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"callus"
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PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation
Lateral root organogenesis plays an essential role in elaborating plant root system architecture. In Arabidopsis, the AP2 family transcription factor PUCHI controls cell proliferation in lateral root primordia. To identify potential targets of PUCHI, we analyzed a time course transcriptomic dataset of lateral root formation. We report that multiple genes coding for very long chain fatty acid (VLCFA) biosynthesis enzymes are induced during lateral root development in a PUCHI-dependent manner. Significantly, several mutants perturbed in VLCFA biosynthesis show similar lateral root developmental defects as puchi-1. Moreover, puchi-1 roots display the same disorganized callus formation phenotype as VLCFA biosynthesis-deficient mutants when grown on auxin-rich callus-inducing medium. Lipidomic profiling of puchi-1 roots revealed reduced VLCFA content compared with WT. We conclude that PUCHI-regulated VLCFA biosynthesis is part of a pathway controlling cell proliferation during lateral root and callus formation.
Journal Article
Global epigenetic analysis revealed dynamic fluctuations in levels of DNA methylation and histone modifications in the calli of Fagopyrum with different capacity for morphogenesis
Buckwheat characterises with high susceptibility to in vitro tissue culture conditions, which have been researched extensively to study a plethora of processes. F. tataricum morphogenic callus (MC) is characterised by its capacity for morphogenesis for up to ten years of culture, displaying an extraordinary level of genome stability, and comprises of proembryogenic cell complexes (PECC),which are the structures resembling somatic embryos arrested on the pre-globular stage. The non-morphogenic callus (NC) that appears on the surface of MC after approximately two years of culture due to endoreduplication cycles, is characterised by aneuploidy, rapid growth rate and high level of oxidative stress. F. esculentum embryogenic callus (EC) has different morphological and histological features, remains stable for up to three years of culture, has a dense, globular structure, and is capable of forming embryoids from the masses of embryogenic cells, but does not produce a non-embryogenic clone. In this work, immunocytochemical analyses revealed dynamic epigenetic changes in Fagopyrum calli. We demonstrated that; decreased level of H3K4me2 seems to be associated with pluripotency acquisition in F. esculentum EC and F. tataricum MC; DNA hypomethylation appears to be connected with the acquisition of the embryogenic potential and PECC reinitiation in F. tataricum MC. Moreover, we observed that H4K16ac and H4K5ac exhibited the highest variability during the course of passage in NC. Elevated levels of these modifications on day zero and day six for H4K16ac and H4K5ac, respectively, seem to be connected with endoreplication peaks, the processes which are characteristic of this callus.Key messageEpigenetic changes accompany the dedifferentiation and re-differentiation processes in long-term callus cultures of Fagopyrum with different capacity for morphogenesis.
Journal Article
Differential Effects of Thidiazuron on Production of Anticancer Phenolic Compounds in Callus Cultures of Fagonia indica
Fagonia indica
, a very important anticancer plant, has been less explored for its in vitro potential. This is the first report on thidiazuron (TDZ)-mediated callogenesis and elicitation of commercially important phenolic compounds. Among the five different plant growth regulators tested, TDZ induced comparatively higher fresh biomass, 51.0 g/100 mL and 40.50 g/100 mL for stem and leaf explants, respectively, after 6 weeks of culture time. Maximum total phenolic content (202.8 μg gallic acid equivalent [GAE]/mL for stem-derived callus and 161.3 μg GAE/mL for leaf-derived callus) and total flavonoid content (191.03 μg quercetin equivalent [QE]/mL for stem-derived callus and 164.83 μg QE/mL for leaf-derived callus) were observed in the optimized callus cultures. The high-performance liquid chromatography (HPLC) data indicated higher amounts of commercially important anticancer secondary metabolites such as gallic acid (125.10 ± 5.01 μg/mL), myricetin (32.5 ± 2.05 μg/mL), caffeic acid (12.5 ± 0.52 μg/mL), catechin (9.4 ± 1.2 μg/mL), and apigenin (3.8 ± 0.45 μg/mL). Owing to the greater phenolic content, a better 2-2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity (69.45 % for stem explant and 63.68 % for leaf explant) was observed in optimized calluses. The unusually higher biomass and the enhanced amount of phenolic compounds as a result of lower amounts of TDZ highlight the importance of this multipotent hormone as elicitor in callus cultures of
F. indica
.
