Callus Tissue Research Articles

An <i>in vitro Arabidopsis thaliana</i> Root Structural and Functional Characterization

Two models of Arabidopsis thaliana rhizogenesis in vitro were studied: 1) from callus and 2) leaf explants petioles on the Murashige and Skoog one-tenth strength hormone-free nutrient medium and also with the addition of the growth regulator such as indole-3-butyric acid. Morphological and anatomical studies show significant changes in the structure of the roots formed de novo in vitro from callus tissue, while the organs from leaf explant petioles were similar to those formed from the seed (primary). By Sabinin-Kolosov method, a decrease in the percentage of active root surface was established. Occurrence of the structural changes during in vitro rhizogenesis and their effect on root functionality are discussed.

Effects of plant growth regulators on callogenesis and embryogenesis in sarnav and desiree potato (Solanum tuberosum L.) varieties

Somatic embryos play a pivotal role in the production of high-quality potatoes and seed breeding. This study focused on determining the concentrations of 1-naphthaleneacetic acid (NAA) and 6-benzyl amino purine (BAP) in the formation of callus tissue and callus induction. Our goal was to assess the efficiency of potato explants with the highest potential for somatic embryo production. To achieve this, we cultivated Sarnav and Desiree potato varieties under in vitro tissue culture conditions, utilizing the obtained tissue cultures for subsequent experiments. The MS nutrient media were enriched with NAA and BAP at ratios of 1.5: 1, 1: 1.5, and 1: 1 mg/L, along with NAA concentrations of 1.5, 1, or 2 mg/L. Somatic embryogenesis experiments were conducted using various MS nutrient media, enriched with BAP and GA3 at concentrations of 1: 0.5, 0.4: 0.1, 0.5: 0.2, and 0.1: 0.1 mg/L of plant growth regulators. During the course of the study, diverse callus formations were observed in both leaf and internodal stem explants. Among the nutrient media, the M2 medium enriched with 1: 1.5 mg/L of NAA and BAP yielded the highest callus formation rates: 92% for the Desiree variety and 100% for the Sarnav variety, specifically in internodal stem explants. Notably, the index of embryo formation in leaf explants selected for somatic embryogenesis within the SE4 medium was 70% for the Sarnav variety and 65% for the Desiree variety. The inclusion of BAP and GA3 at a ratio of 0.1: 0.1 mg/l in the SE4 nutrient medium resulted in somatic embryogenesis in 80% of calli for the Sarnav variety and 78% for the Desiree variety. These findings underscore the potential for regenerating plants through somatic embryogenesis in the Sarnav potato variety, a significant development with implications for genetic transformation studies involving this particular variety.

Myosin heavy chain 2 (MYH2) expression in hypertrophic chondrocytes of soft callus provokes endochondral bone formation in fracture

AimsMuscle–bone interactions during fracture healing are rarely known. Here we investigated the presence and significance of myosin heavy chain 2 (MYH2), a component of myosin derived from muscles, in fracture healing. Main methodsWe collected five hematoma and seven soft callus tissues from patients with distal radius fractures patients, randomly selected three of them, and performed a liquid chromatography–mass spectrometry (LC-MS) proteomics analysis. Proteomic results were validated by histological observation, immunohistochemistry, and immunofluorescence for MYH2 expression. These findings were further confirmed in a murine femoral fracture model in vivo and investigated using various methods in vitro. Key findingsThe LC-MS proteomics analysis showed that MYH proteins were enriched in human soft calluses compared to hematoma. Notably, MYH2 protein is upregulated as high rank in each soft callus. The histological examination showed that MYH2 expression was elevated in hypertrophic chondrocytes within the human soft callus. Consistent with human data, Myh2 were significantly co-localized with Sox9 in hypertrophic chondrocytes of murine femoral fracture, in comparison to pre-hypertrophic and proliferating chondrocytes. Soluble MYH2 protein treatment increased MMP13 and RUNX2 expression in chondrocytes. In soluble MYH2 treatment, proliferation of chondrocytes was not altered, but the osteogenic and chondrogenic features of chondrocytes increased and decreased during differentiation, respectively. SignificanceThese findings indicate the potential of soluble MYH2 protein as a promising therapeutic strategy for promoting endochondral bone formation in chondrocytes following fracture.

