ICAR - National Institute for Research on Commercial Agriculture

Collaborating Centre Principal Investigator (CC-PI)

Dr. K Prabhakara Rao

Senior Scientist

Principal Investigator involved in the project

Dr. K Prabhakara Rao

Senior Scientist

Co-Principal Investigators (Co-PIs)

Dr. K. Sarala

Principal Scientist & Head

Dr. M. Sheshu Madhav

Director

Dr. Anindita Paul

Scientist

Target Traits in Tobacco

Research Methods & Expected Outcomes

Tobacco Specific Nitrosamines (TSNAs), potent carcinogens formed from nicotine and related alkaloids, are a major health concern. Their formation is largely influenced by specific genes involved in alkaloid metabolism. To reduce TSNA accumulation, CRISPR/Cas9-based editing was employed with guide RNAs designed to target these regulatory genes. Off-target predictions were evaluated using Cas-OFFinder, CRISPOR, and CCTop. The validated gRNA constructs were cloned into the binary vector pKSE401 and introduced into Agrobacterium strain EHA105 for transformation of Burley tobacco via the leaf disc method.

A total of 32 transformants were generated with one set of constructs and 27 with another multiplexed construct. PCR screening confirmed the presence of Cas9 and guide RNAs, with 18 and 16 positive events, respectively. Sequencing revealed successful edits in several lines, including insertions at the target sites. The transformants have been advanced to the screen house for further analysis, and additional transformation work is ongoing.