Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds ... more Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds on more than 400 plant species and transmits more than 100 plant viruses. Aphid infestation is mostly managed by insecticides that cause heavy environmental contamination and insect resistance. Cloning of plant derived insecticidal genes to develop transgenic plants under suitable promoter is a promising technology. In the present study, ASAL (MN820725) was isolated from native garlic and cloned in plant transformation vector, pGA482 through Agrobacterium mediated tobacco transformation. PCR of genomic DNA of transgenic tobacco plants using gene specific primers confirmed the presence of asal gene of 546 bp. To detect the integration of gene Southern blot analysis was conducted that revealed stable integration of asal gene while, gene expression was analyzed through qRT-PCR that showed variable expression of asal gene in transgenic tobacco plants. Efficacy of asal gene was evaluated thro...
Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that as... more Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S -adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibiti...
International Journal of Agriculture and Biology, 2010
... M. TEHSEEN AZHAR 1, MEHBOOB-UR-REHMAN, SHAHEEN AFTAB, YUSUF ZAFAR AND SHAHID MANSOOR 1 ... Ma... more ... M. TEHSEEN AZHAR 1, MEHBOOB-UR-REHMAN, SHAHEEN AFTAB, YUSUF ZAFAR AND SHAHID MANSOOR 1 ... Maximum number of tolerant plant against CLCuD was observed in the families of NIBGE-115 × transgenic Coker-312 expressing antisense rep, whilst minimum ...
The present study was carried out to assess the effect of growth regulators in three different co... more The present study was carried out to assess the effect of growth regulators in three different combinations on mass propagation of currently grown three potato cultivars cv. Desiree, Kuroda and Cardinal. Varietal response on In vitro regeneration under different hormonal combinations. For callus induction, internodes of potato cultivars were cultured on modified Murashige & Skoog (MS) medium, added with different growth hormonal combinations. Callusing frequency for all these treatments and cultivars were recorded and in callus induction medium (CIM1) explants showed significantly higher callus formation as compared to two other combinations. For shoot induction, calli were cultured on Murashige & Skoog (MS) modified medium, supplemented with different hormonal combinations. Shoot induction medium (SIM3) gave best shoot induction frequency as compared to other media combinations. On the same media, an average number of shoots per explant were obtained for cultivar Desiree which is s...
Abstract Genetic transformation was carried out by using biolistic gun method. The hypocotyl deri... more Abstract Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) ...
Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and p... more Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1 mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.
Agricultural pest infestation is as old as domestication of food crops and contributes a major sh... more Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. Transgenic production of Vip3A, an insecticidal protein from Bacillus thuringiensis, effectively controls lepidopteran pests. A synthetic vip3A gene was evaluated its efficacy against Spodoptera litura (cotton leafworm), Spodoptera exigua (beet armyworm), Spodoptera frugiperda (fall armyworm), Helicoverpa armigera (cotton bollworm), Helicoverpa zea (corn earworm), Heliothis virescens (tobacco budworm), and Manduca sexta (tobacco hornworm). In artificial diet assays, the Vip3A concentration causing 50% mortality was H. zea > H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. In contrast, on vip3A transgenic tobacco the order of resistance (time until 50% lethality) was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua > S. frugiperda. There was no significant correlatio...
Methods in molecular biology (Clifton, N.J.), 2019
Rapid growth in the genetic transformation of plants is the outcome of versatile transformation m... more Rapid growth in the genetic transformation of plants is the outcome of versatile transformation methods, explant nature, and media regimes. Modern biotechnologists have now a toolkit embraced with different plant transformation methods to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information are created by proper custom synthesis/amplification of DNA sequences from natural sources, modification during gene cloning, and choice of regulatory sequences and delivered to plants via different plant transformation techniques. Cotton is known by different names like king of fiber crops, white gold, etc., due to its socioeconomic involvement in society livelihood. So cotton is the host of several transgenes delivered for the purpose of trait development of improvement outcompeting its wild counterparts. At present most of the cotton adopted by farmers is biotech and contributes significantly in meeting farmers and industry demands. I...
Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme w... more Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.
RNA interference (RNAi) technology has been successfully applied in stacking resistance against v... more RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.
The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cel... more The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed...
Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds ... more Globally, aphid, Myzus persicae is an economically significant, polyphagous crop pest that feeds on more than 400 plant species and transmits more than 100 plant viruses. Aphid infestation is mostly managed by insecticides that cause heavy environmental contamination and insect resistance. Cloning of plant derived insecticidal genes to develop transgenic plants under suitable promoter is a promising technology. In the present study, ASAL (MN820725) was isolated from native garlic and cloned in plant transformation vector, pGA482 through Agrobacterium mediated tobacco transformation. PCR of genomic DNA of transgenic tobacco plants using gene specific primers confirmed the presence of asal gene of 546 bp. To detect the integration of gene Southern blot analysis was conducted that revealed stable integration of asal gene while, gene expression was analyzed through qRT-PCR that showed variable expression of asal gene in transgenic tobacco plants. Efficacy of asal gene was evaluated thro...
Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that as... more Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S -adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibiti...
International Journal of Agriculture and Biology, 2010
... M. TEHSEEN AZHAR 1, MEHBOOB-UR-REHMAN, SHAHEEN AFTAB, YUSUF ZAFAR AND SHAHID MANSOOR 1 ... Ma... more ... M. TEHSEEN AZHAR 1, MEHBOOB-UR-REHMAN, SHAHEEN AFTAB, YUSUF ZAFAR AND SHAHID MANSOOR 1 ... Maximum number of tolerant plant against CLCuD was observed in the families of NIBGE-115 × transgenic Coker-312 expressing antisense rep, whilst minimum ...
