Cytomixis (i.e., chromatin migration between meiocytes) has been detected in many plant species, but not in Medicago sativa spp. In the present study we report the identification of a few cytomictic alfalfa plants. Those plants, the "mother plants," were selfed and crossed with a normal control plant. Microsporogenesis analysis was performed on the mother plants, on the S(1) and F(1) plants, and on controls. The S(1) and F(1) plants, like the mother plants, were found to be cytomictic. Single or multiple chromatin bridges between two or more meiocytes were observed almost exclusively in prophase I. Some completely empty meiocytes were also observed. In addition to cytomixis, other meiotic abnormalities were found. Control plants showed an almost regular meiosis. The highest values of cytomixis were observed in the mother plants, and the lowest in their F(1) progenies. Variability of cytomixis in the F(1) plants is probably due to a heterozygotic condition of the parents for this trait. No significant correlation was found between cytomixis and pollen viability, even if the cytomictic plants showed low values of pollen viability.

TBP (tubulin-based polymorphism) is a new molecular marker based tool that relies on the presence of intron-specific DNA polymorphisms of the plant beta-tubulin gene family. The multifunctional and essential role of the tubulin proteins is reflected in the conservation of regions within their primary amino acid sequence. The ubiquitous nature of this gene family can be exploited using primers that amplify the first intron of different beta-tubulin isotypes, revealing specific fingerprints. The method is rapid, simple, and reliable and does not require preliminary sequence information of the plant genome of interest. The ability of TBP to discriminate between accessions and species in oilseed rape, coffee, and lotus is shown. In all cases, TBP was able to detect specific genetic polymorphisms in the context of a simplified and readily appreciable pattern of DNA amplification. The application of TBP for assessing genetic diversity and genome origins in disseminated plant landraces rather than in highly inbred cultivated species is also discussed.

In the genus Medicago, it is known that 2n gametes have been important in the evolution and breeding of cultivated alfalfa, which is a natural polysomic polyploid (2n=4x=32), however little is known on the frequency of male and female 2n gametes in diploid relatives of alfalfa. To obtain data on the frequency of 2n gametes, more than 12,000 2x-4x and 4x-2x crosses were made in 1982 at Madison (USA). Diploid parents in crosses were from four populations of M. coerulea, two of M. falcata and one diploid population of cultivated M. sativa which was derived by haploidy. The tetraploid seed parent in the crosses was a male-sterile M. sativa clone and vigorous tetraploid M. sativa plants were used as pollen parents. Each of 274 diploid plants was utilized both as male and as female. Of the 548 cross combinations, 266 crosses produced variable quantities of seeds which were sown in 1983 in a greenhouse at Perugia (Italy); the plants were subsequently space transplanted in the field in 1984. The identification of ploidy level of these genotypes was made on the basis of morphological characters, plant fertility, pollen stainability and chromosome counts.Of the 515 plants analyzed, the majority behaved as normal tetraploids indicating that many diploid plants produced 2n gametes. Diplogynous and diplandrous gamete production was not correlated with each other, which indicated a different genetic control of 2n sporogenesis in the 2 sexes. Only 4 F1 triploid plants confirmed the presence of a very effective triploid block in alfalfa. In consequence, bilateral sexual polyploidization is a more likely alternative for the origin of tetraploid alfalfa than triploid bridges. The present study showed that it is possible to efficiently identify genotypes able to produce high frequencies of 2n gametes within natural populations of diploids Medicago that are useful in alfalfa breeding.

Gametes with the somatic chromosome number (2n 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 (2n = 2x = 16) clone H33 of alfalfa (Medicago sativa L.), which produces both male and female 2n gametes. Normal diploid and tetraploid plants were analyzed as controls. The analysis of macrosporogenesis indicated that done H33 produced ≈19% 2n 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 2n 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 3n microspore; moreover some rare 4n 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 2n gametes at high frequency.

Cultivated alfalfa (Medicago sativa L.) is a polysomic polyploid with tetrasomic inheritance. In this crop the hybridization of gametes with the somatic chromosome number (2n gametes) produced by diploid hybrids is a valuable method to maximize heterozygosity. For this purpose it is necessary to obtain diploids which produce high frequencies of 2n gametes. The results of the present paper indicate that phenotypic recurrent selection was efficient in increasing the production of male and female 2n gametes in diploid alfalfa. Two distinct selections were carried out, one for 2n pollen and the other for 2n egg production and two cycles of phenotypic recurrent selection were performed. The percentage of plants producing 2n gametes and the frequency of 2n gametes per plant were improved by selection. Realized heritabilities over the two cycles of recurrent selection were 39% and 60% for 2n pollen and 2n egg production, respectively; on the whole, the responses to the selection indicate that both male and female 2n gamete production are controlled by major and minor genes.