Fig. 1 In planta genetic transformation of Jatropha. Germinating seeds were infected with Agrobacterium tumefaciens strain GV 3101 harboring plant expression vector pRI101-AN carrying selectable marker gene NPT II. The infected seeds were transferred to sterile sand medium filled in pots and grown in laboratory (Fig. 1a). Further, confirmed transgenic plants are being grown in containment (Fig. 1b).

a b

Fig. 2 Structure prediction of curcin precursor gene.

FGENESH 2.6 tool Number of predicted genes 1: in +chain Number of predicted exons 1: in +chain Score: 30.280566

Feature Start End Score ORF Len CDS 11 940 36.15 11 – 940 930 Poly A 973 1.06

Predicted mRNA sequence encoded by one exon (11 – 940); 930 bp, chain +

Fig. 3 Motif scanning of the predicted amino acid sequence using MyHits program

Fig. 3 Motif scanning of the predicted amino acid sequence using MyHits program

Fig. 4 Sequence of the custom designed precursor silencing fragment (860 nt) cloned in the binary vector (pRI101-AN). Sequence in sense (334 nt; red font color) and antisense (334 nt; blue font color) orientation is separated by intronic sequence (192 nt; black font color).

TAAACCTCTAGTTGGTTTTATCGAAATGGTTCCAGAGGCAGCAAGATTCAAATATATTGAGAAAAAAGTATTAACTCAAATTAGCAAAACCTTTAGGCCGCGTGGTGACATAATTAGCCTTGAGAACAACTGGGGAGACCTCTCTTATCAAATACAGAAATCTGTAAATGGTGTATTTCTGAAGCCAGTTCAATTGCAACGTGAAAACTATACCAATATCCTAGTGAACAATGTCACCCAAGTAACAGGTCTCATGGGAGTCTTGTTGAATGCAGTCAATTACAAAGTCTCAATGGAAGAAATTATTTTCAACTACCAAAAGTGGCTGCCATGGCTTAAGTTTCTGCTTCTACCTTTGATATATATATAATAATTATCATTAATTAGTAGTAATATAATATTTCAAATATTTTTTTCAAAATAAAAGAATGTAGTATATAGCAATTGCTTTTCTGTAGTTTATAAGTGTGTATATTTTAATTTATAACTTTTCTAATATATGACCAAAATTTGTTGATGTGCAGAGCCATGGCAGCCACTTTTGGTAGTTGAAAATAATTTCTTCCATTGAGACTTTGTAATTGACTGCATTCAACAAGACTCCCATGAGACCTGTTACTTGGGTGACATTGTTCACTAGGATATTGGTATAGTTTTCACGTTGCAATTGAACTGGCTTCAGAAATACACCATTTACAGATTTCTGTATTTGATAAGAGAGGTCTCCCCAGTTGTTCTCAAGGCTAATTATGTCACCACGCGGCCTAAAGGTTTTGCTAATTTGAGTTAATACTTTTTTCTCAATATATTTGAATCTTGCTGCCTCTGGAACCATTTCGATAAAACCAACTAGAGGTTTA

Fig. 5 Result for minimum free energy (-650.20 Kcal/mol) structure of the curcin precursor gene silencing cassette

Fig. 6 Mountain plot representation of the MFE structure, the thermodynamic ensemble of RNA structures, and the centroid structure of the curcin precursor gene silencing cassette. Additionally the positional entropy for each position is presented.