Cilostazol promotes blood vessel formation and bone regeneration in a murine non-union model

Non-unions represent a major complication in trauma and orthopedic surgery. Many factors contribute to bone regeneration, out of which an adequate vascularization has been recognized as crucial. The phosphodiesterase-3 (PDE-3) inhibitor cilostazol has been shown to exert pro-angiogenic and pro-osteogenic effects in a variety of preclinical studies. Hence, we herein investigated the effects of cilostazol on bone regeneration in an atrophic non-union model in mice. For this purpose, a 1.8 mm femoral segmental defect was stabilized by pin-clip fixation and the animals were treated daily with 30 mg/kg body weight cilostazol or saline (control) per os. At 2, 5 and 10 weeks after surgery the healing of femora was analyzed by X-ray, biomechanics, photoacoustic imaging, and micro-computed tomography (µCT). To investigate the cellular composition and the growth factor expression of the callus tissue additional histological, immunohistochemical and Western blot analyses were performed. Cilostazol-treated animals showed increased bone formation within the callus, resulting in an enhanced bending stiffness when compared to controls. This was associated with a more pronounced expression of vascular endothelial growth factor (VEGF), a higher number of CD31-positive microvessels and an increased oxygen saturation within the callus tissue. Furthermore, cilostazol induced higher numbers of tartrate-resistant acidic phosphate (TRAP)-positive osteoclasts and CD68-positive macrophages. Taken together, these findings demonstrate that cilostazol is a promising drug candidate for the adjuvant treatment of atrophic non-unions in clinical practice.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

THE OBTAINING RHODIOLA ROSEA L. BIOLOGICALLY ACTIVE COMPOUNDS BY BIOTECHNOLOGICAL METHODS (OVERVIEW)

Due to its rich chemical composition including unique biologically active compounds the gold root (Rhodiola rosea L.) is still of interest to researchers all over the world. However, deficiency of the raw material base and metabolome variability are determination the modern development ways of pharmacologically active substances in vitro obtaining. The article summarizes and systematizes the information about of Rhodiola rosea biologically active compounds obtaining, which are most often obtaining from callus tissues. Callus tissue is usually obtaining from leaf explants, and Murashige and Skoog medium with plant growth regulators in various combinations and concentrations is most commonly used for its induction. At the same time, the tissue growth efficiency and target compounds accumulation depends on the intact plant genotype, the precursors presence of these compounds in the medium, elicitors, as well as external influence factors. Recently, a promising direction is the transgenic hairy roots cultivation, which reveals new aspects of synthesis, accumulation and secondary metabolites production regulation. Biosynthesis genetic regulation of significant secondary metabolites is also a promising direction. Numerous studies have demonstrated that salidroside biosynthesis depends on the expression of the TyrDC gene encoding tyrosinedecarboxylase. Consequently, understanding the molecular and genetic mechanisms opens up opportunities for its regulation and metabolic engineering. In this regard, biotechnological methods may be a priority to obtaining salidroside, rosin and their derivatives at higher or at least comparable levels with wild type or cultivated plants.
 Among the various biotechnological strategies used to increase the accumulation of salidroside and glycosides of cinnamic alcohol in cells and cultures of Rhodiola organs, the approach with the addition of the metabolic precursors to the nutrient medium proved to be the most effective. In vitro cultures, a significant increase in the production of rosin and its derivatives was observed when precursors (cinnamic alcohol, cinnamic acid and cinnamic aldehyde) were added to the medium.

