High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration

Abstract

An improved method for the Agrobacterium infiltration of epicotyl segments of 'Pineapple' sweet orange [Citrus sinensis (L.) Osbeck] and 'Swingle' citrumelo [Citrus paradisi Macf. X Poncirus trifoliata (L.) Raf.] was developed in order to increase transformation frequency. Sonication-assisted Agrobacterium-mediated transformation (SAAT), vacuum infiltration, and a combination of the two procedures were compared with conventional Agrobacterium-mediated inoculation method ('dipping' method). It was observed that the morphogenic potential of the epicotyl segments decreased as the duration of SAAT and vacuum treatments increased. Transient GUS expression was not affected by the different SAAT treatments, but it was significantly enhanced by the vacuum infiltration treatments. The highest transformation efficiencies were obtained when the explants were subjected to a combination of SAAT for 2 s followed by 10 min of vacuum infiltration. PCR and Southern blot analysis of the uidA gene were used to confirm the integration of the transgenes. The transformation frequencies achieved in this study (8.4% for 'Pineapple' sweet orange and 11.2% for 'Swingle' citrumelo) are the highest ones reported for both cultivars.

Improving Infection Efficiency of Agrobacterium to Immature Cotyledon Explants of Japanese Apricot (Prunus mume) by Sonication Treatment

“…Several researchers reported that sonication treatment significantly decreased the morphogenic potential or shoot proliferation of explants (de Oliveira et al, 2009;Meurer et al, 1998). In this study, we investigated the influence of sonication treatment on SEG from immature cotyledons of P. mume.…”

“…Sonication treatment has been successfully applied to soybean (Glycine max) (Santarém et al, 1998), loblolly pine (Pinus taede) (Tang et al, 2001), black locust (Robinia pseudoacacia) (Zaragozá et al, 2004), and flax (Linum usitatissimum) (Beranová et al, 2008), and attempted in some fruit trees such as citrus (C. sinensis and C. paradisi × Poncirus trifoliate) (de Oliveira et al, 2009) and banana (Musa spp.) (Subramanyam et al, 2011).…”

Citrus Transformation: Challenges and Prospects

“…Typically, most regenerated transformed shoots are either placed directly in soil containing rooting hormone or on rooting media containing varying levels (0 to 1.0 mg/l) of the auxin naphthaleneacetic acid (NAA) which promotes root development (de Oliveira et al, 2009;Gutierrez-E et al, 1997;Luth & Moore, 1999;Moore et al, 1992). Some researchers have gotten better results by first transferring the shoots to hormone-free media to eliminate the cytokinin benzyl aminopurine (BA) from the regeneration media before placing on NAA containing media.…”

Recent Advances in Fruit Species Transformation

“…Today, application of these modifications solely or in combination with other approaches has made it possible to transfer foreign DNA via Agrobacterium even to monocots (Hiei et al, 1994;Ishida et al, 1996;Hensel et al, 2011) which were initially transformed with direct gene transfer methods since Agrobacterium is not a natural host. Following its first successful usage in soybean and Ohio buckeye (Trick & Finer, 1997), SAAT was also applied recently to fruit species including orange (Oliveira et al, 2009); banana (Subramanyam et al, 2011) and grapevine . In the last paper the developed efficient methodology that combined SAAT with vacuum infiltration allowed to obtain reporter gene expression in different newly formed organs such as stems, petioles and leaves.…”