Towards Agrobacterium-mediated transformation of Hippeastrum x chmielii Chm. Identification of appropriate selection media

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1 Towards Agrobacterium-mediated transformation of Hippeastrum x chmielii Chm. Identification of appropriate selection media Agnieszka Ilczuk, Aleksandra Łukaszewska Warsaw Agricultural University Department of Ornamental Plants Nowoursanowska 166 PL Warsaw Poland ilczuk@alpha.sggw.waw.pl Traud Winkelmann, Margrethe Serek Institute of Floriculture, Tree Nursery Science and Plant Breeding Section Floriculture University of Hannover Herrenhaeuser Str.2 D Hannover, Germany Keywords: breeding, cefotaxim, gene transfer, glufosinate, in vitro, phosphinothricin, regeneration Abstract Hippeastrum species (Amaryllidaceae) have significant floricultural importance and are one of the major bulb crops in the commercial market. Amaryllidales are monocotyledonous plants that have been shown to be generally recalcitrant to molecular genetic manipulation. Successful plant transformation requires efficient regeneration and selection system and these were developed for a new hybrid Hippeastrum x chmielii Chm. This hybrid shows very vigorous growth and flowers without undergoing a quiescent phase. In preparation of transformation experiments the aim of this study was to establish appropriate media for selecting transgenic cells/regenerants. Therefore, the effect of different concentrations of glufosinate (syn. phosphinothricin) on plant regeneration and propagation were analyzed. Glufosinate will be used in transformation experiments to select the cells expressing the pat gene. In addition the influence of cefotaxim, an antibiotic used for suppression of agrobacteria after co-culture, on the selective efficiency of glufosinate was tested. Two different kinds of explants were used: first, young flower stalk segments cultured on MS medium with 2 mg/l 2iP and 0.2 mg/l NAA, and second quarters of in vitro bulblets on MS medium with 0.5 mg/l BA and 0.1 mg/l NAA. Glufosinate totally inhibited bulblet formation for both types of explants even at low concentrations of 1.0 mg l -1 to 2.0 mg l -1. Therefore, this level was found to be suitable for being used in the selection medium. The addition of 500 mg/l cefotaxim did neither inhibit regeneration nor did it interfere with the selective efficiency in glufosinate containing medium. The results obtained in this study are the basis for establishing an Agrobacterium-mediated transformation system. By genetic transformation Hippeastrum x chmielii Chm. can be improved regarding flower longevity or disease resistance. INTRODUCTION In recent years the interest of flower bulbs plants as ornamental crops has increased significantly and as a consequence the cultivation area has also become larger in many countries. Hippeastrum (Amaryllidaceae), more commonly known as Amaryllis, is a genus of flower bulbs comprising about 60 to 70 species. More than 300 varietes are

2 known, presenting a vast range of colours and colour combinations (Wahi and Bhattacharjee, 1986; Okubo, 1993). In the Department of Ornamental Plants at Warsaw Agricultural University, in 1993 Prof. Henryk Chmiel obtained a new interesting hybrid named Hippeastrum x chmielii Chm.. Clones of this new creation grow more vigorously, bloom abundantly every 3-4 months in one season. Their flowers are smaller than in Hippeastrum hybridum but their inflorescences are easier to use in floral compositions. In particular clones the perianth can be white, red, red-orange or bicolor with marked radial pharynx. Hippeastrum x chmielii can be grown for cut flowers or as pot plants (Chmiel et al., 2002). Monocotyledonous plant have been generally considered to be outside the host range of