Double Staining of Plant Materials in Bulk

Ovaries and ovules of Oryza sativa and Zea mays were collected between 9-30 and 10-30 AM, fixed in formalin-acetic-alcohol, stained in Delafield's hematoxylin for 2-4 hr, dehydrated through graded ethanol, counterstained for 3-4 hr either in light green, orange G or fast green (0.05-0.1%) at the 1:1 alcohol-xylene stage and embedded. A few ovaries were hydrolysed in 1 N HCI for 25 min at 60 C, stained in leuco basic fuchsin for 60-90 min, rinsed 3 times with a mixture of: 10% Na₂S₂O₅, 1; N HC1, 1; and distilled water, 18; washed repeatedly in distilled water, dehydrated through graded ethanol, counter-stained for 3-4 hr either with light or fast green (0.05-0.1%) at the 1:1 alcohol-xylene stage and embedded. Microtome sections were cut, ribbons mounted, dried, paraffin removed with xylene, and mounted in balsam. Uniformly stained preparations resulted and the dilute stains gave vivid color contrasts. Large numbers of ovules and ovaries can be processed in a short time, and reliable percentages of viable embryo sacs in normal, sterile and semisterile plants obtained.