Development of Regeneration and Transformation Protocol for Local Sudan’s wheat (Triticum aestivum L) Cultivars

Abstract

The objective of this study was to optimize a simple and efficient Agrobacterium-mediated genetic transformation protocol for two locally grown wheat cultivars (Bohain and Elnielain) using mature embryos. Transformation was carried out with disarmed A. tumefaciens strain EHA105 harbouring a binary vector pCAMBIA1305.2. Conditions for in vitro regeneration of Agrobacterium-infected explants were optimized using Murashige and Skoog (MS) medium supplemented with various concentrations and/or combinations of plant growth regulators (PGRs) and Hygromycin. Results indicated that 70 and 80% of the embryogenic explants of Bohain and Elnielain, respectively, have survived when exposed to Agrobacterium inoculums of 0.1 O.D600 and selection on medium containing 15 (Elnielain) or 25 (Bohain) mg/l of Hygromycin. The putative transformed explants were regenerated on MS medium containing 4.0 mg/l 2,4-D for shoot initiation, transferred to MS medium with 4.0 mg/l 2,4-D plus 1.0 mg/l Zeatin for Shoot elongation, then maintained on MS medium supplemented with a combination of 1.0 mg/l of each of 2,4-D, Zeatin and gibrellic acid (GA3). This protocol resulted in transformation efficiency of 20% for Bohain and 23% for Elnielain. GUS expression was observed in transformed shoots but never in the control plants. PCR amplification of DNA extracted from the transformed tissues demonstrated the generation of the expected amplicon, corresponding to and neomycin phosphotransferase II (nptII), hygromycin phosphotransferase II (hptII), cauliflower mosaic virus-35S (CaMV-35S) promoter and nopaline synthase (NOS) terminator genes. This result strongly verifies the successful transformation of the two locally wheat cultivars and paves the way for problem solving-applications encompassing these or other Sudanese wheat cultivars of economic importance.