An improved cleared-pistil technique for rapid in toto observation of embryo sac malformation in cassava (Manihot esculenta Crantz)

The benzyl benzoate-four-and-a-half (BB-4 1/2) clearing fluid and a technique involving additional methyl salicylate treatment were evaluated for rapid analysis of embryo sac development of cassava ovules to elucidate the embryological aspects of low hybrid seed production in this genus. Pistils were fixed in Farmer's fixative (95 percent ethanol: glacial acetic acid equal 3:1), dissected ovules were cleared in BB-4 1/2 and observed with Nomarski's differential interference contrast microscopy (DIC). The ovules were then treated in a mixture of methyl salicylate and BB-4 1/2 (1:1, v/v) and re-examined with DIC. These two treatments differed significantly in their ability to produce distinct embryo sac features. Abnormal embryo sacs showed irregularities at both pre- and post-anthesis developmental stages. The embryo sacs were devoid of nucleate cells, significantly reduced in volume 112.5 percent of normal), collapsed or non-existent. Incomplete development of the megasporangium, with a characteristic lack of embryo sac, accounted for 9.1 percent malformation at pre-anthesis. Upon control and open pollination, absence of nucleate cells amounted to 6.7 percent and of collapsed embryo sacs to 3.3 percent. Malformed embryo sacs showed normal development of the nucellus as well as the inner and outer integuments. There was no correlation between ovule size and embryo sac volume (r(2) = 0.038 equal ovule length and r(2) = 0.001 equal ovule width) up till 7 DACP in regular ovules. Ovules analyzed with the advanced method showed improved images of embryo sac components, embryo division and defined embryo sac wall, thereby enhancing accurate determination of embryo sac volume. The methyl salicylate treatment yielded more accurate interpretations of morpho-structural features of cassava embryo sacs than benzyl benzoate alone. The embryological basis of low seed production in cassava is presented