Transformation and regeneration of pineapple

We have developed efficient methods for plant regeneration, via both organogenesis and embryogenesis, of Smooth Cayenne pineapple, Ananas comosus (L.) Merr. A range of different types of embryogenic tissues has been developed with varying properties in terms of growth rate and state of development (Firoozabady and Moy, 2004). Two of the embryogenic systems, namely friable embryogenic cell clusters (ECCs) and chunky non-dispersible embryogenic tissues (ETs) have been used for transformation of pineapple. The tissues were cocultivated for 2-3 days with Agrobacterium tumefaciens disarmed strain C58 carrying a binary vector containing either surB gene conferring resistance to chlorsulfuron or the nptII gene conferring resistance to geneticin (G418). After cocultivation and a recovery period, tissues were selected on media containing chlorsulfuron or G418. On average, about 50 or 120 independent transgenic lines were obtained from each gram of ECCs or ETs, respectively, inoculated with Agrobacterium. Transformed embryogenic tissues were transferred to maturation media to form somatic embryos, which subsequently produced transgenic pineapple plants. Transformation has been confirmed by GUS assay, polymerase chain reaction, and by Southern hybridization. Thousands of plants from independently transformed lines were transferred to the greenhouse and to the field to evaluate clonal fidelity and somaclonal variation.