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9 recordsLinked to original sources
PubMed2026

An effective method for isolation and regeneration of Solanum tuberosum mesophyll protoplasts for transgene-free genome editing.

An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.

Callus induction↗
PubMed2026

Integrated metabolomics, transcriptional, and physicochemical analysis reveals key metabolites and genes associated with somatic embryogenesis in Phyllostachys pubescens.

Phyllostachys pubescens (Moso bamboo) is a significant perennial crop species that provides valuable nutritional and industrial uses, as well as carbon sequestration. Due to its remarkable growth rate, bamboo offers an ideal system for studying organogenesis, particularly in monocots. Somatic embryogenesis (SE) serves as a useful technique for crop breeding and improvement. SE in moso bamboo (Phyllostachys pubescens) remains challenging due to limited knowledge of its transcriptional and metabolomic reprogramming. To address this, we optimized callus initiation (MS + 18.1 µM 2,4-D + 8.5 µM picloram), callus proliferation (MS + 12.5 µM 2,4-D + 8.5 µM picloram), and somatic embryogenesis (MS + 1.1 µM 2,4-D + 3.3 µM metatopolin), using nodal segments as explants. UHPLC-Q-TOF-MS-based metabolite profiling revealed distinct biochemical trajectories across developmental stages of P. pubescens. NEC (non-embryogenic callus) was enriched in flavonoids, alkaloids, and saponins, while in-vitro shoots showed flavonoids and glycosides enrichment, and ex-vitro shoots showed high accumulation of glycosides and terpenoids. In contrast, EC (embryogenic callus) showed elevated levels of fatty acid derivatives (α-ESA, 26-Methyl Nigranoate), phytoalexins (Wyerone acid), sesquiterpene (Alpha-santalal, Beta-guaiene), flavonoid glycosides, and plant hormones (Cis-Zeatin, Gibberellin A45), indicating a metabolically active state supporting somatic embryogenesis. Similarly, genes and transcription factors controlling cell differentiation and embryogenesis were upregulated during SE. This study provides a comprehensive resource to facilitate future genomic and genetic investigations aimed at deciphering the molecular basis of organogenesis and advancing research on somatic embryogenesis in bamboo.

Plant Somatic Embryogenesis Techniques↗
PubMed2026

Establishment of an efficient Agrobacterium-mediated genetic transformation protocol for Saccharum officinarum using Black Cheribon as a model genotype.

Efficient Agrobacterium-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (Saccharum spp.). Saccharum officinarum is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for S. officinarum was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in Agrobacterium culture, concentration of AS for infection, Agrobacterium concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in S. officinarum transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of Bar protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of S. officinarum germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.

Agrobacterium↗
PubMed1977

Pentose-shunt oxidation in the periosteal cells in healing fractures.

The activity of pentose-shunt dehydrogenases is very low in periosteal cells of normal rat metatarsals, but increases one day post-fracture and rises linearly over the next two days. By four days post-fracture, the distribution of this activity along the bone shows two centres of high activity: the first in the region of proliferation to form callus and the second at the site where new bone is first seen, one day later. The high rate of generation of NADPH would be expected to reduce glutathione; reduced glutathione has been shown to inhibit alkaline phosphatase activity in these cells.

Alkaline Phosphatase↗
PubMed1975

Morphological and biochemical studies during differentiation and calcification of fracture callus cartilage.

Differentiation and calcification of cartilage of a fracture callus morphologically, ultrastructurally, and histochemically resembles cartilage of growing epiphyseal plate. The fracture callus includes the various cartilage cell types found in the epiphyseal plate. Proliferating and hypertrophic cartilage had higher activities of cytochrome oxidase, alkaline phosphatase and glutamate aspartate transaminase than fibrocartilage. Enzymes controlling glycogen synthesis and glycolysis had higher levels of activity in fibrocartilage than in hypertrophic cartilage. Lysosomal enzymes, catalase, 6-phospho-gluconic acid and glucose 6-phosphate dehydrogenase were uniformly distributed. Alkaline phosphatase was associated with extracellular vesicles found in hypertrophic cartilage. EM dense granules were found in mitochondria in hypertrophic cartilage. There was an increase of total lipids in hypertropic and calcified cartilage as compared to resting cartilage.

Alcian Blue↗
PubMed1979

Bone healing in rabbits after compression osteosynthesis: a comparative study between the radiological and histological findings.

