Theor Appl Genet (1995) 90:135-141 9 Springer-Verlag 1995 O. Calderini 9 A. Mariani Megagametophyte organization in diploid alfalfa meiotic mutants producing 4n pollen and 2n eggs Received: 25 May 1994 / Accepted: 9 August 1994 Abstract Megagametogenesis was studied in five diploid alfalfa mutants producing 4n pollen and 2n eggs, using a stain-clearing technique. All mutants produced embryo sacs with a variable number of supernumerary nuclei both at the early (bi- and tetra-nucleate) and at the late (eight-nucleate) stages of development. The presence of supernumerary nuclei is considered to be a consequence of the production of coenocytic mega- spores. The production of 2n eggs was confirmed through cytological investigation by means of the diam- eter of the egg-cell nucleolus. The frequency of 2n eggs was lower than the frequency of binucleated macro- spores as previously determined. This discrepancy may be due to environmental effects but also to the fact that binucleated macrospores may degenerate or may, after two mitotic divisions, give rise to eight-nucleated em- bryo sacs counted as normals. Key words Alfalfa 9Megagametogenesis 9 Supernumerary nuclei 9 2n eggs 9 Meiotic mutants Introduction In the Medicago sativa complex a meiotic mutation causes the production of 4n pollen ("jumbo pollen"). Previous research has established that this mutation is due to a single recessive gene, named 'Tp", which causes the failure of postmeiotic cytokinesis during micro- sporogenesis (McCoy and Smith 1983; Pfeiffer and Bingham 1983). Jumbo pollen mutants behave essential- Communicated by F. Salamini O. Calderini 9 A. Mariani ([]) Istituto di Ricercbe sul Miglioramento Genetico delte Piante Foraggere del C. N. R., via della Madonna Alta 130, 06128 Perugia, Italy ly as male sterile, but they have normal female fertility and can also produce a high frequency of 2n eggs (McCoy and Smith 1983). Therefore, they can be used as mother plants to obtain tetraploids via unilateral or bilateral sexual polyploidization. During a selection programme aimed at increasing the frequency of 2n gametes in an experimental diploid population of alfalfa, five plants producing jumbo pollen and 2n eggs at high frequency were identified (Veronesi et al. 1990). An analysis of micro- and macro-sporogenesis performed on these plants revealed that they produced tetranu- cleated microspores and jumbo pollen due to the failure of the post-meiotic cytokinesis, and bi-, tri- and tetra- nucleated macrospores due to the lack of cytokinesis after the first- and/or second meiotic division. The fusion of nuclei in the binucleated macrospores resulted in the production of 2n macrospores of the SDR type, which were recognized on the basis of nucleolar dimensions. Observations on clone H25 indicated that it produced embryo sacs with supernumerary nuclei (Mariani et al. 1993). Based on these findings the present research was conducted: (1) to verify the production of macro- gametophytes (MGs) with supernumerary nuclei in all five jp clones; (2) to determine if such abnormal MGs derive from coenomegaspores; and (3) to further confirm the production of 2n eggs through cytological investigation. Materials and methods The jumbo pollen plants analyzed (H21, H23, H25, H27, H29) are the same as reported in a previous study (Mariani et al. 1993). Buds of these plants were fixed in FAA and ovules were prepared according to the technique proposed by Stelly et al. (1984) which proved effective in observing alfalfa micro- and macro-sporogenesis (Tavoletti et al. 1991). The analysis of the MGs developmental stages was performed on 1482 ovules, ranging from 324 in H29 to 466 in H23. Unreduced egg cells were identified by their nucleolar dimensions. For this purpose 547 of the 1482 ovules were used. The nucleolar-size dis- tributions ofjp plants were compared to those of diploid and tetrap- loid controls (100 measurements each).