The Effect of Environmental Conditions on the Expression of Disease in Cultured Tissues of Brassica napus ssp. oldfera Infected with Leptosphaeria maculans

Abstract

AbstractAs a basis for devising an in vitro screening programme, culture conditions were optimized so that tissue cultures from two resistant cultivars of Brassica napus ssp. oleifera (Mikado, Bienvenu) and two susceptible cultivars (Lesira, Ceres) could be differentiated using a disease scoring scheme, when inoculated with Leptosphaeria maculans. Tissues inoculated included thin cell layer explants from soil‐grown plants and in vitro‐grown shoot cultures and callus tissue formed on such explants. The period of incubation and the incubation temperature were of importance in the development of differential disease reactions. Increasing temperature generally resulted in an increase in infection and too great an incubation period resulted in total overgrowth of the tissue. Increasing concentrations (1 × 10−6 M‐1 ×10−4 M) of the auxins 1‐naphthylacetic acid (NAA), 2,4‐dichlorophenoxyacetic acid (2,4‐D) and mdole‐3‐acetic acid (IAA) in the culture medium, resulted in a decrease in disease score of the thin cell layer (TCL) explants from soil‐grown plants. The cytokinins examined 6‐benzyl‐aminopurine (BAP) and 6‐4‐hydroxy‐3‐methyl‐2‐enylaminopurine (zeatin), reduced the extent of infection of the TCL explants when used in combination with the auxin NAA. Medium containing NAA at a concentration of 1 × 10−6 M in combination with BAP at a concentration of 1× 10−6 or 1 × 10−4 M allowed differentiation of the disease reactions of the resistant and susceptible cultivars, when the explants were incubated for 10 days at 20 °C after inoculation. Similar conditions of incubation and the addition of NAA (1 × 10−6 M) combined with BAP (1 × 10−6 M) to the medium also allowed the differentiation of the disease reactions on TCL explants from stems of in vitro shoot cultures of the cultivars Mikado and Lesira. Increasing concentrations of the auxin NAA and the cytokinin BAP resulted in a reduction in the mean disease score of the callus tissue produced on TCL explants from soil‐grown plants, and NAA (1 × 10−5 M) combined with BAP (1 × 10−6 or 1 × 10−5 M) allowed differentiation of resistance and susceptibility in callus tissues when incubated for 5 days at 20 °C. 2,4‐D did not allow differentiation of the cultivars. This was in contrast to the inoculation of callus tissue attached to TCL explants of in vitro shoot cultures, where combinations of 2,4‐D and BAP at concentrations of 1 × 10−6 M allowed differentiation of the resistant and susceptible cultivars.These findings provide a basis for designing selection protocols of value in both traditional as well as in vitro breeding programmes to select lines of oilseed rape with resistance/novel resistance to L. maculans.