Tissue culture independent transformation of the forage crop sunnhemp (Crotalaria juncea L.): an easy method towards generation of transgenics.

A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes 'O' and 'A22' and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D "O" and "A22" and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 µg and 8 µg (per gm of fresh leaf) of the bivalent ID antigen "O" and "A22" . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp.