Premium
In vitro direct plant regeneration and A grobacterium ‐mediated transformation of lucerne ( M edicago sativa L.)
Author(s) -
Kumar S.,
Tiwari R.,
Chandra A.,
Sharma A.,
Bhatnagar R. K.
Publication year - 2013
Publication title -
grass and forage science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.716
H-Index - 56
eISSN - 1365-2494
pISSN - 0142-5242
DOI - 10.1111/gfs.12009
Subject(s) - explant culture , shoot , biology , agrobacterium , hypocotyl , agrobacterium tumefaciens , transformation (genetics) , botany , meristem , murashige and skoog medium , germination , horticulture , in vitro , gene , biochemistry
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.