Improved Agrobacterium-mediated genetic transformation of GNA transgenic sugarcane

Abstract

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81-745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.

Agrobacterium-mediated in planta genetic transformation of sugarcane setts

“…To date, sugarcane has been transformed by either Agrobacterium tumefaciens or Biolistics methods (Manickavasagam et al 2004;Zhangsun et al 2007;Altpeter and Oraby 2010;Joyce et al 2010;Khamrit et al 2012). The transformed plants were regenerated using direct, indirect organogenesis or somatic embryogenesis (Arencibia et al 1998;Manickavasagam et al 2004;Attia et al 2005;Lakshmanan et al 2006;Eldessoky et al 2011;Taparia et al 2012).…”

Genetic Engineering of Saccharum

“…To date, the markers used in selecting for transformed plants include the hygromycin phosphotransferase gene ( hph ) or the bialaphos resistance bar gene (Table 11.1 ). However, more recently, the neomycin phosphotransferase II ( npt II) gene has been successfully shown to be ef fi cient in selecting transformed sugarcane callus (Zhangsun et al 2007 ) . Transformed shoots of the commercial variety Q117 were only regenerated when calli were inoculated with an Agrobacterium strain harboring a plasmid with the npt II gene as opposed to the one with the hph gene, suggesting that the selection system is important for producing transgenic shoots (Joyce et al 2010 ) .…”

Advances in Transgenic Research for Insect Resistance in Sugarcane

“…High GC content in all three of these transgenes, i.e. up to 68.0% in bar gene (Zhang et al 2007), 56.7% in aprotinin (Shantaram 1999) and 48.5% in cry1Ab (Arvinth et al 2010) could be the reason for their stable expression after repeated vegetative propagation. The role of high GC content and codon usage in sugarcane transgene expression was clearly demonstrated by Weng et al (2006Weng et al ( , 2010 who reported a progressive increase in the Cry1Ac content and corresponding insect resistance by increasing the GC content from the native 37.4% to 47.5% and 54.8%.…”

“…The four factors of: (1) strength of the promoters that drive the candidate gene, (2) gene silencing, (3) GC content and codon usage, and (4) somaclonal variation largely influence the success of transgenic lines (Zhang et al 2007). The rice polyubiquitin (RUBIQ2) promoter seems to be the strongest for sugarcane (Zhang et al 2007), but the maize ubi-1 promoter has been most commonly used for insect resistant genes in sugarcane (Table 1). Although the maize ubi-1 promoter is constitutive, it does not express the transgene to the same level in all plant organs, e.g.…”