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At least 19 recordsLinked to original sources
PubMed2026

Dual-gRNA CRISPR/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration.

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of 'Valencia' and hybrid genotypes (CsH1-CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L-1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in 'Valencia' and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.

CRISPR/Cas9↗
PubMed1977

Pituitary enhancement of Wolffian lens regeneration in vitro: spatial and temporal requirements.

When irido-corneal complexes (ICC's) from eyes of adult newts (Notophthalmus viridescens) are cultured on top of pituitary glands they produce advanced lens regenerates in up to 90% of the cases (this report and Connelly et al., '73). To determine if the iris and pituitary gland must be in direct contact with each other in order to produce this enhancement of lens regeneration, ICC's were cultured next to pituitary glands or separated from them by a Nuclepore filter. Cultures behaved as ICC's cultured in the absence of the pituitary gland and produced few advanced lens regenerates. To determine how long the iris and pituitary must be in contact for the enhancement to occur, ICC's were explanted and 5, 10 or 15 days later pituitary glands were placed beneath the dorsal iris pupillary margin. There was a progressive decrease in the number of advanced lens regenerates formed in cultures receiving pituitary glands later than five days after initial explantation of the iris. The conclusions drawn from these experiments are that the iris and pituitary gland must be in extensive direct contact during the first five to ten days in culture in order for pronounced enhancement of lens regeneration to occur.

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↗
PubMed2026

Establishment of an in vitro culture and regeneration protocol for the native Chilean grass Polypogon australis Brong.

Polypogon australis Brong. is a native Chilean grass frequently found colonizing metal-rich mine tailings, yet it lacks an established in vitro regeneration system to support controlled physiological and biotechnological studies. Here, we report a reproducible protocol for seed germination, callus induction, and plant regeneration using coleoptile-mesocotyl explants. Surface-sterilized seeds were germinated on Murashige and Skoog (MS) medium supplemented with sucrose, achieving a cumulative germination percentage of 47.67&#x2009;&#xb1;&#x2009;3.15% after 15 days. The coleoptile-mesocotyl explant proved highly responsive to culture on callus induction medium (CIM) supplemented with dicamba, resulting in a callus induction frequency of 30.55&#x2009;&#xb1;&#x2009;11.96% after 3-5 weeks. Induced calli were predominantly embryogenic, with embryogenic calli representing 65.42&#x2009;&#xb1;&#x2009;8.61% of the total callus population. Embryogenic calli regenerated complete plantlets with a regeneration efficiency of 45.0&#x2009;&#xb1;&#x2009;23.3%. Organogenic structures, including primary shoots and roots, developed directly from embryogenic calli maintained on callus induction medium (CIM) supplemented with dicamba, without transfer to a specialized regeneration medium containing organogenesis-promoting growth regulators. After the initiation of organogenesis, cultures were exposed to a 16&#xa0;h light/8&#xa0;h dark photoperiod while remaining on CIM, and regenerated plantlets were subsequently transferred to MS+10&#xa0;S medium for further growth and elongation. This study establishes the first complete in vitro regeneration system for P. australis, providing a practical framework for future physiological studies, large-scale propagation, genetic transformation, and genome engineering applications in this ecologically relevant native Chilean grass.

Regeneration↗
PubMed2026

A stable transformation platform in pomegranate uncovers PgMYB10 as a key regulator of anthocyanin biosynthesis.

