ESTIMATING ALFALFA SOMACLONAL VARIATION IN AXILLARY BRANCHING PROPAGATION AND INDIRECT SOMATIC EMBRYOGENESIS BY RAPD FINGERPRINTING

The incidence of somaclonal variation was estimated by RAPD fingerprin ting in in vitro cultured plantlets of the highly regenerative genotyp e A70-34 (synonym RL34) of Medicago sativa L. (2n = 4x = 32) from the cultivar Rangelander Plantlets obtained by axillary branching propagat ion on growth-regulator-free medium were compared with those obtained through indirect somatic embryogenesis, i.e., from somatic embryos reg enerated from callus proliferated from petiole tissues. The initial do nor plant, maintained in pot in the greenhouse, was used as the contro l. All evaluated genotypes were maintained in vitro after tissue sampl ing for DNA extraction and polymorphism analysis. Plantlets derived fr om axillary branching propagation exhibited no variant for any of the 75 RAPD markers obtained using eight different 10-mer primers, even af ter 12 repeated monthly subcultures. In contrast. the RAPD fingerprint s of 9 of 39 plantlets regenerated by indirect somatic embryogenesis d iffered from that of the donor for at least one primer and one polymor phic amplification product. The eight primers generated 19 new RAPD ma rkers in the somaclonal variants that were not found in the donor plan t fingerprints, while 24 RAPD markers present in the donor plant finge rprints were not scored in the somaclonal variants. One of the indirec t somatic embryogenesis-regenerated plants displayed 13 polymorphic ba nds, but it did not survive in vitro culture after the second transfer to fresh medium. Most of the somaclonal variants displayed one to fiv e polymorphic bands. Moreover six of nine somaclonal variants were pol ymorphic, with respect to the donor plant, with two or more primers. A xillary 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 so maclonal variants in tissue culture.