Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants

Naqvi, Rubab Zahra; Asif, Muhammad; Saeed, Muhammad; Asad, Shaheen; Khatoon, Asia; Amin, Imran; Mukhtar, Zahid; Bashir, Aftab; Mansoor, Shahid

doi:10.3389/fpls.2017.00055

Cited by 24 publications

(23 citation statements)

References 26 publications

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“…A double gene ( Cry1Ac + Cry2Ab ) construct was developed from an already available triple gene ( Cry1Ac + Cry2Ab + EPSPS ) construct (GenBank accession No. KX880509) in a pSB187 vector 2 having hygromycin selection. For ease in cotton transformation and tissue culture, the construct was re-cloned into a vector with the nptII gene for kanamycin selection.…”

Section: Methodsmentioning

confidence: 99%

“…Confirmed clone (pGA482-12R) was transformed into Agrobacterium tumefaciens strain LBA4404. Insertion into Agrobacterium was confirmed by colony PCR using Cry1Ac , Cry2Ab and nptII primer sets 2 (Table 1)…”

Section: Methodsmentioning

confidence: 99%

“…Production of cotton is liable to biotic stresses including insect pests attack as well as abiotic factors like temperature, drought and salinity. Chewing insect pests include pink bollworm ( Pectinophora gossypiella ), armyworm ( Spodoptera littoralis ), cotton bollworm ( Helicoverpa armigera ) and spotted bollworm ( Earias insulana ) 1,2 . Farmers use chemical insecticides for controlling this menace of insect pests, but most of the pesticides applied by farmers are carcinogenic and neurotoxic in nature and are harmful to humans and other beneficial organisms.…”

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests

Siddiqui

Asif

Asad

et al. 2019

Sci Rep

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Cotton is the main fiber producing crop globally, with a significant impact on the economy of Pakistan. Bt cotton expressing a Cry1Ac gene is grown over a large area in Pakistan, however, there is a major concern that bollworms may develop resistance. Here we have used a durable resistance strategy against bollworms by developing a double gene construct containing Cry1Ac and Cry2Ab (pGA482-12R) for cotton transformation. Both Cry toxin genes have been cloned in the same T-DNA borders and transferred successfully into cotton via Agrobacterium-mediated transformation. Both genes are expressed in transgenic cotton plants and is likely to help breeders in developing new cotton cultivars by incorporating these genes in cotton lines having no Bt genes or expressing Cry1Ac gene (Mon 531). Positive transgenic cotton was identified by PCR using specific primers for the amplification of both Cry1Ac and Cry2Ab genes. Cry1Ac and Cry2Ab expression was confirmed with an immunostrip test and quantified using ELISA that showed significant spatio-temporal expression of Cry2Ab ranging from 3.28 to 7.72 µg/g of the tissue leaf. Insect bioassay with army worm ( Spodoptera litura ) was performed to check the efficacy of NIBGE (National Institute for Biotechnology and Genetic Engineering) double gene transgenic cotton plants and up to 93% insect mortality was observed.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests

Siddiqui

Asif

Asad

et al. 2019

Sci Rep

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“…Pink bollworm ( Pectinophora gossypiella ) has developed resistance against single Bt gene (Cry1Ac). Insect resistant transgenic cotton expressing more than one insecticidal genes can provide durable and better control of insect pests 15 . Insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct has shown resistance against bollworms and armyworm 16 .…”

Section: Introductionmentioning

confidence: 99%

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct

Asif

Siddiqui

Naqvi

et al. 2021

Sci Rep

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Bt cotton expressing Cry1Ac is being cultivated in Pakistan. It has been observed that pink bollworm may have developed resistance against single Bt gene (Cry1Ac). For durable resistance, insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct was developed. There was a need to characterize NIBGE-1601 event for intellectual property rights protection. The Presence of NIBGE Cry1Ac and NIBGE Cry2Ab genes was checked in NIBGE-1601 cotton plants through PCR, while there was no amplification using primers specific for Monsanto events (MON531, MON15985, MON1445). Using genome walking technology, NIBGE-601 event has been characterized. Event-specific primers of NIBGE-1601 were designed and evaluated to differentiate it from other cotton events mentioned above. NIBGE-1601 event detection primers are highly specific, therefore, can detect NIBGE 1601 event at different conditions using single or multiplex PCR. In the qualitative PCR, using NIBGE-1601 event specific primers, 0.05 ng was the limit of detection for NIBGE-1601double gene cotton genomic DNA. Thus event characterization and development of event-specific diagnostics will help in breeding new cotton varieties resistant to cotton bollworms.

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“…2018). More recently, transgenic crops expressing multiple Bt and non-Bt insecticidal proteins have been developed (Naqvi et al . 2017).…”

Section: Introductionmentioning

confidence: 99%

Comparison of in vitro and in planta toxicity of Vip3A for lepidopteran herbivores

Khan

Jander²,

Mukhtar

et al. 2019

Preprint

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Agricultural pest infestation is as old as domestication of food crops and contributes a major 24 share to the cost of crop production. Transgenic production of Vip3A, an insecticidal protein 25 from Bacillus thuringiensis, effectively controls lepidopteran pests. A synthetic vip3A gene was 26 evaluated its efficacy against Spodoptera litura (cotton leafworm), Spodoptera exigua (beet 27 armyworm), Spodoptera frugiperda (fall armyworm), Helicoverpa armigera (cotton bollworm), 28 Helicoverpa zea (corn earworm), Heliothis virescens (tobacco budworm), and Manduca sexta 29 (tobacco hornworm). In artificial diet assays, the Vip3A concentration causing 50% mortality 30 was H. zea > H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. In 31 contrast, on vip3A transgenic tobacco the order of resistance (time until 50% lethality) was M. 32 sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua > S. frugiperda. There was 33 no significant correlation between the artificial diet and transgenic tobacco effects. Notably, the 34 two insect species that are best-adapted for growth on tobacco, M. sexta and H. virescens, 35 showed the greatest tolerance of vip3A-transgenic tobacco. This may indicate synergistic effects 36 of Vip3A and endogenous plant defense mechanisms, e.g. nicotine, to which M. sexta and H. 37 virescens would have greater resistance. Together, our results show that artificial diet assays are 38 a poor predictor of Vip3A efficacy in transgenic plants, lepidopteran species vary in their 39 sensitivity to Vip3A in diet-dependent manner, and that host plant adaptation of the targeted 40 herbivores should be considered when designing transgenic plants for pest control.41 42

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Development of a Triple Gene Cry1Ac-Cry2Ab-EPSPS Construct and Its Expression in Nicotiana benthamiana for Insect Resistance and Herbicide Tolerance in Plants

Cited by 24 publications

References 26 publications

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests

Development and evaluation of double gene transgenic cotton lines expressing Cry toxins for protection against chewing insect pests

Development of event-specific detection method for identification of insect resistant NIBGE-1601 cotton harboring double gene Cry1Ac-Cry2Ab construct

Comparison of in vitro and in planta toxicity of Vip3A for lepidopteran herbivores

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