Characterization of alfalfa (Medicago sativa L.) plants regenerated from selected NaCl tolerant cell lines

Selection of stable, NaCl tolerant alfalfa (Medicago sativa L.) cell lines was accomplished by a step-up selection procedure, whereby cell lines originally selected for tolerance at 0.5% NaCl were subsequently selected at 1.0% NaCl. Sodium chloride tolerant cell lines retained tolerance following four subcultures (16 weeks) on control media (0% NaCl). Plants were regenerated from selected NaCl tolerant cell lines of three initial genotypes, one diploid (2n=2x=16) and two tetraploids (2n=4x=32). In addition, plants were regenerated from control cell lines maintained on 0% NaCl media for the same duration. Plants regenerated from NaCl tolerant cell lines were characterized by extensive somaclonal variation compared to plants regenerated from control lines. Morphologically, all plants regenerated from NaCl tolerant cell lines are abnormal and many (44.7%) were extreme dwarfs (maximum height of 5 cm). The grossly aberrant phenotypes prevented an in-depth characterization of many of the plants regenerated from NaCl tolerant cell lines. Most plants regenerated from NaCl tolerant cell lines had unbalanced polyploid chromosome sets with the most extreme cytogenetic variant having 106 chromosomes. In contrast, 98.5% of the plants regenerated from control cell lines were euploid (85% were tetraploid, 15% were octoploid). Isozyme phenotypes of the plants from NaCl tolerant cell lines were also extensively altered, compared to plants from control cell lines. In vitro NaCl tolerance was maintained following plant regeneration for nine of the 12 regenerants tested. Importantly, whole plant NaCl tolerance was expressed in two of the seven regenerated plants tested at the whole plant level; however, only one of these plants has flowered and is both male and female sterile; the other plant has never flowered. Although NaCl tolerant alfalfa cell lines are efficiently selected, the extensive somaclonal variation that accompanied the selection was a deterrent to successful recovery of heritable NaCl tolerance.