Background

Differential centrifugation has substantially enriched available toolkits in cell biology and biochemistry. For example, decades ago, Bensley and Hoerr successfully isolated mitochondria from guinea pig and rabbit liver tissue [1]; later, Chen and Lehninger refined such procedures in higher plants [2]. Around the same time, Walker developed a method for efficiently isolating chloroplasts using sorbitol as osmotic support, enabling successful carbon fixation studies [3]. Later, isopycnic centrifugation protocols allowed for the recovery of highly pure organelle preparations with good yields, albeit requiring substantial quantities of starting material (~100 g) [4]. While these experiments sparked research on isolated organelles, most protocols required starting material quantities in the gram range [5].

During the 1990s, the successful introduction of optimized procedures to isolate DNA, known as minipreps, standardized DNA isolation and manipulation in thousands of laboratories around the world. This allowed the rapid isolation of DNA from bacteria, filamentous microorganisms, and even plants [6–9]. These preparations are typically purchased as kits offering different isolation scales (i.e., minipreps, midipreps, and even maxipreps). Kits specifically designed to isolate various biological materials, including mitochondria, nuclei, or chloroplasts, are also available at different scales and costs. However, most of these kits are either expensive or difficult to obtain in several countries. An effective chloroplast isolation protocol should require minimal biological and laboratory materials. In addition, organelle isolation should be completed as quickly as possible to ensure functional chloroplasts for downstream applications.

The purpose of this protocol is to provide easy-to-follow instructions for isolating intact chloroplasts from leaves in an inexpensive manner, using standard equipment and materials. It requires only small amounts of starting biological material and has the potential for portability (i.e., in-field isolations). The isolated chloroplasts are suitable for western blotting, microscopy, and omics analyses.