by transformation of a mutant with a maize gene, Nature 330 (1987) [19] A.R. van der Krol, P.E. Lenting, J. Veenstra, I.M. van der Meer, R.E. Koes, A.G.M. Gerats, J.N.M. Mol, A.R. Stuitje, An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation, Nature 333 (1988)

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