Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

Assessing the efficacy of aqueous garlic extract, sodium nitrite and sodium thiosulfate against prolonged oral cyanide exposure in rabbits.

This study was aimed to compare the efficacy of aqueous garlic extract, sodium nitrite (SNT), sodium thiosulfate (STS) and hydroxocobalamin against oral cyanide exposure in rabbits. For this purpose, forty two adult male rabbits were divided randomly into 7 groups of 6 animals (A-G) each. Rabbits in group A were offered feed only and served as negative control, while the rabbits in group B received feed plus potassium cyanide (KCN) at 3mg/kg orally and were kept as positive control. Animals in group C received feed, KCN and intraperitoneal injection (IP) of aqueous garlic extract at 500mg/kg. Rabbits in group D were given feed, KCN and IP injection of STS at 600mg/kg. Members in group E received feed, KCN and IP injection of both aqueous garlic extract at 500mg/kg and SNT at 20mg/kg. Animals in group F were given feed, KCN and IP injection of both STS at 600mg/kg and SNT at 20mg/kg, while the rabbits in group G received feed, KCN and IP injection of hydroxocobalamin at 300mg/kg. The treatments were given to respective groups for 40 days. The efficacy of the antidotes was measured on the basis of changes in biochemical profile of rabbits in each group. In this study, hydroxocobalamin was found to be significantly more effective cyanide (CNI) antidote than garlic, STS, SNT plus garlic extract, or SNT and STS, either alone or in combination. A combination of SNT and garlic extract was the second most effective CNI antidote. The efficacy of garlic alone was significantly higher than STS alone or in combination with SNT. The efficacy of combined SNT and STS was superior to STS alone in treating rabbits with CNI toxicity. In conclusion, aqueous garlic extract alone or in combination with STS can effectively be used against cyanide toxicity.

Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant.

The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to 'dead heart' contains no toxins in cane juice.

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.