Callus formation and plant regeneration from protoplasts of sunflower calli and hypocotyls

Abstract

Sunflower (cv. Girapac SH222) protoplasts were obtained from 4-7 day-old hypocotyls and cotyledons and from two-month old calli. Higher yields of protoplasts were achieved with medium El (KCl 25g dm<sup>-3</sup>, CaCl<sub>2</sub> 2g dm<sup>-3</sup>, MES 0.7 g• dm<sup>-3</sup>, pH 5.5) and the combination of Driselase Fluka 0.2%, Macerozyme Onozuka 0.2% and Cellulase Onozuka R10 0.2%. Hypocotyls gave the highest yields of protoplasts, followed by cotyledons and calli. Protoplasts were cultivated in liquid and on solid media using both L4M (Burrus et al., 1991) and V-KM (Bokelman and Roest, 1983) media. Culture on solid M1 medium (L4M medium supplemented with NAA 3.0 mg•dm<sup>- 3</sup>, 2,4-D 0.1 mg•dm<sup>-3</sup> and BA 1.0 mg•dm <sup>-3</sup>) gave a good planting efficiency with the development of many white-green colonies. These colonies gave rise to small calli which were transferred to MSmod medium (MS medium supplemented with KCI 5 g•dm<sup>-'</sup>, and polyvinylpyrrolidone (PVP, 4 g• dm<sup>-3</sup>) containing benziladenine (BA, 0.5 mg•dm<sup>-3</sup>), naphtaleneacetic acid (NAA, 0.5 mg•dm<sup>-3</sup>) and giberelic acid (GA3 0.1 mg•dm<sup>-3</sup>). After two weeks, calli were transferred to MSmod medium containing BA 1.0 mg•dm<sup>-3</sup>, NAA 0.1 mg•dm<sup>-3</sup>, and GA3 0.1 mg•dm<sup>-3</sup> for shoot formation. Shoots were excised and induced to root in MSmod supplemented with BA 0.1 mg•dm<sup>-3</sup>, NAA 1.0 mg•dm<sup>-3</sup>, and GA3 0.1 mg•dm<sup>-3</sup>. Plantlets were then transferred to sterilised vermiculite for greenhouse acclimation.