Agrobacterium-Mediated Transformation with Chitinase and Glucanase genes against Aspergillus flavus in Georgia Peanuts
Disciplines
Bacteriology | Biotechnology | Integrative Biology | Pathogenic Microbiology | Plant Biology
Abstract (300 words maximum)
Aflatoxin contamination caused by Aspergillus flavus poses a major threat to global peanut (Arachis hypogaea) production and food safety. The development of genetically engineered peanut varieties with enhanced fungal resistance provides a promising strategy for mitigating aflatoxin accumulation. This project focuses on development of transgenic peanut plants via Agrobacterium-mediated transformation using chitinase and β-1,3-glucanase genes, and quantification of aflatoxin B1 (AFB1). Peanut embryos of Georgia-12Y strain were co-cultivated with A. tumefaciens using binary vectors harboring the respective genes under the control of a constitutive promoter. Transformed embryos were cultured on Murashige and Skoog (MS) media containing 3mg/L of 2,4-D for callus induction, followed by shoot and root regeneration on media supplemented with 1mg/L Benzyladenine (BA) and 1mg/L Indole-3-Butyric Acid (IBA), respectively. Untreated samples served as control and experiments were done in replicates. Experimental set up was incubated at 28°C for 14 days. Selection was performed using 25mg/L kanamycin and 250mg/L cefotaxime to identify putative transformants and suppress bacterial overgrowth. Quantification of AFB1 will be done by LC-MS.
So far, our results demonstrated successful callus induction and regeneration of shoots and roots that survived antibiotic selection, suggesting potential transgene integration. Confirmation of stable integration of the resistant genes by Polymerase Chain Reaction (PCR) and expression of these two proteins by Western Blot are in progress. Subsequently, quantification of AFB1 will indicate the extent of resistance conferred by these resistant genes. Our molecular and biochemical data will ultimately indicate the role of these PR proteins against A. flavus defense mechanisms.
This research not only advances current understanding of host–pathogen interactions in peanuts but also contributes to the development of sustainable genetic strategies to reduce aflatoxin B1 contamination in peanut production in Georgia and other peanut growing states.
Use of AI Disclaimer
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Academic department under which the project should be listed
CSM – Molecular and Cellular Biology
Primary Investigator (PI) Name
Dr. Premila Achar
Agrobacterium-Mediated Transformation with Chitinase and Glucanase genes against Aspergillus flavus in Georgia Peanuts
Aflatoxin contamination caused by Aspergillus flavus poses a major threat to global peanut (Arachis hypogaea) production and food safety. The development of genetically engineered peanut varieties with enhanced fungal resistance provides a promising strategy for mitigating aflatoxin accumulation. This project focuses on development of transgenic peanut plants via Agrobacterium-mediated transformation using chitinase and β-1,3-glucanase genes, and quantification of aflatoxin B1 (AFB1). Peanut embryos of Georgia-12Y strain were co-cultivated with A. tumefaciens using binary vectors harboring the respective genes under the control of a constitutive promoter. Transformed embryos were cultured on Murashige and Skoog (MS) media containing 3mg/L of 2,4-D for callus induction, followed by shoot and root regeneration on media supplemented with 1mg/L Benzyladenine (BA) and 1mg/L Indole-3-Butyric Acid (IBA), respectively. Untreated samples served as control and experiments were done in replicates. Experimental set up was incubated at 28°C for 14 days. Selection was performed using 25mg/L kanamycin and 250mg/L cefotaxime to identify putative transformants and suppress bacterial overgrowth. Quantification of AFB1 will be done by LC-MS.
So far, our results demonstrated successful callus induction and regeneration of shoots and roots that survived antibiotic selection, suggesting potential transgene integration. Confirmation of stable integration of the resistant genes by Polymerase Chain Reaction (PCR) and expression of these two proteins by Western Blot are in progress. Subsequently, quantification of AFB1 will indicate the extent of resistance conferred by these resistant genes. Our molecular and biochemical data will ultimately indicate the role of these PR proteins against A. flavus defense mechanisms.
This research not only advances current understanding of host–pathogen interactions in peanuts but also contributes to the development of sustainable genetic strategies to reduce aflatoxin B1 contamination in peanut production in Georgia and other peanut growing states.