Transformation and regeneration of pineapple

Abstract

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.

Cloning and Molecular Characterization of a SERK Gene Transcriptionally Induced During Somatic Embryogenesis in Ananas comosus cv. Shenwan

“…SE is an effective way for plant propagation and genetic transformation (Kumar et al 2005;Firoozabady et al 2006;Wu et al 2008). An understanding of the molecular mechanism of SE in cultures will be a benefit for the establishment of a more effective regeneration system and valuable for the study of cell differentiation and embryo development.…”

Production of transgenic pineapple (Ananas cosmos (L.) Merr.) plants via adventitious bud regeneration

“…This protocol eliminates the need for establishing and maintaining callus cultures, either before or after the transformation events, and thus reduces the possibility for somaclonal variations among the produced transgenic lines. For most of the pineapple transformation methods previously described (Espinosa et al 2002;Firoozabady et al 2006;Ko et al 2006;Yabor et al 2006), calli or embryogenic cell clusters/tissues (ECC/ET; Firoozabady et al 2006) were used as starting materials for transformation. The undifferentiated cultures required 3-5 mo for establishment prior to Agrobacterium infection or bombardment.…”

“…The undifferentiated cultures required 3-5 mo for establishment prior to Agrobacterium infection or bombardment. After transformation, the cultured cells/tissues were either regenerated immediately (Espinosa et al 2002;Yabor et al 2006) or placed under selection for additional 4-8 mo before regeneration (Firoozabady et al 2006;Ko et al 2006). In a method described by Graham et al (2001), leaf bases were used for Agrobacterium infection.…”

“…Both Agrobacterium-mediated and biolistic transformation of pineapple have been previously reported (Graham et al 2001;Sripaoraya et al 2001;Espinosa et al 2002;Firoozabady et al 2006;Ko et al 2006;Yabor et al 2006). The present report describes a new method for Agrobacterium-mediated transformation of pineapple based on direct formation of adventitious buds from leaf bases and stem discs.…”

“…The present report describes a new method for Agrobacterium-mediated transformation of pineapple based on direct formation of adventitious buds from leaf bases and stem discs. Establishment and maintenance of undifferentiated cultures, either before or after the transformation events, were time-consuming integral steps in most of the other studies (Graham et al 2001;Espinosa et al 2002;Firoozabady et al 2006;Ko et al 2006;Yabor et al 2006). The use of undifferentiated cultures also results in a high frequency of somaclonal variations among regenerated pineapple plants (Wakasa 1979;Rangan 1984;Wakasa 1989;Firoozabady and Moy 2004).…”