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The modified cry1Ab and cry1Ac insecticidal genes of Bacillus thuringiensis (Bt) under the control of two different constitutive promoters have been introduced into chickpea (Cicer arietinum L.) by Agrobacterium-mediated transformation of pre-conditioned cotyledonary nodes. 118 stable transformed T 0 plants as independent transformation events were obtained expressing individual cry1Ab, cry1Ac or both pyramided genes for their co-expression driven by either cauliflower mosaic virus 35S promoter with duplicated enhancer (CaMV35S) or synthetic constitutive promoter (Pcec) and their combinations. Integration and inheritance of transgenes in T 0 and T 1 population of transgenic chickpea plants were determined by PCR, RT-PCR and Southern hybridization. Results of Southern hybridization showed single copy integration of cry1Ab or cry1Ac genes in most of the transgenic plants developed with either single or pyramided genes and reflected Mendelian inheritance of transgenes in T 1 progeny. Real time PCR of pyramided transgenic plants clearly showed differential expression of transcripts for both the genes driven by CaMV35S and Pcec promoters. Quantitative assessment of Bt Cry toxins by ELISA of T 0 transgenic chickpea plants showed expression of toxin ranging from 5 to 40 ng mg -1 of total soluble protein (TSP) in leaves of transgenic plants. Insect bioassay performed with transgenic plants showed relatively higher toxicity for plants expressing Cry1Ac protein as compared to Cry1Ab to Helicoverpa armigera. Pyramided transgenic plants with moderate expression levels (15-20 ng mg -1 of TSP) showed high-level of resistance and protection against pod borer larvae of H. armigera as compared to high level expression of a single toxin. These results have shown the significance of pyramiding and co-expression of two Cry toxins for efficient protection against lepidopteran pests of chickpea.

Ever since the discovery of the role of bacteriophytochrome (BphP) in inducing carotenoid synthesis in Deinococcus radiodurans in response to light the role of BphPs in other non-photosynthetic bacteria is not clear yet. Azospirillum brasilense, a non-photosynthetic rhizobacterium, harbours a pair of BphPs out of which AbBphP1 is a homolog of AtBphP1 of Agrobacterium tumefaciens. By overexpression, purification, biochemical and spectral characterization we have shown that AbBphP1 is a photochromic bacteriophytochrome. Phenotypic study of the ΔAbBphP1 mutant showed that it is required for the survival of A. brasilense on minimal medium under red light. The mutant also showed reduced chemotaxis towards dicarboxylates and increased sensitivity to the photooxidative stress. Unlike D. radiodurans, AbBphP1 was not involved in controlling carotenoid synthesis. Proteome analysis of the ΔAbBphP1 indicated that AbBphP1 is involved in inducing a cellular response that enables A. brasilense in regenerating proteins that might be damaged due to photodynamic stress.

Mature embryonic axes were used for chickpea (Cicer arietinum L.) regeneration via somatic embryogenesis. Qualitative and quantitative estimation of protein profile during somatic embryogenesis by SDS-PAGE and densitometric analysis showed differential expression of various storage proteins at different stages of somatic embryo development, which was compared with the profile of developing seeds. Total protein content in somatic embryos of chickpea increased from globular stage [2.9 μg mg -1 (f.m.)] to cotyledonary stage [4.8 μg mg -1 (f.m.)] and then started decreasing during onset of maturation and germination [up to 1.5 μg mg -1 (f.m.)]. Differential expression of seed storage proteins, late embryogenesis abundant (LEA) proteins and proteins related with stress response were documented at different stages of somatic embryogenesis. Germinating somatic embryos showed degradation products of several seed storage proteins and the appearance of new polypeptides (76.8, 67.6, 49.9 and 34.2 kDa), which were absent during differentiation of somatic embryos. A low molecular mass (17.7 kDa) polypeptide was uniformly present during all stages of somatic embryogenesis and it may belong to a group of stress-related proteins. This study describes the expression of true seed storage proteins like legumin, vicilin, convicilin and their subunits at different stages of somatic embryogenesis, which may serve as excellent markers for embryogenic pathway of regeneration in chickpea.

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.