ICAR- Indian Institute of Maize Research

Collaborating Centre Principal Investigator (CC-PI)

Dr. Krishan Kumar

Sr. Scientist

Principal Investigators involved in the project

Dr. Krishan Kumar

Sr. Scientist

Co-Principal Investigators (Co-PIs)

Dr. Mamta Gupta

Sr. Scientist

Dr. Alla Singh

Sr. Scientist

Dr. Bhupender Kumar

Sr. Scientist

Dr. Mukesh Choudhary

Sr. Scientist

Dr. Abhijit Kumar Das

Sr. Scientist

Dr. Pravin Kumar Bagaria

Sr. Scientist

Target Traits in Maize

Research Methods & Expected Outcomes

Target gene sequences from the B73 temperate maize inbred line were retrieved from MaizeGDB (https://www.maizegdb.org/) and used for designing specific primers for the isolation of coding sequences (CDS) from Indian maize genotypes DMRH 1308 and BML6. Briefly, total RNA was isolated from 10-day-old seedlings, and cDNA was synthesized. These target regions were amplified using gene-specific primers, and Sanger sequencing of the amplicons was performed. The CDS sequence information generated from the selected cultivars was then used to design guide RNAs (gRNAs) towards the 5′ end of the coding region with minimal off-targets using CRISPR Direct (https://crispr.dbcls.jp/) and CHOP-CHOP (https://chopchop.cbu.uib.no/) tools. The designed gRNAs were cloned into the pRGEB32-BAR vector (Hunter, 2021) obtained from Addgene, USA. The gRNA insertion/cloning was confirmed by colony PCR, restriction digestion, and Sanger sequencing. So far, two constructs have been developed, and two more are under progress. The successfully assembled construct is currently being used for genetic transformation into nodal explant-derived callus from DMRH 1308 using both Agrobacterium-mediated and biolistic transformation methods.