Journal
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
Volume 22, Issue 20, Pages -Publisher
MDPI
DOI: 10.3390/ijms222010909
Keywords
tropical japonica; gene editing; phytoene desaturase (PDS); rice (Oryza sativa L.); CRISPR/Cas9
Funding
- Agriculture and Food Research Initiative [2020-6701331811]
- USDA National Institute of Food and Agriculture
- Texas AM University
- Texas A&M AgriLife Research
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Optimization of callus induction from mature seeds, combined with both Agrobacterium-mediated and biolistic transformation, has led to successful gene editing in the high-yielding U.S. tropical japonica rice cultivar Presidio. This study achieved a regeneration efficiency of 33% and showed promising albino phenotypes in the edited PDS gene. Sanger sequencing revealed various mutations at the gRNA target sites, paving the way for more efficient gene editing in tropical japonica rice varieties.
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.
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