The Isolation of Peroxisomes, Mitochondria and Chloroplasts from Leaves of Spinach Beet (Beta vulgaris L. ssp. vulgaris)

Attempts to isolate peroxisomes, mitochondria and broken chloroplasts from leaves of Beta vulgaris using classical sucrose homogenising media and gradients gave poor results. A homogenising medium consisting of 0.5 M sorbitol and 2.5%, Ficoll in 0.05 M Tris‐HCl buffer, pH 7.4 (SF medium) greatly increased catalase (peroxisome) sedimentation. Density gradients made from mixtures of SF medium and sucrose (SFS gradients) gave good separation of the three organelles. Best results were obtained with the pellet sedimenting between 1000 ×g (20 min) and 6500 ×g (20 min). The majority of catalase, glycolate oxidase and NADH‐glyoxylate reductase activity in the gradient was restricted to the peroxisomes. Electron microscopy of the gradient fractions showed the isolated peroxisomes were intact and had retained the appearance they had in vivo. The isolation methods described should prove useful with other plant tissue where isolation of cell fractions has hitherto been difficult.