Estimating alfalfa somaclonal variation in axillary branching propagation and indirect somatic embryogenesis by RAPD fingerprinting

The incidence of somaclonal variation was estimated by RAPD fingerprinting in in vitro cultured plantlets of the highly regenerative genotype A70-34 (synonym RL34) of Medicago sativa L. (2n = 4x = 32) from the cultivar Rangelander Plantlets obtained by axillary branching propagation on growth-regulator-free medium were compared with those obtained through indirect somatic embryogenesis, i.e., from somatic embryos regenerated from callus proliferated from petiole tissues. The initial donor plant, maintained in pot in the greenhouse, was used as the control. All evaluated genotypes were maintained in vitro after tissue sampling for DNA extraction and polymorphism analysis. Plantlets derived from axillary branching propagation exhibited no variant for any of the 75 RAPD markers obtained using eight different 10-mer primers, even after 12 repeated monthly subcultures. In contrast. the RAPD fingerprints of 9 of 39 plantlets regenerated by indirect somatic embryogenesis differed from that of the donor for at least one primer and one polymorphic amplification product. The eight primers generated 19 new RAPD markers in the somaclonal variants that were not found in the donor plant fingerprints, while 24 RAPD markers present in the donor plant fingerprints were not scored in the somaclonal variants. One of the indirect somatic embryogenesis-regenerated plants displayed 13 polymorphic bands, but it did not survive in vitro culture after the second transfer to fresh medium. Most of the somaclonal variants displayed one to five polymorphic bands. Moreover six of nine somaclonal variants were polymorphic, with respect to the donor plant, with two or more primers. Axillary branching propagation proved to be a safe clonation technique, as far as genetic stability of the proliferated tissues is concerned, and RAPD markers were an efficient tool for the early detection of somaclonal variants in tissue culture.