Optimizing Agrobacterium-Mediated Transformation and CRISPR-Cas9 Gene Editing in the tropical japonica Rice Variety Presidio.

Backki Kim,Marco Molina-Risco,Oneida Ibarra,Endang M Septiningsih,Mayra Faion-Molina,Michael J Thomson

doi:10.3390/ijms222010909

Abstract

Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent the majority of rice production in the U.S. and South America. The current study has optimized a protocol using callus induction from mature seeds with both Agrobacterium-mediated and biolistic transformation of the high-yielding U.S. tropical japonica cultivar Presidio. Gene editing efficiency was tested by evaluating knockout mutations in the phytoene desaturase (PDS) and young seedling albino (YSA) genes, which provide a visible phenotype at the seedling stage for successful knockouts. Using the optimized protocol, transformation of 648 explants with particle bombardment and 532 explants with Agrobacterium led to a 33% regeneration efficiency. The YSA targets had ambiguous phenotypes, but 60% of regenerated plants for PDS showed an albino phenotype. Sanger sequencing of edited progeny showed a number of insertions, deletions, and substitutions at the gRNA target sites. These results pave the way for more efficient gene editing of tropical japonica rice varieties.

Highlights

Recent advances in CRISPR/Cas-based gene editing have led to a renewed interest in optimizing plant transformation and regeneration techniques to enable more rapid development of gene edited lines for crop improvement
We report the optimization of the rice transformation and regeneration protocol of Presidio with mature seeds and confirmation of efficient CRISPR/Cas9 gene editing by creating knockout mutants of phytoene desaturase (PDS) and young seedling albino (YSA) using both
Amplicons obtained from Presidio were sequenced and compared with the Nipponbare reference genome: sequenced regions showing identical sequences were used to design two guide RNAs for each gene target

Summary

Introduction

Recent advances in CRISPR/Cas-based gene editing have led to a renewed interest in optimizing plant transformation and regeneration techniques to enable more rapid development of gene edited lines for crop improvement. Biolistic-based delivery has fewer constraints on the target species, but often results in multiple insertions or insertion of partial DNA fragments. Both delivery methods require slow, labor-intensive, and often complex sets of species- and genotype-specific protocols for in vitro tissue culture and regeneration from transformed explants. In vitro plant transformation, tissue culture, and regeneration techniques have made tremendous progress over the past few decades, there are still several bottlenecks that hamper faster progress, including low frequency of transformed events, long periods of time needed for tissue culture and regeneration, and low precision of biolistic-mediated transformation [4]

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Discussion

Conclusion