Transformation of the Forage Crop Sunnhemp (Crotalaria juncea L.) Through Tissue Culture Independent Method

Abstract

This chapter looked at the possibility of using a forage variety of Sunnhemp (Crotalaria juncea) for expressing the bivalent FMDV genes. Sunnhemp (Crotalaria juncea L.) belonging to the family Fabaceae is having wide range of  application in  as fibre crop, fodder crop and green manure.. Recombinant vector and bacterial hosts pCAMBIA vector 2301 derived from the pPZP vectors were used in this experiment. After standardization of transformation conditions, the coyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The neomycin phosphotransferase (nptII) gene and the bivalent 1D gene of the two main FMDV serotypes, "O" and "A22," were used as the markers for protocol optimisation. On the cotyledon-ary node, the embryo axes were randomly punctured and co-cultivated with Agrobacterium. The germ- lings were then allowed to grow under standard growth conditions in to mature fertile plants. 60 T₀ plants were established from 3 separate experiments. Three hun- dred 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 \(\mu\)g and 8 \(\mu\)g respectively (per gm of fresh leaf) of the bivalent ID antigen “O” and “A₂₂” . The transformants can be recovered by overcoming the problems of recalcitrancy to tissue culture conditions, generation of somaclonal variations etc. The method can also overcome genotype specificity barrier and thus, can be used for transformation of other Sunnhemp cultivars.