A method to isolate the female sexual cells of Larix decidua for cytological studies

Abstract

A cytological squash technique to study megasporogenesis, egg formation, and proembryo formation in Larix decidua was developed. The middle lamellae of the cells were dissolved and the cytoplasm was cleared by treating the tissues with several combinations of glycerol, water, dimethyl sulfoxide, sodium bicarbonate, and potassium hydroxide at high temperatures. The nucelli, meiocytes, coenocytes, central cells, or eggs were excised in a drop of toluidine blue stain solution on a microscope slide. A coverslip was added and slight pressure was applied. Meiotic chromosomes stained darkly and displayed several structural details well. Metaphase 1 bivalents separated easily and clearly showed their chiasmata. Since meiocyte excision from nucelli was about 90% successful, the degree of synchrony in meiosis within cones could be determined. A few aborting tetrads were observed. Division of the central cell and egg formation could be followed in detail.