Agrobacterium. Evidence for actual transformation in monocotyledonous bulbous plants was reported in Lilium (Cohen and Meredith, 1992; Langeveld et al., 1995; Hoshi et al., 2004), Agapanthus (Suzuki et al., 2001), Tulipa (Wilmink et al., 1992; Chauvin et al., 1999), Narcissus (Sage and Hammatt, 2002), Gladiolus (Kamo, 1997; Chauvin et al., 1999), Muscari (Suzuki and Nakano, 2002) and Allium (Kondo et al., 2000; Zheng et al., 2001; Eady et al., 2003). We are not aware of any report on either agroinfection or transformation in the genus Hippeastrum. The purpose of this study was to establish appropriate media for selection of transgenic cells/regenerants and to develop a transformation protocol thereafter. MATERIAL AND METHODS All experiments were carried out with Hippeastrum x chmielii Chm. Clone 18. In vitro culture of hippeastrum floral stem segments (Ilczuk and Witomska, 2003, 2004) and quarters of in vitro bulblets (Witomska and Ilczuk, 2004) were chosen because, to the best of our knowledge, they are the most effective true-to-type regeneration or propagation system for this species. Experiment with Young Floral Stalks Adult bulbs (15 18 cm) were harvested and kept for six months at 9 o C. After that bulbs were forced about 1-2 weeks at 25 o C. Only bulbs with visible buds were used in this experiment. Floral stems, usually 3-6 cm long measured from the base of the bulb to the inflorescence, were sterilized as follows: 15 s in 70% ethanol, 10 min in NaOCl (1 % active chlorine) solution and rinsing three times in sterile tap water for 1, 2, and 5 min. After sterilization, young floral stems were cut into mm think discs which were cultured with their basal ends downward on a bulb regeneration MS medium (Murashige and Skoog, 1962) with: MS salts, MS vitamins lacking peptone, 2.0 mg l -1 2iP (dimethylallylaminopurine), 0.2 mg l -1 NAA (naphthalene acetic acid), 30 g l -1 sucrose, 8.0 g l -1 agar (Agar Agar Serva), ph 5,7. Experiment with in vitro Bulblets The in vitro bulblets used in this experiment were 5-8 mm in diameter, mean fresh weight 0, 164 g.the bulblet without leaves and root was cut vertically into quarters. Explants were incubated on bulb regeneration MS medium (Murashige and Skoog, 1962) with MS salts, MS vitamins lacking peptone, 0.5 mg l -1 BA (benzylaminopurine), 0.1 mg l -1 NAA, 30 g l -1 sucrose, 8.0 g l -1 agar, ph 5.7

3 Selective Agent Glufosinate was chosen to be used for later selection of the cells expressing the pat gene. It was included in the media to give a final concentration of: 1.0, 1.5, 2.0, and 3.0 mg l -1. Additionally, 500 mg l -1 cefotaxim, an antibiotic used for suppression of agrobacteria after co-culture, were added to the regeneration medium after three days. Both agents were filter sterilized and given to the medium after autoclaving. Explants were subcultured onto fresh medium every 3-4 weeks. Culture Conditions and Evaluation Regeneration took place at 25 o C, under white fluorescent light (30 µmol m -1. s -2 for 16 hours per day). After 1, 2 and 3 months the percentage of explants forming callus and bulblets as well as the number of bulblets per explant were recorded. Each treatment included 25 explants and each experiment was repeated twice. Results were analyzed with Statgraphics 4.1 and the LSD 0.05 was used to compare the means. RESULTS AND DISCUSSION Successful plant transformation requires efficient regeneration and selection systems. In the transformation protocols of bulbous crops different antibiotics were used as selective agents like kanamycin (Chauvin et al., 1997, 1999) and hygromycin (Chauvin et al., 1997; Langeveld et al., 1997; Eady and Lister, 1998; Suzuki and Nakano, 2002; Hoshi et al. 2004). On the other hand herbicide active agents were applied in other studies like