Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

Puspito, Agung Nugroho; Rao, Abdul Qayyum; Hafeez, Muhammad Nadeem; Iqbal, Muhammad Shahzad; Bajwa, Kamran Shehzad; Ali, Qurban; Rashid, Bushra; Abbas, Muhammad; Latif, Ayesha; Shahid, Ahmad Ali; Nasir, Idrees Ahmad; Husnain, Tayyab

doi:10.3389/fpls.2015.00943

Cited by 45 publications

(45 citation statements)

References 25 publications

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“…The expression level of cry1Ac protein varied among different transgenic plants of both cultivars ( Figure 6). The variability in cry1Ac expression in transgenic crops has previously been reported (Olsen at al., 2005;Puspito et al, 2015;Anayol et al, 2016) and has been attributed to the regulatory nature of 35S promoter (Sunilkumar et al, 2012). The cry toxin amount varied among transgenic plants in T0 and T1 generations (Figures 6 and 10).…”

Section: Discussionmentioning

confidence: 99%

Development of transgenic tobacco lines with pyramided insect resistant genes

Bakhsh¹,

Dinç²,

Hussain³

et al. 2018

Turk J Biol

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Insect pests are among the major constraints rendering drastic decreases in crop yield. The expression of stacked insecticidal genes in crops can lead to resistance durability and can delay the development of resistance in target insect pests. The present study was designed to introduce an insect resistance trait in locally cultivated Turkish tobacco cultivars (Basma and Nail) with pyramided insecticidal genes. Agrobacterium strain LBA4404 harboring plasmid pKGH4 with cry1Ac and cry2A genes under the control of 35S promoter was used to infect leaf discs of both cultivars; plasmid also contained uidA within the T-DNA region for earlier screening of putative transformants. The overall transformation efficiency was calculated as 30.7% and 18.8% in Basma and Nail, respectively. PCR results confirmed the integration of cry1Ac, cry2A, uidA, and nptII genes in 40 plants of Basma and 16 plants of Nail. ELISA results showed variation in expression of cry1Ac protein among transgenic plants varying from 0.017 to 0.607 µg/g of fresh tissue. Bioassay results with potato tuber moth (Phthorimea operculella Zeller) showed significant mortality of the targeted pest on primary transformants. Furthermore, T1 transgenic progeny exhibited the inheritance of T-DNA in Mendelian as well as non-Mendelian fashion. The results revealed that lines can serve as a source of germplasm in tobacco breeding programs.

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Section: Discussionmentioning

confidence: 99%

Development of transgenic tobacco lines with pyramided insect resistant genes

Bakhsh¹,

Dinç²,

Hussain³

et al. 2018

Turk J Biol

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“…Here, in the present study we have introduced EPSPS successfully in the triple gene vector for herbicide tolerance, which will help the farmers in future for the control of weeds in cotton. A previous study (Puspito et al, 2015) has shown the efficacy of similar genes for insect and herbicide tolerance using co transformation of different vectors in cotton. Our results support the conclusion of previous studies (Awan et al, 2015; Puspito et al, 2015) and additionally provide novel insights by introducing triple gene construct in a single T-DNA.…”

Section: Discussionmentioning

confidence: 99%

“…A previous study (Puspito et al, 2015) has shown the efficacy of similar genes for insect and herbicide tolerance using co transformation of different vectors in cotton. Our results support the conclusion of previous studies (Awan et al, 2015; Puspito et al, 2015) and additionally provide novel insights by introducing triple gene construct in a single T-DNA. This triple gene construct will allow the breeders to improve their efficiency for developing homozygous lines as all the three genes being in a single T-DNA border will integrate in genome and inherit together.…”

Section: Discussionmentioning

confidence: 99%

“…Different studies have shown the development of single gene or double gene constructs for bt genes developed under 35S promoter for cotton transformation. Single gene constructs for herbicide tolerance gene are also reported to be cloned for transformation purposes (Chen et al, 2006; Bakhsh et al, 2012; Muzaffar et al, 2015; Puspito et al, 2015). …”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2017

