Elevating seed oil content in a polyploid crop by induced mutations in <i>SEED FATTY ACID REDUCER</i> genes

Karunarathna, Nirosha L.; Wang, Haoyi; Harloff, Hans-Joachim; Jiang, Lixi

doi:10.1111/pbi.13381

Cited by 77 publications

(61 citation statements)

References 69 publications

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“…CRISPR/Cas9 induced mutagenesis can be employed to induce the simultaneous knockout of multiple targets within a gene family (Karunarathna et al, 2020 ; Sashidhar et al, 2020 ). Targeting a conserved gene family poses some challenges regarding the design of the gRNAs as well as the screening of edited genotypes and off-target effects.…”

Section: Resultsmentioning

confidence: 99%

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

et al. 2020

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Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

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Section: Resultsmentioning

confidence: 99%

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

et al. 2020

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“…Numerous backcrossing are required in the development of an EMS‐mutant line with a desired trait and reduced background mutation load. CRISPR–Cas9 mutagenesis is clearly superior in this matter because a desired mutation can be incorporated into a genome without a background mutation load (Karunarathna et al ., 2020). The application of CRISPR–Cas9‐induced mutations in plant breeding is not hampered in countries with less restrictive legislation, such as China, North America and Australia, where CRISPR–Cas9‐mediated plants have not been classified as genetically modified organisms.…”

Section: Discussionmentioning

confidence: 99%

Creation of male‐sterile lines that can be restored to fertility by exogenous methyl jasmonate for the establishment of a two‐line system for the hybrid production of rice (Oryza sativa L.)

Pak

Wang

Kim³

et al. 2020

Plant Biotechnology Journal

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Male sterility is widely used in the production of hybrid seeds in rice, but the use of genic male sterility is limited because of the high labour cost for maintaining male‐sterile lines. Previous studies using T‐DNA insertional mutagenesis demonstrated that disrupting the expression of oxophytodienoic acid reductase 3 (OPR3), which is involved in the jasmonate biosynthesis pathway, results in a kind of male sterility that can be restored to fertility by exogenous jasmonate in Arabidopsis. Here, we created male‐sterile mutations by editing the second and fourth exons of OsOPR7 in rice through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated system 9. The induced mutagenesis at these exons resulted in 31.8% and 23.9% male‐sterile plants in the T0 generation, respectively. We screened male‐sterile lines that can be restored to fertility by exogenous methyl jasmonate in the T0, T1 and T2 rice populations and characterized the anther and agronomic traits of the transgenic plants. Results showed the successful generation of male‐sterile lines through the silencing of OsOPR7, the orthologous gene of Arabidopsis OPR3, in a field crop, paving the way for the establishment of a two‐line system for rice hybrid production. The system consists of a male‐sterile line that can be maintained by spraying methyl jasmonate and a restoring line that confers pollen.

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“…As such, potential target genes for breeding using this approach are limited to those that have a negative role with respect to the particular trait chosen for improvement. This strategy has been used to successfully improve a large number of desirable traits, such as those related to seed quality, nutritional status and disease resistance, for example, in a wide range of crop species (Table 2; e.g., Ma et al, 2015;Morineau et al, 2017;Nekrasov et al, 2017;Li, Wang, et al, 2018;Okuzaki et al, 2018;Do et al, 2019;Karunarathna et al, 2020;Subedi et al, 2020;Tian et al, 2020). Similarly, the knock-down of target gene expression can be achieved by targeting cis-regulatory regions rather than coding sequences using this same technology (e.g., Rodr ıguez-Leal et al, 2017;, which allows for the fine-tuning of transcriptional/functional activity, while larger targeted deletions of genomic loci can also be incurred through the use of two sgRNAs simultaneously (e.g., Zhou et al, 2014).…”

Section: A Crispr Platforms Not Requiring the Prolonged Presence Of mentioning

confidence: 99%

Genetic Variation and Unintended Risk in the Context of Old and New Breeding Techniques

Singer

Laurie

Bilichak

et al. 2021

Critical Reviews in Plant Sciences

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For thousands of years, humans have been improving crops to better suit their needs. These enhancements are driven by changes in the genetic makeup of the plant. While this was initially unintentional, there has been a steady push to increase the pace and precision of crop breeding, something that has occurred alongside a growing understanding of genetics and an escalating capacity to thoroughly assess genomes at the molecular level. With the advent and rapid uptake of molecular breeding techniques, such as transgenics and genome editing over the past few decades, there has been much trepidation regarding the possibility of off-target effects derived from unanticipated mutations at loci other than those intended for alteration, and the unintended risks that this might confer. These concerns persist regardless of the fact that a growing number of studies indicate that the occurrence of off-target mutations derived from newer biotechnological breeding techniques are negligible compared to what is observed with many conventional breeding approaches, and even spontaneously from one generation to the next. Given the impending food security crisis that we are facing in the short-term, there is a critical need to implement a wide range of breeding tools as a means of meeting growing demand, withstanding climate change-related pressures, increasing nutrition, and providing environmental benefits. While food safety is clearly of the utmost importance, now is certainly not the time to prevent the use of particular breeding technologies based on unfounded doubts. Therefore, in this review, we attempt to shed light on these apprehensions by putting purported "risks" into the context of plant breeding as a whole by comparing frequencies of spontaneous mutations with those (both anticipated and unanticipated) that occur through various conventional and biotechnological breeding approaches, including transgenics and genome editing. We then consider how these changes may, or may not, translate into unanticipated risk, and discuss the current global regulatory asynchrony surrounding genome edited crops.

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Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes

Cited by 77 publications

References 69 publications

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Creation of male‐sterile lines that can be restored to fertility by exogenous methyl jasmonate for the establishment of a two‐line system for the hybrid production of rice (Oryza sativa L.)

Genetic Variation and Unintended Risk in the Context of Old and New Breeding Techniques

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