Female sterility associated with heavy callose deposition in the nucellus of ovules at anthesis was studied in an F₁ progeny of two alfalfa plants displaying 5 and 81% ovule sterility, respectively. Transgressive segregation was observed and 100% sterile plants were found. Cytological analyses were conducted on three representative plants: F₁7, which displayed 100% sterility and pistil development block; F₁48, which displayed 97-100% sterility and 18-55% pistil development block; F₁22, fertile control, displaying 0-3% callosized ovules and normal pistil development. Optical microscopy was used to examine sectioned ovules from an early bud stage to the mature embryosac stage, following toluidine blue and aniline blue staining. Cleared whole ovules at the same stages stained with aniline blue were also studied.

Ovule development of the sterile and fertile plants did not differ until a pre-meiotic stage at which callose deposition was observed in nucellar cell walls in both sterile and fertile developing ovules. At the meiotic stage, only thin callose layers between the elements of the diads and tetrads were present in the fertile control while, at the same stage, much callose was found in the nucellar cell walls of sterile ovules in which meiosis did not occur. Nucellar cell growth and development were abnormal in sterile plants: megaspore mother cells generally differentiated but never underwent meiosis. In the nucellus of sterile ovules, several cells with callosized walls enlarged to fill part of the space that in normal ovules was occupied by the megaspore and developing embryosac. Integument development appeared normal in both sterile and fertile plants. No callose was observed in fertile ovules after meiosis, while callose remained until the anthesis stage in the sterile ones. The absence of meiosis makes this female sterility trait interesting for studying the genetic control of megasporogenesis in alfalfa.