Sildenafil, a phosphodiesterase-5 inhibitor, stimulates angiogenesis and bone regeneration in an atrophic non-union model in mice

Non-union formation represents a major complication in trauma and orthopedic surgery. The phosphodiesterase-5 (PDE-5) inhibitor sildenafil has been shown to exert pro-angiogenic and pro-osteogenic effects in vitro and in vivo. Therefore, the aim of the present study was to analyze the impact of sildenafil in an atrophic non-union model in mice. After creation of a 1.8 mm segmental defect, mice femora were stabilized by pin-clip fixation. Bone regeneration was analyzed by means of X-ray, biomechanics, photoacoustic and micro-computed tomography (µCT) imaging as well as histological, immunohistochemical and Western blot analyses at 2, 5 and 10 weeks after surgery. The animals were treated daily with either 5 mg/kg body weight sildenafil (n = 35) or saline (control; n = 35) per os. Bone formation was markedly improved in defects of sildenafil-treated mice when compared to controls. This was associated with a higher bending stiffness as well as an increased number of CD31-positive microvessels and a higher oxygen saturation within the callus tissue. Moreover, the bone defects of sildenafil-treated animals contained more tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and CD68-positive macrophages and exhibited a higher expression of the pro-angiogenic and pro-osteogenic markers cysteine rich protein (CYR)61 and vascular endothelial growth factor (VEGF) when compared to controls. These findings demonstrate that sildenafil acts as a potent stimulator of angiogenesis and bone regeneration in atrophic non-unions.

The accordion technique enhances bone regeneration via angiogenesis factor in a rat distraction osteogenesis model.

Objective: The purpose of this study was to observe the effect of the accordion technique (AT) during the distraction phase on chondrogenesis and bone regeneration in a rat femoral distraction osteogenesis (DO) model, and investigate its potential mechanism for reducing the total treatment time of DO. Methods: Fifty-four male Sprague-Dawley (SD) rats that were specific-pathogen-free (SPF) were subjected to DO surgery on the right femur. The distraction rate was 0.5mm/day for 10days, following a latency period of 5days. Rats were randomly divided into Control (no AT, n = 18), Group LA (low amplitude with AT, n = 18), and Group HA (high amplitude with AT, n = 18) according to different AT protocols in the distraction phase. Rats were respectively euthanized by anesthesia overdose at 2, 4 and 6weeks of the consolidation phase, and the femurs were harvested. Digital radiography, micro-computed tomography (micro-CT), biomechanical tests, and histomorphological analysis were used to assess the quality of regenerated bone in the distraction area. Results: Digital radiographic, micro-CT, biomechanical tests, and histological analysis revealed an increase in early-stage callus formation (p < 0.05) and improved blood supply to the callus tissue in Group LA, as compared to both the Control and Group HA. The enhanced differentiation of fibrous and cartilaginous tissue into bone tissue was also observed in Group LA, leading to improved strength and stiffness (p < 0.05) of the regenerated bone at 6weeks of the consolidation phase. The angiogenic (hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF), p < 0.05) and osteogenic (runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and osteopontin (OPN), p < 0.05) biomarkers were higher expressed in Group LA at 2 and 4weeks of consolidation phase, whereas decreased at 6 weeks of consolidation phase. Conclusion: The application of AT with low amplitude during the distraction phase can enhance chondrogenesis and bone regeneration by activating the angiogenesis factor pathway and upregulating the expression of osteogenic-related biomarkers such as HIF-1α, VEGF, RUNX2, OCN, and OPN.

Flavonoid Metabolome-Based Active Ingredient Mining and Callus Induction in Catalpa bungei C. A. Mey

