Reduction of fertile regenerants from protoplasts of Triticum tauschii (Coss.) Schmal.

Four suspension cell lines generated from two accessions of Triticum tauschii (Coss.) Schmal. (Aegilops squarrosa, 2n = 2x = 14, DD genome) were used to develop an efficient protocol for producing fertile regenerants from protoplasts. Protoplasts were isolated from each cell line by incubating fine cell aggregates (<500 µm in diameter) in a solution containing 3% Cellulase ‘Onozuka’ RS, 0.5% Macerozyme R10 and 0.2% Pectolyase Y23. Cell division occurred when the protoplasts were cultured at a density of 1.0–1.5 x 106 protoplasts mL–1 in half-strength MS medium supplemented with 1.1 mg L–1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.6 M glucose and 1.2% agarose. The first cell divisions were observed after 5–7 days. Cell colonies were observed after 14 days and these grew quickly into large clumps when transferred to half-strength MS medium supplemented with 2.2 mg L-1 2,4-D, 30 g L–1 sucrose and solidified with 0.25% Phytagel. The colonies produced somatic embryos within 21–28 days of transfer to this medium. Somatic embryos were transferred to hormone-free MS medium for regeneration into plantlets. Although many regenerants produced shrivelled seeds, 9 of 16 were fertile and produced normal seeds.