Somatic Embryogenesis in Trifolium: Protein Profiles Associated with High-and Low-Frequency Regeneration

A one-step regeneration system was developed for Trifolium rubens and Trifolium pratense using petiole segments of shoot cultured in vitro. Isogenic cell lines of T. rubens showing high- or low-frequency somatic embryogenesis (18 and 0.2 embryos per explant respectively) were isolated from a single seedling genotype by successive cycles of selection and subculture. Variation in the frequency of embryogenesis apparently arose by somaclonal variation in callus cultures, and in basal callus regions of shoot cultures. Using two-dimensional gel electrophoresis, the polypeptide translational profiles of cultured petioles from T. rubens isolines were examined over a 20-day interval covering the induction period for somatic embryogenesis. Two different genotypes of T. pratense, showing high frequency or zero somatic embryogenesis in the same culture system, were also included in the protein analysis. Two polypeptides exhibiting appropriate behavior for association with somatic embryogenesis were detected in the three regenerating lines. These same two components were also detected in the non-regenerating T. pratense genotype. In this latter genotype, however, they were found to exhibit a translational pattern different from that expected of proteins involved in somatic embryogenesis.