Improvement of the in vitro regeneration and Agrobacterium-mediated genetic transformation of Medicago sativa L.

Abstract

Alfalfa is a fodder crop that accounts for one of the best sources of protein and is widely cultivated around the world. In vitro regeneration of alfalfa has been studied earlier; however, most of the studies were almost intervened with callus formation. In this study, 3 explant sources (cotyledonary node, hypocotyl, and root crown) of two Turkish cultivars (Nimet and Savas) were excised from young seedlings. Explants were subjected to different concentrations of BAP, BAP-IBA, and TDZ to evaluate the direct in vitro regeneration potential of selected plant parts. Moreover, we transformed the alfalfa plant with pBin19 harboring 35s.GUS-INT_35s.nptII construct to investigate the transformation efficiency and regeneration frequency after bacterial inoculation. The transformation was carried out by Agrobacterium tumefaciens strain GV2260. The highest mean number of regenerated shoots per explant was recorded as 8.5, 6.66, and 6.33 shoots per explant after cotyledonary node explants were treated with BAP (0.40 mg/L), BAP-IBA (1.25-0.06 mg/L), and TDZ (0.55 mg/L), respectively. The highest gene transformation frequency was 9.52% and 6.19% based on PCR and GUS assays. The regeneration frequency was decreased by up to 48.1% under kanamycin selection pressure. The effect of cultivar on shoot regeneration frequency, mean number of regenerated shoots, and gene transformation efficiency was significant. This study contributes to in vitro regeneration of alfalfa crop and its genetic transformation which could be utilized in future gene transformation studies.