Improved Transformation and Regeneration of Indica Rice: Disruption of SUB1A as a Test Case via CRISPR-Cas9.

Yuya Liang,Backki Kim,Endang M Septiningsih,Julia Bailey-Serres,Sudip Biswas

doi:10.3390/ijms22136989

Abstract

Gene editing by use of clustered regularly interspaced short palindromic repeats (CRISPR) has become a powerful tool for crop improvement. However, a common bottleneck in the application of this approach to grain crops, including rice (Oryza sativa), is efficient vector delivery and calli regeneration, which can be hampered by genotype-dependent requirements for plant regeneration. Here, methods for Agrobacterium-mediated and biolistic transformation and regeneration of indica rice were optimized using CRISPR-Cas9 gene-editing of the submergence tolerance regulator SUBMERGENCE 1A-1 gene of the cultivar Ciherang-Sub1. Callus induction and plantlet regeneration methods were optimized for embryogenic calli derived from immature embryos and mature seed-derived calli. Optimized regeneration (95%) and maximal editing efficiency (100%) were obtained from the immature embryo-derived calli. Phenotyping of T1 seeds derived from the edited T0 plants under submergence stress demonstrated inferior phenotype compared to their controls, which phenotypically validates the disruption of SUB1A-1 function. The methods pave the way for rapid CRISPR-Cas9 gene editing of recalcitrant indica rice cultivars.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 reagents, including the promoterguide (g)RNA and promoter-Cas9 gene constructs in a single vector [10], are delivered into the host plant where the gRNA-Cas9 complex guides the position of Cas9 protein cleavage in the host genome through non-homologous end-joining (NHEJ) or homologous recombination (HR) [11]
To optimize callus induction medium (CIM) for mature seeds, several medium components were modified from the previous japonica and indica rice tissue culture protocols [15,19]
The results of our study show that it is possible to obtain high regeneration and high editing efficiency in Ciherang-Sub1 using CRISPR-Cas9 with immature embryos as explants through Agrobacterium-mediated transformation

Summary

Introduction

The clustered regularly interspaced short palindromic repeats-Cas nuclease (CRISPRCas9) technology for genome editing has become a powerful tool for gene-targeted crop improvement. This technique has been applied to various crops, including cereals, such as wheat [1,2,3], corn [4,5,6], and rice [7,8,9]. The CRISPR-Cas reagents, including the promoterguide (g)RNA and promoter-Cas gene constructs in a single vector [10], are delivered into the host plant where the gRNA-Cas complex guides the position of Cas protein cleavage in the host genome through non-homologous end-joining (NHEJ) or homologous recombination (HR) [11]. Agrobacterium-mediated transformation via T-DNA insertion is the most common and stable method to deliver transgene vectors into a host plant [12]

Objectives

Methods

Results

Conclusion