Effect of plant growth regulators and agar concentration on shoot multiplication and hyperhydricity of Anthyllis barba-jovis

The increase of agar concentration and the addition of auxin into the shoot proliferation medium were tested for their effectiveness in eliminating hyperhydricity in Anthyllis barba-jovis L. during micropropagation. Single node explants of adult- or seedling-origin were cultured on MS medium supplemented with 4.0 mg L^‑1 6-benzyladenine (BA) in combination with 0.0, 0.1 or 0.5 mg L^‑1 1-naphthaleneacetic acid (NAA) solidified with 8.0 or 12.0 g L^‑1 agar. Hormone free-MS medium with 8.0 g L^‑1 agar, used as control, produced the lowest shoot number and length (1.0 shoot explant^‑1, 0.6-0.8 cm), and no hyperhydricity. Both explant types responded in a similar way in all treatments. MS medium with BA solidified with 8 g L^‑1 agar induced the highest shoot proliferation (12.5 shoots explant^‑1, 1.8 cm shoot length) but more than 75% of the shoots were hyperhydric. The addition of NAA (independently of concentration) into the BA-medium, or the increase of agar concentration resulted in reduced hyperhydricity, and the combination of both almost eliminated it. In BA-NAA-media, the highest number of normal shoots was produced when BA was combined with 0.1 mg L^‑1 NAA and 12.0 g L^‑1 agar (7.6 or 7.9 shoots explant^‑1 of adult and seedling origin, respectively). Both agar increased and NAA reduced slightly shoot length (1.0-1.2 cm). Therefore, increasing agar concentration to 12.0 g L^‑1 and supplementing the medium with 4.0 mg L^‑1 BA and 0.1 or 0.5 mg L^‑1 NAA, resulted in the elimination of hyperhydricity and in the best shoot multiplication response.