Callus Formation and Plant Regeneration from Protoplast Derived from Cotyledons and Hypocotyls of Radish (Raphanus sativus L.) and Other Cruciferous Plants

Abstract

Protoplasts were isolated from radish hypocotyls and cotyledons of 4- to 7-day-old seed-ling with three enzyme mixtures, three concentrations of mannitol and two incubation peri-ods. The most effective conditions for protoplast isolation were to shake sliced 5-day-old cotyledons at 27°C for 2 hr in the enzyme solution containing 2% Cellulase Y-C, 0.2% Pec-tolyase Y-23, CPW salts and 0.3 M mannitol. Protoplasts isolated from 16 cruciferous cul-tivars under the most effective condition were compared with other two enzyme mixtures and two materials; cotyledon and hypocotyl. Although the isolation efficiency differed depend-ing on cultivars, the best condition was as same as radish. As the initial plating medium for cell division of protoplasts, Pelletier's B medium (1983) was used, which supplemented 1 mg. liter ^-1 both of 2- (1-naphthyl) acetic acid (NAA) and N- (phenylmethyl) -1H-purine-6-amine (BA), 0.25 mg•liter^-1 2, 4-dichlorophenoxy acetic acid (2, 4-D), 0.5 M mannitol and 20 g•liter^-1 glucose. Then C medium supplemented with 0.2 mg•liter^-1 NAA, 1 mg•liter^-1 BA, 0.1 mg•liter^-1 2, 4-D, 0.02 mg•liter^-1 gibberellic acid₃ (GA₃) and 2 mg•liter^-1 zeatin was added every 710 days. Colonies of about 2 mm in diameter were formed after 4 weeks, and transferred to E medium supplmented with 1 mgliter^-1 NAA and BA, 0.1 mg•liter^-1 2, 4-D, 1 mg•liter^-1 3- (3-indolyl propanoic acid (IPA), 0.02 mg•liter^-1 GA₃ and 2 mgliter ^-1 zeatin. Colonies developed into calli after more 1 month at 25°C, 16 hr daylength, and they were transferred to E medium supplemented with same phytohormones or 1 mg•liter^-1 BA. Regenerated shoots were excised and transferred to Murashige and Shoog (MS) medium, and most of shoots rooted. Callus was formed from protoplasts of all cultivars, but plating efficiency differed depending on the cultivars. Shoot regeneration was not achieved in the turnip cultivar 'Shogoin Ookabu' and in all radish cultivars except 'Moriguchi'.