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Alfalfa is one of the most productive forage species and a major component of feed for dairy and beef cattle in North America.  In addition, alfalfa provides ecosystem services through improving soil nitrogen condition, being pollinator sources, and providing potential biofuel.  In the semiarid northern Great Plains, alfalfa establishment and persistence are generally poor due to grazing, disease, drought and cold conditions.  However, Medicago sativa subsp. falcata (yellow-flowered alfalfa) has showed its capability to produce adventitious shoots on the roots, which presents a potential source of vegetative regeneration.  This ability could be considered as favorable traits for increasing stress tolerance.  The objective of this study was to identify promising new PIs of Medicago sativa subsp. falcata that have capacity to regenerate from roots.  Seven falcata PIs were randomly selected from the National Plant Germplasm System and Persist II (M. sativa) as control.  About 20+ healthy 1-yr old plants for each entry were selected.  Each root was cut into segments 6cm long, originating 1cm or 7cm below the cotyledonary node.  Four root segments of each entry were planted upright in 15 cm dia. plastic pot filled with Miracle Gro potting soil.  Pots maintained in a greenhouse with 16h light/8h dark photoperiod, and 24±3°C temperature, and misted daily.  Number of root segments producing adventitious shoots counted weekly for 19 weeks following planting.  Six PIs populations produced adventitious shoots from roots.  The frequency of generating adventitious shoos from roots ranged from 3.8% to 57% among the 6 populations.  There were 66.7% to 100% produced adventitious shoots survived and developed into new individual plants.  Some of these survived plants successfully reproduced seeds.  Such discovery would be useful for the potential materials for development of germplasm suitable for grazing persistence, drought, cold tolerance and disease resistance.