CRISPR/CAS 9 mediated mutagenesis of the eukaryotic translation initiation factor gene of Nicotiana tabacum for the induction of Potato virus Y tolerance

Author

Nhidza, Tanaka.

Title

CRISPR/CAS 9 mediated mutagenesis of the eukaryotic translation initiation factor gene of Nicotiana tabacum for the induction of Potato virus Y tolerance

Abstract

One of the most significant viral infections affecting tobacco in Zimbabwe is the Potato Virus Y (PVY.). Currently, the most common method for controlling PVY is through the use of pesticides, which can have harmful effects on the environment and human health. By increasing resistance in plants, the need for pesticides could be reduced, leading to a more sustainable and eco-friendly approach to managing PVY. This study aimed at producing a PVY resistant tobacco genotype from a line ONC via Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR) mediated gene editing of the eukaryotic translation-initiation factors (eIF4E). The research utilized databases and web tools in the form of NCBI and CRISPOR to design the guide RNA (gRNA) DNA template. The gRNA DNA template was ligated into the pChimera plasmid and cloned in chemically competent E.coli. The gRNA insert was transferred as a construct into the binary vector pCas9-TPC and cloned in Agrobacterium tumefaciens for leaf disc transformation. The obtained construct was transformed into tobacco variety ONC leaf discs which developed into plantlets in vitro. The plantlets multiplied and after hardening were transferred to the greenhouse in a fumigated vermiculite/pine bark media. The plants displayed vigorous leaf development after 5 days with an increase in size. The Agrobacterium was also utilised in agroinfiltration of potted ONC plantlets and the genomic DNA of edited and wild type ONC plant was extracted and sequenced using the Applied BiosystemsR Genetic analyzer and alignment of the sequences showed a single nucleotide deletion at the 3’end. These sequences were used to generate a phylogenetic tree which illustrated the evolutionary relationships between different tobacco samples based on the sequencing of the eIF4e region. The close relationship between ONC Transformed and ONC Wild Type implies that the modification in the ONC Transformed sample altered the eIF4e region to some extent. It can be concluded that CRISPR/Cas9 technology can be utilised to mediate mutagenicity in the plant genome to induce PVY resistance.

Date

June 2024

Publisher

BUSE

Keywords

CRISPR,
 

PVY,

eIF4,

Nicotiana tabacum (Tobacco),

Agrobacterium.

Supervisor

Dr. G. Zvobgo,