title: Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume Astragalus melilotoides Pall.
abstract: An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74 +/- 0.6x10(5)/g FW) and viability (87.07 +/- 2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3-7 days following culture initiation. The highest division frequency (9.86 +/- 0.68%) and plating efficiency (1.68 +/- 0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l alpha-naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3 +/- 4.1%) and organogenesis (21.6 +/- 0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.
authors.affiliation: School of Life Science, Lanzhou University, 730000 Lanzhou, P.R. China. housw@lzu.edu.cn
authors.forename: S W
authors.initials: SW
authors.lastname: Hou
authors.forename: J F
authors.initials: JF
authors.lastname: Jia
mesh_terms: D027883:Astragalus Plant / Q000196:embryology*; D046508:Culture Techniques / Q000379:methods*; D011523:Protoplasts / Q000166:cytology / Q000502:physiology*; D012038:Regeneration; D012639:Seeds / Q000196:embryology / Q000254:growth & development