Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts.

With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.