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CRISPR/Cas9-mediated introduction of a single base mutation in SOC1, a transcription factor that regulates flowering time, results in late-bolting phenotypes in lettuce. Lettuce is a widely consumed leafy vegetable crop. One of the molecular approaches that can increase leaf yield of lettuce is to delay the onset of flowering. Flowering time or time-to-bolting is not only a valuable trait for lettuce, but also a sought-after phenotype for other leafy vegetable crops. This is because delayed flowering enables more extensive vegetative growth, which leads to higher leaf numbers, and possibly larger leaves. Here, we deployed the most recent gene-editing technique to reduce the expression of SOC1, which is a gene that encodes one of several transcription factors that regulate the onset of flowering in plants. By inducing a single base mutation in SOC1 through Cas9 protein-gRNA ribonucleoproteins (RNPs) system, we showed that the time to first flower bud formation in lettuce is longer than that of wild type. In addition, expression of the floral regulatory genes including LsLFY, LsFUL, LsAPL1, and LsAPL2, was lower in the SOC1 gene edited plants than that of the wild type. The gene-editing technique established in this study could be directly applied for diverse quality improvement of lettuce by direct RNP transfer from protoplasts. Furthermore, it is expected that direct RNP transfer from protoplasts can be used as a useful mean for developing various gene edited crops.

Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efficient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7 ± 2.5% and 52 ± 7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confirmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efficient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

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.

We developed an efficient plant regeneration technique for turmeric (Curcuma longa L.) via somatic embryogenesis derived from embryogenic cell suspensions cultured from petioles. We initiated in vitro callus cultures from the plantlets of turmeric cultured on Murashige and Skoog (MS) medium. Then leaf, petiole, stem, and root explants of in vitro-grown plantlets were cultured on Schenk and Hildebrandt (SH). The stem and root explants formed white nodular calluses at a frequency of 86.7% and 85.8%, respectively, when cultured on SH medium; however, we used the calluses derived from the root explants for initiating a cell-suspension culture. In this study, the actively growing cell suspension culture comprised small, highly cytoplasmic cell aggregates and large vacuolated cells. On being transferred to a basal SH medium (without growth regulators), a few of these calluses differentiated into somatic embryos, which indicated that the initial calluses were embryogenic cells. We stimulated additional shoot differentiation by adding 0.5 mg L−1N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ) and various concentrations of N6-benzyladenine (BA) to the embryogenic cell suspension. The highest frequency at which shoot primordia developed from embryogenic cell clusters was 70% (when cultured in the dark on the supplemented MS medium), with more than 83.3% of regenerated shoot primordia producing roots. We successfully transplanted rooted plantlets into a soil mixture of sterile vermiculite and potting soil (1:1) and grew them to maturity in a growth chamber, achieving a survival rate of > 95%. To our knowledge, this is the first report of complete plant regeneration of turmeric from embryogenic cell-suspension cultures. The embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and gene manipulation for improving turmeric quality.

We aimed to develop a novel technology capable of rapidly selecting mutant plant cell lines. Salt resistance was chosen as a rapid selection trait that is easily applicable to protoplast-derived cell colonies. Mesophyll protoplasts were cultured in a medium supplemented with 0, 50, 100, 150, 200, 250, and 300 mM NaCl. At NaCl concentrations &ge, 100 mM, cell colony formation was strongly inhibited after 4 weeks of culture. Tobacco protoplasts irradiated with 0, 50, 100, 200, and 400 Gy were then cultured to investigate the effects of radiation intensity on cell division. The optimal radiation intensity was 50 Gy. To develop salt-resistant tobacco mutant plants, protoplasts irradiated with 50 Gy were cultured in a medium containing 100 mM NaCl. The efficiency of cell colony formation from these protoplasts was approximately 0.002%. A salt-resistant mutant callus was selected and proliferated in the same medium and then transferred to a shoot inducing medium for adventitious shoot formation. The obtained shoots were then cultured in a medium supplemented with 200 mM NaCl and developed into normal plantlets. This rapid selection technology for generating salt-resistant tobacco mutants will be useful for the development of crop varieties resistant to environmental stresses.

