Use of the four-and-a-half clearing technique to study gymnosperm embryology: Cunninghamia lanceolata

Since its introduction in 1971, the four-and-a-half clearing technique has been widely applied to the study of ovule and female gametophyte development in flowering plants as an alternative to the more arduous paraffin section methods. The technique has undergone several modifications that have broadened its application in studies of Angiosperm embryology. To date, however, the technique has not been successfully applied to embryological features of Gymnosperms. Dark coloration caused by naturally occurring substances and by-products of fixation render the clearing fluid ineffective, and special pretreatment methods used to remove dark substances in Angiosperm ovules have little or no effect on Gymnosperm material. In the technique reported here, paraffin sections of ovules and young seeds of Cunninghamia lanceolata 80-120 microns thick are cleared in benzyl benzoate-4 1/2 clearing fluid and examined with phase contrast optics. Observations of the mature female gametophyte in these cleared preparations are compared with those obtained from 10 microns sections, stained with safranin and fast green, and examined with bright-field optics. Although contrast and definition are more pronounced in stained sections than in cleared ones, the differences would not alter one's interpretation of characteristic structural features. The thick, cleared section offers an advantage over the thin, stained one in that many structural entities are contained within a single section rather than spread through several serial sections. The time required for clearing thick sections is much shorter than that required for making permanent stained preparations.