Cytological Analysis of Macro‐ and Microsporogenesis of a Diploid Alfalfa Clone Producing Male and Female 2<i>n</i> Gametes

Abstract

Gametes with the somatic chromosome number (2 n gametes) are important in germplasm transfer and in the evolution of polyploidy. In this work a stain‐clearing methodology was applied to the cytological study of both macrosporogenesis and microsporogenesis in the diploid (2 n = 2x = 16) clone H33 of alfalfa ( Medicago sativa L.), which produces both male and female 2 n gametes. Normal diploid and tetraploid plants were analyzed as controls. The analysis of macrosporogenesis indicated that done H33 produced ≈19% 2 n macrospores of the second division restitution (SDR) type as a consequence of the absence of cytokinesis in the chalazal dyad. The analysis of microsporogenesis showed clone H33 produced a high frequency of dyads with two 2 n microspores. About 64% of these dyads were of the first division restitution (FDR) type, as a consequence of parallel or nearly parallel spindles at metaphase II; the other dyads originated by abnormal cytokinesis at the end of meiosis and therefore they could be either FDR or SDR type. Abnormal cytokinesis was also responsible for the production of dyads with one n and one 3 n microspore; moreover some rare 4 n microspores originated by the total lack of cytokinesis. Clearing methodology proved very effective in alfalfa for the study of both macro‐ and microsporogenesis. It can therefore be efficiently used for the analysis of meiotic mutants that produce male and/or female 2 n gametes at high frequency.