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.

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&minus, 1 Zeatin and 0.1 mg L&minus, 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.

The efficient plant regeneration system from embryogenic cell suspension cultures of Gynura procumbens (Lour.) Merr. is described. Leaf, stem and petiole explants were cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) in various concentrations (0, 0.1, 0.3, 1.0 and 3.0 mg l−1). Leaf, stem and petiole explants formed pale-yellow nodular callus and off-white calluses at a frequency of 100% when cultured on MS medium supplemented with more than 1 mg l−1 of 2,4-D after 4 weeks incubation. However, only 20% of pale-yellow nodular callus derived from petiole explants developed into white embryonic structures. Upon transfer to MS basal medium without growth regulators, these white embryonic structures differentiated into somatic embryos. Embryogenic cell suspension cultures were initiated from petiole-derived pale-yellow nodular calluses. More than 73.2% of regenerated plantlets via somatic embryogenesis produced roots on MS medium supplemented with 0.1 mg l−1 α-naphthaleneacetic acid and 1 mg l−1 indole-3-butyric acid (IBA), respectively. Rooted plantlets were successfully transplanted to soil mixture of sterile vermiculite and potting soil (1:1) and grown to maturity in a growth chamber, achieving a survival rate of > 95%. The plant regeneration system from embryogenic cell suspension cultures of G. procumbens established in this study could be applied as an alternative for mass proliferation as well as molecular breeding for quality improvement of G. procumbens.

The purpose of this study is to establish the rapid discrimination system of cybrid callus lines by Fourier transform infrared (FT-IR) spectroscopy without genetic fingerprinting analysis. Genomic DNA isolated from two parental lines (Brassica oleracea var. capitata and Raphanus sativus L.) and their cybrid callus lines were analyzed by FT-IR spectroscopy in the spectral region from 4000 to 400 cm−1. Several spectral differences between the two parental lines were detected in the frequency regions of N–H stretching (amide I), C=O stretching vibrations (amide II), and PO2− ionized asymmetric and symmetric stretching. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used to discriminate cybrid cell lines from the two parental species at the genomic DNA level. PLS-DA analysis provided more clear discrimination between the two parental lines and their progeny cybrid lines in the score plot. PCA loading values also showed that obvious spectral differences played a significant role in discrimination between the two parental lines and their cybrid lines. These spectral differences might be directly related to subtle changes in the base functional groups and backbone structures of genomic DNA. Considering these results, this technique could provide a research foundation for the FT-IR spectral-based diagnosis, selection, and discrimination of parental lines and their cybrids. Furthermore, this technique could be applied in the hybrid seed industry for rapid screening with high heterogeneity.

Ultraviolet (UV)-irradiation causes an overproduction of matrix metalloproteinases (MMPs) and collagen (COL) degradation causing skin sunburn, inflammation, or photoaging in human. In this study, we prepared callus from Tiarella polyphylla D. Don ( T. polyphylla) stem and its phytochemical profiles were analyzed using HPLC-MWD. The effects of T. polyphylla callus extract evaluated against on UVB-induced damage in human foreskin fibroblast (Hs68). Hs68 was exposed to UVB in the presence or absence of T. polyphylla callus extract at concentrations of 100 and 250 µg/mL. Cell damage caused by UVB was inhibited by T. polyphylla callus extract, which was tested by cell viability and caspase 3 activity in Hs68 cells. Further experiment revealed that T. polyphylla extract suppressed the level of MMP-1, but increased the level of type I procollagen. In addition, T. polyhylla callus extract inhibited UVB-mediated COL (-1 and -3) protein degradation and MMP (-1, 2, and -3) overexpression in Hs68. These results suggest that T. polyphylla callus extract has considerable potential as a cosmetic ingredient with anti-aging effects.

Aims We developed a new rapid and reliable method for identifying bacteria using a combination of Fourier transform infrared (FT-IR) spectroscopy of bacterial genomic DNA and multivariate analysis. Methods and results FT-IR spectra of genomic DNA from four type strains of Pseudomonas spp., three type strains of Escherichia spp. and two type strains of Bacillus spp. were analysed in the 4000-400 cm-1 region. Spectral differences were found in the frequency regions of N-H stretching (amide I), C=O stretching vibrations (amide II) and PO2 - ionized asymmetric and symmetric stretching. Partial least squares discriminant analysis of the FT-IR spectra showed that the microbial strains could be discriminated by hierarchical clustering analysis. Conclusions FT-IR spectral analysis of bacterial genomic DNA has potential for the rapid identification of bacteria at the genus and species levels. Significance and impact of the study This study reports a new bacterial identification method using multivariate analysis of FT-IR spectra of bacterial genomic DNA.

To determine whether metabolite fingerprinting for whole cell extracts based on Fourier transform infrared (FT-IR) spectroscopy can be used to discriminate and compare metabolic equivalence, standard medicinal parts from four medicinal plants (Cynanchum wilfordii Hemsley, Atractylodes japonica Koidz, Polygonum multiflorum Thunberg and Astragalus membranaceus Bunge) and their in vitro-produced adventitious roots were analyzed by FT-IR spectroscopy. The principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) from the FT-IR spectral data showed that the whole metabolic pattern from Cynanchum wilfordii was highly similar to Astragalus membranaceus. However, Atractylodes japonica and Polygonum multiflorum showed significantly different metabolic patterns. Furthermore, adventitious roots from Cynanchum wilfordii and Astragalus membranaceus also showed similar metabolic patterns compared to their standard medicinal parts. These results clearly show that mass proliferation of adventitious roots may be applied to aquire novel supply of standard medicinal parts from medicinal plants. However, the whole metabolic pattern from adventitious roots of Atractylodes japonica and Polygonum multiflorum were not similar to their standard medicinal parts. Furthermore, FT-IR spectroscopy combined with multivariate analyses established in this study may be applied as an alternative tool to discriminate the whole metabolic equivalence from several standard medicinal parts. Thus, we suggest that these metabolic discrimination systems may be applied for metabolic standardization of herbal medicinal resources.

The conditions for efficient tuber production from suspension cultures of Pinellia ternata cells which is one of medicinal herbs were established and succinic acid in tubers propagated in vitro was determined. Leaf explants formed white nodular structures and off-white calluses at a frequency of 90.6 % when cultured on Murashige and Skoog medium supplemented with 0.54 μM α-naphthaleneacetic acid (NAA); however, this frequency declined substantially with increasing NAA concentration, up to 16.2 μM, at which the frequency reached zero percent. In combination treatments with 4.44 μM 6-benzyladenine (BA) and NAA, however, the frequency of white nodular structure and off-white callus formation from leaf explants did not decrease, even at 16.2 μM NAA. Suspension cultures of P. ternata cells were established from leaf-derived off-white calluses in MS liquid medium containing 5.4 μM NAA and 4.44 μM BA. Upon plating onto MS basal medium, over 90 % of cell aggregates gave rise to microtubers and developed into plantlets. Regenerated plantlets were transplanted in potting soil and grown to maturity in a growth chamber, with a survival rate of >90 %. The highest succinic acid content in suspension culture-derived microtubers was 45 g/kg of extract. Compared with wild P. ternata medicinal tubers, the succinic acid content was very similar. The in vitro P. ternata microtuber proliferation system established in this study is thus an efficient alternative for the mass production of medicinal tubers.

To determine whether or not FT-IR spectroscopy could be used for taxonomic and metabolic discrimination of African yam lines, tuber samples from African and Asian yam species were subjected to FT-IR. Most remarkable spectral differences between African and Asian yams were found in the 1750-1700 cm(-1) region, polysaccharide (1200-900 cm(-1)) and protein/amide I and II (1700-1500 cm(-1)) regions of FT-IR spectra. A hierarchical dendrogram based on partial least square-discriminant analysis (PLS-DA) of FT-IR data from 7 African yam species show phylogenetic relationship. In addition, the content of dioscin, a steroidal saponin found in yam tuber, was predicted using a PLS regression model with regression coefficient R(2)=0.7208 indicated that prediction model had average accuracy. Thus, considering these results we suggest that FT-IR combined with multivariate analysis could be applied as a novel tool for metabolic evaluation and high-throughput screening of African yam lines with higher content of dioscin.

