Agrobacterium rhizogenes-mediated transformation and regeneration of the legume Astragalus sinicus (Chinese milk vetch)

Abstract

A system for the Agrobacterium rhizogenes-mediated transformation and root and then shoot regeneration of the legume species Astragalus sinicus (Chinese milk vetch) has been developed . A. rhizogenes DC-AR2 strain harboring the binary vector pBI121, carrying the uidA gene encoding GUS activity, was used to transform seedlings in wound sites. Transformation was monitored by detection of mikimopine and histochemical β-glucuronidase (GUS) activity. GUS expression was found in approximately 44% of the seedlings 15 days after infection and 46% of the aerial roots tested were GUS positive while all of the GUS positive roots also contained mikimopine, which is a characteristic of strain DC-AR2. When the transformed roots were cultured in vitro, sporadic shoot development occurred after culture periods ranging from 1 to 2 months and transgenic A. sinicus plantlets were established. The transformation of the regenerated plantlets was further confirmed by histochemical GUS activity and by Southern blot analysis. The resulting plants exhibited the ‘Ri plasmid’ syndrome such as small thin leaves and short internodes while cultured roots grew rapidly and were plagiotropic. This transformation and plant regeneration system will be useful as a model system for testing the expression of genes and their effect on a leguminous plant.