The relationship between the radiological and histological findings was studied in a series of rabbit osteotomies and fractures stabilized with plates with and without longitudinal compression. When compression was employed, the gap varied between 10 and 200 micrometers and no radiologically visible callus was seen. After 4 weeks the smallest gaps of 10--30 micrometers were filled histologically with structureless material, while the larger gaps contained 'cortical' bone derived from periosteal and endosteal proliferation. The degree of union could not be assessed from X-ray examination, as there was no correlation between the histological and radiological appearances. When no compression was employed, the gap varied between 200--1200 micrometers; radiologically visible periosteal callus and filling of the gap indicated that consolidation had appeared in four weeks. Gaps of 200--500 micrometers were filled with bone derived from the periosteum and endosteum of lamellar structure which had invaded the original cortical bone ('plugging'). When the gaps were more than 500 micrometers, islands of cartilage and connective tissue were found. In all cases studied, many 'anchoring osteons' passed from the endosteal and periosteal callus into the original cortical bone. It is concluded that a 'compression internal fixation' of cortical bone will not necessarily secure union, as histologically confirmed, even when the formation of radiologically visible callus is suppressed. The lack of periosteal callus appears to be the result of a small gap and rigid fixation, rather than related to strains induced in the bone.

Animals↗
PubMed2026

Development of an Efficient Regeneration and Agrobacterium-Mediated Transformation Protocol for Hosta 'Light Star' Using the RUBY Reporter Gene.

Hosta plantaginea is a perennial shade-tolerant herb of the Liliaceae family, with high ornamental and urban greening value. Hosta 'Light Star' is a newly developed ornamental cultivar with yellow-margined leaves and lilac flowers, but no efficient in vitro regeneration or genetic transformation system has been established for this cultivar to date. In this study, we established a highly efficient in vitro regeneration system for Hosta 'Light Star,' and developed an Agrobacterium-mediated genetic transformation protocol using the RUBY visual reporter gene for non-invasive screening of positive transformants. The optimal callus induction medium was MS&#x2009;+&#x2009;2&#xa0;mg/L 6-BA&#x2009;+&#x2009;0.3&#xa0;mg/L NAA&#x2009;+&#x2009;0.05&#xa0;mg/L 2, 4-D, with a callus induction rate of 53.33% for leaf explants (the optimal explant for sterile seedlings). The optimal adventitious bud proliferation medium was MS&#x2009;+&#x2009;2&#xa0;mg/L 6-BA&#x2009;+&#x2009;0.1&#xa0;mg/L NAA, with a proliferation coefficient of 5.87. The optimal rooting medium was 1/2 MS&#x2009;+&#x2009;0.5&#xa0;mg/L NAA&#x2009;+&#x2009;0.5&#xa0;mg/L IBA, with a 100% rooting rate. The optimal transplant substrate was perlite:vermiculite&#x2009;=&#x2009;2:1, with a 100% transplant survival rate after acclimatization. For Agrobacterium-mediated transformation, the optimal infection parameters were as follows: Agrobacterium suspension OD600&#x2009;=&#x2009;0.6, infection time of 10&#xa0;min, and 200&#xa0;&#x3bc;M acetosyringone; the optimal selection conditions were 300&#xa0;mg/L cefotaxime for bacteriostasis and 30&#xa0;mg/L hygromycin for transformant screening. The final stable transformation efficiency was 2.50% (95% CI 1.23-3.77%), with an escape rate of 16.13%. Transgenic plants showed distinct purplish-red coloration in roots, stems, and leaves, with significantly higher betacyanin accumulation than wild-type plants (p&#x2009;<&#x2009;0.05). Stable integration and expression of the RUBY gene were confirmed by PCR, RT-PCR, and RT-qPCR. This study establishes the first efficient regeneration and Agrobacterium-mediated transformation system for Hosta 'Light Star,' and validates the feasibility of the RUBY reporter gene as a visual marker for Hosta transformation. This system provides a solid technical platform for functional genomic studies, CRISPR/Cas9-mediated gene editing, and molecular breeding of ornamental traits in Hosta.

Transformation, Genetic↗
PubMed1979

[New bone formation in an autologous spongy graft: an experimental morphological study].

Up to 5 days after an transplantation of autogenous cancellous bone in a defect of the ulna of dog which was stabilized by osteosynthesis no remarkable osteoblastic cell activity can be made out by light microscopy using the semithin sectioning technique. On the other hand, there is an obvious increase of osteogenic cells which normally only occur in small numbers in adult dog's cancellous bone of the iliac crest. Only after 7 days, numerous typical osteoblasts can be detected in association with transplanted cancellous chips, osteoblasts which seem to originate from the osteogenic cells by mitotic divisions. One week after the test's commencement, an extensive revascularisation of the transplant can be noticed as well. The osteoblastic cell activity and the forming of new bone are obviously dependent on a sufficient vascular supply in autogenous cancellous graft. The high osteogenic power of an autogenous cancellous graft is based on the ability of survival of the osteogenic cells, which proliferate and differentiate into osteoblasts in the course of one week, wherever its are close enough to functioning capillaries.

Animals↗