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Insect pest complex, cotton leaf curl disease and weeds pose major threat to crop production worldwide, including Pakistan. To address these problems, in the present study a triple gene construct harboring Cry1Ac, Cry2Ab, and EPSPS cassettes has been developed for plant specifically in cotton transformation against lepidopteron insect-pests and weeds. Nicotiana benthamiana (tobacco) was used as a model system for characterization of this triple gene construct. The construct has been assembled in plant expression vector and transformed in N. benthamiana. In six transgenic tobacco lines the integration of Cry1Ac-Cry2Ab-EPSPS in tobacco genome was checked by PCR, while successful protein expression of all the three genes was confirmed through immunostrip assay. Efficacy of Cry1Ac and Cry2Ab was evaluated through insect bioassay using armyworm (Spodoptera littoralis). These transgenic tobacco plants showed significant insect mortality as compared to control plants during insect bioassay. Three out of six tested transgenic lines L3, L5, and L9 exhibited 100% mortality of armyworm, while three other lines L1, L10, and L7 showed 86, 80, and 40% mortality, respectively. This construct can readily be used with confidence to transform cotton and other crops for the development of insect resistant and herbicide tolerant transgenic plants. The transgenic crop plants developed using this triple gene construct will provide an excellent germplasm resource for the breeders to improve their efficiency in developing stable homozygous lines as all the three genes being in a single T-DNA border will inherit together.

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“…An approved local Gossypium hirsutum variety, CEMB-066, was obtained from the CEMB research repository, University of the Punjab, Pakistan, and used for all transformation experiments. Cotton seeds were transformed with Agrobacterium-mediated transformation by the shoot apex cut method as reported by [20]. An empty vector without any transgene was also transformed into the same variety as a mock transformation.…”

Section: Plant Transformation and Gfp Fluorescencementioning

confidence: 99%

Comparative analysis of Constitutive and fiber-specific promoters under the expression pattern of Expansin gene in transgenic Cotton

et al. 2020

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Promoters are specified segments of DNA that lead to the initiation of transcription of a specific gene. The designing of a gene cassette for plant transformation is significantly dependent upon the specificity of a promoter. Constitutive Cauliflower mosaic virus promoter, CaMV35S, due to its developmental role, is the most commonly used promoter in plant transformation. While Gossypium hirsutum (Gh) being fiber-specific promoter (GhSCFP) specifically activates transcription in seed coat and fiber associated genes. The Expansin genes are renowned for their versatile roles in plant growth. The overexpression of Expansin genes has been reported to enhance fiber length and fineness. Thus, in this study, a local Cotton variety was transformed with Expansin (CpEXPA1) gene, in the form of two separate cassettes, each with a different promoter, named as 35SEXPA1 and FSEXPA1 expressed under CaMV35S and GhSCFP promoters respectively. Integration and Spatiotemporal relative expression of the transgene were studied in an advanced generation. GhSCFP bearing transgene expression was significantly higher in Cotton fiber than other plant parts. While transgene with CaMV35S promoter was found to be continually expressing in all tissues but the expression was lower in fiber than that expressed under GhSCFP. The temporal expression profile was quite interesting with a gradual increasing pattern of both constructs from 1DPA (days post anthesis) to 18DPA and decreased expression from 24 to 30 DPA. Besides the relative expression of promoters, fiber cellulose quantification and fluorescence intensity were also observed. The study significantly compared the two most commonly used promoters and it is deduced from the results that the GhSCFP promoter could be used more efficiently in fiber when compared with CaMV35S which being constitutive in nature preferred for expression in all parts of the plant.

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Transformation and Evaluation of Cry1Ac+Cry2A and GTGene in Gossypium hirsutum L.

Cited by 45 publications

References 25 publications

Development of transgenic tobacco lines with pyramided insect resistant genes

Development of transgenic tobacco lines with pyramided insect resistant genes

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

Comparative analysis of Constitutive and fiber-specific promoters under the expression pattern of Expansin gene in transgenic Cotton

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