An efficient genetic transformation platform enables functional validation of PgMYB10, identifying it as a master regulator governing anthocyanin biosynthesis in pomegranate. Limited availability of stable genetic transformation systems restricts functional genomics research in pomegranate. Here, we established efficient in vitro regeneration and Agrobacterium tumefaciens-mediated transformation systems for 'Taishanhong' pomegranate using stem segment explants. Optimized medium combinations produced high-frequency regeneration: a 93.3% shoot-induction rate on MS medium with 1.5&#xa0;mg/L 6-benzylaminopurine (BAP), 0.6&#xa0;mg/L 1-naphthaleneacetic acid (NAA) and 30.0&#xa0;mg/L adenine sulfate (ADS); a proliferation coefficient of 5.4 on MS medium&#xa0;supplemented with 0.8&#xa0;mg/L BAP and 0.3&#xa0;mg/L indole&#x2011;3&#x2011;butyric acid (IBA); effective shoot-strengthening on MS&#xa0;medium containing 1.2&#xa0;mg/L BAP, 0.3&#xa0;mg/L NAA and 0.2&#xa0;mg/L gibberellic acid (GA&#x2083;); and a rooting rate of 95.3% on half-strength MS medium with 1.5&#xa0;mg/L IBA and 0.5&#xa0;mg/L NAA. For transformation, precultured explants were immersed with A. tumefaciens suspension (OD&#x2086;&#x2080;&#x2080; = 0.8) containing 20.0&#xa0;mg/L acetosyringone (AS) for 30&#xa0;min. After four days of dark co-culture, sequential antibiotic screening with 30&#xa0;mg/L kanamycin and bacteriostatic treatment with 400&#xa0;mg/L timentin yielded a stable average transformation efficiency of 17.5% in 'Taishanhong' pomegranate. Subsequent functional analysis revealed that overexpression of PgMYB10 induced pigment accumulation in leaves and stems. In three independent transgenic lines, maximum anthocyanin content and PgMYB10 transcript levels were 5.4-fold and 27.2-fold higher than in wild-type plants, respectively. Six anthocyanin biosynthetic genes (PgCHS, PgCHI, PgF3H, PgDFR, PgANS, and PgUFGT) were markedly upregulated, demonstrating that PgMYB10 positively controls anthocyanin biosynthesis. This transformation system provides a reliable technical platform for functional genomic studies in pomegranate, and PgMYB10 represents as a promising candidate gene for molecular breeding aimed at improving fruit pigmentation.

Anthocyanins↗
PubMed1979

Continuation of fast axonal transport in regenerating axons in vitro.

A previous study by McLean and co-workers reported that regenerating axons of the rabbit vagus nerve were unable to sustain axonal transport in vitro for several months after nerve injury. In contrast, we found that sensory axons of the rat sciatic nerve were able to transport 3H-labeled protein into their regenerating portions distal to the site of injury within a week after injury when placed in vitro. Transport in vitro was not significantly less than transport in axons maintained in vivo for the same period. Transport occurred in the medium that was used by the McLean group, but was significantly reduced in calcium-free medium. When axon regeneration was delared, only small amounts of activity were present in the nerve distal to the site of injury, showing that labeled protein normally present in that part of the nerve was associated with axons and was not a result of local precursor uptake by nonneural elements in the sciatic nerve. We were not able to explain the failure of McLean and co-workers to demonstrate transport in vitro in regenerating vagus nerve, but we conclude that there is no general peculiarity of growing axons that makes them unable to sustain transport in vitro.

Animals↗
PubMed2026

Magnetic nanoparticle-mediated genetic transformation and gene editing system in loquat (Eriobotrya japonica).

Loquat (Eriobotrya japonica Lindl.) is a valuable subtropical fruit tree whose genetic improvement has been significantly constrained by the absence of an efficient genetic transformation system. Although Agrobacterium-mediated transformation is the most widely used method, it proves ineffective in loquat due to the species' recalcitrance to in vitro regeneration. Pollen-based transformation offers a promising alternative by bypassing the need for tissue culture. However, the pollen wall poses a major physical barrier to the uptake of exogenous DNA. In this study, we investigated magnetic nanoparticle (MNP)-mediated transformation as a novel strategy for loquat. We confirmed that loquat pollen contains tricolporate apertures with diameters ranging from 3.0 to 5.0 &#x3bc;m, which are structurally suitable for the entry of MNPs-DNA. Based on this finding, we developed and optimized a transformation protocol using polyethyleneimine-coated Fe3O4 nanoparticles to deliver genetic material into loquat pollen grains. Using this approach, we successfully generated stable transgenic loquat lines, including both overexpression and gene-edited mutants. To our knowledge, this is the first report of successful MNP-mediated pollen transformation in a woody plant species. This work establishes a robust and efficient genetic transformation platform for loquat, providing a valuable tool for functional genomics and molecular breeding, as well as a potentially applicable strategy for other recalcitrant woody plants.

