(The results of the molecular analysis is summarized in Table1) It is still preferred to use host strains like EHA105 with higher copy number ofVir Ggene (Meurer et al.1998) which may help in achieving increased efficiency. == Fig. to mature fertile plants. 60 T0plants were established from 3 individual experiments. Three hundred seeds from the 60 T0plants were sown to raise the T1generation 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 impartial transgenic lines 24 and 37, showed elevated levels of expression of 12 g and 8 g (per gm of fresh leaf) of the bivalent ID antigen O and A22 . The results showed that this transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt ofAgrobacterium tumefaciensmediated transformation of Sunnhemp. The protocol can generate whole herb transformants with relative ease and should be compatible to all genotypes of Sunnhemp. Keywords:Sunnhemp,Agrobacterium tumefaciens, FMDV -1D gene, nptII gene markers,in plantatransformation == Introduction == Till recently legumes have been branded as recalcitrant for transformation, as regenerationin vitrois highly genotype specific and requires a lot of skill. Besides the frequency of transformants generated was very low to make any significant impact. Thus the need of the hour is usually to develop techniques which used the totipotent cells of the legumes to generate transformants. The most popular and Onalespib (AT13387) user friendly amongst the recent techniques is the inplanta transformation byAgrobacterium tumefaciens. A number of legumes (Trieu et al.2000; Olhoft et al.2001etc.) have been transformed by this procedure. These developments have further enhanced the importance of forage legumes for exploitation in development of edible vaccines for immunization against animal diseases like Rinderpest disease (Prasad et al.2004), foot and mouth disease (Dus Santos et al.2005,2002) as these crops are generally used as protein rich fodder. Crotalaria junceacommonly known as Sunnhemp belonging to thepapilionaceaefamily originated in India (Purseglove1981). Species of this genus are wide spread throughout tropical, subtropical Onalespib (AT13387) and to smaller extent temperate countries. It is used as forage for cattle owing to the large amounts of water soluble gums and proteins in their seeds (Pandey and Srivastava1990). Although there are few reports of regenerationin vitroearlier, (Ohara et al.2000) the viability of transgenics of this plant has not been exploited as it is a legume. In the present study, we have looked at the possibility of using a forage variety of Sunnhemp (Crotalaria juncea) for expressing the bivalent FMDV genes. The FMDV viral capsid consists of 60 copies of 4 structural proteins termed as VP1 (1D), VP2 (1B), VP3 (1C) and VP4 (1A). The immune-dominant epitopes are located around the 1D with in the G-H loop (140-160aa) and C terminal end (205-210aa) (Meloen and Barteling1986). Thus ID gene has been exploited as the ideal candidate for development of edible vaccines (Carrillo et al.1998) for foot and mouth disease (FMD). FMDV exists as 7 antigenically distinct serotypes (O, A, C, Asia 1 and SAT 13) with no cross Onalespib (AT13387) reactivity among them (Ogra et al.2001). In India 3 of them (O, Rabbit Polyclonal to SPI1 A, Asia1) are in circulation and 2 of these (O and A) are responsible for large number of outbreaks. Hence vaccines against these are necessary to avoid major economic losses. With this objective in mind we have selected 1D gene of FMDV serotypes O and A in the present study. == Materials and methods == == Recombinant vector and bacterial hosts == pCAMBIA vector 2301 derived from the pPZP vectors (Hajdukiewick et al.1994) were used in this experiment. The generally employed GUS reporter gene is not ideal for legume transformation as the legumes show an endogenous GUS like activity. For this reason, we have used another gene construct namely, the bivalent 1D gene of the two major FMDV serotypes O and A22 as one of the markers. (The detailed.