TY - JOUR
T1 - Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties
AU - Pradhan, Subrata
AU - Chakraborty, Anirban
AU - Sikdar, Narattam
AU - Chakraborty, Saikat
AU - Bhattacharyya, Jagannath
AU - Mitra, Joy
AU - Manna, Anulina
AU - Dutta Gupta, Snehasish
AU - Sen, Soumitra Kumar
N1 - Funding Information:
The authors extend their sincere appreciation for Partha Das and Sona Dogra for their assistance in generating the transgenic rice lines related to this study, Sudarshan Maity for maintenance and rearing of the genetic lines under outdoor cultural conditions. pX6-GFP plasmid DNA was a generous gift from Dr. N-H Chua, Rockefeller University, USA. Financial assistance from National Agricultural Innovation Project, Indian Council of Agricultural Research (NAIP/ICAR) in terms of grant support to Advanced Laboratory for Plant Genetic Engineering and fellowship to SP is thankfully acknowledged. Finally, the authors extend their sincere gratitude to the distinguished editor and the anonymous reviewers, whose incisive comments have helped immensely to improve the clarity of the manuscript.
Publisher Copyright:
© 2016, Springer-Verlag Berlin Heidelberg.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2016/10/1
Y1 - 2016/10/1
N2 - 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) fromBacillus thuringiensis.The selectable marker gene and the redundant transgene(s) were eliminated through Cre/loxmediated recombination and genetic segregation to make consumer friendlyBt-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 sativaindica 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.
AB - 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) fromBacillus thuringiensis.The selectable marker gene and the redundant transgene(s) were eliminated through Cre/loxmediated recombination and genetic segregation to make consumer friendlyBt-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 sativaindica 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.
KW - Cre/lox recombination
KW - Field assay
KW - Green-tissue specific expression
KW - Marker free transgenic rice
KW - Vegetative insecticidal protein (Vip)
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U2 - 10.1007/s00425-016-2535-1
DO - 10.1007/s00425-016-2535-1
M3 - Article
C2 - 27165311
AN - SCOPUS:84966586576
SN - 0032-0935
VL - 244
SP - 789
EP - 804
JO - Planta
JF - Planta
IS - 4
ER -