Response surface methodology mediated optimization of phytosulfokine and plant growth regulators for enhanced protoplast division, callus induction, and somatic embryogenesis in Angelica Gigas Nakai.

BACKGROUND: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas. RESULTS: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L- 1), kinetin (KN, 0, 0.5, and 1.0 mg L- 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L- 1 2,4-D + 0.5 mg L- 1 KN + 2 nM PSK, 0.5 mg L- 1 2,4-D + 0.9 mg L- 1 KN and 90 nM PSK, and 1.5 mg L- 1 2,4-D and 1 mg L- 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration. CONCLUSIONS: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.

Development of late-bolting plants by CRISPR/Cas9-mediated genome editing from mesophyll protoplasts of lettuce.

KEY MESSAGE: 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.

TSA Promotes CRISPR/Cas9 Editing Efficiency and Expression of Cell Division-Related Genes from Plant Protoplasts.

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.

The Effect of Sodium Butyrate on Adventitious Shoot Formation Varies among the Plant Species and the Explant Types.

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.

Temporal and Spatial Expression Analysis of Shoot-Regeneration Regulatory Genes during the Adventitious Shoot Formation in Hypocotyl and Cotyledon Explants of Tomato (CV. Micro-Tom).

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.

Effects of TSA, NaB, Aza in Lactuca sativa L. protoplasts and effect of TSA in Nicotiana benthamiana protoplasts on cell division and callus formation.

Whole-plant regeneration via plant tissue culture is a complex process regulated by several genetic and environmental conditions in plant cell cultures. Recently, epigenetic regulation has been reported to play an important role in plant cell differentiation and establishment of pluripotency. Herein, we tested the effects of chemicals, which interfere with epigenetic regulation, on the plant regeneration from mesophyll protoplasts of lettuce. The used chemicals were histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (NaB), and the DNA methyltransferase inhibitor azacytidine (Aza). All three chemicals increased cell division, micro-callus formation and callus proliferation in lettuce protoplasts. Cell division increased by more than 20% with an optimal treatment of the three chemicals. In addition, substantial increase in the callus proliferation rates was observed. In addition, TSA enhances cell division and adventitious shoot formation in the protoplast culture of Nicotiana benthamiana. The regenerated tobacco plants from TSA-treated protoplasts did not show morphological changes similar to the control. TSA increased histone H3 acetylation levels and affected the expression of CDK, CYCD3-1, and WUS in tobacco protoplasts. Thus, we investigated the effect of TSA, NaB, and Aza on Lactuca sativa L. protoplasts and the effect of TSA on cell division and callus formation in Nicotiana benthamiana protoplasts, which facilitates plant regeneration from mesophyll protoplasts. Furthermore, these chemicals can be directly applied as media additives for efficient plant regeneration and crop improvement in various plant species.

Efficient Plant Regeneration System from Leaf Explant Cultures of Daphne genkwa via Somatic Embryogenesis.

This study aimed to establish an efficient plant regeneration system from leaf-derived embryogenic structure cultures of Daphne genkwa. To induce embryogenic structures, fully expanded leaf explants of D. genkwa were cultured on Murashige and Skoog (MS) medium supplemented with 0, 0.1, 0.5, 1, 2, and 5 mg·L-1 2,4-dichlorophenoxyacetic acid (2,4-D), respectively. After 8 weeks of incubation, the highest frequency of embryogenic structure formation reached 100% when the leaf explants were cultivated on MS medium supplemented with 0.1 to 1 mg·L-1 2,4-D. At higher concentrations of 2,4-D (over 2 mg·L-1 2,4-D), the frequency of embryogenic structure formation significantly declined. Similar to 2,4-D, indole butyric acid (IBA) and α-naphthaleneacetic acid (NAA) treatments were also able to form embryogenic structures. However, the frequency of embryogenic structure formation was lower than that of 2,4-D. In particular, the yellow embryonic structure (YES) and white embryonic structure (WES) were simultaneously developed from the leaf explants of D. genkwa on culture medium containing 2,4-D, IBA, and NAA, respectively. Embryogenic calluses (ECs) were formed from the YES after subsequent rounds of subculture on MS medium supplemented with 1 mg·L-1 2,4-D. To regenerate whole plants, the embryogenic callus (EC) and the two embryogenic structures (YES and WES) were transferred onto MS medium supplemented with 0.1 mg·L-1 6-benzyl aminopurine (BA). The YES had the highest plant regeneration potential via somatic embryo and shoot development compared to the EC and WES. To our knowledge, this is the first successful report of a plant regeneration system via the somatic embryogenesis of D. genkwa. Thus, the embryogenic structures and plant regeneration system of D. genkwa could be applied to mass proliferation and genetic modification for pharmaceutical metabolite production in D. genkwa.

Anti-Inflammatory Effects of Weigela subsessilis Callus Extract via Suppression of MAPK and NF-κB Signaling.

Weigela subsessilis is used in folk medicine to treat pain and allergic syndromes in Korea. However, the antibacterial and anti-inflammatory activities of W. subsessilis callus extract remain unexplored. In this study, we aimed to evaluate the W. subsessilis callus of pharmacological activity. Therefore, we first established in vitro calluses of W.subsessilis via plant tissue culture methods. We then evaluated the antioxidant and anti-inflammatory effects of W. subsessilis callus extract in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. The W. subsessilis callus extract showed antioxidant and anti-inflammatory effects. These effects were regulated via suppression of mitogen-activated protein kinase signaling through LPS-induced translocation of nuclear factor kappa B (NF-κB) p65 from the cytoplasm to the nucleus. W. subsessilis callus extract also showed antibacterial and anti-inflammatory activities in Propionibacterium acnes-treated HaCaT keratinocyte cells. These results indicate that W. subsessilis callus extract has antioxidant, antibacterial and anti-inflammatory activities, suggesting its possible application in the treatment of inflammatory disorders.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus.

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.

Anti-Inflammatory Activities of an Extract of In Vitro Grown Adventitious Shoots of Toona sinensis in LPS-Treated RAW264.7 and Propionibacterium acnes-Treated HaCaT Cells.

Toona sinensis has been traditionally used to treat dysentery, enteritis, flatulence, and itchiness. However, the existence of anti-inflammatory effects of T. sinensis on Propionibacterium acnes-induced skin disease is unknown. In vitro cultures of plant cells and tissues produced under controlled conditions offer a continuous production platform for plant natural products including pigments and anti-inflammatory agents. In this study, we determine the anti-inflammatory activities of an extract of in vitro grown adventitious shoots of T. sinensis on P. acnes, the etiologic agent of skin inflammation. The extract of T. sinensis showed antioxidant and anti-inflammatory activity in LPS-treated RAW264.7 cells. It also had antibacterial activity and anti-inflammatory effects on P. acnes-treated HaCaT cells. In addition, these effects were regulated by suppression of the mitogen-activated protein kinase (MAPK) pathways. These results suggesting the potential application of adventitious shoots of T. sinensis grown with an in vitro proliferation system as a medicine for treating P. acnes-induced inflammatory skin disease.

Development of a Rapid Selection System for Salt-Resistant Mutants of Nicotiana benthamiana through Protoplast Culture after Gamma Irradiation.

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 ≥ 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.

Rapid metabolic discrimination and prediction of dioscin content from African yam tubers using Fourier transform-infrared spectroscopy combined with multivariate analysis.

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.