Tandem combination of Trigonella foenum-graecum defensin (Tfgd2) and Raphanus sativus antifungal protein (RsAFP2) generates a more potent antifungal protein.

Plant defensins are small (45 to 54 amino acids) positively charged antimicrobial peptides produced by the plant species, which can inhibit the growth of a broad range of fungi at micro-molar concentrations. These basic peptides share a common characteristic three-dimensional folding pattern with one α-helix and three ß-sheets that are stabilized by eight disulfide-linked cysteine residues. Instead of using two single-gene constructs, it is beneficial when two effective genes are made into a single fusion gene with one promoter and terminator. In this approach, we have linked two plant defensins namely Trigonella foenum-graecum defensin 2 (Tfgd2) and Raphanus sativus antifungal protein 2 (RsAFP2) genes by a linker peptide sequence (occurring in the seeds of Impatiens balsamina) and made into a single-fusion gene construct. We used pET-32a+ vector system to express Tfgd2-RsAFP2 fusion gene with hexahistidine tag in Escherichia coli BL21 (DE3) pLysS cells. Induction of these cells with 1 mM IPTG achieved expression of the fusion protein. The solubilized His6-tagged recombinant fusion protein was purified by immobilized-metal (Ni2+) affinity column chromatography. The final yield of the fusion protein was 500 ng/µL. This method produced biologically active recombinant His6-tagged fusion protein, which exhibited potent antifungal action towards the plant pathogenic fungi (Botrytis cinerea, Fusarium moniliforme, Fusarium oxysporum, Phaeoisariopsis personata and Rhizoctonia solani along with an oomycete pathogen Phytophthora parasitica var nicotianae) at lower concentrations under in vitro conditions. This strategy of combining activity of two defensin genes into a single-fusion gene will definitely be a promising utility for biotechnological applications.

Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata.

A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, ß-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of T(0) and T(1) plants. Rooted T(0) and T(1) shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting.