Presentation Information
[P03-444]Metabolic Reprogramming of Steroidal Glycoalkaloid Biosynthesis in Potato via CYP88B1 Genome Editing
○Takaya Inoue1, Shuhei Yasumoto1, Hikaru Seki1,2, Toshiya Muranaka2 (1. Grad. Sch. Eng., Univ. Osaka (Japan), 2. OTRI, Univ. Osaka (Japan))
Keywords:
genome editing,potato,steroidal glycoalkaloids,TALEN,transient gene expression
[Purpose]
The potato (Solanum tuberosum) is a major crop, ranking fourth in global production. Its sprouts and green tubers contain toxic steroidal glycoalkaloids (SGAs), solanine and chaconine, which can cause food poisoning. Therefore, developing SGA-free varieties is crucial. The enzyme CYP88B1 contributes to SGA synthesis. Our previous study demonstrated that genome editing of CYP88B1 using Transcription Activator-Like Effector Nucleases (TALENs) significantly reduces SGA levels and leads to the accumulation of steroid saponins, which are valuable as raw materials for steroidal drugs. However, the CYP88B1 genome-edited lines contain transgenes to express TALENs, making them difficult to use commercially. In this study, we aimed to create transgene-free, genome-edited potato strains that produce steroid saponins rather than SGAs by knocking out CYP88B1.
[Method]
Agrobacterium-mediated transient expression of artificial nucleases has been reported as a method to generate transgene-free genome-edited plants. Stem segments of potato cv. Sayaka were inoculated with A. tumefaciens carrying a TALEN-expression vector targeting CYP88B1 and co-cultured for three days. Segments were then transferred to a shoot regeneration medium without antibiotics and cultivated for two months with periodic medium changes. Mutagenesis was initially screened by heteroduplex mobility assay (HMA). To assess T-DNA integration, we performed genomic PCRs using primer pairs for the exogenous fragment.
[Results]
The targeted mutagenesis was initially screened using HMA. Out of 1650 regenerated shoots, one mutant candidate was identified. Quantification of SGAs, α-solanine, and α-chaconine present in leaves and stems of in vitro-cultured shoots and in tubers harvested from soil-grown plants was performed by LC-MS, revealing a significant reduction of SGAs compared to the wild-type control. To evaluate T-DNA integration in the genome-edited plant, we conducted genomic PCRs using seven primer pairs; however, none of the primer pairs produced clear amplification of exogenous fragments.
[Consideration]
Although the metabolic flux appears to be redirected from SGA biosynthesis toward steroidal saponin production, the accumulation of steroidal saponins has not yet been quantitatively evaluated. This will be assessed using LC-MS in future studies. While genomic PCR suggests the absence of transgenes, more comprehensive analyses, such as whole-genome sequencing and k-mer analysis, are required for definitive validation.
[Conclusion]
Agrobacterium-mediated transient expression of artificial nucleases is valuable for generating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. However, enhancing its efficiency remains a future challenge. If the mutant generated in this study is enriched with beneficial steroidal saponins, it could serve as a new resource for steroid production.
[Acknowledgements]
This work was supported by Cabinet Office, Government of Japan, programs for Bridging the gap between R&d and the IDeal society (society 5.0) and Generating Economic and social value (BRIDGE) on “Creation of industrial plants using simultaneously modified genome editing technology” Grant Number JPJ013009, and the GteX Program Japan Grant Number JPMJGX23B0.
The potato (Solanum tuberosum) is a major crop, ranking fourth in global production. Its sprouts and green tubers contain toxic steroidal glycoalkaloids (SGAs), solanine and chaconine, which can cause food poisoning. Therefore, developing SGA-free varieties is crucial. The enzyme CYP88B1 contributes to SGA synthesis. Our previous study demonstrated that genome editing of CYP88B1 using Transcription Activator-Like Effector Nucleases (TALENs) significantly reduces SGA levels and leads to the accumulation of steroid saponins, which are valuable as raw materials for steroidal drugs. However, the CYP88B1 genome-edited lines contain transgenes to express TALENs, making them difficult to use commercially. In this study, we aimed to create transgene-free, genome-edited potato strains that produce steroid saponins rather than SGAs by knocking out CYP88B1.
[Method]
Agrobacterium-mediated transient expression of artificial nucleases has been reported as a method to generate transgene-free genome-edited plants. Stem segments of potato cv. Sayaka were inoculated with A. tumefaciens carrying a TALEN-expression vector targeting CYP88B1 and co-cultured for three days. Segments were then transferred to a shoot regeneration medium without antibiotics and cultivated for two months with periodic medium changes. Mutagenesis was initially screened by heteroduplex mobility assay (HMA). To assess T-DNA integration, we performed genomic PCRs using primer pairs for the exogenous fragment.
[Results]
The targeted mutagenesis was initially screened using HMA. Out of 1650 regenerated shoots, one mutant candidate was identified. Quantification of SGAs, α-solanine, and α-chaconine present in leaves and stems of in vitro-cultured shoots and in tubers harvested from soil-grown plants was performed by LC-MS, revealing a significant reduction of SGAs compared to the wild-type control. To evaluate T-DNA integration in the genome-edited plant, we conducted genomic PCRs using seven primer pairs; however, none of the primer pairs produced clear amplification of exogenous fragments.
[Consideration]
Although the metabolic flux appears to be redirected from SGA biosynthesis toward steroidal saponin production, the accumulation of steroidal saponins has not yet been quantitatively evaluated. This will be assessed using LC-MS in future studies. While genomic PCR suggests the absence of transgenes, more comprehensive analyses, such as whole-genome sequencing and k-mer analysis, are required for definitive validation.
[Conclusion]
Agrobacterium-mediated transient expression of artificial nucleases is valuable for generating non-transgenic mutant plants without the need to segregate out transgenes through sexual reproduction. However, enhancing its efficiency remains a future challenge. If the mutant generated in this study is enriched with beneficial steroidal saponins, it could serve as a new resource for steroid production.
[Acknowledgements]
This work was supported by Cabinet Office, Government of Japan, programs for Bridging the gap between R&d and the IDeal society (society 5.0) and Generating Economic and social value (BRIDGE) on “Creation of industrial plants using simultaneously modified genome editing technology” Grant Number JPJ013009, and the GteX Program Japan Grant Number JPMJGX23B0.
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