In vitro generation of somaclonal variant plants of sugarcane (Saccharum spp. hybrids) for tolerance to toxins produced by Fusarium sacchari.

Abstract

The fungus Fusarium sacchari (Butler) Gams causes stem rot in sugarcane especially in association with the stem borer Eldana saccharina Walker. Sugarcane plants tolerant to F. sacchari PNG40 were obtained by chemical mutagenesis and in vitro selection during somatic embryogenesis and plantlet regeneration on media containing F. sacchari culture filtrates (CF), followed by selection in the greenhouse. Somaclonal variants tolerant to F. sacchari PNG40 CF were established by treatment of calli with ethyl methanesulphonate (EMS) and various selection treatments. Investigations were conducted to test the effect of varying CF concentrations and the culture developmental stages (embryo maturation, embryo germination and plantlets) that were most effective in screening calli and plants. Incorporation of CF (0-100 ppm) in the media, at either embryo maturation or germination stages, resulted in significant callus necrosis, and consequent decreased plantlet yield. The highest callus necrosis of 95.55 ± 0.9 % and the lowest plant yield of 1.4 ± 0.45 plants/0.2 g were obtained after inclusion of 100 ppm CF in the germination medium compared with 61.5 ± 3.8 % and 43.8 ± 5.6 plants/0.2 g in the maturation medium, respectively. Exposure of whole plants with trimmed roots to 0-1500 ppm CF resulted in inhibition of root re-growth, with the 1500 ppm CF treatment having the greatest negative effect. Subsequent treatments involved immersing in vitro plantlets in varying concentrations of F. sacchari conidial suspensions. This resulted in 33.3 % and 100 % mortality with 103 and 105 conidia/ml treatments, respectively. Control and EMS-treated calli and potentially tolerant regenerated plants were selected using the established CF and inoculation treatments. Plants from EMS treatments displayed more varying root length. More plants with increased root growth, in the presence of CF, were produced from these treatments than from non-EMS treatments, indicating the ability of EMS to induce somaclonal variation. These putative tolerant plants were inoculated with PNG40 and those selected using CF in vitro were symptomless whilst the positive controls (plants unexposed to CF) were symptomatic. Re-isolation of Fusarium from the inoculated plants and identifying isolates as PNG40 using ISSR analysis confirmed tolerance of the asymptomatic plants and the fungus as the causal agent of the observed symptoms. This confirmed that tolerance to CF correlates to tolerance to F. sacchari PNG40. Future work includes testing stability of tolerance in the field and after sexual reproduction, and use of this protocol to produce plants that permit endophytic PNG40 colonisation towards biological control of E. saccharina.