Genome editing of Ideal Plant Architecture-1 (IPA1) gene for enhancing tillering in wheat (Triticum aestivum L.)

Islam, Asif

Genome editing of Ideal Plant Architecture-1 (IPA1) gene for enhancing tillering in wheat (Triticum aestivum L.)

Date

2023

Authors

Islam, Asif

Publisher

Punjab Agricultural University

Abstract

Wheat is a global staple cereal, and breeding initiatives aim to increase wheat yield to meet consumption and supply needs. Increasing wheat tillers is a key factor in crop yield. Understanding the ideal wheat plant structure helps explain tiller regulation. Ideal Plant Architecture IPA1 gene in rice enhances number of productive tillers. CRISPR/Cas9 based genome editing with Agrobacterium-induced transformation targeting IPA1 wheat orthologs was undertaken in the present study. The guide RNAs (gRNAs) were engineered to target exon 1 of the IPA1 homeologs TraesCS7A02G246500, TraesCS7B02G144900, and TraesCS7D02G245200, simultaneously and subjected to an in-vitro cleavage assay, which identified gRNA 7ABDE1 as a potential candidate. The gRNA cleaved the 1190 bp amplicon containing exon 1 into two 1050 and 139 bp fragments. The gRNA was then injected into the AGL1 strain of Agrobacterium together with the JD633 vector, which delivered the sgRNA and Cas9 components into the plant system. Sanger sequencing with target-specific primers validated the construct assembly's transformation into the wheat genome. A total of 500 wheat immature embryos excised 14 days post-anthesis were used to introduce the Cas9-cassette and after two cycles of Hygromycin selection, 85 putative transformed calli are in the regeneration media. In addition, 54 previously generated GE0 plants for IPA1 gene were characterized for the presence of Cas9 cassette and 284 GE1 plants derived from these GE0 plants were phenotypically investigated and subjected to Sanger sequencing for putative mutations in the target gene. Confirmed genome edited plants will be further studied for the effect of mutations on the plant architecture.

Citation

Islam, Asif (2023). Genome editing of Ideal Plant Architecture-1 (IPA1) gene for enhancing tillering in wheat (Triticum aestivum L.) (Unpublished M.Sc. thesis). Punjab Agricultural University, Ludhiana, Punjab, India.