In vitro culture and genetic transformation of selected ancestral and commercial sugarcane germplasm.

Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

Sugarcane is an economically important crop and its high demand has necessitated the use of biotechnology methods to produce and accelerate the production of desirable genotypes. One such method is genetic transformation. However, as sugarcane is a highly polyploid crop, which originated from interspecific crosses between Saccharum spontaneum and S. officinarum, efforts to transform it are inhibited by transgene promoter silencing. As ancestral lines have a simpler genetic makeup than modern varieties, they may be useful to test promoter function. Intrinsic to the generation of transgenic plants is the ability to produce plants from specific species and varieties, for which an indirect method of regeneration is needed. Consequently, the first objective of this study was to determine a high yielding protocol for somatic embryogenic calli. The second was to transform such calli and produce regenerated plants to assess transgene expression. A preliminary study was conducted using eight ancestral varieties to determine which were the most responsive in culture. Leaf roll disks were cultured on 5 mg.1¯¹ 2, 4-D and callus production was assessed. Based on these results and the availability of plant material, S. spontaneum Nigeria 1, S. spontaneum Nigeria 2, S. spontaneum Coimbatore, S. officinarum NG 77-69, and S. officinarum Black Cheribon and the commercial polyploid variety NCo376 were selected and tested on 11 different callus induction media. The S. spontaneum variety that generated the highest percentage of leaf disks that produced callus and plant yield was Nigeria 1 (61 % and 259 plants/10 disks, respectively), whilst the S. officinarum variety was Black Cheribon (75 % and 90 plants/10 disks, respectively). The best media for both comprised of MS salts and vitamins, 20 g.1¯¹ sucrose, 0.5 g.1¯¹ casein hydrolysate 5 mg.1¯¹ 2, 4-D and 8 g.1¯¹ agar. NCo376 produced the most amount of callus (93 %) when cultured on media containing 3 mg.1¯¹ 2, 4-D and gave a final yield of 450 plants/10 disks. Based on the yields obtained above and the availability of plant material, the varieties S. spontaneum Nigeria 1 and S. officinarum NG77-69 were selected for genetic transformation studies. Calli of these varieties as well as that of NCo376 were microprojectile bombarded with either pEmuKN + pAHC27 or pEmuKN + pR₁₁F¯. Following bombardment, the calli were cultured onto paromomycin-containing (1 ml.1¯¹) selection media and regenerated plants were obtained after 8-12 weeks. Transgene integration into the plant genome was assessed using PCR and qPCR techniques, and indicated that all NCo376 plantlets contained the GUS and npt II transgenes. However, only 4 out of 5 and 2 out of 3 S. officinarum NG77-69 plants transformed with pAHC27 and pR₁₁F¯- respectively, and 6 out of 10 S. spontaneum Nigeria 1 plants transformed with pR₁₁F¯- contained these transgenes. The transformation efficiencies achieved for NCo376, for the constructs pAHC27 and pR₁₁F¯- was 0.27 and 0.33 transgenic plants/blast, respectively. For NG77-69 it was 0.27 and 0.13 transgenic plants/blast, whilst that of Nigeria 1 was 0.20 and 0.40 transgenic plants/blast. Stable transgene expression in acclimatized plants was then assessed using a histochemical GUS assay and none of the plants expressed the GUS gene.