Improved shoot regeneration protocol for hairy roots of the legume Astragalus sinicus

Abstract

An improved protocol for shoot regeneration from hairy roots transformed by Agrobacterium rhizogenes of the legume species Astragalus sinicus (Chinese milk vetch) has been developed. The A. rhizogenes strain DC-AR2 harboring the binary vector pBI121 which carries the uidAgene encoding β-glucuronidase activity and the kanamycin resistance gene nptII, was used to transform cut ends of plantlet hypocotyls. Transformed hairy roots were selected on medium containing 75 μg ml⁻¹ kanamycin, and transformation was monitored by detection of the opine mikimopine, histochemical β-glucuronidase activity, the polymerase chain reaction, and Southern blot analysis. The cytokinins benzylaminopurine, kinetin, and thidiazuron suppressed the growth of 8-month and 3-year-old hairy roots, but were necessary for adventitious shoot formation that could occur with some lines. Putative somatic embryos developed from transformed roots on medium with 7.5-10.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. Light did not affect shoot regeneration from transformed hairy roots. This transformation and shoot regeneration system should be useful for testing gene expression quickly and be amenable to studies of shoot morphogenesis and interactions with rol gene expression.