Development of a CRISPR-Cas9 Editing System Using D14 in Chickpea (Cicer arietinum)

Abstract

Chickpea is one of the world’s most prevalent legume crops, with millions of tonnes grown annually. However, chickpea crops are vulnerable to massive yield loss caused by climate change and pest infestation. With the advancement of genomic sequencing and refined techniques for genetic editing, scientific approaches can be readily applied to identify biological traits that can help improve yield. This study focused on chickpea branching morphology and its regulation by the strigolactone (SL) pathway. The SL receptor D14 was targeted for mutagenesis through CRISPR-Cas9 editing. Chickpea seedlings underwent Agrobacterium tumefaciens-mediated T-DNA transformation and whole-plant regeneration in order to introduce the CRISPR-Cas9 gene constructs into the plant genome. In order to investigate sgDNA expression strength, two different T-DNA vectors were developed. One expressed multiple different D14 sgDNA fragments on individual promoter and terminators, while the other expressed those sgRNA under the same promoter and terminator with each sgRNA separated by a pre-tRNAGly sequence. However, chickpea was found to be highly resistant to in vitro regeneration, specifically at the rooting stage. A new protocol was thus developed for the hypocotyl-mediated regeneration of Agrobacterium-transformed chickpeas which utilized a 100 µM IBA root shock and frequent liquid medium cycling in order to generate roots. PCR genotypic analysis and Sanger sequencing was unable to confirm any putative D14 transformants, and it was concluded that future adjustments should be made in order to troubleshoot transformation efficiency and selection by antibiotics. With an efficient transformation vector, the applications of this method can be applied not only to D14, but to the multitudes of other unexplored chickpea genes.