DNA-free genome editing for ZmPLA1 gene via targeting immature embryos in tropical maize

Abstract

Doubled haploid (DH) production accelerates the development of homozygous lines in a single generation. In maize, haploids are widely produced by the use of haploid inducer Stock 6, earlier reported in 1959. Three independent studies reported haploid induction in maize which is triggered due to a 4 bp frame-shift mutation in matrilineal ( ZmPLA1 ) gene. The present study was focused on the generation of mutants for ZmPLA1 gene in maize inbred line LM13 through site-directed mutagenesis via CRISPR/Cas9-mediated ribonucleoprotein (RNP) complex method to increase the haploid induction rate. Three single guide RNAs (sgRNAs) for the ZmPLA1 gene locus were used for transforming the 14 days old immature embryos via bombardment. 373 regenerated plants were subjected to mutation detection followed by Sanger’s sequencing. Out of three putative mutants identified, one mutant depicted one base pair substitution and one base pair deletion at the target site.

Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol

“…Despite the growing number of publications on improved maize transformation and GE technologies, successful reports on tropical maize lines have been less numerous than those on their temperate counterparts ( Rangari et al, 2023 ; Souza et al, 2017 ; reviewed by Hernandes-Lopes et al, 2023 ; Yadava et al, 2017 ). Thus, in the present study, we applied the MR-based approach previously used by Aesaert et al (2022) for optimized B104 transformation and GE to different tropical maize lines.…”