The present study was carried out to assess the effect of growth regulators in three different co... more The present study was carried out to assess the effect of growth regulators in three different combinations on mass propagation of currently grown three potato cultivars cv. Desiree, Kuroda and Cardinal. Varietal response on In vitro regeneration under different hormonal combinations. For callus induction, internodes of potato cultivars were cultured on modified Murashige & Skoog (MS) medium, added with different growth hormonal combinations. Callusing frequency for all these treatments and cultivars were recorded and in callus induction medium (CIM1) explants showed significantly higher callus formation as compared to two other combinations. For shoot induction, calli were cultured on Murashige & Skoog (MS) modified medium, supplemented with different hormonal combinations. Shoot induction medium (SIM3) gave best shoot induction frequency as compared to other media combinations. On the same media, an average number of shoots per explant were obtained for cultivar Desiree which is s...
Abstract Genetic transformation was carried out by using biolistic gun method. The hypocotyl deri... more Abstract Genetic transformation was carried out by using biolistic gun method. The hypocotyl derived embryogenic calli (explants) of cotton (Gossypium hirsutum L.) cv. Cocker-312 were transformed with a recombinant pGreen II plasmid, in which both, bar (selection marker) ...
Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and p... more Biosafety is a multidisciplinary approach that encompasses social, societal, ethical issues and policies for the regulations of genetically modified (GM) organisms. The potential health risks associated with GM sugarcane containing AVP1 gene confers resistance against drought and salinity were evaluated by animal feeding studies and some genotoxicity assays. Acute and sub-chronic toxicity examinations were carried out via oral dose administration of GM sugarcane juice supplemented with the normal diet (modified from certified rodent standard diet) on Wistar rats. AVP1 protein concentration in sugarcane juice was 1mg/1 mL. Biochemical, haematological blood analyses were performed and the results revealed that there were non-significant differences among all the treatment groups; GM sugarcane juice, non-GM sugarcane juice and the control group (normal diet and water). Genotoxicity assessment based on the comet assay and the micronucleus assay data exhibited that AVP1 GM sugarcane was not genotoxic or cytotoxic in rat's peripheral blood. These research findings supported the conclusion that GM AVP1 sugarcane was non-toxic in experimental animals. Therefore, data generated through this research work would be helpful for the commercial release of GM AVP1 sugarcane.
Agricultural pest infestation is as old as domestication of food crops and contributes a major sh... more Agricultural pest infestation is as old as domestication of food crops and contributes a major share to the cost of crop production. Transgenic production of Vip3A, an insecticidal protein from Bacillus thuringiensis, effectively controls lepidopteran pests. A synthetic vip3A gene was evaluated its efficacy against Spodoptera litura (cotton leafworm), Spodoptera exigua (beet armyworm), Spodoptera frugiperda (fall armyworm), Helicoverpa armigera (cotton bollworm), Helicoverpa zea (corn earworm), Heliothis virescens (tobacco budworm), and Manduca sexta (tobacco hornworm). In artificial diet assays, the Vip3A concentration causing 50% mortality was H. zea > H. virescens > S. exigua > H. armigera > M. sexta > S. frugiperda > S. litura. In contrast, on vip3A transgenic tobacco the order of resistance (time until 50% lethality) was M. sexta > H. virescens > S. litura > H. zea > H. armigera > S. exigua > S. frugiperda. There was no significant correlatio...
Methods in molecular biology (Clifton, N.J.), 2019
Rapid growth in the genetic transformation of plants is the outcome of versatile transformation m... more Rapid growth in the genetic transformation of plants is the outcome of versatile transformation methods, explant nature, and media regimes. Modern biotechnologists have now a toolkit embraced with different plant transformation methods to generate specific and targeted genetic variation for performance improvement of crop plants. Genetic information are created by proper custom synthesis/amplification of DNA sequences from natural sources, modification during gene cloning, and choice of regulatory sequences and delivered to plants via different plant transformation techniques. Cotton is known by different names like king of fiber crops, white gold, etc., due to its socioeconomic involvement in society livelihood. So cotton is the host of several transgenes delivered for the purpose of trait development of improvement outcompeting its wild counterparts. At present most of the cotton adopted by farmers is biotech and contributes significantly in meeting farmers and industry demands. I...
Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme w... more Glyphosate quashes the synthesis of 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) enzyme which intercedes the functioning of shikimate pathway for the production of aromatic amino acids. Herbicide resistant crops are developed using glyphosate insensitive EPSPS gene isolated from Agrobacterium sp. strain CP4, which give farmers a sustainable weed control option. Intentions behind this study were to design and characterize the synthetic herbicide resistant CP4-EPSPS gene in a model plant system and check the effectiveness of transformed tobacco against application of glyphosate. Putative transgenic plants were obtained from independent transformation events, and stable plant transformation, transgene expression and integration were demonstrated respectively by PCR, qRT-PCR and Southern hybridization. Gene transcript level and gene copy number (1-4) varied among the tested transgenic tobacco lines. Herbicide assays showed that transgenic plants were resistant to glyphosate after 12 days of spraying with glyphosate, and EPSPS activity remained at sufficient level to withstand the spray at 1000 ppm of the chemical. T1 plants analyzed through immunoblot strips and PCR showed that the gene was being translated into protein and transmitted to the next generation successfully. This codon optimized synthetic CP4-EPSPS gene is functionally equivalent to the gene for glyphosate resistance available in the commercial crops and hence we recommend this gene for transformation into commercial crops.
RNA interference (RNAi) technology has been successfully applied in stacking resistance against v... more RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.
The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cel... more The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed...
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