Seeds as biofactories for the production of biopharmaceutical protein have been reported mostly in cereals such as maize and rice. The use of sorghum as a biofactory has not been reported, although sorghum has many features that are similar to maize and has potential as biofactory. Therefore, the aim of the present study was to develop strategies for seed specific GFP expression, to serve as a foundation for future production of pharmaceutical proteins in sorghum. For this, research conducted in this thesis has considered the three major stages required for the successful generation of sorghum transgenics harbouring the gfp gene: i) efficient system for sorghum tissue culture, ii) efficient system for gene delivery into sorghum and iii) availability of the functional regulatory element capable to direct GFP expression into protein bodies of sorghum endosperm.
In this study, and as also reported by others in the literature, sorghum was found to be very recalcitrant to tissue culture manipulation and genetic transformation. In vitro performance was very much genotype dependent and using the right genotype was found to be crucial for successful recovery of transgenics. The highly reputable sorghum inbred line, Tx430, was found to be superior to the conventionally used sorghum genotypes, 296B and SA281, in Godwin Laboratory. In-house tissue culture media developed was efficient for successful callus induction and regeneration of genotype Tx430.
The two most popular methods for gene delivery are Agrobacterium-mediated transformation (AMT) and particle bombardment (PB). AMT was first trialled because it offers insertion of larger DNA segments with minimal rearrangement and copies of transgenes. These are difficult to achieve following PB, which incorporates plasmid DNA fragments into the plant genome in a random fashion. Using the optimized AMT protocol developed in this study, we have demonstrated efficient transfer of Ubi-gfp gene cassette into sorghum genotype Tx430, based on GFP expression 50 days post-infection. However, the survival and regeneration frequencies of the agroinfected explants were still regarded as low. Explant necrosis due to hypersensitive reaction toward agroinfection was inevitable. Therefore, AMT requires further media development and experimentation if it is to be a valid approach. On the other hand, the PB protocol developed in this study successfully regenerated sorghum transgenics harbouring the constructs pMB-Ubi-gfp, pMB-Ubi-ss-gfp, pMB-α-kaf-gfp and pMB-α-kaf-ss-gfp (see description below) with transformation efficiencies of 13.9%, 2.8%, 5.6% and 2.8% respectively. Stable integration of the transgene in T0 plants was confirmed by PCR and Southern blot analysis.
The α-kafirin promoter coupled with the signal peptide (SP) encoding gene, the signal sequence (ss), was successfully isolated from genomic DNA of sorghum genotype 296B using a PCR-based approach. The α-kaf promoter contains endosperm specificity-determining motifs, prolamin-box, the O2-box 1, CATC and TATA boxes required for α-kafirin gene expression in sorghum seeds. Evaluation of the α-kaf promoter and ss functionality required the construction of appropriate gene cassettes comprising fusion of the isolated regulatory element and a reporter gene. For this purpose, gfp was selected. Using pMB-Ubi-gfp as a plasmid DNA backbone and overlapping PCR approach, three plasmid constructs were successfully developed: i) pMB-Ubi-ss-gfp, ii) pMB-α-kaf-gfp and iii) pMB-α-kaf-ss-gfp. The developed constructs were subjected to GFP transient assay using sorghum and maize explants. Results from the assay have demonstrated that the α-kaf promoter and SP were functional. The promoter drove seed specific GFP expression while SP::GFP fusion assembled and folded properly, preserving the fluorescent properties of GFP in all tissues tested except the sorghum leaf.
The functionality of the α-kaf promoter and SP was further tested in its stably transformed native host. Microscopic examination and GFP-ELISA demonstrated that the α-kaf promoter regulates spatial and temporal GFP expression in sorghum. The promoter strength was comparable to the Ubi promoter in driving GFP expression in seeds. Further, through pMB-Ubi-ss-gfp transgenics, the SP was shown to be functional, directing a high level of GFP accumulation in sorghum seeds. Therefore the α-kaf promoter and SP have potential in biotechnological applications for seed specific protein expression.