Chapter 21 Methods for Mesophyll and Bundle Sheath Cell Separation

Abstract

Publisher Summary This chapter describes the methods for the separation of mesophyll and bundle sheath cells from maize seedlings. The resultant cells, which can be easily manipulated, are physiologically active. The overall purity and viability are usually greater than 95%. Physical destruction method for bundle sheath cell isolation is used to purify intact bundle sheath strands from which bundle sheath protoplasts, nuclei, and chloroplasts can be isolated if desired. Mesophyll cells are broken during the grinding process. Although the suspension of the first grinding is enriched for mesophyll chloroplasts and nuclei, it is heavily contaminated with bundle sheath chloroplasts and nuclei. The grinding is usually performed with a Polytron homogenizer for its efficiency and flexibility. For large batch isolation, a Cuisinart food processor can be used instead of manual cutting to first chop the leaves into small pieces. Enzymatic digestion method allows the simultaneous isolation of mesophyll protoplasts and bundle sheath strands. To obtain pure bundle sheath strands by grinding, it is important to remove the thick midvein and cut leaves to 1- to 3-mm strips for efficient grinding to eliminate the contamination of mesophyll cells.