CULTURE OPTIMIZATION FOR PERENNIAL RYEGRASS PROTOPLASTS

The effects of conditioned medium, O-acetylsalicylic acid (ASA) and antioxidants on the plating efficiency and plant regeneration of suspension-derived protoplasts were investigated with the aim to improve the procedure for protoplast culture in perennial ryegrass. Conditioning of the protoplast culture medium was indispensable for the proliferation of microcalli from protoplasts derived from young, regeneration-competent suspension cultures and, in addition, enhanced microcallus formation from protoplasts from older suspension cultures. The addition of ASA to the protoplast culture medium improved the plating efficiency of protoplasts, but not the regeneration frequency. The antioxidants, vitamins C, E and n-propyl gallate, did not improve the culture of perennial ryegrass protoplasts. However, the addition of other antioxidants (superoxide dismutase, catalase, glutathione, glutathione peroxidase and phospholipase A2), involved in the reduction of peroxides, to the protoplast isolation medium and the culture medium increased the plating efficiency.