Journal Article
Green synthesized iron oxide nanoparticles as a potential regulator of callus growth, plant physiology, antioxidative and microbial contamination in Oryza sativa L
In tissue culture, efficient nutrient availability and effective control of callus contamination are crucial for successful plantlet regeneration. This study was aimed to enhance callogenesis, callus regeneration, control callus contamination, and substitute iron (Fe) source with FeO-NPs in Murashige and Skoog (MS) media. Nanogreen iron oxide (FeO-NPs) were synthesized and well characterized with sizes ranging from 2 to 7.5 nm. FeO-NPs as a supplement in MS media at 15 ppm, significantly controlled callus contamination by (80%). Results indicated that FeCl
3
-based FeO-NPs induced fast callus induction (72%) and regeneration (43%), in contrast FeSO
4
-based FeO-NPs resulted in increased callus weight (516%), diameter (300%), number of shoots (200%), and roots (114%). Modified media with FeO-NPs as the Fe source induced fast callogenesis and regeneration compared to normal MS media. FeO-NPs, when applied foliar spray, increased Plant fresh biomass by 133% and spike weight by 350%. Plant height increased by 54% and 33%, the number of spikes by 50% and 265%, and Chlorophyll content by 51% and 34% in IRRI-6 and Kissan Basmati, respectively. Additionally, APX (Ascorbate peroxidase), SOD (Superoxide dismutase), POD (peroxidase), and CAT (catalase) increased in IRRI-6 by 27%, 29%, 283%, 62%, while in Kissan Basmati, APX increased by 70%, SOD decreased by 28%, and POD and CAT increased by 89% and 98%, respectively. Finally, FeO-NPs effectively substituted Fe source in MS media, shorten the plant life cycle, and increase chlorophyll content as well as APX, SOD, POD, and CAT activities. This protocol is applicable for tissue culture in other cereal crops as well.
Journal Article
Genotype-dependent induction of embryogenic callus and programmed cell death in Korean pine
Background
The strong genotype dependence of embryogenic callus (EC) induction limits the high-frequency regeneration of Korean pine (
Pinus koraiensis
) via somatic embryogenesis (SE).
Methods
We examined the morphological and histocytological features of various callus types using external morphology, microstructure, and ultrastructure analyses. To assess their embryogenic potential, we conducted somatic embryogenesis (SE) experiments. Additionally, we investigated programmed cell death (PCD) events associated with genotype-dependent embryogenic callus (EC) formation in Korean pine by performing TUNEL assays, detecting caspase-3 and vacuolar processing enzyme (VPE) activity, and measuring H
2
O
2
and O
2
•−
levels.
Results
Obvious cell death was observed in the cell ultrastructure of LEC and NEC induced by recalcitrant genotypes. In LEC cells, we found typical characteristics of autolytic PCD, such as vacuole fusion, vacuolation of cells, invagination and rupture of the plasma membrane, few cytoplasm and organelles. In the NEC, degrading nucleus, withered cytoplasm and invagination and rupture of the plasma membrane were detected in early stage, but vacuole fusion was not observed. In addition, we observed DNA fragmentation into small fragments in LEC and NEC. Significant changes in the activities of caspase-3 and VPE, reactive oxygen species (ROS) levels, especially for H
2
O
2
, have been identified in LEC. However, in NEC, only the significant changes in the O
2
•−
level and the caspase-3 activity were observed.
Conclusion
PCD was present in NEC and LEC cells in recalcitrant genotypes of Korean pine, which may be responsible for the loss or reduction of embryonic capacity of induced Korean pine callus. Our novel findings on PCD help elucidate the underlying causes of the difficulty in inducing EC from recalcitrant genotypes of Korean pine from a novel perspective.
Journal Article
Effect of explant type and plant growth regulators on callus induction, growth and secondary metabolites production in Cnidium officinale Makino
This study reports the effect of explant type and plant growth regulators (PGRs) on callus induction in Cnidium officinale. Compared to stem, root explant showed maximum percent callus formation of 75% on Murashige and Skoog (MS) medium containing 2.3 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.2 µM benzyladenine (BA). At 30th day of callus culture on the said medium, callus fresh weight was sevenfold higher than other tested PGRs treatments. It was noted that MS medium supplemented with 27.1 µM 2,4-D showed the highest 0.30 mg g−1 DW of total phenols, while total flavonoids content reached to a maximum of 0.05 mg g−1 DW on the MS medium supplemented with 4.5 µM 2,4-D and 2.2 µM BA. Conversely, maximum (83.9%) DPPH free radical scavenging activity was observed in calli grown on the MS medium supplemented with 2.3 µM 2,4-D and 2.2 µM BA. The high-performance liquid chromatography (HPLC) analysis revealed higher phthalide content in callus than intact roots of in vitro plants. While 3-butylidenephthalide content in callus was comparable to the intact shoots and roots of in vitro grown C. officinale. The concentrations of 2,4-D played a significant role in the production of phthalide and 3-butylidenephthalide. Additional measures are recommended to further enhance their production in vitro.Graphical abstract
Journal Article
Promotive effect of phytosulfokine - peptide growth factor - on protoplast cultures development in Fagopyrum tataricum (L.) Gaertn
Background
Fagopyrum tataricum
(Tartary buckwheat) is a valuable crop of great nutritional importance due to its high level of bioactive compounds. Excellent opportunities to obtain plants with the high level or the desired profile of valuable metabolites may be provided by in vitro cultures. Among known in vitro techniques, protoplast technology is an exciting tool for genetic manipulation to improve crop traits. In that context, protoplast fusion may be applied to generate hybrid cells between different species of
Fagopyrum
. To apply protoplast cultures to the aforementioned approaches in this research, we established the protoplast-to-plant system in Tartary buckwheat.