Catalpa bungei C. A. Mey is a unique and precious multi-purpose tree species that possesses great timber-related, ornamental and medicinal values. In this study, MS, N6 and DKW were used as basic media, and different concentrations of 6-BA and NAA were added for callus induction. The induction rate and total flavonoid content of callus tissue showed that the best callus induction medium was DKW + 2.0 mg·L−1 6-BA + 0.5–1.0 mg·L−1 NAA. The leaves of different bark phenotypes of C. bungei C. A. Mey and the callus tissue extracted from young leaves of C. bungei C. A. Mey were used as experimental materials to construct metabolomic profiles of widely targeted flavonoids. Based on the metabolomic databases, the predominant flavonoids were screened from the callus tissues. Eight flavonoid metabolites increased in callus, and diosmetin-7-O-rutinoside (diosmin) was the flavonoid constituent with the shortest retention time, most efficient detection and best medicinal functions among these 8. The optimal medium for callus induction was supplemented with different concentrations of elicitors (salicylic acid SA and yeast extract YE). The optimal elicitor and the amount to be added were determined by analyzing the induction rate of callus, as well as the total contents of flavonoids and diosmin. The addition of SA and YE in appropriate amounts increased the total flavonoid content in the callus, but only the addition of YE promoted the formation of diosmin in the callus. The optimal medium formulation to promote the formation of callus was DKW + 2.0 mg·L−1 6-BA + 1.0 mg·L−1 NAA + 30 g·L−1 sucrose + 6.8 g·L−1 agar + 10 μmol·L−1 SA. The medium formulation to optimally increase the content of geraniol glucoside was DKW + 2.0 mg·L−1 6-BA + 1.0 mg·L−1 NAA + 30 g·L−1 sucrose + 6.8 g·L−1 agar + 200 mg·L−1 YE. The results of the present study will provide a scientific basis for the subsequent increase in the content of the active components of the suspension cells via the addition of elicitors, and for the production of diosmin in factory settings.

CD51 labels periosteal injury-responsive osteoprogenitors.

The periosteum is a critical source of skeletal stem and progenitor cells (SSPCs) that form callus tissue in response to injury. There is yet to be a consensus on how to identify SSPCs in the adult periosteum. The aim of this study was to understand how potential murine periosteal SSPC populations behave in vivo and in response to injury. We evaluated the in vivo differentiation potential of Sca1-CD51+ and Sca1+CD51+ cells following transplantation. In vitro, the Sca1+CD51+ population appears to be more primitive multipotent cells, but after transplantation, Sca1-CD51+ cells showed superior engraftment, expansion, and differentiation into chondrocytes and osteoblasts. Despite representing a clear population with flow cytometry, we identified very few Sca1+CD51+ cells histologically. Using a periosteal scratch injury model, we successfully mimicked the endochondral-like healing process seen in unstable fractures, including the expansion and osteochondral differentiation of αSMA+ cells following injury. CD51+ cells were present in the cambium layer of resting periosteum and expanded following injury. Sca1+CD51- cells were mainly localized in the outer periosteal layer. We found that injury increased colony-forming unit fibroblast (CFU-F) formation in the periosteum and led to rapid expansion of CD90+ cells. Several other populations, including Sca1-CD51+ and CD34+ cells, were expanded by day 7. Mice with enhanced fracture healing due to elevated Notch signaling mediated by NICD1 overexpression showed significant expansion of CD51+ and CD34hi cells in the early stages of healing, suggesting these populations contribute to more rapid healing. In conclusion, we demonstrate that periosteal injury leads to the expansion of various SSPC populations, but further studies are required to confirm their lineage hierarchy in the adult skeletal system. Our data indicate that CD51+ skeletal progenitor cells are injury-responsive and show good engraftment and differentiation potential upon transplantation.

Genome-Wide Identification and Expression Analysis of BrGeBP Genes Reveal Their Potential Roles in Cold and Drought Stress Tolerance in Brassica rapa.

The GLABROUS1 Enhancer Binding Protein (GeBP) gene family is pivotal in regulating plant growth, development, and stress responses. However, the role of GeBP in Brassica rapa remains unclear. This study identifies 20 BrGeBP genes distributed across 6 chromosomes, categorized into 4 subfamilies. Analysis of their promoter sequences reveals multiple stress-related elements, including those responding to drought, low temperature, methyl jasmonate (MeJA), and gibberellin (GA). Gene expression profiling demonstrates wide expression of BrGeBPs in callus, stem, silique, and flower tissues. Notably, BrGeBP5 expression significantly decreases under low-temperature treatment, while BrGeBP3 and BrGeBP14 show increased expression during drought stress, followed by a decrease. Protein interaction predictions suggest that BrGeBP14 homolog, At5g28040, can interact with DES1, a known stress-regulating protein. Additionally, microRNA172 targeting BrGeBP5 is upregulated under cold tolerance. These findings underscore the vital role of BrGeBPs in abiotic stress tolerance. Specifically, BrGeBP3, BrGeBP5, and BrGeBP14 show great potential for regulating abiotic stress. This study contributes to understanding the function of BrGeBPs and provides valuable insights for studying abiotic stress in B. rapa.

The Method and Effect of Landscape Tree Surgery

樹木對居住在城市的人類生活影響巨大，不僅能美化環境、過濾空氣和水、更能激發正向情緒，展現各城市於文化、歷史、地理、生態等特色。因此城市中樹木的健康和風險問題更應被關注，以避免樹木感染災害及無預警倒伏或枝條斷落等公共安全問題。&lt;br&gt;本研究目的主要是進行藉由「景觀樹木VTA健康檢查診斷評估表」探討經由「改良式樹木外科手術」的腐朽處理，包括傷口腐朽清創去除、高溫碳化保護及塗佈傷口保護藥劑處理的9個案例的分析，探討其是否能有效的抑止腐朽菌和促進樹木生長勢的恢復。&lt;br&gt;研究結果顯示，使用｢景觀樹木VTA健康檢查診斷評估表」，評估改良式樹木外科手術前和後的樹木的生長狀況，是可行但有其限制，術後若達五年以上的生長恢復期，則VTA評估表的分數和樹木健康程度才會有呈現出顯著關係。若恢復期不及五年，則需再配合觀察腐朽傷口癒傷組織的形成、傷口面積的減少、無流出組織液等來判定「改良式樹木外科手術｣對樹木生長幫助。&lt;br&gt;樹木外科手術的成效，在國內外的各方專家學者有許多不同的看法，這些歧異或許是因為植物種類不同、環境風土差異、動植物生態差異、各家所使用處理方法不同。後續研究可以針對這些歧異度如何影響外科手術的成效來進行探討。Trees have a huge impact on human being living in cities. They can not only beautify the environment, filter air and water, but also stimulate positive emotions and represent the cultural, historical, geographical, ecological, and other characteristics of each city. Therefore, more attentions should be paid to the health and risk issues of trees in cities to avoid tree infection disasters and public safety issues such as unexpected tree falling without warning or branch breakage. &lt;br&gt;The purpose of this study is mainly to explore the decay treatment through ''modified tree surgery'' through the ''Landscape Tree VTA (Visual Tree Assessment) Form'', including wound decay debridement and removal, high-temperature carbonization protection, and wound protection agent application. An analysis of 9 cases was conducted to explore whether it can effectively inhibit decay bacteria and promote the recovery of tree growth. &lt;br&gt;The research results show that using the '' Landscape Tree VTA Form '' to evaluate the growth status of trees before and after modified tree surgery is feasible but has its limitations. If there is a growth recovery period of more than five years after surgery, then there will be a significant relationship between the score of the VTA and the health of the tree. If the recovery period is less than five years, it is necessary to observe the formation of callus tissue from the decayed wound, the reduction of the wound area, and the absence of tissue fluid leakage to determine whether the ''modified tree surgery'' will help the tree growth. &lt;br&gt;Experts and scholars worldwide have many different opinions on the effectiveness of tree surgery. These differences may be due to different plant species, different environmental conditions, different animal and plant ecology, and different treatment methods used by each company. Follow-up research could explore how these differences affect surgical outcomes.

Tissue-specific stilbene accumulation is an early response to wounding/grafting as revealed by using spatial and temporal metabolomics.

