Protoplasts as a tool for isolating functional chloroplasts from leaves

Leaf protoplasts from various grasses can be used for isolatingchloroplasts with high photosyndietic activity. The protoplastswere stable for more than 20 hr during which time chloroplastscould be isolated from protoplasts without any loss of originalCO₂ fixation capacity (100–157 µmoles/mg chl-hr).Using Triticum aestiuum to optimize assay conditions, the pHoptimum for CO₂ fixation by the chloroplasts isolated from protoplastswas between 8.2 and 8.6. Magnesium (0.75 mM) was required formaximum CO₂ fixation by the isolated chloroplasts and sodiumascorbate in the medium allowed a more linear increase in CO₂fixation with time. Based upon ¹⁴CO₂ fixation and ferricyanide-dependentoxygen evolution as criteria of intactness, chloroplasts fromprotoplasts exhibited a high degree of intactness compared tothose obtained by mechanical grinding. Chloroplasts isolatedfrom grass leaves by mechanical grinding had a relatively lowcapacity for endogenous CO₂ fixation and required addition ofribose-5-phosphate and ADP for maximum activity. (Received September 8, 1975; )