bialaphos (Watad et al., 1998; Lipsky et al., 2002) and glufosinate (Chauvin et al., 1997; Eady et al., 2003). In the experiments reported in this paper, we studied the reaction of Hippeastrum floral stem segments and quarters of in vitro bulblets in the presence of cefotaxim and glufosinate. The control treatments (without glufosinate) demonstrated that the formation of bulblets in Hippeastrum took place in both propagation systems, but in much higher rates from in vitro bulblets (Tables 1 and 2). For both explants types the adding of 500 mg/l cefotaxime to regeneration media had no negative effects on bulblet formation and it did not interfere with the selective efficiency in the glufosinate containing media (Tables 1, 2; Fig. 1, 2). Other reports on monocotyledonous plants showed, that they are tolerant to cefotaxim. The same concentration of 500 mg l -1 cefotaxim as in the present study was used in the selection media for Muscari armeniacum (Suzuki and Nakano, 2002) and Alsteroemeria (Akutsu et al., 2004), but for Lilium only 300 mg l -1 (Hoshi et al., 2004) and for Gladiolus only mg l -1 were used as reported by Kamo (1997). The effect of glufosinate on bulblet formation was very marked in both experiments. At 1.0 mg l -1 glufosinate, the explants from young flower stalks remained alive for a few weeks, but did not show bulb regeneration. Therefore this level was found to be suitable for being used in the selection medium (Table 1, Fig. 1). In the second experiment with quarters of in vitro bulblets a concentration of 2.0 mg l -1 glufosinate successfully inhibited growth of tissues and bulblet formation (Table 2, Fig. 2). As reported by Chauvin et al. (1997), addition of glufosinate in the selection media totally inhibited regeneration for both Tulipa and Gladiolus even at low concentrations. This preliminary study has been undertaken to determine a strategy and suitable conditions for further investigations in transformations experiments. By Agrobacterium-

4 mediated transformation, of Hippeastrum x chmielii Chm. can be improved regarding flower longevity or disease resistance. Literature cited Akutsu, M.., Ishisaki, T. and Sato, H Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes. Mol. Breeding 13: Chauvin, J.E., Hamann, H., Cohat J. and Le Nard, M Selective agents and marker genes for use in genetic transformation of Gladiolus grandiflorus and Tulipa gesneriana. Acta Hort. 430: Chauvin, J.E., Marhadour, S., Cohat, J. and Le Nard, M. 1999: Effects of gelling agents on in vitro regeneration and kanamycin efficiency as a selective agent in plant transformation procedures. Plant Cell, Tissue and Organ Culture 58: Chmiel, H., Ilczuk, A. and Łukaszewska, A All-round merits of new Hippeastrum hybrid. Flower Tech. 5 (2): Cohen, A. and Meredith, C.P Agrobacterium-mediated transformation of Lilium. Acta Hort. 352: Eady,C.C. and Lister, C.E. 1998: A comparison of four selective agents for use with Allium cepa L. immature embryos and immature embryo-derived cultures. Plant Cell Rep. 18: Eady, C., Davis, S., Farrant, J., Reader, J. and Kenel, F Agrobacterium tumefaciens-mediated transformations and regeneration of herbicide resistant onion (Allium cepa) plants. Ann. appl. Biol. 142: Hoshi, Y., Kondo, M., Mori, S., Adachi, Y., Nakano, M. and Kobayashi, H Production of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep. 22: Ilczuk, A. and Witomska, M Effect of bulb chilling and growth regulators on the in vitro regeneration from shoot explants in Hippeastrum x chmielii Chm. Z. Probl. Post. Nauk Roln. 491: Ilczuk, A. and Witomska, M Effect of bulb chilling and growth regulators on the in vitro organogenesis in Hippeastrum x chmielii Chm. Biotech. 2 (65): Kamo, K Factors affecting Agrobacterium tumefacies-mediated gusa expression and opine synthesis in Gladiolus. Plant Cell Rep. 16: Kondo, T., Hasegawa, H. and Suzuki, M. 2000: Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer. Plant Cell Rep. 19: Langeveld, S.A., Gerrits, M.M., Derks, A.F.L.M., Boonekamp, P.M. and Bol, J.F Transformation of lily by Agrobacterium. Euphytica 85 (1-3): Langeveld, S.A., Marinova, S., Gerrits, M.M., Derks A.F.L.M. and Boonekamp, P.M Genetic transformation of lily. Acta Hort. 430: 290 pp. Lipsky, A., Cohen, A., Gaba, V., Kamo, K., Gera, A. and Watad, A Transformation of Lilium longiflorum plants for Cucumber Mosaic Virus resistance by particle bombardment. Acta Hort. 568: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays with tabacco cultures. Physiol. Plant. 15: Okubo, H Hippeastrum (Amaryllis)., in: The Physiology of flower bulbs. Le Nard M., De Hertogh A.A. (red.). Elsevier, Amsterdam:

5 Sage, D. and Hammatt, N Somatic embryogenesis and transformation in Narcisuss pseudonarcissus cultivars. Acta Hort. 570: Suzuki, S. and Nakano, M Agrobacterium-mediated production of transgenic plants of Muscari armeniacum Leichtl. ex Bak. Plant Cell Rep. 20: Suzuki, S., Supaibulwatana, K., Mii, M. and Nakano M Production of transgenic plants of Lilioceuos ornamental plant Agapanthus praecox spp. orientalis (Leighton) via Agrobacterium-mediated transformation of embryogenic calli. Plant Sci. 161: Wahi, S.D. and Bhattacharjee, S.K Correlation and path-coefficient analysis in Hippeastrum hybridum. South Indian Hort. 34 (4): Watad, A.A., Yun, D.-J., Matsumoto, T., Niu, X., Wu, Y., Kononowicz, A.K., Bressan, R.A. and Hasegawa, P. M Microjectile bombardment-mediated transformation of Lilium longiflorum. Plant Cell Rep. 17: Wilmink, A. and Dons, J.J.M Selective agents and marker genes for use in transformation of monocotyledonous plants. Plant Mol. Biol. Rep. 11: Wilmink, A., Ven, de B.C.E. and Dons, J.J.M Expression of the GUS-gene in monocot tulip after introduction by particle bombardment and Agrobacterium. Plant Cell Rep. 11: Witomska, M. and Ilczuk, A Formation of adventitious bulblets in vitro on scale explants in Hippeastrum x chmielii Chm. Biotech. 2 (65): Zheng, S.J., Khrustaleva, L., Henken, B., Sofiari, E., Jacobsen, E., Kik, C. and Krens, F.A Agrobacterium tumefaciens-mediated transformation of Allium cepa L.: the production of transgenic onions and shallots. Mol. Breeding 7: Tables Table 1. Effect glufosinate (gluf.) and cefotaxim (cef.) on regeneration of Hippeastrum x chmielii Chm. - explants from flower stalks Medium Percentage of explants* brown unchanged with callus with bulblets Number of bulblets per explant** time (months) MS a 0.6a MS+500 mg/l cef. MS+1mg/l gluf mg/l cef. MS+1.5 mg/l gluf mg/l cef b 0.5a * total number of explants per treatment: 25 = 100% ** means followed by the same letter do not differ at 5% level of significance

6 Table 2.: Effect of glufosinate (gluf.) and cefotaxim (cef.) on regeneration of Hippeastrum x chmielii Chm.- explants from quarters of in vitro bulblets. Percentage of explants* Number of bulblets brown unchangend with bulblets per explant** Medium time (months) MS a 1.2 a 1.5 a MS+ 500 mg/l cef. MS+ 1 mg/l gluf mg/l cef. MS+ 2 mg/l gluf mg/l cef. MS+ 3 mg/l gluf mg/l cef b 1.4 b 2.0 b * total number of explants per treatment: 25 = 100% ** means followed by the same letter do not differ at 5% level of significance Figures a b c d Fig. 1.: Explants from flower stems cultured for one month on: a/ MS medium; b/ mg/l cefotaxim; c/ + 1 mg/l glufosinate mg/l cefotaxim; d/ mg/l glufosinate mg/l cefotaxim a b c d e Fig. 2.: Explants from quarters of in vitro bulblets cultured for one month on: a/ MS medium; b/ mg/l cefotaxim; c/ + 1 mg/l glufosinate mg/l cefotaxim; d/ + 2 mg/l glufosinate mg/l cefotaxim; e/ + 3 mg/l glufosinate mg/l cefotaxim