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Main Conclusion: Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Syn vip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Syn vip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (syn bar) gene. Marker gene excision was achieved in hybrids with up to 41.18 % recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII + cre-syn bar), were established in subsequent generation through genetic segregation.

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61 % mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

Main Conclusion: Ectopic expression of a deletion mutant ( ndv200 ) of Bacillus thuringiensis vip3BR gene in tobacco plant provided almost complete protection against major crop pests cotton boll worm ( Helicoverpa armigera ), black cut worm ( Agrotis ipsilon ) and cotton leaf worm ( Spodoptera littoralis ). Whereas vip3BR transgenic tobacco plant failed to protect themselves from these insects and showed resistance towards cotton leaf worm only. An analogous form of the Bacillus thuringiensis vip3Aa insecticidal toxin gene, named vip3BR, was identified and characterized, and exhibited similar attributes to the well-known Vip3Aa toxin. Vip3BR possessed broad-spectrum lepidopteran-specific insecticidal properties effective against most major crop pests of the Indian subcontinent. A Vip3BR toxin protein N-terminal deletion mutant, Ndv200, showed increased insecticidal potency relative to the native toxin, which conferred efficacy against four major crop pests, including cotton boll worm (Helicoverpa armigera), black cut worm (Agrotis ipsilon), cotton leaf worm (Spodoptera littoralis), and rice yellow stem borer (Scirpophaga incertulas). Ligand blot analysis indicated the Ndv200 toxin recognized the same larval midgut receptors as the native Vip3BR toxin, but differed from receptors recognized by Cry1A toxins. In the present study, we tested the prospect of the vip3BR and ndv200 toxin gene as candidate in development of insect-resistant genetically engineered crop plants by generating transgenic tobacco plant. The study revealed that the ndv200 mutant of vip3BR insecticidal toxin gene is a strong and prospective candidate for the next generation of genetically modified crop plants resistant to lepidopteran insects.

Issues relating to sustenance of the usefulness of genetically modified first generation Bt crop plants in the farmer's field are of great concern for crop scientists. Additional biotechnological strategies need to be in place to safeguard the possibility for yield loss of Bt crop by other lepidopteran insects that are insensitive to the Cry1A toxin, and also against the possibility for emergence of resistant insects. In this respect, Cry2A toxin has figured as a prospective candidate to be the second toxin to offer the required protection along with Cry1A. In the present study, the entomocidal potency of Cry2A toxin was enhanced through knowledge-based protein engineering of the toxin molecule. Deletion of 42 amino acid residues from the N-terminal end of the peptide followed by the replacement of Lys residues by nonpolar amino acids in the putative transmembrane region including the introduction of Pro resulted in a 4.1-6.6-fold increase in the toxicity of the peptide against three major lepidopteran insect pests of crop plants.

Generation of insect-resistant, transgenic crop plants by expression of the insecticidal crystal protein (ICP) gene of Bacillus thuringiensis (Bt) is a standard crop improvement approach. In such cases, adequate expression of the most appropriate ICP against the target insect pest of the crop species is desirable. It is also considered advantageous to generate Bt-transgenics with multiple toxin systems to control rapid development of pest resistance to the ICP. Larvae of yellow stem borer (YSB), Scirpophaga incertulas, a major lepidopteran insect pest of rice, cause massive losses of rice yield. Studies on insect feeding and on the binding properties of ICP to brush border membrane receptors in the midgut of YSB larvae revealed that cryIAb and cryIAc are two individually suitable candidate genes for developing YSB-resistant rice. Programs were undertaken to develop Bt-transgenic rice with these ICP genes independently in a single cultivar. A cryIAc gene was reconstructed and placed under control of the maize ubiquitin 1 promoter, along with the first intron of the maize ubiquitin 1 gene, and the nos terminator. The gene construct was delivered to embryogenic calli of IR64, an elite indica rice cultivar, using the particle bombardment method. Six highly expressive independent transgenic ICP lines were identified. Molecular analyses and insect-feeding assays of two such lines revealed that the transferred synthetic cryIAc gene was expressed stably in the T2 generation of these lines and that the transgenic rice plants were highly toxic to YSB larvae and lessened the damage caused by their feeding.

Necessity for the production of transgenic crop plants of India, capable of expression of insecticidal Bt protein in plant to combat lepidopteran pest damage has been strongly felt. Bacillus thuringiensis Kurstaki HD73 crystal protein coded by CryIA(c)73 gene has been found to be sufficiently effective against the major pests of jute and chickpea. An attempt to isolate the gene and make its use in a chimaeric gene construct for expression in plant was carried out. The plasmid CryIA(c)73 gene was cloned and tailored at the 3' end. The expression of the truncated gene was monitored in the minicell systems of E. coli. The entomocidal property was found to be fully retained by the gene product. Deletion of the nucleotides at the 5' end was carried out and chimaeric gene construct of cryIA(c)73 was made in such a way that it was fused in frame with GUS gene under the control of the caMV 35S promoter with Nos polyadenylated terminus. Such a chimaeric gene construct was used as the passenger of a Ti plasmid derived plant vector with kanamycin gene (NPTII) as the additional plant marker. Transformation through infection of tobacco and mustard plant cells in culture was carried out. Plants regenerated from the transformed cells showed the presence of GUS gene indicating the expression of the cloned fused gene. Also, Northern analysis established the presence of cryIA(c)73 gene transcripts in the transgenic plants.