Agronomic evaluation and molecular characterisation of the acetolactate synthase gene in imazapyr tolerant sugarcane (Saccharum hybrid) genotypes.

KEY MESSAGE: Mutagenesis had no effect on number of stalks/plot, stalk height, fibre and sucrose content of mutants. Imazapyr tolerance is likely due to a S622N mutation in the acetolactate synthase gene. The herbicidal compound imazapyr is effective against weeds such as Cynodon and Rottboellia species that constrain sugarcane production. This study aimed to compare agronomic characteristics of three imazapyr tolerant mutants (Mut 1, Mut 6 and Mut 7) with the non-mutated N12 control after 18 months of growth, and to sequence the acetolactate synthase (ALS) gene to identify any point mutations conferring imazapyr tolerance. There were no significant differences in the number of stalks/plot, stalk height, fibre and sucrose contents of the mutants compared with the N12 control. However, Mut 1 genotype was more susceptible to the Lepidopteran stalk borer, Eldana saccharina when compared with the non-mutated N12 (11.14 ± 1.37 and 3.89 ± 0.52% internodes bored, respectively), making Mut 1 less desirable for commercial cultivation. Molecular characterisation of the ALS gene revealed non-synonymous mutations in Mut 6. An A to G change at nucleotide position 1857 resulted in a N513D mutation, while a G to A change at nucleotide position 2184 imposed a S622N mutation. Molecular dynamics simulations revealed that the S622N mutation renders an asparagine side chain clash with imazapyr, hence this mutation is effective in conferring imazapyr tolerance.

Assessing the Likelihood of Gene Flow From Sugarcane (Saccharum Hybrids) to Wild Relatives in South Africa.

Pre-commercialization studies on environmental biosafety of genetically modified (GM) crops are necessary to evaluate the potential for sexual hybridization with related plant species that occur in the release area. The aim of the study was a preliminary assessment of factors that may contribute to gene flow from sugarcane (Saccharum hybrids) to indigenous relatives in the sugarcane production regions of Mpumalanga and KwaZulu-Natal provinces, South Africa. In the first instance, an assessment of Saccharum wild relatives was conducted based on existing phylogenies and literature surveys. The prevalence, spatial overlap, proximity, distribution potential, and flowering times of wild relatives in sugarcane production regions based on the above, and on herbaria records and field surveys were conducted for Imperata, Sorghum, Cleistachne, and Miscanthidium species. Eleven species were selected for spatial analyses based on their presence within the sugarcane cultivation region: four species in the Saccharinae and seven in the Sorghinae. Secondly, fragments of the nuclear internal transcribed spacer (ITS) regions of the 5.8s ribosomal gene and two chloroplast genes, ribulose-bisphosphate carboxylase (rbcL), and maturase K (matK) were sequenced or assembled from short read data to confirm relatedness between Saccharum hybrids and its wild relatives. Phylogenetic analyses of the ITS cassette showed that the closest wild relative species to commercial sugarcane were Miscanthidium capense, Miscanthidium junceum, and Narenga porphyrocoma. Sorghum was found to be more distantly related to Saccharum than previously described. Based on the phylogeny described in our study, the only species to highlight in terms of evolutionary divergence times from Saccharum are those within the genus Miscanthidium, most especially M. capense, and M. junceum which are only 3 million years divergent from Saccharum. Field assessment of pollen viability of 13 commercial sugarcane cultivars using two stains, iodine potassium iodide (IKI) and triphenyl tetrazolium chloride, showed decreasing pollen viability (from 85 to 0%) from the north to the south eastern regions of the study area. Future work will include other aspects influencing gene flow such as cytological compatibility and introgression between sugarcane and Miscanthidium species.

In vitro generation of somaclonal variant plants of sugarcane for tolerance to Fusarium sacchari.

KEY MESSAGE : A combination of in vitro culture and mutagenesis using ethyl methanesulfonate (EMS) followed by culture filtrate-mediated selection produced variant sugarcane plants tolerant and resistant to Fusarium sacchari. Eldana saccharina is a destructive pest of the sugarcane crop in South Africa. Fusarium sacchari PNG40 (a fungal strain harmful to E. saccharina) has the potential to be an endophytic biological control agent of the stalk borer. However, the fungus causes Fusarium stalk rot in sugarcane. In the current study, sugarcane plants tolerant and resistant to F. sacchari PNG40 were produced by exposing embryogenic calli to the chemical mutagen ethyl methanesulfonate (EMS), followed by in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF). The incorporation of 100 ppm CF in the culture media at the embryo maturation stage, at germination, or at both, resulted in callus necrosis and consequent reduced plantlet yield. Subsequent trimming of the roots of regenerated plants and their exposure to 1,500 ppm CF served as a further selection treatment. Plants produced from EMS-treated calli displayed improved root re-growth in the presence of CF pressure compared with those from non-treated calli. The tolerance of CF-selected plants was confirmed in greenhouse tests by inoculation with F. sacchari PNG40, re-isolation of Fusarium spp. from undamaged tissue of asymptomatic plants and establishment of the identity of fungal isolates as PNG40 using molecular analysis. The restriction of PNG40 presence to the inoculation lesion in some plants suggested their resistance to the fungus. Genotypes exhibiting symptomless endophytic colonization by PNG40 were identified and will be utilised for testing biological control strategies against E. saccharina.