Induction Of Tetraploid Lotus Japonicus And Interspecific Hybridization With Super-Root-Derived Lotus Corniculatus Regenerants

Super-roots (SR), a unique in vitro cloning system of fast and continuously growing roots, has been isolated from self-incompatible Lotus corniculatus L. The objective of this research was to develop self-fertile SR plants by hybridizing SR forms of tetraploid L. corniculatus (2n = 4x = 24) with cholchicine-doubled forms of the diploid, self-fertile L. japonicus (Regel) K. Larsen (2n = 2x = 12) species. Tetraploid forms of L. japonicus 'Miyakojima' MG-20 (MG-20) were induced by treating seeds with 0, 0.05, 0.1, and 0.2% (w/v) colchicine solutions for 12 or 24 h. The rate of tetraploidization as determined by flow cytometry was most efficient at the lowest colchicine concentration for both treatment durations. Induced tetraploids of MG-20 had thicker stems with larger flowers and leaves compared with diploid forms of MG-20. Seeds produced by tetraploid MG-20 were also larger than those of the diploids; however, pod lengths and grain numbers were reduced. Two putative F-1-interspecific hybrid plants were recovered, using embryo rescue, from hand-pollinated crosses between tetraploid forms of MG-20 and SR. The F-1-interspecific hybrids displayed morphological features that were intermediate between the parental species. The root-growth rate of hybrid-derived root cultures was slightly reduced compared with the original SR. The hybrid plants are self-compatible and able to produce seeds. The interspecific hybrids obtained in this study demonstrate the potential to combine self-fertility and SR traits in Lotus forage breeding programs.