Results
In this work, cellulase and pectinase activity enabled protoplast isolation from non-morphogenic and morphogenic callus (MC), reaching, on average, 2.3 × 10
6
protoplasts per g of fresh weight. However, to release protoplasts from hypocotyls, the key step was the application of driselase in the enzyme mixture. We showed that colony formation could be induced after protoplast embedding in agarose compared to the alginate matrix. Protoplasts cultured in a medium based on Kao and Michayluk supplemented with phytosulfokine (PSK) rebuilt cell walls, underwent repeated mitotic division, formed aggregates, which consequently led to callus formation. Plating efficiency, expressing the number of cell aggregate formed, in 10-day-old protoplast cultures varied from 14% for morphogenic callus to 30% for hypocotyls used as a protoplast source. However plant regeneration via somatic embryogenesis and organogenesis occurred only during the cultivation of MC-derived protoplasts.
Conclusions
This study demonstrated that the applied protoplast isolation approach facilitated the recovery of viable protoplasts. Moreover, the embedding of protoplasts in an agarose matrix and supplementation of a culture medium with PSK effectively stimulated cell division and further development of Tartary buckwheat protoplast cultures along with the plant regeneration. Together, these results provide the first evidence of developing a protoplast-to-plant system from the MC of
Fagopyrum tataricum
used as source material. These findings suggest that Tartary buckwheat’s protoplast cultures have potential implications for the species’ somatic hybridization and genetic improvement.
Journal Article
The mode of interfragmentary movement affects bone formation and revascularization after callus distraction
Callus distraction is sometimes associated with a delay in the maturation process and serious complications. It is believed that these complications are often caused by instability of the bone segment fixation. Typical fixation devices, such as ring-fixators, show significant deformations in all directions under external loading and muscle forces. This leads to axial compression and tension as well as shear movements in the healing area. Herein we investigated the hypothesis that the direction of interfragmentary movement after callus distraction affects the bone formation and revascularization during the maturation process. Two custom fixator systems were designed to apply a protocol of lateral callus distraction and subsequent cyclic stimulation of the regenerate tissue. One fixator system was used to apply either compressive or tensile stimulation while the other was used to apply shearing stimulation. The fixators were applied to the tibial surface of the right hind leg of sheep specimens. During lateral callus distraction, a titanium plate was elevated by 0.275 mm perpendicular to the long axis of the bone twice daily, resulting in a 5.5 mm gap at the end of the ten-day distraction phase. Following a seven-day consolidation phase, the regenerate in the gap between tibial cortex and titanium plate was stimulated once daily by cyclic movement for 120 cycles. The stimulation was applied for 18 days with amplitudes of 0.6 mm in compression (Group C) or tension (Group T), or a 1.0 mm shear amplitude (Group S). Seven weeks postoperatively the specimens were analyzed for quantity of bone formation, the presence of cartilage and fibrous tissue, and blood vessel density. There was a significantly higher blood vessel density (4.6 ± 1.6%) in Group C than in Group T (1.2 ± 0.4%) or Group S (1.0 ± 0.5%) (p < 0.01). The amount of bone was significantly higher in Group C (25.6% ± 13.0%) than in Group T (13.5 ± 4.9%) (p < 0.05). Group S showed a similar amount of bone (14.0 ± 10.7%) to Group T. The results show that bone formation and revascularization are dependent on the direction of interfragmentary movement and that the cyclic compression best stimulates the healing process.
Journal Article
A rare clinical presentation of plantar keratoderma
A 56-year-old male agricultural worker from a tropical region presented with progressive thickening and painful callus-like lesions on the right sole over the past six months. He reported no history of trauma or systemic illness. Initially, he used over-the-counter moisturisers and emollients, which offered minimal relief. Over time, the lesions worsened, causing painful fissures and difficulty walking. A course of topical antifungals and steroids provided no lasting improvement. Clinical examination revealed diffuse hyperkeratosis with yellowish plaques and deep fissures on the soles. Based on history and findings, a diagnosis of plantar keratoderma was made. Plantar keratoderma is a disorder characterised by abnormal thickening of the plantar skin due to hyperkeratosis. It may be hereditary or acquired, commonly affecting individuals exposed to chronic mechanical stress, especially those walking barefoot or working in agriculture in tropical climates. Typical presentation includes painless or painful thickened soles, fissures, and restricted mobility. If untreated, the condition can lead to secondary infections and chronic discomfort. Management involves regular mechanical debridement, keratolytic agents such as salicylic acid or urea creams, and, in severe cases, systemic retinoids. Identifying any underlying cause or systemic association is important for long-term control. Unlike minor calluses, plantar keratoderma requires consistent dermatological care to prevent complications and improve quality of life.
Journal Article