Grafting is widely used in horticulture. Shortly after grafting, callus tissues appear at the graft interface and the vascular tissues of the scion and rootstock connect. The graft interface contains a complex mix of tissues, we hypothesised that each tissue has its own metabolic response to wounding/grafting and accumulates different metabolites at different rates. We made intact and wounded cuttings and grafts of grapevine, and then measured changes in bulk flavonoid, phenolic acid and stilbenoid concentration and used metabolite imaging to study tissue-specific responses. We show that some metabolites rapidly accumulate in specific tissues after grafting, for example, stilbene monomers accumulate in necrotic tissues surrounding mature xylem vessels. Whereas other metabolites, such as complex stilbenes, accumulate in the same tissues at later stages. We also observe that other metabolites accumulate in the newly formed callus tissue and identify genotype-specific responses. In addition, exogenous resveratrol application did not modify grafting success rate, potentially suggesting that the accumulation of resveratrol at the graft interface is not linked to graft union formation. The increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms), and potentially increases antioxidant activity and antifungal capacities.

A Common Molecular Signature Indicates the Pre-Meristematic State of Plant Calli.

In response to different degrees of mechanical injury, certain plant cells re-enter the division cycle to provide cells for tissue replenishment, tissue rejoining, de novo organ formation, and/or wound healing. The intermediate tissue formed by the dividing cells is called a callus. Callus formation can also be induced artificially in vitro by wounding and/or hormone (auxin and cytokinin) treatments. The callus tissue can be maintained in culture, providing starting material for de novo organ or embryo regeneration and thus serving as the basis for many plant biotechnology applications. Due to the biotechnological importance of callus cultures and the scientific interest in the developmental flexibility of somatic plant cells, the initial molecular steps of callus formation have been studied in detail. It was revealed that callus initiation can follow various ways, depending on the organ from which it develops and the inducer, but they converge on a seemingly identical tissue. It is not known, however, if callus is indeed a special tissue with a defined gene expression signature, whether it is a malformed meristem, or a mass of so-called "undifferentiated" cells, as is mostly believed. In this paper, I review the various mechanisms of plant regeneration that may converge on callus initiation. I discuss the role of plant hormones in the detour of callus formation from normal development. Finally, I compare various Arabidopsis gene expression datasets obtained a few days, two weeks, or several years after callus induction and identify 21 genes, including genes of key transcription factors controlling cell division and differentiation in meristematic regions, which were upregulated in all investigated callus samples. I summarize the information available on all 21 genes that point to the pre-meristematic nature of callus tissues underlying their wide regeneration potential.

Fındıkta Dilciksiz Aşı Tekniğinde Kaynaşmanın Anatomik ve Histolojik Gelişimi

The anatomical and histological development of the graft union in splice grafting in hazelnut (Corylus avellana L.) was examined. One-year suckers were used as rootstock and scion materials. Grafting was done manually in the first week of December. The grafted plants were kept for 28 days at 26-28 °C and 70-80% relative humidity conditions. The cross and longitudinal sections with a thickness of 12-20 microns taken from the graft union area by a rotary microtome 14, 18, 21, 26, 32, 52 ve 140 days after grafting were examined microscopically. Early callus proliferation from rootstock and particularly scion two weeks after grafting was generally slow. The initial cambial differentiations in callus tissues were seen in sections 18 days after grafting. Cambial continuity between rootstock and scion was established 32 days after grafting. The sections in the following periods exhibited that the graft partners were in vascular relationship. The development of the graft union was successfull with all its stages. It was observed that the amount of callus tissue proliferated during the first two weeks after grafting directed the subsequent development of the union. In this respect, it is thought that developing methods that encourage callus formation in the early periods of the union will increase the success in related studies. The anatomical and histological examinations revealed that rootstock and scion thicknesses that are very close to each other and accordingly well matching of graft partners from cambial zones affect the development of union positively and prepare a suitable basis for early vascular differentiation.

Optimization of Callus Induction and Shoot Regeneration from Tomato Cotyledon Explants.

