White sweetclover (Melilotus alba Desr.) is an autogamous diploid with more than 70 years' worth of biochemical and genetic investigations that serve as the foundation for the current research. All sweetclover species studied to date are diploid with n=8 chromosomes (Smith, Gorz 1965). Melilotus species are easy to grow, and exhibit a short seed-to-seed cycle time (3 months).
A large number of biochemical studies have been performed on members of this genus because they produce significant quantities of coumarin, the simplest of all flavonoids. More is probably known about the biochemistry of flavonoid synthesis in sweetclover than in any other legume because of efforts to find cultivars that do not cause sweetclover-bleeding disease (Smith, Gorz 1965). Improper curing or ensiling of sweetclover hay leads to the formation of dicoumarol, a compound that interferes with vitamin K activation of prothrombin, which is necessary in. blood clotting. This hemorrhagenic agent, given the trade name Dicumarol, is used to prevent blood clots after surgery. Warfarin, probably the best-known anticoagulant, is a derivative of dicoumarol.
In the process of searching for coumarin mutants, a large number of other mutants were generated by Gorz and Haskins, Goplen, Micke, and others. Sweetclover produces a broad spectrum of flavonoids and related molecules, many of which have already been extensively studied. Gorz and Haskins and co-workers identified genes important for seed color (Y/y; Specht et al. 1976) and for seed and seedling color (C/c; Gorz, Haskins 1975). It is very likely that plants with pale seeds or seedlings are flavonoid mutants. Goplen (1992) found that a single recessive gene controlled the white sepal trait in sweetclover, and Goplen and Micke also uncovered a large number of floral mutants (Goplen 1976; Schiebe, Micke 1965). We also have obtained a large number of mutants in our screens of EMS- and neutron bombardment-generated M2 plants generated by Rneen and LaRue (1988) from the U389 line (Hirsch et al. 2000). This report describes some of these mutants as well as additional ones generated from the U390 line.
2.1. Isolation of mutants. Seeds from the wild-type Melilotus alba (Desr.) line U389 were mutagenized as described earlier (Kneen, LaRue 1988). Mutants of the U390 line were obtained by treatment with EMS (Hirsch, LaRue, unpublished). The U390 line, a wild-type dwarfed U389 (Gengenbach et al. 1969), is approximately the same size and has a similar flowering time as Arabidopsis (Figure 1). M2 seeds were planted in sand-vermiculite (1:1), watered with a medium without N, and inoculated with the wild-type Sinorhizobium meliloti strain Rml021 to look for
2. Materials and Methods symbiotic mutants. The Antho" and non-nodulating white sweetclover mutants were derived from Nod+Fix+U389 plants. The inflorescence mutants were derived from the U390 line.
2.2. Genetic analysis. The T280 line was crossed to the wild-type U390 line, and Fi hybrid seeds resulted. The Antho" mutants were crossed to the wild-type U389 line to obtain Fi hybrids. The F] flowers were selfed to produce the F2 generation. From the segregating F2 populations, F3 plants were obtained and analyzed for their phenotypes.
2.3. Plant growth. Sweetclover seeds of the non-nodulating mutants were surface-sterilized briefly in 95% ethanol, followed by immersion in commercial bleach for 45 min. The seeds were then washed five times with sterile water, placed on 0.8% Phytagar (GiboBRL), and grown in the dark for 48 h.
2.4. Inoculation studies. The wild-type S. meliloti strain Rml021 was used as inoculum. Liquid cultures were grown shaking in an incubator at 30°C, washed once with sterile water, and re-suspended to approximately 108 cells per ml. For the mycorrhizal studies, aseptic spores of Glomus intraradices (Premier Tech, Quebec) were used for inoculating the roots. The plants were grown in a sand-vermiculite mixture watered with one-strength Hoagland's medium minus P.
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