Regeneration of whole plants from protoplasts of Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful regeneration of whole plants from protoplasts of Gracilaria gracilis (Stackhouse) Steentoft, Irvine and Farnham. Protoplasts were isolated and purified using a previously optimized protocol. Protoplasts with regenerated cell walls divided to produce callus-like cell masses which showed the presence of uniseriate, filamentous outgrowths. Bud outgrowth from callus masses was associated with a distinct change in colour intensity at the point of outgrowth. Ultimately, whole plants were regenerated from the callus-like cell masses with overall yields of approximately two to three whole plants per 10(4) protoplasts seeded. Two distinctive patterns of regeneration were observed. In the first case, protoplasts regenerated slowly to produce plants which resembled the parent plants, exhibiting slender, branched thalli. Growth rates of regenerated seaweed were similar to that of wild-type G. gracilis cultured under the same conditions with 115 g of seaweed cultured from 15 individually regenerated plants over a year. In the second case, protoplasts regenerated rapidly to produce plants which remained small with thalli that were thick and unbranched and had a limited life span. These results provide an important foundation for the development of a successful tissue culture system for G. gracilis.