Cultivated tomato (Solanum lycopersicum L.) is one of the most important horticultural crops in the world. The optimization of culture media for callus formation and tissue regeneration of different tomato genotypes presents numerous biotechnological applications. In this work, we have analyzed the effect of different concentrations of zeatin and indole-3-acetic acid on the regeneration of cotyledon explants in tomato cultivars M82 and Micro-Tom. We evaluated regeneration parameters such as the percentage of callus formation and the area of callus formed, as well as the initiation percentage and the number of adventitious shoots. The best hormone combination produced shoot-like structures after 2-3 weeks. We observed the formation of leaf primordia from these structures after about 3-4 weeks. Upon transferring the regenerating micro-stems to a defined growth medium, it was possible to obtain whole plantlets between 4 and 6 weeks. This hormone combination was applied to other genotypes of S. lycopersicum, including commercial varieties and ancestral tomato varieties. Our method is suitable for obtaining many plantlets of different tomato genotypes from cotyledon explants in a very short time, with direct applications for plant transformation, use of gene editing techniques, and vegetative propagation of elite cultivars.

Shoot Culture of Ocimum sp. and Its Phytochemical Profile

The success of the in vitro culture technique is influenced by many factors, including the type of explants, culture medium and exogenous plant growth regulators (PGR). These factors greatly influence the speed and effectiveness of plant regeneration as well as the profile of the phytochemical compounds produced. Therefore, this study aims to determine the effect of the PGR combination on the growth response of in vitro node explants as well as the phytochemical profiles of Ocimum sp. Node explants obtained from 2 weeks old in vitro seedlings were cultured on MS medium with the addition of PGR, namely BAP and Kinetin (0.2 and 5 mg/l) combined with NAA (0.1 and 0.2 mg/l) and synthetic cytokinins alone namely Thidiazuron (TDZ) (1, 3, and 5 mg/l). The growth response of the explants and the potential for regeneration were observed for 8 weeks of culture. Effects of adding activated charcoal (AC) to root media were observed on growth of plantlets aged 2 weeks. Phytochemical profile of In vitro shoot was analyzed using GC-MS and LC-MS to be compared with its profile in in vitro callus tissue. The results showed that MS medium with the addition of a combination of cytokinin and auxin was able to induce shoot regeneration in node explants of Ocimum sp. in vitro. The Kinetin/NAA combination produced better shoot height growth, while the BAP/NAA combination produced a higher leaves number. Thidiazuron at all concentrations was able to induce shoots that were more likely to form rosettes. The addition of AC to the rooting medium did not have a positive effect on the response of shoot and plantlet growth. Chromatographic screening showed different profiles of secondary compounds in the callus and shoot tissues of Ocimum sp. in vitro. Callus composed of actively dividing cells do not produce some of the secondary compounds as produced by in vitro shoots. This shows the difference in the potential of cells or tissues in synthesizing secondary metabolites.

Characterization and Potential Function Analysis of the SRS Gene Family in Brassica napus.

SRS (SHI-related sequence) transcription factors play a crucial role in plant growth, development, and abiotic stress response. Although Brassica napus (B. napus) is one of the most important oil crops in the world, the role of SRS genes in B. napus (BnSRS) has not been well investigated. Therefore, we employed a bioinformatics approach to identify BnSRS genes from genomic data and investigated their characteristics, functions, and expression patterns, to gain a better understanding of how this gene family is involved in plant development and growth. The results revealed that there were 34 BnSRS gene family members in the genomic sequence of B. napus, unevenly distributed throughout the sequence. Based on the phylogenetic analysis, these BnSRS genes could be divided into four subgroups, with each group sharing comparable conserved motifs and gene structure. Analysis of the upstream promoter region showed that BnSRS genes may regulate hormone responses, biotic and abiotic stress response, growth, and development in B. napus. The protein-protein interaction analysis revealed the involvement of BnSRS genes in various biological processes and metabolic pathways. Our analysis of BnSRS gene expression showed that 23 BnSRS genes in the callus tissue exhibited a dominant expression pattern, suggesting their critical involvement in cell dedifferentiation, cell division, and tissue development. In addition, association analysis between genotype and agronomic traits revealed that BnSRS genes may be linked to some important agronomic traits in B. napus, suggesting that BnSRS genes were widely involved in the regulation of important agronomic traits (including C16.0, C18.0, C18.1, C18.2 C18.3, C20.1, C22.1, GLU, protein, TSW, and FFT). In this study, we predicted the evolutionary relationships and potential functions of BnSRS gene family members, providing a basis for the development of BnSRS gene functions which could facilitate targeted functional studies and genetic improvement for elite breeding in B. napus.

