Characterisation of Simple In Vitro Cultures of Striga hermonthica Suitable for Metabolic Studies

Abstract: The aim of this study is to develop simplified models for standardised screenings of xenobiotics, especially targeted against mannitol production, to control the harmful parasite, S. hermonthica. Chlorophyllous protoplasts and calli were produced from the young leaves of the parasite. Best yield from protoplast isolation was obtained when leaf segments were incubated at 30 °C, in the light, under shaking in an enzyme cocktail containing 2 % cellulase Onozuka R10, 0.1 % Pectolyase and sorbitol 1 M as the osmoticum. Oxygen exchange measurements, as well as labelling experiments with ¹⁴C-bicarbonate, emphasised a significant decrease in photosynthetic capacity of protoplasts, mannitol remaining, however, a major primary product of photosynthesis. Initiation of cell cultures was unsuccessful and instability of protoplasts prevents their standardised utilisation for herbicide screening. In contrast, globular calli produced first on MS medium containing 0.5 mg L^-1 NAA, 2.5 mg L^-1 BAP and 2 % sucrose were stable for two years, after monthly subculturing on fresh medium. Sucrose substitution by mannose in the medium did not change kinetic growth and stability. Potential autotrophy was lost for calli by increasing exogenous sugar level. Biochemical analyses and labelling experiments with ¹⁴C-bicarbonate or ¹⁴C-sucrose or -mannose showed that carbon partitioning is modified in calli, in comparison with young leaves or protoplasts, sucrose or mannose accumulation being favoured in sucrose- or mannose-fed calli, respectively. However, carbon flow towards mannitol was more marked in calli growing on high mannose medium. Stability and preservation of an active mannitol biosynthetic pathway allows planning of xenobiotic assays with calli as a simplified model for Striga hermonthica.