Steroidal glycoalkaloids (SGAs) are specialized metabolites found in members of Solanum species, and are also known as toxic substances in Solanum food crops such as tomato (Solanum lycopersicum), potato (Solanum tuberosum), and eggplant (Solanum melongena). SGA biosynthesis can be divided into two main parts: formation of steroidal aglycones, which are derived from cholesterol, and glycosylation at the C-3 hydroxy group. This review focuses on recent studies that shed light on the complete process of the aglycone formation in SGA biosynthesis and structural diversification of SGAs by duplicated dioxygenases, as well as the development of non-toxic potatoes through genome editing using these findings.

Genetic improvement of soybean seed traits is important for developing new varieties that meet the demand for soybean as a food, forage crop, and industrial products. A large number of soybean genome sequences are currently publicly available. This genome sequence information provides a significant opportunity to design genomic approaches to improve soybean traits. Genome editing represents a major advancement in biotechnology. The production of soybean mutants through genome editing is commonly achieved with either an Agrobacterium-mediated or biolistic transformation platform, which have been optimized for various soybean genotypes. Currently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system, which represents a major advance in genome editing, is used to improve soybean traits, such as fatty acid composition, protein content and composition, flavor, digestibility, size, and seed-coat color. In this review, we summarize the recent advances in the improvement of soybean seed traits through genome editing. We also discuss the characteristics of genome editing using the CRISPR/Cas9 system with transformation platforms.

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

Genome editing is highly useful for crop improvement. The method of expressing genome-editing enzymes using a transient expression system in Agrobacterium, called agrobacterial mutagenesis, is a shortcut used in genome-editing technology to improve elite varieties of vegetatively propagated crops, including potato. However, with this method, edited individuals cannot be selected. The transient expression of regeneration-promoting genes can result in shoot regeneration from plantlets, while the constitutive expression of most regeneration-promoting genes does not result in normally regenerated shoots. Here, we report that we could obtain genome-edited potatoes by positive selection. These regenerated shoots were obtained via a method that combined a regeneration-promoting gene with the transient expression of a genome-editing enzyme gene. Moreover, we confirmed that the genome-edited potatoes obtained using this method did not contain the sequence of the binary vector used in Agrobacterium. Our data have been submitted to the Japanese regulatory authority, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), and we are in the process of conducting field tests for further research on these potatoes. Our work presents a powerful method for regarding regeneration and acquisition of genome-edited crops through transient expression of regeneration-promoting gene.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze–thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F₁ seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

Genome editing is a promising method for simultaneously mutagenizing homoeologs in the three subgenomes of wheat (Triticum aestivum L.). However, the mutation rate via genome editing must be improved in order to analyze gene function and to quickly modify agronomic traits in wheat. Here, we examined the Cas9-induced mutation rates in wheat plants using two promoters for single guide RNA (sgRNA) expression and applying heat treatment during Agrobacterium tumefaciens-mediated transformation. Using the TaU6 promoter instead of the OsU6 promoter from rice (Oryza sativa L.) to drive sgRNA expression greatly improved the Cas9-induced mutation rate. Moreover, a heat treatment of 30°C for 1 day during tissue culture increased the Cas9-induced mutation rate and the variety of mutations obtained compared to tissue culture at the normal temperature (25°C). The same heat treatment did not affect the regeneration rates of transgenic plants but tended to increase the number of transgene integration sites in each transgenic plant. These results lay the foundation for improving the Cas9-induced mutation rate in wheat to enhance research on gene function and crop improvement.

In general, plant organ size is determined using cell number and expansion. In our previous study, we generated soybean (Glycine max) mutants of the PEAPOD (PPD) genes GmPPD1 and GmPPD2 using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 system. Some of these mutants exhibited extremely abnormal phenotypes, such as twisted pods and limited seeds. These phenotypes were attributed to the frameshift mutation in both GmPPD loci. In this study, the physiological and molecular biological properties of mutant plants with two knocked-out GmPPD loci (ppd-KO) were characterized. The ppd-KO mutant exhibited a delayed growth phase from the time of development of the unifoliolate leaves to that of first trifoliolate leaves and a stay-green phenotype, which were not observed in the other mutants of soybean or ppd mutants of other plant species. Gene expression analysis revealed considerably decreased expression of SPIRAL1-LIKE 5 (GmSP1L5), mainly causing the twisted pod phenotype observed in the ppd-KO mutant. The relationship between PPD and SP1L5 has not been previously reported, and in this study, we showed that that loss of PPD functioning affects SP1L5 expression in soybean. In this study, we revealed that the decrease in PPD function contributed to organ enlargement and that complete knockout of PPD has a negative effect on soybean organogenesis.