Cold and Chemical Pretreatments to aid Chromosome Counts in a Grass Leaf Squash Technique Incorporating Hot Pectinase Maceration

Leaf buds, comprising the basal 3-5 mm of the youngest leaves attached to short stems, were dissected out of fast-growing young tillers of certain grasses, including Festuca, Lolium and Phalaris spp. and various hybrids. They were kept overnight in distilled water at 0-2 C, treated in a mixture of equal parts by volume of saturated aqueous solutions of 5,7-dibromo-8-hydroxyquinoline containing a surfactant (Tween 80), and 1-bromo-naphthalene for 3-4 hr at 0-2 C, and fixed in Newcomer's fluid. The rinsed samples were hydrolysed in 1 N HCl for 8 min at 60 C and Feulgen stained for 1 hr. After rinsing, the buds were macerated in a filtered 3% solution of Pectinol 100-D (Rohm and Haas) in 0.1 M acetate buffer at pH 4.5 for 10 min at 60 C. Squashes were made in 45% acetic acid. The combined cold and chemical pretreatments resulted in strongly contracted, easily counted metaphase chromosomes, while intact cells with full chromosome complements were more readily retained during squashing after enzyme maceration at 60 C than at room temperature.