title: Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.
abstract: 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.
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India. subratapradhan.bio@gmail.com.
authors.forename: Subrata
authors.initials: S
authors.lastname: Pradhan
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Anirban
authors.initials: A
authors.lastname: Chakraborty
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Narattam
authors.initials: N
authors.lastname: Sikdar
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Saikat
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Jagannath
authors.initials: J
authors.lastname: Bhattacharyya
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Joy
authors.initials: J
authors.lastname: Mitra
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Anulina
authors.initials: A
authors.lastname: Manna
authors.affiliation: Department of Agricultural and Food Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Snehasish
authors.initials: S
authors.lastname: Dutta Gupta
authors.affiliation: Advanced Laboratory for Plant Genetic Engineering, Indian Institute of Technology Kharagpur, Kharagpur, 721302, India.
authors.forename: Soumitra Kumar
authors.initials: SK
authors.lastname: Sen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015153:Blotting, Western; D005786:Gene Expression Regulation; D006790:Host-Parasite Interactions; D007300:Insect Control / Q000379:methods; D007313:Insecta / Q000502:physiology; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides / Q000378:metabolism; D012275:Oryza / Q000235:genetics* / Q000469:parasitology; D010935:Plant Diseases / Q000235:genetics* / Q000469:parasitology; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D007306:Insecticides; C447139:Vip3A protein, Bacillus thuringiensis; D012273:Ribulose-Bisphosphate Carboxylase
keywords: Cre/lox recombination; Field assay; Green-tissue specific expression; Marker free transgenic rice; Vegetative insecticidal protein (Vip)