We established a high throughput screening system of African yam tuber lines which contain high contents of total carotenoids, flavonoids, and phenolic compounds using ultraviolet-visible (UV-VIS) spectroscopy and Fourier transform infrared (FT-IR) spectroscopy in combination with multivariate analysis. The total carotenoids contents from 62 African yam tubers varied from 0.01 to 0.91 μg·g -1 dry weight (wt). The total flavonoids and phenolic compounds also varied from 12.9 to 229 μg·g -1 2 ) between predicted values and estimated values of total carotenoids, flavonoids and phenolic compounds were 0.83, 0.86, and 0.72, respectively. These results showed that quantitative predictions of total carotenoids, flavonoids, and phenolic compounds were possible from FT-IR spectra of African yam tuber lines with higher accuracy. Therefore we suggested that quantitative prediction system established in this study could be applied as a rapid selection tool for high yielding African yam lines.

Fourier transform infrared spectroscopy (FT-IR) spectroscopy in combination with multivariate analysis was used to discriminate two different F1 hybrid lines from their parental inbred lines. Genomic DNA was isolated from leaves of three parental lines and two F1 hybrid lines of Brassica campestris subsp. pekinensis. Purified genomic DNA was analyzed by FT-IR spectroscopy in the spectral region from 4,000 to 400 cm−1. FT-IR spectra confirmed typical spectral differences between the frequency regions of N–H stretching (amide I) and C=O stretching vibrations (amide II) as well as PO2− ionized asymmetric and symmetric stretching. Principal component analysis was able to discriminate between F1 hybrid progenies depending on their parental lineages, even though they share the same maternal background. Partial least squares discriminant analysis gave a more clear discrimination between the two parental lines and their hybrid progenies. These FT-IR spectral differences might be directly related to subtle changes in the base functional group and backbone structures of genomic DNA. Considering these results, this technique could provide a solid research foundation for FT-IR spectral-based rapid diagnosis, selection, and discrimination of parental lines from their progenies. Furthermore, this technique could be applied to test purity in the hybrid seed industry.

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l−1 of α-naphthaleneacetic acid (NAA) and 1 mg l−1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.

This study describes the effect of myo-inositol on sustained cell division and plant regeneration from cotyledon-derived protoplast of cabbage (Brassica oleracea var. capitata). Freshly isolated protoplasts were cultured in modified Murashige and Skoog (MS) medium removed ammonia ions and containing thiamine HCl, myo-inositol, 2,4-D, BA, sucrose and several concentrations of myo-inositol (2, 4, 6, 8, 10% (w/v)) as an osmotic stabilizer. After 3 weeks of culture in the dark at , the plating efficiency of cabbage protoplasts reached to when cultured in modified MS medium supplemented with 2,4-D, BA, sucrose and 8% (w/v) of myo-inositol at a density of protoplasts/ml. Rapidly growing cell colonies after 3 weeks of culture were transferred to the same culture medium removed osmoticum. To induce shoot regeneration from calluses, calluses with about 2 mm in diameter were transferred to the MS medium containing BA and NAA. After further three weeks of incubation onto the medium in the light, green shoots were formed on the surface of calluses at a frequency of 30%. Upon transfer to half-strength MS basal medium, roots were formed onto the bottom of regenerated shoots without auxin treatments. These regenerated plantlets were successfully acclimatized to soil transfer, grown to normal mature plants. The cabbage protoplast culture system established in this study could be applied for production of somatic hybrids or cybrids by asymmetric protoplast fusion and mass proliferation of elite somatic clones of cabbage.