HORTSCIENCE 39(6) OCTOBER 2004 1208 Genetic Transformation of Garlic (Allium sativum L.) by Particle Bombardment Alejandrina Robledo-Paz, 1 José Luis Cabrera-Ponce, 2 Víctor Manuel Villalobos-Arámbula, 3 Luis Herrera-Estrella, 3 and Alba Estela Jofre- Garfias 4 Departamento de Ingeniería Genética de Plantas, Centro de Investigación y de Estudios Avanzados del I.P.N. Km, 9.6 libramiento Norte Carretera Irapuato- León, Apdo. Postal 629, 36500 Irapuato, Gto, México Additional index words. biolistic, transgenic plants, somatic embryogenesis, genetic engineering, tissue culture Abstract. Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Em- bryogenic calluses, derived from garlic cultivar ‘GT96-1’, were bombarded with plasmid DNA containing genes coding for hygromycin phosphotransferase and β-glucuronidase. Putatively transformed calluses were identified in the bombarded tissue after 4 months of selection on 20 mg·L –1 hygromycin B. The transgenic nature of the selected material was demonstrated by GUS histochemical assay and Southern blot hybridization analysis, and twenty transgenic plants were regenerated. Garlic (Allium sativum L.), family Alliaceae (Takhtahan, 1997), is a monocotyledonous crop used worldwide both as a condiment and for its medicinal properties. This crop is susceptible to numerous diseases caused by fungi, viruses, nematodes and insect pests. Garlic does not read- ily produce seeds, and is propagated vegetatively with a low multiplication rate. This mode of reproduction facilitates disease transfer (Novak, 1990) and prevents improvement of garlic by traditional breeding methods. Both Agrobacterium and particle bombard- ment, have proven to be very useful techniques for the generation of transgenic plants of both dicotyledoneous and monocotyledoneus spe- cies (Bommineni et al., 1993; Bower and Birch, 1992; Cabrera-Ponce et al., 1995, 1997; Casas et al., 1993; Chee and Slightom, 1992; Christou, 1995; Eady, 2001; Eady et al., 2000; Fromm et al., 1990; Hiei et al., 1994; Ishida et al., 1996; Kihara et al., 1998; McCabe et al., 1988; Russell et al., 1993; Thomas et al., 1994; Vain et al., 1998). The use of genetic engineering techniques to obtain cultivars of garlic harbour- ing genes for pathogen resistance will surely have an impact on garlic production. induce the development of somatic embryos. Medium C was prepared with the salts and vitamins of MS medium and 1.0 mg·L –1 of N6-benzyladenine (BA) plus sucrose 0.058 M and Phytagar 7 g·L –1 . Cultures were incu- bated at 24 °C under 16 h daily illumination (50 μmol·m –2 ·s –1 ). Embryogenic calluses were cultured for 6 weeks on this medium and then transferred to medium D (similar to medium C but devoid of growth regulators). DNA (plasmids). The plasmid used for the bombardment experiments (pWRG1515) contains the hygromycin phosphotransferase (hph) and the b-glucuronidase (gusA) genes under control of the cauliflower mosaic virus 35 S (CaMV35S) promoter and the 3' region of the nopaline synthase gene (3'NOS) (Christou et al., 1991). The plasmid was amplified in E. coli, and extracted and purified by alkaline lysis as described by Birnboim and Doly (1979). Purified DNA was resuspended in sterile distilled water and adjusted to 1 μg·μL –1 for bombardments. Bombardment procedure. The plasmid was precipitated onto M10 (0.73 μm) tungsten particles as described by Tomes et al. (1995) using the following mixture: 50 μL of tung- sten particle suspension (15 mg·mL –1 ); 5 μL pWRG1515 (1 μg·μL –1 ); 50 μL 2.5 M CaCl 2 and 20 μL 0.1 M spermidine. Mixtures were sonicated briefly and then centrifuged at 10,800 g n for 10 s. The super- natant was removed and the pellet was resus- pended in 60 μL absolute ethanol and aliquots of 10 μL were delivered to each macrocarrier. Embryogenic calluses were bombarded us- ing a PDS-1000 He particle delivery system (Sanford et al., 1991) at 800 PSI rupture disk