Microprojectile bombardment was used to introduce DNA into embryogenic callus of garlic (Allium sativum L.) and produce stably transformed garlic plants. Embryogenic 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.
Alejandrina Robledo-Paz, José Luis Cabrera-Ponce, Víctor Manuel Villalobos-Arámbula, Luis Herrera-Estrella, and Alba Estela Jofre-Garfias
Salvador Guzmán-González, Pedro Valadez-Ramírez, Rosa-Edith Robles-Berber, Laura Silva-Rosales, and José-Luis Cabrera-Ponce
Biolistic genetic transformation of plants with viral genes is a method for controlling plant virus diseases; however, optimization of the particle bombardment parameters according to the transformation system is a key factor for an appropiate transgene expression and, therefore, a stronger resistance mechanism in transgenic plants. In order to optimize biolistic parameters, somatic papaya (Carica papaya L.) cv. Maradol embryo masses were bombarded with the CAMBIA 1301 plasmid construction that contains the coat protein gene (CP) of the papaya ringspot virus isolate of Colima, Mexico, driven by the double constitutively CaMV 35S promoter and flanked for the GUS and hygromycin (hpt) resistance genes. Particle bombardment protocol was carried out using the Helios™ Gene Gun device (BioRad) and the manufacturer's instruction manual. Helium pressure (50, 100, and 150 psi) and gold particle size (0.6, 1.0, and 1.6 μm) were evaluated. Five days after bombardment, somatic embryo clusters were used for GUS transient expression and, during 2 months, were selected into 50, 75, and 150 mg·L-1 hygromycin-containing media to its later CP-PCR detection. Results showed that 50 psi and 1.0 μm were the two optimal values for the assayed analyses. This is the first report of genetic transformation of papaya using the Helios™ Gene Gun device as a new tool compared to conventional PDS-1000/He.