In vitro direct plant regeneration and Agrobacterium-mediated transformation of lucerne (Medicago 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 6d 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 2mgL(-1) TDZ and 1mgL(-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.