In vitro direct plant regeneration and A grobacterium ‐mediated transformation of lucerne ( M edicago sativa L.)

In vitro direct plant regeneration of lucerne was achieved by simultaneous application of thidiazuron ( TDZ ) and 6‐benzyladenine ( BA ) in Murashige and Skoog ( MS ) medium. Seedlings were germinated and grown for 6 d on growth regulator–containing MS medium. The shoot tip, consisting of the apical meristem along with parts of the cotyledonary leaves and hypocotyl, was then cultured on a medium containing the growth regulator(s). Adventitious budding of the shoot tip was promoted synergistically by treatment with TDZ and BA , and a maximum of thirty‐five shoots per explant was obtained on a medium supplemented with 2 mg L −1 TDZ and 1 mg L −1 BA . Plant regeneration frequency varied from 67 to 93%, and five Indian lucerne cultivars responded well to the regeneration protocol. The Agrobacterium ‐mediated transformation frequency from co‐cultivated explants was 13% following multiple shoot induction. Southern analysis of the T 0 plants and T 1 progenies confirmed stable inheritance of the hpt marker gene. Agrobacterium infection of the explant caused a significant reduction in the plant regeneration frequency (23%) and the number of shoots induced (11) when compared with uninfected explants. A single shoot tip provided sufficient material to regenerate and establish twenty‐seven lucerne plants, whereas only nine plants could be regenerated from an Agrobacterium co‐cultivated explant. This transformation protocol could represent a valuable improvement over existing ones for lucerne.