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.

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.

Responses of meristematic callus cells of two Cynodon dactylon genotypes to aluminium.

Responses to Al3+ of embryogenic callus cells of an Al-sensitive (Al-S) and Al-resistant (Al-R) Cynodon dactylon genotype were evaluated with regard to Al3+ toxicity and resistance. A chemical equilibrium speciation model (MINTEQA2) was used to ensure the availability of the Al3+ ion in culture media, which was supplied as 0.08-2.3 mM Al3+ for 2-8 weeks. Increasing Al3+ concentration and exposure time had a greater negative impact on the Al-S than on the Al-R genotype, in terms of callus growth rate and frequency of non-embryogenic cells. Exposure to 0.8 mM Al3+ for 2 weeks resulted in an 88% reduction in the Al-S meristematic cell number, whereas that of the Al-R genotype remained unaffected. In addition, the Al-S cells accumulated three times more Al in the nucleus than did the Al-R cells, suggesting that Al interfered with mitosis. The Al-R cells appeared to exclude Al3+ from its cells through an increase in extracellular pH (4.34 in Al-R and 4.08 in Al-S) and by the immobilisation of Al in the cell wall (33% more in Al-R). The results showed that by studying the cellular responses to Al3+ it is possible to discriminate between the Al-S and Al-R C. dactylon genotypes.