Eriobotrya↗
PubMed2025

Efficient and versatile rapeseed transformation for new breeding technologies.

Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.

Brassica napus↗
PubMed1978

The role of myonuclei in muscle regeneration: an in vitro study.

It is well established that during muscle regeneration, the satellite cells which are in a state of mitotic arrest, can initiate cell division to produce myoblasts which subsequently fuse to form myotubes. However, whether myonuclei, contained within damaged myotubes, or "freed" as a result of the trauma, play any role in muscle regeneration remains unresolved. In myogenic cultures, it is possible to obtain renewed myogenesis when initial cultures are sub-cultured. The aim of this study, was to obtain evidence of the participation by myonuclei of primary cultures in myogenesis which occurs subsequently in secondary cultures. In culture, myonuclei can be labelled with H3-thymidine and their ultimate fate, either as "free" myonuclei or myonuclei associated with disrupted myotubes can be followed unequivocally. Three types of experiments are performed: (i) Primary myogenic cultures containing only myotubes are subcultured. (ii) Primary myogenic cultures containing myotubes with labelled myonuclei are disrupted and subcultured. (iii) Primary myogenic cultures containing myotubes with unlabelled myonuclei are mixed with labelled mononucleated myogenic cells and sub-cultured. In all instances no evidence of myogenesis from myonuclei is obtained. It is concluded that myonuclei, which were rendered postmitotic during myogenesis, remain so when muscle is disrupted and cannot re-enter the mitotic cycle.

Cell Fractionation↗
PubMed1979

Studies on the cytodynamics of human endometrial regeneration. III. In vitro short-term incubation historadioautography.

The proliferation kinetics of endometrial regeneration were assessed by tracing nucleoprotein precursor, radiothymidine. Increased isotope uptake occurred by cycle day 3 and was confined to the gland cells of denuded stratum basale (basal layer) and persistent uterine lining, bordering peripheral areas of denudation. This increase resulted in the formation of flattened surface epithelial cells, which initially were devoid of significant deoxyribonucleic acid synthesis. Such alterations are consistent with changes in cells involved in ameboid migration. Maturation of migrating cells was accompanied by a significant rise in labeling indices and replication at cycle days 3 and 4. By cycle day 5, endometrial repair was complete. The findings indicate that, following physiologic loss of stratum functionale, endometrial healing involves both migration and replication of surface cells, which originate from the gland stumps of the residual basal layer and the persistent surface epithelium adjacent to the periphery of denuded endometrium.

Adult↗
PubMed1977

Factors causing release of ribosomal subunits from isolated nuclei of regenerating rat liver in vitro.

Incubation medium II causes release of ribosomal subunits from isolated prelabeled nuclei of regenerating rat liver in vitro (Sato, T., Ishikawa, K. and Ogato, K. (1976) Biochim. Biophys. Acta 000, 000-000). The effects of individual components of this medium on release of subunits were studied and the following results were obtained. 1. Dialyzed cytosol was effective in causing release of total labeled RNA, but its effect on release of labeled ribosomal subunits was rather lower than that of low molecular yeast RNA. Spermidine inhibited the release of total labeled RNA as well as that of labeled ribosomal subunits. 2. Low molecular yeast RNA was the most effective component for inducing release of labeled ribosomal subunits. Homologous ribosomal RNA was as effective as yeast RNA. Cytoplasmic ribosomes, prepared by washing with solution of high salt concentration, and their subunits were also effective. 3. Transfer RNA was not so effective as yeast RNA and ribosomal RNA and even after heat treatment it had little effect. 4. Among the homopolyribonucleotides tested, polyuridylic acid had a strong effect but polyadenylic acid, polycytidylic acid and polyinosinic acid had no effect. 5. The effects of yeast RNA and polyuridylic acid in causing release of labeled ribosomal subunits were dependent upon their concentrations in the reaction mixture. The characteristics of the factors which cause release of labeled ribosomal subunits in vitro are discussed on the basis of the results.