Metformin regulates bone marrow stromal cells to accelerate bone healing in diabetic mice.

Diabetes mellitus is a group of chronic diseases characterized by high blood glucose levels. Diabetic patients have a higher risk of sustaining osteoporotic fractures than non-diabetic people. The fracture healing is usually impaired in diabetics, and our understanding of the detrimental effects of hyperglycemia on fracture healing is still inadequate. Metformin is the first-line medicine for type 2 diabetes (T2D). However, its effects on bone in T2D patients remain to be studied. To assess the impacts of metformin on fracture healing, we compared the healing process of closed-wound fixed fracture, non-fixed radial fracture, and femoral drill-hole injury models in the T2D mice with and without metformin treatment. Our results demonstrated that metformin rescued the delayed bone healing and remolding in the T2D mice in all injury models. In vitro analysis indicated that compromised proliferation, osteogenesis, chondrogenesis of the bone marrow stromal cells (BMSCs) derived from the T2D mice were rescued by metformin treatment when compared to WT controls. Furthermore, metformin could effectively rescue the impaired detrimental lineage commitment of BMSCs isolated from the T2D mice in vivo as assessed by subcutaneous ossicle formation of the BMSC implants in recipient T2D mice. Moreover, the Safranin O staining of cartilage formation in the endochondral ossification under hyperglycemic condition significantly increased at day 14 post-fracture in the T2D mice receiving metformin treatment. The chondrocyte transcript factors SOX9 and PGC1α, important to maintain chondrocyte homeostasis, were both significantly upregulated in callus tissue isolated at the fracture site of metformin-treated MKR mice on day 12 post-fracture. Metformin also rescued the chondrocyte disc formation of BMSCs isolated from the T2D mice. Taken together, our study demonstrated that metformin facilitated bone healing, more specifically bone formation and chondrogenesis in T2D mouse models.

FEATURES OF GROWTH AND INULIN CONTENT IN CALLUS CULTURES Cichorium intybus L. in vitro

In vitro callus cultures of common chicory (Cichorium intybus L.) were obtained and their growth and biochemical characteristics depending on the hormonal composition of the MS medium and the spectral composition of light were studied. The study of the effect of light culture on callus tissue formation and inulin accumulation in it was carried out in opaque grow tents with radiation aligned with the flux density of pho- tosynthetic photons and different ratios of its levels in the region of 660 nm (R, red) and 730 nm (FR, far red). The control variant was placed under white linear fluorescent lamps. The resulting cultures were character- ized by high proliferative activity and the capability for morphogenesis. It has been established that the inter- action of two factors—the presence of auxins in the nutrient medium (IAA or NAA at a concentration of 7.5 mg/L in combination with BAP 0.5 mg/L) and cultivation under light culture conditions (FR R, FR = R, FR R)—had a significant impact on the biosynthetic potential of cell cultures. In the obtained cultures, a study of the quantitative content of inulin was carried out. It has been shown that the high content of inulin (7.55–7.95%) in callus cultures was on the MS medium in combination with IAA at FR R illumination. This is probably due to the fact that well proliferating and highly morphogenic callus tissue was formed under these conditions. The obtained results confirm the hypothesis about the specificity of cultured cells to in vitro synthesize and accumulate secondary metabolites in dedifferentiated cells and the dependence of this process on factors of chemical and physical nature.