Abstract
Abstract Walnut (Juglans regia L.) is an economically valuable tree species globally, renowned for its nutritious nuts and quality timber. However, walnut breeding is significantly constrained by inherent biological factors, and an efficient and reliable genome-editing system has yet to be established. In this study, we developed an optimized walnut genome-editing platform by systematically selecting superior receptor from 30 walnut cultivars using genotype-dependent direct somatic embryogenesis and regeneration systems. Walnut cultivar HT-14 exhibited the highest embryogenic induction (53.33%) and regeneration efficiency (85.33%), and 35S: RUBY was effectively expressed in somatic embryos of the HT-14 genotype, proving it ideal receptor material for genetic transformation. Additionally, 12 walnut-specific endogenous Pol III promoters (JrU3 and JrU6) were cloned and validated for their ability to significantly enhance CRISPR/Cas9-editing efficiency by targeting the walnut phytoene desaturase gene (JrPDS). Compared to commonly used exogenous promoters (AtU6-26 and BpU6-6), these native promoters, the JrU3-chr3 promoter achieving an editing efficiency of 58.82%, significantly increased mutation efficiencies in walnut. Furthermore, endogenous promoters promoted higher frequencies of homozygous and biallelic mutations and greater mutation diversity. Collectively, this study establishes a robust and efficient genome-editing platform for walnut, providing essential technical support for functional genomics research and accelerating the precision breeding walnut varieties. These findings also offer valuable methodologies and insights into genome-editing applications in other perennial woody plants.
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Claims 8
Claim · model-written summary
Walnut cultivar HT-14 had the highest embryogenic induction (53.33%) among the cultivars screened.
- Taxon
- Walnut (Juglans regia L.)
Juglans regiae0 - Material
- HT-14e1
- Outcome
- embryogenic inductione1
- Value
- 53.33%e1
- Comparator
- 30 walnut cultivarse2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e1“Walnut cultivar HT-14 exhibited the highest embryogenic induction (53.33%) and regeneration efficiency (85.33%)”
e2“systematically selecting superior receptor from 30 walnut cultivars”
Claim · model-written summary
Walnut cultivar HT-14 had the highest regeneration efficiency (85.33%) among the cultivars screened.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Material
- HT-14e1
- Outcome
- regeneration efficiencye1
- Value
- 85.33%e1
- Comparator
- 30 walnut cultivarse2
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e1“Walnut cultivar HT-14 exhibited the highest embryogenic induction (53.33%) and regeneration efficiency (85.33%)”
e2“systematically selecting superior receptor from 30 walnut cultivars”
Claim · model-written summary
The 35S:RUBY construct was effectively expressed in somatic embryos of the HT-14 genotype.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Material
- HT-14e3
- Organ / stage
- somatic embryose3
- Outcome
- 35S: RUBYe3
- Direction
- effectively expressede3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e3“35S: RUBY was effectively expressed in somatic embryos of the HT-14 genotype”
Claim · model-written summary
Twelve walnut-specific endogenous Pol III promoters significantly enhanced CRISPR/Cas9 editing efficiency when targeting JrPDS.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Treatment
- walnut-specific endogenous Pol III promoterse4
- Gene
- JrPDSe4
- Outcome
- CRISPR/Cas9-editing efficiencye4
- Direction
- significantly enhancee4
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e4“12 walnut-specific endogenous Pol III promoters (JrU3 and JrU6) were cloned and validated for their ability to significantly enhance CRISPR/Cas9-editing efficiency by targeting the walnut phytoene desaturase gene (JrPDS)”
Claim · model-written summary
Endogenous promoters significantly increased walnut mutation efficiencies compared with commonly used exogenous promoters.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Treatment
- these native promoterse5
- Outcome
- mutation efficienciese5
- Direction
- significantly increasede5
- Comparator
- commonly used exogenous promoters (AtU6-26 and BpU6-6)e5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e5“Compared to commonly used exogenous promoters (AtU6-26 and BpU6-6), these native promoters, the JrU3-chr3 promoter achieving an editing efficiency of 58.82%, significantly increased mutation efficiencies in walnut”
Claim · model-written summary
The JrU3-chr3 promoter achieved an editing efficiency of 58.82% in walnut.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Gene
- JrU3-chr3 promotere5
- Outcome
- editing efficiencye5
- Value
- 58.82%e5
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e5“Compared to commonly used exogenous promoters (AtU6-26 and BpU6-6), these native promoters, the JrU3-chr3 promoter achieving an editing efficiency of 58.82%, significantly increased mutation efficiencies in walnut”
Claim · model-written summary
Endogenous promoters promoted higher frequencies of homozygous and biallelic mutations than exogenous promoters.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Treatment
- endogenous promoterse6
- Outcome
- frequencies of homozygous and biallelic mutationse6
- Direction
- highere6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e6“Furthermore, endogenous promoters promoted higher frequencies of homozygous and biallelic mutations and greater mutation diversity”
Claim · model-written summary
Endogenous promoters promoted greater mutation diversity in walnut.
- Taxon
- Walnut (Juglans regia L.)Juglans regiae0
- Treatment
- endogenous promoterse6
- Outcome
- mutation diversitye6
- Direction
- greatere6
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Walnut (Juglans regia L.)”
e6“Furthermore, endogenous promoters promoted higher frequencies of homozygous and biallelic mutations and greater mutation diversity”