Chapter 8 Isolation and Characterization of Plant Nuclei

Abstract

Publisher Summary This chapter discusses the isolation and characterization of plant nuclei. A number of procedures and variations on these procedures have been described for isolating plant nuclei. A wide range of homogenization buffers containing stabilizing agents such as sucrose, glycerol, or hexylene glycol and divalent cations have been employed along with filtration protocols and centrifugation strategies for isolating this plant organelle. Two additional treatments are often employed to aid in the isolation and purification of plant nuclei. One of these treatments involves immersing embryos, whole seedlings, or excised organs for a few minutes in cold diethylether and subsequent removal of this solvent. This treatment appears to facilitate the disruption of tissues and cells, and it also affects the integrity and stability of nuclei released during cell disruption. The second treatment employs a nonionic detergent—such as Triton X-100—for lysing contaminating organelles. Different plant organs and tissues present different levels of difficulty in purifying intact nuclei. The plant cell wall presents a substantial barrier to isolating fragile organelles, such as nuclei. In some cases (e.g., wheat and rice germ), the nuclei appear to be relatively stable and can be easily isolated without rupture. In other cases (e.g., soybean hypocotyl or plumule and cauliflower curd), the nuclei are fragile, and these plant materials generally require an ether treatment to isolate intact, relatively undamaged nuclei.