Introduction

There has been several advances in the area of alfalfa cytogenetics in the past 10 years. Prior to this there have been comprehensive chapters written on the cytogenetics of alfalfa including Lesins and Gillies (1972) Stanford et al . (1972), McCoy and Bingham (1988 and 1991) and McCoy and Echt (1992). The most recent progress can be characterized by the advancements in the use of computerized image analysis, chromosome banding, and in situ hybridization.

Bauchan and Campbell (1994) developed and utilized a computerized image analysis system to critically measure, analyze and construct the karyotype of diploid alfalfa. Since that time the development of faster computers (20 Mhz to 600 Mh z) with larger memory (512KB to 128MB RAM) has allowed for increased resolution (1048 x 960 pixels to 2096 x 1920 pixels) and direct connections with Windows based software such as spread sheets for data analysis and presentation programs for production o f pictures for analysis and publication. These advances have made it possible for even more precise karyotypic investigations and several cells per hour can be analyzed rather than one or two per day.

Johnson et al. (1984) and McCoy and Bingham (1988) stated that their attempts to band the chromosomes of Medicago were either unsuccessful or produced bands at the centromeres only and thus this technique did not aid in the advan cement of Medicago cytogenetics. Masoud et al. (1991) showed that the chromosomes of diploid M. sativa ssp. sativa (L.) L. & L. cv. CADL (cultivated alfalfa at the diploid level) had additional bands. B auchan and Hossain (1997) perfected the C-banding technique and extensively applied the technique to prove that there are additional bands and developed a standardized karyotype for diploid ssp. falcata Arcengeli and ssp. coerulea Schmalh.. Bauchan and Hossain (1998a) were the first to develop N-banding for the genus Medicago and this was the first successful utilization of N-banding to identify individual chromosomes of a dicot plant. Falistocco et al. (1995) published a karyotype of tetraploid M. sativa ssp. sativa showing that the individual chromosomes could be identified and added additional proof that alfalfa is an autotetraploid.

Schaff et al.(1990) utilized molecular cytogenetic techniques on alfalfa chromosomes. They used enzymatic techniques (strepavidin-horseradish peroxidase complex) to label a specific gene ($-tubulin) and in situ hybridize the gene to alfalfa chromosomes to identify the location of the gene on two chromosomes. Calderini et al. (1996) used fluorescent stains [4', 6-diamidion-2-phenylindole (DAPI) and chromomycin A₃ CMA₃₎] to band the chromosomes of alfalfa showing that the nucleolar organizing regions (NOR) were high in GC content. Cluster et al. (1996) and Calderini et al. (1996) have used dual fluorescent staining (rhodamine and DAPI) for fluorescent in situ hybridization (FISH) techniques to label the 18S gene of rDNA and identify the number and location of the genes on the chromosomes.