Animals↗
PubMed1979

In vivo and in vitro studies on endothelial regeneration.

Endothelial regeneration was studied in rabbit aorta after intra-arterial balloon catheterization. Most of the regenerated endothelium originated from existing branches which was assessed by the Evans-blue uptake pattern and confirmed by transmission and scanning electron microscopy. Glucocorticoid treatment enhanced re-endothelialization whereas hyperlipemic diet inhibited. Sera from minipigs fed an atherogenic diet consistently have less ability than sera from control pigs to stimulate in vitro the regeneration of wounded endothelium-like monolayers of 3T3-B cells. The deficiency is probably due to an inhibitor which appears and disappears with changes in the diet.

Animals↗
PubMed1977

Crude transfer-factor preparations stimulate trypsinized human lymphocytes to form rosettes with sheep red cells.

The binding sites for sheep red cells (E) on human lymphocytes are trypsin-sensitive but regenerate in vitro on incubation at 37 degrees C. The rate of this regeneration was increased in the presence of dialysates of human leucocyte extracts (DLE). Thus incubation of trypsinized lymphocytes for 3 hr in appropriate dilutions of DLE resulted in a 2- to 6-fold increase of E-binding activity above that observed in medium-incubated control lymphocytes. Dialysates prepared from human thymus and brain similarly accelerated recovery of E-binding activity whilst dialysates of human fibroblasts and liver cells were inactive. The regeneration of trypsinized membrane immunoglobulin was slightly delayed in the presence of DLE. These findings indicate that leucocyte dialysates contain an activity which preferentially stimulates the regeneration of a T-lymphocyte membrane component. It is suggested that this activity may account for the immunological restoration observed in some patients with T-lymphocyte deficiency after injection of leucocyte dialysates.

Humans↗
PubMed1977

Characterization of ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two different in vitro systems.

The ribonucleoprotein particles released from isolated nuclei of regenerating rat liver in two in vitro systems were studied and the following results were obtained. 1. When the isolated nuclei of regenerating rat liver labeled in vivo with [14C] orotic acid were incubated in medium containing ATP and an energy-regenerating system (medium I) release of labeled 40-S particles was observed. Analysis of these 40-S particles showed that they contained heterogeneous RNA but no 18 S or 28 S ribosomal RNAs and their buoyant density in CsCl was 1.42-1.45 g/cm3, suggesting that they were nuclear informosome-like particles released during incubation. 2. When the same nuclei were incubated in the same medium fortified with dialyzed cytosol, spermidine and yeast RNA (medium II), release of labeled 60-S and 40-S particles was observed. Using CsCl buoyant density gradient centrifugation, two components were found in the labeled ribonucleoprotein particles released from nuclei in this medium. The labeled 60-S particles were found to contain 28-S RNA as the main component and their buoyant density in CsCl was 1.61 g/cm3, suggesting that they were labeled large ribosomal subunits. The labeled 40-S particles contained both 18 S RNA and heterogeneous RNA and they formed two discrete bands in CsCl, at 1.40 and 1.56 g/cm3, suggesting that they contained small ribosomal subunits and nuclear informosome-like particles. 3. These results clearly indicate that addition of dialyzed cytosol, spermidine and low molecular yeast RNA to medium I causes the release of ribosomal subunits or their precursors from isolated nuclei in the in vitro system.

Animals↗