Agrobacterium - Mediated Transformation of Citrus Cell Suspension Cultures

An efficient protocol for the genetic transformation of cell suspension cultures of several citrus cultivars using Agrobacterium is described. Cell suspension cultures of Citrus sinensis ‘Hamlin’, ‘Valencia’ and ‘OLL8’ (an early maturing ‘Valencia’ like somaclone), Citrus unshiu ‘Okitsu wase’ and Citrus reticulata ‘Ponkan’ and ‘W. Murcott’ were transformed using A. tumefaciens EHA105. It was determined that hygromycin was a better selection antibiotic than kanamycin and efficient transformation results were obtained only when an Agrobacterium strain containing hptII as a selectable marker was utilized. The EGFP protein was visualized in transgenic embryos within 6 weeks of transformation. Several transgenic plants were obtained using this protocol and hptII and egfp gene integration was confirmed. Amenability of cell suspension cultures to transformation using Agrobacterium would allow the transformation of any cultivar that can be induced in vitro as embryogenic cell masses, including special seedless sweet oranges or Satsuma mandarins and other difficult-to-transform cultivars of the mandarin/tangerine or lemon group. INTRODUCTION In recent years, there has been a major thrust in citrus improvement research due to competition in international citrus markets and increasing disease, pest and abiotic pressures on citriculture (Grosser et al., 2000). Several strategies exist for the genetic improvement of citrus including conventional breeding and genetic transformation (Pena et al., 2007). Currently, genetic transformation as a tool for citrus improvement is gaining in popularity. This method of citrus improvement is especially useful in cases where it is not possible to engineer a particular trait of interest to an otherwise elite cultivar using conventional breeding (Dutt et al., 2010). Different methods have been used for incorporation of transgenes into citrus. These methods include Agrobacterium-mediated transformation using juvenile in vitro epicotyl segments (Moore et al., 1992; Dutt and Grosser, 2009), mature internode segments obtained from greenhouse-grown plants (Cervera et al., 1998; Almeida et al., 2003) or embryogenic callus obtained from unfertilized ovules (Li et al., 2002). Direct incorporation of DNA into protoplasts using electroporation (Niedz et al., 2003) or PEG mediated (Fleming et al., 2000; Omar et al., 2007) have also been reported. Of the above, the most popular method for transformation of a wide range of citrus cultivars is the Agrobacterium-mediated transformation using juvenile epicotyls as explants. This technique, although popular, has several drawbacks in that it cannot be used in cultivars that do not produce seeds like seedless sweet oranges, lemons or Satsuma mandarins. Also, several seedy cultivars in the genus citrus, including specialty cultivars in the mandarin/tangerine group remain difficult to transform using this method. In this study, we investigate an alternate method of transformation using cell suspension cultures. Proc. II IS on Citrus Biotechnology Eds.: A. Gentile and S. La Malfa Acta Hort. 892, ISHS 2011