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.

KEYWORDS:

• Crispr/Cas9
• haploid induction
• immature embryos
• maize
• Ribonucleoprotein (RNP) complex

Abbreviations

T1	=	First Transgenic generation
LM13	=	Ludhiana maize 13
bp	=	Base pair
CRISPR	=	Clustered Regularly Interspaced Short Palindromic Repeats
Cas9	=	CRISPR-associated protein 9

Acknowledgments

The funding provided by the Department of Biotechnology, Ministry of Science and Technology, Government of India, in the form of various grants is gratefully acknowledged. G.H-G acknowledges the award of National Talent Scholarship by Indian Council of Agricultural Research (ICAR-NTS).

Authors Contribution

Conceptualization of research (Priti Sharma, Yogesh Vikal, Gagandeep Singh); Designing of the experiments (Priti Sharma, Yogesh Vikal); Contribution of experimental materials (Yogesh Vikal, Gagandeep Singh); Execution of field/lab experiments and data collection (Sagar K Rangari, Manjot Kaur, Harjot Kaur, Nidhi Uppal); Analysis of data and interpretation (Sagar K Rangari, Manjot Kaur); Preparation and editing of the manuscript (Sagar K Rangari, Harjot Kaur, Priti Sharma).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplemental data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21645698.2023.2197303

Additional information

Funding

The work was supported by the Department of Biotechnology, Ministry of Science and Technology, India [BT/GET/119/SP25803/2018]; ICAR-NTS [EDN/1/25/2015].