Wajeeha Khan
Ayub Agricultural Research Institute, Pakistan
Abstract Title:
CRISPR/Cas9-Mediated targeted editing and molecular validation of StMAPK19 in potato (solanum tuberosum L.) for functional analysis of salt stress signaling
Biography:
Wajeeha Khan is a Scientific Officer at the Vegetable Research Institute, Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan, and serves as the Project Manager of a tissue culture research project. Her research focuses on vegetable breeding, genetics, hybrid seed production, and the application of molecular breeding approaches for developing climate-resilient and high-yielding vegetable cultivars. She actively engages in international research collaborations and is committed to advancing sustainable agriculture through scientific innovation and crop improvement.
Research Interests:
Salinity stress poses a significant constraint to sustainable potato seed production, necessitating the development of molecular tools for precision trait improvement. Mitogen-Activated Protein Kinases (MAPKs) play central roles in abiotic stress signal transduction; however, functional validation of specific MAPK members in potato remains limited. This study aimed to establish and molecularly validate genome-edited lines targeting StMAPK19 using CRISPR/Cas9 technology.
The StMAPK19 gene was identified through bioinformatic analysis, and target-specific guide RNAs were designed using in silico tools to ensure high specificity and minimal off-target effects. Primers and target sites were computationally validated prior to construct development. CRISPR/Cas9 knockout vectors were assembled for loss-of-function analysis, while parallel overexpression constructs were designed to facilitate gain-of-function studies.
Recombinant plasmids were introduced into Agrobacterium tumefaciens, followed by Agrobacterium-mediated transformation of potato explants and subsequent tissue culture-based regeneration. Putative transformants were screened, and molecular validation was performed through RNA extraction, cDNA synthesis, and RT-PCR analysis to confirm transgene integration and expression.
To verify genome editing events, next-generation sequencing data (FASTQ/BAM files) from transformed lines were analyzed using Integrative Genomics Viewer (IGV). Clear insertion–deletion (indel) mutations were detected at the targeted StMAPK19 locus, confirming successful CRISPR-mediated mutagenesis. Stable edited lines were identified, establishing a validated platform for further functional characterization of MAPK-mediated salt stress responses.
This work demonstrates the successful implementation of a CRISPR/Cas9-based genome editing pipeline in potato and provides foundational resources for integrating precision molecular breeding approaches into seed potato improvement programs.