pressure. Embryogenic calluses (300 mg of fresh weight) were placed in the center of each petri plate containing medium B, and the sample chamber was evacuated to 0.07 atm. The distance between the rupture membrane and the macrocarrier was 1.2 cm, flight distance was 1.2 cm, and calluses were placed at 7.0 cm from the microparticle launching point. GUS assays. Transient gusA gene expres- sion was assayed 24 h after bombardment. Calluses were incubated in 100 μL reaction buffer containing 0.5 mg·mL –1 of 5-bromo-4- chloro-3-indolyl β-D-glucuronide (X-gluc) at 37 °C (McCabe et al., 1988). Blue spots were counted using a stereo microscope. Selection of transgenic clones. Bombarded calluses were maintained on medium B for 9 d before being transferred to medium B plus 20 mg·L –1 of hygromycin B. Tissues were subcultured on fresh medium until control (nonbombarded tissues) did not grow (16 weeks), and proliferation of putative transgenic embryogenic calluses was observed. Resistant embryogenic calluses were subcultured on medium C with hygromycin B to induce embryo development. Regener- ated plantlets were transferred to medium D plus hygromycin B to obtain shoot and root elongation. Southern blot analysis. Total genomic DNA from transgenic and wild type calluses were isolated following the method of Shure et al. (1983). DNA (20 μg) was digested with EcoRI Onion and garlic are among commercially important crops for which it has been possible, although difficult, to obtain transgenic plants. To date there are only a few reports of transient expression (Barandiaran et al., 1998; Eady et al., 1996) and stable genetic transformation of these species (Kondo et al., 2000, Myers and Simon, 1998). Here we describe a reliable and efficient protocol to obtain transgenic garlic plants employing callus tissue derived from root explants as target tissues for transformation by particle bombardment. Materials and Methods Induction of somatic embryogenesis. Cloves of garlic cultivar ‘GT96-1’ were sepa- rated from the bulb and peeled before treatment with a chlorinated solution (1.8% active chlo- rine) for 20 min. Treated cloves were rinsed three times in sterilized distilled water. Disinfected cloves were placed on a sprout- ing medium prepared with half the concentra- tion of salts and vitamins found in Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) and supplemented with 0.029 M sucrose and 7 g·L –1 Phytagar (Gibco, BRL), pH 5.8 (medium A). The cloves were subsequently incubated at 24 ± 2 °C under 16 h daily illumination (50 μmol·m –2 ·s –1 ). After three days, 3-mm-long root tip segments were cut with a scalpel and cultured in 100 × 15-mm petri dishes (15 segments per plate) containing medium B to induce embryogenic calluses. This medium was prepared with salts from Chu et al. (1975) (medium N6), vitamins from Eriksson (1965), 0.058 M sucrose, 6 mM L-proline, 8 g.L –1 Phytagar, as well as 1.0 mg·L –1 of 2,4-D. Root tips were cultured for 8 weeks at 24 ± 2 °C in complete darkness. Embryogenic calluses were either maintained on medium B or subcultured onto medium C to HORTSCIENCE 39(6):1208–1211. 2004. Received for publication 10 Feb. 2003. Accepted for publication 9 Dec. 2003. We want to thank Claudia Geraldine León Ramírez, Nélida Vázquez Sánchez and Rosa María Adame Alvarez for their technical assistance. This work was supported by Colegio de Postgraduados, Montecillo, México and The National Council for Science and Technology (CONACYT, México) by means of a Graduate Scholarship to ARP and a grant (28534N, awarded to AEJG). 1 Former graduate student. Current address: Instituto de Recursos Genéticos y Productividad, Colegio de Postgraduados. Km. 36.5 Carretera México-Texcoco. Apdo. Postal 56230, Montecillo, Edo, México; e-mail arobledo@colpos.mx. 2 Research assistant. 3 Full profesor. 4 Associated professor. To whom correspondence should be addressed; ajofre@ira.cinvestav.mx.