Enhancing the competence for plant regeneration in tissue culture studies is an important issue not only for efficient genetic transformation of commercial crops but also for the reproducibility of scientific reports. In this study, we investigated optimization of several tissue culture conditions including plant growth regulators, types and ages of explants, culture densities, and plant position in order to improve the competence of adventitious shoot formation of the tomato (Solanum lycopersicum cv. Micro-Tom). In addition, we examined the differential expression of D-type cyclin (CYCD3-1) and several shoot regeneration regulatory genes from hypocotyl and cotyledon explants of tomato during shoot organogenesis. A treatment of 1 mg L⁻¹ Zeatin and 0.1 mg L⁻¹ Indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium containing 3% sucrose was optimal for adventitious shoot formation from hypocotyl and cotyledon explants. The younger explants exhibited more shoot formation regardless of explant types. Additionally, those closest to the shoot apical meristem produced more shoots compared to the other regions in the hypocotyl and the cotyledon explants. Gene expression of CYCD3-1, SHOOT MERISTEMLESS (STM), and cytokinin dependent WUSCHEL (WUS) was significantly higher in younger explants than in older ones. Furthermore, an increase in CYCD3-1, STM, and WUS expression was evident at the distal part of hypocotyls and the proximal part of cotyledons compared to other regions. These differential gene expression profiles exhibited good agreement with the results of shoot formation obtained from diverse explants of tomato. These results suggest that temporal and spatial gene expression of shoot regeneration regulatory genes plays an important role in enhancing the competence and the reproducibility of adventitious shoot formation from tomato explants.

Wheat is highly susceptible to heat stress, which significantly reduces grain yield. In this study, we used RNA-seq technology to analyze the transcript expression at three different time-points after heat treatment in three cultivars differing in their susceptibility to heat stress: Jopum, Keumkang, and Olgeuru. A total of 11,751, 8850, and 14,711; 10,959, 7946, and 14,205; and 22,895, 13,060, and 19,408 differentially-expressed genes (log2 fold-change > 1 and FDR (padj) < 0.05) were identified in Jopum, Keumkang, and Olgeuru in the control vs. 6-h, in the control vs. 12-h, and in the 6-h vs. 12-h heat treatment, respectively. Functional enrichment analysis showed that the biological processes for DEGs, such as the cellular response to heat and oxidative stress—and including the removal of superoxide radicals and the positive regulation of superoxide dismutase activity—were significantly enriched among the three comparisons in all three cultivars. Furthermore, we investigated the differential expression patterns of reactive oxygen species (ROS)-scavenging enzymes, heat shock proteins, and heat-stress transcription factors using qRT-PCR to confirm the differences in gene expression among the three varieties under heat stress. This study contributes to a better understanding of the wheat heat-stress response at the early growth stage and the varietal differences in heat tolerance.

The selection of wheat varieties with high arabinoxylan (AX) levels could effectively improve the daily consumption of dietary fiber. However, studies on the selection of markers for AX levels are scarce. This study analyzed AX levels in 562 wheat genotypes collected from 46 countries using a GWAS with the BLINK model in the GAPIT3. Wheat genotypes were classified into eight subpopulations that exhibited high genetic differentiation based on 31,926 SNP loci. Eight candidate genes were identified, among which those encoding F-box domain-containing proteins, disease resistance protein RPM1, and bZIP transcription factor 29 highly correlated with AX levels. The AX level was higher in the adenine allele than in the guanine alleles of these genes in the wheat collection. In addition, the AX level was approximately 10% higher in 3 adenine combinations than 2 guanine, 1 adenine, and 3 guanine combinations in genotypes of three genes (F-box domain-containing proteins, RPM1, and bZIP transcription factor 29). The adenine allele, present in 97.46% of AX-95086356 SNP, exhibited a high correlation with AX levels following classification by country. Notably, the East Asian wheat genotypes contain high adenine alleles in three genes. These results highlight the potential of these three SNPs to serve as selectable markers for high AX content.

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 μM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco (Nicotiana benthamiana) and tomato (Solanum lycopersicum). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin (CYCD3-1) and shoot-regeneration regulatory gene WUSCHEL (WUS) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli.