Improved Method of Enzyme Digestion for Root Tip Cytology

Abstract

Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.